Intestinal cholecystokinin and leptin signaling and the regulation of glucose production By Brittany Anne Rasmussen A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Physiology University of Toronto © Copyright by Brittany Anne Rasmussen (2015)
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Intestinal cholecystokinin and leptin
signaling and the regulation of glucose production
By
Brittany Anne Rasmussen
A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy
Graduate Department of Physiology University of Toronto
© Copyright by Brittany Anne Rasmussen (2015)
ii
Title: Intestinal cholecystokinin and leptin signaling and the regulation of glucose
production
Degree: Doctor of Philosophy
Year of Convocation: 2015
Full Name: Brittany Anne Rasmussen
Department: Physiology, University of Toronto
General Abstract
The number of individuals affected by diabetes is on the rise due in part to lifestyle
and/or genetic factors. Diabetes and obesity are characterized by a disruption in glucose
homeostasis due in part to an elevation in glucose production (GP). It is of utmost importance to
understand the regulation of GP in normal, obese and diabetic settings in hopes to unveil
therapeutic targets that lower blood glucose concentrations in diabetes and obesity.
The small intestine has also been documented to regulate glucose homeostasis
independent of changes in food intake although the intestinal signaling mechanism(s) remain
largely unknown. Specifically, the duodenum senses an increase in lipids and triggers release of
CCK and activates the CCK1 receptor in the duodenum to lower GP via a gut-brain-liver axis.
However, the downstream intestinal CCK1 receptor signaling effectors remain unknown. In
study 1 of this thesis, the signaling molecule PKA was shown, for the first time to our
knowledge, to lie downstream of the duodenal CCK1 receptor to trigger vagal afferent firing
and a gut-brain-liver axis to lower GP. Importantly, direct activation of duodenal PKA lowered
GP and bypassed duodenal CCK resistance in high fat fed rats.
Like the duodenum, the distal part of the small intestine, the jejunum, also senses lipids
to lower GP via a gut-brain-liver axis, but whether hormonal action mediates this GP-lowering
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effect of the jejunum remains unknown. In study 2 of this thesis, a novel GP-lowering effect of
leptin in the jejunum was described. Specifically, jejunal leptin activated the long form leptin
receptor and PI3K to lower GP in normal, high fat fed or uncontrolled diabetic rodents via a
neuronal network, and contributed to the early anti-diabetic effect of bariatric surgery.
In conclusion, this doctoral thesis demonstrates that independent activation of duodenal
CCK-PKA and jejunal leptin-PI3K signaling potently lowers GP in normal, high-fat fed and
diabetic rodents via a gut-brain-liver neuronal axis. Thus, targeting hormonal (i.e., CCK and
leptin) signaling in the small intestine represents a potential therapeutic strategy to lower GP and
restore glucose homeostasis in diabetes and obesity, and may mimic the anti-diabetic effect of
bariatric surgery.
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Acknowledgements
I would first like to thank my supervisor Dr. Tony Lam who has constantly challenged
me throughout my studies to help me become an independent and critical thinker. I appreciate
all the effort you have made in helping me become the best I can be academically and for your
support in my career decisions. I am also thankful for my committee members, Dr. Adria Giacca
and Dr. Khosrow Adeli who have taught me to think on the spot, think critically, and search for
the best explanation. Their knowledge and wisdom continues to inspire me throughout every
committee meeting and presentation I give on my research. I would not be where I am today
without the support of the Lam Lab. I have met some incredible people along this journey who
have acted not only as mentors but also as great friends. I would like to especially thank Dr.
Danna Breen, for all of the laughs we shared as well as challenging me to become a better
student. I would also like to thank the rest of the “gut” team, Clémence Côté, Melika Zadeh
Tahmesabi, Dr. Frank Duca, Sophie Hamr and Paige Bauer. You have been an amazing team to
work with and have also provided moral support throughout my studies. I seriously could not
have done it without you guys! I would also like to thank the rest of the lab members, Penny
Wang, Elena Burdett, Claire Yang, Patricia Mighiu, Dr. Jessica Yue, Dr. Beatrice Filippi, Mona
Abraham, Mary LaPierre, and Beini Wang. It has been wonderful getting to know each and
everyone of you and your help and support was always appreciated. My family is of utmost
importance to me and I would like to extend a big thank you to my parents, Rodney and Patricia
Rasmussen, who have never questioned my career choices and have supported me with in any
decisions I have made. Also to by brothers Ryan and Jordi (and Loy and John) and sister Lauren
for their continued support throughout my academic career. Lastly, to my husband Oliver. All I
can say is that I would have never made it to this point without you and I love you very much.
You have always provided comfort and support and I am forever grateful to have you in my life.
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Table of Contents Introduction ...................................................................................................................... 1 Chapter 1
1.1 Obesity and Diabetes ................................................................................................................................ 1 1.2 The small intestine and the regulation of metabolic homeostasis ................................................... 6
1.2.1 Local gut-brain paracrine effect versus the endocrine effect of gut-derived hormones ............... 6 1.3 Gastrointestinal Peptides ......................................................................................................................... 9
1.3.1 Gastric Peptides .................................................................................................................................................... 9 1.3.2 Proximal Intestinal Peptides ........................................................................................................................... 15 1.3.3 Distal Intestinal Peptides ................................................................................................................................. 20
1.4 Small intestine control of glucose production through a gut-brain-liver neuronal axis ......... 30 1.4.1 Duodenal lipid sensing and CCK secretion triggers a gut-brain-liver axis to lower glucose production ........................................................................................................................................................................ 30 1.4.2 Jejunal nutrient sensing triggers a gut-brain-liver axis to lower glucose production .................. 37
1.5 Bariatric surgery, gut hormones and intestinal nutrient sensing ................................................ 39 1.5.1 Bariatric surgical procedures and changes in gut hormones ................................................................ 39 1.5.2 Duodenal jejunal bypass surgery, nutrient sensing and beyond ......................................................... 44
1.6 Summary of Introduction ..................................................................................................................... 49 1.6 Rationale and Significance of the Studies ......................................................................................... 49 1.7 General Hypothesis ................................................................................................................................ 51 1.8 Specific Aims ........................................................................................................................................... 51
General Methods .......................................................................................................... 52 Chapter 22.1 Animals ..................................................................................................................................................... 52
2.1.1 High Fat Feeding Animal Model .................................................................................................................. 52 2.2 Surgical Procedures ............................................................................................................................... 52
2.2.1 Vessel Cannulation ............................................................................................................................................ 53 2.2.2 Intestinal Cannulation ....................................................................................................................................... 53
2.3 Pancreatic Euglycemic (Basal Insulin) Clamp Technique ............................................................ 54 2.4 Protein Assay ........................................................................................................................................... 55 2.5 Biochemical Analyses ............................................................................................................................. 56
2.5.1 Plasma Glucose ................................................................................................................................................... 56 2.5.2 Plasma Glucose Tracer Specific Activity .................................................................................................. 56 2.5.3 Plasma Insulin ..................................................................................................................................................... 57
2.6 Calculations ............................................................................................................................................. 58 2.7 Statistical Analysis .................................................................................................................................. 58
Study 1 ............................................................................................................................. 60 Chapter 33.1 Abstract .................................................................................................................................................... 61 3.2 Introduction ............................................................................................................................................. 62 3.3 Materials and Methods .......................................................................................................................... 64
3.3.1 Animal Preparation ............................................................................................................................................ 64 3.3.2 Animal Surgeries ................................................................................................................................................ 64 3.3.3 Intraduodenal Infusions and Treatments .................................................................................................... 66 3.3.4 Pancreatic Euglycemic (Basal Insulin) Clamp Technique in Rats .................................................... 66 3.3.6 PKA Activity Assay .......................................................................................................................................... 68 3.3.7 PCR methods ....................................................................................................................................................... 69 3.3.9 Biochemical Analysis ....................................................................................................................................... 71 3.3.10 Calculations and Statistical Analysis ........................................................................................................ 71
3.4 Results ....................................................................................................................................................... 72 3.4.1 Direct activation of PKA lowers glucose production ............................................................................ 72 3.4.2 Activation of PKA lowers glucose production via a vagal afferent firing ...................................... 73
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3.4.3 Activation of NR1-containing NMDA receptors is required for duodenal PKA to lower glucose production ........................................................................................................................................................ 74 3.4.4 Duodenal PKA activation requires brain to liver communication to lower glucose production ............................................................................................................................................................................................. 76 3.4.5 CCK lowers glucose production via PKA activation ............................................................................. 76 3.4.6 The CCK1 receptor fails to activate PKA after short term high fat feeding .................................. 77
3.5 Discussion ................................................................................................................................................. 78 Study 2 ............................................................................................................................. 97 Chapter 4
4.1 Abstract .................................................................................................................................................... 98 4.2 Introduction ............................................................................................................................................. 99 4.3 Materials and Methods ........................................................................................................................ 100
4.3.1 Animal Preparation ......................................................................................................................................... 100 4.3.2 Animal Surgeries ............................................................................................................................................. 103 4.3.3 Intraintestinal infusions and treatments ................................................................................................... 105 4.3.4 Pancreatic (Basal Insulin) Euglycemic Clamp Technique ................................................................ 106 4.3.5 Rat [3–3H] glucose infusion protocol (non-clamped conditions) .................................................... 107 4.3.6 Fasting and refeeding protocol ................................................................................................................... 108 4.3.7 Gut tissue collection and preparation for western blotting and enzymatic activity assay ...... 108 4.3.8 Western blotting .............................................................................................................................................. 109 4.3.9 RNA extraction, reverse transcription and PCR methods ................................................................. 110 4.3.10 PI3K Activity Assay ............................................................................................................................... 112 4.3.11 Biochemical Analysis .................................................................................................................................. 113 4.3.12 Calculations and Statistical Analysis ..................................................................................................... 114
4.4 Results ..................................................................................................................................................... 115 4.4.1 Jejunal leptin requires jejunal leptin receptor activation to lower glucose production ............ 115 4.4.2 A STAT3-independent and PI3K-dependent signaling pathway is required for jejunal leptin to lower glucose production via a neuronal network .......................................................................................... 117 4.4.3 Jejunal leptin’s action remain intact in high fat fed or diabetic rats .............................................. 119 4.4.4 The antidiabetic effect of DJB surgery is mediated by jejunal leptin action ............................... 121
4.5 Discussion ............................................................................................................................................... 122 Summary and Conclusions ...................................................................................... 144 Chapter 5
5.1 Summary of Studies in this Thesis .................................................................................................... 144 5.2 General Summary ................................................................................................................................ 145 5.3 General Conclusion .............................................................................................................................. 145
General Discussion .................................................................................................... 147 Chapter 66.1 Do nutrient sensing mechanisms interact with both CCK and leptin? .................................... 147 6.2 What other intestinal hormones share similar signaling mechanisms as CCK and leptin? 149
6.2.1 PKA ..................................................................................................................................................................... 149 6.2.2 PI3K ..................................................................................................................................................................... 150
6.3 What is the cellular location of CCK-PKA and leptin-PI3K signaling in the intestine? ...... 151 6.4 What is the relevance of CCK and leptin signaling in disease models? ................................... 153
Limitations of the Studies ........................................................................................ 156 Chapter 7
Future Directions ....................................................................................................... 159 Chapter 8 References .................................................................................................................... 164 Chapter 9
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List of Abbreviations
AA Arachidonic acid ACC Acetyl-CoA carboxylase ACS Acyl-CoA synthetase AG Acylated-ghrelin AMPK Adenosine monophosphate activate protein ANOVA Analysis of variances ARC Arcuate nucleus ATP Adenosine trisphosphate B Bound fraction B0 Total binding BB-dp Diabetes-prone BioBreeding rat BBB Blood brain barrier BMI Body mass index BPD/DS Bilio-pancreatic diversion/duodenal switch BSA Bovin serum albumin cAMP Cyclic adenosine monophosphate cAMP-GEFII; Epac2
cAMP guanine nucleotide exchange factor II Exchange protein directly activated by cAMP 2
CaSR CCK1 receptor
Calcium sensing receptor Cholecystokinin 1 receptor
CCK2 receptor Cholecystokinin 2 receptor CD36 Cluster determinant 36 CCK Cholecystokinin CNS Central nervous system CPT-1 Carnitine almitoyltrasnferase-1 DAG diacylglyercol db/db mouse Long form leptin receptor knock out mouse DJB Duodenal jejunal bypass DPP-IV Dipeptidyl peptidase IV DVC Dorsal vagal complex Ex-4 Exendin-4 Ex-9 Exendin-9 Fa/fa rat Lean koletsky rat Fak/fak rat Obese koletsky rat FFA FTO
Free fatty acids Fat mass and obesity associated gene
GCGR GWAS
Glucagon receptor Genome wide association studies
GHSR Growth hormone secretagogue receptor 1a GIP Glucose-dependent insulinotropic peptide GIPR Glucose-dependent insulinotropic peptide receptor GLP-1 GLP-1R
Glucagon like peptide-1 Glucagon like peptide-1 receptor
GLP-2 Glucagon-like peptide-2 GLP-2R Glucagon-like peptide-2 receptor GLUT2 Glucose transporter 2
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GOAT Ghrelin O-acyltransferase gp130 Class I cytokine receptor family GPCR G-protein coupled receptor GPR120 G-protein coupled receptor 120 GPR40 G-protein coupled receptor 40 GSIS Glucose stimulated insulin secretion HFD High fat diet HRP Horseradish peroxidase i.c.v Intracerebroventricular i.p. Intraperitoneal IP3 Inositol triphosphate IRS1 Insulin receptor substrate 1 KATP channels ATP-sensitive potassium channels LAGB Laparoscopic adjustable gastric band LCFA Long chain fatty acids MAPK Mitogen activated protein kinase MUNC18-1 mammalian uncoordinated-18 1 NMDA receptor N-methyl-D-aspartate receptor NPY Neuropeptide y NTS Nucleus of the solitary tract OAG 1-oleoyl-2-acetyl-sn-glycerol Ob-Ra Leptin receptor isoform A Ob-Rb; Leprb Long form leptin receptor Ob-Re Leptin receptor isoform E OXN oxyntomodulin PC Prohormone convertase PI3K Phosphatidylinositol-3-OH kinase PKA Protein kinase A PKC Protein kinase C PLC Phospholipase C POMC Pro-opimelanocortin PYY Peptide YY Ra Rate of appearance Rd RC RIA
Rate of disappearance Regular chow Radioimmunoassay
RYGB Roux-en-Y gastric bypass SD rat Sprague dawley rat SG Sleeve gastrectomy SGLT-1 Sodium glucose transporter-1 SLR Soluble leptin receptor SNARE Soluble NSF Attachment Protein REceptor STAT3 Signal transduction and activator of transcription -3 STAT3 PI Signal transduction and activator of transcription -3 peptide
inhibitor STC-1 cell line Secretin tumor cell-1 cell line STZ Streptozotocin TBST Tris buffered saline-tween VAMP2 Vesicle-associated membrane protein-2
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List of Tables
Table 2.1 Diet content of the regular chow and lard-oil enriched high fat diet. .......................... 59
Table 3.1 Plasma insulin and glucose concentrations of the groups receiving an intraduodenal infusion during basal and clamp conditions. ....................................................................... 94
Table 3.2. Plasma insulin and glucose concentrations of the groups receiving both an intraduodenal infusion and DVC infusion during basal and clamp conditions ................... 95
Table 3.3 Plasma insulin and glucose concentrations of the groups receiving an intraduodenal infusion during basal and clamp conditions. ....................................................................... 96
Table 4.1 Plasma insulin and glucose concentrations of groups receiving intrajejunal infusions during the basal and clamp conditions .............................................................................. 143
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List of Figures
Figure 1.1 Site of synthesis and secretion of gastrointestinal peptide hormones. …………….....8
Figure 1.2 Duodenal and jejunal nutrient sensing mechanisms trigger a gut-brain-liver neuronal axis to lower glucose production. ………………………………………………………………47
Figure 3.1 Schematic representation of working hypothesis – duodenal Sp-CAMPS activates PKA to lower glucose production, which is abolished upon co-infusion of Sp-CAMPS and H-89 or Rp-CAMPS, and experimental design. .................................................................. 81
Figure 3.2 Duodenal PKA activation lowers glucose production. .............................................. 82
Figure 3.3 Schematic representation of working hypothesis – duodenal PKA activation increases vagal afferent firing ............................................................................................................. 83
Figure 3.4 Direct activation of duodenal PKA increases the spontaneous discharge rate of the mesenteric nerve and inhibits spinal afferent firing of the duodenum. ............................... 84
Figure 3.5 Schematic representation of working hypothesis – duodenal PKA activation triggers a neuronal network to lower glucose production and experimental design. ........................ 85
Figure 3.6 Duodenal PKA activation lowers glucose production through a neuronal network. . 86
Figure 3.7 Schematic representation of working hypothesis – duodenal PKA activation lowers glucose production through a gut-brain-liver neuronal axis and experimental design. ....... 87
Figure 3.8 Duodenal PKA activation lowers glucose production through activation of the DVC NR1- containing NMDA receptor and hepatic innervation. ................................................ 88
Figure 3.9 Schematic representation of working hypothesis – Duodenal CCK requires PKA activation to lower glucose production and experimental design. ....................................... 89
Figure 3.10 Duodenal CCK requires PKA activation to lower glucose production. ................... 90
Figure 3.11 Schematic representation of working hypothesis – Duodenal CCK fails to suppress glucose production upon high fat feeding, which is rescued upon PKA activation and experimental design ............................................................................................................. 91
Figure 3.12 Duodenal CCK fails to activate duodenal PKA and lower glucose production after three days of high fat feeding. ............................................................................................. 92
Figure 3.13 Duodenal Sp-CAMPS activates duodenal PKA activity and lowers glucose production in high fat diet fed rats. ...................................................................................... 93
Figure 4.1 Schematic representation of the working hypothesis – Gastric leptin activates the intestinal long form leptin receptor to activate a PI3K-dependent and STAT-3 independent signaling axis to lower glucose production through a neuronal network. ......................... 126
Figure 4.2 Leptin receptor expression in intestinal tissue. ........................................................ 127
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Figure 4.3 Jejunal leptin administration lowers glucose production. ........................................ 128
Figure 4.4 Jejunal leptin lowers glucose production independent of changes in portal and circulating leptin levels. ..................................................................................................... 129
Figure 4.5 Leptin activates leptin receptors to lower glucose production in rats (chemical approach). .......................................................................................................................... 130
Figure 4.6 Leptin activates leptin receptors to lower glucose production in lean fa/fa rats but not in fak/fak (Kolestky) long form leptin receptor deficient rats (molecular approach). ........ 131
Figure 4.7 Jejunal leptin activates leptin receptors to lower glucose production in C57BL/6 but not db/db mice (molecular approach). ............................................................................... 132
Figure 4.8 Jejunal leptin lowers glucose production in C57BL/6 independent of changes in circulating leptin levels. ..................................................................................................... 133
Figure 4.9 Jejunal leptin lowers glucose production through a STAT3-independent and PI3K dependent pathway. ........................................................................................................... 134
Figure 4.10 Jejunal and duodenal leptin activate intestinal STAT3, and only jejunal leptin activates jejunal PI3K. ....................................................................................................... 135
Figure 4.11 Jejunal leptin lowers glucose production through a neuronal network. ................. 136
Figure 4.12 Jejunal leptin lowers glucose production in high fat diet fed rats. ......................... 137
Figure 4.13 Jejunal leptin lowers glucose production in high fat diet fed rodents independent of a rise in plasma leptin levels. ............................................................................................. 138
Figure 4.14 Jejnual leptin lowers plasma glucose levels and glucose production in uncontrolled diabetic rodents independent of changes in plasma insulin and glucagon levels. ............. 139
Figure 4.15 Jejunal leptin lowers plasma glucose levels and glucose production in uncontrolled diabetic rodents independent of a rise in plasma leptin levels. .......................................... 140
Figure 4.16. Schematic of duodenal-jejunal bypass (DJB) surgery and jejunal catheter placement. .......................................................................................................................... 141
Figure 4.17 Jejunal leptin action mediates the rapid anti-diabetic effect of DJB surgery. ........ 142
Figure 5.1 Summary of duodenal and jejunal hormonal signaling that triggers a neuronal network to lower glucose production ................................................................................ 146
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Published Manuscripts that Contributed to this Thesis
Review papers: Rasmussen, BA*, Breen, DM* and Lam, TK. Lipid sensing in the gut, brain and liver. Trends Endocrinol Metab 23, 49-55, 2011 *Equal contribution Permission to reproduce portions of the above manuscript has been obtained from the copyright owner: Elsevier Limited Rasmussen, BA*, Breen, DM*, Côté, CD, Jackson, M, and Lam, TK. Nutrient sensing mechanisms in the gut as therapeutic targets for diabetes. Diabetes 62, 3005-3013, 2013 *Equal contribution Permission to reproduce portions of the above manuscript has been obtained from the copyright owner: American Diabetes Association Côté, CD*, Zadeh-Tahmasebi, M*, Rasmussen, BA, Duca, FA, and Lam, TK. Hormonal Signaling in the gut. J Biol Chem, 289, 11642, 2014 *Equal contribution Permission to reproduce portions of the above manuscript has been obtained from the copyright owner: American Society for Biochemistry and Molecular Biology Study 1 (Chapter 3): Rasmussen, BA, Breen, DM, Luo, P, Cheung, GW, Yang, CS, Sun, B, Kokorovic, A, Rong, W, and Lam, TK. Duodenal activation of cAMP-dependent protein kinase induces vagal afferent firing and lowers glucose production in rats. Gastroenterology 142, 834-843, 2012 Permission to reproduce portions of the above manuscript has been obtained from the copyright owner: Elsevier Limited Study 2 (Chapter 4): Rasmussen, BA*, Breen, DM*, Duca, FA, Côté, CD, Zadeh Tahmasebi, M, Filippi, BM, and Lam, TK. Jejunal leptin-PI3K signaling lowers glucose production. Cell Metabolism 19, 1-7, 2014 *Equal contribution Permission to reproduce portions of the above manuscript has been obtained from the copyright owner: Elsevier Limited Other studies contributing to the completion of this thesis: Breen, DM, Yue, JT, Rasmussen, BA, Kokorovic, A, Cheung, GWC, and Lam, TK. Duodenal PKC-δ and cholecystokinin signaling axis regulates glucose production. Diabetes 60, 3148-3153, 2011
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Breen, DM, Rasmussen BA, Kokorovic, A, Rennian, W, Cheung, GW, and Lam, TK. Jejunal nutrient sensing is required for duodenal-jejunal bypass surgery to rapidly lower glucose concentrations in uncontrolled diabetes. Nature Medicine 18, 950-955, 2012 Duca, FA, Côté, CD, Rasmussen, BA, Zadeh-Tahmasebi, M, Rutter, GA, Filippi, BM, and Lam, TK. Metformin activates a duodenal Ampk-dependent pathway to lower hepatic glucose production. Nature Medicine In Press Côté, CD*, Rasmussen, BA*, Duca, FA*, Zadeh-Tahmasebi, M, Baur, JA, Daljeet, M, Breen, DM, Filippi, BM, and Lam TK. Duodenal Sirt1 activation reverses insulin resistance through a neuronal network in rats. Nature Medicine In Press *Equal contribution
1
Chapter 1Introduction
1.1 Obesity and Diabetes
The incidence of individuals who are obese or overweight has more than doubled since
1980 with 2.1 billion people worldwide being either overweight (BMI ≥ 25) or obese (BMI ≥
30) as of 20131. More alarmingly, the number of children and adolescents (ages 2-19) that are
obese or overweight has risen since 1980 in both boys and girls in developing and developed
countries1, which will likely persist into adulthood2. Obesity is a serious risk factor for life
threatening co-morbidities such as cardiovascular disease, hypertension, type 2 diabetes, cancer
and premature mortality3 and costs the Canadian Healthcare System an average of $5.5 billion
annually4. Given that the number of obese and overweight individuals is predicted to increase1,
it is of utmost importance to understand the pathogenesis of this disease in hopes to reduce its
associated health and economic burdens.
Under normal physiological conditions mammals achieve a remarkably stable body
weight by maintaining energy homeostasis by matching overall energy intake and expenditure
over long periods of time. This tight homeostatic regulation is traditionally believed to involve a
complex integration of acute and chronic metabolic, neural and hormonal factors. For example,
postprandial release of gut hormones activate a local paracrine gut-brain axis5 and gut-brain-
brown fat neuronal axis6 to acutely regulate food intake and energy expenditure, respectively,
where circulating endocrine chronic signals such as insulin and leptin control the overall
metabolic state of adipose stores7,8 as well as feeding9 and energy expenditure10 via the central
nervous system (CNS). Obesity is caused by a shift in energy balance, favoring increased energy
2
intake and decreased expenditure due to disruptions in the aforementioned gut-brain food intake
axis11–16, as well as central insulin17–21 and leptin resistance22–28. Contributors to these defects
include genetic, environmental and/or social factors. For example, studies have demonstrated
that humans with mutations in genes such as leptin29 (long term energy regulator) or the
melanocortin 4 receptor gene30 (regulates gut peptides release31 thus may acutely regulate
energy homeostasis, as well as chronically regulate adipose stores32) are obese, demonstrating a
monogenic effect on the development of obesity. However, genetic predisposition to obesity in
most individuals is polygenic whereby the presence of genetic variations in multiple genes
contributes to its development. For example, common gene variations associated with increased
BMI are beginning to be uncovered by the genome-wide association studies (GWAS)33 such as
in the MC4R gene and the fat mass and obesity associated gene (FTO)34 (although the FTO link
to obesity is recently debated35). However, given the rapid rise in the incidence of obesity over
the last 50 years, it is unlikely that genetic changes are the main culprit for the obesity pandemic,
and indeed, identical twins are discordant for obesity36. As such, it is more likely that changing
environmental and lifestyle factors are the primary engines of the current pandemic. For
example, daily stress as well as social habits and cues all play a significant role in both daily
consumption (or overconsumption in the case of obesity including increased intake of energy
dense foods) as well as decreased energy expenditure (or reduced physical activity in the case
for obesity, although still debated37,38). Taken together, obesity is likely resultant of a complex
interplay of environmental factors and each individual’s genetic susceptibility to these factors,
which leads to a disruption in energy homeostasis.
Despite the complex and multifactorial etiology of this disease, recent advancements in
the understanding of the pathogenesis of obesity has led to the development of drug therapies
aimed at reducing energy intake and/or increasing energy expenditure. Many have demonstrated
moderate to substantial weight loss such as Orlistat (which inhibits pancreatic lipase and thus
3
lipid absorption from the gut thereby reducing caloric intake) and sibutramine (a serotonin
reuptake inhibitor which reduces energy intake and increases energy expenditure39) among
others. However, these therapeutic options come with mild side effects such as uncontrolled and
oily bowel movements (seen with orlistat treatment) as well as more serious side effects such as
cardiovascular problems (i.e., serotonin releasing agents fenfluramine, dexfenfluramine and
sibutramine, which have now been removed from the market) or depression (such as rimonabant
which activates cannabinoid CB1 receptors in the brain)40,41. Given the limited success and
potential risks of obesity drug treatments, currently the most successful weight loss intervention
is gastric bypass surgery, which has shown significant and sustained clinical improvements such
as decreased body weight and food intake (while the effects on energy expenditure are currently
debated42). However these surgeries are extremely invasive and have various surgical
complications and at times a need for reoperation43. Thus, it remains of utmost importance to
continue to dissect the mechanism(s) underlying the regulation of body weight to unveil
potential targets to develop successful therapies without associated risks and side effects.
Interestingly, surgical intervention techniques44 aimed at weight loss improve
hyperglycemia, the hallmark of diabetes, highlighting the pathological interconnectivity of the
two diseases. Indeed, as mentioned previously, obesity is a primary cause of type 2 diabetes, as
80-90% of type 2 diabetic individuals are obese/overweight in Canada alone45. Diabetes affects
an alarming amount of people, estimated at 382 million worldwide by the International Diabetes
Federation46. More worryingly, similar to obesity, the number of children and adolescents
affected by the disease has also risen47,48. Within Canada, more than 9 million individuals live
with diabetes or prediabetes and it is estimated that diabetes will cost the Canadian healthcare
system $16.9 billion a year by 202049. Understanding the regulation of glucose homeostasis in
both a normal and diabetic setting will begin to uncover targets to restore the regulation of
glycemia.
4
Similar to energy homeostasis, under normal conditions, glucose homeostasis is tightly
regulated by controlling the rate of glucose production and uptake in both the fasted and fed
state. In the fasting state, glucose levels are maintained by the hormone glucagon, which after its
secretion from pancreatic α cells binds to its receptor on the liver to increase glucose
production50. This works in an opposite fashion to the hormone insulin, produced from
pancreatic β-cells, which prevents hyperglycemia through a suppression of glucose production
and the stimulation of glucose uptake51. In the fasting state, insulin levels remain low as to not
counteract the effect of glucagon and to prevent peripheral glucose uptake. Thus, through a
counterbalance of these two hormones, circulating glucose levels will rise under fasting
conditions to ensure sufficient energy for distribution to various organs.
In direct contrast, intake of a meal leads to exogenous sources of glucose entering the
system from absorption of glucose from the intestinal lumen, changing the state from fasting to
fed conditions. In this fed condition, there now exists both endogenous and exogenous sources
of glucose and the regulation of glucose homeostasis shifts to ensure circulating glucose levels
are not too high. The control of nutrient delivery into the small intestine is based on the gastric
emptying rate which is a major physiological determinant of postprandial glycemia after a meal,
accounting for ~35% of peak glucose concentrations after ingestion of oral glucose in healthy
volunteers52,53. In addition to promoting secretion of incretin hormones, glucose then enters the
circulation to trigger the first phase insulin response whereby insulin is rapidly secreted to reach
an initial short lived peak within 5 to 7 minutes, lasting around 10-15 minutes54. Following this
initial first phase of insulin secretion, the second phase is characterized by a steady and long-
lasting increase in plasma insulin concentrations54 where glucagon secretion is suppressed as to
not counteract the effects of insulin. In addition, nutrient induced gut peptide release locally
activates a gut-brain-liver axis to regulate glucose production, which will be described in detail
later in this introduction. Together, these mechanisms ensure that endogenous glucose
5
production is suppressed and peripheral glucose uptake is stimulated to account for the
exogenous glucose entering the system and ensure glucose concentrations are maintained in
their normal homeostatic range.
Defects in the aforementioned homeostatic regulation of glucose levels result in fasting
hyperglycemia in type 1 and 2 diabetes. As type 1 diabetes is characterized by an autoimmune
response that destructs insulin producing pancreatic β-cells55, medications aim to increase
insulin levels to lower plasma glucose levels. Type 2 diabetes, the current focus of this thesis, is
characterized by increased glucose production, peripheral insulin resistance (possibly caused
through an increase in circulating fatty acids seen with obesity56), reduced/altered insulin
secretion, and elevated glucagon levels57,58. Given that fasting hyperglycemia in type 2 diabetes
is largely due to an increase in the rate of glucose production59, development of drug therapies
aimed at reducing glucose production may prove efficacious. Indeed, metformin, the most
widely prescribed type 2 diabetic drug, reduces hyperglycemia via a reduction in glucose
production60, however treatment has been associated with gastrointestinal discomfort and lactic
acidosis60. More recently, incretin based therapies, aimed at increasing the levels and action of
gut-derived incretin hormones have proven successful to lower glucose levels61 and body
weight41. Even more effective than pharmacological interventions for glucose control is bariatric
surgery62,63, which lowers glucose production64 and glycemia in association with changes in gut
peptides65. Taken together, the early anti-diabetic effect of these drugs and bariatric surgery
highlights the role of the intestine in the development of obesity and diabetes and its therapeutic
potential as a target site to lower glucose levels to reduce the risk of diabetic complications. The
focus of this current thesis is to characterize the gluco-regulatory role of gut-derived peptides
and how they contribute to the pathogenesis of obesity and diabetes.
6
In summary, the small intestine has been viewed as an organ that acutely regulates
feeding, nutrient digestion and metabolism via nutrient induced gut peptide release and
subsequent activation of a local paracrine gut-brain axis as well as through an endocrine fashion
activating peripheral and/or CNS targets. While the ability of gut derived hormones to regulate
glycemia via direct tissue action has been largely studied, only more recently has it been
demonstrated that gut-derived peptides locally trigger a gut-brain axis to acutely regulate
glycemia, similar to feeding regulation. In the following section, the contribution of the local
gut-brain axis versus direct tissue endocrine action of gut derived hormones on feeding and
glucose regulation will be discussed.
1.2 The small intestine and the regulation of metabolic homeostasis
1.2.1 Local gut-brain paracrine effect versus the endocrine effect of gut-derived hormones
The gastrointestinal tract is the first point of contact between nutrients and the host
whereby initiation of negative feedback mechanisms to maintain metabolic homeostasis first
takes place. The gastrointestinal tract relays information of an incoming meal, such as the size
and composition via both gastric and intestinal signals, which are integrated within the CNS,
more specifically the hindbrain, to ultimately reduce food intake. More specifically, the
hindbrain integrates signals of both chemical (endocrine) and neural origin (via
mechanoreceptors or local activation of neurons innervating the intestine). For example, nutrient
delivery into the stomach can be sensed by mechanoreceptors that detect tension66, stretch67 and
volume68, and these mechanical signals are relayed to the brain via spinal and spinal nerves. In
addition to gastric mechanical signals, both the stomach and small intestine can send chemical
signals to relay nutritional status via secretion of gut-derived hormones. More specifically, the
inner lining of the GI tract houses a single layer of epithelial cells containing specialized
enteroendocrine (EEC) cells, which express nutrient sensing elements on the apical side.
7
Following a meal, activation of the nutrient sensing elements leads to the triggering of
intracellular signaling pathways, which results in the depolarization of the cell membrane
causing the release of gut hormones expressed within the EEC. These hormones can directly
enter the bloodstream to act within the periphery to control food intake and pass through the
leaky blood brain barrier to target the central nervous system directly. In contrast, these gut
peptides can act in a paracrine fashion to activate their corresponding receptors on vagal
afferent terminals innervating the small intestine to trigger a gut-brain neuronal axis to acutely
control feeding and metabolism. Indeed, intestinal nutrient infusions reduce food intake within
minutes suggesting activation of local intestinal signals is required for the acute effects on
feeding rather than postabsorptive affects69.
Similar to appetite regulation, nutrient induced secretion of gut-derived hormones has
been demonstrated to regulate glycemia via endocrine actions via either inhibition or stimulation
of the release of insulin or glucagon directly or via their action within the CNS to control
glucose production or pancreatic hormone release70. In addition, nutrients in the preabsorptive
state can activate sensing mechanisms in the small intestine via local gut peptide release and
subsequent activation of a gut-brain negative feedback system to regulate gastric emptying71 and
thus control the rate of glucose entry into the blood, as well as inhibit glucose production by the
liver69 to acutely regulate glucose levels. The purpose of the current thesis is to dissect the local
paracrine effect of gut-derived hormones on the regulation of glucose homeostasis.
The next focus will review the paracrine versus endocrine metabolic regulatory
mechanisms of specific hormones such as from the stomach: leptin and ghrelin, from the
duodenum/jejunum: CCK and glucose-dependent insulinotropic peptide (GIP) (and possibly L
cell derived hormones), and from the ileum: glucagon-like peptide-1/2 (GLP-1/2),
oxyntomodulin (OXN), and peptide YY (PYY) (Figure 1.1).
8
Figure 1.1 Site of synthesis and secretion of gastrointestinal peptide hormones
Shown is the site of synthesis as well as secretion of both stomach and small intestinal derived hormones. The stomach produces and secretes ghrelin and leptin. The duodenum and jejunum synthesize and secrete CCK and GIP. It is currently debated whether the duodenum contains L cells and thus synthesizes and secretes GLP-1/2, OXN and PYY, which are more commonly thought to arise from the ileum. Adapted from Côté, CD*& Zadeh-Tahmasebi, M* et al. Hormonal Signaling in the gut. J Biol Chem, 289, 11642, 2014 *Equal contribution. Permission to reproduce this figure has been obtained from the copyright owner: American Society for Biochemistry and Molecular Biology
9
1.3 Gastrointestinal Peptides
1.3.1 Gastric Peptides
1.3.1.1 Ghrelin
Ghrelin is a 28 amino acid peptide expressed in various tissues such as the stomach,
intestine, pituitary, pancreas, kidney, lung, ovaries and brain72. The stomach is the major source
of ghrelin, which is synthesized in endocrine X/A cells of the gastric mucosa73 and is highly
concentrated in the fundic region74,75. In order to become active, ghrelin must undergo multiple
cleavage steps starting as a 117 amino acid pre-prohormone. First, the removal of a secretory
signal peptide at its N-terminus and cleavage at its C-terminus by prohormone convertase
(PC)1/3 is needed to result in a prohormone76,77. Second, ghrelin undergoes esterification by an
acyl-transferase, ghrelin O-acyltransferase (GOAT)78 becoming acylated-ghrelin (AG), which
accounts for approximately 10-20% of circulating ghrelin79. It is traditionally believed that only
after acylation by GOAT that ghrelin binds to its widely expressed receptor, the growth
hormone secretagogue receptor 1a (GHSR)80, however recent data suggests that des-acyl ghrelin
(originally believed to be a non-active form) may also bind to the GHSR to exert biological
effects81. With respect to nutrient induced regulation of ghrelin secretion, glucose, amino acids,
and lipids can all suppress ghrelin secretion. However, carbohydrates are its most potent
suppressor, followed by proteins and lipids82,83.
After its secretion, ghrelin acts as a “hunger hormone”. This is due to the fact that an
increase in ghrelin levels has been associated with timing of a meal in both rodents and
humans84,85, peaking at meal initiation followed by a postprandial decrease back to baseline72.
Indeed, peripheral and central administration of ghrelin increases food intake in both rodents
and humans86–88 which is abolished upon intraperitoneal (i.p.) co-administration of a GHSR
antagonist or anti-ghrelin immunoglobulin, instead resulting in a decrease in food intake89,90.
Given these findings, it is evident that ghrelin may act in an endocrine and/or paracrine fashion.
10
Indeed, ghrelin can cross the blood brain barrier (BBB) where it is thought to activate different
regions of the brain to control food intake, including the hypothalamus91,92 and brain stem93,94
which has been widely studied (recently reviewed in95). Alternatively, vagal afferents that
innervate the stomach express the ghrelin receptor and in both rodents and humans, and
vagotomy (which eliminates communication between the stomach and brain) abolishes the
ability of ghrelin to increase food intake96,97, demonstrating a local paracrine effect of ghrelin on
feeding regulation. Thus, it is evident that ghrelin induces feeding, whether through its intestinal
and/or brain action. Likewise, ghrelin also down regulates receptor expression for anorexigenic
peptides such as PYY, GLP-1, and CCK98,99, further emphasizing its orexigenic effects.
Not only does ghrelin regulate feeding, but it also plays a role in the maintenance of
blood glucose levels in the fasting condition. In rodents, administration of AG can cause a rapid
inhibition of glucose stimulated insulin secretion (GSIS)100, through its direct action on the
pancreas101. This is strengthened by the findings that blockade of AG102 and the GHSR101
improves the insulin response to a glucose challenge, and that the GHSR is located in pancreatic
islets103–107. These findings have been translated to humans, where administration of higher
doses of AG suppressed GSIS108. In contrast to its peripheral actions, central ghrelin works in an
opposite fashion by acting as a positive regulator of insulin secretion81,109. This implies that
peripheral AG action may counteract the hyperinsulinemic action of central AG. However,
future studies are needed to better understand the direct versus indirect effects of ghrelin on
GSIS. In addition to glucose regulation via pancreatic β cell insulin release, α cell secretion of
glucagon can also regulate glycemia. Traditionally ghrelin is thought to have no affect on
glucagon secretion110, however recent literature suggests the presence of the GHSR in α cells in
mice and demonstrates secretion of glucagon following AG administration to mouse islets111. In
contrast to the regulation of insulin secretion, central ghrelin does not appear to affect glucagon
levels109.
11
In addition to the regulation of insulin secretion, studies suggest that ghrelin may also
affect insulin sensitivity. Indeed, ghrelin and ghrelin receptor knock out mice exhibit decreased
body weight and insulin levels, while being more insulin sensitive112. In line with these findings,
administration of AG in humans caused insulin resistance in association with a rise in
circulating FFA113,114. In contrast to these results, in mice undergoing the hyperinsulinemic
euglycemic clamp studies, AG administration improved peripheral, but not hepatic insulin
sensitivity115. Additionally, both a positive and negative correlation between ghrelin levels and
the incidence of type 2 diabetes and insulin resistance has been reported in humans116–119. Thus,
these studies collectively suggest that the role of ghrelin in the regulation of insulin sensitivity
remains controversial, with recent studies still claiming opposing results120,121.
In contrast to the controversial findings on the regulation of insulin sensitivity by
ghrelin, it is commonly accepted that ghrelin can increase gastric emptying thus increasing the
amount of glucose that can be absorbed by the duodenum and subsequently enter into the
circulation122,123. This effect of ghrelin may be mediated by neuronal mechanisms, as the ability
of ghrelin to increase gastric emptying was abolished when neural communications were
negated by surgical or chemical techniques124 demonstrating that ghrelin can locally regulate
glycemia in the presence of luminal glucose. Thus, in addition to alleviating the inhibition of
ghrelin on GSIS, an increase in the gastric emptying rate stimulated by ghrelin will increase
nutrient release into the duodenum to cause secretion of gut peptides to inhibit further ghrelin
release to prevent an increase in glucose absorption and resultant hyperglycemia.
Taken together, these studies suggest that while ghrelin may act within the periphery to
regulate food intake and glucose homeostasis, its local regulatory actions may be of equal
importance. This suggests that local gut-derived hormonal signals may indeed play an integral
role in mediating metabolic homeostasis, which is a common theme amongst the different
peptide hormones secreted within the gastrointestinal tract.
12
1.3.1.2 Leptin
In contrast to the well-known stomach derived hormone ghrelin, leptin is less studied in
regards to its synthesis and secretion from the stomach. Although it is conventionally believed
that the hormone leptin is mainly produced by adipose tissue125 and circulates in proportion to
fat mass126, leptin is also produced in the stomach127 as well as the placenta128, skeletal
muscle129, and mammary epithelium130. No matter where its site of secretion, leptin’s effects are
mediated through its long form leptin receptor (Ob-Rb; Leprb) belonging to the class I cytokine
receptor family (also known as the gp130 receptor family)131,132. There are six isoforms of the
receptor that have been identified and termed A-F, including Leprb. All six isomers of the
receptor share the same 805 amino acids at the N-terminus and are products of the db gene133.
The smallest of the receptors is the isoform E (Ob-Re) which does not contain a transmembrane
or cytoplasmic domain but rather is a soluble binding protein, also known as the soluble leptin
receptor (SLR)132. Interestingly, the transcript for this isoform has not been detected in humans,
likely due to differences in post-translational processing134,135. The remaining 5 isoforms contain
a transmembrane domain and a short intracellular portion. It is generally believed that Leprb is
the only isoform capable of signaling by activating molecules such as signal transduction and
activator of transcription-3 (STAT3) and insulin receptor substrate 1/phosphatidylinositol-3-OH
kinase (PI3K) signaling through phosphorylation of tyrosine residues on the receptor136,137.
However, this has recently been challenged by the findings that Ob-Ra may have signaling
capacity138.
Within the stomach, leptin has been localized to pepsinogen secreting gastric chief cells
mainly in the fundic region127,139, as well as in a small number of epithelial cells of the
stomach140. The concentration of leptin in the stomach has been estimated to be around half that
found in adipose tissues in rats of the same age141. In contrast, in humans, the amount has been
found to be double142, however a direct comparison is difficult to make with differences in
13
sampling between humans and rats. Interestingly, due to the fact that leptin is localized within
different cells of the stomach, it can undergo both endocrine127,139 and exocrine143 secretion.
Indeed, it has been demonstrated that refeeding with a diet containing carbohydrates, protein
and lipids stimulates leptin secretion into the gastric juices within the stomach in both rats127 and
humans139 suggesting that all class of nutrients may cause release of gastric leptin. This
secretion is rapid, where leptin content in the stomach has been shown to rapidly decline within
20 minutes of refeeding127. The exocrine secretion of leptin has been demonstrated by the
finding that leptin from the stomach lumen survives the acidic gastric environment as it has been
measured and detected in the duodenal juice after refeeding143. It is hypothesized that leptin is
synthesized within gastric chief cells at the level of the rough endoplasmic reticulum separately
from the Leprb, where the receptor first undergoes maturation to the soluble leptin receptor
isoform (SLR; Ob-Re) and is then bound to leptin at the level of the secretory granule144. Thus,
upon nutrient stimulation of leptin release, leptin is complexed to its SLR to increase its survival
in the gastric juices. In contrast to these findings, human studies suggest that leptin secreted into
the intestine was not found to be associated with macromolecules142 (such as the SLR) and the
SLR has been suggested to bind to leptin and antagonize its effects145,146. Given that the SLR-
leptin complex does not affect unbound leptin induced activation of the Leprb146, it remains to be
assessed whether the amount of free versus bound leptin is regulated by nutritional status. Such
findings will clarify how leptin reaches the intestinal lumen intact.
In regards to food intake regulation, studies mostly focus on adipocyte derived leptin and
demonstrate that its central actions can control feeding9. Given that leptin must cross the BBB147
and modulate feeding through release of brain neuropeptides and subsequent changes in gene
expression, this satiety effect is considered to be effective over a long-term period. In contrast,
gastric leptin is thought to modulate short-term satiation through activation of gastric and/or
intestine nerve endings. This is based on the findings that Leprb is found to be expressed on cells
14
of, or vagal afferents innervating the stomach and small intestine144,148–151. It may be that vagal
afferent Leprb play a more important role in food intake regulation as selective deletion of these
receptors in vagal afferents resulted in increased food intake and weight gain152, which is not
seen with their deletion in epithelial cells153,154. Nonetheless, given that leptin makes its way to
the duodenal lumen, it may work in concert with other gut peptides to regulate feeding. In fact,
leptin has been demonstrated to cause secretion of CCK, which results in a positive feedback
loop to increase the amount of leptin released143. In addition, both Leprb149 and CCK1
receptors155 are expressed on vagal afferent neurons, and activate common targets to trigger
vagal firing, whereby leptin enhances CCK induced satiety156,157. Taken together, these findings
suggest a cooperative and synergistic mechanism for CCK and gastric derived leptin to regulate
short-term satiation.
Similar to feeding regulation, the glucoregulatory role of leptin has been mostly studied
in the hypothalamus158. However, given its role in CCK secretion, it also plays a role in
regulating gastric emptying. Thus, leptin indirectly (through CCK action) regulates glucose
homeostasis by inhibiting the amount of nutrients entering into the intestinal lumen to be
absorbed into the circulation159. Given that CCK has been shown to acutely regulate glucose
homeostasis through a gut-brain-liver axis (described in detail below), it remains to be
elucidated whether intestinal leptin action has a similar glucoregulatory role. Leptin also
regulates GLP-1 secretion160, which affects glucose homeostasis through a variety of
mechanisms and will be described later. However, it should be noted that these studies do not
directly implicate gastric derived leptin in these effects, as leptin was administered i.p..
Conversely, a direct role for gastric derived leptin on intestinal regulation of glucose
homeostasis comes from the finding that leptin regulates glucose absorption as luminal leptin
administration reduced the recruitment of the sodium glucose transporter-1 (SGLT-1) to the
apical membrane161, thus impeding glucose absorption within the intestine.
15
In addition to the stomach derived peptides ghrelin and leptin, the small intestine
secretes a variety of hormones to trigger both local and peripheral signaling to regulate both
feeding and glucose homeostasis, which will be described in detail below.
1.3.2 Proximal Intestinal Peptides
1.3.2.1 Cholecystokinin
In 1928, Ivey and Oldberg discovered the gut peptide CCK and implicated its role in
stimulating gall bladder contractions162. CCK is predominantly found within intestinal I cells in
the proximal intestine (duodenum and jejunum), but also within the enteric and central nervous
system and pancreas163. The 115 amino acid prepro-CCK polypeptide must undergo multiple
posttranslational modifications including sulfation of its C terminus via protein tyrosine
sulfotransferase, multiple cleavage steps via endoprotease and carboxypeptidase E and
amidation via amidating enzyme to generate CCK-8, the shortest and biologically active form of
CCK164. All forms of nutrients (glucose, lipids, and proteins) have all been shown to stimulate
CCK secretion165–168. More specifically, the breakdown of triglycerides into long chain fatty
acids (LCFA) is required for lipids to stimulate CCK secretion169–171, and individual amino acids
such as phenylalanine172 and tryptophan173 can stimulate CCK release. The mechanisms
initiating CCK release remain largely unknown, but have begun to be uncovered. For instance,
the involvement of the lipid transporter, cluster determinant 36 (CD36) and G-protein coupled
receptors (GPCRs), such as GPR40 are required174,175 and protein kinase C (PKC), a
serine/threonine kinase may be involved for lipid induced secretion of CCK as the LCFA oleic
acid has been shown to release CCK in vitro through activation of PKC176. PKC may then
activate Soluble NSF Attachment Protein REceptor (SNARE) proteins or accessory proteins, as
PKC induces insulin secretion in pancreatic β cells through mammalian uncoordinated-18-1
(MUNC18-1) and vesicle-associated membrane protein-2 (VAMP2)177, and stimulates CCK
16
secretion in vitro through VAMP2178. More recently, the involvement of immunoglobulin-like
domain containing receptor-1 on the basolateral surface of I cells has been shown to mediate fat
stimulated CCK secretion179, which may elevate calcium levels and cause docking of SNARE
proteins for CCK release. After secretion from intestinal I cells, CCK plays an important role
pertaining to the digestion and absorption of nutrients through stimulation of pancreatic
secretion, excretion of bile from the gall bladder, and delaying gastric emptying, which enables
the intestine to effectively digest nutrients180–184.
Studies have demonstrated that CCK also plays an important role in mediating hunger
suppression in rodents, primates, and humans185–193 through a neuronal network194–196. This
appetite suppressive effect of CCK is mediated through the CCK receptor, of which there are
two known isoforms, namely the CCK-1 receptor (predominantly expressed in the
gastrointestinal tract) and the CCK-2 receptor (predominantly expressed in the brain).
Activation of the CCK-1 receptor is necessary for lipids to lower food intake167,197–199 through
vagal afferent firing200,201 and subsequent hindbrain N-methyl-D-aspartate (NMDA) receptor
activation202 involving mitogen activated protein kinase (MAPK) signaling203. Thus, like the
previously described peptides, CCK activates a local paracrine gut-brain axis to control feeding.
In fact, as mentioned, CCK may not work alone but through its interaction with leptin by
affecting the capacity of vagal afferent neurons to regulate expression of transcription factors156.
As to which isoform mediates these effects remains debated as while CCK-8 is the biologically
active form, it has also been suggested that CCK-33 and CCK-58 may play a role in food intake
through reducing meal size and prolonging the intermeal interval, possibly due to their long half
life204,205.
Although these findings suggest a local paracrine vagal mediated pathway for CCK-
induced satiation, there are studies that suggest that CCK may induce satiety through an
endocrine action at the level of the brain. This is based on the following findings: 1) the CCK-2
17
receptor is also expressed within the hindbrain and hypothalamus, 2) microinjections of CCK
into various hypothalamic nuclei decreases food intake206,207, and 3) lesions of the hindbrain
attenuate CCK-induced satiation208. However, the findings that infusion of CCK into the celiac
artery (which directly supplies the GI tract) significantly reduced food intake in comparison to
infusion the jugular vein209 suggests that the energy intake suppressive effect of CCK is likely
via activation of a local paracrine gut-brain axis, rather than an endocrine effect in the brain.
In addition to its food intake suppressive effects, it is generally believed that CCK
regulates GSIS to regulate glucose homeostasis. However reports on CCK-induced GSIS have
mixed findings, some demonstrating an effect210–219, where in others, this effect is absent220–224.
This may be due to the differences in the dose of CCK administered in these studies suggesting
that CCK’s effects may be pharmacological rather than physiological. Nonetheless, an
intravenous CCK infusion results in a drop in plasma glucose levels in conjunction with
biphasic insulin secretion225 and CCK deficient mice have impaired insulin secretion226. This
effect is likely due to CCK interaction with its receptor, as an i.p. injection of a CCK1 receptor
antagonist negated CCK-induced insulin release227 and may be mediated via its endocrine action
on pancreatic β cells, where its receptor is expressed 228. It is suggested that CCK may potentiate
GSIS through activation of G proteins and the classical phospholipase C (PLC) system229. This
involves an increase in the production of inositol triphosphate (IP3) and diacylglycerol (DAG)
which activates PKC230 and increases intracellular calcium levels227 to induce insulin secretion.
Alternatively, after PKC activation, CCK may activate phospholipase A2 that forms arachidonic
acid (AA)231 to cause insulin release, which has been demonstrated to be independent of
changes in calcium levels232. A recent study also suggests that CCK may regulate insulin
sensitivity226 although the mechanism(s) involved are unknown and require further
investigation.
18
CCK also regulates glucose homeostasis through modulating gastric emptying. This has
been demonstrated in humans, where CCK modulates postprandial glycemia by delaying gastric
emptying of nutrients from the stomach into the proximal gut180. As with many other functions
of CCK, its effect on gastric emptying is mediated through the CCK1 receptor233–237. Further
evidence suggests that CCK may not need to bind to its CCK1 receptor located on the pyloric
sphincter, but rather may act in a paracrine fashion and directly activate CCK1 receptors present
on gastric vagal afferents to delay gastric emptying238. Intestinal motility and pyloric pressure239
are also affected by CCK, further demonstrating the ability of CCK to regulate nutrient transit in
the intestine and subsequent absorption into the circulation. Thus, through regulating GSIS and
gastric emptying, CCK can regulate postprandial glucose homeostasis.
More recently the ability of CCK to activate a gut-brain-liver axis to acutely regulate
glucose production has been demonstrated (described in detail below). However, the signaling
cascade required for such regulation remains to be determined. Given that pancreatic CCK1
receptor signaling is well known, perhaps a similar signaling cascade exists at the level of the
gut to regulate glucose homeostasis, which is a focus of the current thesis. Another duodenal
hormone, GIP, also shares similar pancreatic effects as CCK and will be described in detail
below.
1.3.2.2 Glucose-dependent Insulinotropic Polypeptide
GIP is a single 42-amino acid peptide that is derived from a 153-amino acid precursor
through post-translational processing of proGIP240. This peptide was originally observed to
inhibit gastric acid secretion and motility in dogs241 and is highly expressed in K cells of the
duodenum and jejunum242. In the fasting condition, GIP circulates at low levels but becomes
elevated upon feeding by glucose243–247, proteins248 and fats, where in regards to fats, long chain
triglycerides are the most potent stimulator of GIP release249. The ability of various nutrients to
19
stimulate GIP release appears to be species dependent, where fat is a more potent stimulator
than carbohydrates in humans, and in contrast, carbohydrates are more potent than fat in rodents
and pigs250. The release of GIP by nutrients involves nutrient associated receptors or transporters
such as GPR40, GPR120, SGLT-1251,252.
Interestingly, GIP does not regulate food intake, which has been confirmed in
humans253,254. GIP is most commonly known and demonstrated as an incretin hormone. An
incretin hormone (INtestine seCRETion INsulin) is defined as a gut-derived hormone that
induces a greater insulin secretory response upon an oral glucose load in comparison to an
intravenous glucose infusion of the same amount. A direct infusion of GIP enhances GSIS in
healthy humans and rats by affecting the early-phase of insulin release255–259. The insulinotropic
affect of GIP is mediated via its endocrine action on pancreatic islets as GIP receptors (GIPR)
are expressed on pancreatic β cells260. Upon activation of the GIPR, there is an increase in cyclic
adenosine monophosphate (cAMP) levels261 which activate downstream mediators, protein
kinase A (PKA) and cAMP guanine nucleotide exchange factor II (cAMP-GEFII; Epac2)262.
Both of these downstream molecules are involved in a variety of intracellular functions such as
altered ion channel activity that leads to an increase in calcium levels and enhanced insulin
exocytosis. More specifically, activation of PKA leads to adenosine triphosphate (ATP)-
sensitive potassium (KATP) channels closure and subsequent depolarization of the plasma
membrane263. This depolarization event leads to the opening of voltage gated calcium channels,
which allows the entry of calcium to further increase intracellular calcium levels through
mobilization from intracellular stores264 via PKA and Epac2. This increase in calcium results in
insulin secretion through calcium dependent exocytosis. Given that CCK and GIP both regulate
GSIS through activation of GPCR pathway(s), and that duodenal CCK regulates glucose
production through its receptor (discussed below), it remains to be investigated whether GIP
20
may also regulate glucose homeostasis via local paracrine activation of a gut-brain axis
through activation of common downstream signaling.
In addition to insulin secretion, GIP also stimulates the secretion of glucagon255,265 likely
through a direct action on pancreatic islets as the GIPR is found to be expressed on pancreatic α
cells260. In humans, it has been demonstrated that GIP up regulates glucagon levels during
fasting and hypoglycemic conditions266. This suggests that this hormone has diverging glucose
dependent effects with opposite actions on the two main pancreatic hormones, therefore acting
as a bi-functional blood glucose level stabilizer.
In contrast to the other gastrointestinal peptides discussed, GIP does not regulate the rate
of gastric emptying from the stomach254,267. However, a recent study suggest that GIP inhibits
intestinal glucose absorption and intestinal motility through a somatostatin mediated pathway268.
This is contrast to previous findings that GIP increases SGLT-1 expression and thus increases
glucose absorption from the small intestine269. In addition, another previous study also reports
that similarly to GLP-1, GIP also regulates intestinal motility263 which may affect glucose
absorption. Thus, while GIP may regulate glycemia via its effect on intestinal glucose
absorption, it is likely that GIP does not activate a gut-brain axis directly to regulate glycemia
given that its receptor is not expressed in vagal afferents270.
More commonly known for its incretin action is the hormone GLP-1 that is found in the
more distal intestine, and will be described in detail below.
1.3.3 Distal Intestinal Peptides
1.3.3.1 Glucagon-like peptide-1
GLP-1 is a posttranslational product of proglucagon which is mainly found to be
expressed in L cells271 in the distal intestine (the ileum and colon) but is also found in the
CNS272 and pancreas273. There are two forms of GLP-1, GLP-1(7-36)NH2 and GLP-1(7-
21
37)274,275 that arise from enzymatic cleavage of the proglucagon by prohormone convertase 1/3
to become active and then enter the lymphatic system or bloodstream to exert their actions276.
GLP-1 has a very short half-life (1-2 minutes)277 due to its rapid degradation by the enzyme
dipeptidyl peptidase IV (DPP-IV)278, and as such it is debated whether a rapid neuronal
mechanism may be needed for GLP-1 action. Moreover, the secretion of GLP-1 is biphasic,
with an early phase of secretion, followed by prolonged second phase279. GLP-1 binds to its
receptor (GLP-1R), a GPCR280 that is found to be expressed in many tissues281, to exert its wide
variety of physiological effects.
Given that nutrients do not likely reach the distal intestine within the time frame of GLP-
1 secretion, it is suggested that intestinal hormones may signal via vagal nerves to cause its
secretion282. However, L cells are also found in the proximal small intestine, and may contribute
to the secretion of GLP-1 from the intestine into the circulation271. Given that CCK activates a
neuronal network to regulate glucose homeostasis at the level of the duodenum (described in
detail below), and that GLP-1 may be released in the duodenum and activate GLP-1Rs on vagal
afferents283 to increase vagal firing284,285, it remains to be investigated whether GLP-1 and CCK
work together to regulate this axis. Nonetheless, all forms of nutrients cause GLP-1 secretion
and their mechanism of secretion have been studied. Glucose-mediated secretion may be
mediated by sweet taste receptors or SGLT-1286,287, although glucose sensing and signaling
mechanisms in the intestine are still under debate288. For lipids, similar to what is seen for the
release of other peptide hormones, the hydrolysis of triglycerides to release LCFAs is required
for GLP-1 release289, and may be mediated by fatty acid transport protein 4290, GPR40291, and
GPR120292. As mentioned earlier, LCFA entry into the duodenum also stimulates CCK
secretion, which has also been implicated in GLP-1 release as blockade of CCK1 receptor
signaling during fat intake abolished the rise of GLP-1 in humans289. The mechanism of amino
acid induced secretion of GLP-1 remains largely unknown, but a recent studies suggests the
22
involvement of G-protein coupled receptor (GPCR) 6A293. Beyond nutrient stimulation, GLP-1
may mediate its own secretion through an autoregulatory loop294 or be stimulated by bile acids
via a TGR5-dependent pathway295,296.
After entering the circulation, GLP-1 is widely known for its food intake suppressive
effects in both rodents and humans, which are mediated both through peripheral and central
mechanisms. Its peripheral effects are likely mediated via activation of vagal afferents297 where
its receptor is expressed283. This is demonstrated by the fact that peripheral exendin-9 (Ex-9)
administration abolishes the food intake suppressive effects of peripheral GLP-1298, and surgical
or chemical inactivation of the vagus attenuates GLP-1R activation and satiety299. However, the
involvement of the vagus nerve in mediating GLP-1 anorectic effects has been recently
challenged and may be due to CNS GLP-1R signaling300. Indeed, GLP-1 may act in various
regions of the brain including the hindbrain and hypothalamus and studies have demonstrated
that an injection of Ex-9 into the third ventricle abolishes the ability of GLP-1 to decrease food
intake301–303. Further, direct hindbrain administration of Exendin-4 (Ex-4; GLP-1 analog)
reduces food intake304 which is mediated by PKA/MAPK signaling305. However, a connection
between the suppressive effects on food intake of both the peripheral and central GLP-1R may
exist as peripheral GLP-1 administration fails to affect food intake after abolishment of a vagal-
brainstem-hypothalamic pathway299.
In addition to the food intake suppressive effect of GLP-1, GLP-1 also belongs to the
family of incretin hormones and potentiates GSIS through either a paracrine or endocrine
fashion, similar to GIP. Indeed, intestinal GLP-1 reaches the pancreas via the portal vein, but
blockade of vagal activation attenuates GSIS, indicating that GLP-1 may act within a paracrine
fashion to stimulate insulin release306. Moreover, GLP-1 also stimulates GSIS through binding
to its GLP-1R expressed on the β-cell307. Similar to its effects in the brain, increased AC activity
occurs after binding of GLP-1 to its receptor, resulting in formation of cAMP which
23
subsequently increases PKA activity308. Additionally, increased cAMP concentrations results in
the activation of cAMP-GEFII or Epac2262. Both proteins alter ion channel activity, which leads
to the closure of KATP-channels through phosphorylation of the SUR1 subunit309. Interestingly,
GLP-1 has been shown to sensitize the KATP channels to ATP, as less ATP is required for
closure of the channels310. The closure of these channels shifts the membrane potential and
opens internal voltage-gated calcium channels, increasing intracellular calcium levels through
release from intracellular calcium stores311 and the number of readily releasable insulin
secretory vesicles. In addition to PKA activation, other pathways have been implicated such as a
calmodulin mediated pathway. Briefly, a calmodulin inhibitor reversed the actions of GLP-1 on
KATP channels and subsequent depolarization of the membrane312.
In contrast to the similarity of GLP-1 and GIP to increase insulin secretion, GLP-1
inhibits glucagon secretion313 to regulate circulating glucose levels, which is glucose
dependent314–316. This has been demonstrated in isolated rat islets and is proposed to be
mediated by somatostatin secretion from pancreatic δ cells by GLP-1 and subsequent binding to
the somatostatin receptor-2317. This effect is likely paracrine in nature as treatment with
somatostatin antibodies abolished the inhibitory effect of GLP-1 on glucagon secretion317. In
addition, GLP-1Rs are not found to be expressed on α cells307 which strengthens the hypothesis
that this inhibition is likely through an indirect mechanism. This may be through its action on δ
cells, although the findings of GLP-1R expression on δ cells is inconsistent318. Moreover, this
effect of GLP-1 is likely in hypoglycemic conditions, when glucagon levels are high in order to
increase circulating glucose levels. This is based on findings that during a hypoglycemic clamp
in humans, the inhibitory effect of GLP-1 on glucagon secretion was lost when circulating
glucose levels were near or just below normal fasting levels319.
In addition to its direct effect on the pancreas, GLP-1 also regulates glucose
homeostasis via extrapancreatic mechanisms. Given that the portal vein is exposed to higher
24
levels of active GLP-1, it is not surprising that GLP-1 acts within this region to regulate
glucose disposal, given the existence of a hepatoportal glucose sensor, and the finding that
portal infusion of glucose with exendin-9 attenuated the ability of portal glucose to increase
glucose clearance320. In line with these findings, an increase in intestinal incretin action via
selective inhibition of intestinal and not systemic DPP-IV is also sufficient to enhance glucose
tolerance in association with increased vagal firing285. Thus, GLP-1R activation within the
portal vein triggers a portal-brain-muscle neuronal axis to control glucose disposal. Given these
findings, it remains to be addressed whether intestinal GLP-1 locally activates its receptors on
vagal afferents innervating the small intestine to trigger a gut-brain-liver axis, like CCK, to
regulate glucose production.
Although unknown for intestinal GLP-1 action, within the arcuate nucleus of the
hypothalamus, GLP-1 has been demonstrated to regulate glucose production321. In addition,
central GLP-1 signaling has also been shown to reduce insulin stimulated muscle glucose
utilization under hyperglycemic conditions to favor hepatic glycogen storage322. However, the
role of CNS GLP-1 signaling (as well as vagal signaling) in the regulation of glucose
homeostasis has recently been challenged, similar to feeding regulation. This is due to the
recent finding that liraglutide, a long acting GLP-1 agonist, still has glucose lowering effects in
mice lacking the GLP-1R in either the vagus nerve or brain300. Given that this study utilized
mice lacking the GLP-1R from birth, additional studies are warranted to dissect the role of both
vagal and brain GLP-1R signaling in the regulation of glycemia.
GLP-1 also regulates glucose homeostasis through its ability to control GI motor
functions through the “ileal brake” where the rate of specific unabsorbed nutrients reach the
distal intestine is controlled. In humans, intravenous GLP-1 administration slows gastric
emptying in a dose-dependent manner which is likely mediated via vagal afferents323. In
addition, GLP-1 also controls pressure waves in the duodenum and can alter pyloric pressure,
25
effects that are abolished upon Ex-9 administration324–326. Thus, GLP-1 alters the amount of
nutrients entering the small intestine, another way in which it regulates glucose homeostasis.
A close relative to GLP-1, GLP-2, is also secreted from L cells, and its involvement in
glucose and food intake regulation is described below.
1.3.3.2 Glucagon-like peptide-2
GLP-2 is a 33 amino acid peptide that is created by posttranslational cleavage of
proglucagon at the same time as GLP-1, and is also found to be located in intestinal L cells. It is
believed that GLP-2 is secreted upon nutrient ingestion327, along with GLP-1. Therefore, GLP-
2’s secretion is likely predominantly mediated by long chain fatty acids282,290,291,328. However, as
discussed for GLP-1, other L cell secretagogues include glucose and bile acids329,330 as well as
peptide hormones such as GIP and leptin may also cause GLP-2 secretion. In regards to its
secretion profile, GLP-2 also exhibits a biphasic pattern of secretion with an acute increase that
occurs rapidly followed by a more delayed and prolonged response327,328,331. After secretion,
GLP-2 mediates its effects through binding to its receptor (GLP-2R), a GPCR, and activates a
diverse set of downstream signaling molecules such as PKA332.
GLP-2 is widely known for its intestinal growth actions such as affecting barrier
function and intestinal protection333. However, its effect on suppressing food intake have been
debated. For instance, its peripheral effects have been demonstrated in mice where an i.p
injection of GLP-2 reduced short term feeding, which was abolished when a GLP-2R antagonist
was co-administered334. However, this same effect was not demonstrated in humans335,336. Thus
the peripheral effects of GLP-2 on food intake remain to be resolved. Rather, it is more widely
accepted that the food intake suppressive effects of GLP-2 are mediated centrally. Indeed,
multiple regions of the brain express the GLP-2R including hypothalamic and
extrahypothalamic regions337 and it has been demonstrated that an intracerebroventircular (i.c.v)
26
GLP-2 infusion inhibits food intake337,338 through hypothalamic neuron signaling339. This food
intake suppression is likely through a relay between the hypothalamus and hindbrain338.
As mentioned previously, the main site of action for GLP-2 is within the GI tract where
GLP-2 affects intestinal nutrient transit. It has been demonstrated to inhibit gastric
emptying,340,341 enhance gastric capacity by decreasing gastric fundic tone342 and reduce
intestinal transit in vivo343. This inhibitory effect may be mediated by central signaling by GLP-
2, as neuronal specific deletion of the GLP-2R in hypothalamic nuclei resulted in accelerated
gastric emptying339. However, GLP-2’s effects on gastric emptying are not as potent as seen for
GLP-1341, but nonetheless, its affect on intestinal transit of nutrients ultimately affects glucose
homeostasis.
In contrast to GLP-1, GLP-2 is not an incretin, and as such it does not affect GSIS by
pancreatic β cells. However, it has been suggested that GLP-2 does affect glucagon secretion
from α cells through activation of its receptor in rats344 suggesting that it may play a role in the
regulation of glucose homeostasis. In line with these findings in rats, the GLP-2R has been
detected in α cells in humans344 and an exogenous GLP-2 administration rapidly increased
plasma glucagon levels345. In contrast, other studies have not been able to detect the GLP-2R in
murine islets and did not see an increase in glucagon levels after GLP-2 administration346. Thus,
the importance of GLP-2R signaling in the control of glucagon secretion requires further
clarification as differences are detected among different species.
In addition to its effects described above, it is recently suggested that GLP-2 may play a
role in regulating insulin sensitivity through central GLP-2R. This was demonstrated by the
findings that GLP-2R deletion in the hypothalamic pro-opimelanocortin (POMC) expressing
neurons impaired glucose tolerance and hepatic insulin sensitivity and GLP-2R activation in
these neurons activated PI3K/Akt signaling which was required for GLP-2 to regulate hepatic
glucose production347. These findings uncover a new role for GLP-2 in the regulation of glucose
27
homeostasis in addition to its known affects on intestinal growth. Given these findings, future
investigation into whether GLP-2 regulates glucose production at the level of the small intestine
is warranted, which has been demonstrated for the gut-derived hormone CCK (please see
below).
Another L-cell derived hormone is OXN, which has been suggested to regulate
metabolic homeostasis as described below.
1.3.3.3 Oxyntomodulin
OXN is a 37 amino acid peptide derived from glucagon whose structure was elucidated
in 1981348. Similar to GLP-1 and GLP-2, OXN is also found in L-cells of the small intestine349
and its secretion is stimulated upon nutrient ingestion, with GLP-1350. There is no clear
demonstration of the existence of an OXN receptor, and it is more widely accepted that OXN
binds to the GLP-1R or glucagon receptor (GCGR) to exert its effects351. Thus, similar to that
postulated for GLP-1, local OXN activation of the GLP-1R may regulate glucose homeostasis
similar to that seen for CCK within the intestine. Interestingly, in regards to the GLP-1R, OXN
activates downstream molecules β-arrestin 2 and results in an increase in cAMP levels but has
less preference to MAPK signaling352. This suggests that OXN and GLP-1 may differ in their
downstream signaling and in vivo effects.
Similar to many of the peptide hormones already discussed, OXN has food intake
suppressive effects both peripherally and centrally depending on the species studied. Indeed,
OXN administration dose-dependently inhibited food intake in rats353 and humans354. In
contrast, peripheral OXN administration in mice did not affect feeding. However, i.c.v
administration of OXN transiently inhibited food intake likely through its activation of the GLP-
1R as the food intake suppressive effects of OXN are abolished in GLP-1R knock out mice355.
Thus, the food intake suppressive effects of OXN remain to be clarified.
28
Moreover, the ability of OXN to regulate glucose homeostasis remains largely unknown,
but some studies do suggest such a role. For instance, OXN has been demonstrated to regulate
gastric emptying in humans356, but these findings were not seen in mice357. In regards to GSIS,
OXN has been shown to increase cAMP levels in association with insulin secretion in mice357,
and similar results have been demonstrated in rats358. Additional studies are warranted to
address the glucoregulatory role of OXN.
The last L-cell derived metabolic regulatory hormone is PYY, which is described in
detail below.
1.3.3.4 Peptide YY
PYY is a 36 amino acid peptide that is secreted from L cells together with the previously
described peptides GLP-1/2 and OXM359. In the circulation, there are two forms of PYY arising
from its cleavage leading to PYY(1-36)NH2 and PYY(3-36)NH2360,361 by DPP-IV278, with
PYY(3-36)NH2 being the major circulating from of PYY. Both carbohydrates and lipids
stimulate its release. In regards to lipids, the conversion to LCFA170,362 is required to stimulate
PYY release and may involve GPCRs as demonstrated for GLP-1 release. In regards to
carbohydrates, the mechanisms described for GLP-1 may also be involved as described
previously. CCK can also stimulate PYY release in humans suggesting that a neuronal axis
exists between the duodenum and ileum363. PYY is also similar to GLP-1 in terms of its
secretion profile which is biphasic, resulting in an increase in PYY levels in as short as 15
minutes which peak after 1-2 hours after a meal364. After secretion, PYY(1-36)NH2 binds to its
Y receptor subtypes Y1, Y2, Y4 and Y5 receptors, where PYY(3-36)NH2 binds only to Y2 and
Y5359,365.
Similar to other peptide hormones discussed, peripheral and central PYY(3-36)NH2
administration has been demonstrated to regulate feeding. PYY(3-36)NH2’s peripheral affects
29
are likely mediated via vagal afferents where the Y2 receptor is expressed366, as vagotomy
abolished exogenous PYY(3-36)NH2 induced c-fos activation in the ARC and did not affect
feeding299. Furthermore, direct PYY(3-36)NH2 administration is hypothesized to inhibit ARC
neuropeptide Y (NPY) neurons to suppress feeding, as PYY(3-36)NH2 binding to Y2 receptors
resulted in reduced NPY release through a reduction in cAMP production and neurotransmitter
exocytosis367. Thus, PYY(3-36)NH2 may inhibit food intake through a neuronal gut-brain axis.
Given that CCK regulates feeding and glucose homeostasis through a gut-brain and gut-brain-
liver axis, respectively, it remains to be addressed whether PYY may also regulate glucose
homeostasis through activation of its receptor expressed on vagal afferents, in addition to
feeding.
In contrast to many of the other peptides discussed, PYY(1-36)NH2 inhibits GSIS. This
is likely by direct action on the pancreas as suggested by several studies: 1) Pyy knockout mice
exhibit hyperinsulinemia in both fasted and fed states368, 2) studies have demonstrated in
isolated islets that direct PYY(1-36)NH2 administration reduced GSIS in a dose dependent
manner369,370 and 3) mice lacking the Y1 receptor hypersecrete insulin371. Moreover, it is
suggested that PYY(3-36)NH2 does not directly affect GSIS372 as neither Y2R and Y5R are
detected in murine islets371,372. Interestingly, PYY(3-36)NH2 instead may stimulate insulin
secretion through increasing GLP-1 levels372. Thus, the ability of PYY to regulate insulin
secretion remains controversial but if in fact PYY inhibits GSIS, it may be that it works in an
opposite fashion to other gut peptides to prevent hypoglycemia.
In contrast to the findings that PYY may inhibit GSIS and thus elevate glucose levels, it
may inhibit gastric emptying, suggesting that it could play a role in ensuring glucose levels do
not get too high after a meal. This has been demonstrated in various species, including
monkeys373 and humans, where PYY(3-36)NH2 is more effective in comparison to PYY(1-36)
NH2374,375. In addition to gastric emptying inhibition, PYY also regulate intestinal motility,
30
suggesting that PYY regulates entry and subsequent movement of nutrients in the intestinal
tract.
Taken together, the introduction to the above gastrointestinal peptide hormones
demonstrates that local hormonal signals induce neuronal activation to regulate metabolic
homeostasis. The mechanism of local hormonal signaling to trigger a gut-brain-liver axis,
specifically, to regulate glucose production will be described below.
1.4 Small intestine control of glucose production through a gut-brain-liver neuronal
axis
Modified from: Rasmussen, BA*, Breen, DM* and Lam, TK. Lipid sensing in the gut, brain and liver. Trends Endocrinol Metab 23, 49-55, 2011 *Equal contribution (Review)
As mentioned above, upon nutrient entry, the small intestine secretes a variety of
peptides that regulate glucose homeostasis and food intake through a variety of mechanisms.
Throughout this introduction, it has become apparent that, in addition to their indirect effects,
many studies already point to direct hormonal signaling in the gut to regulate feeding and
glucose regulation. In line with this hypothesis, the gut-derived hormone CCK can locally
activate a gut-brain-liver neuronal network to lower glucose production. In the following
section, the neuronal axis triggered by nutrient sensing and subsequent peptide hormone
secretion will be discussed in detail as applicable.
1.4.1 Duodenal lipid sensing and CCK secretion triggers a gut-brain-liver axis to lower
glucose production
1.4.1.1 LCFA!LCFA-CoA
After ingestion of a meal, dietary lipids accumulate in the small intestine. The most
prominent forms of lipid in a typical western diet include triglycerides, cholesterol,
phospholipids and LCFA, where triglycerides are the primary form of lipid. Triglycerides are
31
emulsified through secretion of bile salts from the gallbladder. Pancreatic lipase secretion from
the exocrine pancreas breaks down triglycerides to fatty acids and monoglycerides to be
absorbed by enterocytes. Lipid sensing is then said to begin with the absorption of triglycerides
in the intestinal lumen. Fatty acids are transported into enterocytes via CD36, which functions as
a fatty acid transporter in the proximal sections (duodenum and jejunum) of the small
intestine174. Sensors of FFA in the small intestine include GPCRs such as GPR40 and GPR120,
whose ligands both include medium- and long-chain saturated and unsaturated FFA. Both
GPR40 and GPR120 have been shown to play a role in FFA-stimulated secretion of gut derived
hormones291,292, as discussed previously.
Once inside the cell, LCFAs are metabolized by acyl-CoA synthetase (ACS) to form
LCFA-CoA, which is mediated via the specific ACS isoform, ACS3376. After conversion,
LCFA-CoA have two fates: 1) LCFA-CoA and monoglycerides are recombined into
triglycerides and packaged into chylomicrons for exocytosis, or alternatively, 2) LCFA-CoA are
then transported into the mitochondria via carnitine almitoyltransferase-1 (CPT-1) to undergo β-
oxidation. The process through which β-oxidation occurs at the level of the intestine is similar to
what occurs in the brain377 and liver378.
Regardless of the fate of LCFA-CoA, its formation is required for intestinal lipids to
lower glucose production379. This has been demonstrated by an intraduodenal infusion of
Intralipid (a soybean emulsion of mono- and polyunsaturated fatty acids), which was able to
lower hepatic glucose production during the pancreatic basal insulin clamp technique. Of note,
Intralipid was infused at a rate that ensured the effects observed were in the pre-absorptive
state380. Co-infusion of Intralipid with triacsin C (an inhibitor of ACS3381) prevented the
suppression on glucose production induced by upper intestinal lipids, suggesting that conversion
of LCFA to LCFA-CoA is essential for upper intestinal lipids to lower hepatic glucose
production. The suppression of glucose production was mediated by a gut-brain-liver axis as 1)
32
interruption of the neuronal connection between the gut and brain by co-infusion of tetracaine,
or performing subdiaphragmatic vagotomy or gut vagal deafferentation surgical procedures, also
blocked the ability of LCFA to suppress glucose production 2) administration of MK-801
(NMDA ion channel blocker) into the nucleus of the solitary tract (NTS) abolished the LCFA
induced inhibition of glucose production and 3) infusion of Intralipid into rats that had received
hepatic vagotomy (a surgical technique that interrupts the connection between the brain and
liver) also abolished the ability of fats to lower glucose production379. All of the above effects
were independent of weight loss, consistent with all the studies discussed below. In line with
these findings, a recent study showed that activation of NMDA receptors within the dorsal vagal
complex (DVC) by the agonist glycine decreases glucose production382 strengthening the
existence of a gut-brain (at the level of the DVC and activation of NMDA receptors)-liver axis
to regulate glucose production (Figure 1.2).
The ability of upper intestinal lipids to regulate glucose homeostasis has recently been
shown to be mediated by PKC-δ activation383 and CCK-1 receptor activation384. The importance
of both of these molecules in mediating upper intestinal lipid regulation of glucose production is
shown by the inability to regulate plasma glucose levels in fasting/refeeding experiments upon
inhibition of either PKC-δ383 or CCK-1 receptor activation384. These experiments highlight the
possible downstream mediators of intestinal lipid sensing and will be discussed in the following
sections.
1.4.1.2 PKC-δ! CCK
It is evident from the previous section that changes in the availability of LCFA-CoA
regulate glucose homeostasis. Although this is well established, the associated signaling
mechanisms downstream need to be elucidated and studies suggest that PKC, a serine/threonine
kinase, is a potential molecule to mediate the effect of lipid. The PKC family consists of at least
33
10 isoforms, which are divided into three subfamilies based upon their second messenger
requirements385. Conventional PKCs (α, βI, βII, and γ) require both calcium and the lipid DAG
for activation whereas novel PKCs (δ, ε, η, and θ) require DAG but not calcium. Unlike both
conventional and novel PKCs, the atypical PKCs (ζ, ι, and λ) require only phospholipids/lipids
and not calcium or DAG for activation. As there are several different PKC isoforms and PKC
protein expression varies between different tissues, it is not surprising that numerous biological
functions have been ascribed to PKC activity.
As mentioned previously, short-term accumulation of LCFA-CoA in the duodenum
lowers glucose production through a gut-brain-liver neuronal axis379. However the necessary
step(s) that mediate this effect on glucose production were not elucidated in that study. Given
that most PKC isoforms are present in the small intestine and the pattern of expression between
rodents and humans is similar, with a few exceptions386,387, a potential role for PKC in
regulating glucose production is warranted. In fact, activation of duodenal mucosal PKC-δ was
found to be sufficient and necessary for lipid sensing to regulate glucose production383. In brief,
an intraduodenal infusion of 1-oleoyl-2-acetyl-sn-glycerol (OAG, PKC activator), during the
pancreatic basal insulin clamp, lowered glucose production, which was blocked by co-
administration of a PKC-δ inhibitor or an adenovirus expressing the dominant negative form of
PKC-δ. Furthermore, this effect was shown to be mediated by a gut-brain-liver neuronal axis as
administration of tetracaine or NTS MK-801, and hepatic vagotomy all prevented PKC-δ
activation from decreasing glucose production (Figure 1.2).
The above results discussed suggest that duodenal lipid sensing and subsequent
activation of PKC-δ occur in the fasting state as the pancreatic clamp technique is conducted in
animals that have undergone 5 hours of fasting. What remains in question is whether these
mechanisms are activated during refeeding. Thus, a more physiological assessment of the role of
34
intestinal lipid sensing mechanisms in the regulation of glucose homeostasis involves the use of
fasting/refeeding experiments. During fasting/refeeding, circulating glucoregulatory hormones
are changed at will while plasma glucose levels rise, and this elevation of plasma glucose is
counteracted by an inhibition of hepatic gluconeogenesis388. Direct inhibition of PKC-δ382 in the
duodenum during a fasting/refeeding experiment disrupts glucose homeostasis causing plasma
glucose levels to rise. This observation strengthens the role of PKC-δ in the regulation of
glucose homeostasis. This is also true for CCK-1 receptor inhibition384 which will be discussed
in more detail below.
What is the signaling pathway(s) downstream of PKC-δ that is required for lipids to
lower glucose production? As discussed briefly in section 1.3.2.1, in vitro studies report a
mechanistic link between PKC and CCK as the LCFA oleic acid activates PKC to stimulate the
release of CCK in the secretin tumor cell (STC)-1 cell line176,390. Furthermore, in addition to
PKC-δ being the downstream effector of lipid sensing, activation of duodenal CCK was also
found to be sufficient and necessary for lipid sensing to regulate glucose production384, which
will be discussed in detail in the following section. Based on all of these findings, this
relationship was addressed using both pharmacological and molecular approaches to inhibit
PKC-δ and illustrated that duodenal PKC-δ stimulation requires CCK-1 receptor activation to
lower hepatic glucose production during a pancreatic basal insulin clamp. However, duodenal
PKC-δ is not required for CCK to decrease glucose production391. Whether PKC isoforms other
than PKC-δ play a role either upstream or downstream of CCK in the regulation of glucose
production remains possible and warrants further investigation. Thus, it is proposed that PKC-δ
is upstream of CCK and stimulates CCK release, subsequently leading to the activation of the
CCK-1 receptor to regulate glucose homeostasis. The mechanistic link between PKC-δ
activation and the stimulation of CCK release remains unknown. However, also discussed in
35
section 1.3.2.1, the potential involvement of SNARE proteins (i.e. Munc18-1 and VAMP-2)
could be the subject of future studies. This is due to the fact that PKC-δ has been shown to
enhance insulin secretion coupled with increased phosphorylation of Munc18-1 in pancreatic β-
cells177 and VAMP-2 mediates CCK secretion in STC-1 cell lines178.
1.4.1.3 CCK!CCK-1 receptor
As discussed in the previous section, CCK lies downstream of PKC-δ and is an
important mediator of duodenal lipid sensing391. A recent study reported that CCK in the
duodenum lowers glucose production through a neuronal network and is downstream of
lipids384. Briefly, a CCK-8 infusion into the duodenum during the pancreatic basal insulin clamp
lowered glucose production. This glucose production suppression effect was abolished upon co-
administration of CCK-8 with the CCK-1 receptor blocker MK-329 as well as infusion of CCK-
8 in CCK-1 receptor knockout rats. In addition, co-infusion of lipids with MK-329 abolished the
ability of the lipid administration within the duodenum to lower glucose production. The gut-
brain axis was also defined, as co-administration of CCK-8 with the anesthetic tetracaine
abolished the glucose production suppression effect of CCK-8. These data suggest that duodenal
CCK-8 stimulates the vagal afferent to lower glucose production and is the downstream
mechanism of lipid sensing (Figure 1.2). Furthermore, inhibition of the NMDA receptor
through administration of MK-801, or hepatic vagotomy surgery blocked the ability of duodenal
CCK-8 administration to lower glucose production384. This finding suggests that activation of
NMDA receptors and subsequent neuronal relay to the liver is required for intestinal lipid
sensing/CCK-1 receptor activation to lower glucose production.
Taken together, the data mentioned provides evidence for the existence of a duodenal
lipid ! LCFA ! LCFA-CoA ! PKC-δ ! CCK ! CCK-1 receptor pathway that triggers a
gut-brain-liver axis to lower hepatic glucose production (Figure 1.2).
36
1.4.1.4 Effects of High Fat Feeding on the Gut-Brain-Liver Axis
As discussed, several advances have recently been made that have uncovered part of the
downstream signaling mechanisms of intestinal lipids in normal rodents. Interestingly, the
signaling pathway mentioned above fails to lower glucose production in rodents fed a high fat
diet for 3 days, a model of diet-induced hepatic392,393 and hypothalamic394 insulin resistance,
discussed in more detail below. Thus, these studies have allowed us to get closer to identifying
the location of the defect induced by high-fat feeding.
First, as mentioned previously, PKC-δ activation was found to lie downstream of
duodenal lipids to activate the gut-brain-liver neuronal axis to lower glucose production in
normal rodents383. As in the case of duodenal lipid infusion, high-fat feeding also prevented
direct stimulation of PKC-δ, through duodenal OAG infusion, from lowering glucose
production391. These findings suggest that the signaling defect does not lie within the inability of
lipid to trigger signaling events like PKC-δ activation. Similar to PKC-δ, CCK activation was
also demonstrated to be required for duodenal lipids to lower glucose production384. Again, rats
overfed with a HFD completely failed to respond to duodenal CCK-8 to lower glucose
production384. These findings are consistent with the fact that duodenal PKC-δ activation is
upstream of CCK signaling, and that diet-induced CCK resistance is postulated to lie within the
downstream signaling cascade of CCK-1 receptors384, as direct activation of duodenal mucosal
PKC-δ still fails to overcome intestinal CCK resistance to lower glucose production391. Taken
together, these findings strengthen the argument that duodenal lipid resistance lies downstream
of the CCK/CCK-1 receptor signaling cascade (Figure 1.2). However, the exact location of
resistance at the level of the duodenal CCK-1 receptor still remains to be explored. Thus, it is
essential to investigate the downstream signaling mechanisms of the CCK1 receptor to begin to
37
uncover where the resistance lies to uncover possible ways to restore the functionality of this
axis, a focus of the current thesis.
In addition to the duodenum, gut peptides are also found more distally in the jejunum, as
previously described (i.e. CCK and GIP). This suggests that hormonal signaling in the jejunum
could play a role in regulating glucose homeostasis. First, what is known in regards to the role
of nutrient sensing in the jejunum and the regulation of glucose homeostasis will be discussed in
detail below.
1.4.2 Jejunal nutrient sensing triggers a gut-brain-liver axis to lower glucose production
Modified from: Rasmussen, BA*, Breen, DM*, Côté, CD, Jackson, M, and Lam, TK. Nutrient sensing mechanisms in the gut as therapeutic targets for diabetes. Diabetes 62, 3005-3013, 2013 *Equal contribution (Review)
It is traditionally believed that nutrients reach the distal gut only in malabsorptive
conditions395. However, during the early phases of food ingestion, nutrients have been shown to
reach the distal intestine in both animals396–399 and humans400,401 suggesting that the more distal
intestine may also regulate glucose production through a gut-brain-liver neuronal axis. In fact, a
recent study demonstrates such an axis exists in the jejunum402, and that the jejunum shares
similar nutrient sensing mechanisms as the duodenum, which will be discussed in detail below.
First, the ability of the jejunum to sense lipids was tested. Similar to the findings in the
duodenum, a jejunal Intralipid infusion lowered glucose production during the pancreatic basal
insulin euglycemic clamp technique. This was abolished upon co-infusion of an ACS inhibitor
suggesting that the conversion of LCFA to LCFA-CoA is also required for the jejunum to lower
glucose production402 (Figure 1.2). These effects were independent of weight loss, which is
consistent for all of the findings in the study. What still remains unknown is the downstream
signaling pathway of jejunal lipid sensing. This may require CCK or other gut derived
hormones, which is a focus of the current thesis.
38
Next the ability of the jejunum to sense glucose to lower glucose production was tested.
Indeed, a direct glucose infusion into the jejunum lowered glucose production during the
pancreatic clamp. This required glucose uptake into intestinal cells as co-infusion of glucose
with phlorizin, a SGLT inhibitor, abolished the ability of glucose to lower glucose production
(Figure 1.2)402. Importantly, a direct glucose infusion into the portal vein at the same
concentration did not affect the glucose kinetics, suggesting that infusion of glucose into the
jejunum activates local signaling mechanisms to lower glucose production402. Similar to lipids,
the downstream hormonal signaling involved in jejunal glucose-induced suppression of glucose
homeostasis remains to be assessed. However, this may also involve CCK or other gut derived
hormones, which is a focus of this current thesis.
The involvement of a gut-brain-liver axis was then tested for jejunal nutrient sensing.
Similar to the duodenum, blockade of gut to brain signaling via infusion of the anesthetic
tetracaine abolished both lipid and glucose-induced suppression of glucose production402.
Further, NTS administration of MK-801 or hepatic vagotomy negated the glucose production
suppression effects of lipids and glucose402. Thus, a gut-brain-liver neuronal axis also exists for
jejunal nutrient sensing as seen in the duodenum.
Given that the direct disruption of duodenal nutrient sensing mechanisms results in a
dysregulation of glucose homeostasis during a fasting and refeeding protocol, the same
experimental procedure was performed with or without blockade of jejunal nutrient sensing
mechanisms to address the relevance of jejunal nutrient sensing. Interestingly, blockade of either
nutrient (lipid and glucose) sensing in the jejunum did not disrupt glucose homeostasis during
the refeeding study402. This suggests that nutrient sensing mechanisms in the jejunum may
become apparent under conditions of disrupted nutrient flow such as when sections of intestine
are surgically removed for either cancer or bariatric surgical procedures. Thus, the ability of
jejunal nutrient sensing mechanisms to regulate glucose homeostasis was tested after duodenal-
39
jejunal bypass surgery. This surgical technique, as well as the findings of the involvement of
jejunal nutrient sensing to mediate the beneficial effects of this surgery, will be discussed in
greater detail below. Before such discussion, the different types of bariatric surgical procedures
as well as changes in gut peptide hormone secretion associated with bariatric surgery will be
reviewed first.
1.5 Bariatric surgery, gut hormones and intestinal nutrient sensing
1.5.1 Bariatric surgical procedures and changes in gut hormones
Bariatric surgery encompasses many surgical procedures that are either restrictive in
nature (i.e. gastric banding or vertical sleeve gastrectomy) by altering the stomach size or
nutrient flux into the stomach, or in addition to changing the stomach size, bypass sections of
the small intestine thus altering the amount of nutrients entering the stomach and the intestinal
tract (i.e. Roux-en-Y gastric bypass). Bariatric surgery was primarily used as a weight loss
procedure for obese subjects (BMI > 35). Indeed, these surgical procedures have profound
weight loss effects, where gastric banding results in ~20% weight loss, and Roux-en-Y gastric
bypass (RYGB) results in ~25% weight loss403–405. In addition to the dramatic weight loss
effects of these surgeries, surgeons noticed that many patients with type 2 diabetes who had
undergone the surgery for morbid obesity experienced complete diabetes remission. Indeed,
bariatric surgery normalizes glucose levels in type 2 diabetes, and these effects have been shown
to be independent of weight loss406. Excitingly, one study indicates that bariatric surgery caused
diabetes remission, and this effect is still present even after 6 years407. In addition, bariatric
surgery has been demonstrated to reduce the risk of developing diabetes by 80% over 7 years408.
Given such success, there has been a world-wide effort to i) better understand which surgical
procedures have the best results and ii) begin to uncover the mechanism(s) of the surgery in
hopes to discover molecular candidates that can be targeted to mimic the beneficial effects.
Many scientists have focused on changes in gut hormone secretion after surgery as potential
40
candidates for mediators in both the weight loss and glucose lowering effects. Indeed, changes
in gut hormone profiles have been demonstrated which, in addition to the four main surgical
procedures currently used in patients, are described below.
1.5.1.1 Common types of bariatric surgical procedures and beneficial outcomes
1.5.1.1.1 Laparoscopic adjustable gastric band surgery
The laparoscopic adjustable gastric band (LAGB) surgery is characterized by the
insertion of a synthetic band just below the gastro-esophageal junction that creates a gastric
pouch. In order to control the amount of food entering the stomach, the band size can be
changed through inflation or deflation409. Thus patients can limit their caloric intake and delay
gastric emptying into the small intestine. Typically used for its weight loss effects, LAGB is
shown to cause substantial body weight loss, however this depends on the starting BMI410. This
surgical procedure has very little complication associated with it compared to the other surgical
interventions described below and is the safest of all of procedures. In addition to its weight loss
effects, patients with mild obesity and type 2 diabetes underwent remission following LAGB410.
However, not all patients experience weight loss with this surgery, a common complaint for
LAGB.
1.5.1.1.2 Sleeve Gastrectomy
The sleeve gastrectomy (SG) procedure involves removal of 80% of the stomach
creating a small stomach pouch. This procedure originated from another form of bariatric
surgery, the bilio-pancreatic diversion/duodenal switch, as surgeons noticed that substantial
weight loss occurred before the second part of the procedure was performed409. This procedure
has substantial weight loss effects for both morbidly obese and extremely obese patients411. The
potential risk associated with this particular procedure is B12 deficiency409 but initial data
suggests that diabetes remission has occurred for some patients412.
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1.5.1.1.3 Roux-en-Y Gastric Bypass Surgery
Roux-en-Y gastric bypass (RYGB) surgery is the most commonly used bariatric surgical
procedure and accounts for ~60% of bariatric surgical procedures conducted in the United
States411. This procedure was developed in the 1970s and was modified to its current form.
There are two components to this surgical procedure: a restrictive and malabsorptive
component. The restrictive element of the procedure involves reducing the size of the stomach
by creating a gastric pouch out of the upper portion of the stomach. The malabsorptive
component involves changing the intestinal tract as follows: the jejunum is divided into two
limbs, the upper bilio-pancreatic limb and a lower limb (also called the Roux limb). The Roux
limb is brought up and connected to the restricted stomach, which results in bypassing nutrient
entry into the duodenum and proximal jejunum. The bilio-pancreatic limb is then connected to
the Roux limb through a distal jejunostomy, and delays the interaction of food coming into
contact with pancreatic enzymes and bile409. This is one of the most complicated procedures
surgically, but has substantial effects on diabetes remission410. However, one of the negative
consequences of this surgery is “dumping syndrome” (as the pyloric sphincter is removed)
which encompasses a group of symptoms including weakness and abdominal discomfort and
sometimes increased bowel evacuation after ingestion of a meal. Interestingly, although more
invasive surgically, there are less complications associated with RYGB in comparison to LAGB.
1.5.1.1.4 Bilio-pancreatic diversion/duodenal switch
The bilio-pancreatic diversion/duodenal switch (BPD/DS) was developed by the
combination of two different surgical procedures413–415. The restrictive component of this
surgery involves partial removal of the stomach as well as a change in stomach curvature. In
contrast to RYGB, this surgical procedure keeps the pyloric sphincter of the stomach intact
which eliminates dumping syndrome as a complication. Similar to RYGB, the malabsorptive
42
component of this surgery involves the separation of food to digestive enzymes and bile. Further
down the intestinal tract, these separated intestinal components are rejoined into a common tract
where food, bile and enzymes join allowing for limited fat absorption. This is the least common
form of bariatric surgical procedures conducted even though there are substantial weight loss
effects, even greater than RYGB416. In addition to weight loss, there is excellent diabetes
resolution410. However, this procedure has the largest mortality rates and poses the greatest risk
for nutritional deficiencies409.
1.5.1.2 Changes in gut hormones
The resolution of diabetes following gastric bypass surgeries is thought to be explained
by the “foregut/hindgut” hypothesis. The foregut hypothesis states that by excluding the
proximal small intestine, there is a reduction in some negative/anti-incretin hormone, which
consequently improves glucose control. The hindgut hypothesis states that by excluding the
proximal portion of the small intestine, there is an increase in the secretion of distal
hormones417. Indeed, many studies focus on GLP-1 levels after bypass surgery. After RYGB
there is an increase in circulating GLP-1 levels418–420 which is thought to be a potential mediator
of the weight loss and glucose lowering effect of this surgery, and this increase is higher than in
patients who received gastric banding421. However, not all studies are consistent in their findings
in regards to GLP-1 levels, which may be due to the fact that the precision of GLP-1 assays can
vary, or that GLP-1 measurements were taken during fasting conditions422–424. Moreover,
changes in PYY levels have been seen after RYGB surgery in comparison to other surgeries,
and have been shown to increase within 2 days and remain elevated up to 24 months after
surgery425. Similar to GLP-1, the increase in PYY levels is greater in individuals who have
received RYGB in comparison to other forms of bypass surgery421. Another hormonal change
seen after both RYGB and SG is a reduction in circulating ghrelin levels426,427 which is not
43
surprising given that both surgical techniques have a stomach-reducing component to the
surgery. However, other studies saw an increase in ghrelin levels following RYGB428 and thus
the involvement of ghrelin in gastric bypass surgery remains controversial. In addition to GLP-
1, PYY and ghrelin, other studies in humans suggest that GIP may be involved. However, the
results among different studies are not consistent, some demonstrating and increase429, decrease
or no changes of postprandial levels of GIP430. Circulating leptin levels have also been assessed
in patients are RYGB and have consistently been found to be lower after surgery, likely due to a
decrease in body fat431,432. Thus, it is evident that the exact gut hormone profile found after
gastric bypass surgery remains controversial and warrants further investigation.
It is clear that data collected in human studies is limited to correlative findings and
remains inconclusive. Thus, the use of animal models helps to dissect the potential mechanisms
responsible for the resolution of diabetes after bariatric surgery. However, similar to findings in
humans, studies in rodents are also controversial with different findings amongst different
groups. In regards to RYGB surgery, it is suggested that changes in GLP-1 may mediate the
beneficial effects of the surgery433. However, the finding that RYGB still has beneficial effects
in GLP-1R knock out rodents434 questions this hypothesis. However, another group suggests
that rodents may have different responses to gastric bypass surgery due to differences in GLP-1
responsiveness435. Thus GLP-1 may indeed play a role but is likely not the sole mediator of the
beneficial effects of the surgery. Moreover, an increase in PYY concentrations has also been
suggested to improve glucose homeostasis436, although studies in Y receptor knockout models
are lacking. Therefore, the relative contribution of GLP-1 and PYY signaling in mediating the
beneficial effects of RYGB remains unresolved. Moreover, similar conflicting results are seen
after SG surgery where changes in both GLP-1 and ghrelin are postulated to mediate the
beneficial effects of this surgery437. However, the use of receptor knock out models suggests
otherwise, as this surgical procedure still has its beneficial effects in both ghrelin438 and GLP-
44
1R439 knock out rodents. Thus, the exact mechanisms through which these surgeries exert their
beneficial effects are still largely unknown. It is likely a combination of different hormonal
signaling tied in with the complexity of diabetes that creates difficulties in searching for
nonsurgical tools to recapitulate the effects of these surgeries.
Given the fact many forms of bariatric surgery change the anatomy of the intestinal tract,
and the findings that intestinal nutrient sensing mechanisms trigger a gut-brain-liver axis
independent of weight loss, intestinal nutrient sensing may be involved in mediating some of the
beneficial effects of bariatric surgery. In order to test this hypothesis, a surgical procedure that
only modifies the intestinal tract is needed, which will be described in more detail below.
1.5.2 Duodenal jejunal bypass surgery, nutrient sensing and beyond
Modified from: Rasmussen, BA*, Breen, DM*, Côté, CD, Jackson, M, and Lam, TK. Nutrient sensing mechanisms in the gut as therapeutic targets for diabetes. Diabetes 62, 3005-3013, 2013 *Equal contribution
Duodenal jejunal bypass (DJB) surgery involves repositioning the intestinal tract without
restriction or exclusion of the stomach. More specifically, this procedure first involves exclusion
of the duodenum and proximal jejunum, and connection of the distal jejunum to the stomach.
Thus, nutrients from the stomach bypass the duodenum and enter directly into the jejunum. This
surgical procedure has been shown to have glucose lowering effects in non-obese rodents440 and
in non-obese or mild-obese humans with type 2 diabetes441–444, independent of weight loss. This
experimental form of bypass surgery is conducted in order to tease out the stomach restricting
effects from the intestinal specific effects. Thus, what remains in question is whether intestinal
nutrient sensing mechanisms are mediating the glucose lowering effects of this surgery.
In this regard, DJB surgery was conducted in two different models of non-obese
uncontrolled insulin deficient diabetes402. The first model involved injection of streptozotocin
(STZ), a cytotoxic agent that selectively kills pancreatic β cells and reduces insulin
45
concentrations by ~80%. Due to low insulin levels, these rodents display fasting and fed
hyperglycemia. DJB surgery in this model rapidly lowered plasma glucose levels which was
independent of changes in circulating insulin and glucagon levels402. Similar results were seen in
the insulin deficient, nonobese diabetes-prone BioBreeding (BB-dp) rats that spontaneously
develop type 1 diabetes. Importantly, the glucose lowering effect induced by this surgery is
mediated by jejunal nutrient sensing mechanisms (Figure 1.2). This is demonstrated by the fact
that 2 days after DJB surgery, blocking jejunal nutrient sensing mechanisms during a fasting and
refeeding study resulted in a rise in glucose levels402. To further confirm these findings, jejunal
nutrient infusions were conducted in STZ rodents without DJB surgery, which lowered plasma
glucose levels as well as glucose production. Importantly, these findings are consistent with
those seen in mild-obese type 2 diabetic humans441. That is, the glucose response during a
refeeding study in these rodents while blocking nutrient-sensing mechanisms402 closely
resembled the glucose response during an oral glucose tolerance test in patients before
surgery441. This suggests that nutrient sensing mechanisms may a play a role in the glucose
lowering effect of bariatric surgery in humans. Whether gut-derived hormones play a role in
mediating the early anti-diabetic effects of DJB surgery, in addition to nutrient sensing
mechanisms, is currently unknown, which is a focus of this current thesis.
Given that jejunal nutrient sensing has been demonstrated to trigger a gut-brain-liver
neuronal axis to lower glucose production, it is likely that such an axis is activated after DJB
surgery to lower glucose production402. This is consistent with other groups’ findings that
demonstrate that DJB64 or a variant of DJB445 lower blood glucose concentrations and glucose
production through the CNS in type 2 diabetic rodents. As discussed above, DJB surgery
lowered glucose levels in insulin deficient rodents suggesting that an increase in circulating
insulin levels does not account for the early glucose lowering effect402. Furthermore, the glucose
production lowering effect seen in obese type 2 diabetic rodents was independent of an
46
improvement in insulin action64. However, it is important to note that these findings do not
exclude the possibility that DJB surgery lowers blood glucose concentrations via increased
insulin-dependent or independent glucose uptake in a more long term setting, as it has been
suggested that an improvement in β cell function occurs after surgery in obese type 2 diabetic
subjects446.
As stated above for RYGB, it is thought that changes in insulin secretion are accounted
for by changes in circulating GLP-1, although this is controversial447. Complicating matters
more, in STZ induced uncontrolled diabetic rats, DJB surgery lowered glucose levels in
association with a rise in circulating GLP-1 levels. However, in BB-dp rats with DJB surgery,
the rapid glucose lowering effect was seen without changes in GLP-1 concentrations. Other
studies also reports no changes in GLP-1 after DJB in Zucker Diabetic Fatty rats437, and Goto-
Kakizaki rats448, similar to the findings in BB-dp rodents. Moreover, bariatric surgery still has
profound effects on glucose tolerance in high fat fed rodents deficient of GLP-1Rs, as discussed
previously. Thus, the relative contribution of GLP-1 in mediating the glucose lowering effect of
DJB surgery remains to be resolved. The focus of this current thesis is to address whether other
gut-derived hormones play a role in the improvement in glucose regulation following DJB
surgery.
In addition to increasing distal gut peptide secretion, altering the intestinal tract during
this surgical procedure also alters the mixing of bile with nutrients in the proximal small
intestine and thus may alter bile acid levels. Bile acids have recently been implicated in playing
a role in glucose homeostasis through their effects on glucose production and increased glucose-
induced insulin secretion330,449. Bile acids are postulated to play a role in mediating the
beneficial effects of RYGB in dogs450 and an increase in circulating bile acids has been detected
in humans450 after bypass surgery. Indeed, DJB and other bariatric surgical procedures
47
performed in rodents have been associated with an increase in bile acids levels64,451, and the
glucoregulatory as well as body weight regulatory effects of SG were abolished in nuclear
receptor FXR knockout mice452. It still remains in question whether bile acids action is required
for the rapid glucoregulatory effects of DJB surgery.
Interestingly, less invasive procedures that mimic DJB surgery have been developed
such as the duodenal endoluminal sleeve, which involves inserting a flexible tube that inhibits
the interaction of nutrients with the duodenum and has been shown to have similar effects on
glucose regulation451. This is a step in the right direction in regards to finding less invasive ways
to lower glucose levels in diabetes. Nonetheless, a lot of work still remains in order to uncover
the mechanisms of this surgery. By doing so, we may be able to find noninvasive target
strategies to improve the lives and outcomes for patients who are diabetic and/or obese.
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Figure 1.2 Duodenal and jejunal nutrient sensing mechanisms trigger a gut-brain-liver neuronal axis to lower glucose production. Upon lipid entry into the duodenum a LCFA-CoA ! PKC-δ ! CCK ! CCK1 receptor signaling pathway activates vagal afferents to signal to the NTS to activate NMDA receptors to lower glucose production. This duodenal pathway is abolished upon high fat feeding for three days. Like the duodenum, the more distal intestine, the jejunum, is capable of sensing both glucose and lipids to trigger a neuronal network to lower glucose production, which is required for the early anti-diabetic effect of DJB surgery. Adapted from Breen, DM* and Rasmussen, BA* et al. (2013) Diabetes 62, 3005-3013 *Equal contribution. Permission to reproduce this figure has been obtained from the copyright owner: American Diabetes Association
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1.6 Summary of Introduction
Diabetes and obesity are characterized by a variety of factors including the dysregulation
in food intake and glucose homeostasis. The intestine is the first line of defense against nutrient
excess and activates local hormonal signaling to regulate glucose levels as well as satiety. More
recently, the duodenum has been demonstrated to sense lipids to trigger the release of CCK to
lower glucose production via a gut-brain-liver axis. However, this signaling pathway is impaired
upon short-term high fat feeding, suggesting duodenal CCK resistance. The downstream
signaling of the CCK-1 receptor to trigger this neuronal network remains unknown as well as
whether direct activation of these signaling molecules could bypass CCK resistance. Like the
duodenum, the jejunum is also capable of sensing nutrients and has been shown to activate a
gut-brain-liver neuronal axis and mediate the glucose lowering effect of DJB surgery. Whether
other gastrointestinal hormones trigger a similar axis and contribute to this glucoregulatory
effect also warrants future investigation.
1.6 Rationale and Significance of the Studies
Diabetes is a worldwide epidemic with the number of individuals affected by the disease
increasing at an alarming rate, in large part due to the combination of genetic and lifestyle
factors453. Diabetes and/or obesity are often characterized by hepatic insulin resistance,
reduced/altered insulin secretion, muscle insulin resistance and increased glucose production,
where fasting hyperglycemia in type 2 diabetes has been shown to be due largely to an increase
in glucose production59. Chronic hyperglycemia can lead to diabetic complications such as
neuropathy, nephropathy, and retinopathy454. Thus, uncovering novel mechanisms that lower
glucose production in diabetes or obesity will unveil therapeutic targets to lower glucose levels
and reduce the risk of diabetic complications.
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It has been demonstrated that a duodenal lipid ! LCFA ! LCFA-CoA ! PKC-δ !
CCK ! CCK-1 receptor pathway triggers a gut-brain-liver axis to lower hepatic glucose
production379,383,384,391. Furthermore, direct administration of CCK-8 into the duodenum fails to
lower glucose production in rats fed a high-fat diet384. Although the site(s) of this defect remains
unclear, evidence confirms that it is located downstream of CCK release, as both lipid
administration379 and PKC-δ activation391 (both of which stimulate CCK release) still fail to
lower glucose production. Whether this resistance lies at the level of the CCK-1 receptor and/or
within the signaling cascade of the receptor currently remains to be explored. The purpose of
Study 1 in this thesis was to address the downstream signaling of the CCK-1 receptor, namely
PKA, to regulate glucose production, and to determine whether direct activation of PKA can
bypass duodenal CCK resistance in rodents fed a high fat diet for 3 days.
Like the duodenum, the jejunum is capable of sensing nutrients to trigger a gut-brain-
liver neuronal axis to lower glucose production402. These nutrient sensing mechanisms become
apparent after DJB surgery, whereby the influx of nutrients into the jejunum lowers glucose
levels. Whether hormonal action mediates this glucose production-lowering effect remains
unknown. The purpose of Study 2 in this thesis was to address whether leptin (produced by the
stomach) action in the intestine triggers a neuronal network to lower glucose production and
whether intestinal leptin action mediates the glucose lowering effect of DJB surgery.
The pancreatic (basal insulin) euglycemic pancreatic clamp technique in combination with
intestinal infusion of various compounds was performed in both normal and diseased rats. These
studies provide evidence for PKA as a duodenal target to lower glucose production in high fat
diet fed rodents and for the possible role of jejunal leptin signaling in mediating the beneficial
effects of DJB surgery.
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1.7 General Hypothesis
Independent CCK and leptin signaling in the intestine triggers a neuronal network to lower
glucose production.
1.8 Specific Aims
This thesis consists of two studies that examined intestinal hormonal signaling involvement
in the regulation of glucose production and whether these signaling axes remain intact in
diseased settings.
Study 1. To determine whether duodenal PKA activation plays a role in the CCK1 receptor
mediated decrease in glucose production and whether direct activation of duodenal PKA
bypasses duodenal CCK-resistance acquired upon high fat feeding.
Study 2. To investigate whether leptin activates jejunal Leprb-mediated signaling
pathway(s) to regulate glucose production via the central nervous system and whether enhanced
gastric leptin action in the jejunum contributes to the glucose-lowering effect of DJB surgery in
non-obese uncontrolled diabetes.
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Chapter 2General Methods
2.1 Animals All animal study protocols were reviewed and approved by the Institutional Animal Care
and Use Community of the University Health Network. For all studies, adult male (8-week old)
male Sprague-Dawley (SD) rats (~300g) were obtained from Charles Rivers Laboratories
(Montreal, Quebec, Canada). Rats were individually housed and maintained on a standard 12-
12h light dark cycle, and had ad libitum access to water and rat chow (Harlan Teklad 6%
mouse/Rat diet; composition: 49% carbohydrate, 33% protein and 18% fat; total calories
provided by digestible nutrients: 3.1 kcal/g). Rats were given at least 5 days to acclimatize upon
arrival before surgeries were performed.
2.1.1 High Fat Feeding Animal Model
A subgroup of male SD rats were placed on a lard-oil enriched high fat diet ad libitum
for three days after intestinal and vascular catheter implantation (see Table 1 for high fat diet
and standard chow composition; Ren’s Pet Depot, ON, Canada). Rats that were hyperphagic and
consumed more calories as rats on regular chow were used for the clamp experiments. These
rats have previously been shown to develop hepatic392,393 and hypothalamic394 insulin resistance
and duodenal Intralipid379 and CCK384 resistance.
2.2 Surgical Procedures
Rats were first anesthetized with an i.p. cocktail of (60-90 mg/kg) ketamine (Ketalean;
Bimeda-MTC, Cambridge, Ontario) and (8-10mg/kg) Xylazine (Rompun; Bayer) before
performing surgical procedures described below. All surgical procedures were preceded through
53
shaving both the abdominal and neck area and cleaning with 70% ethanol and 10% povidone-
iodine (Betadine solution, ON, Canada) before incisions were made. Recovery from surgical
procedures was ensured through monitoring body weight gain and food intake for 4-6 days after
the surgery.
2.2.1 Vessel Cannulation
Indwelling catheters were made with polyethylene tubing (PE 50, Clay Adams, Boston,
MA) with a cuff extension (15mm, internal diameter of 0.02 inches) of Silastic tubing (Dow
Corning, Midland, MI). After blunt dissecting through the muscle layers, the carotid artery was
isolated from connective tissue and the vagus nerve. Using a 4-0 silk thread, the exposed vessel
was ligated at the cranial end. At the caudal end, another thread was loosely tied and the two
ligatures were pulled taut. A small incision was made into the vessel wall. The indwelling
catheter was then inserted past the overlap and the catheter was secured through tightening the
loose ligature. Blood withdrawal and infusion were tested from the catheter. The same
procedure was conducted for the right internal jugular vein. After insertion, the catheters were
tunneled subcutaneously with a 16G needle and filled with a 10% heparin mixture (saline with
1000 U/ml of heparin) to maintain patency of the cannula and closed with a metal pin until the
day of the procedure.
2.2.2 Intestinal Cannulation
Duodenal and jejunal cannulation surgeries were performed as described379,384,402. Three
to four days before the clamp studies, exposure of the gastrointestinal tract within the
peritoneum was conducted through a laparotomy incision made on the ventral midline as well as
the abdominal muscle wall. After identifying the pyloric sphincter, the duodenum was identified
as 1.5 cm distal to the sphincter. In separate rats, the jejunum was identified as 8–10 cm from
the Ligament of Treitz. With a 21-gauge needle, a small hole was made on the ventral aspect of
54
the duodenum or jejunum (in a region with the least vascularization to minimize bleeding) to
allow insertion of an intestinal catheter made of silicone tubing (0.04 in ID, 0.085 in. OD; Sil-
Tec, Technical Products, USA) with a 0.2 cm extension of smaller silicone tubing (0.025 in ID,
0.037 in. OD; Sil-Tec, Technical Products, USA). To ensure the cannula was placed in the
lumen of the duodenum, the cannula was flushed with saline. In order to ensure the catheter
remained in place after surgery, it was anchored to the outer serosal surface of the duodenum or
jejunum with 3M adhesives (Vetbond) and a 0.5 cm2 piece of Marlex mesh sewn to the surface
with a 6-0 silk suture. Through the laparotomic incision, the proximal portion of the catheter
exited the abdominal cavity and the abdominal wall was closed with a 4-0 silk suture. At the
back of the neck, a 2 cm midline incision was made in the skin, rostral to the interscapular area,
and the cannula was tunneled subcutaneously to exit the incision. This 2 cm incision was sewn
closed with 4-0 silk sutures and the proximal portion of the cannula was closed with a metal pin.
The cannula was flushed daily with 0.1 ml of saline to ensure patency on the day of the clamp
studies.
2.3 Pancreatic Euglycemic (Basal Insulin) Clamp Technique
The night before the in vivo clamp experiments, the rats were restricted to ~57 kcal to
ensure the same post-absorptive nutritional status. The total length of the experiment was 200
minutes. At t = 0, a primed-continuous infusion of [3–3H] glucose (Perkin Elmer, MA, USA; 40
µCi bolus; 0.4 µCi/ min) was initiated and maintained until t = 200 min to assess glucose
kinetics based on the tracer-dilution methodology. Blood samples were collected in heparinized
tubes at 10 minute intervals and subjected to centrifugation at 6000 rpm to separate the plasma
and plasma glucose was measured as described below (2.8.1 Plasma Glucose) to obtain basal
glucose readings (t = 60-90 min). At t = 90 min until the end of the experiment (t = 200) a
pancreatic (basal insulin) clamp was initiated by providing a continuous insulin (1.2
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mU/kg/min; porcine insulin; Sigma-Aldrich, St. Louis, MO, USA) and somatostatin (3
µg/kg/min; Bachem, CA, USA) infusion to inhibit endogenous insulin and glucagon secretion.
After initiation of the pancreatic clamp, a variable infusion of a 25% glucose solution (45%
glucose; Sigma-Aldrich, St. Louis, MO, USA) was provided to maintain basal plasma glucose
levels (t = 60-90 min) and adjusted every 10 minutes if needed. From t = 150 to t = 200,
intraduodenal or intrajejunal infusions (0.01 ml/min) were performed. Additional samples were
obtained at the 10-minute intervals for the determination of [3–3H] glucose specific activity,
insulin, and leptin levels (see 2.8.2-2.8.4 for details). Rats were anesthetized at the end of the
experiments through a direct infusion of ketamine into the jugular vein and portal plasma
samples were taken followed by tissue collection. Tissues were freeze-clamped in situ with steel
tongs pre-cooled in liquid nitrogen. All tissue samples were stored at –80 ºC and plasma
samples were stored –20 ºC until use. The Harvard Apparatus PHD 2000 infusion pumps (MA,
USA) were used for all infusions during the clamp.
2.4 Protein Assay
The Thermo Scientific Pierce 660nm Protein Assay (Thermo Scientific, IL, USA) was
used to measure the protein concentration of different tissue samples with BSA used as a
standard. This assay is a colorimetric assay based on the binding of a dye-metal complex to
protein under acidic conditions, which causes a shift in the dye’s maximum absorption,
measured at 660nm. The color produced by the assay is stable and the color increases in
intensity with increasing protein concentration. The tissue samples were aliquoted for the
protein assay, thawed, vortexed and kept on ice. The samples were diluted 1:20 with distilled
water in an eppendorf tube. Standards were prepared using stock BSA (2 mg/ml) diluted with
distilled water to prepare a curve ranging from 0 to 2 mg/ml. 10 µl of the BSA standards were
transferred to a 96 microwell plate in duplicate. Then, 10 µl of the diluted tissue samples were
56
added to the plate in duplicate. 150 µl of the Thermo Scientific Pierce 660nm Protein Assay
Reagent was added to each well and allowed to change color. After 5 minutes, the plate was
transferred to a spectrophotometer and the absorbance was read at 660nm. Through
interpolation, the protein concentrations of the tissue samples were determined.
2.5 Biochemical Analyses
2.5.1 Plasma Glucose
The measurements of plasma glucose concentrations were conducted by the glucose
oxidase methods using a GM9 Analox Glucose Analyzer (Analox Instruments, Lunenburg,
MA). Blood samples were collected into heparinized tubes and centrifuged at 6000 rpm to
separate the plasma. Upon calibration of the analyzer with a provided standard, a 10 µl D-
glucose containing plasma sample was pipetted into the reaction well containing a solution with
glucose oxidase and oxygen. The following reaction occurs after injection of a sample:
β-D-Glucose + O2 Glucose oxidase
D-gluconic acid +H!O!
The rate of oxygen consumption is proportional to the amount of glucose in the plasma sample.
A polarographic sensor measures the rate of oxygen consumption to determine the plasma
glucose concentration. More specifically, the partial pressure of oxygen in the sample is
measured as Clark-type amperometric oxygen electrodes are immersed in the sample and a
potential is applied between them that reduces dissolved oxygen at the working electrode.
Results are obtained within 20 seconds of inserting the sample into the apparatus.
2.5.2 Plasma Glucose Tracer Specific Activity
50 µl of plasma was used to determine the specific activity of [3-3H] in the plasma. The
samples were first deproteinized by the addition of 100 µl of Ba(OH)2 and ZnSO4 followed by
vortexing and centrifugation at 6000 rpm for 5 minutes at 4°C. The supernatant of each sample
was transferred to scintillation vials and evaporated to dryness to remove tritiated water (since
57
tritium on the C-3 position of glucose is lost to water during glycolysis). Thus, radioactivity
represents the [3-3H] glucose in the plasma only. Scintillation fluid (Bio-Safe Scintillation
Cocktail, Research Products International Corp., Mount Prospect, IL, USA) was added to the
dried sample to detect the radioactive signal and counted in a LS6500 Multipurpose Scintillation
Counter (Beckman, USA).
2.5.3 Plasma Insulin
A radioimmunoassay (RIA) was used to determine plasma insulin concentrations using a
rat insulin kit (100% specificity) from Linco research (St. Charles, MO). The antigen-antibody
binding principle is used in the RIA. Briefly, the amount of insulin present in the plasma sample
is in competition for binding to antibodies raised against insulin (guinea pig anti-rat insulin
antibody) with a labeled tracer antigen (125I labeled insulin). Thus the amount of radiolabeled
125I-labeled insulin that binds is in reverse proportion to the amount of known standards and the
amount of insulin in the plasma sample. Separation of the 125I-labeled insulin and unbound
fractions is conducted through the use of a double antibody solid phase.
Specifically, a 2-day protocol as per the supplier’s instructions was used. First, the
generation of standard curve is constructed with the use of 50 µl of standards with a range of
known concentrations (0.1, 0.2, 0.5, 1.0, 2.0, 5.0 and 10.0 ng/ml). Then 50 µl of the plasma
samples was pipetted into appropriate tubes and the addition of 50 µl of 125I-labeled insulin and
50 µl of the rat insulin antibody is added to both the standards and samples, and were vortexed.
1.0 ml of precipitating reagent is added after overnight incubation at 4°C followed by vortexing
and incubation at 4°C for 20 minutes. To pellet the bound insulin, the samples were then
centrifuged. A gamma counter (Perkin Elmer 1470) is used to count the radioactivity of the
pellet. The radioactivity counts (B) for the standards and samples are expressed as a percentage
of the mean counts of total binding reference tubes (B0):
58
% total binding=%BB0
= Standard or sample
B0 x 100%
A standard curve is constructed by plotting the % !!!
for each standard against the known
concentration. Through interpolation, the concentration of the insulin samples was determined.
2.6 Calculations
During the pancreatic clamp experiments, a radioactive [3–3H] glucose tracer was
infused at a constant rate to allow for equilibration of the tracer glucose with the glucose in the
body. After equilibration, using the steady state formula, glucose production and uptake can be
determined. That is, in the steady state basal condition, the rate of glucose uptake (Rd) is equal
to the rate of glucose appearance (Ra) or rate of endogenous glucose appearance. Thus, using
the steady state formula, the Ra and Rd can be can be determined by the following equation:
Ra=Rd=Constant tracer infusion rate ( µCimin )
Specific activity ( µCimg )
During the pancreatic clamp where an exogenous glucose infusion is given to maintain
euglycemia, glucose production is calculated by subtracting the exogenous glucose infusion rate
from the Rd:
Ra=Rd-Glucose Infusion Rate
2.7 Statistical Analysis
Data are presented as means + SEM. When a comparison was made between two
groups, an unpaired Student’s t-test was performed. Where comparisons were made across
more than two groups, analysis of variances (ANOVA) was performed, and if significant, this
was followed by Tukey’s post-hoc test, which enabled comparisons of all treatment groups. A
probability of P < 0.05 was accepted as significant. The statistical software program Prism
(GraphPad Software Inc., CA, USA) was used for statistical calculations.
59
Table 2.1 Diet content of the regular chow and lard-oil enriched high fat diet.
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Chapter 3Study 1
Duodenal Activation of cAMP-Dependent Protein Kinase Induces Vagal Afferent Firing and Lowers Glucose Production in Rats
Modified From: Rasmussen, BA, Breen, DM, Luo, P, Cheung, GW, Yang, CS, Sun, B, Kokorovic, A, Rong, W, and Lam, TK. (2012) Duodenal activation of cAMP-dependent protein kinase induces vagal afferent firing and lowers glucose production in rats. Gastroenterology 142, 834-843 Permission to reproduce portions of the above manuscript has been obtained from the copyright owner: Elsevier Limited
61
3.1 Abstract
Background and Aims: The duodenum detects a rise in nutrients to maintain energy and
glucose homeostasis. However, the signaling and neuronal mechanisms involved still remain
unknown. In the present study, we examined whether activation of adenosine 3’,5’-cyclic
monophosphate (cAMP)-dependent protein kinase A (PKA) in the duodenum lies downstream
of CCK to trigger vagal afferent firing and regulate glucose production. Methods: We
selectively activated duodenal PKA in rats through a duodenal infusion of a PKA activator (Sp-
CAMPs) and assessed changes in glucose kinetics during the pancreatic (basal insulin)
euglycemic clamps and vagal afferent firing. To assess whether duodenal PKA signaling is
required for glucose regulation, PKA activation induced through infusion of Sp-CAMPS or a
CCK1 receptor agonist (CCK-8) was blocked through co-infusion of two independent cell-
permeable PKA inhibitors H-89 and Rp-CAMPs. We also tested whether a neuronal network is
required and if the gluco-regulatory effects of duodenal PKA activation remain intact in rats fed
a high fat diet. Results: In normal rats, an intraduodenal infusion of Sp-CAMPs increased both
PKA activation and vagal afferent firing and lowered glucose production. Co-infusion of Sp-
CAMPs with H-89 or Rp-CAMPs (PKA inhibitors) negated the metabolic and neuronal effects
of duodenal PKA activation. The metabolic effects were also negated upon co-infusion with
tetracaine, inhibition (both molecular and pharmacologic) of NR1-containing NMDA receptors
within the DVC, or hepatic vagotomy. Duodenal CCK-8 infusion failed to lower glucose
production upon duodenal PKA inhibition, whereas duodenal CCK resistance in high fat diet fed
rats was bypassed upon duodenal Sp-CAMPs administration, which activated PKA and lowered
glucose production. Conclusions: A neural glucoregulatory function of duodenal PKA signaling
was identified.
62
3.2 Introduction
It is approximated that a staggering 220 million people have type 2 diabetes with almost
half of this population living in China455,456. Diabetes and/or obesity are often characterized by
hepatic insulin resistance, reduced/altered insulin secretion, muscle insulin resistance and
increased glucose production, where fasting hyperglycemia in type 2 diabetes has been shown to
be due largely to an increase in glucose production. Chronic hyperglycemia can lead to diabetic
complications such as neuropathy, nephropathy, and retinopathy454. Thus, uncovering novel
mechanisms that lower glucose production in diabetes or obesity will unveil therapeutic targets
to lower glucose levels and reduce the risk of diabetic complications.
An acute rise in nutrients is detected by the duodenum to trigger negative feedback
systems to maintain peripheral homeostasis457. The absorption and metabolism of pre-absorptive
lipids, through activation of biochemical pathways within the duodenum concurrently inhibits
glucose production and food intake15. The underlying mechanisms of duodenal lipid metabolism
induced suppression of glucose production and food intake remain elusive. However the
secretion of CCK from the duodenal I-cells, and subsequent binding of CCK to its gut CCK1
receptors, are sufficient and necessary for lipids to trigger in parallel a gut-brain and a gut-brain-
liver axis to lower appetite163,167,198,458,459 and glucose production384, respectively. In addition to
glucose production regulation, CCK plays a role in digestion and improves nutrient absorption
by stimulating pancreatic amylase secretion, promotes bile release from the gall bladder, and
delays gastric emptying184. Importantly, the physiological relevance of duodenal CCK action in
glucose regulation is highlighted by the findings that either molecular or pharmacological
inhibition of duodenal CCK1 receptor signaling during refeeding disrupts glucose
homeostasis384.
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The CCK1 receptor is a G-protein coupled receptor460 which is mostly expressed in the
gut. Classical G-protein coupled receptor signaling involves both PKA and PLC signaling
activated by upstream G-proteins Gαs and Gαq, respectively. The CCK1 receptor signaling
pathways have been studied in the pancreatic acinar cell461 and both signaling pathways have
been described to mediate the direct CCK/CCK1 receptor signaling cascade in pancreatic
secretions461–464. To date, the signaling pathway mediating the duodenal CCK1 receptor induced
suppression of glucose production remains unknown. Whether PKA activation mediates CCK1
receptor signaling in the duodenum to regulate peripheral glucose homeostasis will be tested in
the current study.
Duodenal CCK-resistance is acquired in response to short term high fat
feeding5,384,458,459. Although the site(s) of this defect remains unclear, evidence confirms that for
glucose production regulation it is located downstream of CCK release, as both lipid
administration379 and PKC-δ activation382 (both of which stimulate CCK release) still fail to
lower glucose production. Thus, studies aimed at uncovering the duodenal CCK1 receptor
signaling cascade (i.e., PKA signaling) that regulates glucose production in normal and high-fat
fed rats will begin locating the molecular defects that occur in duodenal CCK-resistance. Such a
finding will potentially uncover novel signaling molecules within the duodenum that could be
targeted in diabetes and obesity to restore glucose homeostasis.
In the current study, we propose that direct activation of the duodenal PKA signaling
pathway is necessary for the CCK/CCK1 receptor and sufficient to trigger vagal afferent firing
to activate a neuronal network to lower glucose production in rats in vivo.
64
3.3 Materials and Methods
3.3.1 Animal Preparation
Male SD rats weighing between 280-300g were obtained and maintained as described in
General Methods Section 2.1.
3.3.1.1 High Fat Diet Feeding
A subgroup of rats were fed a lard oil enriched high fat diet for three days. Rats that
were hyperphagic underwent the clamp studies. Please refer to General Methods 2.1.1 for details
on high fat feeding.
3.3.2 Animal Surgeries
3.3.2.1 Intestinal and vascular cannulation
Rats were anesthetized and a duodenal catheter was inserted 0.5 cm proximal to the
pyloric sphincter. A subgroup of rats underwent jejunal catheter placement (8-10 cm from the
Ligament of Treitz). After the intestinal cannulation, the jugular vein and carotid artery were
cannulated. Please refer to the General Methods Section 2.2.1 and 2.2.2 for details regarding
these surgical procedures.
3.3.2.2 Selective Hepatic Branch Vagotomy
A subgroup of rats underwent hepatic vagotomy surgery as previously
described379,384,402. On the ventral midline a laparotomy incision was made, followed by an
incision through the abdominal muscle wall to exposure the gastrointestinal tract within the
peritoneum. The stomach was gently retracted using sterile saline soaked cotton gauze to reveal
the descending esophagus and ventral subdiaphragmatic trunk. A 6-0 suture with a needle was
used to make a small puncture within the bottom portion of the stomach to allow easy
visualization throughout the surgery. The hepatic vagus of the ventral subdiaphragmatic vagal
trunk was transected by microcautery. This disrupts neural communication between the liver
65
and brain. This also results in a slight decrease in the innervations to the intestine as the hepato-
duodenal sub-branch supplies a small portion of the intestine. Intestinal and vascular
cannulation surgeries were performed immediately after the vagotomy surgery.
3.3.2.3 Stereotaxic Surgery
For a subgroup of rats, implantation of a bilateral catheter targeting the nucleus of the
solitary tract within the dorsal vagal complex was performed. Specifically, after rats were
anesthetized, they were mounted onto a stereotaxic apparatus (David Kopf Instruments,
Tunjunga, CA) with ear bars and a nose piece set at +5.0mm. For implantation into the NTS, 26-
gauge stainless steel double guide cannulae were inserted using the following coordinates: 0.0
mm on the occipital crest, 0.4 mm lateral to the midline for both sides, and 7.9 mm below the
skull surface. The double guide cannula was secured with cyanoacrylate glue (HRS Scientific,
QC, Canada) and electric ortho-jet powder liquefied with Ortho-jet Acrylic liquid (Central
Dental, ON, Canada). After the glue and powder had hardened, the double guide cannula was
closed with a dummy cannula and dust cap (HRS Scientific, QC, Canada) until the day of the
experiment. Rats were given a week of recovery time after the stereotaxic surgery before
intestinal and vascular cannulation surgeries were performed.
3.3.2.3.1 Adenoviral Infection in the DVC after Stereotaxic Surgery
Immediately after the stereotaxic surgery, and before placement of the dummy cannula
and dust cap, a subgroup of rats received 3 µL of adenovirus (adenovirus containing short
hairpin RNA NR1: 4.0 x1011 plaque-forming units/mL; mismatch, 4.0 x 1011 plaque-forming
units/mL) per side of the cannulae over a 30-second injection using Hamilton syringes
(Hamilton Company, NV, USA). In order to prevent backflow, microsyringes were left in the
cannula for 20 minutes before removal. After removal, the dummy cannula and dust cap were
placed in and on the guide cannula, respectively, until the day of the clamp experiment. We
66
have previously verified that direct injection of this adenovirus into the DVC knocked down the
NR1 subunit of the NMDA receptors382,465.
3.3.3 Intraduodenal Infusions and Treatments
The following substances were infused into the duodenum through a duodenal catheter during
the pancreatic clamp (t = 150-200) at a rate of 0.01 ml/min:
(1) saline
(2) Sp-cAMPS (PKA agonist; 30 µmol/L; Tocris Bioscience, Ellisville, MO, USA); a
subgroup of rats also received this treatment in the jejunum during the pancreatic clamp.
(3) H-89 (PKA antagonist; 12 µmol/L; Tocris Bioscience, Ellisville, MO, USA)
(4) Rp-CAMPS (PKA antagonist; 12 µmol/L; Tocris Bioscience, Ellisville, MO, USA)
(5) tetracaine (local anesthetic; 0.015 mg/min; Sigma-Aldrich, St. Louis, MO, USA)
(6) CCK-8 (35 pmol /kg/min; Sigma-Aldrich, St. Louis, MO, USA)
(7) MK-329 (CCK1 receptor antagonist; 1.6 µg/kg/min; Tocris Bioscience, Ellisville, MO,
USA)
Reagents #3, and 5-7 were dissolved in DMSO, #2 and #4 in distilled water. The rates for Sp-
CAMPs, H-89 and Rp-CAMPs were based on dose-response studies. The dose for the anesthetic
tetracaine and CCK-8 were based on previous studies379,384.
3.3.4 Pancreatic Euglycemic (Basal Insulin) Clamp Technique in Rats
Please refer to the General Methods section 2.3 for a detailed description of the clamp
procedure. Rats were restricted the night before the clamp experiment. A primed-continuous
constant infusion of [3–3H] glucose was given throughout the experiment (t = 200) to reach
steady state. The pancreatic clamp was then initiated at t = 90 where insulin (1.2 mU/kg/min)
and somatostatin (3 µg/kg/min) were infused at a constant rate. Blood samples were taken to
determine if a variable 25% glucose infusion was needed to maintain euglycemia. At t = 150, a
67
duodenal infusion at 0.01 ml/min was conducted. A subgroup of rats received a jejunal infusion
from t = 150-200 minutes at 0.01 ml/min. Another group of rats received an MK-801 (NMDA
receptor antagonist; dissolved in 0.9% NaCl) infusion (0.03 ng/min; Sigma-Aldrich, St. Louis,
MO, USA) into the NTS at t = 90 until t = 200 at a rate of 0.006 µl/min using the CMA/400
syringe microdialysis infusion pump (Chromatographysciences, Montreal, QC, Canada), in
addition to the duodenal infusion.
3.3.5 Electrophysiological Ex Vivo Recordings of Duodenum Preparation
Just below the sphincter of Oddi (~5 cm long), the duodenum was removed from
anesthetized (80mg/kg pentobarbital i.p) male SD rats (200-250g). This duodenal tissue was
immediately placed in a recording chamber and subsequently perfused with oxygenated (95%
O2 + 5% CO2) Krebs’ solution (composition: NaCl 120 mM; KCl 5.9 mM, NaH2PO4 1.2 mM;
MgSO4 1.2 mM; NaHCO3 15.4 mM; CaCl2 2.5 mM; glucose 11.5 mM) at room temperature
with a Miniplus 3 perfusion pump (World Precision Instruments (WPI), USA) as previously
described466,467. Both ends of the duodenal segment were cannulated with two Genie syringe
pumps (WPI) connected in parallel to an intraluminal inflow cannula through a T-piece
connector to allow for perfusion of Krebs solution or test solutions through the lumen. In 15-
minute intervals, intraluminal infusions (9 mL/h) were conducted. Ramp distensions (up to 60
mm Hg) were performed using a three-way tap on the intraluminal outlet cannula, which was
closed while Krebs’ solution was perfused. Using a suction electrode, a branch of mesenteric
nerves, containing both spinal and vagal afferents, was dissected and pressure was recorded via
a pressure amplifier (NL 108, Digitimer, UK) and nerve activity was recorded with a Neurolog
headstage (NL100, Digitimer), and amplified (NL104) and filtered (NL 125, band pass 200-
3000 Hz). A Micro 1401 interface and Spike2 software (Cambridge Electronic Design, UK)
were used to acquire the nerve signal. An oscilloscope (Tektronix TDS 210) was used to display
whole nerve activity. The spontaneous afferent nerve discharge and distension-induced activity
68
were allowed to become stable, and then the intraluminal infusion solution was switched to 30
µmol/L Sp-CAMPS (in Krebs’ solution, 9 ml/h for 30 minutes; Tocris Bioscience, Ellisville,
MO, USA). In a separate set of experiments, co-infusion of Sp-CAMPS with H-89 (12 µmol/L)
was performed.
3.3.6 PKA Activity Assay
PKA activity in duodenal samples taken directly after the clamp studies was measured
with the PepTag Assay Kit (Promega, Madison, WI) with minor modifications. 1 g of frozen
duodenal tissue was homogenized with a motor and pestle in ice-cold PKA extraction buffer
containing 25 mmol/L Tris-HCl (pH 7.4), 0.5 mmol/L EDTA & EGTA, 0.5 mmol/L ethylene
glycol-bis(β -aminoethylether)-N,N,N’,N’-tetraacetic acid, 10 mmol/L β-mercaptoethanol, and
3X Complete Mini EDTA-Free Protease Inhibitor Cocktail Tablet (Roche Diagnostics, Laval,
QC, Canada) and transferred to eppendorf tubes. The homogenates were centrifuged at 12,300
rpm for 5 minutes at 4°C. The supernatant was transferred to new eppendorf tubes and the
protein concentration was measured as described in the General Methods section 2.4. 5 µg of
protein was used for the reaction, which contained the PKA reaction 5X buffer, A1 peptide,
PKA activator 5X solution, Peptide protection solution, the sample and water. The positive
control contained all solutions described with the catalytic subunit of PKA provided by the kit
where the negative control contained no subunit or sample. All reagents excluding the sample or
catalytic subunit was first pipetted and incubated in a 30°C water for 1 min. Then the duodenal
lysates or PKA catalytic subunit (positive control) was added and the reaction was begun for 30
min at 37°C.The reaction was stopped by transferring the tubes to a 95°C heating block for 10
min. Then, 1 µl of 80% glycerol was added to the samples. The samples were then run on a
0.8% agarose gel (0.4 g of agarose dissolved in 50 mL of 50mM Tris-HCl (pH 8.0)) at 100 V for
15 minutes. The gel fluorescence was analyzed with a BioRad Molecular Imager Gel Doc XR+
69
Imaging System (BioRad, Hercules, CA, USA). Data were analyzed using ImageJ (National
Institutes of Health software).
3.3.7 PCR methods
3.3.7.1 Tissue Preparation and RNA Extraction
An equal number of male SD rats were placed on a standard chow diet and a lard-oil
enriched high fat diet for 3 days ad libitum. Approximately 100 mg of fresh duodenal whole
tissue was collected and utilized. The tissues were homogenized using a mortar and pestle,
which were cooled by liquid nitrogen. Following homogenization, RNA was isolated using the
TRIzol method (Invitrogen-Life Technologies). 1.0 mL of TRIzol reagent was added to each
sample and vortexed to allow for the tissue homogenates to dissolve. Insoluble material from the
homogenate was then removed by centrifugation at 12,000 g for 10 minutes at 4ºC. The RNA-
containing supernatant was collected and allowed to incubate for 5 minutes at room temperature
to permit complete dissociation of the nucleoprotein complex. 0.2 mL of chloroform was added
to the samples, vortexed and incubated for 3 minutes at room temperature. The samples were
centrifuged at 12,000 g for 15 minutes at 4ºC. Following centrifugation, the mixture separated
into a lower red, phenol-chloroform phase, an interphase and a colourless upper aqueous phase.
The upper aqueous phase was collected as RNA remains exclusively in this phase. 0.5 mL of
isopropanol was added to the sample, vortexed and incubated at room temperature for 10
minutes. The samples were centrifuged at 12,000 g for 10 minutes at 4ºC to precipitate the RNA.
After removing the supernatant, 1mL of 75% ethanol was added to wash the RNA pellet. The
mixture was vortexed and centrifuged at 7,400 g for 5 minutes at 4ºC. The supernatant was
removed and the RNA pellet was allowed to dry for 10 minutes. The RNA pellet was
reconstituted in RNase-free water and incubated at 55ºC for 10 minutes. Measurement of the
optical density (OD) was performed to quantify RNA content at 260 and 280 nm using 2 µl of
70
sample with a NanoDrop 1000 spectrophotomoter (Thermo Fisher Scientific, Mississauga, ON,
Canada). The ratio of 260/280 should be between 1.8 and 2 for RNA. RNA concentration
(µg/ml) was then calculated as:
RNA concentration = OD260 x dilution factor x 40
3.3.7.2 cDNA synthesis and PCR
First-strand cDNA was synthesized from 2 µg total RNA using the SuperScript™III
reverse transcriptase protocol (Invitrogen Life Technologies, Carlsbad, CA, USA). The
RNA/primer mixture was prepared in 0.5 mL tubes with the following: 2 µg of total RNA,
Oligo(dT)30 (50 µM), dNTP mix (10 mM), and DEP–treated water. The tubes were incubated at
65 °C for 5 min and immediately transferred to 55 °C. The cDNA synthesis mix was then made
as follows: DEPC-treated water, 10X RT buffer, 25 mM MgCl2, 0.1 M DTT, RNaseOUT™
Recombinase RNase Inhibitor, and SuperScript™III RT. The cDNA Synthesis mix was
prewarmed to 55 °C. The cDNA synthesis mix was added to each sample incubating at 55 °C
and incubated for 50 min total. The reaction was terminated at 85 °C and the tubes were chilled
on ice. After brief centrifugation and collection of the reaction, RNase H was added to each tube
and incubated at 37 °C for 20 min before proceeding to PCR. A PCR mix was prepared (totaling
50 µl) with the following reagents: Phusion High-Fidelity DNA polymerase (Thermo Scientific,
IL, USA), 5X buffer, dNTP (10 mM), primers (13µM), cDNA (aliquots of 5 µL and 1 µL of
cDNA product were used for PCR for CCK1 receptor and β-actin respectively), and water.
PCR amplification was performed with a S1000 Thermal Cycler (Biorad, Hercules, CA, USA)
with an initial cycle of 95 °C for 3 min, followed by 30 cycles each at 95 °C for 30 s
(denaturing), 56 °C for 30 s (annealing), and 72 °C for 40 s (extension). The final extension step
was executed at 72 °C for 7 min. The sequence of the primers for the CCK1 receptor were as
follows: 5′-TGAACTCGGACTGGAAAATGAGAC-3′ for the forward primer and
71
5′-GCATAGCGTCACTTGGCAACAG-3′ for the reverse primer. The sequence of the primers
for β-actin were as follows: 5'-TGAGACCTTCAACACCCCAGCC-3' for the forward primer
and 5'-GAGTACTTGCGCTCAGGAGGAG-3' for the reverse primer. The expected
amplification product sizes for CCK1 receptor and β-actin were 563 bp and 642bp, respectively.
A negative control reaction that contained all the PCR components, except the target cDNA,
was included in each PCR assay. β-actin was used as a control for PCR efficiency. A 1%
agarose gel was prepared by combining 100 ml of 1 x TBE (Tris, boric acid, and EDTA), 1g of
agarose and ethidium bromide. The PCR final products were electrophoresed at 90V until
separation. A DNA ladder was included in the gel for determination of product size. Gels were
visualized under ultra violet light with a BioRad Molecular Imager Gel Doc XR+ Imaging
System (BioRad, Hercules, CA, USA). The band intensities for CCK1 receptor density was
quantified by densitometry with the Quantity One 1-D Analysis Software (BioRad, Hercules,
CA, USA) and normalized to those of the housekeeping gene, β-actin.
3.3.9 Biochemical Analysis
Please refer to the General Methods section 2.6.1-2.6.3 for details on biochemical
analyses. Plasma glucose concentrations were determined using a GM9 Analox Glucose
Analyzer (Analox Instruments, Lunenbertg, MA). Radioactivity of plasma glucose was
conducted as described. Plasma insulin levels were measured using a radioimmunoassay (Linco
Research, St Charles, MO).
3.3.10 Calculations and Statistical Analysis
Values are represented as the mean ± SEM. The basal conditions were averaged between
t = 60-90 minutes and the clamp conditions were averaged between t = 180-200 minutes. For the
electrophysiological ex vivo recordings of duodenal preparations, the mesenteric afferent nerve
activity was recorded as a mean discharge rate (impulses/sec). ANOVA was used to determine
72
statistical differences between groups followed by a Tukey’s post hoc test. A probability of P <
0.05 was considered significant.
3.4 Results
3.4.1 Direct activation of PKA lowers glucose production
In order to first evaluate whether activation of PKA within the duodenum regulates
glucose production, we infused the cell-permeable PKA activator Sp-CAMPS directly into the
duodenal lumen of normal rodents during the pancreatic (basal insulin) euglycemic clamp
studies (Figure 3.1 A and B). During the pancreatic clamp studies, where plasma insulin was
maintained at basal levels (t=60-90 min) (Table 3.1) an intraduodenal Sp-CAMPS
administration increased the glucose infusion rate which was needed to maintain euglycemia
(Figure 3.2A). This was due secondarily to an inhibition in the rate of glucose production
(Figure 3.2B and C) while the rate of glucose uptake remained unchanged (Figure 3.2 D).
Importantly, a direct infusion of Sp-CAMPS into the jejunum failed to lower glucose production
(clamp glucose production: 12.2 +/- 2.0 mg/kg/min; n = 5), suggesting that duodenal Sp-
CAMPS administration activated duodenal PKA to lower glucose production. Next, co-infusion
of Sp-CAMPS with two independent cell-permeable PKA inhibitors H89 or Rp-CAMPS was
conducted (Figure 3.1A and B). Inhibition of PKA activation through co-infusion of
intraduodenal Sp-CAMPS with either H89 or Rp-CAMPS abolished the ability of duodenal Sp-
CAMPS to increase the glucose infusion rate (Figure 3.2A) and decrease glucose production
(Figure 3.2B and C). In order to confirm the specificity of these treatments to activate duodenal
PKA, from duodenal tissues taken immediately after the termination of the clamp studies we
assessed PKA activity. The A1 peptide is phosphorylated by PKA and thus a higher the ratio of
phospho(P)-A1/A1 reflects a higher degree of PKA activation. Intraduodenal Sp-CAMPS
induced duodenal PKA activity (Figure 3.2E) and this activation was fully blocked by co-
73
infusion with Rp-CAMPS (Figure 3.2E). These data suggest that direct activation of duodenal
PKA is sufficient to lower glucose production.
3.4.2 Activation of PKA lowers glucose production via a vagal afferent firing
We next assessed whether duodenal PKA lowers glucose production through activation
of a neuronal network. We first evaluated, in an ex vivo duodenal preparation, the effect of an
intraluminal infusion of Sp-CAMPS on mesenteric neuronal discharge rate (Figure 3.3). In this
regard, a branch of the mesenteric nerves consisting of both vagal and spinal afferents was
dissected and recorded using a suction electrode466. An intraduodenal infusion of Sp-CAMPS
resulted in a gradual rise in the spontaneous discharge rate of the mesenteric nerve (Figure
3.4A) The peak discharge rate during intraduodenal Sp-CAMPS administration was 116.8 +
25.4 imp/s and this reflected a significant increase of 27 + 7 % (Figure 3.4B) compared with the
average basal discharge rate (92.0 + 20.8 imps/s). We next addressed whether this increase in
mesenteric neuronal discharge rate was due to activation of PKA through co-infusion of Sp-
CAMPS with the PKA inhibitor H89. An intraduodenal infusion of H89 alone did not have any
effect on the spontaneous firing rate but abolished the ability of Sp-CAMPS to increase
mesenteric neuronal discharge rate (Figure 3.4C). These results suggest that direct activation of
duodenal PKA increases the spontaneous firing rate of the duodenal mesenteric nerve.
We next investigated whether changes in vagal and/or spinal firing resulted in changes in
the mesenteric neuronal firing. This was through measuring distension-induced duodenal
afferent activity in response to duodenal PKA activation. In brief, ramp distension results in
biphasic increases in duodenal afferent nerve discharge of which the initial phase of the
response (Figure 3.4D; rectangle) is a rapid increase in afferent discharge at the beginning of
distension, reflecting an activation of low threshold mechanoreceptors. The second phase
(Figure 3.4D; arrow) is an accelerated increase in afferent activity when the intraluminal
74
pressure reaches ~20 mmHg, reflecting an activation of high threshold mechanoreceptors. It is
generally thought that firing of vagal afferents is due to activation of low threshold
mechanoreceptors that encode innocuous (physiological) mechanical stimulation (i.e., gastric
emptying) whereas spinal afferents are mostly triggered by activation of high threshold
mechanoreceptors that encode noxious stimulation (i.e., over distension due to obstruction)467.
We report that intraduodenal administration of Sp-CAMPS inhibited the high threshold
mechanosensory responses (Figure 3.4E). Together with the overall increase in duodenal
mesenteric spontaneous afferent firing induced by duodenal PKA activation, our results strongly
suggest that an intraduodenal Sp-CAMPS administration activates duodenal spontaneous vagal
afferent firing but inhibits the spinal afferent.
We next delineated the functional relevance of the change in duodenal ex vivo vagal
afferent firing induced by duodenal PKA activation by assessing the neuronal network that is
required in glucose regulation induced by duodenal Sp-CAMPS. In this regard, during the clamp
studies we co-infused intraduodenal Sp-CAMPS with the local anesthetic tetracaine (Figure 3.5
A and B). Infusion of tetracaine alone into the duodenum did not affect the glucose kinetics
(Figure 3.6A-D) but abolished the ability of intraduodenal Sp-CAMPS to increase the glucose
infusion rate and lower glucose production independent of changes in plasma insulin and
glucose levels as well as glucose uptake during the clamps (Figure 3.6A-D;Table 3.2). Thus,
duodenal innervation of vagal afferent nerves is required for intraduodenal Sp-CAMPS to lower
glucose production.
3.4.3 Activation of NR1-containing NMDA receptors is required for duodenal PKA to
lower glucose production
The vagal afferent nerves that innervate the small intestine terminate at the level of NTS
within the DVC. To address the requirement of NMDA receptor activation in duodenal Sp-
75
CAMPS induced suppression of glucose production, we next inhibited NMDA receptor-
mediated neuronal transmission in the DVC via direct NTS-targeted administration of the
NMDA receptor blocker MK-801 (Figure 3.7A and B). A MK-801 infusion into the DVC
alone did not affect glucose kinetics (Figure 3.8A-D) but negated the ability of duodenal Sp-
CAMPS to increase the glucose infusion rate and lower glucose production (Figure 3.8A-C).
This blockade effect of DVC MK-801 occurred independent of changes in the rate of glucose
uptake (Figure 3.8D) and plasma insulin levels (Table 3.2).
The NMDA receptor is composed of the NR1 and NR2 subunits465 and direct activation
of either the NR1 or NR2 subunit within the DVC is sufficient to lower glucose production382.
To address the role of the NR1 subunit in duodenal PKA induced suppression in glucose
production, we knocked down the NR1 subunit expression of the NMDA receptors in the DVC.
To this end, we injected an adenovirus expressing the shRNA of NR1 vs. mismatch (mm)
control into the DVC (Figure 3.7A) and the rats subsequently underwent duodenal infusion and
pancreatic clamp studies (Figure 3.7B). We have previously confirmed the specificity of the
NR1 knock-down within the DVC using the same adenoviral injection protocol382. An
intraduodenal Sp-CAMPS administration increased the glucose infusion rate and lowered
glucose production in DVC mm-injected rats (Figure 3.8A-C). The ability of duodenal Sp-
CAMPS infusion to alter glucose kinetics was negated in DVC shRNA-NR1-injected rats
(Figure 3.8A-C). Together with the pharmacological loss-of-function studies, these results
indicate that activation of the NR1-containing NMDA receptor within the DVC mediates the
vagal afferent neuronal signal(s) ignited by duodenal PKA activation to lower glucose
production.
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3.4.4 Duodenal PKA activation requires brain to liver communication to lower glucose
production
We next tested whether brain to liver communication is required for duodenal PKA
activation to lower glucose production, we repeated the intraduodenal Sp-CAMPS infusion
clamp studies in rats that received hepatic vagotomy, a surgical procedure which abolishes brain
to liver communication (Figure 3.7A). An intraduodenal Sp-CAMPS administration failed to
increase the glucose infusion rate and lower glucose production in hepatic vagotomized rats
(Figure 3.8A-C). Together with the duodenal ex vivo data, these in vivo results together indicate
that activation of duodenal PKA is sufficient to increase vagal afferent firing to trigger a gut-
brain-liver axis to lower glucose production.
3.4.5 CCK lowers glucose production via PKA activation
A direct duodenal CCK-8 administration activates CCK1 receptors to trigger a neuronal
network to lower glucose production, an effect that is abolished in rodents fed a high fat diet for
three days384. In order to locate the downstream potential defect(s) of CCK signaling in the
duodenum causing duodenal CCK resistance, we first addressed whether binding of CCK to its
CCK1 receptor results in PKA activation to lower glucose production. An intraduodenal
infusion of CCK-8 with either PKA inhibitor, H89 or Rp-CAMPS, was performed while plasma
insulin and glucose levels were maintained at basal levels during the clamps (Figure 3.9A and
B; Table 3.3). Consistent with previous findings384, intraduodenal CCK-8 increased the
glucose infusion rate required to maintain euglycemia due to an inhibition of glucose production
(Figure 3.10A-C) rather than changes in glucose uptake (Figure 3.10D). Co-infusion of CCK-8
with either H89 or Rp-CAMPS abolished the ability of duodenal CCK-8 to increase the glucose
infusion rate and lower glucose production (Figure 3.10A-C). Importantly, in duodenal tissues
taken immediately after the pancreatic clamp studies, duodenal CCK-8 administration activated
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PKA (Figure 3.10E), which was reversed by co-infusion of the PKA inhibitor Rp-CAMPS
(Figure 3.10E). To ensure activation of PKA lies downstream of the CCK1 receptor, we next
co-infused Sp-CAMPS with a CCK1 receptor antagonist MK-329 which did not abolish the
ability of Sp-CAMPS to lower glucose production (Figure 3.10B and C), indicating that PKA
activation lies downstream of CCK. Given these findings, we next tested whether direct
activation of PKA bypasses duodenal CCK resistance to lower glucose production in rats fed
with a high fat-diet (Figure 3.11A).
3.4.6 The CCK1 receptor fails to activate PKA after short term high fat feeding
Rats were fed with a lard-oil enriched diet (Table 2.1) for 3 days and rats that were
hyperphagic underwent the intraduodenal infusion clamp studies (Figure 3.11A). Intraduodenal
CCK-8 failed to increase duodenal PKA activity (Figure 3.12E) and the glucose infusion rate
(Figure 3.12A), and also failed to lower glucose production (Figure 3.12B and C) in high fat-
fed rats. Glucose uptake was comparable among groups (Figure 3.12D). The inability of
duodenal CCK-8 to regulate glucose homeostasis was independent of changes in duodenal
CCK1 receptor expression upon high fat feeding (Figure 3.13F), suggesting that CCK-
resistance lies within the signaling pathway(s). In this regard, direct activation of duodenal PKA
via Sp-CAMPS increased the glucose infusion rate (Figure 3.13A), lowered glucose production
(Figure 3.13B and C) and activated duodenal PKA (Figure 3.13E) in high fat-fed rats. Glucose
uptake was unaltered (Figure 3.13D). These results suggest that direct activation of duodenal
PKA bypasses CCK-resistance to lower glucose production and that duodenal CCK-resistance
likely arises from the inability of the CCK1 receptor-coupled signaling cascade to activate PKA
in high-fat fed rats.
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3.5 Discussion
This current study set out to elucidate the duodenal CCK/CCK1 receptor-signaling
cascade that regulates glucose production. We here demonstrated that direct activation of
duodenal PKA was sufficient to lower glucose production in vivo in parallel to an induction of
spontaneous vagal afferent firing in an ex vivo duodenum preparation. The in vivo functional
relevance of the increased vagal afferent firing was illustrated by the following findings when: i)
inhibition of neuronal innervation of the duodenum ii) DVC NR1-containing NMDA receptors
and iii) hepatic vagal transmission all negated the ability of duodenal PKA activation to lower
glucose production. Moreover, in normal rats, duodenal CCK/CCK1 receptor signaling requires
PKA activation to lower glucose production. Excitingly, direct activation of duodenal PKA
bypassed CCK resistance to lower glucose production in rodents fed a high fat diet. The
physiological relevance in glucose regulation of duodenal PKA signaling remains to be
clarified. However, these data collectively illustrate that duodenal PKA activation triggers vagal
afferent firing to activate NR1 containing NMDA receptors to lower glucose production in
normal and high fat fed rodents. Thus, administration of PKA agonists into the duodenum could
potentially help to restore glucose homeostasis in diabetes and obesity.
It has been previously demonstrated that vagal afferents innervating the small intestine
express the CCK1 receptor468. We are currently limited by technology to locate the exact site of
duodenal PKA activation. However our data strongly suggests that activation of the duodenal
CCK1 receptor G-protein coupled PKA signaling triggers vagal afferent firing to lower glucose
production. It remains to be clarified how PKA mediated signaling pathway(s) trigger vagal
afferent firing to inhibit glucose production. Given that the anesthetic tetracaine, a voltage gated
sodium channel inhibitor, abolished the ability of duodenal PKA activation to lower glucose
production, voltage gated sodium channels represent a potential downstream mediator.
Supporting this finding, PKA activation potentiates sodium current in the brain469 and argues for
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a role of duodenal voltage-gated sodium channels in mediating duodenal signal(s) to regulate
glucose production.
In addition to the findings described above, we also demonstrated that activation of
duodenal PKA inhibits mechanosensory spinal afferent firing. Although the underlying
mechanisms in the regulation of pain remain elusive, studies aimed to dissect the neuronal and
signaling mechanisms that control pain tend to focus on the control of spinal afferent firing as
changes in spinal afferent firing directly modulate pain470. For the first time, our study indicates,
that duodenal PKA signaling can regulate spinal afferent firing. Although this hypothesis
remains to be validated, an important potential implication arises in the context of the current
study, as direct delivery of a PKA agonist into the duodenum would lower glucose production in
the absence of pain induction.
We further discovered that direct activation of duodenal PKA can lower glucose
production in high fat diet fed rodents to bypass CCK resistance. This suggests that a duodenal
CCK-8 administration fails to activate the CCK-1 receptor and subsequent downstream
mediators, such as PKA, through an inability of G-protein coupled signaling to activate AC and
increase cAMP formation. This is consistent with the findings that high fat feeding reduces the
activity of AC and subsequent cAMP formation in the liver471. In addition to the PKA pathway,
the duodenal CCK/CCK1 receptor activates a PLC-dependent signaling pathway to regulate
pancreatic secretions461–464. The role of PLC in mediating the duodenal CCK effect on glucose
production remains to be clarified but our preliminary data suggests an involvement of duodenal
PLC signaling since an intraduodenal co-infusion of the PLC inhibitor U 73122 (200 µM) with
CCK-8 negated the ability of CCK-8 to inhibit glucose production. The rate of glucose
production during the clamp was 11.1 +/- 1.5 mg/kg/min (CCK-8 + U73122; n= 6) vs. 10.8 +/-
0.7 (U73122 alone; n=5) or 6.0 +/- 0.4 (CCK-8 alone; n=8). Finally, given that duodenal protein
kinase C-δ signaling is necessary for duodenal lipid sensing383 and sufficient to trigger CCK1
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receptors391 to regulate glucose production, we propose duodenal lipids activate a PKC-δ ->
CCK1 receptor -> cAMP-PKA dependent sequential signaling cascade to trigger vagal afferent
firing and inhibit glucose production in normal rats. Importantly, direct activation of
downstream signaling of CCK1 receptor (i.e., PKA), but not upstream (i.e., PKC-δ391), bypasses
duodenal CCK-resistance to inhibit glucose production in high-fat fed rats.
In summary, we have demonstrated, for the first time to our knowledge, that activation
of duodenal PKA ignites vagal afferent firing and triggers a neuronal network to lower glucose
production. In addition, activation of duodenal PKA is required for CCK to lower glucose
production in normal rats and bypasses CCK-resistance in high-fat fed rats to lower glucose
production. These data highlight a previously unappreciated role of duodenal PKA signaling in
neural regulation of glucose homeostasis.
As discussed in the introduction, CCK and leptin may interact to regulate feeding. Given
that CCK regulates glucose production, the purpose of Study 2 of this thesis was to address
whether intestinal leptin action, like CCK, also regulates glucose production through a neuronal
network.
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Figure 3.1 Schematic representation of working hypothesis – duodenal Sp-CAMPS activates PKA to lower glucose production, which is abolished upon co-infusion of Sp-CAMPS and H-89 or Rp-CAMPS, and experimental design.
A) Proposed model for duodenal PKA to lower glucose production. Infusion of Sp-CAMPS (PKA agonist) activates duodenal PKA and such activation is prevented upon co-infusion with either PKA inhibitor H-89 and Rp-CAMPS. B) Schematic representation of experimental design: on Day 1, intravenous and duodenal catheters were implanted in male SD rats (280-300g). Rats were given 4 to 5 days of recovery until the pancreatic clamp studies where duodenal infusions of saline, Sp-CAMPS ± H-89 or Rp-CAMPS were administered.
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Figure 3.2 Duodenal PKA activation lowers glucose production.
(A and B) During the pancreatic clamp (t = 180-200) an intraduodenal Sp-CAMPS infusion (30 µmol/L) increased the glucose infusion rate (A, *P < 0.05 vs other groups) and decreased glucose production (B, *P < 0.05 vs other groups). Co-infusion of Sp-CAMPS with H-89 or Sp-CAMPS abolished the effects of Sp-CAMPS on A) the glucose infusion rate and B) glucose production. C) Suppression of glucose production during the clamp period (t = 180-200) expressed as the percent reduction from the basal state (t = 60-90) glucose production (*P < 0.05 vs other groups). D) The rate of glucose uptake remained unchanged amongst all groups. E) Duodenal PKA activity assessed with the PepTag assay. Duodenal Sp-CAMPS infusion significantly increased the amount of phosphorylated A1 peptide versus nonphosphorylated A1 peptide (*P < 0.01 vs other groups). SAL, n = 10; Sp-CAMPS n = 9; H-89 n = 5; Sp-CAMPS + H-89 n = 6; Rp-CAMPS n = 5; Sp-CAMPS + Rp-CAMPS n = 5. Values are shown ± SEM.
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Figure 3.3 Schematic representation of working hypothesis – duodenal PKA activation increases vagal afferent firing
Proposed model for duodenal PKA activation to increase vagal afferent firing. Infusion of Sp-CAMPS into the duodenum increases the spontaneous discharge rate of the mesenteric nerve which is inhibited upon co-administration with H-89.
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Figure 3.4 Direct activation of duodenal PKA increases the spontaneous discharge rate of the mesenteric nerve and inhibits spinal afferent firing of the duodenum.
A) An intraluminal infusion of Sp-CAMPS increases the spontaneous discharge rate of the duodenal mesenteric nerve. The top panel represents nerve activity and the bottom panel represents discharge frequency. B) An intraluminal infusion of Sp-CAMPS increased the spontaneous discharge rate of the mesenteric nerve, represented as normalized nerve activity (*P < 0.05 vs control). C) An intraluminal infusion of Sp-CAMPS with H-89 abolished the increase in afferent discharge. D) Distension-evoked biphasic activation of the duodenal afferent nerves ± Sp-CAMPS administration. Pressure increase and discharge frequency are shown in top and bottom panels, respectively. The rectangles indicate activation of low-threshold mechanoreceptors and the arrows indicate activation of high-threshold mechanoreceptors. Intraduodenal Sp-CAMPS administration inhibited high-threshold mechanosensory responses. E) Sp-CAMPS infusion inhibited the high-threshold mechanosensory responses, represented as a change in discharge rate in comparison to the basal discharge rate (**P < 0.05 vs control). N = 6 per group. Values are shown ± SEM.
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Figure 3.5 Schematic representation of working hypothesis – duodenal PKA activation triggers a neuronal network to lower glucose production and experimental design.
A) Proposed model for a neuronal network activated by duodenal PKA activation. The local anesthetic tetracaine abolishes neuronal innervation of the duodenum. B) Schematic representation of experimental design. Tetracaine was co-infused with Sp-CAMPS during the pancreatic clamp.
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Figure 3.6 Duodenal PKA activation lowers glucose production through a neuronal network.
(A and B) An intraduodenal infusion of Sp-CAMPS increased the glucose infusion rate (A, *P < 0.01 vs other groups) and decreased glucose production (B, *P < 0.01 vs other groups). This was abolished upon co-infusion with tetracaine. C) Suppression of glucose production during the clamp period (t = 180-200) expressed as the percent reduction from the basal state (t = 60-90) glucose production (*P < 0.001 vs other groups). D) The rate of glucose uptake remained unchanged in all groups. SAL, n = 10; Sp-CAMPS n = 9; tetracaine, n = 5; Sp-CAMPS + tetracaine, n = 5. Values are shown ± SEM.
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Figure 3.7 Schematic representation of working hypothesis – duodenal PKA activation lowers glucose production through a gut-brain-liver neuronal axis and experimental design.
A) Proposed model for PKA induced activation of a gut-brain-liver axis. MK-801, a potent NMDA receptor antagonist, blocks activation of the receptors. A separate group of rats received a viral injection of an adenovirus expressing short hairpin RNA-NR1 versus a mismatch control to knockdown expression of the NR1 subunit of the NMDA receptor. Another group of rats received hepatic vagotomy surgery, which abolishes communication between the brain and liver. An intraduodenal Sp-CAMPS infusion failed to lower glucose production in the presence of a MK-801 infusion in the DVC, rats injected with shRNA NR1, or rats subjected to hepatic vagotomy. B) Experimental protocol. Stereotaxic surgeries were performed on male SD rats (~250-300g) 7 days prior to duodenal and vascular cannulations. A subgroup of rats received an adenovirus injection at the same time of the stereotaxic surgery. During the clamp studies, an intraduodenal Sp-CAMPS infusion was given ± MK-801 infusion.
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Figure 3.8 Duodenal PKA activation lowers glucose production through activation of the DVC NR1- containing NMDA receptor and hepatic innervation.
(A and B) An intraduodenal Sp-CAMPS infusion increased the glucose infusion rate (A, *P < 0.001, # P < .001 vs other groups) and decreased glucose production (B, *P < 0.001, #P < 0.001 vs other groups). Rats that received a DVC MK-801 administration, hepatic vagotomy surgery or injection of shRNA-NR1 failed to respond to a duodenal Sp-CAMPS infusion. C) Suppression of glucose production during the clamp period (t = 180-200) expressed as the percent reduction from the basal state (t = 60-90) glucose production (*P < 0.001, # P < 0.01 vs other groups). D) The rate of glucose uptake remained unchanged in all groups. SAL, n = 10; Sp-CAMPS; DVC-MK-801 n = 5; Sp-CAMPS + DVC-MK-801 n = 5; DVC-shRNA NR1 n = 7; DVC-mistmatch n = 5; HVAG n = 6; Sp-CAMPS + HVAG n = 5. Values are shown as mean ± SEM. HVAG: Hepatic vagotomy
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Figure 3.9 Schematic representation of working hypothesis – Duodenal CCK requires PKA activation to lower glucose production and experimental design.
A) Proposed model for the necessity of PKA activation for duodenal CCK to lower glucose production. H-89 and Rp-CAMPS are PKA inhibitors, and MK-329 is a CCK1 receptor inhibitor. Intraduodenal CCK fails to lower glucose production upon co-infusion with either PKA inhibitor H-89 or Rp-CAMPS. PKA activation lies downstream of the CCK1 receptor as a Sp-CAMPS infusion lowers glucose production in the presence of the CCK1 receptor MK-329. B) Experimental protocol. 4 to 5 days after duodenal and vascular cannulation, the clamp studies were conducted where CCK-8 ± H-89 or Rp-CAMPS and Sp-CAMPS ± MK-329 were given.
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Figure 3.10 Duodenal CCK requires PKA activation to lower glucose production.
(A and B) An intraduodenal CCK-8 infusion increased the glucose infusion rate (A, *P < 0.01 versus other groups) and decreased glucose production (B, *P < 0.05 versus other groups). In contrast, coinfusion with either H-89 or Rp-CAMPS abolished the effects of CCK-8. A Sp-CAMPS infusion increased the glucose infusion rate (A, *P < 0.01 versus other groups) and decreased glucose production (B, *P < 0.05 versus other groups) in the presence of MK-329. C) Suppression of glucose production during the clamp period expressed as the percentage decrease from basal (*P < 0.01 versus all groups). E) A duodenal CCK-8 infusion significantly increased the amount of phosphorylated A1 peptide versus nonphosphorylated A1 peptide (*P < 0.05 versus all groups). SAL n = 10; CCK-8 n = 8; H-89 n = 5; CCK-8 + H-89 n = ; Rp-CAMPS n = 5; CCK-8 + Rp-CAMPS n = 5. Values are shown as mean ±SEM.
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Figure 3.11 Schematic representation of working hypothesis – Duodenal CCK fails to suppress glucose production upon high fat feeding, which is rescued upon PKA activation and experimental design
A) Proposed model to determine whether duodenal CCK-8 can suppress glucose production in response to high fat feeding for 3 days. Rats were placed on a lard-oil enriched high fat diet for 3 days and then the clamp studies were performed. Duodenal CCK-resistance is bypassed upon PKA activation. B) Experimental protocol. Rats were placed on regular chow for 4 days and then switched to a lard-oil enriched high fat diet for 3 days until the pancreatic clamp study where an intraduodenal infusion of CCK-8 or Sp-CAMPS was given.
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Figure 3.12 Duodenal CCK fails to activate duodenal PKA and lower glucose production after three days of high fat feeding.
A) Intraduodenal CCK-8 increased the glucose infusion rate (A, * P < 0.05, versus other groups) and decreased glucose production (B, *P < 0.05 versus other groups) in regular chow fed rats. After high fat feeding for 3 days, rats failed to respond to intraduodenal CCK-8. C) Suppression of glucose production during the clamp period expressed as a percentage decrease from basal (C, *P < 0.01 versus other groups). D) Glucose uptake remained unchanged among the groups. E) PKA activation in tissues taken after the clamp studies. Intraduodenal infusion of CCK-8 in HFD rats failed to increase PKA activity. SAL RC n = 10; SAL HFD n = 5; CCK-8 RC n = 8; CCK-8 HFD n = 6. Values are shown as mean ± SEM
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Figure 3.13 Duodenal Sp-CAMPS activates duodenal PKA activity and lowers glucose production in high fat diet fed rats.
(A and B) Intraduodenal Sp-CAMPS increased the glucose infusion rate (A, *P < 0.05 versus other groups) and decreased glucose production (B, *P < 0.05 versus other groups) in rats fed with RC. The glucose infusion rate was increased (A, #P < 0.01 versus other groups) and decreased glucose production (a, #p < 0.01 vs. other groups) in rats fed HFD. C) Suppression of glucose production during the clamp period expressed as a percentage decrease from basal (C, *P < 0.05 versus other groups, #P < 0.05). E) Duodenal Sp-CAMPS infusion in rats fed a HFD significantly increased the amount of phosphorylated A1 peptide versus nonphosphorylated A1 peptide (E, *p < 0.01 versus HFD SAL). F) CCK1 receptor expression was comparable in both regular chow and high fat fed rats. SAL RC n = 10; SAL HFD n = 5; Sp-CAMPS RC n = 9; Sp-CAMPS HFD n = 9. Values are shown as mean ± SEM.
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Table 3.1 Plasma insulin and glucose concentrations of the groups receiving an intraduodenal infusion during basal and clamp conditions.
Values are expressed as means ± SEM. (Basal: 60-90 min; Clamp: 180-200 min).
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Table 3.2. Plasma insulin and glucose concentrations of the groups receiving both an intraduodenal infusion and DVC infusion during basal and clamp conditions
Values are expressed as means ± SEM. (Basal: 60-90 min; Clamp: 180-200 min). HVAG, hepatic vagotomy.
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Table 3.3 Plasma insulin and glucose concentrations of the groups receiving an intraduodenal infusion during basal and clamp conditions.
Values are expressed as means ± SEM. (Basal: 60-90 min; Clamp: 180-200 min).
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Chapter 4Study 2
Jejunal Leptin-PI3K signaling lowers glucose production Modified From: Rasmussen, BA*, Breen, DM*, Duca, FA, Côté, CD, Zadeh Tahmasebi, M, Filippi, BM, and Lam, TK. (2014) Jejunal leptin-PI3K signaling lowers glucose production. Cell Metabolism 19, 1-7 *Equal contribution Permission to reproduce portions of the above manuscript has been obtained from the copyright owner: Elsevier Limited
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4.1 Abstract
Background and Aims: The fat derived hormone leptin binds to its hypothalamic receptors to
regulate glucose homeostasis. Leptin is also synthesized in the stomach and binds to its
receptors expressed in the intestine. Given that recent studies report jejunal nutrient sensing is
necessary for DJB to lower glucose production and plasma glucose levels in uncontrolled
diabetic rodents with insulin-deficiency, we sought to determine whether intestinal leptin
regulates glucose production through similar mechanisms as the brain in normal, and disease
models and whether jejunal leptin action mediates the glucose-lowering effect induced by DJB
in insulin-deficient uncontrolled diabetes. Methods: In rats and mice, we administered leptin
into the jejunum for 50 min and evaluated changes in glucose production during pancreatic
clamps in vivo. Molecular and chemical loss-of-function approaches targeting intestinal leptin
receptor-mediated signaling were utilized to assess the underlying mechanisms involved in
normal and diabetic (with or without DJB) rodents. Results: Intrajejunal leptin infusion
activated jejunal PI3K and STAT3 and lowered glucose production in normal rats and mice
independent of changes in circulating leptin and insulin levels. The glucose production-lowering
effect induced by jejunal leptin was negated in leptin receptor deficient fak/fak rats and db/db
mice, or upon co-infusion with a leptin receptor antagonist. Interestingly, blockade of jejunal
PI3K and not STAT-3 signaling negated jejunal leptin to lower glucose production in normal
rats, while the metabolic effect of leptin was also seen in insulin deficient STZ-induced
uncontrolled diabetic (independent of changes in glucagon levels) and insulin resistant HFD
rodents. Lastly, blockade of jejunal leptin action disrupted glucose homeostasis during refeeding
in uncontrolled diabetic rodents that received DJB. Conclusions: These data unveil a novel
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glucoregulatory site of leptin action and suggest that enhancing leptin-PI3K signaling in the
jejunum lowers plasma glucose concentrations in normal and diabetic conditions.
4.2 Introduction
Since the discovery of leptin125, there has been a large effort by scientists to evaluate the
physiological impact of hypothalamic leptin signaling9. Indeed, the brain plays an important role
in mediating the action of leptin by binding to the hypothalamic Leprb and activating
downstream signaling molecules, STAT3 and/or PI3K to regulate energy balance9,472. In
addition to regulating energy balance, activation of hypothalamic PI3K and STAT3 via a central
leptin infusion improves insulin sensitivity473 and lowers glucose production474 in high-fat fed
rodents. Central leptin also lowers plasma glucose levels in non-obese STZ-induced insulin-
deficient uncontrolled diabetic rodents by inhibiting glucose production in association with a
drop in plasma glucagon levels475,476. Thus it is evident that since its discovery, many questions
in regards to hypothalamic leptin action have been uncovered, but much work still remains to
uncover the neurocircuitry and metabolic impact of hypothalamic leptin action. Moreover,
whether leptin action regulates metabolism in extra-hypothalamic sites remains in question, but
studies are beginning to uncover such sites as hindbrain leptin signaling lowers food intake477.
In addition to adipocytes, it is believed that leptin is also produced by gastric chief
cells127,144 and acts on the Leprb expressed in the intestine and/or on vagal afferents that
innervate the intestine149–151 to regulate various intestinal processes. For example, gastric leptin
is secreted in response to nutrient ingestion478 and has been shown to work in collaboration with
other gut peptides to modulate feeding479. In addition to food intake regulation, leptin also helps
to maintain the intestinal environment as mice480 and humans481 with mutations in the leptin
receptor have higher susceptibility to intestinal infection. Furthermore, leptin regulates intestinal
lipid482 and carbohydrate483 absorption and increases neuronal activity of the NTS484. Given that
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STAT3 and PI3K are expressed in the intestine and/or on the vagal terminals that innervate the
intestine151,157,485, and that intestinal leptin receptor signaling regulates various intestinal
functions, intestinal leptin receptor signaling may also regulate glucose homeostasis. However,
such a hypothesis has not yet been tested in vivo.
Given that hypothalamic leptin triggers a neurocircuitry to control glucose
homeostasis25,486 while in parallel nutrient sensing in the small intestine triggers a gut-brain axis
to regulate glucose homeostasis402,487 we hypothesize that intestinal leptin activates a leptin
receptor-PI3K and/or STAT3-dependent pathway to regulate glucose homeostasis through a
neuronal network. In addition, jejunal nutrient sensing mechanisms are required for DJB surgery
to lower plasma glucose levels and glucose production in non-obese uncontrolled diabetic
rodents with insulin deficiency402. Given that hypothalamic brain leptin action has a similar
effect in uncontrolled diabetes475,476 we then evaluated whether the glucoregulatory control of
intestinal leptin action is intact in uncontrolled diabetic or high-fat fed rodents and necessary for
the rapid anti-diabetic effect of DJB.
4.3 Materials and Methods
4.3.1 Animal Preparation
Normal SD rats (280-300g) were maintained as described in General Methods section
2.1. 18 week old (~25-30g) C57BL/6J mice were obtained from Jackson laboratories (Bar
Harbor, Maine, USA). Mice were housed in groups of four and maintained on a standard 12-12h
light dark cycle, and had ad libitum access to water and rodent chow (Harlan Teklad 6%
mouse/Rat diet; 49% carbohydrate, 33% protein and 18% fat; total calories provided by
digestible nutrients: 3.1 kcal/g). Mice were given at least 5 days to acclimatize upon arrival
before surgeries were performed.
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4.3.1.1 Koletsky Rat
The Koletsky rat (fak/fak) was used as a model deficient of the long form leptin
receptors. The obese phenotype results from a spontaneous autosomal recessive nonsense
mutation on chromosome 5 producing a mutated leptin receptor488,489. Both lean (fa/fa) and
obese Koletsky rats (fak/fak) were obtained from Charles River at the age of 8 weeks. Koletsky
(fak/fak) rats weigh nearly the same as their lean littermates until ~4-6 weeks of age (~250-300g)
and then become hyperphagic and rapidly gain weight and become obese. Young adult (+6
weeks of age) Koletsky (fak/fak) rats are also hypertensive, hyperinsulinemic, hyperlipidemic,
and display only a marginal elevation in post-prandial glucose levels (6.2 vs. 5.2 mM) but with
normal fasting glucose levels, indicating only mild glucose intolerance490,491. Lean and obese
Koletsky rats were monitored at 4:00pm daily for body weight and food intake. Due to the fact
that obese Koletsky rats are hyperphagic, food intake was restricted to just below the average
amount of food consumed by the lean Koletsky rats as described473 in order to maintain a body
weight of ~300 g that was comparable to the lean control and male Sprague-Dawley rats. After
5 weeks, intravenous and jejunal cannulation surgeries were performed.
4.3.1.2 db/db mouse
The obese leptin receptor deficient male db/db mice from Jackson Laboratories were
used as an additional model of long form leptin receptor deficiency. Diabetes that results in
these mice arises from a recessive, autosomal single-gene mutation on chromosome 4, with
complete penetrance492–494. These mice become obese around 3-4 weeks, with elevations of
blood glucose levels at 4 to 8 weeks. Thus, affected mice are polydipsic, polyuric and
hyperphagic495. db/db mice were obtained at ~ 6-7 weeks of age. In order to age and weight
match the lean 18 week old C57BL/6J control mice (25-30g) to the obese db/db mice, the obese
db/db mice were monitored for body weight and food intake daily until achieving a body weight
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of ~30 g (~ extra 12 weeks). Since obese db/db mice are hyperphagic, their food intake was
restricted daily in order to maintain body weight near ~30 g. At 18 weeks of age, jugular vein
and jejunal cannulation surgeries were performed.
4.3.1.3 High fat diet feeding
A subgroup of rats were fed a lard oil enriched high fat diet for three days. Rats that
were hyperphagic underwent the clamp studies. Please refer to General Methods 2.1.1 for details
on high fat feeding.
4.3.1.4 Streptozotocin induced uncontrolled diabetes
A subgroup of SD rats were injected with 65 mg/kg STZ (Sigma-Aldrich, St. Louis,
MO, USA). STZ is a diabetogenic agent that is cytotoxic to pancreatic β cells and is used to
induce uncontrolled diabetes in rodents496. Through the glucose transporter 2 (GLUT2)497 the
deoxyglucose moiety of STZ enters the pancreatic β cells and its cytotoxic effect are through its
nitrosurea moiety. STZ was first weighed out and transferred to a light protective conical tube.
Before injection, the powder was dissolved in 0.9% saline and immediately injected i.p. to
induce diabetes in rats. STZ was administered 5–6 d before sham or DJB surgery or 4 days
before jejunal and vascular surgeries. Injected rats had ad libitum access to food and water. The
rats were monitored daily for blood glucose levels with a glucometer (Contour Blood Glucose
Meter, Bayer Inc., Toronto, ON, Canada) to ensure they were hyperglycemic. We included only
rats that were hyperglycemic (plasma glucose levels > 300 mg/dl) in the study.
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4.3.2 Animal Surgeries
4.3.2.1 Intestinal and vascular cannulation
4.3.2.1.1 Rats
3-4 days before the clamp studies, SD and Koletsky rats were anesthetized and a jejunal
catheter was inserted 8-10 cm from the Ligament of Treitz. A subgroup of rats underwent
duodenal catheter placement (0.5 cm proximal to the pyloric sphincter.). After the intestinal
cannulation, the jugular vein and carotid artery were cannulated. Refer to General Methods
Section 2.2.1 and 2.2.2 for details regarding these surgical procedures in rats.
4.3.2.1.2 Mice
The surgical procedures conducted in mice were similar to that described for rats in
General Methods Section 2.2. 2-3 days before the clamp studies, C57BL/6 mice or db/db mice
were anesthetized with an i.p. cocktail of (60-90 mg/kg) ketamine (Ketalean; Bimeda-MTC,
Cambridge, Ontario) and (8-10mg/kg) Xylazine (Rompun; Bayer). Exposure of the
gastrointestinal tract within the peritoneum was conducted through a laparotomy incision made
on the ventral midline as well as the abdominal muscle wall. After identifying the pyloric
sphincter, the jejunum was identified to be 4-6 cm from the Ligament of Treitz. With a 25-gauge
needle, a small hole was made on the ventral aspect of the jejunum (in a region with the least
vascularization to minimize bleeding) to allow insertion of an intestinal catheter made of
polyethylene tubing (PE 10, Clay Adams, Boston, MA) with a 0.1 cm extension of smaller
silicone tubing (0.012 in ID, 0.025 in. OD; Sil-Tec, Technical Products, USA). To ensure the
cannula was placed in the lumen of the jejunum, the cannula was flushed with saline. It was then
anchored to the outer serosal surface of the jejunum with 3M adhesives (Vetbond) and a 0.2 cm2
and piece of Marlex mesh sewn to surface with a 6-0 silk suture. Through the laparotomic
incision, the proximal portion of the catheter excited the abdominal cavity and the abdominal
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wall was closed with a 6-0 silk suture. At the back of the neck, a 1 cm midline incision was
made in the skin, rostral to the interscapular area, and the cannula was tunneled subcutaneously
to exit the incision. This 1 cm incision was sewn closed with 6-0 silk sutures and the proximal
portion of the cannula was closed with a knot until the day of the experiment. For jugular vein
cannulation, an indwelling catheter made with polyethylene tubing (PE 10, Clay Adams,
Boston, MA) with a cuff extension (10 mm, internal diameter of 0.012 inches) of Silastic tubing
(Dow Corning, Midland. MI) was inserted for infusion purposes. Briefly, after blunt dissection
through the muscle layer, the jugular vein was teased out and two 7-0 silk sutures were used to
prevent blood flow. After a small incision into the vessel wall, the catheter was inserted. After
insertion, the catheters were tunneled subcutaneously and filled with a 0.2% heparin mixture to
maintain patency of the cannula, which was tunneled with an 18 G needle. The cannula was
closed through knotting until the day of the procedure.
4.3.2.2 Duodenal-jejunal bypass surgery
DJB surgery was conducted in Study 2 as previously described in STZ (65 mg/kg)
injected rats402,440. The rats were fasted the night before the surgery to ensure no food remained
in the digestive tract. A midline laparotomy incision was made on the ventral flank of the rat to
expose the gastrointestinal tract. After locating the stomach and proximal duodenum, blood
vessels innervating both areas were sutured with 4-0 silk sutures to ensure no bleeding occurred
during the surgery. The stomach was clamped proximal to the pyloric sphincter with a metal
curved clamp and blunted small end scissors were used to separate the duodenum containing the
gastric sphincter from the stomach. The gastric sphincter duodenal stump was closed with 6-0
silk sutures using the purse-suture technique. 15 cm from the pyloric sphincter, a transection
was made to separate the distal duodenum/most proximal portion of the jejunum from the
remaining jejunum and digestive system. The distal section of the jejunum was then
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anastomosed to the stomach (lumen to lumen) with a 6-0 suture. 12 cm distal from the new
jejunum/stomach connection, a small 1 cm hole was cut using blunted small scissors where the
distal duodenum/most proximal jejunum section was anastomosed. The gastrointestinal tract
was returned to the abdomen and wet with saline. The sham surgery consists of the same
procedure except that all the transactions and cuts were sutured back together. The rodents were
monitored daily after surgery for recovery via food intake and body weight. Blood glucose
concentrations were also monitored each day after surgery in both sham and DJB rats (please
see the General Methods section 2.6.1 for plasma glucose measurements).
4.3.3 Intraintestinal infusions and treatments
The following substances were infused through a jejunal or duodenal catheter as described
during the pancreatic clamp from t = 150-200 at a rate of 0.01 ml/min in rats or from t = 120-
170 at 2 µl/min in mice:
(1) saline
(2) leptin (6.7 ng/min; R & D systems, Minneapolis, MN, USA)
(3) soluble leptin receptor (SLR) (binds to leptin to prevent binding to the Leprb 1 µg/min; R
& D systems, Minneapolis, MN, USA)
(4) STAT3 PI (STAT3 peptide inhibitor; 15 pmol/min; Calbiochem, Millipore, Billerica,
MA, USA)
(5) wortmannin (PI3K antagonist; 0.002 nmol/min, Sigma-Aldrich, St. Louis, MO, USA)
(6) LY294002 (PI3K antagonist; 0.2 nmol/min Sigma-Aldrich, St. Louis, MO, USA)
(7) tetracaine (local anesthetic; 0.01 mg/min Sigma-Aldrich, St. Louis, MO, USA)
Solution #2-4 was dissolved in saline while solution #5-7 was dissolved in 5% dimethyl
sulfoxide (DMSO). In mice, saline or leptin (6.7 ng/min) was infused intrajejunally. The dose
chosen for leptin (6.7 ng/min) for the intraintestinal infusions was selected based on the gastric
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emptying rate of leptin in rats143 and also based on the amount of leptin that was administered
centrally in rats that lowered glucose production474. In a separate set of experiments in both rats
and mice, intravenous leptin was infused at the same rate (0.01 ml/min for rats; 2 µl/min for
mice), duration (50 min) and dose (6.7 ng/min) as performed for the intrajejunal leptin infusions
in conjunction with receiving intrajejunal saline infusion (0.01 ml/min for rats; 2 ul/min for
mice).
4.3.4 Pancreatic (Basal Insulin) Euglycemic Clamp Technique
4.3.4.1 Pancreatic (Basal Insulin) Euglycemic Clamp Technique in Rats
Please refer to the General Methods section 2.3 for a detailed description of the clamp
procedure. After an overnight food restriction, rats received a primed-continuous constant
infusion of [3–3H] glucose, which was given throughout the experiment (t = 200) to reach
steady state. The pancreatic clamp was then initiated at t = 90 where insulin (1.2 mU/kg/min)
and somatostatin (3 µg/kg/min) were infused at a constant rate. Blood samples were taken to
determine if a variable 25% glucose infusion was needed to maintain euglycemia. At t = 150, a
jejunal infusion (please refer to 4.3.3) at 0.01 ml/min was conducted and maintained until the
end of the experiment (t = 200). In a subgroup a rats, a duodenal infusion of leptin was
performed at 0.01 ml/min. In a separate set of experiments, in addition to a jejunal saline
infusion, an intravenous leptin infusion was performed from t = 150 to t = 200 at the same equal
dose and duration as the intrajejunal leptin infusion.
4.3.4.2 Pancreatic (Basal Insulin) Euglycemic Clamp Technique in Mice
After an overnight food restriction, a primed-continuous intravenous infusion of [3–3H]-
glucose (1 µCi bolus, 0.1 µCi/min; Perkin Elmer) was initiated at the beginning of the
experiment (t = 0 min) and maintained until completion of the study (t = 170) to assess glucose
kinetics under steady state conditions using the tracer dilution methodology. A pancreatic (basal
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insulin)-euglycemic clamp was started through a constant infusion of insulin (1.4 mU/kg/min)
and somatostatin (8.3 µg/kg/min) from t = 60 until t = 170. Every 10 minutes, via tail sampling,
blood glucose readings were conducted (please see the General Methods section 2.5.1 for
plasma glucose measurements) and a variable infusion of 10% glucose solution was started and
periodically adjusted (every 10 min from t = 60–170 min) to maintain plasma glucose levels
similar to the basal state (t = 50 and 60 min). Jejunal infusions (2 µl/min) were initiated at 120
min and continued for the remaining 50 min until t = 170. In a separate set of experiments, in
addition to a jejunal saline infusion, an intravenous leptin infusion was conducted at equal dose
and duration jejunal leptin infusion from t = 120 to t = 170 min. Plasma samples for the
determination of [3–3H] glucose specific activity (please see the General Methods section 2.5.2)
were obtained at 10–min intervals during the basal period (50 and 60 min) and at the end of the
jejunal infusion period (150-170 min). At the end of the experiment, mice were anesthetized and
tissue samples were removed and immediately immersed in liquid nitrogen. All tissue samples
were stored at –80 ºC until use.
4.3.5 Rat [3–3H] glucose infusion protocol (non-clamped conditions)
These studies were performed in a group of STZ-injected rats 9-10 days after the STZ
injection (65 mg/kg) and 4 days after jejunal and vascular cannulation surgeries. Only if the rats
were hyperglycemic (plasma glucose concentrations > 300 mg/dl) were they then included in
the subsequent infusion studies. Rats were restricted to ~56 kcal the night before the experiment.
The total experimental time for the in vivo infusion experiments was 140 minutes. At t = 0, a
primed-continuous infusion of [3–3H] glucose (40 µCi bolus; 0.4 µCi/min) was initiated and
maintained until the end of the experiment (t = 140 min) to assess glucose kinetics under steady
state conditions using the tracer dilution methodology. At t = 90 min, an intrajejunal infusion of
saline or leptin was initiated and continued for the remaining 50 min. In a separate set of
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experiments, intravenous leptin was infused at the same dose and duration as intrajejunal leptin
infusion and was initiated at 90 min and continued for the remaining 50 min of the experiment.
Plasma samples for the determination of [3–3H] glucose specific activity (please see the General
Methods section 2.5.2) were obtained at 10–min intervals during the basal period (60-90 min)
and at the end of the jejunal infusion period (130 and 140 min). At the end of the experiments,
rats were anesthetized and tissue samples were removed and immediately immersed in liquid
nitrogen. All tissue samples were stored at –80 ºC until use.
4.3.6 Fasting and refeeding protocol
Fasting and refeeding experiments were conducted in the STZ-injected rats that received
either SHAM or DJB surgery in conjunction with jejunal catheter placement. The experiment
took place 2 days after the surgery. The night before the experiment (5:00pm), the rats were
fasted for 24 hours. At 4:50pm (t = -10), the day of the experiment, baseline glucose
measurements (please see General Methods section 2.5.1 for details regarding plasma glucose
readings) were taken via tail sampling. Then, a continuous intrajejunal infusion (Harvard
Apparatus PHD 2000 infusion pumps) of either (i) saline or (ii) SLR (1 µg/min; the same dose
as given in the clamp studies) was initiated and lasted throughout the course of the experiment
until t = 50 to match the treatment during the clamp studies. At t = 0, rats were allowed to
consume a regular chow diet ad libitum. Blood glucose levels and food intake were measured
throughout the course of the experiment in 10-minute intervals until completion (t = 50).
4.3.7 Gut tissue collection and preparation for western blotting and enzymatic activity
assay
Separation of the jejunal or duodenal mucosal layer (~100 mg) from the jejunal or
duodenal smooth muscle layer (~150 mg) was conducted immediately after removal from
anesthetized animals at the termination of the clamp studies. The separation was done in a petri
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dish filled with 0.9% saline on ice with a spatula. The separated layers were transferred to
separate eppendorf tubes and stored at -80°C until use. The tissues were transferred to ice the
day of the western blot or enzymatic activity assay and lysed on ice with a handheld blender in
6.3 µl per 1mg of tissue of a buffer containing: 50 mM Tris–HCl (pH 7.5), 1 mM EGTA, 1 mM
EDTA, 1 % (w/v) Nonidet P40, 1 mM sodium orthovanadate, 50 mM sodium fluoride, 5 mM
sodium pyrophosphate, 0.27 M sucrose, 1 µM Dithiotritolo (DTT) and protease inhibitor
cocktail (Roche Diagnostics, Laval, QC, Canada). After homogenization, the tissues were spun
at 12,000 rpm for 15 minutes at 4ºC. The supernatant was transferred to new eppendorf tubes
and the protein concentration of each homogenized tissue was determined with the Pierce 660
nm protein assay as described in the General Methods section 2.4.
4.3.8 Western blotting
Intestinal tissues were removed, separated into the mucosal and smooth muscle layer,
homogenized and processed as described in section 4.3.6 above. 100 µg of protein was thawed,
vortexed and then subjected to sodium dodecyl sulfate- polyacrylamide gel electrophoresis on
an 8% polyacrylamide gel for 90 minutes at 100V. After electrophoresis separation, in transfer
buffer the protein was transferred to nitrocellulose membranes. The membranes were incubated
for 1 hour at room temperature with Tris buffered saline-Tween (TBS-T) containing 5% (w/v)
BSA. The membranes were then immunoblotted in the same buffer for 16 hours at 4 °C with the
indicated primary antibodies (diluted to 1:1000 for pSTAT3 and total STAT3; Cell Signaling
Technology, Danvers, MA, USA). The blots were then washed 5 times with TBS-T for 30
minutes at room temperature to remove the primary antibody and incubated with secondary
horseradish peroxidase (HRP)-conjugated rabbit IgG antibody (Cell Signaling Technology,
Danvers, MA, USA) for P-STAT3 and total STAT3 (diluted 1:4000) in 5% skim milk for 1 hour
at room temperature. After repeating the washing steps, the signal was detected with the
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enhanced chemiluminescence reagent (Thermo Scientific, IL, USA). Immunoblots were
exposed to x-ray film and developed using a film automatic processor (SRX-101; Konica
Minolta Medical), and films were scanned with the GS-800 Calibrated Densitometer (BioRad,
Hercules, CA, USA). The phosphorylation level of STAT3 was quantified by densitometry with
the Quantity One 1-D Analysis Software (BioRad, Hercules, CA, USA) and normalized for the
corresponding total protein level. As the ability of leptin to stimulate STAT3 phosphorylation
was found to be the same in both the mucosal and smooth muscle layer of jejunum and
duodenum, we have simply presented leptin-induced STAT3 phosphorylation in the jejunal and
duodenal mucosa.
4.3.9 RNA extraction, reverse transcription and PCR methods
4.3.9.1 RNA Extraction
The total RNA from rat duodenal and jejunal mucosa was extracted by using the
PureLink RNA Mini Kit from Ambion. First, a RNase free work area was ensured. Of note,
RNase free tubes and pipette tips were used throughout the extraction. Briefly, ~70 mg was
homogenized in lysis buffer (provided with the kit) containing 1% 2-mercaptoethanol using the
rotor-stator homogenizer. The lysate was then centrifuged at 12,000 X g for 2 minutes.
Following centrifugation, one volume of 70% ethanol was added to the tissue homogenate and
mixed. 700 µl of the sample was transferred to the spin cartridge and centrifuged at 12,000 x g
for 15 seconds at room temperature. The flow-through was then discarded and this process was
repeated 3 times. 700 µl of wash Buffer I followed by 500 µl of wash buffer II was added
individually and the same process was conducted as described for the 70% ethanol. The
membrane with the attached RNA was allowed to dry for 1-2 minutes. Recovery was conducted
through the addition of RNase-Free water to the spin cartridge and allowed to incubate at room
temperature for 1 minute. After centrifugation, the purified RNA was stored at –80 ºC until use.
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Measurement of the optical density (OD) was performed to quantify RNA content at 260 and
280 nm using 2 µl of sample with a NanoDrop 1000 spectrophotometer (Thermo Fisher
Scientific, Mississauga, ON, Canada). The ratio of 260/280 should be between 1.8 and 2 for
RNA. RNA concentration (µg/ml) was then calculated as:
RNA concentration = OD260 x dilution factor x 40
4.3.9.2 Reverse transcription and polymerase chain reaction
The reverse transcription and subsequent PCR were preformed with the QIAGEN
OneStep RT-PCR kit that allowed performing both the reactions in a single PCR programme.
After thawing of all reactants, a master mix was made on ice containing the Omniscript and
Sensiscript Reverse Transcriptase and the HotStarTaq DNA Polymerase (provided by the kit), a
dNTP mix with a final concentration of 400 µM each, the Q-reagent (provided by the kit) and
the primers (0.6 µM each). A total amount of 1µg of RNA was incubated in this reaction
mixture. In controls, reverse transcriptase was omitted. The PCR reaction was performed in a 50
µl volume using a S1000 Thermal Cycler (Biorad, Hercules, CA, USA) as follow: 30’ at 50°C
(reverse transcription), 15’ at 95°C (initial PCR activation), three step cycling (40 cycles) of 45’
at 94°C, 1’ at 50°C and 1’ at 72°C and a final extension for 10’ at 72°C. The sequence of the
forward primer used was 5’- ATGAAGTGGCTTAGAATCCCTTCG-3’ and that of the reverse
was 5’-ATATCACTGATTCTGCATGCT-3’ (ACGT Corporation, Toronto, ON, Canada) as
previously described for the long form leptin receptor149.
A 1.3% agarose gel was prepared by combining 100 ml of 1 x TBE (Tris, boric acid, and
EDTA), 1.3g of agarose and 4 µl of RedSafe Nucleic Acid Staining Solution (INtRON
Biotechnology,). 25 µl of the PCR product containing 5X Gel Loading Dye (New England
Biolabs) was then run through the gel from the negative to positive electrode at ~90 V. DNA
ladders were included in the gel for determination of product size. Bands were visualized under
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ultra violet light with a BioRad Molecular Imager Gel Doc XR+ Imaging System (BioRad,
Hercules, CA, USA).
4.3.10 PI3K Activity Assay
4.3.10.1 Immunoprecipitation of PI3K and Assay Reaction
PI3K activity was measured with the PI3K activity ELISA: Pico kit (echelon, K-1000s;
Salt Lake City, UT, USA). On day 1 of the experiment, PI3K was immunoprecipitated from 500
µg of protein, prepared as described above in 4.3.7, with 5 µl of an anti-p89 PI3K subunit
antibody (Millipore; Billerica, MA, USA). A negative control was included with no addition of
the antibody. The lysate was incubated for 1 hour at 4°C in a rotating wheel (Mini Labroller,
Diamed lab supplies, Edison, NJ, USA) with the antibody. Next, 25 µl of 25% protein A/G
beads (Santa Cruz, CA, USA) were added to the lysate and was incubated for an additional 2
hours at 4°C in a rotating wheel. The beads were then spun at 8000 rpm for 1 min at 4°C and the
supernatant was kept and frozen for later use if needed. The remaining pellet underwent a
washing protocol: washed 3 times with 500 µl of the lysis buffer (please refer to 4.3.7) + 0.1M
NaCl, three times with 500 µl of 0.1 M Tris-HCl pH 7.4, 5 mM LiCl and 0.1 mM sodium
orthovanadate and twice with 500 µl of 10 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM EDTA
and 0.1 mM sodium orthovanadate. The KBZ reaction buffer was then prepared by combining
the KBZ buffer with 1M DTT, 10mM ATP and ultrapure water, according to the number of
samples in the assay. The last wash was then aspirated and the beads were re-suspended in 30 µl
of the KBZ reaction buffer. Then, a 100 µM PI(3,4,5)P2 (PIP2) working solution was prepared
with the addition of distilled water to the substrate vial. 30 µl of the PIP2 substrate was also
added to the beads. A positive control was added by combining 5 µl of purified PI3K with KBZ
reaction buffer and PIP2 substrate. The reaction was conducted through incubation of the
reaction containing eppendorf tubes at 37°C for 4 hours. After 4 hours, the reaction was stopped
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through addition of 90 µl of the Kinase Stop Solution provided by the kit. The tubes were then
centrifuged at 8000 rpm for 2 min at 4°C. The supernatant was transferred to clean tubes and
stored at -20°C.
4.3.10.2 ELISA Incubation and Detection
The following were prepared on ice: a standard curve buffer (KBZ reaction buffer +
PIP2 substrate + K-EDTA), PIP3 standard stock at 3.6 µM (distilled water into PIP3 vial), and
TBS-T buffer (distilled water + 10x TBS-T buffer). The stopped kinase reactions described
above were thawed on ice and PIP3 standards and controls for the ELISA for prepared (360 n,
120 nM, 40 nM, 13.3 nM, 4.4 nM, no enzyme control, no lipid control). 60 µl of the standards,
no enzyme control and no lipid control, and stopped kinase reactions were transferred to the
incubation plate provided by the kit. Then, 60 µl of the PIP3 detection buffer was added to each
well where the plate was sealed and incubated for 1 hour at room temperature on a plate shaker.
100 µl from each well was transferred to the detection plate provided by the kit. The detection
plate was sealed and incubated for 1 hour at room temperature on a plate shaker. After
aspiration, the wells were washed 3 times with 200 µl of TBS-T and 100 µl of secondary
detector solution was added to each well and the plate was incubated for 30 min. Each well was
aspirated and 300 µl of TMB solution was added. Color was allowed to develop for 15 minutes
in a dark space. The reaction was stopped by adding 50 µl of 1N H2SO4 stop solution. The plate
was transferred to a spectrophotometer and the absorbance was read at 450nm. The statistical
software program Prism (GraphPad Software Inc., CA, USA) was used for data analysis. Data
was presented as fold increase over the saline treated samples.
4.3.11 Biochemical Analysis
Please refer to the General Methods section 2.6.1-2.6.3 for details on biochemical
analyses. Plasma glucose concentrations were determined using a GM9 Analox Glucose
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Analyzer (Analox Instruments, Lunenbertg, MA). Radioactivity of plasma glucose was
conducted as described. Plasma insulin levels were measured using a radioimmunoassay (Linco
Research, St Charles, MO).
4.3.11.1 Plasma Leptin
Peripheral and portal plasma leptin levels were measured using a rat Leptin RIA kit
(Linco Research, St. Charles, MO). This RIA kit follows the same principle as that described in
the 2.5.3 Plasma insulin section. Briefly, leptin from peripheral and portal plasma samples or
standards compete for binding to a guinea pig anti-rat leptin antibody against 125I-labeled leptin.
The amount of radiolabeled 125I-labeled leptin binds in reverse proportion to the known
standards and the amount of leptin in the plasma sample. Separation of the 125I-labeled leptin
and unbound fractions is conducted through the use of a double antibody solid phase.
Specifically, a three day protocol as per the supplier’s instructions was used. 50 µl of
peripheral and portal plasma samples and standards in a range of concentrations (0.78, 1.56,
3.125, 6.25, 12.5, 25 ng/ml) were prepared and 50 µl of the anti-leptin antibody was added. The
tubes were vortexed and allowed to incubate at room temperature overnight. After the first day,
50 µl of 125I-labeled leptin was added and samples were vortexed and allowed to incubate
overnight at room temperature. 500 µl of precipitating reagent was added followed by vortexing
and incubation at 4°C for 20 minutes. The samples were then centrifuged to pellet the bound
leptin and the radioactivity of this pellet was counted by a gamma counter (Perkin Elmer 1470).
The final concentration of leptin in the samples was conducted through construction of a
standard curve and interpolation.
4.3.12 Calculations and Statistical Analysis
Data are represented as mean ± SEM. Data between t = 60-90 (rats) and t = 50-60 (mice)
were averaged for basal conditions and data between t = 150-200 (rats) and t = 120-170 (mice)
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were averaged for clamp conditions. Statistical difference between two groups was determined
via unpaired Student’s t-test. When comparisons were made across more than two groups,
ANOVA was performed, and if significant, this was followed by Tukey’s post-hoc test, which
enabled comparisons of all treatment groups.
4.4 Results
4.4.1 Jejunal leptin requires jejunal leptin receptor activation to lower glucose production
We first assessed whether Leprb is expressed in duodenal and jejunal tissues via PCR
technology. Consistent with previous reports97,103,410,411,416, PCR analyses revealed Leprb
expression in both the duodenum and jejunum of normal rats (Figure 4.2). To assess whether
stimulation of the Leprb in the small intestine, which is classically known to mediate the
metabolic effects of leptin, possesses the ability to regulate glucose production, we subjected
fully recovered conscious healthy rats to a pancreatic (basal insulin) clamp when leptin was
administered directly into the duodenum or jejunum during the final 50 min of the experiment
(Table 4.1; The infusion-clamp experiments lasted a total of 200 min; basal period is averaged
from 60-90 min and clamp period is averaged from 180-200 min) (Figure 4.1). Interestingly,
despite the presence of the Leprb in the duodenum, an intraduodenal leptin infusion did not
affect glucose metabolism as evident by the fact that the glucose infusion rate (Figure 4.3A),
glucose production (Figure 4.3B and C) and glucose uptake (Figure 4.3D) remained
comparable to the vehicle control. In contrast, a continuous intrajejunal leptin infusion for 50
min led to a higher glucose infusion rate (Figure 4.3A) and lower glucose production (Figure
4.3B and C) compared to saline. No changes in glucose uptake (Figure 4.3D) and plasma
insulin and glucose levels (Table 4.1) were detected.
Next, we wanted to confirm that this effect of leptin is specific to the jejunum, as it could
be argued that the decrease in glucose production induced by intrajejunal leptin administration is
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due to a leakage of leptin into the portal and subsequent peripheral circulation resulting in
activation of extra-intestinal leptin receptors (i.e. in the hypothalamus). We measured leptin
levels in plasma samples taken from both portal and peripheral blood at the end of the 50 min
gut infusion period. Importantly, an intrajejunal infusion of leptin for 50 min did not elevate
plasma leptin levels during the clamps (averaged from 180-200 min) (Figure 4.4A) and did not
alter portal leptin levels at the end of the experiments (Figure 4.4A). To alternatively ensure
changes of glucose production in response to an intrajejunal leptin infusion occurred
independent of changes in circulating leptin levels, we performed an intravenous administration
of leptin at an equal dose and duration as the intrajejunal leptin infusion. An intravenous leptin
administration for 50 min given at the equal dose as the intrajejunal leptin infusion did not alter
glucose metabolism (Figure 4.4B-D). Thus, an intrajejunal (but not intraduodenal) infusion of
leptin lowers glucose production independently of changes in plasma leptin, insulin and glucose
levels. Subsequent clamp studies were conducted to delineate the downstream effectors involved
in leptin’s effects in the jejunum.
To determine whether the binding of leptin to the Leprb in the jejunum is responsible for
lowering glucose production, we co-infused leptin with a SLR previously shown to rapidly bind
to leptin and antagonize its effects499. An intrajejunal infusion of the SLR alone for 50 min did
not alter glucose metabolism (Figure 4.5A-D) but fully negated the effect of leptin (Figure
4.5A-D). To further evaluate that the Leprb is required for jejunal leptin-sensing to lower
glucose production, we performed the same set of jejunal leptin infusion experiments in a Leprb
deficient rodent model, the obese (fak/fak) Koletsky rat versus the lean Fa/Fa control. First, an
intrajejunal leptin 50 min infusion led to a higher glucose infusion rate (Figure 4.6A) and lower
glucose production (Figure 4.6B and C) with no changes in glucose uptake (Figure 4.6D)
compared to saline in lean Fa/Fa rats. When placed on a regular chow diet ad lib, fak/fak rats are
hyperphagic and rapidly increase body weight compared to their lean Fa/Fa littermates500. To
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match their body weight to the Fa/Fa control rats, we limited the food intake of the fak/fak rats
similar to that described473 and assessed jejunal leptin action in weight- and age-matched leptin
receptor deficient fak/fak rats. An intrajejunal leptin infusion failed to alter glucose metabolism
compared to saline in the leptin receptor deficient fak/fak rats (Figure 4.6A-D).
We next tested jejunal leptin action in the leptin receptor-deficient obese db/db mice.
We first developed a murine intrajejunal infusion model to directly activate jejunal sensing
mechanisms as performed in the rat, by implanting a catheter directly into the jejunum of mice.
We then subjected fully recovered conscious mice to a pancreatic (basal insulin) clamp while
leptin was administered continuously and directly into the jejunum for 50 min. Similar to what
was discovered in rats, an intrajejunal leptin infusion in healthy C57Bl6/J mice led to a higher
glucose infusion rate (Figure 4.7A) and lower glucose production (Figure 4.7B and C) with no
changes in glucose uptake (Figure 4.7D) compared to saline. Importantly, this glucose lowering
effect was independent of a rise in plasma leptin levels since intravenous leptin infusion
administered at an equal dose and duration as intrajejunal leptin infusion did not alter glucose
metabolism in healthy C57Bl6/J mice (Figure 4.8A-D). Importantly, intrajejunal leptin infusion
was unable to alter the glucose infusion rate (Figure 4.7A) and glucose production (Figure 4.7B
and C) in the weight- and age-matched leptin receptor deficient db/db mice, confirming our
observations in the obese Koletsky rats. These leptin receptor loss-of-function experiments
demonstrate that activation of jejunal leptin receptors by preabsorptive leptin lowers glucose
production in rats and mice.
4.4.2 A STAT3-independent and PI3K-dependent signaling pathway is required for
jejunal leptin to lower glucose production via a neuronal network
Although STAT3474 and PI3K473 are required for central leptin to regulate glucose, and
both signaling molecules are expressed in the intestine and/or in vagal afferent nerves151,157,485,
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the functional impact of the intestinal leptin-STAT3/PI3K signaling is unknown. To address the
role of jejunal STAT3, we co-infused the STAT3 peptide inhibitor (STAT3 PI) with leptin
directly in the jejunum for 50 min. In contrast to the hypothalamus474, STAT3 PI failed to negate
the effect of jejunal leptin compared to saline in rats (Figure 4.9A-D). A higher jejunal STAT3
phosphorylation (Y705) with leptin compared to saline was detected (Figure 4.10A) in the
tissues taken immediately after the clamp studies. Co-infusion of leptin with STAT3 PI negated
the elevation of STAT3 phosphorylation (Y705) (Figure 4.10A) but did not prevent the gluco-
regulatory effect (Figure 4.9A-D). Thus, jejunal leptin activates local STAT3 but such
activation is not required for the gluco-regulatory impact of jejunal leptin.
To investigate the role of jejunal PI3K, we co-infused two independent PI3K inhibitors,
LY294002 or wortmannin, with leptin into the jejunum for 50 min. An intrajejunal infusion of
LY294002 or wortmannin alone did not affect whole-body glucose metabolism (Figure 4.9A-
D) but fully abolished the effect of leptin (Figure 4.9A-D). PI3K activity was assessed from
jejunal tissues taken immediately after the clamp studies. An intrajejunal leptin infusion led to a
higher jejunal PI3K activity compared to saline (Figure 4.10B), and this activation was negated
by co-infusion with LY294002 (Figure 4.10B). In the jejunal tissues that were obtained
immediately after an intrajejunal leptin plus SLR infusion where the SLR negated the effect of
leptin (Figure 4.5A-D), an intrajejunal leptin administration failed to activate PI3K as well
(Figure 4.10B), confirming jejunal PI3K is a target of the leptin receptor. Of note, an
intrajejunal leptin administration did not activate duodenal PI3K activity (Figure 4.10C) or
hypothalamic STAT3 (Figure 4.10D) in the same rats where jejunal PI3K was activated
(Figure 4.10C). Further, an intraduodenal leptin infusion activated duodenal STAT3 (Figure
4.10E) but not duodenal PI3K (Figure 4.10F). Although it is tempting to speculate that the
inability of intraduodenal leptin infusion to lower glucose production (Figure 4.3A-D) is due to
an absence of PI3K activation, future studies are needed to address this possibility as well as the
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reasons behind the inability of duodenal leptin to activate PI3K. Nonetheless, selective
activation of the jejunal leptin receptors by presabsorptive leptin triggers jejunal PI3K to lower
glucose production.
Given that jejunal nutrient-sensing ignites afferent neuronal signals to lower glucose
production402, a neuronal network is a potential downstream effector of jejunal leptin signaling.
We infused the topical anesthetic tetracaine locally into the jejunum for 50 min to inhibit
neurotransmission of the nerves that innervate the jejunum402 while leptin was infused. An
intrajejunal infusion of tetracaine alone did not affect whole-body glucose metabolism (Figure
4.11A-D) but was sufficient to fully reverse the ability of intrajejunal leptin administration to
increase the exogenous infusion rate (Figure 4.11A) and lower glucose production (Figure
4.11B and C) during the clamp while glucose uptake (Figure 4.11D) remained comparable
within groups. Thus, neuronal transmission is required for jejunal leptin to lower glucose
production.
4.4.3 Jejunal leptin’s action remain intact in high fat fed or diabetic rats
Recent studies highlight leptin as an anti-diabetic therapy501,502 in which leptin’s effect is
attributed to the brain475,476. In parallel, direct administration of i.c.v leptin is effective to lower
glucose production in 3d high-fat fed rats474. We have previously characterized this 3 day high
fat fed model as having duodenal CCK resistance384,487 as well as insulin resistance393. Thus, we
tested the effectiveness of jejunal leptin action in this same 3d high-fat fed model. An
intrajejunal leptin infusion for 50 min led to a higher glucose infusion rate (Figure 4.12A) and
lowered glucose production (Figure 4.12B and C) compared to saline with no changes in
glucose uptake (Figure 4.12D) in high-fat fed rats. The gluco-regulatory effect of jejunal leptin
action in high-fat feeding was comparable to that observed in regular chow fed rats (Figure
4.12A-D). We again tested whether this effect of jejunal leptin in high fat diet rats is
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independent of a rise in circulating leptin levels. Indeed, an intravenous leptin infusion
administered at an equal dose and duration as an intrajejunal leptin infusion did not alter glucose
metabolism in 3d high-fat fed rats (Figure 4.13A-C). These findings indicate that the ability of
jejunal leptin to lower glucose production in this model is specific to the jejunum in the current
experimental conditions. These findings suggest that agonism of jejunal leptin signaling may
represent a novel therapeutic approach for obesity and diabetes.
We next investigated jejunal leptin action in non-obese insulin-deficient uncontrolled
diabetic rats. We injected the rats with STZ to elevate plasma glucose concentrations and
glucose production to ~340 mg dl-1 (Figure 4.14A) and ~23 mg kg-1 min-1 (Figure 4.14B)
respectively, and reduce insulin concentrations by ~80% (Figure 4.14D). An intrajejunal
infusion of leptin for 50 min was effective to induce a reduction in plasma glucose levels
(Figure 4.14A) and glucose production (Figure 4.14B) in non-clamped conditions in
comparison to saline. Consistent with our findings in the various models used in this study, an
intravenous leptin infusion at the same dose and duration did not lower plasma glucose levels or
glucose production in the same diabetic model (Figure 4.15 A and B). These findings indicate
that the ability of jejunal leptin to lower plasma glucose levels and glucose production in this
non-obese insulin-deficient uncontrolled diabetic rodent model is specific to the jejunum. This
glucose- and glucose production-lowering effect of jejunal leptin was independent of changes in
circulating glucagon (Figure 4.14C) and insulin (Figure 4.14D) levels as well. We reason that
if jejunal leptin action (like jejunal nutrient sensing402) lowers plasma glucose levels and glucose
production in insulin-deficient uncontrolled diabetic rats, jejunal leptin action (like jejunal
nutrient sensing) may mediate the rapid early anti-diabetic effects of DJB surgery.
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4.4.4 The antidiabetic effect of DJB surgery is mediated by jejunal leptin action
We next investigated whether gastric leptin action in the jejunum contributes to the
glucose-lowering effect of DJB surgery in non-obese STZ-induced uncontrolled diabetes. In
order to test this hypothesis, we inhibited jejunal leptin action in uncontrolled diabetic rats that
received DJB while monitoring plasma glucose levels during refeeding. The use of refeeding as
our experimental design is based on the fact that refeeding will cause secretion of leptin from
the gastric chief cells into the lumen127,143 and makes its way to the jejunum (as the duodenum is
bypassed) to subsequently bind to the Leprb in the jejunum144 and possibly lower plasma
glucose levels in diabetic rodents that have received DJB surgery. First, DJB or sham surgery
was performed as described440 in STZ-induced uncontrolled diabetic rats (Figure 4.16).
Consistent with our previous study402, DJB exerted a rapid reduction of fed plasma glucose
levels compared to sham in STZ-induced diabetic rats within 2 d after surgery (Figure 4.17A).
This effect was not associated with changes in plasma insulin (Figure 4.17B) or glucagon
(Figure 4.17C) levels nor changes in food intake (Before surgery STZ-SHAM 27g ± 0.7 and
STZ-DJB 31g ± 1.3, 2d after surgery STZ-SHAM 4g ± 1.3 and STZ-DJB 5g ± 1.4) or body
weight (Before surgery STZ-SHAM 301g ± 11.2 and STZ-DJB 314g ± 4.3, 2d after surgery
STZ-SHAM 291g ± 14.5 and STZ-DJB 305g ± 8.8).
After confirming our model, we then tested whether jejunal leptin sensing mediates this
early improvement of glycemia induced by DJB in uncontrolled diabetes. We inserted a jejunal
catheter, targeting the same location where the jejunal leptin receptor antagonist SLR infusion
negated the effect of leptin (Figure 4.3A-D), in conjunction with DJB in STZ-diabetic rats
(Figure 4.16). To this end, we then carried out a fasting-refeeding experiment in these rodents
to promote gastric leptin secretion127 while infusing the SLR directly into the jejunum to disrupt
jejunal leptin signaling (Figure 4.16) in STZ-diabetic rats 2 d after DJB. We monitored plasma
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glucose levels for 50 min during refeeding in an attempt to match the duration of the intrajejunal
leptin infusion during the clamp studies (Figure 4.3A-D). Consistent with a glucose-lowering
effect of DJB in STZ-induced diabetic rats, the glucose control stimulated by jejunal leptin
sensing during refeeding was intact in STZ-DJB diabetic rats that received intrajejunal saline
infusion as compared to STZ-SHAM (Figure 4.17D, top panel). This glucose control was
independent of changes in food intake (Figure 4.17E). In contrast, an intrajejunal infusion of
the SLR into STZ-DJB rats disrupted the glucose control observed in the STZ-DJB intrajejunal
saline infused rats during refeeding, resulting in elevated plasma glucose concentrations (Figure
4.17D, bottom panel). This marked elevation of plasma glucose levels seen with intrajejunal
SLR infusion occurred independently of changes in food intake (Figure 4.17E) but did not
reach that of STZ-SHAM jejunal saline infused rats (Figure 4.17D, top panel). Nonetheless,
these findings illustrate that gastric leptin action in the jejunum contributes to the rapid (2 d)
glucose-lowering effect induced by DJB in uncontrolled diabetes.
4.5 Discussion
Previous studies focus on the brain as a primary tissue mediating leptin’s effects on the
regulation of glucose homeostasis158. Our discovery revises the traditional view of leptin action
and suggests that, in addition to the brain, leptin triggers a jejunal signaling pathway to lower
glucose production. The physiological relevance of intestinal leptin action remains to be
clarified as (i) jejunal but not duodenal leptin action lowers glucose production and (ii) short-
term inhibition of jejunal leptin receptor-mediated action (via 50 min intrajejunal SLR or PI3K
inhibitors infusion) per se did not alter glucose production. These unknowns however are
balanced with current findings reporting that selective activation of jejunal leptin-PI3K lowers
glucose production in rats and mice.
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The current study demonstrates that activation of the jejunal leptin receptor is required
for preabsorptive leptin to activate PI3K to lower glucose production, which was confirmed
through the use of two molecular knockout and chemical inhibitory approaches. STAT5 and
SOCS3 have also been demonstrated to be downstream signaling molecules of the leptin
receptor in the hypothalamus136,503 and both are expressed in the intestine151,156. It remains to be
assessed whether these are potential targets of jejunal leptin action. We also made the interesting
discovery that jejunal-leptin PI3K, and not STAT3 signaling lowers glucose production. Thus,
the role of intestinal STAT3 activation warrants future investigation. In regards to a potential
effector of jejunal PI3K activation, voltage gated sodium channels remain a possibility as co-
infusion of leptin with the anesthetic tetracaine (an inhibitor of voltage gated sodium channels)
abolished leptin’s glucose production suppression effects. In line with this hypothesis, one study
suggests that PI3K alters sodium conductance504. To our knowledge, no similar studies exist for
STAT3. In addition, the current study at best narrows down the site of leptin-PI3K signaling to
the jejunum (i.e., jejunal mucosa and/or the vagal nerves that innervate the jejunum). The exact
site in the GI tract (i.e. cell type involvement) remains to be addressed. Nonetheless, it is clear
that neuronal innervation is required for the gluco-regulatory effect of jejunal leptin.
Previous studies have demonstrated that intestinal peptide hormones, like leptin, regulate
glucose homeostasis via a neuronal axis. For example, the peptide hormone CCK is secreted in
the duodenum upon lipid ingestion and activates PKA to regulate glucose production through a
neuronal network384,487. In addition, selective inhibition of intestinal DPP-IV, the enzyme which
rapidly degrades GLP-1, leads to activation of vagal afferents to improve glucose tolerance in
diet-induced obese rodents285. These findings, together with the discovery of leptin in the
current study, indicate that peptide hormones bind to their receptors expressed in the GI tract to
trigger the CNS to regulate glucose homeostasis. Interestingly, leptin in the brain has been
shown to enhance the action of intestinal CCK to decrease feeding9,477 and possibly at the level
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of the intestine157,505 where both the CCK-1 receptor155 and the Leprb149 are expressed. Thus,
jejunal leptin and CCK may act additively or synergistically to regulate glucose production,
although it would be important to first assess whether CCK action in the jejunum (like in the
duodenum) regulates glucose production.
Given that a jejunal leptin infusion lowers glucose production in high-fat fed rats, jejunal
leptin signaling may have therapeutic relevance. It has been previously demonstrated that leptin
resistance occurs after 3 days of high fat feeding392, but a direct infusion of leptin into the
brain474 and jejunum still lower glucose production. However, it remains to be assessed whether
jejunal leptin action through a neuronal network remains intact in chronic obese models.
Moreover, both central and peripheral leptin administration in STZ-induced insulin deficient
uncontrolled diabetic rodents normalizes plasma glucose concentrations in association with
lowering hyperglucagonemia475,476,501. We here demonstrate that jejunal leptin in this same
rodent model lowers (but does not normalize) plasma glucose concentrations through an
inhibition of glucose production, which was independent of changes in circulating glucagon
levels. However, it should be noted that glucagon levels were not elevated in this model to begin
with. This is consistent with the previous findings that a jejunal glucose and lipid infusion
lowered glucose levels in this same model, also independent of changes in glucagon levels. In a
more chronic model of uncontrolled diabetes402, the involvement of glucagon action in
mediating jejunal leptin’s effects may be more apparent.
Upon intake of nutrients into the gastrointestinal tract, there is release of different
peptides hormones364,506,507 including gastric leptin127. There is still much debate over the role of
gut peptides involvement in the glucose lowering effect of DJB with studies demonstrating
conflicting results64,402,445. Nonetheless, the involvement of bile acids is also becoming apparent
and it is suggested that they may also contribute to these beneficial effects450. Given that after
DJB surgery the route of delivery of nutrients from the stomach is redirected into the jejunum,
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this would stimulate gastric leptin release, which would make its way to the jejunum to possibly
activate jejunal leptin receptors to mediate the rapid anti-diabetic effect of DJB. We here
discovered that upon blocking leptin receptor signaling through infusion of a SLR during
refeeding in uncontrolled diabetic rodents with DJB disrupted the glucose control albeit this
blockade did not elevate glucose levels to the same extent as seen in uncontrolled diabetic
rodents who had received sham surgery. This suggests that leptin does not work alone to lower
glucose concentrations after DJB surgery but requires additional mechanisms. It is worth
examining whether jejunal leptin converges with nutrient sensing mechanisms as this may
uncover the additional mechanisms that are required. In addition to the uncontrolled diabetic
rodent model used in this study, it remains to be clarified whether jejunal leptin action mediates
the anti-diabetic effects of other types of bariatric surgery in obese and diabetic models.
In conclusion, we here unveil that leptin action in the jejunum activates a jejunal leptin-
receptor-PI3K dependent pathway to lower glucose production. This effect of jejunal leptin
remains intact in high-fat fed or uncontrolled diabetic rodents and mediates the rapid anti-
diabetic effect of DJB surgery. Taken together, these findings suggest that jejunal leptin
signaling may be targeted as a novel therapeutic strategy to lower glucose levels in diabetes.
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Figure 4.1 Schematic representation of the working hypothesis – Gastric leptin activates the intestinal long form leptin receptor to activate a PI3K-dependent and STAT-3 independent signaling axis to lower glucose production through a neuronal network.
Proposed model for intestinal leptin to lower glucose production. The soluble leptin receptor (SLR) is a leptin receptor inhibitor, which binds leptin and prevents its binding to the receptor. Koletsky rats and db/db mice are long form leptin receptor deficient rodents. Signal transducer and activator of transcription 3 peptide inhibitor (STAT3 PI) is a STAT3 inhibitor and LY294002 and wortmannin are PI3K inhibitors. Tetracaine is a local anesthetic that prevents neuronal activation. An intestinal leptin infusion fails to lower glucose production upon blockade of leptin receptor, PI3K and vagal afferent activation.
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Figure 4.2 Leptin receptor expression in intestinal tissue.
PCR analysis of the long form leptin receptor in both duodenal and jejunal mucosa tissue. –RT: negative control run in the absence of reverse transcriptase. A 375bp project was amplified by primers for the long form leptin receptor.
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Figure 4.3 Jejunal leptin administration lowers glucose production.
(A and B) During the pancreatic clamp (t = 180-200) an intrajejunal leptin infusion (6.7 ng/min) increased the glucose infusion rate (A, **P < 0.01 vs. other groups) and decreased glucose production (B, **P < 0.01 vs. other groups). C) Suppression of glucose production during the clamp period (t = 180-200) expressed as the percent reduction from the basal state (t = 60-90) glucose production (**P < 0.01 vs other groups). D) The rate of glucose uptake remained unchanged amongst all groups. Values are shown as mean ± s.e.m. n = 5–7 rats per group; n = 7–8 mice per group.
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Figure 4.4 Jejunal leptin lowers glucose production independent of changes in portal and circulating leptin levels.
A) Plasma leptin levels before (basal) and at the end of the camp (t = 180-200) during a jejunal saline or leptin infusion. Portal leptin levels were taken at the termination of the clamp studies (t = 200). (B and C) During the pancreatic clamp (t = 180-200) an intrajejunal leptin infusion (6.7 ng/min) increased the glucose infusion rate (A, **P < 0.01 vs. other groups) and decreased glucose production (B, **P < 0.01 vs. other groups). An intravenous leptin infusion at the same dose and duration did not affect the B) glucose infusion rate or C) glucose production. D) The rate of glucose uptake remained unchanged amongst all groups. Values are shown as mean + s.e.m. n = 5–7 rats per group.
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Figure 4.5 Leptin activates leptin receptors to lower glucose production in rats (chemical approach).
(A and B) During the pancreatic clamp (t = 180-200) an intrajejunal leptin infusion (6.7 ng/min) increased the glucose infusion rate (A, **P < 0.01 vs. other groups) and decreased glucose production (B, **P < 0.01 vs. other groups). Co-infusion of the SLR negated the ability of jejunal leptin to A) increase the glucose infusion rate and B) lower glucose production. C) Suppression of glucose production during the clamp period (t = 180-200) expressed as the percent reduction from the basal state (t = 60-90) glucose production (**P < 0.01 vs. other groups). D) The rate of glucose uptake remained unchanged amongst all groups. Values are shown as mean ± s.e.m. n = 5–7 rats per group. SLR, soluble leptin receptor.
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Figure 4.6 Leptin activates leptin receptors to lower glucose production in lean fa/fa rats but not in fak/fak (Koletsky) long form leptin receptor deficient rats (molecular approach).
(A and B) During the pancreatic clamp (t = 180-200) an intrajejunal leptin infusion (6.7 ng/min) increased the glucose infusion rate (A, **P < 0.01 vs. other groups) and decreased glucose production (B, **P < 0.01 vs. other groups) in lean fa/fa rats but not fak/fak rats. C) Suppression of glucose production during the clamp period (t = 180-200) expressed as the percent reduction from the basal state (t = 60-90) glucose production (**P < 0.01 vs. other groups). D) The rate of glucose uptake remained unchanged amongst all groups. Values are shown as mean ± s.e.m. n = 5–7 rats per group.
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Figure 4.7 Jejunal leptin activates leptin receptors to lower glucose production in C57BL/6 but not db/db mice (molecular approach).
(A and B) During the pancreatic clamp (t = 150-170) an intrajejunal leptin infusion (6.7 ng/min) increased the glucose infusion rate (A, ***P < 0.001 vs. other groups) and decreased glucose production (B, **P < 0.01 vs. other groups) in C57BL/6 but not db/db mice. C) Suppression of glucose production during the clamp period (t = 150-170) expressed as the percent reduction from the basal state (t = 50-60) glucose production (**P < 0.01 vs other groups). D) The rate of glucose uptake remained unchanged amongst all groups. Values are shown as mean ± s.e.m. n = 7-8 mice per group.
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Figure 4.8 Jejunal leptin lowers glucose production in C57BL/6 independent of changes in circulating leptin levels.
(A and B) During the pancreatic clamp (t = 150-170) an intrajejunal leptin infusion (6.7 ng/min) increased the glucose infusion rate (A, ***P < 0.001 vs. other groups) and decreased glucose production (B, **P < 0.01 vs. other groups). An intravenous leptin infusion at the same dose and duration did not affect the A) glucose infusion rate or B) glucose production. D) The rate of glucose uptake remained unchanged amongst all groups. Values are shown as mean + s.e.m. n = 7-8 mice per group.
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Figure 4.9 Jejunal leptin lowers glucose production through a STAT3-independent and PI3K dependent pathway.
(A and B) During the pancreatic clamp (t = 180-200) an intrajejunal leptin infusion (6.7 ng/min) increased the glucose infusion rate (A, **P < 0.01 vs. other groups) and decreased glucose production (B, **P < 0.01 vs. other groups). Co-infusion of LY294002 or wortmannin negated the ability of jejunal leptin to A) increase the glucose infusion rate and B) lower glucose production. A STAT3 PI co-infusion with leptin did not abolish leptin’s effects. C) Suppression of glucose production during the clamp period (t = 180-200) expressed as the percent reduction from the basal state (t = 60-90) glucose production (**P < 0.01 vs. other groups). D) The rate of glucose uptake remained unchanged amongst all groups. Values are shown as mean ± s.e.m. n = 5–7 rats per group. STAT3 PI, signal transducer and activator of transcription 3 peptide inhibitor LY, LY294002, Wort, wortmannin
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Figure 4.10 Jejunal and duodenal leptin activate intestinal STAT3, and only jejunal leptin activates jejunal PI3K.
A) The level of phosphorylation of STAT3 analyzed by western blot analysis and expressed as a fold increase over saline in the jejunum obtained from rats at the end of the clamp studies (*P < 0.05 vs. other groups). B) PI3K activity measured in the jejunum at the end of the clamp expressed as a fold increase over a jejunal saline infusion (*P < 0.05 vs. other groups). The increase in PI3K activity was abolished upon co-infusion with LY or the SLR. C) PI3K activity in the jejunum or duodenum at the end of the clamp with jejunal saline or jejunal leptin infusion expressed as fold increase over saline. D) The level of STAT3 phosphorylation/total STAT3 in the hypothalamus obtained at the end of the clamp studies in rats that received a jejunal saline or leptin infusion. Analyzed by western blot and expressed as fold increase over saline. (E and F) The level of phosphorylation of STAT3/total STAT3 (E, *P < 0.05 vs. saline) analyzed by western blot and expressed as fold increase over saline and F) PI3K activity in duodenal tissues obtained at the end of the rat clamp studies that received a duodenal saline or leptin infusion. Values are shown as mean + s.e.m. n = 5–7 rats per group. STAT3 PI, signal transducer and activator of transcription 3 peptide inhibitor LY, LY294002, SLR, soluble leptin receptor.
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Figure 4.11 Jejunal leptin lowers glucose production through a neuronal network.
(A and B) During the pancreatic clamp (t = 180-200) an intrajejunal leptin infusion (6.7 ng/min) increased the glucose infusion rate (A, **P < 0.01 vs. other groups) and decreased glucose production (B, **P < 0.01 vs. other groups). Co-infusion of tetracaine negated the ability of jejunal leptin to A) increase the glucose infusion rate and B) lower glucose production. C) Suppression of glucose production during the clamp period (t = 180-200) expressed as the percent reduction from the basal state (t = 60-90) glucose production (**P < 0.01 vs. other groups). D) The rate of glucose uptake remained unchanged amongst all groups. Values are shown as mean ± s.e.m. n = 5–7 rats per group.
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Figure 4.12 Jejunal leptin lowers glucose production in high fat diet fed rats.
(A and B) During the pancreatic clamp (t = 180-200) an intrajejunal leptin infusion (6.7 ng/min) increased the glucose infusion rate (A, **P < 0.01 vs. other groups) and decreased glucose production (B, **P < 0.01 vs. other groups) in both regular chow and high fat diet fed rats. C) Suppression of glucose production during the clamp period (t = 180-200) expressed as the percent reduction from the basal state (t = 60-90) glucose production (**P < 0.01 vs. other groups). D) The rate of glucose uptake remained unchanged amongst all groups. Values are shown as mean ± s.e.m. n = 5–7 rats per group.
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Figure 4.13 Jejunal leptin lowers glucose production in high fat diet fed rodents independent of a rise in plasma leptin levels.
(A and B) During the pancreatic clamp (t = 180-200) an intrajejunal leptin infusion (6.7 ng/min) increased the glucose infusion rate (A, ***P < 0.001 vs. other groups) and decreased glucose production (B, **P < 0.01 vs. other groups) in high fat diet fed rats. An intravenous leptin infusion at the same dose and duration did not affect the A) glucose infusion rate or B) glucose production. D) The rate of glucose uptake remained unchanged amongst all groups. Values are shown as mean + s.e.m. n = 5-6 rats per group.
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Figure 4.14 Jejunal leptin lowers plasma glucose levels and glucose production in uncontrolled diabetic rodents independent of changes in plasma insulin and glucagon levels.
(A and B) In non-clamped conditions, a jejunal leptin infusion (6.7 ng/min) decreased plasma glucose levels (* P < 0.05 vs. saline) and glucose production (* P < 0.05 vs. saline). C) Plasma glucagon levels during a jejunal saline or leptin infusion. D) Plasma insulin levels during a jejunal saline or leptin infusion. Values are shown as mean + s.e.m. n = 5-6 rats per group. STZ, streptozotocin.
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Figure 4.15 Jejunal leptin lowers plasma glucose levels and glucose production in uncontrolled diabetic rodents independent of a rise in plasma leptin levels.
(A and B) In non-clamped conditions, a jejunal leptin infusion (6.7 ng/min) decreased plasma glucose levels (* P < 0.05 vs. saline) and glucose production (* P < 0.05 vs. saline). An intravenous leptin infusion at the same dose and duration did not affect the A) plasma glucose levels or B) glucose production. Values are shown as mean + s.e.m. n = 5-6 rats per group. STZ, streptozotocin.
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Figure 4.16. Schematic of duodenal-jejunal bypass (DJB) surgery and jejunal catheter placement.
Point A: Proximal to the pyloric sphincter, a cut is made and the duodenal stump is closed off. Point B: The jejunum is reconnected to the stomach and a jejunal catheter was inserted into the lumen. Point C: The distal end of duodenal stump connected to the distal end of the jejunum/proximal ileum.
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Figure 4.17 Jejunal leptin action mediates the rapid anti-diabetic effect of DJB surgery.
A) Fed plasma glucose levels obtained from STZ-induced diabetic rats 2 days after sham or DJB surgery (* P < 0.05 vs. SHAM). DJB exerts a rapid glucose lowering effects 2 days after the DJB surgery. (B and C) Fed plasma insulin B) and glucagon C) levels before STZ injection, after STZ injection (before surgery) and 2d after surgery (C, ** P < 0.01 vs. other groups). (D, top panel) Plasma glucose levels during refeeding in STZ-diabetic rats with DJB vs. SHAM surgery and infused with intrajejunal saline. (D, bottom panel) Plasma glucose levels during refeeding in STZ-diabetic rats with DJB surgery and infused with intrajejunal saline or the SLR (φ STZ-DJB-jejunal saline vs. STZ-SHAM-jejunal saline, * STZ-DJB-jejunal saline vs. STZ-DJB-jejunal SLR, *P < 0.05, **P < 0.01, *** P < 0.001, ****P < 0.0001 (same for both symbols)). Blockade of leptin signaling through an SLR infusion results in dysregulated glucose homeostasis during refeeding. E) Accumulated food intake during the refeeding protocol. Values are shown as mean ± s.e.m. n = 5–6 rats per group. SLR, soluble leptin receptor, STZ, streptozotocin.
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Table 4.1 Plasma insulin and glucose concentrations of groups receiving intrajejunal infusions during the basal and clamp conditions
Data are means ± SEM (basal: t = 60-90 min, clamp: 180-200 min).
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Chapter 5Summary and Conclusions
5.1 Summary of Studies in this Thesis
Previous studies in our laboratory have demonstrated the existence of duodenal lipid !
PKC-δ ! CCK ! CCK-1 receptor signaling pathway that triggers a gut-brain-liver neuronal
axis to lower glucose production, which is abolished in rodents fed a high fat diet for three days.
This demonstrates that these rodents acquire duodenal CCK resistance upon short term high fat
feeding. To begin locating the potential site(s) of resistance, the first study of this thesis
delineated the downstream signaling pathway of the CCK1 receptor. In pancreatic acinar cells
the CCK1 receptor has been demonstrated to signal through PKA but it is unknown whether the
duodenal CCK1 receptor shares this same signaling pathway. In this regard, we utilized the
pancreatic (basal insulin) euglycemic clamp technique to address whether direct activation of
duodenal PKA signaling lowers glucose production. We demonstrated that direct duodenal PKA
activation ignites vagal afferent firing to activate NR1 containing NMDA receptors within the
DVC to lower glucose production and lies downstream of the CCK1 receptor. Interestingly,
direct activation of duodenal PKA in rodents fed a high fat diet bypassed CCK resistance and
lowered glucose production. This study provides evidence that CCK resistance arises, in part,
from the inability of the CCK1 receptor to activate the downstream signaling molecule PKA.
Similar to the duodenum, nutrient infusion into the jejunum (both lipids and glucose)
triggers a gut-brain-liver axis to lower glucose production, and contributes to the glucose
lowering effect of DJB surgery. It remains unknown whether gastrointestinal peptides can also
trigger this same neuronal network and also play a similar role in DJB surgery. Although most
studies focus on adipocyte-derived leptin and its glucoregulatory effects within the CNS, leptin
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is produced in gastric chief cells of the stomach and regulates various intestinal functions. Given
these findings, the focus of study 2 was to address whether leptin in the jejunum regulates
glucose production and contributes to the glucose lowering effect of DJB surgery. In this regard,
through the use of the pancreatic (basal insulin) clamp technique, we first demonstrated that a
jejunal leptin administration lowered glucose production through a long form leptin receptor-
PI3K dependent and STAT3-independent manner, which required a neuronal network. We
further demonstrated that jejunal leptin action remains intact in both STZ induced uncontrolled
diabetic rodents as well as in high fat fed rats. Lastly, we demonstrated that gastric derived
leptin may contribute to the glucose lowering effect of DJB surgery as blockade of leptin
signaling during refeeding in diabetic rodents who received DJB surgery resulted in a
dysregulation in glucose homeostasis. Thus, this study demonstrates a glucoregulatory role of
leptin within the intestine and suggests that enhancing leptin-PI3K signaling in the jejunum may
lower plasma glucose concentrations in diabetes.
5.2 General Summary
This doctoral thesis demonstrates that independent activation of the duodenal CCK-
PKA and jejunal leptin-PI3K signaling axis lowers glucose production in normal, high-fat fed
and diabetic rodents via a gut-brain-liver neuronal axis (Figure 5.1).
5.3 General Conclusion
Through the two studies in this thesis, we have demonstrated that independent local
duodenal CCK and jejunal leptin signaling, in contrast to peripheral effects of intestinal
hormonal signaling discussed in the general introduction, regulates glucose production through a
neuronal network. This opens a new area of research whereby targeting local hormonal
signaling, in addition to peripheral hormonal affects, could trigger the nervous system to
regulate glucose homeostasis.
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Figure 5.1 Summary of duodenal and jejunal hormonal signaling that triggers a neuronal network to lower glucose production
In Study 1 we demonstrated that the duodenal CCK1 receptor signals through PKA to trigger vagal afferent firing and activated NR1 containing NMDA receptors within the DVC to lower glucose production. Importantly, direct activation of duodenal PKA bypassed CCK-resistance in short-term high fat fed rodents. Moreover, Study 2 demonstrated that jejunal leptin triggers a leptin receptor ! PI3K signaling axis to lower glucose production, which remained intact in both high fat fed and STZ rodents. Further, jejunal leptin action mediates the early anti-diabetic effects of DJB surgery.
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Chapter 6General Discussion
6.1 Do nutrient sensing mechanisms interact with both CCK and leptin?
Following lipid entry into the duodenum and uptake into intestinal cells, LCFA are
metabolized into LCFA-CoA by ACS to activate PKC-δ to cause CCK secretion to subsequently
activate a CCK1-receptor ! PKA signaling cascade to lower glucose production508. Intralipid is
an emulsion containing different fatty acids with varying degrees of saturation. Thus, it remains
in question whether individual fatty acids within this emulsion cause CCK secretion to lower
glucose production. Interestingly, it has been demonstrated that individual fatty acids may
differentially regulate gastrointestinal processes. For instance, intestinal infusions of individual
monounsaturated versus polyunsaturated fatty acids differ in their effectiveness to reduce food
intake509 possibly through differences in their ability to slow gastric emptying510 and release of
gut peptides511. Moreover, the monounsaturated fatty acid, oleic acid, has been demonstrated to
cause secretion of CCK in the STC-1 cell line176. Thus, it remains to be addressed whether
individual fatty acids affect glucose production and cause CCK secretion and PKA signaling or
whether different signaling mechanisms are involved.
In addition to lipids, both glucose and proteins have been demonstrated to cause the
release of CCK. In regards to proteins, individual amino acids such as phenylalanine172 and
tryptophan173 can stimulate CCK release. Phenylalanine has been shown to reduce food intake in
association with an increase in circulating CCK levels in human patients512. The mechanism
upstream of CCK release by amino acids may involve a calcium sensing receptor (CaSR) that
was originally found in parathyroid cells. This receptor has recently been found in CCK
secreting cells and has been shown to stimulate CCK release in conjunction with an increase in
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intracellular calcium levels513. Thus, whether amino acids trigger CCK release to regulate
glucose production remains to be uncovered and may involve the CaSR receptor. In addition,
glucose uptake into cells via SGLT-1 has been shown to be required for jejunal glucose sensing
to regulate glucose production402. Although it remains unknown whether glucose administration
into the duodenum lowers glucose production, SGLT-1 is expressed in the proximal intestine514.
This suggests that if the duodenum senses an increase in glucose entry into the intestine to lower
glucose production, uptake into intestinal cells and subsequent release of CCK may play a role.
In regards to gastric leptin secretion, it has been demonstrated that the presence of
nutrients in the stomach causes the release of leptin which makes it way into the
duodenum127,143. However, these studies conducted refeeding experiments with rat chow diet
that encompasses carbohydrates, lipids and proteins. Thus, this suggests that all forms of
nutrients can cause leptin release from the stomach. However, what remains unknown is
whether any of these nutrients are more potent stimulators of leptin release, as certain
carbohydrates are more potent than others515. Given that study 2 demonstrates a beneficial
glucoregulatory effect of leptin after bypass surgery, it would be of interest to determine which
nutrient causes the greatest release of leptin from the gastric chief cells in the stomach.
Moreover, once gastric leptin is secreted and makes its way to the jejunum to activate
Leprb receptors, nutrients will also be found within the small intestine. As discussed in section
1.4.2 of the general introduction, intestinal nutrients (glucose and lipids) have been shown to
regulate glucose levels in both normal and uncontrolled diabetic conditions402. Whether glucose
and lipid sensing mechanisms converge at the level of the intestine remains unknown. However,
within the hypothalamus, convergence of glucose and lipid sensing occurs via an adenosine
monophosphate activated protein (AMPK) – malonyl-CoA – CPT-1 pathway516. More
specifically, hypothalamic glucose is converted to acetyl-CoA, which is subsequently converted
to malonyl-CoA via acetyl-CoA carboxylase (ACC). AMPK is a negative regulator of ACC,
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which when inhibited, allows for inhibition of CPT-1 by malonyl-CoA, which disrupts the
transfer of LCFA-CoA into the mitochondria to undergo β-oxidation. These processes together
sense hypothalamic glucose and lipids to regulate glucose production516. Interestingly, leptin has
been shown to inhibit hypothalamic AMPK activity to decrease feeding517 and AMPK518 and
CPT-1519 are expressed in the intestine. This raises the possibility that lipid sensing mechanisms,
and possible convergence with glucose sensing mechanisms, in the intestine are required for
leptin action to regulate glucose homeostasis.
6.2 What other intestinal hormones share similar signaling mechanisms as CCK
and leptin?
6.2.1 PKA
Study 1 demonstrates that a duodenal CCK! CCK1 receptor ! PKA signaling cascade
exists to regulate glucose production. In addition to the CCK1 receptor, PKA is also activated
by many different GPCRs. Specifically, the GLP-1R is known to signal through a PKA
mediated pathway in the β cell308 as well as the brain305. It is traditionally believed that GLP-1 is
secreted from L cells within the ileum to exert its effects. However, as discussed in section
1.3.3.1 of the Introduction, it is now currently debated whether L cells are also found in the
proximal small intestine271. Given that GLP-1R are expressed on vagal afferents270, perhaps
GLP-1 within the duodenum also activates PKA to regulate glucose homeostasis. In line with
this finding, inhibition of DDP-IV, the enzyme that rapidly degrades GLP-1, within the small
intestine through administration of an oral DDP-IV inhibitor, activates vagal afferents and
improves glucose tolerance285. Thus, it remains possible that GLP-1 mediated activation of PKA
in the small intestine may contribute to the regulation of glucose homeostasis.
In addition to GLP-1, other intestinal hormone receptors are GPCRs that signal through
PKA. GIP has been shown to activate PKA to regulate GSIS263. However, the GIPR is
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expressed in the intestine261 but is not found to be expressed in vagal afferents270 suggesting that
GIP may not activate PKA within vagal afferents. In contrast, the GLP-2 receptor also activates
PKA to regulate GSIS332 and has been found to be expressed in vagal afferents and increase
activity within the NTS upon activation520. Given that L cells may exist in the proximal
intestine, GLP-2 may signal through duodenal PKA, but whether GLP-2 triggers a neuronal
network to regulate glucose production remains to be investigated.
6.2.2 PI3K
Study 2 demonstrated that activation of PI3K is required for jejunal leptin to lower
glucose production. Interestingly, in addition to activating PKA and PLC, CCK has also been
shown to activate PI3K in pancreatic acinar cells521. Thus it may be possible that CCK signaling
in the duodenum shares common signaling with jejunal leptin by activating PI3K to lower
glucose production. Although a duodenal leptin administration failed to activate PI3K and
subsequently did not lower glucose production, the leptin receptor is a tyrosine kinase associated
receptor that requires autophosphorylation of tyrosine residues to cause activation of
downstream molecules. This is different from GPCR signaling which is mediated by G proteins.
Thus, it remains possible that the CCK1 receptor could also signal via PI3K to regulate glucose
production. Moreover, the GLP-2R has been shown to signal through PI3K in the brain347.
Whether this signaling occurs in the intestine remains unknown, but given that activation of the
GLP-2R on vagal afferents signals to the NTS as described above, GLP-2 could signal through
PI3K in addition to PKA to trigger a neuronal network like CCK and leptin. Similar to GLP-2,
the GLP-1R has also been found to signal via PI3K in the brain to regulate food intake304. Given
that GLP-1R signaling in the upper intestine may regulate glucose homeostasis, it remains to be
addressed whether GLP-1R signaling via PI3K triggers a similar axis as CCK and leptin.
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6.3 What is the cellular location of CCK-PKA and leptin-PI3K signaling in the
intestine?
The current set of studies in this thesis set out to dissect intestinal hormone signaling
mechanisms and the regulation of glucose production. Study 1 demonstrated that PKA lies
downstream of CCK to trigger a gut-brain-liver axis to lower glucose production where study 2
demonstrated that jejunal leptin administration activates PI3K to trigger a neuronal network (and
likely a gut-brain-liver axis previously demonstrated for jejunal nutrient sensing) to lower
glucose production. However, the current set of data does not address the exact location of these
hormonal signaling pathways. What is known from these studies is that these signaling
pathways occur in the mucosa and/or smooth muscle layer of the small intestine as the samples
measured for activation of downstream signaling molecules were conducted in these layers.
Given the complexity of the small intestinal tract in terms of cell types, it remains to be
addressed whether these signaling cascades take place in intestinal cells such as enteroendocrine
cells, epithelial cells or exclusively in vagal afferents that innervate both the smooth muscle and
mucosal layer of the small intestine.
In regards to PKA signaling, study 1 measured PKA activity from the whole tissue
encompassing both the mucosal and smooth muscle layer. However, the findings that activation
of PKA lowered glucose production in the presence of the CCK1 receptor inhibitor, MK-329,
demonstrates that PKA lies downstream of the CCK1 receptor and not upstream. It has been
demonstrated that the CCK1 receptor is expressed on vagal afferents innervating the small
intestine522. Thus, given our findings as well as the findings of CCK1 receptor expression, it
remains likely that PKA signaling occurs in vagal afferent neurons. To better confirm this
hypothesis, anterograde labeling could be used, a technique that involves injection of an
antibody into the nodose ganglion that travels down the vagus nerve to the small intestine, in
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order to visualize the innervation through immunohistochemistry155. Through co-staining for
PKA, whether PKA is found within vagal afferent neuronal cells could be determined.
Long form leptin receptor expression is more complex in the small intestine as it has
been found to be expressed in many different cell types including epithelial cells480, CCK and
GLP-1 secreting cells143,160 and vagal afferents innervating the small intestine149–151. Given this
complexity of expression, the intestinal effects of leptin could be mediated by any one of these
various cell types. In study 2, we measured long form leptin receptor expression and PI3K
activity separately in the mucosal and smooth muscle layer and detected leptin receptor
expression and an increase in PI3K activity in both layers. Unfortunately, this does not narrow
down the cell type responsible for mediating leptin-induced activation of PI3K and subsequent
regulation of glucose production. However, two recent studies153,154 begin to narrow down the
cell type that may not be responsible for jejunal leptin’s effects. In brief, these groups generated
epithelial cell specific long form leptin receptor knock out mice through the crossing of villin-
Cre mice with Leprflox/flox mice. Interestingly, these specific knock out mice were no different
from the control mice in terms of food intake and body weight regulation. Given these findings,
it may be that epithelial cell long form leptin receptor activation is not responsible for mediating
the glucoregulatory effect of jejunal leptin. In contrast to these findings, a recent paper
demonstrated that deletion of the long form leptin receptor in vagal afferent neurons leads to
hyperphagia and obesity. These findings demonstrate that vagal afferent long form leptin
receptors, rather than receptors in epithelial cells, play a role in mediating leptin’s effects152. In
order to better address the hypothesis that long form leptin receptors specifically in vagal
afferent neurons regulate glucose homeostasis as a follow up to Study 2 in the current thesis, the
pancreatic clamp technique could be performed in both knock out mice with a jejunal leptin
infusion, which would be expected to lower glucose production in epithelial cell long form
leptin receptor knock out if leptin receptor and subsequent PI3K activation within epithelial
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cells is not required for jejunal leptin’s glucoregulatory effect and would be abolished in mice
with a deletion of the long form leptin receptor in vagal afferents. In fact, the latter technique
could be utilized for PKA signaling (using CCK1 receptorflox/flox mice) to confirm PKA
activation within vagal afferent neuronal cells is required for CCK to lower glucose production.
Furthermore, as suggested for PKA, the visualization of leptin receptors in vagal afferents could
be conducted through the use of anterograde tracing and immunohistochemistry.
6.4 What is the relevance of CCK and leptin signaling in disease models?
A common theme throughout this thesis is to begin to dissect local intestinal hormonal
signaling mechanisms in the regulation of glucose production in both normal and diabetic/obese
settings in hopes to unveil therapeutic targets to lower blood glucose concentrations in diabetes
and obesity. Indeed, our previous studies suggest that even short term exposure of the duodenum
to a diet high in fat content causes early onset insulin resistance and disrupts the ability of
duodenal lipids to activate the CCK1 receptor to lower glucose production. In study 1, we
uncovered a possible site of duodenal CCK resistance, which is the inability of the CCK1
receptor to activate PKA. As such, one possible therapeutic target to overcome this resistance
after high fat feeding is to directly activate PKA to lower glucose levels.
In fact, the therapeutic potential of targeting the gastrointestinal tract, and subsequently
PKA signaling to regulate glucose homeostasis in diabetes and obesity has recently been
established for metformin and resveratrol (metformin is the most widely prescribed type 2
diabetic drug, and resveratrol is a polyphenolic compound well known for its insulin sensitizing
effects). In regards to metformin, in the same three day high fat diet model used in the current
thesis, an acute metformin infusion for 50 minutes during the basal insulin euglycemic
pancreatic clamp lowered glucose production via a neuronal network523. This was independent
of its direct effect on the liver60 as a metformin infusion at the same dose and duration into the
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portal vein failed to lower glucose production in the 50 minute time frame. This preabsorptive
effect of metformin required GLP-1R and subsequent PKA activation as co-infusion of
metformin with exendin-9 (GLP-1R antagonist) or Rp-CAMPS negated the metformin induced
suppression of glucose production (Figure 5.1). Similarly, in another study, resveratrol was
infused for 50 minutes during a hyperinsulinemic euglycemic pancreatic clamp in the same
three day high fat fed rodent model and improved hepatic insulin sensitivity and lowered
glucose production524. This effect was also dependent on PKA activation, as seen with
metformin (Figure 5.1). Thus, these studies demonstrate that anti-diabetic compounds such as
metformin and resveratrol utilize a GLP-1R-PKA signaling cascade to lower glucose levels in
an early onset insulin resistant, duodenal CCK resistant, high fat diet rodent model. More
importantly, metformin and resveratrol were still effective to regulate glycemia in 28 day high
fat diet induced obese and insulin resistant rodents, as well as in
nicotinamide/streptozotocin/high fat diet fed induced type 2 diabetic rodents suggesting that
PKA signaling remains intact in more chronic disease models. This strengthens the idea that
PKA signaling could be targeted in diabetes and obesity to lower glucose levels.
Another potential therapeutic strategy to lower blood glucose concentrations is the use of
gastric bypass surgery, which is commonly performed in obese patients and has been shown to
have beneficial effects on glucose homeostasis. Surgical procedures such as RYGB and DJB
surgery bypass the proximal duodenum and connect the jejunum to the stomach, where RYGB
also alters the stomach size. Thus, it is likely that these surgical procedures bypass the
duodenum that is unresponsive to lipids (and possibly other nutrients) and directs nutrient flow
into the jejunum in which hormonal signaling likely remains intact. This is suggested in study 2,
as a direct leptin administration into the jejunum activated a leptin receptor-PI3K signaling
cascade to lower glucose production in high fat fed or uncontrolled diabetic rodents via a
neuronal network, and contributed to the early anti-diabetic effect of bariatric surgery. In line
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with these findings, a recent study reported increased intestinal PI3K activation after RYGB
surgery525. Whether nutrient sensing and/or other hormones lie upstream or downstream of
jejunal leptin signaling to regulate glucose levels after DJB surgery remains to be addressed.
However, study 2 suggests that alternate mechanisms are required for the rapid lowering of
glycemia after DJB surgery given the findings that disrupting jejunal leptin signaling in STZ-
DJB rats during refeeding did not disrupt glucose control to the extent of that seen in STZ-sham
rats. Given that leptin has been demonstrated to cause GLP-1 secretion160, and the finding that
DJB surgery in STZ rodents elevated plasma GLP-1 levels 2 days after surgery402, GLP-1 may
play a role in mediating the rapid glucose lowering effect (Figure 5.1). However, in the
autoimmune diabetes-prone Biobreeding rat, DJB surgery rapidly lowered glucose levels
independent of a rise in GLP-1 levels402 and thus the functional relevance of GLP-1 in
mediating the rapid anti-diabetic effect of DJB surgery remains to be clarified. Dissecting
whether jejunal leptin signaling converges with nutrient sensing mechanisms402 may shed light
on additional mechanisms required (Figure 5.1). In fact, a recent study suggests that after
RYGB surgery, the number of CCK secreting I cells increases in the roux and common limbs526.
Although study 1 demonstrated that PKA activation in the jejunum did not regulate glucose
production, CCK signaling in the jejunum may still be relevant as in addition to PKA, duodenal
CCK also signals through PLC. It is worth investigating whether nutrient sensing in the
jejunum402 causes CCK release to work in concert with leptin to regulate glucose levels after
DJB surgery, as leptin has been previously shown to enhance the ability of CCK to decrease
food intake156. In summary, our results, in combination with other group’s findings, suggest that
activation of jejunal leptin-PI3K signaling (and possibly other hormonal pathways) may mimic
the anti-diabetic effect of bariatric surgery.
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Chapter 7Limitations of the Studies
1. One of the major limitations to the current studies is that we do not demonstrate the cell
type where CCK and leptin activate downstream molecules PKA and PI3K, respectively.
As mentioned in the general discussion, it is likely that PKA signaling occurs in vagal
afferent neuronal cells where the CCK1 receptor is expressed522, which remains to be
confirmed. Furthermore, we currently cannot rule out the possibility that leptin acts on
leptin receptors on epithelial480, enteroendocrine143 or neuronal cells149–151 as the long
form leptin receptor has been found to be expressed on each cell type. The current study
only suggests that PI3K activation occurs within both the mucosal and smooth muscle
layer. Thus, as discussed above, it would be of interest to utilize either visualization
techniques or epithelial/vagal specific knock out models to address the exact location of
PKA and PI3K signaling triggered by CCK and leptin, respectively.
2. In study 2, the use of whole body long form leptin receptor knock out models was used
to address the involvement of the receptor in mediating the effects of jejunal leptin on
glucose production suppression. In addition to these models, we also utilized co-infusion
of leptin with the SLR, which binds leptin and prevents it from binding to its receptor.
We are currently lacking a molecular intestinal specific knock down approach for the
long form leptin receptor. Due to the time constraints of this thesis, the production of an
adenovirus expressing shRNA to the long form leptin receptor was not conducted.
However, this could be done as a future experiment as we have previously developed
adenoviruses expressing shRNA to MEK1527.
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3. In both studies, an intestinal infusion of various compounds was given for 50 minutes.
Given this long duration of infusion, it is possible that although we aim to target specific
regions of the small intestine (either the duodenum or jejunum) these compounds may
leak into other regions during the clamp studies. In study 1, we did address this in
regards to a Sp-CAMPs (PKA activator) infusion. Sp-CAMPs was given directly into the
jejunum during the pancreatic clamp, which had no effect on the glucose kinetics
suggesting that PKA activation was specific to the duodenum to lower glucose
production during a duodenal Sp-CAMPS infusion (and not due to a leak into the
jejunum). We did not examine whether CCK-8 administration into the jejunum triggers a
neuronal network to lower glucose production, which could be addressed by giving a
direct jejunal CCK-8 infusion during the pancreatic clamp studies. In regards to leptin,
we first gave a duodenal leptin infusion and did not see any changes in the glucose
kinetics even though the receptor is expressed in the duodenum. We then demonstrated
that a direct jejunal leptin infusion lowered glucose production, confirming that a
duodenal leptin administration did not leak into the jejunum during the clamp studies.
Whether a jejunal leptin infusion travels to the ileum to regulate glucose production
remains unknown. This region of the small intestine has yet to be studied in regards to
triggering a gut-brain-liver axis although the long form leptin receptor is said to be
expressed in L cells in the ileum160, suggesting that such an axis could exist for leptin in
this region. This could also be addressed by a direct infusion of leptin into the ileum
during the pancreatic clamp.
4. The pancreatic clamp technique is used to address whether activation of intestinal
hormonal signaling regulates glucose production in the absence of changes in circulating
glucoregulatory hormones, which does not directly address the physiological relevance
of such signaling. The physiological relevance of such activation can be addressed
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through the use of a refeeding non-clamp protocol and an intravenous glucose tolerance
test where circulating glucoregulatory hormones are allowed to change at will. For the
fasting and refeeding protocol, PKA activation or leptin receptor signaling could be
inhibited to see whether this results in a dysregulation in glycemia. In addition, an
intravenous glucose tolerance test could be conducted with infusion of Sp-CAMPS into
the duodenum or leptin into the jejunum, to address whether activation of either
signaling pathway improves glucose tolerance and whether inhibiting these pathways
disrupts the improvement. Together these techniques would demonstrate that CCK-PKA
and leptin-PI3K signaling regulates glucose homeostasis in a more physiological setting.
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Chapter 8Future Directions
1. It has previously been demonstrated that duodenal lipid metabolism is required for CCK
release and subsequent activation of a gut-brain-liver axis to lower glucose production.
Study 1 extends these findings by demonstrating that PKA activation is required for the
glucose production lowering effect of CCK. Given that other nutrients also lead to CCK
release as discussed in 1.3.2.1, these nutrients may trigger CCK release to activate a gut-
brain-liver axis through activation of PKA. If so, this would suggest that CCK acts as a
converging point for duodenal nutrients to regulate glucose homeostasis. Furthermore, it
would also be of interest to then establish whether diets high in sugar or protein also
cause duodenal CCK resistance, as this would demonstrate that a balanced diet is
integral to maintain the regulation of glucose homeostasis by intestinal nutrient sensing
mechanisms. It would also be of interest to determine whether leptin requires lipid-
sensing mechanisms to regulate glucose homeostasis, which has been suggested for
leptin in regards to the regulation of food intake at the level of the hypothalamus.
2. Another future direction, and major limitation to the current studies, is that the exact
localization of peptide hormone signaling is at best narrowed down to the smooth muscle
and/or mucosal layer of the small intestine. Given that both of the leptin receptor149–151
and CCK1 receptor528 are expressed on vagal afferents innervating the small intestine, as
described previously, our current hypothesis is that upon binding of these hormones to
their receptors, downstream signaling events are activated to increase vagal afferent
firing. In order to better address the exact location of signaling, we could first use
anterograde tracing of vagal afferent neurons combined with immunohistochemistry to
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co-localize the molecules of interest with specific cell types. To assess the requirement
of various cell types the following could be conducted: i) create Nav1.8-Cre Leprflox/flox
or CCK1 receptorflox/flox mice to knock out the leptin receptor or CCK1 receptor in vagal
afferent neurons or ii) generate an adenovirus expressing shRNA to either the CCK1
receptor or leptin receptor which could be injected into the nodose ganglion. Upon
confirmation of knocking down either receptor from neuronal cells, infusion of CCK-
8/Sp-CAMPS or leptin into the intestine during the pancreatic clamp could be conducted
to address the requirement of CCK1 receptor or leptin receptor activation in vagal
afferent neurons. This would demonstrate that both PKA and PI3K are localized within
vagal afferents, and address which cell type CCK-1 and leptin receptor activation is
required to exert their glucoregulatory effect.
3. Although the studies in this thesis dissect hormonal signaling mechanisms in two
different regions of the small intestine in regards to the control of glucose homeostasis, it
remains to be addressed whether these hormones could work together in order to control
glucose homeostasis as has been demonstrated for the regulation of food intake. Given
that duodenal leptin administration failed to lower glucose production, it is likely that
leptin and CCK do not work together in this region of the small intestine. In regards to
the jejunum, CCK secreting I cells have been located to this region of the small intestine.
Thus, it may be that leptin and CCK work together in the jejunum to lower glucose
production. In order to address this question, it would first have to be determined
whether a CCK-8 administration into the jejunum lowers glucose production during the
pancreatic clamp. Given that in study 1, the administration of the PKA activator Sp-
CAMPS into the jejunum failed to lower glucose production, if CCK does indeed have
an effect in the jejunum, this would be a PKA-independent effect. Rather, jejunal CCK
could activate a CCK1 ! PLC signaling pathway to regulate glucose production as a co-
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infusion of CCK-8 with a PLC inhibitor into the duodenum abolished the suppression in
glucose production. After demonstrating a gluco-regulatory effect of CCK in the
jejunum, a co-infusion of CCK with the SLR or leptin with MK-329 could address
whether these hormones work together to regulate glucose production. It would also be
interesting to address whether co-administration of leptin and CCK have synergistic or
additive effects at the level of the jejunum by direct co-infusion of both hormones during
the pancreatic clamp.
4. The current studies in this thesis begin to look at the downstream signaling molecules for
both the CCK1 receptor and long form leptin receptor. There are other common
downstream signaling molecules activated by these receptors, which also may play a role
in mediating their effects on glucose production regulation. In regards to CCK1 receptor
signaling, study 1 further indicates that a PLC dependent pathway may be activated
downstream of the receptor as blockade of PLC signaling disrupted the ability of a CCK-
8 administration to lower glucose production. Interestingly, CCK also activates PI3K in
pancreatic acinar cells521. Thus, it may be possible that CCK signaling in the duodenum
shares common signaling with jejunal leptin by activating PI3K to lower glucose
production. Co-infusion with the PI3K inhibitors used in study 2 with CCK-8 during the
pancreatic clamp could address the possibility. Furthermore, whether intestinal leptin
activates PLC to cause activation of PI3K remains unknown, as demonstrated in the
brain529. This could also be addressed by co-infusion of leptin with the PLC inhibitor
used in study 1 during the pancreatic clamp.
5. Both studies in this thesis use an early onset model of insulin resistance induced through
short-term high fat feeding to address whether both PKA activation in the duodenum and
a jejunal leptin administration were still able to lower glucose production. This model
has important implications as addressing whether intestinal CCK and leptin action
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remains intact may unveil the intestine as a site of hormone action which can be targeted
to lower glucose production before hyperglycemia occurs. Nonetheless, it still remains
important to address whether these signaling mechanisms also remain intact after longer
term high fat feeding or in genetically obese models. In order to address this question, a
more chronic high fat diet/STZ rodent model of type 2 diabetes530 could be used
whereby intestinal infusions of CCK-8/Sp-CAMPS and leptin could be performed and
plasma glucose levels could be monitored to see whether a glucose lowering effect
remains. For study 1 and 2, we could also utilize diabetic db/db mice to address whether
direct activation of PKA and/or PI3K lowers glucose levels. In addition, in study 2 the
effect of jejunal leptin was tested in the STZ induced uncontrolled diabetic rodent model.
Leptin’s effects remained intact in this model whereby a jejunal leptin administration
lowered plasma glucose levels and glucose production in hyperglycemic rats. This
occurred in the absence of a suppression of glucagon levels seen previously with a CNS
leptin administration. Thus, it remains to be addressed whether a jejunal leptin infusion
would similarly lower glucose levels and production in a more chronic STZ model
through suppression of hyperglucagonemia.
6. In study 2, a duodenal leptin administration failed to lower glucose production. Upon
looking at downstream signaling molecules, we found that duodenal leptin activated
STAT3, but failed to activate PI3K. Given our findings that jejunal leptin activated PI3K
to lower glucose production, we believe that the failure of duodenal leptin to lower
glucose production is due to its inability to activate PI3K. In this regard, it would be of
interest to determine why duodenal leptin fails to activate PI3K. Upon binding of the
leptin to its receptor, there is subsequent phosphorylation of three key tyrosine residues
that are involved in activating different downstream signaling pathways. STAT3 is
activated by Tyr1138 recruitment and subsequent phosphorylation by Jak2 where PI3K is
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activated by Jak2 phosphorylation of IRS158. Thus, examining whether IRS is expressed
in the duodenum, or if IRS fails to be phosphorylated by duodenal leptin administration,
may address why duodenal leptin fails to activate PI3K and regulate glucose production.
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