CNS HORMONAL AND NUTRITIONAL REGULATION OF GLUCOSE … · 2017. 12. 19. · Title: CNS Hormonal and Nutritional regulation of Glucose and Energy homeostasis Name: Mona Anna Abraham
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CNS HORMONAL AND NUTRITIONAL REGULATION OF
GLUCOSE AND ENERGY HOMEOSTASIS
by
Mona Anna Abraham
A dissertation submitted in conformity with the requirements for the degree of Doctor of Philosophy
1.2 The role of the dorsal vagal complex in the regulation of glucose and energy homeostasis ........................................................................................................................18
Supplementary Figure 3. 5 Metabolic effects of DVC and iv infusion of ALX in 3d-HFD rats. 84
Supplementary Figure 3. 6 Metabolic effects of chemical and molecular inhibition of DVC
GlyT1 in obese rats. ...................................................................................................................... 85
Figure 4. 1 Summary of Study 1 and Study 2. .............................................................................. 88
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List of Publications that contributed to this Thesis
Study 1:
Abraham, M.A., Yue, J.T., LaPierre, M.P., Rutter, G.A., Light, P.E., Filippi, B.M., and Lam, T.K. (2014). Hypothalamic glucagon signals through the KATP channels to regulate glucose production. Mol Metab 3, 202-208.
Study 2:
Abraham, M.A.*, Yue, J.T.*, Bauer, P.V., LaPierre, M.P., Wang, P., Duca, F.A., Filippi, B.M., Chan, O., and Lam, T.K. (2016). Inhibition of glycine transporter-1 in the dorsal vagal complex improves metabolic homeostasis in diabetes and obesity. Nat Commun 7, 13501. *Equal contribution
Review Papers:
Abraham, M.A., and Lam, T.K. (2016). Glucagon action in the brain. Diabetologia 59, 1367-1371.
Abraham, M.A., Filippi, B.M., Kang, G.M., Kim, M.S., and Lam, T.K. (2014). Insulin action in the hypothalamus and dorsal vagal complex. Exp. Physiol. 99, 1104-1109.
LaPierre, M.P.*, Abraham, M.A.*, Filippi, B.M., Yue, J.T., and Lam, T.K. (2014). Glucagon and lipid signaling in the hypothalamus. Mamm. Genome 25, 434-441. *Equal contribution
Filippi, B.M.*, Abraham, M.A.*, Yue, J.T., and Lam, T.K. (2013). Insulin and glucagon signaling in the central nervous system. Rev. Endocr. Metab. Disord. 14, 365-375. *Equal contribution
Other studies contributing to the completion of this dissertation:
LaPierre, M.P., Abraham, M.A., Yue, J.T., Filippi, B.M., and Lam, T.K. (2015). Glucagon signalling in the dorsal vagal complex is sufficient and necessary for high-protein feeding to regulate glucose homeostasis in vivo. EMBO Rep 16, 1299-1307.
Filippi, B.M., Bassiri, A., Abraham, M.A., Duca, F.A., Yue, J.T., and Lam, T.K. (2014). Insulin signals through the dorsal vagal complex to regulate energy balance. Diabetes 63, 892-899.
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Chapter 1 Introduction
1.1 Diabetes and Obesity In 1878, the French physiologist Claude Bernard described that ‘All the vital mechanisms,
however varied they may be, have only one object, that of preserving constant the conditions of
the internal environment which make a free and independent life possible’.1,2 This maintenance
of an internal state defended against changes is particularly true for the regulation of blood
glucose levels and body weight. Low levels of plasma glucose would deplete the brain of its only
energy source, leading to seizures, unconsciousness, and death. On the other hand, sustained
elevation of blood glucose can also be fatal as it causes diabetes and associated complications.
Therefore, it is vital for the body to maintain blood glucose at a fairly narrow range of 4-7
mmol/L. Glucose homeostasis is that process of maintaining blood glucose at a steady-state
level. Similarly, in a healthy body, body weight and body fat is also defended against acute
perturbations under the influence of a tightly regulated homeostatic process called energy
homeostasis. For instance, rats when subjected to caloric restriction display significant weight
loss but when returned to free access of food, quickly rebound regaining their initial body weight
within days3,4. Remarkably, this precise nature of body weight regulation is also true in humans,
both lean and obese. In fact, the literature documents the weight gain rate among obese men
(0.04 kg·BW/year) is slower than men with no history of obesity (0.18 kg·BW /year)5.
However, despite these robust homeostatic systems, Type 2 diabetes and obesity are the
two most challenging public health concerns of the 21st century. Where the global prevalence of
diabetes was 9.3% in 2015, this number is predicted to increase to 12.1 % by 20256. Coincident
with this diabetes epidemic, the prevalence rates of obesity has also been escalating, with about
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13% of the world’s adult population obese in 20147. People affected by diabetes and obesity are
predisposed to developing cardiovascular diseases, and cancer8, which in turn reduce their life
expectancy and cost heavily in health care expenses. In Canada alone, the economic burden of
diabetes, which was approximated at $12.2 billion in 2010, is projected to increase by another
$4.7 billion by 20209. Meanwhile, the annual economic burden of obesity in Canada ranges from
$ 4.6 billion to $7.1 billion10. Given the survival and financial crisis caused by these diseases and
the expected rise in the number of affected individuals, the need for therapeutic interventions
aimed at combating the diabetes and obesity epidemic is more than ever today.
In this regard, considerable advances have been made scientifically in understanding the
different mechanisms that provide effective feedback to regulate glucose and energy
homeostasis. It is now recognized that the central nervous system (CNS) plays a critical role in
coordinating and integrating various components of glucose and energy regulation. In particular,
hormonal and nutrient signals from the periphery, relaying the body’s energy status, are detected
by the brain and integrated in CNS pathways to maintain constant blood glucose levels and body
weight stability. It follows that Type 2 diabetes and obesity develops as a result of dysregulation
in the ability of the CNS hormone and sensing pathways to appropriately couple the body’s
energy needs with nutrient intake and endogenous nutrient output. As such, delineating the
mechanisms of CNS hormonal signaling and/or nutrient sensing is vital in understanding and
identifying potential molecular targets to therapeutically restore regulation of feeding behaviour
and glucose homeostasis. Till date, tremendous progress has been made to elucidate the
molecular and cellular pathways, primarily within the hypothalamus and hindbrain, comprising
hormonal action and nutrient sensing circuits (which will be reviewed as follows).
The goal of this dissertation is to characterize novel CNS mechanisms of hormonal
signaling and nutrient sensing involved in the control of glucose and energy balance, thereby
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unveiling potential new therapeutic targets and experimental approaches to improve metabolic
control in diabetes and obesity.
1.2 The role of the hypothalamus in the regulation of glucose and energy homeostasis Of the different anatomical regions in the brain, the hypothalamus in particular is a privileged
CNS site to sense and integrate peripheral signals to regulate metabolic homeostasis. The median
eminence located at the mediobasal hypothalamus (MBH) and adjacent to the arcuate nucleus
(ARC) is a circumventricular organ, which is lined by fenestrated brain endothelium. This
permits circulating molecules to traverse past the blood brain barrier (BBB) and access the
hypothalamic ARC. There are two sets of first order neurons in the ARC, on which peripheral
metabolic hormones such as insulin, leptin, and glucagon and nutrients such as fatty acids and
glucose act: 1) the neurons that produce the anorexigenic (appetite suppressing) neuropeptides,
pro-opiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART), and
2) the neurons that produce the orexigenic (appetite promoting) neuropeptides, agouti-related
peptide (AgRP) and neuropeptide Y (NPY)11. Projections from these first order neurons to
second order neurons in other hypothalamic areas such as the paraventricular hypothalamus,
lateral hypothalamus, and ventromedial hypothalamus or extrahypothalamic areas including the
Given that CNS hormonal and nutritional sensing mechanisms are impaired in models of
obesity and/or diabetes, the question arises whether the effectiveness of DVC glycine to lower
glucose production is intact in settings of insulin resistance, uncontrolled diabetes and DIO. We
are encouraged by the findings that DVC glycine infusion, at the same dose infused in normal
rodents, is still able to lower hepatic lipid production in an acute diet-induced insulin resistance
model139 and that other central nutrient sensing mechanisms, such as those of central lactate are
preserved in an early-onset model of STZ-diabetes110. Further, no studies till date have directly
tested whether DVC glycine sensing can regulate energy homeostasis. However, given that DVC
NMDA receptors play a role in the control of food intake (as described previously) elicited by
hormones such as cholecystokinin143, and that glycine binding acts as a modulator to NMDA
transmission in the DVC, it is plausible that glycine triggers a sensing mechanism via DVC
NMDA receptors to reduce appetite.
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However, glycine as a CNS-active drug suffers from the limitation of having a poor
pharmacokinetic profile144. For instance, following oral administration of glycine, although
elevated glycine levels in the cerebral cortex, there was a concomitant increase in the rate of
glycine uptake and rapid conversion of glycine into serine in brain tissue, thereby limiting brain
exposure to extracellular glycine145. Indeed, studies from the schizophrenia field have reported
the use of glycine administration as an approach to activate NMDA receptor-mediated
transmission in schizophrenic patients. Schizophrenia displays reduced NMDA receptor
function, and therefore increasing NMDA receptor function via pharmacological manipulation is
integral to the treatment for schizophrenia. While there are some encouraging studies reporting
that large doses of glycine can improve the negative symptoms in schizophrenic patients146,147,
there are studies that have failed to confirm the efficacy of glycine as a therapeutic agent148. It is
not clear whether the conflicting evidence is due to low CNS exposure to extracellular glycine.
In fact, the level of extracellular glycine in the CNS is primarily dependent on regulation
by the Na+/Cl−-dependent glycine transporters, GlyT1 and GlyT2149. In particular, GlyT1, which
is expressed on glial cells, is the primary transporter of glycine mediating the uptake of glycine
into cells near NMDA receptors. Thus, blockade of GlyT1 could increase synaptic glycine levels,
thereby potentiating activation of NMDA receptors. Indeed, GlyT1 inhibitors have been tested in
rodents and preliminary clinical studies and have proved to be beneficial in the treatment of
schizophrenia150-153.
Given the unfavourable properties of glycine administration, the question arises as to
whether GlyT1 inhibition in the DVC would be a favourable approach to trigger glycine sensing
in the brainstem and more importantly, whether there is a novel therapeutic potential for DVC
GlyT1 inhibition, in the treatment for diabetes and obesity to lower glucose levels and body
weight via the activation of NMDA receptors.
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1.3 The role of glial cells in the regulation of glucose and energy homeostasis. When glial cells were first discovered in the 1800s, they were viewed as merely “cellular glue”
for the brain, holding neurons together. However, in recent years, there has been accumulating
evidence pointing that far from being passive, glial cells play a critical role in the normal
functioning of the brain including synaptic plasticity, development, neurotransmission and
metabolism154. In fact, astrocytes, a type of glial cells, are emerging as important regulators of
nutrient and energy sensing mechanisms in the CNS, and they do so by expressing specific
hormonal receptors and nutrient transporters. For instance, in the hypothalamus, astrocytic
insulin receptors are indispensible for proper glucose and insulin entry into the brain, in turn
contributing to CNS regulation of systemic glucose and energy homeostasis155. Similarly, leptin
signaling in hypothalamic astrocytes has also been reported to play an important role in the CNS
control of feeding156. At the same time, studies also show that glial specific glucose transporter,
GLUT1 is vital for hypothalamic glucose sensing to regulate peripheral glucose levels109, and
that lipoprotein lipase (LPL) in astrocytes controls lipid uptake in the hypothalamus for central
regulation of body weight and glucose metabolism157. Notably, in addition to transporting
circulating nutrients and expressing hormonal receptors, glial cells can also contribute to
systemic metabolic control via uptake of neurotransmitters from the synaptic cleft. For instance,
astrocytes, which is critical for regulating synaptic transmission by this excitatory amino acid.
Further, glutamate uptake into astrocytes has also been reported to increase glycolysis and lactate
production, in turn modulating nutrient availability for neurons107,158. Therefore, CNS regulation
of nutrient sensing and hormonal signals is, atleast in part, directed by glial cells. However, what
remains to be shown and will be addressed in this dissertation, is whether the glial specific
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glycine transporter, GlyT1 like GLT1, can couple CNS nutrient mechanisms to control systemic
metabolic homeostasis.
1. 4 Summary of Introduction/Rationale of Study 1 and Study 2 Type 2 diabetes and obesity, the two largest public health concerns of today, are progressive
metabolic disorders of glucose and energy homeostasis. Over the last two decades, significant
progress has been made in support of the role the brain plays in regulating peripheral glucose and
energy homeostasis. The two key regions of the CNS: the MBH and the DVC can receive and
integrate information from hormones and nutrients to subsequently direct changes in hepatic
glucose production and feeding behaviour, and they do so by using distinct and sometimes
common receptors and intracellular signalling pathways. AMPK and KATP are two such crucial
intracellular signaling pathways responsible for the metabolic effects of MBH insulin, leptin,
glucose, fatty acids and amino acid leucine to regulate whole-body energy homeostasis and
glucose control. More recently, a novel gluco-regulatory role of MBH glucagon was discovered
to lower glucose production via the PKA pathway. Whether MBH glucagon action is
downstream mediated by AMPK and KATP signaling remains to be investigated. The focus of
Study 1 was to evaluate whether AMPK-mediated lipid sensing and KATP channels are
necessary for MBH glucagon for glucose regulation.
Moreover, the smallest amino acid glycine triggers neurotransmission in the DVC to
regulate metabolic homeostasis including glucose and lipid metabolism using NMDA receptors.
Importantly, DVC glycine sensing can normalize the hypersecretion of lipids induced by 3d
HFD. These findings highlight the therapeutic potential of glycine sensing to lower blood lipids
in individuals with obesity and diabetes. Could there be a novel therapeutic potential for DVC
glycine manipulation in lowering blood glucose and body weight in obese and diabetic
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individuals? Because glycine has poor pharmacokinetics, and because glycine levels in the
CNS are enhanced by GlyT1 inhibition, in Study 2, we investigated whether DVC GlyT1
inhibition could sufficiently trigger glycine sensing to improve glucose and energy
production by Ca2+ -dependent activation of nitric oxide synthase (NOS)215-217, while NO-
mediated activation of cGMP-dependent protein kinase in turn can activate KATP channel
opening218. Whether this coupling of NMDA receptors to KATP channels or vice versa play a role
in CNS regulation of metabolic homeostasis remain to be investigated.
Indeed, NMDA receptors are also present in the hypothalamus but interestingly these
hypothalamic channels are shown to influence energy homeostasis in the opposite direction as in
the DVC. For instance, in the LH, it is the injection of NMDA receptor antagonists that lowers
feeding219 while intrahypothalamic injection of glutamate analogs, that are specific to NMDA
receptors, increases food intake220. Further, deletion of GluN1 or GluN2 subunits from
hypothalamic AgRP neurons cause lowering of food intake and body weight221,222. Nonetheless,
glutamatergic action and NMDA receptors in both the hypothalamus and the brainstem are
important for energy balance control. Of note, there is little evidence on the role of
hypothalamic NMDA receptors in regulating glucose homeostasis.
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The relevance of MBH glucagon action and DVC GlyT1 inhibition in health and disease
Indeed, the prime objective of this dissertation was to unveil novel molecular mechanisms in the
CNS that would serve as therapeutic targets to lower blood glucose, food intake and body weight
gain in diabetes and obesity. As our previous study has demonstrated that hypothalamic glucagon
resistance in the context of high fat feeding is manifested by the inability of glucagon receptor
signaling to activate PKA79, the findings of Study 1 indicate that activating hypothalamic KATP
channels may be therapeutically advantageous. In fact, activation of hypothalamic KATP channels
by oral administration of the KATP channel activator, diazoxide has already been implicated in
lowering glucose production in humans163. This study performed the same euglycemic –
somatostatin clamping technique as in our dissertation work, fixing the gluco-regulatory
hormones in circulation and showed that in healthy individuals, oral diazoxide treatment led to a
30% suppression of glucose production. Additional studies in healthy rats confirmed that
diazoxide’s inhibitory effects on glucose production are negated in the presence of the KATP
channel blocker glibenclamide, suggesting these effects are likely mediated by KATP channels in
the brain163. Furthermore, intranasal administration of insulin at doses that increases the insulin
concentration in the cerebrospinal fluid (CSF) led to a suppression of glucose production during
pancreatic clamps in humans, likely through activation of CNS KATP channels176.
Additionally, our study contributes to the evolving association between increased
glucagon action and a metabolically healthier phenotype- a theme that has been garnering
scientific attention in the recent years. Multiple studies have reported that activation of glucagon
receptors in conjunction with other G protein-coupled receptors is metabolically advantageous in
diabetes and obesity. For example, a triagonist aimed at simultaneous activation of glucagon,
GLP-1 and glucose-dependent insulinotropic (GIP) receptors improved metabolic and glycemic
profiles in obese and diabetic rodents223. In addition, dual activation of glucagon and GLP-1
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receptors normalized glucose tolerance and reduced food intake in mice with diet-induced
obesity224,225. These pre-clinical studies have attributed the beneficial effects of glucagon
primarily to increased energy expenditure and decreased food intake, and its hyperglycemic
effects to be countered by the actions of GLP-1 and/or GIP. Whether these polyagonists reach
the brain and activate central glucagon signalling to improve the diabetogenic effects in these
polyagonist therapeutic strategies remain to be investigated. In the same light, a recent
investigation reports that reduced glucagon suppression 2 hours after an intravenous glucose
challenge is associated with a healthier metabolic phenotype including lower BMI, higher insulin
sensitivity and reduced risk of impaired glucose tolerance226. These findings together with our
study reshape our understanding of glucagon’s physiological role in health and disease.
In Study 2, we took advantage of multiple disease models to test the therapeutic potential
of DVC GlyT1 inhibition/glycine sensing in diabetes and obesity. The fasting hyperglycemic
(type 2 diabetic) model used in our dissertation, arguably is the closest rodent model
recapitulating the pathogenesis of type 2 diabetes in humans. Elegantly described by Samuel et
al.191, a low dose of STZ protected by nicotinamide injection induces partial destruction of beta
cells, thereby preventing beta cell compensation for 7 d HFD-induced insulin resistance but still
maintaining basal insulin levels, consequently leading to fasting hyperglycemia secondary to an
elevation of hepatic glucose production179,180. Other rodent models of diabetes including the
Zucker diabetic fatty and Goto-Kakisaki diabetic rats as well as the db/db mice have their
diabetic characteristics confounded by a rise in glucocorticoid levels, which are not represented
in the hormonal profile of type 2 diabetic patients, and therefore are not accurate models of
clinical diabetes. In comparison, the plasma corticosterone levels are not increased in the
STZ/Nic/HFD model of hyperglycemic rats191. Importantly, we showed that 7d STZ/Nic/HFD
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diabetic rats are glucose intolerant and that infusion of GlyT1 inhibitor into the DVC leads to
improvement of glucose tolerance.
Further, our study indicates that DVC GlyT1 inhibition could also be effective as an early
intervention for insulin resistance and pre-diabetic conditions. While the 3d HFD-model we used
in Study 2 is not a diabetic model, it is a well-established model for hepatic insulin resistance
validated under hyperinsulinemic pancreatic clamp conditions28,227-229. In fact, a recent study
from our own lab testing the insulin sensitizing effects of resveratrol had validated 3d HFD rats
to be insulin resistant in regulating glucose production under hyperinsulinemic clamp conditions,
which could be reversed upon resveratrol infusion into the duodenum179. Whether GlyT1
inhibitors, like resveratrol, sensitizes insulin to regulate glucose production warrants
investigation; but under basal-insulin clamp conditions, our findings conclusively show that
DVC GlyT1 inhibition directly lowers glucose production independent of changes in basal
insulin levels. Further, based on the observation that 3d HFD rats display hyperphagia, a key
driving force of obesity, our data also implicate that DVC GlyT1 inhibition treatments are
effective in regulating glucose production in conditions of early onset diet-induced obesity as
well. However, instead of solely relying on an early onset obesity model (which is not in fact
obese), we also used a 28-d HFD obese model to show that DVC GlyT1 inhibition is effective in
regulating glucose production even at a later stage in diet-induced obesity. Previously, these 28d
HFD obese rats failed to show suppression of glucose production and stimulation of glucose
uptake compared to regular chow-fed rats under insulin-stimulated conditions (i.e.,
hyperinsulinemic clamps)179, thereby indicating hepatic and peripheral insulin resistance.
The therapeutic relevance of GlyT1 inhibition was further corroborated by our data
showing that systemic administration of GlyT1 inhibitors result in similar desirable metabolic
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effects to those of direct DVC GlyT1 inhibition. However, given that GlyT1 and NMDA
receptors are also present in non-neural tissues such as the pancreas, the possible side effects of
using GlyT1 inhibitors and/ or increasing glycine systemically will have to be assessed
cautiously. A previous study reports that inhibition of NMDA receptor transmission in the islets
enhances glucose-stimulated insulin secretion205. It is possible then, because glycine is a co-
agonist of the NMDA receptor, that systemic infusion of GlyT1 inhibitors and thus, elevated
systemic glycine levels would have the undesirable side effect of enhancing NMDA receptor
transmission in the islets and consequently lead to reduced insulin secretion- an effect especially
detrimental for Type 2 diabetic patients. Interestingly though, multiple studies have documented
circulating glycine levels to be inversely associated with Type 2 diabetes risk230-232. A recent
study has in fact validated that glycine treatment results in increased insulin secretion from intact
human islets, and a disruption in this glycine-insulin action contributes to impaired insulin
secretion in Type 2 diabetes233. Of particular note, the effect of glycine to stimulate insulin
secretion was mediated via activation of the glycine receptors (GlyR) in the pancreatic islets
since antagonism of GlyR with strynchnine-prevented glycine induced insulin secretion.
Whether elevation of endogenous glycine levels in the pancreas during systemic GlyT1
inhibition could potentiate NMDA receptors to alter insulin secretion merits future investigation.
The use of divergent and sometimes common signaling pathways by circulating hormones
and nutrients in the CNS to regulate glucose and energy homeostasis.
Recent studies have highlighted this theme that a commonly derived pathway for CNS
hormonal action could regulate both glucose and energy homeostasis. As described in Chapter 1,
insulin action in the MBH and the DVC regulates both energy and glucose homeostasis234. In the
MBH, it does via a PI3K-dependent pathway to regulate glucose and feeding control, whereas in
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the DVC, insulin signals through an ERK-dependent pathway to regulate feeding, body weight
and glucose production122. In regards to CNS glucagon action, a recent study by Quiñones et al.
reported that hypothalamic glucagon requires glucagon receptor and activation of downstream
PKA in the hypothalamic ARC to lower food intake, since icv. co-infusion of the glucagon
receptor antagonist des-His1-[Glu9] glucagon amide or the PKA inhibitor H-89 negated the
ability of central glucagon to decrease feeding235. Of note, these were the same chemical
inhibitors employed to confirm the role of glucagon receptor and PKA in mediating MBH
glucagon action on glucose control79, thereby suggesting that any associated molecular players in
the glucagon receptor–PKA branch are likely to be a part of a common pathway for both feeding
and glucose regulation by CNS glucagon. Moreover, Quiñones et al. also reported changes in
AgRP expression associated with the anorectic action of central glucagon, again consistent with
the observation that MBH glucagon receptors co-localised with AgRP neurons, thereby
demonstrating that in brain glucagon action, AgRP neurons mediate both glucose and feeding
regulation.
However, it appears that not the entire signaling pathway converges for the glycaemic
and satiety effects of hypothalamic glucagon action. Contrary to how MBH glucagon signals to
exert glucose control independent of MBH AMPK as shown in Study 1, the suppressive effect
central glucagon exerts on feeding involved inhibition of AMPK and activation of the
downstream target ACC. Specifically, molecular activation of AMPK in the ARC via injection
of a constitutively active AMPK virus blunted the anorectic effects of central glucagon
injections, whereas we showed that activation of MBH AMPK via the same viral approach had
no effect on the glucose production-lowering effect of MBH glucagon. Consistent with this,
there were decreased AMPK and increased ACC in the ARC associated with the satiety effect of
central glucagon injections, whereas we reported that the glucose-lowering effect of MBH
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glucagon infusions was associated with no differences in pACC/total ACC levels in the MBH. It
is likely these distinct mechanisms by which brain glucagon acts potentially allows for selective
and independent control of glucose and feeding regulation. It is also possible that there are
different populations of glucagon-responsive neurons within the MBH in which glucagon
signaling pathways might be distinct for glucose and energy regulation, and this warrants future
investigation. However, it cannot be overlooked that perhaps the differences in glucagon dose
and administration—a single bolus i.c.v. glucagon injection at a dose of 480 ng for feeding vs 2 h
of constant MBH glucagon infusion with a much lower dose of 3.6 pg for glucose control —
could explain some of the differences in the regulation of molecular targets for feeding and
glucose regulation by brain glucagon. Alternatively, these findings could be due to differences in
the times at which the glucagon-treated tissues were obtained for molecular analysis: changes in
AMPK and ACC, which mediate the anorectic action of hypothalamic glucagon, were measured
1 h after the i.c.v. glucagon single bolus injection vs after 2 h of constant infusion of MBH
glucagon.
Study 2 describes that DVC GlyT1 inhibition plays a role in glucose tolerance and
glucose production in response to elevated extracellular glycine levels and activated NMDA
receptors. Although the involvement of NMDA receptors in the food intake and body weight
regulation by DVC GlyT1 inhibition was not directly shown in our study, we are encouraged by
other reports that show that blockade of NMDA receptors in the DVC enhances feeding236, and
that activation of DVC NMDA receptors is also required for CCK’s vagally mediated
suppression of food intake143,237. Thus, by postulation, DVC NMDA receptors act as the common
downstream mediator for DVC GlyT1 inhibitory control of glucose and energy homeostasis.
Notably, though, other studies have shown the existence of differential mechanisms of CNS
nutrient sensing regulating glucose and energy balance. For instance, activation of the mTOR
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pathway is required for the inhibitory effect of MBH leucine on food intake111, while the
suppressive effect of MBH leucine on glucose production is independent of mTOR113. Whether
mechanisms of DVC GlyT1 inhibition (and DVC glycine sensing) diverge downstream of
NMDA receptors to allow for selective and autonomous control of feeding and glucose
regulation remain to be investigated.
In the pursuit of new pharmacological targets that would treat diabetes and obesity, in
order to design molecules where the main goal is to curb both hyperglycemia and hyperphagia it
becomes important to identify the point of convergence in these CNS fuel-sensing mechanisms
that control both glucose and energy homeostasis. However, it becomes equally important to
distinguish the point of divergence when the goal is to target one homeostatic regulation but not
the other. For instance, a lean diabetic individual would not require lowering body weight, as
opposed to reducing his glucose levels. In the same line, an obese non-diabetic individual would
only want to improve his energy balance as opposed to glucose regulation. Nonetheless, our
studies along with others’ point to the finding that hormonal action and nutrient sensing in the
CNS act via common as well as distinct signaling mechanisms to mediate glucose and energy
metabolism.
4.3 Limitations and Future directions Lack of a direct electrophysiological assessment confirming that MBH KATP channels were
inhibited by our molecular and pharmacological approaches is a major limitation in Study 1 that
concludes MBH glucagon activates KATP channels to lower glucose production. Given that
blockade of CNS KATP channels leads to membrane depolarization and increased electrical
activity, we acknowledge performing complementary patch-clamp studies to record changes in
membrane potential and firing activity, presumably, in AgRP neurons of MBH slices in response
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to DN Kir6.2 (vs GFP) or glibenclamide (vs saline) with glucagon treatment would have greatly
strengthened our findings. An important future direction of Study 1 would also be to show
evidence for a direct PKA-mediated phosphorylation of KATP channels in the hypothalamus. It
has been shown that Kir6.2 can be phosphorylated by PKA at S372 and at S1571 for SUR1 in
pancreatic beta cells164. We could transfect hypothalamic cells lines (e.g. GT1-7, known to
express AgRP) with either wild type Kir6.2 or mutant Kir6.2 cDNA (mutated at S372) together
with either wild type SUR1 or mutant SUR1 cDNA (mutated at S1571), and test their ability to
be phosphorylated after PKA stimulation or glucagon treatment using in vitro phosphorylation
assays.
Further, we also did not measure malonyl- and LCFA-CoA levels in Study 1 that
concludes MBH glucagon works through a lipid-sensing independent mechanism. Interestingly
though, studies show activating or inhibiting the AMPK -> malonly-CoA sensing pathway does
not always translate in changes in LCFA-CoA levels in the hypothalamus55. Exogenous leptin
administration has been shown to increase the levels of malonyl-CoA level without subsequently
affecting the LCFA-CoA levels in the ARC54 whereas ghrelin signaling in the hypothalamus is
known to increase LCFA-CoA levels while inhibiting hypothalamic ACC, which reduces the
malonyl-CoA level65. These studies cast doubt on the reliability of malonyl-CoA and LCFA-
CoA levels as readout for lipid sensing activation. Perhaps then, the strength of Study 1 ought to
be the fact that we did not solely focus on blocking the beginning of the lipid sensing pathway,
we also showed neither inhibition of MBH PKC-δ (blocking the end of the lipid sensing
pathway) affected MBH glucagon, thereby ruling out a lipid sensing dependent mechanism for
MBH glucagon’s effect of glucose homeostasis.
The principal findings of Study 1 were generated using a single manipulation of glucose
homeostasis, the pancreatic basal-insulin clamp. Though closer to physiological conditions than
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the hyperinsulinemic-euglycemic clamp, our findings would be even more convincing in light of
additional methods. Specifically, what is the relevance of this system in the post-prandial state,
where multiple systems are acting on glucose homeostasis in concert? Performing iv GTTs and
mixed-meal tolerance tests would provide further insight and confirmation into the MBH
glucagon signaling axis.
Given the work of polyagonist therapeutic studies targeting glucagon in conjunction with
GLP-1 and GIP receptors in the management of obesity and diabetes223, another important
question is whether concurrent infusions of glucagon, GLP-1 and GIP into the MBH would
result in redundant, additive or synergistic effects in lowering glucose production. This will
begin addressing whether the CNS penetrance of these polyagonists and whether activation of
MBH glucagon signalling plays a role in counteracting the hyperglycemic effects of peripheral
glucagon in these polyagonist therapeutic strategies.
We have identified, for the first time, that DVC GlyT1 inhibition and thus, DVC glycine
sensing plays a critical role in the regulation of energy balance as defined by changes in food
intake and body weight. However, components of energy expenditure (i.e. physical activity and
thermogenesis) are just as important on the energy balance equation. Given that obesity develops
when energy intake exceeds energy expenditure, the goal of any anti-obesity treatment should be
to reduce energy intake, promote energy expenditure, or both. Therefore, to truly repurpose
GlyT1 inhibitors as an effective obesity therapy, it becomes critical to determine the effect of
DVC GlyT1 inhibition on energy expenditure. Interestingly, the DVC as well as the NMDA
receptors in the DVC has directly been implicated in promoting the thermogenic activity of the
brown adipose tissue238,239. For instance, activation of DVC NMDA receptors is shown to
mediate the effect of increasing brown adipose thermogenesis in response to lipid infusion into
the duodenum239. In light of this, we would expect treatment with DVC GlyT1 inhibitors would
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sufficiently activate NMDA receptors in the DVC to increase energy expenditure. Indeed, to
confirm this, our next step would be to monitor rats treated with DVC ALX (vs. saline) and/or
injected with LV-GlyT1 shRNA (vs. LV-MM) using metabolic chambers, as described
previously240.
It should be noted that most of the viral vectors we used to modulate various molecular
targets in our studies including AMPK activity or GlyT1 expression were under the ubiquitous
promoter, cytomegalovirus (CMV), thereby altering gene expression in non-target cells which is
a limitation in our work. In the future, we could use cell-specific promoters such as the neuron
specific enolase (NSE) or glial fibrillary acidic protein (GFAP) to alter our molecular targets
specifically in neuronal cells versus glial cells, respectively241.
With state-of the-art technologies that allow real-time manipulation of genetically defined
neuronal populations, an important future direction would be to map out the hypothalamic and
neuronal circuitry in the DVC involved in the regulation of glucose and energy homeostasis. As
an example, NTS neurons are known to excite brain regions such as the lateral parabrachial
nucleus (PBN) to modulate feeding behaviour. Recently, distinct population of neurons in the
PBN expressing the neuropeptide calcitonin gene-related protein (CGRP) (CGRPPBN) were
identified to lower feeding242,243. It is likely that lateral PBN neurons or CGRPPBN neurons would
be a downstream target mediating the effects of DVC GlyT1 inhibition to lower feeding and
body weight gain, especially given that DVC GlyT1 inhibition activates NMDA receptors and
that glutamatergic signalling activates CGRPPBN neurons244. Interesting future direction would be
to check for c-fos labeling in CGRPPBN neurons following DVC ALX treatments as well as to
check whether optogenetically inactivating CGRPPBN neurons (using the inhibitory
channelrhodopsin protein construct) would abolish the anorectic effects of DVC GlyT1
inhibition.
102
By contrast, our understanding of the glucoregulatory regulatory neurocircuits is far less
defined. However, a recent study employing optogenetic circuitry mapping approaches, revealed
that activating AgRP → LHA projections as well as activation of AgRP → anterior bed nucleus
of the stria terminalis (aBNST)vl projections impair systemic insulin sensitivity245. Clearly, future
studies are needed to know whether these neuronal circuits underlie MBH glucagon or DVC
GlyT1’s ability to alter peripheral glucose homeostasis.
103
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