University of South Florida Scholar Commons Graduate eses and Dissertations Graduate School 3-16-2016 Metabolic erapy for Age-Dependent Impaired Wound Healing Shannon Lynn Kesl Follow this and additional works at: hp://scholarcommons.usf.edu/etd Part of the Physiology Commons is Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate eses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. Scholar Commons Citation Kesl, Shannon Lynn, "Metabolic erapy for Age-Dependent Impaired Wound Healing" (2016). Graduate eses and Dissertations. hp://scholarcommons.usf.edu/etd/6104
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University of South FloridaScholar Commons
Graduate Theses and Dissertations Graduate School
3-16-2016
Metabolic Therapy for Age-Dependent ImpairedWound HealingShannon Lynn Kesl
Follow this and additional works at: http://scholarcommons.usf.edu/etd
Part of the Physiology Commons
This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion inGraduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please [email protected].
Scholar Commons CitationKesl, Shannon Lynn, "Metabolic Therapy for Age-Dependent Impaired Wound Healing" (2016). Graduate Theses and Dissertations.http://scholarcommons.usf.edu/etd/6104
The research included in this dissertation was made possible by support from our funding
sources, including the James A. Haley Veterans Hospital’s Merit Review, the Department of
Molecular Pharmacology and Physiology, Scivation Inc., and the Office of Naval Research
(ONR).
I would like to express my heartfelt gratitude to everyone who has supported me in my
pursuit of my doctoral degree. Firstly, I want to acknowledge the contribution from all of the
ratticans that have sacrificed their lives to further scientific research.
To my mentor, Dr. Dominic D’Agostino thank you for your relentless encouragement,
guidance, and always going above and beyond for me no matter what was going on around you.
You are one of the hardest working people I know. Thank you for always being just an email or
phone call away, and always working diligently through the night for the betterment of our lab,
my career, and my life. Thank you for instilling in me your passion and drive for helping others
through our research and for helping me to realize why our job as scientists is so important. To
my Co-mentor, Dr. Mack Wu, thank you for investing in me when I needed someone and for
your continuous direction and support. To Dr. Lisa Gould, thank you for believing in me as your
first Ph.D. student, for always being there even when we are far apart, for giving me an
opportunity to see the clinical aspect of our research, and especially for challenging me and
making me a better scientist.
To the members of my dissertation committee, Dr. Thomas Taylor-Clark, Dr. Paula
Bickford, Dr. Kenneth Ugen, Dr. Patrick Bradshaw for their scientific guidance and support over
the past five plus years. To my outside chair, Dr. Stanley Stevens Jr., thank you for stepping in
last minute and handling everything efficiently. I wish I had gotten to know you earlier in my
career, but am hopeful for a continued relationship in the future.
To my lab family, Carol Landon, Dr. Jay Dean, Geoffrey Ciarlone, Jacob Sherwood, Dr.
Christopher Rogers, Dr. Csilla Ari, Andrew Koutnik, and Nathan Ward, thank you for making
work such an entertaining but reassuring environment even when I needed to order something
last minute, we have had a midnight time point, or when I have forced you to listen to musicals. I
will be forever grateful for the time we have shared. To Dr. Andrea Trujillo for your continued
guidance and support, thank you for teaching me so much. I want to give a special recognition
and thank you to my wonderful friend and colleague, Dr. Angela Poff, from my whole heart I
wish to thank you for your unwavering love, support, and encouragement, none of this would
have been possible without you.
To my school family, Dr. Franklin Poff, Randi McCallian, Dr. Chase Lambert, and Dr.
Jillian Whelan, who have always been there with wine, excellent dinners, wine, relaxing
company, wine, ridiculous conversation, and wine to ease the stress of this journey. To Shelby
Evans and Marilyn Rodriguez, who always listened when I gushed about science even when they
didn’t understand and thought it was boring, thank you for being there whether near or far. To
Myshell (Michelle) Jung, thank you for being a source of constant inspiration and love even
when we didn’t get to see each other every day or get to braid hair during incubation times. To
my friends and family who have always loved and encouraged me throughout my life, Beth
Webster, Joshua Smith, Katie Rader, Chad Rader, Harper Rader, June Guy, Merle Guy, John
Fry, Michelle Adkins, Kyle Adkins, Travis Adkins, Amanda Fox, James Fox, Keith Waldron,
Jeanette Kesl, and Robert Kesl. To my Presley Rader for always being enthusiastic to learn about
science, watching your face light up when you looked into a microscope for the first time
restored my wonderment for science. To my hippo and chicken, thank you for your
unconditional love and for always being there to melt my stress away with snuggles on the
couch.
To my best friend, my heart, my husband, Jason, I know it has been hard being apart for
the majority of my dissertation work and prep, but know that I felt your unconditional love and
support even from Germany. Thank you for the countless times of reassurance, encouragement,
laughter, and inspiration. I look forward to many more years of our life together. Last but not
least to my mother, Jennifer Fry, thank you for always believing in me and pushing me to
achieve my dreams. None of this would have been possible without your continual words of
encouragement, your unconditional love, and your unwavering support no matter where life has
taken me or how long it has taken me to get there. I am forever grateful.
i
TABLE OF CONTENTS
List of Tables ................................................................................................................................. iv List of Figures ..................................................................................................................................v List of Abbreviations .................................................................................................................... vii Abstract ........................................................................................................................................ xiii Chapter 1: Wound Healing Physiology ...........................................................................................1
1.8.1. Classification of Chronic Wounds .............................................................18 1.8.2. Current Standard of Care, Therapies and Their Disadvantages .................20
1.8.2.1. TIME and Amputation .................................................................21 1.8.2.2. Adjunctive Therapies ...................................................................22
1.9. Age Influence on Chronic Wounds ........................................................................26 1.9.1. Aged Related Changes in Skin Morphology .............................................26 1.9.2. Age Related Changes in the Wound Healing Cascade ..............................27
1.10. Closing Remarks ....................................................................................................30 1.11. References for Chapter 1 .......................................................................................31
Chapter 2: Oral Ketone Supplementation as a Novel Therapy for Age-Dependent Delay Wound
2.2. Ketone Body Synthesis and Metabolism ...............................................................47 2.3. Review of Ketogenic Diet and Therapeutic Uses ..................................................48
2.3.1. Potential Concerns and Side Effects ..........................................................49 2.4. Exogenous Ketone Supplementation .....................................................................50
2.4.1. 1,3-Butanediol ............................................................................................51 2.4.2. MCT Oil .....................................................................................................52 2.4.3. βHB Salt .....................................................................................................54 2.4.4. Combination BMS+MCT ..........................................................................55 2.4.5. Ketone Esters .............................................................................................56 2.4.6. Potential Concerns and Side Effects: Ketoacidosis ...................................57
2.5. Potential Physiology Affected to Enhance Wound Healing ..................................58 2.5.1. Ketones and Inflammation .........................................................................59 2.5.2. Ketones and ROS .......................................................................................60 2.5.3. Ketones and Blood Flow and Angiogenesis ..............................................62 2.5.4. Ketones and Metabolism ............................................................................63 2.5.5. Ketones Suppress Blood Glucose via Increasing Insulin Sensitivity ........64
2.6. Ischemic Wound Healing Model ...........................................................................66 2.7. Use of Primary Human Dermal Fibroblasts in vitro ..............................................69 2.8. Central Hypothesis and Project Goals ...................................................................70 2.9. Closing Remarks ....................................................................................................71 2.10. References for Chapter 2 .......................................................................................72
Chapter 3: Establishing Therapeutic Ketosis (Metabolic Therapy) with Exogenous Ketone
Supplementation ..........................................................................................................86 3.1. Chapter Synopsis ...................................................................................................86 3.2. Effects of Exogenous Ketone Supplementation on Blood Ketones, Glucose,
Triglycerides, and Lipoprotein Levels in Sprague-Dawley Rats ...........................87 3.2.1. Ketone supplementation causes little to no change in triglycerides and
lipoproteins ................................................................................................88 3.2.2. Therapeutic levels of hyperketonemia suppress blood glucose levels .......90 3.2.3. Effects of ketone supplementation on organ weight and body weight
percentage ..................................................................................................92 3.3. Effect of Sustaining Dietary Ketosis Through Exogenous Ketone
Supplementation on the Hippocampal and Serum Metabolome of Sprague-Dawley Rats .........................................................................................................103 3.3.1. Oral administration of ketone supplements elevates blood ketone levels,
increases Krebs cycle intermediates, MCFAs, antioxidants, and adenosine in serum and hippocampal tissue and implications for wound healing ...103
3.4. References for Chapter 3 .....................................................................................113
Chapter 4: Enhancing Wound Healing with Metabolic Therapy ................................................123 4.1. Chapter Synopsis .................................................................................................123 4.2. Dietary Ketone Supplementation Increases Blood Flow and Wound Closure in an
Ischemic Wound Model in Young and Aged Fischer Rats ..................................124
iii
4.2.1. Aged rats metabolize exogenous ketone supplements differently than young rats .................................................................................................125
4.2.2. Hypoglycemic effect of hyperketonemia attenuated with age .................127 4.2.3. Ketone supplementation increases blood flow in both young and aged
rats ............................................................................................................128 4.2.4. Ketone supplementation accelerates wound closure in young and aged
rats ............................................................................................................129 4.2.5. Food- integrated ketone supplementation did not elicit weight loss in
young and aged rats .................................................................................131 4.3. Closing Remarks ..................................................................................................133 4.4. References for Chapter 4 .....................................................................................139
Chapter 5: Potential Mechanisms for Exogenous Ketone Supplementation to Enhance Wound
Healing .......................................................................................................................144 5.1. Chapter Synopsis .................................................................................................144 5.2. Potential Mechanisms of Action for Exogenous Ketone Enhancement of Ischemic
Wound Healing in Young and Aged Fisher Rats .................................................145 5.2.1. Effects of ketone supplementation on inflammation ...............................146 5.2.2. Effects of ketone supplementation on ROS production ...........................150 5.2.3. Effects of ketone supplementation on angiogenesis ................................152 5.2.4. Effects of ketone supplementation on metabolism ..................................153
5.3. Closing Remarks ..................................................................................................154 5.4. References for Chapter 5 .....................................................................................165
Chapter 6: Discussion: Implications for Wound Healing ............................................................172 6.1. Chapter Synopsis .................................................................................................172 6.2. Implications for Wound Healing and Future Directions ......................................172 6.3. References for Chapter 6 .....................................................................................176
Appendix A: Methods and Materials ...........................................................................................179 Appendix B: Copyright Permissions ...........................................................................................198 Appendix C: Published Manuscripts ...........................................................................................202 About the Author ............................................................................................................... End Page
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LIST OF TABLES
Table 3.1 Global metabolism profile of serum and hippocampal tissue following chronic
administration of KE and BMS+MCT .................................................................109 Table A.1 Caloric density of standard rodent chow and dose of ketone supplements .........181
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LIST OF FIGURES
Figure 3.1 Effects of ketone supplementation on triglycerides and lipoproteins ....................96 Figure 3.2 Effects of ketone supplementation on blood βHB .................................................... 97 Figure 3.3 Effects of ketone supplementation on blood glucose ................................................... 98 Figure 3.4 Relationship between blood ketone and glucose levels ............................................... 99 Figure 3.5 Effects of ketone supplementation on organ weight ..............................................100 Figure 3.6 Effects of ketone supplementation on body weight .................................................... 101 Figure 3.7 Effects of ketone supplementation on basal blood ketone and basal blood glucose
levels ....................................................................................................................102 Figure 3.8 Ketone supplementation elevates blood ketone levels in serum and hippocampal
tissues ...................................................................................................................110 Figure 3.9 Ketone supplementation increases medium chain fatty acids in serum and
hippocampal tissue ...............................................................................................110 Figure 3.10 Ketone supplementation increases Kreb’s cycle intermediates ...........................111 Figure 3.11 Ketone supplementation increases antioxidants ..................................................112 Figure 3.12 Ketone supplementation increase Adenosine ......................................................112 Figure 4.1 Effects of ketone supplementation on blood ketone and blood glucose levels ....134 Figure 4.2 Relationship between blood ketone and blood glucose levels attenuated with age ........................................................................................................................134 Figure 4.3 Ketone supplementation increases blood flow in both young and aged rats .......135 Figure 4.4 Ketone supplementation enhances wound closure time in young and aged rats .......................................................................................................................136
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Figure 4.5 Ketone supplementation elevates blood ketones levels but does not affect blood glucose levels .......................................................................................................137
Figure 4.6 Body and Spleen Weights ....................................................................................138 Figure 5.1 Heterogeneity of Aged Ischemic Wound Healing ...............................................155 Figure 5.2 Effects of ketone supplementation on pro-inflammatory and anti-inflammatory
cytokines ..............................................................................................................156 Figure 5.3 Effects of ketone supplementation on growth factors and leptin .........................157 Figure 5.4 Ketone supplementation does not affect M1 to M2 ratio of day 7 ischemic wounds
in aged rats ...........................................................................................................158 Figure 5.5 Ketone supplementation decreases ROS production ...........................................159 Figure 5.6 Ketone supplementation does not affect Antioxidants SOD2 and NQO1 in vitro
and ex vivo ............................................................................................................160 Figure 5.7 Ketone supplementation does not significantly increase blood vessel density in
day 7 aged ischemic wounds ................................................................................161 Figure 5.8 Ketone supplementation increases migration in young and aged HDFs .............162 Figure 5.9 Ketone supplementation increases proliferation in young and aged HDFs .........163 Figure 5.10 Ketone supplementation decreases lactate levels in young and aged HDFs .......164
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LIST OF ABBREVIATIONS
α-SMA ................................................................................................... Alpha-smooth muscle actin βHB ................................................................................................................ Beta-hydroxybutyrate AcAc ............................................................................................................................. Acetoacetate AD ..................................................................................................................... Alzheimer’s disease ADH .......................................................................................................... Aldehyde dehydrogenase ADP ............................................................................................................. Adenosine diphosphate ALS .................................................................................................... Amyotrophic lateral sclerosis AMP ....................................................................................................... Adenosine monophosphate ARE.......................................................................... Antioxidant or electrophilic response element ATP .............................................................................................................. Adenosine triphosphate BD ................................................................................................................... R, S-1, 3- Butanediol BMI ....................................................................................................................... Body Mass Index BMS ....................................................................................................................... Na+/K+ βHB Salt BMS+MCT ....................................................... Na+/K+ βHB Salt: Medium Chain Triglyceride Oil CAT...................................................................................................................................... Catalase CD31 ..................................................................................................... Cluster of differentiation 31 CD68 ..................................................................................................... Cluster of differentiation 68 CD206 ................................................................................................. Cluster of differentiation 206
viii
CMS .............................................................................. Centers for Medicare & Medicaid Services CNS ............................................................................................................ Central Nervous System CRP .................................................................................................................. Cysteine-rich protein CVD .............................................................................................................. Cardiovascular disease DHE ....................................................................................................................... Dihydroethidium DMEM .................................................................................... Dulbecco’s Modified Eagle Medium ECM .................................................................................................................. Extracellular matrix EGF .......................................................................................................... Endothelial growth factor EMT ........................................................................................................... Electromagnetic therapy eNOS ............................................................................................. Endothelial nitric oxide synthase ET-1 .............................................................................................................................. Endothelin-1 ETC ............................................................................................................. Electron transport chain FADH2 .................................................................................................. Flavin adenine dinucleotide FDA ........................................................................................... US Food and Drug Administration FGF ............................................................................................................. Fibroblast growth factor FOXO3A .............................................................................................................. Forkhead box O3a G6P .................................................................................................................. Glucose-6-phosphate G6PDH .................................................................................... Glucose-6-phosphate dehydrogenase GC/MS ............................................................................ Gas chromatography - mass spectrometry GI ............................................................................................................................. Gastrointestinal GLUT ................................................................................................................. Glucose transporter GNG ....................................................................................................................... Gluconeogenesis GSH .................................................................................................................. Reduced glutathione
damaged mitochondria, and decreased cellular proliferation [200].
It has been demonstrated that angiogenesis is decreased with age because of reduced
levels of the angiogenic factors: FGF, VEGF, and TGF-β, impaired vasodilation, proliferation
and migration of angiogenic, microvascular endothelial cells. It is important to note that the
coexistence of hypertension, diabetes, smoking, and hypercholesterolemia exacerbates the
inherent effects of aging that are detrimental to angiogenesis [176, 201].
The metabolic challenge of a wound is exacerbated in elderly patients, who have a higher
prevalence of malnutrition combined with reduced total energy expenditure, basal metabolic rate,
and protein synthesis [17]. Without appropriate energy (ATP) levels, wound healing is
significantly impaired. The ATP deficiency creates an imbalance of energy production versus
utilization. To compensate, anaerobic respiration increases in the wound bed, which only
produces 2 ATP per glucose molecule as compared to the potential 38 ATP made from normal
30
aerobic respiration [41]. Gupta et al. showed that key metabolic enzymes were decreased in aged
rats [51]. It has been illustrated that mitochondria become larger and less numerous with age,
mitochondrial respiratory chain (MRC) enzyme activities decrease as well as mitochondrial
membrane potential [202]. Furthermore, there is a significant reduction in mitochondrial
bioenergetic capacity with advancing age, which has been shown in numerous animal models
and recently in a study with human volunteers [202, 203].
1.10. Closing Remarks
In summary, there is come controversy regarding whether chronological age alone affects
wound healing. However, it is well documented that there is increased ROS production with age,
leading to cellular damage, reduced blood flow leading to the lessening of nutrient exchange,
decreased metabolic activity and ATP production at the wound bed, and unresolved chronic
inflammation. It is easily deduced how these factors may lead to increased prevalence of chronic
wounds in older adults. Most approaches to accelerate wound healing focus on delivery of
growth factors by topical application. The discouraging results of focusing on single molecular
targets are not surprising since wound healing is the product of a complex set of interactions
between numerous factors. It is here that we aim to switch the approach to optimizing the
body’s physiology to reach the wound bed systemically as well as targeting multiple facets of
impaired wound healing with one therapy- exogenous ketone supplementation, which will be
discussed further in the next chapter.
31
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CHAPTER 2: ORAL KETONE SUPPLEMENTATION AS A NOVEL THERAPEUTIC
FOR DELAYED WOUND HEALING
2.1. Chapter Synopsis
In the previous chapter, I provided a thorough overview of chronic wounds and the non-
efficacious therapies that are currently available, especially for the aged population. Here I
provide a rationalization for the use of an oral exogenous ketone supplement as a novel therapy
for wound healing. The ketogenic diet has been used since the 1920s for refractory epilepsy in
children. Though it has been proven to be effective, it is hard to maintain and is limited in a
clinical setting. Recently, it has been shown that ketone bodies themselves may confer
therapeutic benefits, paving the way for an exogenous ketone supplement that does not require
dietary restriction. Ketones have been shown to decrease inflammation, decrease ROS levels,
increase blood flow, and increase ATP hydrolysis and increase metabolism. As previously
described, these are the four underlying age-dependent features we will utilize to determine the
efficacy of our treatment. For these reasons, we hypothesize that exogenous ketone
supplementation will augment wound healing and we propose to test their effects in vivo using
an ischemic wound model in young and aged Fischer 344 rats and in vitro using patient-derived
primary human dermal fibroblasts isolated from discarded skin.
Portions of this chapter have previously been published in “Kesl SL, Poff AM, Ward NP,
47
Fiorelli TN, Ari C, Van Putten AJ, Sherwood JW, Arnold P, D’Agostino DP. Effects of
Exogenous Ketone Supplementation on Blood Ketone, Glucose, Triglyceride, and Lipoprotein
Levels in Sprague-Dawley Rats. Nutrition and Metabolism (2016)”, Poff AM, Kesl SL,
D’Agostino DP “Ketone Supplementation for Health and Disease” Book Chapter, Oxford Press
(Submitted), and “ Trujilio AN*, Kesl SL*, Sherwood J, Wu M, Gould LJ. (2014)
Demonstration of the Rat Ischemic Wound Model. Journal of Visualized Experiments: JoVE ”. *
Indicates that both authors contributed equally to the work. Copies of these articles may be found
in Appendix C. See Appendix B for copyright permissions. Materials and methods presented in
this chapter can be found in Appendix A.
2.2. KetoneBodySynthesisandMetabolism
Ketone bodies are naturally elevated to serve as alternative metabolic substrates for extra-
hepatic tissues during the prolonged reduction of glucose availability, suppression of insulin,
and depletion of liver glycogen, such as occurs during starvation, fasting, vigorous exercise,
calorie restriction, or the ketogenic diet (KD). As blood glucose levels drop, the body goes
through a metabolic shift from glucose-based metabolism towards fatty acid oxidation and
hepatic ketogenesis to generate ATP. As a result, hepatic cells synthesize necessary glucose
through gluconeogenesis (GNG) and β-oxidation metabolizes fats (dietary and stored) to form
Acetyl CoA. During GNG, the oxaloacetate stores are depleted, causing the Acetyl CoA from
fatty acid oxidation to accumulate and not enter the Krebs cycle. The excess Acetyl CoA
produces the ketone bodies beta-hydroxybutyrate (βHB) and acetoacetate (AcAc) via a 3-step
enzymatic process known as ketogenesis. From the liver, these substrates are transported
48
systemically to extra-hepatic tissues (i.e. muscle, brain, and skin) where Acetyl CoA is
reconstructed and used to generate ATP via the Krebs cycle and the ETC [1, 2]. Additionally,
AcAc can be decarboxylated to produce the third ketone body, acetone. Although acetone has
been considered mainly to be a metabolic by-product that is eliminated via the lungs and urine, it
has recently been shown to play an important role in the anticonvulsant properties of the
ketogenic diet [3-7].
2.3. Review of Ketogenic Diet and Therapeutic Uses
The classical ketogenic diet (KD) consists of a 4:1 ratio of fat to protein and carbohydrate
combined, with 80-90% of total calories derived from fat [8]. For most patients this equates to
eating less than 50 grams of carbohydrates and around 100 grams of protein a day [9]. Actual
carbohydrate and protein intake must be individually optimized to maintain therapeutic ketone
production. The macronutrient ratio of the KD induces the metabolic shift towards hepatic
ketogenesis, elevating AcAc and βHB in the blood. Compared to other low carb diets like the
Atkins diet, the ketogenic diet requires only adequate protein. Excess protein intake can fuel
GNG, resulting in glucose synthesis that disables ketogenesis. It is important to emphasize that
not all low carbohydrate diets are ketogenic. The ketogenic diet is really a high-fat diet, and not a
high-protein diet.
Emerging evidence supports the therapeutic potential of the ketogenic diet (KD) for a
variety of disease states, leading investigators to research methods of harnessing the benefits of
nutritional ketosis without the dietary restrictions. The KD has been used as an effective non-
pharmacological therapy for pediatric intractable seizures since the 1920s [10-12]. In a study of
49
150 epileptic children who averaged 400 seizures per month and were on multiple anti-epileptic
medications, 60% reported a significant decrease in seizure frequency [12]. In addition to
epilepsy, the ketogenic diet has elicited significant therapeutic effects for weight loss and type-2
diabetes (T2D) [13]. Several studies have shown significant weight loss on a high fat, low
carbohydrate diet without significant elevations of serum cholesterol [14-21]. Another study
demonstrated the safety and benefits of long-term application of the KD in T2D patients. Patients
exhibited significant weight loss, reduction of blood glucose, and improvement of lipid markers
after eating a well-formulated KD for 56 weeks [22]. Recently, researchers have begun to
investigate the use of the KD as a treatment for acne, polycystic ovary syndrome (PCOS),
Eric Verdin and colleagues have recently demonstrated βHB is an endogenous and
specific inhibitor of class 1 histone deacetylases (HDACs) [126]. Elevated βHB levels by fasting,
61
calorie restriction, and exogenous administration increased global histone acetylation in mouse
tissues and induced the transcription of genes encoding oxidative resistance factors FOXO3A
and MT2 via selective depletion of HDAC 1 and HDAC2. Furthermore, the authors
demonstrated in vivo that exogenous ketone supplementation could prevent oxidative stress.
Mice were pretreated with βHB via a subcutaneous pump for 24 hours prior to receiving an
injection of paraquat, which induces the production and accumulation of ROS. Protein
carbonylation was suppressed by 54%, lipid peroxidation was completely suppressed, and
endogenous antioxidants mitochondrial superoxide dismutase (MnSOD, SOD2) and catalase
(CAT) were elevated in the renal tissue of βHB pre-treated mice.
Regulated ROS levels are critical for optimal wound healing. Normal oxidative
phosphorylation, transient hypoxia, and increased neutrophil and macrophage respiratory bursts
release ROS during wound healing. The increased level of ROS transcends the beneficial effect
and causes additional tissue damage and healing delay. This unquenched ROS production is
prevalent in the aged population. Recently, Moor et al demonstrated age-dependent deficiencies
in the glutathione and SOD2 antioxidant pathways. In an ischemic wound model, wounds from
aged rats had lower SOD2 protein and activity, decreased ratio of reduced/oxidized glutathione,
and decreased glutathione peroxidase activity [127]. These age-exaggerated insufficiencies lead
to excessive inflammation and impaired wound healing.
One of the hallmarks of aging is mitochondrial dysfunction [128]. Electron microscope
images of aged mitochondria show disorganization and degeneration via absent cristae,
vacuolation, and swelling. Treatment with lipoic acid and carnitine stimulates repair of
mitochondrial membranes, returns functionality to the mitochondria, and demonstrates a young
phenotype [129]. Bough et al showed increased mitochondrial biogenesis in rats maintained on a
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KD for 4-6 weeks [130, 131]. Interestingly, exogenous ketone supplementation with a ketone
ester has been shown to induce mitochondrial biogenesis [132]. Mice in this study were fed a
diet from which approximately 30% of calories were derived from the D-β-hydroxybutyrate-R-
1,3-butanediol monoester for one month. The mitochondrial content and expression of electron
transport chain proteins were significantly increased in the intrascapular brown adipose tissue as
compared to control mice, although calorie intake was matched between the two groups. These
experiments support the hypothesis that ketone supplementation could have therapeutic benefits
for an aged population.
2.5.3. Ketones and Blood Flow and Angiogenesis
Restoration of blood flow via angiogenesis is critical for healing a wound, as well as the
integration of skin substitutes. Venous, arterial and diabetic blood flow insufficiencies are major
underlying contributors to chronic wound development. Additionally, most older patients have
hypertension or other comorbidities that affect blood flow. In a study by Hasselbalch and
colleagues, 8 volunteers (4 male, 4 female, 24±4 years of age, normal weight) were given an
infusion of Na+-βHB via antecubital vein at a rate of 4-5 mg/kg/min corresponding to an infusion
of ~350mL/hr for 3-3.5 hours. Global cerebral blood flow was measured via the Kety-Schmidt
technique; ketone supplementation showed a 39% increase in cerebral blood flow [133].
As noted in Chapter 1 of this dissertation, VEGF is the most potent angiogenic factor for
its effects on multiple components of the angiogenesis cascade: endothelial cell mitogenesis,
chemotaxis, and induction of vascular permeability [134, 135]. In addition to VEGF, endothelin-
1 (ET-1) is another cytokine produced in the response to hypoxia and is a crucial promoter of
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cell proliferation, migration and chemotaxis, and angiogenesis in wound healing [136-138]. A
study by Isales and colleagues demonstrated that supplementation of mouse brain microvascular
endothelial cells with βHB and AcAc both independently increased ET-1 and had an added affect
when applied together. βHB but not AcAc increased VEGF levels [139]. Though this area needs
further exploration, this may be one mechanism that oral ketone supplementation may augment
wound healing.
2.5.4. Ketones and Metabolism
In the 1940s Henry Lardy demonstrated that βHB and AcAc had distinct energetic
efficiencies compared to 16 major carbohydrate, lipid, and intermediary metabolites in their
ability to increase bull sperm mobility while simultaneously decreasing oxygen consumption.
[140, 141]. Nearly 50 years later, Richard Veech and colleagues confirmed that ketone bodies
increased metabolic efficiency and elucidated the molecular mechanisms in the working perfused
rat heart [124, 142]. They demonstrated that supplementation of glucose-containing perfusate (10
mM glucose) with 5 mM ketones (4 mM βHB, 1 mM AcAc) increased cardiac hydraulic work
by approximately 25% while simultaneously reducing oxygen consumption [124]. Their study
demonstrated the ketone-mediated increase in metabolic efficiency was facilitated by a reduction
of the mitochondrial NAD couple and an oxidation of the coenzyme Q couple increasing the
energy span between the sites (mentioned previously to decrease ROS production). This results
in an increase in energy released by electrons in the ETC, causing more protons to be pumped
into the inner mitochondrial space. The electrochemical gradient is enhanced, hyperpolarizing
the cell, and increasing the ΔG0 (free enthalpy) of ATP hydrolysis; thus, increasing metabolic
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efficiency. Additionally, ketone bodies were shown to cause a 16-fold elevation in acetyl-CoA
content and increased Krebs cycle intermediates. Furthermore, thermodynamic tables for heat of
combustion, calculated with bomb calorimeter experiments, show that βHB produces more
energy than glucose per carbon molecule [143].
Decreased availability of ATP negatively impacts nearly every aspect of the healing
process [144]. Decreased nutrient and oxygen exchange during wound healing limits ATP
production to fuel wound healing processes. Additionally, the skin predominantly uses anaerobic
respiration, which decreases the potential ATP production by 90% compared to aerobic
respiration [145-147]. Chiang and colleagues developed a new intracellular ATP delivery
technique in which highly fusogenic lipid vesicles (ATP-vesicles) are used to encapsulate
magnesium-ATP (Mg-ATP). When the vesicles come into contact with the cell membrane, they
fuse together and deliver the contents into the cytosol. Using eleven controlled-pairs of adult
nude mice, they demonstrated accelerated wound healing via enhanced development of
granulation tissue, more rapid re-epithelialization, and elevated VEGF levels [31, 63].
2.5.5. Ketones Suppress Blood Glucose via Increasing Insulin Sensitivity
Failure to heal in diabetic lower limbs is associated with hyperglycemia, insulin
resistance, tissue hypoxia, chronic inflammation, oxidative stress, impairment of the immune
system, and metabolic dysfunction [148, 149]. A study by Rubinstein et al. showed that once
diabetes was a controlled, diabetic foot ulcers in 11 of 15 patients healed in 4 to 13 weeks [150].
In addition, diabetic patients with glucose under 200 mg/dl showed a decrease in surgical site
infections after foot and ankle surgery [151]. Recent studies suggest that many of the benefits of
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the KD are due to the effects of ketone body metabolism. Interestingly, in studies on T2D
patients, improved glycemic control, improved lipid markers, and retraction of insulin and other
medications occurred before weight loss became significant, suggesting physiological effects of
ketone metabolism [3, 63]. Exogenous ketone supplements may provide therapeutic benefits for
the underlying hyperglycemia and insulin resistance as reports have demonstrated that oral
ketone administration lowers blood glucose by increasing insulin sensitivity. This could be
critical for older adults as all have demonstrable insulin resistance, even if not diabetic. Richard
Veech has suggested this hypoglycemic effect is the result of ketones activating pyruvate
dehydrogenase (PDH), which enhances insulin-mediated glucose uptake and the production of
acetyl-CoA. In addition, the body regulates ketone production via ketonuria and ketone-induced
insulin release, which shuts off hepatic ketogenesis. The insulin from this process may increase
glucose disposal which, when coupled with PDH activation, could drive down blood glucose
levels. The administration of 5 mM ketone has been shown to increase acetyl-CoA production
16-fold in the glucose-perfused isolated rat heart. Additionally, in this model, ketones and insulin
increased cardiac hydraulic efficiency to a similar degree, approximately 25-35% [124].
Male rats were fed a standard diet with 30% of calories replaced with the R-3-
hydroxybutyrate-R-1,3-butanediol monoester for 14 days. The ketone ester-supplemented diet
induced nutritional ketosis (3.5mM βHB), and both plasma glucose and insulin were decreased
by approximately 50% [132]. Glucose was decreased from 5 mM to 2.8 mM, and insulin was
decreased from 0.54 ng/mL to 0.26 ng/mL. In a similar study by the same group, mice receiving
a KE diet exhibited a 73% increase in the Quantitative Insulin-Sensitivity Check Index
(QUICKI), a surrogate marker of insulin sensitivity, compared to control, calorie-matched mice
[132]. Fasting plasma glucose levels were not altered in these mice, but fasting plasma insulin
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levels were reduced by approximately 85% in the KE-fed mice compared to controls,
demonstrating that exogenous ketones enhance insulin sensitivity [152].
The histone deacetylase inhibitor (HDACI) activity of βHB could also be beneficial in
T2D by altering the direct regulation of HDAC-dependent glucose metabolism and by inducing
resistance to oxidative stress. HDACs regulate the expression of genes encoding many metabolic
enzymes, and HDAC3 knockout animals exhibit reduced glucose and insulin. SAHA, a class I
HDAC inhibitor, has been shown to improve insulin sensitivity and increase oxidative
metabolism and metabolic rate in a mouse model of diabetes [153]. Butyrate, a short-chain fatty
acid that is structurally similar to β-hydroxybutyrate and also acts as a HDACI lowers blood
glucose and insulin levels and improves glucose tolerance and respiratory efficiency [154]. The
vascular dysfunction in T2D is thought to be caused by oxidative stress [155]. HDAC inhibition
prevents renal damage in mouse models of diabetic nephropathy through modulation of redox
mechanisms [156]. Therefore, βHB suppression of oxidative stress through HDAC inhibition
may help restore insulin sensitivity and manage complications of diabetes.
2.6. Ischemic Wound Healing Model
As mentioned previously, a chronic wound is by definition one that entirely fails to heal.
In this respect, there is no standardized animal model that reflects the human chronic wound
environment. However, animal models have been developed that attempt to mimic these
conditions for the purpose of furthering our understanding of the complexity of chronic wounds.
The rat species, often employed due to its wide availability, size and docile nature is used for
wound healing studies as it is large enough to provide a suitable skin area for incisional and
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excisional wounding, imaging and tissue collection [157]. Yet, it should be noted that the skin of
a rat and a human are different morphologically, as rats are loose-skinned animals. This distinct
anatomical characteristic enhances wound contraction over re-epithelialization in rat integument
[157]. Additionally, the presence of a subcutaneous panniculus carnosus muscle in rats,
contributes to healing by both contraction and collagen formation [158, 159]. These very
important morphological distinctions were considered in the optimization of the following rat
ischemic skin wound model used for this dissertation. Additionally, specific modifications were
implemented to decrease wound contraction and reduce the influence of the panniculus carnosus
muscle [160].
In the rat ischemic wound model, a dorsal bi-pedicle 10.5 x 3 cm flap is surgically
created on day 0. A silicone sheet is placed under the panniculus carnosus fascia and above the
paraspinous muscles, limiting revascularization from the underlying tissue, which increases the
duration of flap ischemia. Two 6mm punches are created within the flap, extending just above
the fascia, creating ischemic wounds. Additionally, two 6 mm punches are created lateral to the
flap, where there isn’t silicone sheet, providing control non-ischemic wounds within the same
animal.
One of the main factors in the development of a chronic wound is localized tissue
ischemia (reduced blood flow) contributing to the inability to clear inflammation [160]. In 2005,
Dr. Lisa Gould developed and validated this model as a modification of the ischemic wound
model originally described by Schwartz et al. and subsequently used in modified form by Chen
et al. [161, 162]. The wound model was modified to test the induction of angiogenesis in the
wound bed.
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Wound healing in rats has often been the subject of debate due to their ability to heal
infected wounds and high rate of inter-animal variability [160]. One of the original goals of the
model during its development was to decrease this variation. Modifications to the width of the
flap, reducing the number of wounds with specific placement (centered on the flap with
consistent cranio-caudal location) and introduction of a silicone sheet has accomplished this
goal. Wound healing by contraction has also been reduced and healing by epithelialization, as in
humans, is the measured outcome. Adaptation of the model to a different strain of rat, ie the
F344, has also proven successful and reproduces the degree of ischemia observed using Sprague
Dawley rats.
To achieve consistency with this model while performing multiple surgeries, it was found
that it is important to create the ischemic wounds prior to elevation of the flap for silicone sheet
placement [160]. Additionally, not punching through the panniculus carnosus fascia is critical to
provide a viable wound bed to remain over the silicone. The silicone acts not only to prevent
vascular regrowth but also as a “splint” that reduces wound contraction. The application of the
adhesive and dressings to prevent infection and maintain a moist environment for wound healing
is also important. Product choice can be what is preferred or used in the researcher’s animal
facility. However, it is not uncommon for some of the animals to be able to remove their
dressings, no matter what type of adhesive/dressing combination is used.
The bi-pedicled flap should remain viable throughout a time course of healing which is
approximately 28 days, depending on rat strain and other co-morbidities present. Rarely,
abscesses can form in the flap (particularly near sutures) and seromas may form under the flap.
Fluid can be drained and antibiotics administered if necessary. However, if the flap loses
viability and becomes necrotic it is recommended that that animal no longer be used. Wound
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excision for biochemical analysis does introduce variability due to (1) some normal tissue must
be retained for support (2) the choice of tissue homogenization and preparation for isolation of
RNA, DNA or protein and (3) inherent inter-animal variability [127, 160, 163]. One could
consider this last point a limitation to the model. It was found that reducing the size of the flap
(<2.0 cm) or flap trauma can cause necrosis, indicating that minor variations in technical or
environmental factors such as temperature or stress levels, may also lead to biochemically
detectable variation between wound samples from one rat to another [160].
In summary, this model, with a longitudinal, bi-pedicle flap ranging from 2.0-3.0 cm in
width and a strategically placed silicone sheet, is a reliable model of prolonged tissue ischemia.
Once the user is adept at using the techniques to create a consistent ischemic wound, they should
be able to adapt it to additional ages and species of rodents (mice included). The excisional
wounds can be treated topically, or systemic treatments utilized to further explore the
mechanism(s) involved in chronic wound formation, exacerbated inflammatory responses,
aberrant angiogenesis and delayed wound closure.
2.7. Use of Primary Human Dermal Fibroblasts in vitro
Though immortal cells lines are more abundant and more cost efficient to maintain, in this
dissertation, we utilized primary human dermal fibroblasts isolated from discarded skin.
Compared to immortal cell lines, primary cell lines maintain a physiology that is more closely
identified to in vivo physiology. The caveat is that they must be used at early passage, generally
passage 2-8. Additionally, due to the heterogeneity of patients, the in vitro work was repeated in
at least two different patient-derived cell lines thereby reducing individual confounding. Also,
70
fibroblasts behave differently depending on the location from which they are isolated; therefore,
skin for these studies was collected from similar anatomic locations. The complex extracellular
environment, paracrine, and autocrine interactions and range of cell types involved in wound
repair limits in vitro models to confirm what we see in in vivo models. Additionally, since there
is such a gamut of players and interactions, by focusing on just one cell type we may miss some
of the potential mechanism for our therapy.
2.8. Central Hypothesis and Project Goals
There is a dire need to discover novel and effective treatments for chronic wounds. There
are often multiple mitigating factors that prevent normal wound closure leading to minimally
effective wound therapies as few multifactorial treatments are available.
Exogenous ketone supplements are being developed as an alternative or adjuvant method
of inducing therapeutic ketosis aside from the classic ketogenic diet. It seems clear that these
novel compounds have the potential to offer benefits for both healthy and diseased individuals
alike. It is likely that most, if not all, of the conditions which are known to benefit from the KD
would receive some benefit from exogenous ketone supplementation. Importantly, ketone
supplementation provides a tool for achieving therapeutic ketosis in patients who are unable,
unwilling, or uninterested in consuming a low carbohydrate or ketogenic diet. It may also help
circumvent some of the difficulties associated with KD therapy, as it allows for rapid induction
of ketosis in a dose-dependent fashion, which can be sustained with prolonged consumption.
Simultaneously, it could provide patients with the opportunity to reap the benefits of ketosis
without the practical and social difficulties of a highly restrictive diet.
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Therefore, we hypothesize that exogenous ketone supplementation will improve metabolic
and physiological attributes to promote accelerated healing in the impaired wound environment
associated with aging.. To date, the majority of ketone-based metabolic enhancement strategies
have focused on enhancing brain and heart metabolism; thus, this dissertation proposes a novel
therapy, which will be evaluated in a cutaneous wound-healing model [164, 165]. Although this
dissertation focuses on the underlying features of the aging phenotype, similar molecular deficits
have been described in diabetes and other disorders [166], suggesting that administration of
ketones as an alternative fuel may have broader applications. The studies completed in this
dissertation provide an important step in the potential discovery of effective and novel treatments
for chronic wounds.
2.9. Closing Remarks
In this chapter I have provided scientific rationale for the potential utility of oral ketone
supplementation against age impaired wound healing. Approaching the healing wound from a
metabolic perspective provides an innovative opportunity to stimulate beneficial physiological
and cellular mechanisms that may be lacking or attenuated in the aged population. Chapters 3-5
will consist of the results and a detailed discussion of this dissertation work. A summary of the
findings, closing remarks, and major implications of this data will be discussed in Chapter 6.
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without significantly affecting heart health biomarkers. Additionally we sent serum and
hippocampal samples from KE (5 g/kg) and BMS+MCT (10 g/kg) supplemented rats to
Metabolon Inc. to determine the effects that oral ketone supplementation would have on the
metabolome. Ketone supplements increased Kreb’s cycle intermediates, antioxidants, and
adenosine, which supports our hypothesis that oral ketone supplementation, will enhance wound
healing.
Portions of this chapter have previously been published in “Kesl SL, Poff AM, Ward NP,
Fiorelli TN, Ari C, Van Putten AJ, Sherwood JW, Arnold P, D’Agostino DP. Effects of
Exogenous Ketone Supplementation on Blood Ketone, Glucose, Triglyceride, and Lipoprotein
Levels in Sprague-Dawley Rats. Nutrition and Metabolism (2016)”. A copy of this article may
be found in Appendix C. See Appendix B for copyright permissions. Materials and methods
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presented in this chapter can be found in Appendix A.
3.2. Effects of Exogenous Ketone Supplementation on Blood Ketones, Glucose,
Triglyceride, and Lipoprotein Levels in Sprague-Dawley Rats
Nutritional ketosis induced with the KD has proven effective for the metabolic
management of seizures and potentially other disorders [1-26]. We hypothesized that oral
administration of ketone supplements could safely produce sustained nutritional ketosis (>0.5
mM) without carbohydrate restriction. Thus, we tested the effects of 28-day administration of
five ketone supplements on blood glucose, ketones, and lipids in male Sprague-Dawley rats. The
supplements included: 1,3-butanediol (BD), a sodium/potassium β-hydroxybutyrate (βHB)
mineral salt (BMS), medium chain triglyceride oil (MCT), BMS+MCT 1:1 mixture, and 1,3
butanediol acetoacetate diester (KE). Rats received a daily 5-10g/kg dose of their respective
ketone supplement via intragastric gavage. Weekly whole blood samples were taken for analysis
of glucose and βHB at baseline and 0.5, 1, 4, 8, and 12 hrs post-gavage, or until βHB returned to
baseline. In this section, we present evidence that chronic administration of ketone supplements
can induce a state of nutritional ketosis without the need for dietary carbohydrate restriction with
little or no effect on lipid biomarkers. The notion that we can produce the therapeutic effects of
the KD with exogenous ketone supplementation is supported by our previous study which
demonstrated that acutely administered KE supplementation delays central nervous system
(CNS) oxygen toxicity seizures without the need for dietary restriction [27]. We propose that
exogenous ketone supplementation could provide an alternative method of attaining the
therapeutic benefits of nutritional ketosis, and as a means to further augment the therapeutic
potential of the KD.
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3.2.1 Ketone supplementation causes little to no change in triglycerides and lipoproteins
One common concern regarding the KD is its purported potential to increase the risk of
atherosclerosis by elevating blood cholesterol and triglyceride levels [28, 29]. This topic
remains controversial as some, but not all studies have demonstrated that the KD elevates blood
levels of cholesterol and triglycerides [30-35]. Kwitervich and colleagues demonstrated an
increase in low-density lipoprotein (LDL) and a decrease in high-density lipoprotein (HDL) in
epileptic children fed the classical KD for two years [36]. In that study, total cholesterol
increased by ~130%, and stabilized at the elevated level over the 2-year period. A similar study
demonstrated that the lipid profile returned to baseline in children who remained on the KD for
six years [37]. Children typically remain on the diet for approximately two years then return to a
diet of common fat and carbohydrate ingestion [38]. The implications of these findings are
unclear, since the influence of cholesterol on cardiovascular health is controversial and
macronutrient sources of the diet vary per study. In contrast, more recent studies suggest that the
KD can actually lead to significant benefits in biomarkers of metabolic health, including blood
lipid profiles [39-46]. In these studies, the KD positively altered blood lipids, decreasing total
triglycerides and cholesterol while increasing the ratio of HDL to LDL [42-51]. Although, the
KD is well established in children, it has only recently been utilized as a strategy to control
seizures in adults. In 2014, Schoeler and colleagues reported on the feasibility of the KD for
adults, concluding that 39% of individuals achieved > 50% reduction in seizure frequency,
similar to the results reported in pediatric studies. Patients experienced similar gastrointestinal
adverse events to those previously described in pediatric patients, but they did not lead to
discontinuation of the diet in any patient [52].
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With oral ketone supplementation, we observed a significant elevation in blood βHB
without dietary restriction and with little change in lipid biomarkers (Figure 3.1). Over the 4
week study, MCT-supplemented rats demonstrated decreased HDL compared to controls. No
significant changes were observed in any of the triglycerides or lipoproteins (HDL, LDL) with
any of the remaining exogenously applied ketone supplements. It should be noted that the rats
used for this study had not yet reached full adult body size [53]. Their normal growth rate and
maturation was likely responsible for the changes in triglyceride and lipoprotein levels observed
in the control animals over the 4 week study (baseline data not shown, no significant differences)
[54, 55]. Future studies are needed to investigate the effect of ketone supplementation on fully
mature and aged animals. Overall, our study suggests that oral ketone supplementation has little
effect on the triglyceride or lipoprotein profile after 4 weeks. However, it is currently unknown if
ketone supplementation would affect lipid biomarkers after a longer duration of consumption.
Further studies are needed to determine the effects of ketone supplements on blood triglyceride
and lipoproteins after chronic administration and as a means to further enhance the
hyperketonemia and improve the lipid profile of the clinically implemented (4:1) KD.
LDL is the lipoprotein particle that is most often associated with atherosclerosis. LDL
particles exist in different sizes: large molecules (Pattern A) or small molecules (Pattern
B). Recent studies have investigated the importance of LDL-particle type and size rather than
total concentration as being the source for cardiovascular risk [29]. Patients whose LDL particles
are predominantly small and dense (Pattern B) have a greater risk of cardiovascular disease
(CVD). It is thought that small, dense LDL particles are more able to penetrate the endothelium
and cause in damage and inflammation [56-59]. Volek et al. reported that the KD increased the
pattern and volume of LDL particles, which is considered to reduce cardiovascular risk [47].
90
Though we did not show a significant effect on LDL levels for ketone supplements, future
chronic feeding studies will investigate the effects of ketone supplementation on lipidomic
profile and LDL particle type and size.
3.2.2. Therapeutic levels of hyperketonemia suppress blood glucose levels
We demonstrated that therapeutic ketosis could be induced without dietary (calorie or
carbohydrate) restriction and that this acute elevation in blood ketones was significantly
correlated with a reduction in blood glucose (Figure 3.2-3.4). The BMS ketone supplement did
not significantly induce blood hyperketonemia or reduce glucose in the rats. The KE
supplemented rats trended towards reduced glucose levels; however, the lower dose of this agent
did not lower glucose significantly, as reported previously in acute response of mice (60). MCTs
have previously been shown to elicit a slight hypoglycemic effect by enhancing glucose
utilization in both diabetic and non-diabetic patients [60-62]. Kashiwaya et al. demonstrated that
both blood glucose and blood insulin decreased by approximately 50% in rats fed a diet where
30% of calories from starch were replaced with ketone esters for 14 days, suggesting that ketone
supplementation increases insulin sensitivity or reduces hepatic glucose output [63]. This
ketone-induced hypoglycemic effect has been previously reported in humans with IV infusions
of ketone bodies [64, 65]. Recently, Mikkelsen et al. showed that a small increase in βHB
concentration decreases glucose production by 14% in post-absorptive healthy males [66].
However, this has not been previously reported with any of the oral exogenous ketone
supplements we studied. Ketones are an efficient and sufficient energy substrate for the brain,
and will therefore prevent side effects of hypoglycemia when blood levels are elevated and the
91
patient is keto-adapted. Owen et al. most famously demonstrated this in 1967 wherein keto-
adapted patients (starvation induced therapeutic ketosis) were given 20 IU of insulin. The blood
glucose of fasted patients dropped to 1-2mM, but they exhibited no hypoglycemic symptoms due
to brain utilization of ketones for energy [67]. Therefore, ketones maintain brain metabolism and
are neuroprotective during severe hypoglycemia. The rats in the MCT group had a correlation of
blood ketone and glucose levels at week 4, whereas the combination of BMS+MCT produced a
significant hypoglycemic correlation both at baseline and at week 4. No hypoglycemic
symptoms were observed in the rats during this study. Insulin levels were not measured in this
study; however, future ketone supplementation studies should measure the effects of exogenous
ketones on insulin sensitivity with a glucose tolerance test. An increase in insulin sensitivity in
combination with our observed hypoglycemic effect has potential therapy implications for
glycemic control in T2D [68]. Furthermore, it should be noted that the KE metabolizes to both
AcAc and βHB in 1:1 ratio [27]. The ketone monitor used in this study only measures βHB as
levels of AcAc are more difficult to measure due to spontaneous decarboxylation to acetone;
therefore, the total ketone levels (βHB +AcAc) measured were likely higher, specifically for the
KE (14). Interestingly, the 10 g/kg dose produced a delayed blood βHB peak for ketone
supplements MCT and BMS+MCT. The higher dose of the ketogenic supplements elevated
blood levels more substantially, and thus reached their maximum blood concentration later due
to prolonged metabolic clearance. It must be noted that the dosage used in this study does not
translate to human patients, since the metabolic rate of rats is considerably higher. Future studies
will be needed to determine optimal dosing for human patients.
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3.2.3. Effects of ketone supplementation on organ weight and body weight percentage
Ketone supplementation did not affect the size of the brain, lungs, kidneys or heart of
rats. As previously mentioned, the rats were still growing during the experimental time frame;
therefore, organ weights were normalized to body weight to determine if organ weight changed
independently to growth. There could be several reasons why ketones influenced liver and spleen
weight. The ratio of liver to body weight was significantly higher in the MCT supplemented
animals (Figure 3.5). MCTs are readily absorbed in the intestinal lumen and transported directly
to the liver via hepatic portal circulation. When given a large bolus, such as in this study, the
amount of MCTs in the liver will likely exceed the β-oxidation rate, causing the MCTs to be
deposited in the liver as fat droplets [69]. The accumulated MCT droplets in the liver could
explain the higher liver weight to body weight percentage observed with MCT supplemented
rats. Future toxicology and histological studies will be needed to determine the cause of the
observed hepatomegaly. It should be emphasized that the dose in this study is not optimized in
humans. We speculate that an optimized human dose would be lower due to the higher metabolic
rate of the rats and may not cause hepatomegaly or potential fat accumulation. Nutritional ketosis
achieved with the KD has been shown to decrease inflammatory markers such as TNF-α, IL-6,
IL-8, MCP-1, E-selectin, I-CAM, and PAI-1 [8, 70], which may account for the observed
decrease in spleen weight. As previously mentioned, Veech and colleagues demonstrated that
exogenous supplementation of 5mM βHB resulted in a 28% increase in hydraulic work in the
working perfused rat heart and a significant decrease in oxygen consumption [71-73]. Ketone
bodies have been shown to increase cerebral blood flow and perfusion [74]. Also, ketone bodies
have been shown to increase ATP synthesis and enhance the efficiency of ATP production [14,
68, 73]. It is possible that sustained ketosis results in enhanced cardiac efficiency and O2
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consumption. Even though the size of the heart did not change for any of the ketone
supplements, further analysis of tissues harvested from the ketone-supplemented rats will be
needed to determine any morphological changes and to understand changes in organ size. It
should be noted that the Harlan standard rodent chow 2018 is nutritionally complete and
formulated with high-quality ingredients to optimize gestation, lactation, growth, and overall
health of the animals. The same cannot be said for the standard American diet (SAD). Therefore,
we plan to investigate the effects of ketone supplements administered with the SAD to determine
if similar effects will be seen when the micronutrient deficiencies and macronutrient profile
mimics what most Americans consume.
MCT oil has recently been used to induce nutritional ketosis although it produces dose-
dependent gastrointestinal (GI) side effects in humans that limit the potential for its use to
significantly elevate ketones (>0.5 mM). Despite these limitations, Azzam and colleagues
published a case report in which a 43-year-old-man had a significant decrease in seizure
frequency after supplementing his diet with 4 tablespoons of MCT oil twice daily [75]. An
attempt to increase his dosage to 5 tablespoons twice daily was halted by severe GI intolerance.
Henderson et al. observed that 20% of patients reported GI side effects with a 20g dose of
ketogenic agent AC-1202 in a double blind trial in mild to moderate Alzheimer’s patients [76].
We visually observed similar gastrointestinal side effects (loose stools) in the rats treated with
MCT oil in our study. Rats were closely monitored to avoid dehydration, and gastric motility
returned to normal between 12-24 hrs. Interestingly, the BMS+MCT supplement elevated βHB
similarly to MCT oil alone, without causing the adverse gastrointestinal effects seen in MCT-
supplemented rats. However, this could be due to the fact in a 10 g/kg dose of BMS+MCT, only
5 g/kg is MCT alone, which is less than the 10 g/kg dose that elicits the GI side effects. This
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suggests that this novel combination may provide a more useful therapeutic option than MCT oil
alone, which is limited in its ability to elevate ketones in humans.
Exogenously delivered ketone supplements significantly altered rat weight gain for the
duration of the study (Figure 3.6). However, rats did not lose weight and maintained a healthy
range for their age. Rats have been shown to effectively balance their caloric intake to prevent weight
loss/gain [77-79]. Due to the caloric density of the exogenous ketone supplements (Table 3.1) it is
possible for the rats to eat less of the standard rodent chow and therefore fewer carbohydrates while
maintaining their caloric intake. Food intake was not measured for this study. However, if there
was a significant carbohydrate restriction there would be a significant change in basal blood
ketone and blood glucose levels. As the hallmark to the KD, carbohydrate restriction increases
blood ketone levels and reduces blood glucose levels. Neither an increase in basal blood ketone
levels nor a decrease in basal blood glucose levels was observed in this study (Figure 3.7).
Additionally, if there were an overall blood glucose decrease due to a change in food intake, this
would not explain the rapid reduction (within 30 minutes) in blood glucose correlated with an
elevation of blood ketone levels after an intragastric bolus of ketone supplement (Figures 3.2-
3.4).
Several studies have investigated the safety and efficacy of ketone supplements for disease
states such as AD and Parkinson’s disease, and well as for parenteral nutrition [68, 80-86]. Our
research demonstrates that several forms of dietary ketone supplementation can effectively
elevate blood ketone levels and achieve therapeutic nutritional ketosis without the need for
dietary carbohydrate restriction. We also demonstrated that ketosis achieved with exogenous
ketone supplementation can reduce blood glucose, and this is inversely associated with the blood
ketone levels. Although preliminary results are encouraging, further studies are needed to
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determine if oral ketone supplementation can produce the same therapeutic benefits as the classic
KD in the broad-spectrum of disease states that are currently under investigation. Ketone
supplementation could be used as an alternative method for inducing ketosis in patients who
have previously had difficulty implementing the KD because of palatability issues, gall bladder
removal, liver abnormalities, or intolerance to fat. Additional experiments should be conducted
to see if ketone supplementation could be used in conjunction with the KD to assist and ease the
transition to nutritional ketosis and enhance the speed of keto-adaptation. In this study we have
demonstrated the ability of several ketone supplements to elevate blood ketone levels, providing
multiple options to induce therapeutic ketosis based on patient need. Though additional studies
are needed to determine the therapeutic potential of ketone supplementation, many patients that
previously were unable to benefit from the KD may now have an alternate method of achieving
therapeutic ketosis. Ketone supplementation may also represent a means to further augment
ketonemia in those responsive to therapeutic ketosis, especially in those individuals where
maintaining low glucose is important.
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Figure 3.1 Effects of ketone supplementation on triglycerides and lipoproteins: Ketone supplementation causes little change in triglycerides and lipoproteins over a 4-week study. Graphs show concentrations at 4-weeks of total cholesterol (A), Triglycerides (B), HDL (C), and LDL (D). MCT supplemented rats had significantly reduced concentration of HDL blood levels compared to control (p<0.001)(B). One-Way ANOVA with Tukey’s post hoc test, results considered significant if p<0.05. Error bars represent mean (SD).
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Figure 3.2 Effects of ketone supplementation on blood βHB. (A, B) Blood βHB levels at times 0, 0.5, 1, 4, 8, and 12 hours post intragastric gavage for ketone supplements tested. (A) BMS+MCT and MCT supplementation rapidly elevated and sustained significant βHB elevation compared to controls for the duration of the 4-week dose escalation study. BMS did not significantly elevate βHB at any time point tested compared to controls. (B) BD and KE supplements, maintained at 5 g/kg, significantly elevated βHB levels for the duration of the 4-week study. Two-Way ANOVA with Tukey's post hoc test, results considered significant if p<0.05. Error bars represent mean (SD).
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Figure 3.3 Effects of ketone supplementation on blood glucose. (A, B) Blood glucose levels at times 0, 0.5, 1, 4, 8, and 12 hours (for 10 dose) post intragastric gavage for ketone supplements tested. (A) Ketone supplements BMS+MCT and MCT significantly reduced blood glucose levels compared to controls for the duration of the 4-week study. BMS significantly lowered blood glucose only at 8 hrs/week 1 and 12hrs/week (B) KE, maintained at 5 g/kg, significantly reduced blood glucose compared to controls from week 1-4. BD did not significantly affect blood glucose levels at any time point during the 4-week study. Two-Way ANOVA with Tukey's post hoc test, results considered significant if p<0.05. Error bars represent mean (SD)
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Figure 3.4 Relationship between blood ketone and glucose levels: (A) BMS+MCT (5 g/kg) supplemented rats demonstrated a significant inverse relationship between elevated blood ketone levels and decreased blood ketone levels (r2=0.4314, p=0.0203). (B) At week 4, BMS+MCT (10 g/kg) and MCT (10 g/kg) showed a significant correlation between blood ketone levels and blood glucose levels (r2=0.8619, p<0.0001; r2=0.6365, p=0.0057). Linear regression analysis, results considered significant if p<0.05.
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Figure 3.5 Effects of ketone supplementation on organ weight Data is represented as a percentage of organ weight to body weight. (A, B, D, F) Ketone supplements did not significantly affect the weight of the brain, lungs, kidneys or heart. (C) Liver weight was significantly increased as compared to body weight in response to administered MCT ketone supplement compared to control at the end of the study (day 29) (p<0.001). (E) Rats supplemented with BMS+MCT, MC and BD had significantly smaller spleen percentage as compared to controls (p<0.05, p<0.001, p<0.05). Two-Way ANOVA with Tukey's post-hoc test; results considered significant if p<0.05. Error bars represent mean (SD).
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Figure 3.6 Effects of ketone supplementation on body weight: Rats administered ketone supplements gained less weight over the 4-week period; however, did not lose weight and maintained healthy range for age. KE supplemented rats gained significantly less weight during the entire 4-week study compared to controls. BMS+MCT, BMS, and BD supplemented rats gained significantly less weight than controls over weeks 2-4.MCT supplemented rats gained significantly less weight than controls over weeks 3-4. Two-Way ANOVA with Tukey's post hoc test, results considered significant if p<0.05. Error bars represent mean (SD).
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Figure 3.7 Effects of ketone supplementation on basal blood ketone and basal blood glucose levels: Rats administered ketone supplements did not have a significant change in basal blood ketone levels (A) or basal blood glucose levels (B) for the four week study. Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05. Error bars represent mean (SD)
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3.3. Effect of Sustaining Dietary Ketosis Through Exogenous Ketone Supplementation on
the Serum and Hippocampal Metabolome of Sprague-Dawley rats
In the previous section, we reported that oral administration of ketone supplements
produced nutritional ketosis (>0.5 mM) without carbohydrate restriction and the effects of a 28-
day administration of five ketone supplements on blood glucose, ketones, and lipids in male
Sprague-Dawley rats. We hypothesized that the 28-day administration would affect metabolomic
markers. Serum (~300 µL) and hippocampal tissues for ketone supplements KE and BMS+MCT
were collected on day 28 at 4 hours post-intragastric gavage (peak ketone elevation). Metabolon
analyzed samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and gas
chromatography-mass spectrometry (GC-MS). Both ketone supplements significantly increased
serum Krebs cycle intermediates: citrate, fumarate and malate. KE supplement significantly
increased alpha-ketoglutarate and BMS+MCT supplement significantly increased succinate in
the rat serum. Additionally, both supplements affected medium chain fatty acids, antioxidants,
and adenosine in both serum and hippocampal tissue. Although both forms of ketone
supplementation increased brain and blood ketone levels, the global metabolic profiles were
p<0.05). This observation supports the hypoglycemic effect demonstrated in the previous study
as well as previous studies by Veech and colleagues [87, 88]. As discussed, this hypoglycemic
effect has been attributed to the enhancement insulin sensitivity, which may help diabetic foot
ulcers specifically. As discussed, MCFAs are one of the main components of MCT oil namely
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caprylic acid (C8:0) and Capric acid (10:0); these medium chain fatty acids were significantly
elevated in the serum and hippocampal tissue of BMS+MCT supplemented rats (Figure 3.9).
This demonstrates that the MCT oil is being metabolized into its MCFAs, which are available in
the blood stream and tissues to be used for energy. Interestingly, KE supplemented rats also had
significantly elevated capric acid in the serum and hippocampal tissue.
Krebs cycle intermediates citrate, fumarate and malate were significantly elevated in the
serum of both KE-treated and BMS+MCT-treated animals (Figure 3.10). Additionally, KE
significantly elevated the intermediate α-ketoglutarate and BMS+MCT significantly elevated
succinate. These elevations were not seen in the hippocampal tissue. As discussed, non-wounded
skin predominantly uses anaerobic respiration with only about 30% going to the Krebs cycle; this
is exaggerated during wound healing when hypoxia due to tissue damage shifts the metabolic
profile to only using 4% Krebs cycle [89-92]. However, ketones have been shown to increase
blood flow; thus, increased blood flow would lead to potential increased oxygenation and the
restoration of the non-wounded skin metabolic profile. Even with a 30% Krebs cycle flux, an
increase in Acetyl CoA via ketolysis would produce a net increase in ATP production. Indeed,
Veech and colleagues demonstrated a 16-fold increase in acetyl CoA with a ketone ester
supplementation [88]. Acetyl co-A was significantly elevated via BMS+MCT supplementations
but not KE in the hippocampal tissue, supporting this finding. Levels of pyruvate and lactate
were unchanged in both ketone supplements in both the serum and hippocampal tissue
demonstrating that there wasn’t an increased flux of glycolysis and anaerobic respiration. Even
though there initially wouldn’t be sufficient oxygen for the wound bed to use the intermediates in
the ETC, Krebs cycle intermediates have other anabolic fates that would be important to wound
healing. Citrate is a precursor to fatty acid synthesis; α-ketoglutarate has been shown to scavenge
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H2O2 in cell cultures and is an amino acid synthesis precursor [93]. Krebs cycle intermediates
were shown to be decreased in aged rats; this supplementation may restore these levels to those
of a young person or at least elevate them [94]. Significant amino acid synthesis is supported by
significant serum elevations in the amino acids glycine and serine. Additionally, KE-treated rats
had a significant increase in PPP intermediates ribose-5-phosphate and ribulose/xylulose-5-
phosphate demonstrating an increase flux through the pathway and potentially nucleic acid
synthesis needed for wound healing. From this data, we can speculate that if the ketones were
being used as energy a higher portion of the glucose can be shunted to the PPP instead of being
metabolized for energy.
Surprisingly, oxidative stress (oxidized glutathione, GSSG (serum)) and antioxidant
capacity (reduced glutathione (hippo), carnosine (serum), and anserine (serum)) were elevated,
suggesting a heightened state of oxidative stress and antioxidant defense with ketone
supplementation. As discussed by Moor and colleagues, age alone affects the levels of SOD2 and
glutathione antioxidant profile [95]. Without appropriate oxidative stress, neutrophil respiratory
bursts cannot effectively clear bacteria from the wound bed; however, without appropriate
antioxidant capacity, the reactive oxygen species become damaging to neighboring tissue. This
ketogenic elevation of both oxidative stress and antioxidant defense may help clear bacteria as
well as quench ROS to promote wound healing in an aged phenotype. Additionally, carnosine (β-
alanyl-L-histidine) and anserine (β-alanyl-N-methylhistidine) were elevated by KE and
BMS+MCT supplementation (Figure 3.11). Carnosine is a natural dipeptide widely and
abundantly distributed in excitable tissue. Although its physiologic role is not completely
understood, many beneficial actions have been attributed to carnosine, such as being an
antioxidant, antiglycating and ion-chelating agent, and a free-radical scavenger. Several studies
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have shown that supplementation with carnosine accelerates wound healing [96-100]. Anserine
is a dipeptide containing beta alanine and histidine and has been shown to exhibit equal
antioxidant activity to carnosine by reducing the primary molecular products of lipid
peroxidation [101-103]. A study by Altavilla et al. demonstrated that reduction of lipid
peroxidation restores impaired VEGF expression leading to accelerated wound healing and
angiogenesis in a diabetic wound model [104]. This supports a possible mechanism of action for
ketone supplementation to augment wound healing. However, further experiments need to be
performed to optimize dose as too high of an oxidative state, which can be seen in the KE
supplementation can potentially be damaging, especially in an aged patient.
Commonly known for its role in energy transfer via phosphorylation, adenosine also
plays a role in the regulation of blood flow as a potent vasodilator. Adenosine was significantly
elevated in the serum via KE supplementation and trended towards significance for BMS+MCT;
however, BMS+MCT supplemented rats showed a significant elevation of adenosine in their
hippocampal tissue (KE not significant). Even though BMS+MCT did not show a significant
elevation in the blood stream, this suggests that the BMS+MCT supplemented rat tissues were
more efficient at using it; therefore a larger portion had already been sequestered in the
hippocampal tissue. High levels of AMP could reflect a metabolic insufficiency for the KE
supplement, which may be due to the high dose; further studies optimizing the dose of ketone
supplements are needed. Masino and colleagues have demonstrated that increased adenosine of
the A1 subtype plays a key role in the anticonvulsant success of ketogenic strategies [105-108].
Additionally, several studies have shown that topical adenosine application accelerates wound
healing via adenosine A2A receptors and promoting angiogenesis [109-117].
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A recent study by Sood et al, was the first to document a global metabolomic profile of
diabetic and non-diabetic wounds, 7 days post injury. In non-diabetic mice, 88 of the 129
detected metabolites had a significant response to injury, 85 up regulated and 3 down-regulated.
In diabetic wounds, 81 metabolites had a significant response to injury with 76 up regulated and
5 down regulated. Interestingly, they found 62 unique metabolites that differed between the non-
diabetic and diabetic wound phenotype [118]. From their study, glycine was dysregulated in
diabetic wounds and both ketone supplements significantly increased glycine in the serum (1.3,
1.27 fold change). A recent metabolic profiling of cancer cells has correlated glycine with
increased cell proliferation; however, it remains to be determined whether glycine is essential for
cell proliferation during wound healing [119]. Two recent studies suggest that Kynurenine, a
kynurenate precursor, may play a role in both anti-inflammatory activity and fibroblast
proliferation during wound repair; additionally, it has been shown to be dysregulated in diabetic
wounds [120, 121]. KE supplemented rats showed an increase in kynurenate but didn’t reach
significance (2.61 fold) (0.05<p<0.10). Additionally, metabolite OH-phenylpyruvate was shown
to be dysregulated and KE supplementation significantly enhanced the levels 3.59 fold in the
serum. Little is known about the role during wound healing. Further experiments need to be
conducted to determine the meaning of this observation.
The metabolomics profiling data presented here help us to understand the metabolic
consequences of KE and BMS+MCT administration and offers insights into potential
mechanisms of action by which ketone supplementation may augment wound healing as well as
affect other disease states for future studies.
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Table 3.1 Global metabolism profile of serum and hippocampal tissue following chronic administration of KE and BMS+MCT: Rats received a daily 5-g/kg dose of water (control) (n=11), KE (n=11), or BMS+MCT (n=12) via intragastric gavage for 29 days. On day 29, blood serum (~300 µL) and hippocampal tissues were collected 4-hours post-intragastric gavage (peak ketone elevation) and global metabolomics profiling was performed at Metabolon Inc. using gas and liquid chromatography and tandem mass spectrometry. Tx1=KE and Tx2=BMS+MCT. Results were considered significant when p<0.05 and direction is indicated by red or green arrows. 388 known metabolites were identified in the serum and 290 were identified in the hippocampus. KE significantly increased 106 metabolites and significantly decreased 36 in the serum compared to control; increased 10 and decreased 2 compared to control in the hippocampus (p<0.05, Welch’s two sample t-test). BMS+MCT significantly increased 57 metabolites and significantly decreased 62 metabolites compared to control in the serum; increased 28 and decreased 8 compared to control in the hippocampus (p<0.05). An additional 24 metabolites trended towards significance compared to control for KE and 58 for BMS+MCT (0.05<p<0.10).
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Figure 3.8 Ketone supplementation elevates blood ketone levels in serum and hippocampal tissues: KE and BMS supplemented rats had elevated blood ketone levels in their serum. KE elevated βHB 15.76 fold and AcAc 8.84 fold. BMS+MCT elevated βHB 35.33 fold and AcAc 15.39 fold. KE elevated βHB 2.7 fold in hippocampus and BMS+MCT elevated βHB 3.39 fold.
Figure 3.9 Ketone supplementation increases medium chain fatty acids in serum and hippocampal tissue: KE significantly increased medium chain fatty acid (MFCA) Caprate in the serum (3.15 fold) and in the hippocampus (1.92 fold) (p<0.05). BMS+MCT significantly increased Caprate in the serum (3.98 fold) and in the hippocampus (3.16 fold) (p<0.05). BMS+MCT significantly increased Caprylate in the serum (13.19 fold) and in the hippocampus (3.24) fold (p<0.05).
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Figure 3.10 Ketone supplementation increases Krebs cycle intermediates: KE and BMS+MCT supplemented rats had significantly elevated Krebs cycle intermediates in their serum. Both, KE and BMS+MCT increased citrate (1.99 fold, 2.34 fold) (A), fumarate (1.92, 2.45 fold) (B), and malate (2.03, 2.68 fold) (C) Additionally KE increased alpha-ketoglutarate (1.98 fold) (D) and BMS+MCT increased succinate (1.66 fold) (E). Krebs cycle precursor Acetyl CoA was significantly elevated by BMS+MCT in the hippocampus (1.37 fold) (F). Krebs cycle intermediates were unchanged in hippocampal tissues.
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Figure 3.11 Ketone supplementation increases antioxidants: KE and BMS+MCT significantly increased antioxidants carnosine (3.23, 1.79 fold) and anserine (5.66, 3.70 fold) in the serum (p<0.05). Carnosine and anserine were unchanged in the hippocampus.
Figure 3.12 Ketone supplementation increases Adenosine: KE significantly increased adenosine levels in the serum (9.18 fold) (p<0.05). BMS+MCT significantly increased adenosine levels in the hippocampus (10.95 fold).
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CHAPTER 4: ENHANCING WOUND HEALING WITH EXOGENOUS KETONE
SUPPLEMENTATION
4.1. Chapter Synopsis
In the previous chapter, we determined that exogenous ketone supplementation
significantly elevated blood ketone levels and reduced blood glucose levels in juvenile Sprague-
Dawley rats. In this chapter, we present data demonstrating that both BD and BMS+MCT
ketone supplements elicit age-dependent rapid elevation of blood ketone levels and reduction of
blood glucose levels in young (8 month) and aged (20 month) Fischer 344 rats similar to those
determined in Chapter 3. Additionally, we present data demonstrating the effects of food-
integrated oral ketone supplementation on an ischemic wound-healing model in young and aged
Fischer 344 rats. In the ischemic wound model, the flap placement on the dorsum prevents the
use of oral gavage; therefore, the ketone supplements were mixed into the food and fed ad
libitum. Nonetheless, this allowed continuous delivery of the ketone supplements in smaller
doses as the rats fed throughout the day. Compared to a bolus administration, multiple daily
doses or inclusion in the food may provide more sustained benefits. Additionally, we chose one
synthetic (BD) and one natural (BMS+MCT) ketone supplement for the following studies.
Though KE would seem to be a logical choice to continue with, the Fischer 344 rats refused to
eat the food mixed with this supplement; therefore, BD was used.
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Portions of this chapter have been taken from a manuscript Poff AM, Kesl SL,
D’Agostino DP “Ketone Supplementation for Health and Disease” Book Chapter, Oxford Press
(Submitted). A copy of this article may be found in Appendix C. Materials and Methods
presented in this chapter may be found in Appendix A.
4.2. Dietary Ketone Supplementation Increases Blood Flow and Wound Closure in an
Ischemic Wound Model in Young and Aged Fischer Rats
It has been well established that the ketogenic diet (KD) induces “keto-adaptation”, a
physiologic state characterized by a shift away from glucose metabolism and towards fat and
ketone body metabolism. [1-3]. As discussed in previous chapters, there are many
physiological changes associated with sustained ketosis that may contribute to its multifaceted
therapeutic potential for many disease states including chronic wounds. Thus, we previously
measured blood glucose, ketones, lipids, and other biochemical metabolites in response to
chronic oral ketone administration to show physiological equivalence to KD [4]. Increasing
evidence shows that limited energy and nutrient exchange is associated with age-related
impairment of wound healing. We hypothesized that oral ketone supplementation without
dietary restriction would enhance wound closure in young and aged Fischer rats by improving
blood flow and supplying an alternative energy substrate. In our preliminary studies, we
measured the magnitude and duration of ketosis following administration of a single 6.5g/kg
dose of ketone precursors: 1,3-Butanediol (BD), Na+/K+ βHB salt and medium chain
triglyceride (MCT) oil 1:1 mixture (BMS+MCT), or water in young and aged Fischer 344 rats
(n=6). Substances were administered through an intragastric gavage, and whole blood samples
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(10 µl) were acquired for analysis of glucose and βHB at 0, 0.5, 1, 1.5, 2, 4, 8, 12, and 24 hours
following administration. Following the creation of ischemic wounds, the ketogenic
supplements were added to a standard diet fed ad libitum for 28 days. Laser Doppler imaging of
the ischemic peri-wound tissue every seven days demonstrated significantly increased blood
flow in young rats (n=10) fed BD at day 14 and 28 (p<0.001) and BMS+MCT at day 7, 14, and
28 (p<0.01). In aged rats, blood flow was significantly increased in BD-fed at day 14 and
BMS+MCT-fed at days 7 and 14 (p<0.05). Wound size was significantly smaller in young rats
fed BD and BMS+MCT compared to control at 11 and 14 days following wound creation
(p<0.05). In aged rats, BD-fed wounds were significantly smaller at days 11 and 14 (p<0.05)
and in BMS+MCT-fed at days 11, 14, and 28 (p<0.05). Wound healing improved by three days
in aged BD-fed, seven days in young BMS+MCT-fed, and ten days in aged BMS+MCT-fed
compared to the healing time line of the control animals.
4.2.1 Aged rats metabolize exogenous ketone supplements differently than young rats
In Chapter 3, we studied if oral ketone administration could elicit similar physiological
effects as the KD by determining how blood glucose, ketones, and lipids and other biochemical
metabolites are affected by chronic ketone administration [4]. Here we present evidence that
chronic administration of ketone supplements can induce a state of nutritional ketosis without
the need for dietary carbohydrate restriction, enhance blood flow, and augment wound closure
in young and aged Fischer 344 rats.
Both young and aged rats exhibited elevated ketones within 30 minutes of the bolus
administration for both BD and BMS+MCT supplements and were sustained for 12 hours
(p<0.05). However, BD supplemented aged rats demonstrated a peak elevation of blood βHB
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levels at 8 hours, which was delayed compared to their younger counterparts who had peaked
blood βHB levels at 4 hours post intragastric gavage. Additionally, aged animals supplemented
with either of the ketone supplements demonstrated a ~1.5x higher elevation of blood ΒHB
levels for the same dose compared to the young animals (Figures 4.1A, B). An additional set of
young and aged animals were tested with a 10 g/kg dose of their respective ketone supplements
and a similar pattern emerged, young and aged animals’ blood βHB levels both peaked at 12
hours post gavage (data not shown). Aged animals administered BD appeared sedated, had
increased blood BHB up to 8 mM, and hypoglycemia (<50 mg/dL). We speculated that these
sedative effects might be because BD is metabolized as an alcohol; thus, we decided that an
intragastric gavage at 10 g/kg was not optimal for aged animals. Young animals were able to
tolerate the dose.
The metabolic challenge of a wound is exacerbated in elderly patients [5]. Without
sufficient energy (ATP) levels, wound healing is significantly impaired. As discussed, the ATP
deficiency creates an imbalance of energy production versus utilization (the catabolism to
anabolism ratio) leading to loss of lean body mass (LBM) [6, 7]. Additionally, Gupta and
colleagues demonstrated that metabolic enzymes hexokinase, citrate synthase,
phosphofructokinase, and lactate dehydrogenase were decreased in aged rats. Additionally, the
metabolic enzyme glucose-6-phosphate dehydrogenase exhibited elevated activity and the
beginnings stages followed by a significant decreased activity in a later phase compared to
controls [8]. These age-dependent metabolic changes could explain the difference between the
young and aged rats’ response to acute ketone supplementation. However, ketones have been
shown to increase Kreb’s cycle intermediates, mitochondrial biogenesis, and enhance overall
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metabolic efficiency; thus, this is one potential mechanism of how the prolonged ketone
administration could have enhanced wound healing.
4.2.2. Hypoglycemic effect of hyperketonemia attenuated with age
In Chapter 3, we demonstrated that at baseline and 4 weeks, 4 hours after intragastric
gavage with BMS+MCT (5 g/kg), the elevation of blood ketones was inversely correlated with
the reduction in blood glucose (r2=0.4314, p=0.0203, r2=0.8619, p<0.0001). This correlation
was not observed at any time point for BD supplemented rats [4]. Veech and colleagues have
demonstrated that administration of similar ketone supplement simultaneously decreased blood
glucose and blood insulin by approximately 50%. Our data show the same correlation between
elevated blood ketone levels and reduced blood glucose levels with exogenous BMS+MCT
supplementation in 8-month-old Fischer rats, supporting the work of Veech and confirming our
previous work (Figure 4.2). However, this hypoglycemic effect is not apparent in the 20-month
animals (Figure 4.2). As discussed, metabolic changes that occur during aging may play a role
in diminishing this relationship. Furthermore, insulin resistance has been shown to increase with
age [9]. Veech and colleagues have determined that ketone-induced hypoglycemia occurs via
increasing insulin sensitivity [10-12]. In the older adult, with pre-existing has insulin resistance,
the same ketone-induced effect on insulin sensitivity may not occur or it may not be enough to
cause hypoglycemia. It should be noted that even though there wasn’t a significant correlation
between blood ketone levels and blood glucose levels in aged ketone supplemented rats, there
was suppression of glucose based on the linear regression analysis (data not shown). This
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observation supports the speculation that an age-dependent metabolic change diminishes the
significant correlation but doesn’t completely neutralize the relationship.
Additionally, hyperketonemia was observed throughout the 28-day wound healing study
in the BD supplemented young and aged rats, but was not noted in the BMS+MCT
supplemented rats (Figure 4.5A). In Chapter 3, we demonstrated that ketone supplementation
caused a sustained reduction in glucose over the time course of the intragastric gavage study;
however, this sustained reduction of glucose was not observed over the course of the wound
healing study (Figure 4.5B). In a follow-up chronic feeding study (15 weeks), food-integrated
ketone supplementation resulted in elevated blood ketone levels without affecting the blood
glucose levels throughout the study, which supports our findings in this study (data not shown,
unpublished data).
4.2.3. Ketone supplementation increases blood flow in both young and aged rats
Restoration of blood flow via angiogenesis is critical for healing a wound and to support
revascularization of grafts including tissue engineered skin substitutes. Venous, arterial, and
diabetic blood flow insufficiencies are major underlying contributors to chronic wound
development. Additionally, older patients have a greater prevalence of hypertension, diabetes,
and smoking, which exacerbate the age-dependent angiogenic insufficiencies [13, 14]. In this
study, oral ketone supplementation significantly increased blood flow in young and aged rats
supplemented with BD or BMS+MCT (Figure 5.3B). In the raw Laser Doppler data, darker
colors are indicative of low blood flow/ischemia and lighter colors demonstrate increased blood
flow. The increased blood flow in the ketone-supplemented rats is clearly visible in the raw data
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(Figure 5.3A). This wound-healing model was created to induce ischemia and thus delay
wound healing [15, 16]. Further studies need to be conducted to measure oxygen concentrations
during wound healing to establish that increased blood flow reverses flap ischemia leading to
accelerated wound healing. In a study by Hasselbalch and colleagues, exogenous ketone
supplementation exhibited a 39% increase in cerebral blood flow [17]. Additionally, VEGF, a
potent angiogenic factor, has shown to be elevated with exogenous ketone supplementation [18-
20]. As discussed in Chapter 3, ketone supplementation has been determined to increase the
potent vasodilator, adenosine. These two mechanisms may explain how ketone supplementation
increased blood flow in the ischemic wound model.
4.2.4 Ketone supplementation accelerates wound closure in young and aged rats
As the population continues to age, there is a critical need to develop effective wound
healing therapies. In this study, we have demonstrated that exogenous ketone supplementation
enhanced wound closure by ten days (36% faster) in aged rats supplemented with BMS+MCT
and three days (10% faster) in aged rats supplemented with BD compared to control aged rats
(Figure 4.4). Additionally, young rats supplemented with BMS+MCT healed three days earlier
(14% faster) compared to standard diet fed young rats (Figure 4.4). Future studies are designed
to determine the wound healing effect of topical ketone administration alone and in combination
with the oral exogenous ketone therapy. As is, this therapy could allow patients to benefit from
nutritional ketosis without dietary restriction.
Studies by Nevin and colleagues investigated the influence of a topical application of
virgin coconut oil (VCO) on the healing of dermal wounds in young rats [21-23]. Their results
130
demonstrated a significant beneficial effect of VCO administration on intracellular and
extracellular matrix components compared to controls including increased cross-linking
collagen molecules indicating greater wound tensile strength and increased total DNA of the
dismutase 2, glutathione reductase, glutathione peroxidase) during wound healing, which led to
a decrease in lipid peroxides (MDA). They concluded that the wound healing property of VCO
might be due to its minor biologically active components and antimicrobial fatty acids. Previous
studies have demonstrated that food integrated coconut oil was able to eliminate bacterial
infection and stimulate the immune response [24]. Coconut oil is a natural source of medium
chain triglycerides (MCTs), which have been shown to modulate cellular proliferation, cell
signaling, and growth factor activities as well as elicit ketogenesis as seen in this study and our
previous study [4, 25-27].
The BMS contains potassium and other minerals to prevent sodium overload.
Maintaining an optimal sodium-mineral ratio should help offset any potential adverse effects of
sodium on blood pressure. For example, multiple studies have shown that potassium provides
an antihypertensive effect and protects cardiovascular damage in salt-sensitive hypertension
[28, 29]. It is speculated that this formulation will be especially beneficial for elderly patients
most susceptible to sodium-induced hypertension [11]. The dose of the salt solution is easily
adjusted to benefit the patients’ needs. In this study a 10% ketone salt solution is mixed in a 1:1
ratio with MCT oil, which allows for reduced dosing of each component compared to
administering the compounds individually. This reduces the potential for side effects (sodium-
induced hypertension, gastric side effects, etc.) and results in distinct synergistic blood ketone
profile [30]. A noteworthy observation from our previous study revealed that in rats, MCT alone
131
was more effective than BMS+MCT or BMS alone at inducing ketosis. However, preliminary
human data suggests that the BMS+MCT mixture is most effective at inducing ketosis and
MCT the least. This suggests that there is inter-species variability in the metabolic response to
ketone supplements, which will need to be further characterized to fully understand effects in
humans. In the rats, the BMS+MCT supplement elevated blood ketones similar to that of MCT
alone; however, the gastric side effects were not observed suggesting a potential method for
avoiding this unwanted adverse effect [31].
4.2.5. Food- integrated ketone supplementation did not elicit weight loss in young and
aged rats
The KD is hypothesized to induce weight loss by reducing appetite through the satiety
effect of ketone bodies, reducing lipogenesis and increasing lipolysis, and enhancing metabolic
efficiency with fat and ketone metabolism [32]. Recently, preliminary studies have shown that
exogenous ketone supplementation can also induce weight loss. The administrations of both
βHB and BD have been shown to decrease food intake in rats and pigmy goats [33-37].
Similarly, it is suggested that MCTs increase satiety, resulting in reduced food intake and
weight loss as a consequence of their rapid oxidation into ketone bodies [38-40]. MCTs may
further counteract fat deposition in adipocytes by increasing thermogenesis [41]. Several studies
in animals and humans have revealed increased energy expenditure and lipid oxidation with
MCTs compared to LCTs [42-51]. Ketone esters have also been shown to affect weight in
mice, rats, and humans [20, 52-55]. In Chapter 3, all five ketogenic supplements tested via
intragastric oral gavage (BD, KE, MCT, BMS, BMS+MCT) inhibited weight gain compared to
132
control animals [4]. Similarly, a 15-week chronic feeding study in which KE, BMS, or
BMS+MCT replaced approximately 20% of the diet by weight, fed ad libitum, led to reduced
weight gain compared to control animals (data not shown, unpublished data).
Even though weight loss has been noted in a variety of studies, significant weight loss
was not observed in either young or aged rats of this study (Figure 4.6A). To improve post-
anesthetic appetite, thereby limiting the initial weight loss that is standard with an invasive
surgery, the rats were fasted for 18 hours before surgery. . Weight loss was noted during the
first week after surgery, but did not reach significance in any group, and was similar across all
groups demonstrating that it wasn’t a ketone-mediated effect. Weight was maintained in all
groups for the remainder of the study. This initial fasting may be a reason that the initial weight
loss during the first week post-surgery did not reach significance. In the previously discussed
studies, rats were Sprague Dawley and were either juvenile or still in a growth phase, whereas
in this study the rats were mature or elderly Fischer 344s. Age and strain variability may
account for the lack of weight loss in this study compared to previously discussed experiments.
Particularly in elderly patients where malnutrition may already be present, it is critical to
prevent the wound from parasitizing substrates from the rest of the body for energy production,
resulting in what is called protein-energy malnutrition (PEM). The body catabolizes the
muscle, skin, and bone to support the synthesis of proteins, inflammatory cells, and collagen
needed to fight infection and repair the wound, causing a loss of lean body mass (LBM). As an
individual loses more LBM, wound healing is more likely to be delayed. A loss of more than
15% LBM impairs wound healing, a 30% LBM loss stops wound healing, and a loss of 40%
LBM typically results in death [56-60]. A body in ketosis has been shown to be muscle sparing
as it is adapted to using the readily available fat stores, attenuating the catabolic effects [7]. The
133
weight maintenance seen in this wound healing study may reflect this protein-sparing effect.
Moreover, if ketosis enhances insulin sensitivity, glucose uptake by the insulin-sensitive
skeletal muscle should be increased. Together, these effects would help support muscle tissue
health and function, suggesting another possible mechanism of ketone supplementation in
diminishing LBM and PEM.
4.3 Closing Remarks
It has been debated if chronological age alone affects wound healing. However, with the
aged population showing decreased blood flow leading to the decline of nutrient exchange,
decreased metabolic activity and ATP production at the wound bed, it is clear how these factors
may result in an exacerbation of chronic wounds in older adults. The discouraging results from
studies focusing on single molecular targets are not surprising since wound healing is the
outcome of a complex set of interactions between numerous factors. This is likely one reason
most wound therapies are minimally effective. Our approach utilizes endogenous physiology to
reach the wound bed systemically with one therapy, exogenous ketone supplementation, that
has multiple downstream effects.. Even though there is much work still to be done, this
approach shows promising results as a potential wound therapy.
134
Figure 4.1 Effects of ketone supplementation on blood ketone and blood glucose levels: (A, B) Blood βHB and blood glucose levels at times 0, 0.5, 1, 4, 8, 12, and 24 hours post (6.5 g/kg) intragastric gavage for ketone supplements tested. BMS+MCT and BD supplementation rapidly elevated and sustained significant βHB elevation (p< 0.05) (A) and significantly reduced glucose (p < 0.05) (B) compared to controls in both young (8M) and aged (20M) rats. Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05. Error bars represent mean (SD).
Figure 4.2 Relationship between blood ketone and blood glucose levels attenuated with age: At four hours post intragastric gavage, BMS+MCT (6.5 g/kg) supplemented rats demonstrated a significant correlations between elevated blood ketone levels and decreased blood glucose levels in young (8M) rats (r2=0.4775, p=0.0393); however, correlation was not present in aged (20M) rats (r2=0.0088, p=0.8431). Linear regression analysis, results considered significant if p<0.05. Error bars represent mean (SD).
8M Fisher Rats-BMS+MCT
60 80 100 1200
1
2
3
4
5
Glucose (mg/dL)
BH
B (m
M)
r2=0.4775p=0.0393 *
20M Fisher Rats- BMS+MCT
60 80 100 1200
1
2
3
4
5
Glucose (mg/dL)
BH
B (m
M)
r2=0.0088p=0.8431
135
Figure 4.3 Ketone Supplementation increases blood flow in both young and aged rats: Laser Doppler was used to measure blood flow weekly in the ischemic flap. Raw data from aged rats fed SD, BD and BMS ketone supplements at day 14 demonstrates the difference in blood flow seen by the laser doppler (A). Laser Doppler imaging of the ischemic peri-wound tissue every 7 days for 28 days demonstrated significantly increased blood flow in young rats (n=10 per group) with the BD at day 14 (p<0.0001) and 28 (p=0.0002) and the BMS+MCT at day 7 (p=0.0007), 14(p<0.0001), and 28 (p=0.0036) compared to control. There was an increase in blood flow in the aged rat flaps (n=10 per group) treated with the BD at day 14 (p=0.0039) and BMS+MCT at days 7 (p=0.0305) and 14 (p=0.0008) compared to control. Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05. Error bars represent mean (SD).
136
Figure 4.4 Ketone supplementation enhances wound closure time in young and aged rats: Visualized using Kaplan-Meier survival plot. Wound healing (closure) was determined to be significantly different in the young (n=10 per group) Fischer rats in BD at day 11 (p=0.0493) and in BMS+MCT at day 11 (p=0.0022) and 14 (p=0.0349). In the aged Fischer rats (n=10 per group), the BD was significantly different at day 11 (p=0.0230) and 14 (p=0.0233) and BMS+MCT was significantly different at day 11 (p=0.0115), 14 (p=0.0016) and 28 (p=00010). Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05.
137
Figure 4.5 Ketone supplementation elevates blood ketones levels but does not affect blood glucose levels: (A) Hyperketonemia was sustained for the duration of the wound healing progression in BD supplemented young and aged animals. (B) Ketone supplementation did not significantly reduce blood glucose levels at any point of the healing period. Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05. Error bars represent mean (SD).
138
Figure 4.6 Body and Spleen Weight: (A) Ketone supplementation did not result in significant weight loss through the study in young and aged animals. (B) Though aged animals’ spleen size trended towards splenomegaly, results did not reach significance. Two-Way ANOVA with Tukey’s post-hoc test, results considered significant if p<0.05. Error bars represent mean (SD).
139
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144
CHAPTER 5: POTENTIAL MECHANISMS FOR EXOGENOUS KETONE
SUPPLEMENTATION TO ENHANCE WOUND HEALING
5.1. Chapter Synopsis
In this chapter, we present data defining some potential mechanisms by which exogenous
ketone supplementation augments ischemic wound healing. We focus on four main age
dependent factors: inflammation, ROS production, angiogenesis, and metabolism. Since ketone
supplementation enhanced wound healing, we hypothesized that there would be measurable
physiological changes in wound healing as early three days post-wounding. Periwound tissue
was harvested at day three and day seven post-wounding for mechanistic analysis. Additionally
mechanistic studies were conducted using primary human dermal fibroblast (HDFs) to confirm
ex vivo observations. From these studies we conclude that exogenous ketone supplementation
decreases ROS production, increases migration and proliferation, and decreases lactate
production. Ketone supplementation did not affect inflammatory markers or antioxidants SOD2
and NQO1. Though there were similarities in effects of ketone supplements, BD and BMS+MCT
elicited distinctive mechanistic profiles. We propose further studies at later time points of wound
healing to determine if exogenous ketone supplementation will affect wound healing at a later
phase than we originally hypothesized.
145
5.2. Potential Mechanisms of Action for Exogenous Ketone Enhancement of Ischemic
Wound Healing in Young and Aged Fisher Rats
In the previous chapter, we reported that oral ketone supplementation without dietary
restriction enhanced wound closure and increased blood flow in young and aged Fisher 344 rats.
We hypothesized that exogenous ketone supplementation promoted wound healing via
enhancement of physiological factors such as increasing proliferation, advancing migration,
reducing ROS production, and resolving inflammation. Experiments in vitro with young and
aged primary human dermal fibroblasts supplemented with 5mM βHB for 72 hours ahead of an
oxidative stimulus (100μM tert-butyl-hydrogen peroxide) resulted in significantly decreased
Two-way ANOVA with Tukey’s post-hoc analysis, results were considered significant when
p<0.05. Error Bars Represent mean (SD).
197
A.5. References for Appendix A
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Demonstration of the Rat Ischemic Wound Model. Journal of Visualized Experiments: JoVE ”.
*Indicates that both authors contributed equally in the work.
Permission for inclusion of the aforementioned publication was granted via email (see below).
B.2 Copy Right Permissions for: Kesl SL, Poff AM, Ward NP, Fiorelli TN, Ari C, Van Putten
AJ, Sherwood JW, Arnold P, D’Agostino DP. Effects of Exogenous Ketone Supplementation on
Blood Ketone, Glucose, Triglyceride, and Lipoprotein Levels in Sprague-Dawley Rats. Nutrition
and Metabolism (2016).
199
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APPENDIX C: PUBLISHED MANUSCRIPTS
This appendix contains the original publications for the following references:
Trujilio AN*, Kesl SL*, Sherwood J, Wu M, Gould LJ. (2014) Demonstration of the Rat
Ischemic Wound Model. Journal of Visualized Experiments: JoVE. * Indicates that both authors
contributed equally to the work.
Kesl SL, Poff AM, Ward NP, Fiorelli TN, Ari C, Van Putten AJ, Sherwood JW, Arnold P,
D’Agostino DP. Effects of Exogenous Ketone Supplementation on Blood Ketone, Glucose,
Triglyceride, and Lipoprotein Levels in Sprague-Dawley Rats. Nutrition and Metabolism. 2016.
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RESEARCH Open Access
Effects of exogenous ketonesupplementation on blood ketone, glucose,triglyceride, and lipoprotein levels inSprague–Dawley ratsShannon L. Kesl1*, Angela M. Poff1, Nathan P. Ward1, Tina N. Fiorelli1, Csilla Ari1, Ashley J. Van Putten1,Jacob W. Sherwood1, Patrick Arnold2 and Dominic P. D’Agostino1
Abstract
Background: Nutritional ketosis induced by the ketogenic diet (KD) has therapeutic applications for many diseasestates. We hypothesized that oral administration of exogenous ketone supplements could produce sustainednutritional ketosis (>0.5 mM) without carbohydrate restriction.
Methods: We tested the effects of 28-day administration of five ketone supplements on blood glucose, ketones,and lipids in male Sprague–Dawley rats. The supplements included: 1,3-butanediol (BD), a sodium/potassium β-hydroxybutyrate (βHB) mineral salt (BMS), medium chain triglyceride oil (MCT), BMS + MCT 1:1 mixture, and 1,3butanediol acetoacetate diester (KE). Rats received a daily 5–10 g/kg dose of their respective ketone supplement viaintragastric gavage during treatment. Weekly whole blood samples were taken for analysis of glucose and βHB atbaseline and, 0.5, 1, 4, 8, and 12 h post-gavage, or until βHB returned to baseline. At 28 days, triglycerides, totalcholesterol and high-density lipoprotein (HDL) were measured.
Results: Exogenous ketone supplementation caused a rapid and sustained elevation of βHB, reduction of glucose,and little change to lipid biomarkers compared to control animals.
Conclusions: This study demonstrates the efficacy and tolerability of oral exogenous ketone supplementation ininducing nutritional ketosis independent of dietary restriction.
BackgroundEmerging evidence supports the therapeutic potential ofthe ketogenic diet (KD) for a variety of disease states,leading investigators to research methods of harnessingthe benefits of nutritional ketosis without the dietaryrestrictions. The KD has been used as an effective non-pharmacological therapy for pediatric intractable sei-zures since the 1920s [1–3]. In addition to epilepsy, theketogenic diet has elicited significant therapeutic effects
for weight loss and type-2 diabetes (T2D) [4]. Severalstudies have shown significant weight loss on a high fat,low carbohydrate diet without significant elevations ofserum cholesterol [5–12]. Another study demonstratedthe safety and benefits of long-term application of theKD in T2D patients. Patients exhibited significant weightloss, reduction of blood glucose, and improvement oflipid markers after eating a well-formulated KD for56 weeks [13]. Recently, researchers have begun toinvestigate the use of the KD as a treatment for acne,polycystic ovary syndrome (PCOS), cancer, amyotrophiclateral sclerosis (ALS), traumatic brain injury (TBI) andAlzheimer’s disease (AD) with promising preliminaryresults [14–26].
* Correspondence: [email protected] of Molecular Pharmacology and Physiology, Morsani College ofMedicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC8,Tampa, FL 33612, USAFull list of author information is available at the end of the article
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The classical KD consists of a 4:1 ratio of fat to pro-tein and carbohydrate, with 80–90 % of total calories de-rived from fat [27]. The macronutrient ratio of the KDinduces a metabolic shift towards fatty acid oxidationand hepatic ketogenesis, elevating the ketone bodiesacetoacetate (AcAc) and β-hydroxybutyrate (βHB) in theblood. Acetone, generated by decarboxylation of AcAc, hasbeen shown to have anticonvulsant properties [28–32].Ketone bodies are naturally elevated to serve as alternativemetabolic substrates for extra-hepatic tissues during theprolonged reduction of glucose availability, suppression ofinsulin, and depletion of liver glycogen, such as occursduring starvation, fasting, vigorous exercise, calorierestriction, or the KD. Although the KD has cleartherapeutic potential, several factors limit the efficacyand utility of this metabolic therapy for widespreadclinical use. Patient compliance to the KD can be lowdue to the severe dietary restriction - the diet beinggenerally perceived as unpalatable - and intolerance tohigh-fat ingestion. Maintaining ketosis can be difficultas consumption of even a small quantity of carbohydratesor excess protein can rapidly inhibit ketogenesis [33, 34].Furthermore, enhanced ketone body production and tissueutilization by the tissues can take several weeks (keto-adap-tation), and patients may experience mild hypoglycemicsymptoms during this transitional period [35].Recent studies suggest that many of the benefits of the
KD are due to the effects of ketone body metabolism.Interestingly, in studies on T2D patients, improved gly-cemic control, improved lipid markers, and retraction ofinsulin and other medications occurred before weightloss became significant. Both βHB and AcAc have beenshown to decrease mitochondrial reactive oxygen species(ROS) production [36–39]. Veech et al. have summarizedthe potential therapeutic uses for ketone bodies [28, 40].They have demonstrated that exogenous ketones favorablyalter mitochondrial bioenergetics to reduce the mitochon-drial NAD couple, oxidize the co-enzyme Q, and increasethe ΔG’ (free enthalpy) of ATP hydrolysis [41]. Ketonebodies have been shown to increase the hydraulic effi-ciency of the heart by 28 %, simultaneously decreasingoxygen consumption while increasing ATP production[42]. Thus, elevated ketone bodies increase metabolic effi-ciency and as a consequence, reduce superoxide productionand increase reduced glutathione [28]. Sullivan et al. demon-strated that mice fed a KD for 10–12 days showed increasedhippocampal uncoupling proteins, indicative of decreasedmitochondrial-produced ROS [43]. Bough et al. showed anincrease of mitochondrial biogenesis in rats maintained on aKD for 4–6 weeks [44, 45]. Recently, Shimazu et al. reportedthat βHB is an exogenous and specific inhibitor of class Ihistone deacetylases (HDACs), which confers protectionagainst oxidative stress [38]. Ketone bodies have also beenshown to suppress inflammation by decreasing the
inflammatory markers TNF-a, IL-6, IL-8, MCP-1, E-selectin,I-CAM, and PAI-1 [8, 46, 47]. Therefore, it is thought thatketone bodies themselves confer many of the benefits asso-ciated with the KD.Considering both the broad therapeutic potential and
limitations of the KD, an oral exogenous ketone supple-ment capable of inducing sustained therapeutic ketosiswithout the need for dietary restriction would serve as apractical alternative. Several natural and synthetic ketonesupplements capable of inducing nutritional ketosis havebeen identified. Desrochers et al. elevated ketone bodiesin the blood of pigs (>0.5 mM) using exogenous ketonesupplements: (R, S)-1,3 butanediol and (R, S)-1,3butanediol-acetoacetate monoesters and diester [48]. In2012, Clarke et al. demonstrated the safety and efficacyof chronic oral administration of a ketone monoester ofR-βHB in rats and humans [49, 50]. Subjects maintainedelevated blood ketones without dietary restriction andexperienced little to no adverse side effects, demonstratingthe potential to circumvent the restrictive diet typicallyneeded to achieve therapeutic ketosis. We hypothesized thatexogenous ketone supplements could produce sustainedhyperketonemia (>0.5 mM) without dietary restriction andwithout negatively influencing metabolic biomarkers, suchas blood glucose, total cholesterol, HDL, LDL, and triglycer-ides. Thus, we measured these biomarkers during a 28-dayadministration of the following ketone supplements in rats:naturally-derived ketogenic supplements included mediumchain triglyceride oil (MCT), sodium/potassium -βHBmineral salt (BMS), and sodium/potassium -βHB mineralsalt +medium chain triglyceride oil 1:1 mixture (BMS +MCT) and synthetically produced ketogenic supplementsincluded 1, 3-butanediol (BD), 1, 3-butanediol acetoace-tate diester/ ketone ester (KE).
MethodsSynthesis and formulation of ketone supplementsKE was synthesized as previously described [29]. BMS isa novel agent (sodium/potassium- βHB mineral salt)supplied as a 50 % solution containing approximately375 mg/g of pure βHB and 125 mg/g of sodium/potas-sium. Both KE and BMS were developed and synthesizedin collaboration with Savind Inc. Pharmaceutical gradeMCT oil (~65 % caprylic triglyceride; 45 % capric trigly-ceride) was purchased from Now Foods (Bloomingdale,IL). BMS was formulated in a 1:1 ratio with MCT at theUniversity of South Florida (USF), yielding a final mix-ture of 25 % water, 25 % pure βHB mineral salt and50 % MCT. BD was purchased from Sigma-Aldrich(Prod # B84785, Milwaukee, WI).
Daily gavage to induce dietary ketosisAnimal procedures were performed in accordance withthe University of South Florida Institutional Animal
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Care and Use Committee (IACUC) guidelines (Protocol#0006R). Juvenile male Sprague–Dawley rats (275–325 g,Harlan Laboratories) were randomly assigned to one ofsix study groups: control (water, n = 11), BD (n = 11), KE(n = 11), MCT (n = 10), BMS (n = 11), or BMS +MCT(n = 12). Caloric density of standard rodent chow anddose of ketone supplements are listed in Table 1. Ondays 1–14, rats received a 5 g/kg body weight dose oftheir respective treatments via intragastric gavage.Dosage was increased to 10 g/kg body weight for thesecond half of the study (days 15–28) for all groupsexcept BD and KE to prevent excessive hyperketone-mia (ketoacidosis). Each daily dose of BMS would equal~1000–1500 mg of βHB, depending on the weight of theanimal. Intragastric gavage was performed at the same timedaily, and animals had ad libitum access to standardrodent chow 2018 (Harlan Teklad) for the duration ofthe study. The macronutrient ratio the standard rodentchow was 62.2, 23.8 and 14 % of carbohydrates, proteinand fat respectively.
Measurement and analysis of blood glucose, ketones,and lipidsEvery 7 days, animals were briefly fasted (4 h, wateravailable) prior to intragastric gavage to standardizelevels of blood metabolites prior to glucose and βHBmeasurements at baseline. Baseline (time 0) was imme-diately prior to gavage. Whole blood samples (10 μL)were taken from the saphenous vein for analysis ofglucose and βHB levels with the commercially avail-able glucose and ketone monitoring system PrecisionXtra™ (Abbott Laboratories, Abbott Park, IL). Bloodglucose and βHB were measured at 0, 0.5, 1, 4, 8,and 12 h after test substance administration, or untilβHB returned to baseline levels. Food was returned toanimals after blood analysis at time 0 and gavage. Atbaseline and week 4, whole blood samples (10 μL)were taken from the saphenous vein immediatelyprior to gavage (time 0) for analysis of total choles-terol, high-density lipoprotein (HDL), and triglycerideswith the commercially available CardioChek™ bloodlipid analyzer (Polymer Technology Systems, Inc., In-dianapolis, IN). Low-density lipoprotein (LDL)
cholesterol was calculated from the three measuredlipid levels using the Friedewald equation: (LDL Choles-terol = Total Cholesterol - HDL - (Triglycerides/5)) [51,52]. Animals were weighed once per week to trackchanges in body weight associated with hyperketonemia.
Organ weight and collectionOn day 29, rats were sacrificed via deep isofluraneanesthesia, exsanguination by cardiac puncture, and de-capitation 4–8 h after intragastric gavage, which correlatedto the time range where the most significantly elevatedblood βHB levels were observed. Brain, lungs, liver,kidneys, spleen and heart were harvested, weighed (AWS-1000 1 kg portable digital scale (AWS, Charleston, SC)),and flash-frozen in liquid nitrogen or preserved in 4 %paraformaldehyde for future analysis.
StatisticsAll data are presented as the mean ± standard deviation(SD). Data analysis was performed using GraphPadPRISM™ version 6.0a and IBM SPSS Statistics 22.0. Re-sults were considered significant when p < 0.05. Trigly-ceride and lipoprotein profile data were analyzed usingOne-Way ANOVA. Blood ketone and blood glucosewere compared to control at the applicable time pointsusing a Two-Way ANOVA. Correlation between bloodβHB and glucose levels in ketone supplemented rats wascompared to controls using ANCOVA analysis. Organand body weights were analyzed using One-WayANOVA. Basal blood ketone and blood glucose levelswere analyzed using Two-Way ANOVA. All mean com-parisons were carried out using Tukey’s multiple com-parisons post-hoc test.
ResultsEffect of ketone supplementation on triglycerides andlipoproteinsBaseline measurements showed no significant changesin triglycerides or the lipoproteins (data not shown).Data represent triglyceride and lipoprotein concentra-tions measured after 4 weeks of daily exogenous ketonesupplementation. No significant change in total choles-terol was observed at 4 weeks for any of the ketonetreatment groups compared to control. (Fig. 1a). No
Table 1 Caloric density and dose of ketone supplementsMacronutrient Information Standard Diet Water BMS + MCT BMS MCT KE BD
% Cal from Fat 18.0 0.0 50.0 N/A 100.0 N/A N/A
% Cal from Protein 24.0 0.0 N/A N/A 0.0 N/A N/A
% Cal from Carbohydrates 58.0 0.0 N/A N/A 0.0 N/A N/A
Total Caloric Density (Kcal/g) 3.1 0.0 5.1 1.9 8.3 5.6 6.0
Dose 0–14 Days (g/kg) ad libitum N/A 5.0 5.0 5.0 5.0 5.0
Dose 15–28 Days (g/kg) ad libitum N/A 10.0 10.0 10.0 5.0 5.0
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significant difference was detected in triglycerides forany ketone supplement compared to control (Fig. 1b).MCT supplemented animals had a significant reductionin HDL blood levels compared to control (p < 0.001)(Fig. 1c). LDL levels in ketone-supplemented animals didnot significantly differ from controls (Fig. 1d).
Ketone supplementation causes rapid and sustainedelevation of βHBOver the 28-day experiment, ketone supplements ad-ministered daily significantly elevated blood ketonelevels without dietary restriction (Fig. 2a, b). Naturallyderived ketogenic supplements including MCT (5 g/kg)elicited a significant rapid elevation in blood βHB within30–60 min that was sustained for 8 h. BMS +MCT (5 g/kg) elicited a significant elevation in blood βHB at 4 h,which was no longer significant at 8 h. BMS (5 g/kg) didnot elicit a significant elevation in blood βHB at anytime point. For days 14–28, BMS +MCT (10 g/kg) andMCT (10 g/kg) elevated blood βHB levels within 30 minand remained significantly elevated for up to 12 h. Weobserved a delay in the peak elevation of blood βHB:
BMS +MCT peaked at 8 h instead of at 4 h and MCT at4 h instead of at 1 h. Blood βHB levels in the BMS groupdid not show significant elevation at any time point,even after dose escalation (Fig. 2a). Synthetically derivedketogenic supplements including KE and BD supple-mentation rapidly elevated blood βHB within 30 minand was sustained for 8 h. For the rats receiving ketonesupplementation in the form of BD or the KE, dosagewas kept at 5 g/kg to prevent adverse effects associatedwith hyperketonemia. The Precision Xtra™ ketone moni-toring system measures βHB only; therefore, total bloodketone levels (βHB +AcAc) would be higher than mea-sured. For each of these groups, the blood βHB profileremained consistent following daily ketone supplementa-tion administration over the 4-week duration. (Fig. 2b).
Ketone supplementation causes a significant decrease ofblood glucoseAdministration of ketone supplementation significantlyreduced blood glucose over the course of the study(Fig. 3a, b). MCT (5 g/kg) decreased blood glucose com-pared to control within 30 min which was sustained for
Fig. 1 Effects of ketone supplementation on triglycerides and lipoproteins: Ketone supplementation causes little change in triglycerides andlipoproteins over a 4-week study. Graphs show concentrations at 4-weeks of total cholesterol (a), Triglycerides (b), LDL (c), and HDL (d).MCT supplemented rats had signfiicantly reduced concentration of HDL blood levels compared to control (p < 0.001) (b). One-Way ANOVAwith Tukey’s post hoc test, results considered significant if p < 0.05. Error bars represent mean (SD)
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Fig. 2 Effects of ketone supplementation on blood βHB. a, b Blood βHB levels at times 0, 0.5, 1, 4, 8, and 12 h post intragastric gavage for ketonesupplements tested. a BMS +MCT and MCT supplementation rapidly elevated and sustained significant βHB elevation compared to controls forthe duration of the 4-week dose escalation study. BMS did not significantly elevate βHB at any time point tested compared to controls. b BD andKE supplements, maintained at 5 g/kg, significantly elevated βHB levels for the duration of the 4-week study. Two-Way ANOVA with Tukey’s posthoc test, results considered significant if p < 0.05. Error bars represent mean (SD)
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Fig. 3 Effects of ketone supplementation on blood glucose. a, b Blood glucose levels at times 0, 0.5, 1, 4, 8, and 12 h (for 10 dose) post intragastricgavage for ketone supplements tested. a Ketone supplements BMS +MCT and MCT significantly reduced blood glucose levels compared to controlsfor the duration of the 4-week study. BMS significantly lowered blood glucose only at 8 h/week 1 and 12 h/week 3 (b) KE, maintained at5 g/kg, significantly reduced blood glucose compared to controls from week 1–4. BD did not significantly affect blood glucose levels atany time point during the 4-week study. Two-Way ANOVA with Tukey’s post hoc test, results considered significant if p < 0.05. Error barsrepresent mean (SD)
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8 h at baseline and at week 1. MCT (10 g/kg) likewisedecreased blood glucose within 30 min and lastedthrough the 12 h time point during weeks 2, 3, and 4.BMS +MCT (5 g/kg) lowered blood glucose comparedto control from hours 1–8 only at week 1. BMS +MCT(10 g/kg) lowered blood glucose compared to controlwithin 30 min and remained low through the 12 h timepoint at weeks 2, 3, and 4. Rats supplemented with BMShad lower blood glucose compared to control at 12 h inweek 4 (10) (Fig. 3a). Administration of BD did not sig-nificantly change blood glucose levels at any time pointduring the 4-week study. KE (5 g/kg) significantly low-ered blood glucose levels at 30 min for week 1, 2, 3, and
4 and was sustained through 1 h at weeks 2–4 and sus-tained to 4 h at week 3. (Fig. 3b).
Hyperketonemia suppresses blood glucose levelsAt baseline, 4 h after intragastric gavage, the elevation ofblood ketones was inversely related to the reduction of bloodglucose compared to controls following the administrationof MCT (5 g/kg) (p = 0.008) and BMS+MCT (5 g/kg) (p =0.039) . There was no significant correlation between bloodketone levels and blood glucose levels compared to controlsfor any other ketone supplemented group at baseline(Fig. 4a). At week 4, 4 h after intragastric gavage, there was asignificant correlation between blood ketone levels and
Fig. 4 Relationship between blood ketone and glucose levels: a BMS +MCT (5 g/kg) supplemented rats demonstrated a significant inverse relationshipbetween elevated blood ketone levels and decreased blood ketone levels (r2 = 0.4314, p = 0.0203). b At week 4, BMS +MCT (10 g/kg) and MCT (10 g/kg)showed a significant correlation between blood ketone levels and blood glucose levels (r2 = 0.8619, p < 0.0001; r2 = 0.6365, p = 0.0057). Linear regressionanalysis, results considered significant if p < 0.05
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blood glucose levels compared to controls in MCT (10 g/kg)and BMS+MCT (10 g/kg) (p < 0.0001, p < 0.0001) (Fig. 4b).
Ketone supplementation changes organ weight anddecreases body weightAt day 29 of the study, animals were euthanized andbrain, lungs, liver, kidneys, spleen and heart were har-vested and weighed. Organ weights were normalized tobody weight. Ketone supplementation did not signifi-cantly change brain, lung, kidney, or heart weights
compared to controls (Fig. 5a, b, d, f ). MCT supple-mented animals had significantly larger livers comparedto their body weight (p < 0.05) (Fig. 5c). Ketone supple-ments BMS +MCT, MCT and BD caused a significantreduction in spleen size (BMS +MCT p < 0.05, MCT p< 0.001, BD p < 0.05) (Fig. 5e). Rats administered KEgained significantly less weight over the entire studycompared to controls. BMS +MCT, BMS, and BD sup-plemented rats gained significantly less weight than con-trols during weeks 2 – 4, and MCT animals gained less
Fig. 5 Effects of ketone supplementation on organ weight: Data is represented as a percentage of organ weight to body weight. a, b, d, fKetone supplements did not significantly affect the weight of the brain, lungs, kidneys or heart. c Liver weight was significantly increased ascompared to body weight in response to administered MCT ketone supplement compared to control at the end of the study (day 29) (p < 0.001).e Rats supplemented with BMS +MCT, MCT, and BD had significantly smaller spleen percentage as compared to controls (p < 0.05, p < 0.001, p < 0.05).Two-Way ANOVA with Tukey’s post-hoc test; results considered significant if p < 0.05. Error bars represent mean (SD)
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weight than controls at weeks 3 – 4 (Fig. 6). Increasedgastric motility (increased bowel evacuation and changesto fecal consistency) was visually observed in rats sup-plemented with 10 g/kg MCT, most notably at the 8 and12-h time points. All animals remained in healthy weightrange for their age even though the rate of weight gainchanged with ketone supplementation [53–54]. Food in-take was not measured in this study. However, there wasnot a significant change in basal blood glucose or basalblood ketone levels over the 4 week study in any of therats supplemented with ketones (Fig. 7).
DiscussionNutritional ketosis induced with the KD has proveneffective for the metabolic management of seizures andpotentially other disorders [1–26]. Here we presentevidence that chronic administration of ketone supple-ments can induce a state of nutritional ketosis withoutthe need for dietary carbohydrate restriction and withlittle or no effect on lipid biomarkers. The notion thatwe can produce the therapeutic effects of the KD withexogenous ketone supplementation is supported by ourprevious study which demonstrated that acutely admin-istered KE supplementation delays central nervoussystem (CNS) oxygen toxicity seizures without theneed for dietary restriction [29]. We propose thatexogenous ketone supplementation could provide analternative method of attaining the therapeutic
benefits of nutritional ketosis, and as a means tofurther augment the therapeutic potential of the KD.
Ketone supplementation causes little to no change intriglycerides and lipoproteinsOne common concern regarding the KD is its purportedpotential to increase the risk of atherosclerosis by elevat-ing blood cholesterol and triglyceride levels [55, 56]. Thistopic remains controversial as some, but not all, studieshave demonstrated that the KD elevates blood levels ofcholesterol and triglycerides [57–62]. Kwitervich and col-leagues demonstrated an increase in low-density lipopro-tein (LDL) and a decrease in high-density lipoprotein(HDL) in epileptic children fed the classical KD fortwo years [27]. In this study, total cholesterol in-creased by ~130 %, and stabilized at the elevated levelover the 2-year period. A similar study demonstrated thatthe lipid profile returned to baseline in children whoremained on the KD for six years [63]. Children typicallyremain on the diet for approximately two years then re-turn to a diet of common fat and carbohydrate ingestion[64]. The implications of these findings are unclear, sincethe influence of cholesterol on cardiovascular health iscontroversial and macronutrient sources of the diet varyper study. In contrast to these studies, the majority of re-cent studies have suggested that the KD can actually leadto significant benefits in biomarkers of metabolic health,including blood lipid profiles [65–72]. In these studies, the
Fig. 6 Effects of ketone supplementation on body weight: Rats administered ketone supplements gained less weight over the 4-week period;however, did not lose weight and maintained healthy range for age. KE supplemented rats gained significantly less weight during the entire4-week study compared to controls. BMS +MCT, BMS, and BD supplemented rats gained significantly less weight than controls over weeks 2–4.MCTsupplemented rats gained significantly less weight than controls over weeks 3–4, Two-Way ANOVA with Tukey’s post hoc test, results consideredsignificant if p < 0.05. Error bars represent mean (SD)
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KD positively altered blood lipids, decreasing total triglyc-erides and cholesterol while increasing the ratio of HDLto LDL [68–77]. Although, the KD is well-established inchildren, it has only recently been utilized as a strategy tocontrol seizures in adults. In 2014, Schoeler and col-leagues reported on the feasibility of the KD for adults,concluding that 39 % of individuals achieved > 50 % reduc-tion in seizure frequency, similar to the results reported inpediatric studies. Patients experienced similar gastrointes-tinal adverse advents that have been previously describedin pediatric patients, but they did not lead to discontinu-ation of the diet in any patient [78].With oral ketone supplementation, we observed a sig-
nificant elevation in blood βHB without dietary restric-tion and with little change in lipid biomarkers (Fig. 1).Over the 4 week study, MCT-supplemented rats demon-strated decreased HDL compared to controls. No signifi-cant changes were observed in any of the triglycerides orlipoproteins (HDL, LDL) with any of the remaining ex-ogenously applied ketone supplements. It should benoted that the rats used for this study had not yetreached full adult body size [79]. Their normal growthrate and maturation was likely responsible for the
changes in triglyceride and lipoprotein levels observed inthe control animals over the 4 week study (baseline datanot shown, no significant differences) [80, 81]. Futurestudies are needed to investigate the effect of ketonesupplementation on fully mature and aged animals.Overall, our study suggests that oral ketone supplemen-tation has little effect on the triglyceride or lipoproteinprofile after 4 weeks. However, it is currently unknown ifketone supplementation would affect lipid biomarkersafter a longer duration of consumption. Further studiesare needed to determine the effects of ketone supplementson blood triglyceride and lipoproteins after chronicadministration and as a means to further enhance thehyperketonemia and improve the lipid profile of the clinic-ally implemented (4:1) KD.LDL is the lipoprotein particle that is most often associ-
ated with atherosclerosis. LDL particles exist in differentsizes: large molecules (Pattern A) or small molecules(Pattern B). Recent studies have investigated the im-portance of LDL-particle type and size rather thantotal concentration as being the source for cardiovascularrisk [56]. Patients whose LDL particles are predominantlysmall and dense (Pattern B) have a greater risk of
Fig. 7 Effects of ketone supplementation on basal blood ketone and basal blood glucose levels: Rats administered ketone supplements did nothave a significant change in basal blood ketone levels (a) or basal blood glucose levels (b) for the four week study. Two-Way ANOVA with Tukey’spost-hoc test, results considered significant if p < 0.05. Error bars represent mean (SD)
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cardiovascular disease (CVD). It is thought that small,dense LDL particles are more able to penetrate the endo-thelium and cause in damage and inflammation [82–85].Volek et al. reported that the KD increased the patternand volume of LDL particles, which is considered to re-duce cardiovascular risk [73]. Though we did not show asignificant effect on LDL levels for ketone supplements,future chronic feeding studies will investigate the effectsof ketone supplementation on lipidomic profile and LDLparticle type and size.
Therapeutic levels of hyperketonemia suppress bloodglucose levelsWe demonstrated that therapeutic ketosis could beinduced without dietary (calorie or carbohydrate) restric-tion and that this acute elevation in blood ketones wassignificantly correlated with a reduction in blood glucose(Figs. 2, 3 and 4). The BMS ketone supplement did notsignificantly induce blood hyperketonemia or reducedglucose in the rats. The KE supplemented rats trendedtowards reduced glucose levels; however, the lower doseof this agent did not lower glucose significantly, as re-ported previously in acute response of mice [59]. MCTshave previously been shown to elicit a slight hypoglycemiceffect by enhancing glucose utilization in both diabeticand non-diabetic patients [86–88]. Kashiwaya et al. dem-onstrated that both blood glucose and blood insulindecreased by approximately 50 % in rats fed a diet where30 % of calories from starch were replaced with ketone es-ters for 14 days, suggesting that ketone supplementationincreases insulin sensitivity or reduced hepatic glucoseoutput [89]. This ketone-induced hypoglycemic effect hasbeen previously reported in humans with IV infusions ofketone bodies [90, 91]. Recently, Mikkelsen et al. showedthat a small increase in βHB concentration decreases glu-cose production by 14 % in post-absorptive health males[92]. However, this has not been previously reported withany of the oral exogenous ketone supplements we studied.Ketones are an efficient and sufficient energy substrate forthe brain, and will therefore prevent side effects ofhypoglycemia when blood levels are elevated and the pa-tient is keto-adapted. This was most famously demon-strated by Owen et al. in 1967 wherein keto-adaptedpatients (starvation induced therapeutic ketosis) weregiven 20 IU of insulin. The blood glucose of fasted pa-tients dropped to 1–2 mM, but they exhibited nohypoglycemic symptoms due to brain utilization of ke-tones for energy [93]. Therefore, ketones maintain brainmetabolism and are neuroprotective during severehypoglycemia. The rats in the MCT group had a correl-ation of blood ketone and glucose levels at week 4,whereas the combination of BMS +MCT produced a sig-nificant hypoglycemic correlation both at baseline and atweek 4. No hypoglycemic symptoms were observed in the
rats during this study. Insulin levels were not measured inthis study; however, future ketone supplementation stud-ies should measure the effects of exogenous ketones oninsulin sensitivity with a glucose tolerance test. Anincrease in insulin sensitivity in combination with ourobserved hypoglycemic effect has potential therapy impli-cations for glycemic control in T2D [40]. Furthermore, itshould be noted that the KE metabolizes to both AcAcand βHB in 1:1 ratio [29]. The ketone monitor used in thisstudy only measures βHB as levels of AcAc are more diffi-cult to measure due to spontaneous decarboxylation toacetone; therefore, the total ketone levels (βHB+AcAc)measured were likely higher, specifically for the KE [14].Interestingly, the 10 g/kg dose produced a delayed bloodβHB peak for ketone supplements MCT and BMS +MCT.The higher dose of the ketogenic supplements elevatedblood levels more substantially, and thus reached theirmaximum blood concentration later due to prolongedmetabolic clearance. It must be noted that the dosage usedin this study does not translate to human patients, sincethe metabolic physiology of rats is considerably higher.Future studies will be needed to determine optimal dosingfor human patients.
Effects of ketone supplementation on organ weight andbody weight percentageKetone supplementation did not affect the size of thebrain, lungs, kidneys or heart of rats. As previously men-tioned, the rats were still growing during the experimen-tal time frame; therefore, organ weights were normalizedto body weight to determine if organ weight changed in-dependently to growth. There could be several reasonswhy ketones influenced liver and spleen weight. The ra-tio of liver to body weight was significantly higher in theMCT supplemented animals (Fig. 5). MCTs are readilyabsorbed in the intestinal lumen and transported directlyto the liver via hepatic portal circulation. When given alarge bolus, such as in this study, the amount of MCTsin the liver will likely exceed the β-oxidation rate, caus-ing the MCTs to be deposited in the liver as fat droplets[94]. The accumulated MCT droplets in the liver couldexplain the higher liver weight to body weight percentageobserved with MCT supplemented rats. Future toxicologyand histological studies will be needed to determine thecause of the observed hepatomegaly. It should be empha-sized that the dose in this study is not optimized inhumans. We speculate that an optimized human dosewould be lower and may not cause hepatomegaly or po-tential fat accumulation. Nutritional ketosis achieved withthe KD has been shown to decrease inflammatory markerssuch as TNF-α, IL-6, IL-8, MCP-1, E-selectin, I-CAM, andPAI-1 [8, 46], which may account for the observeddecrease in spleen weight. As previously mentioned,Veech and colleagues demonstrated that exogenous
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supplementation of 5 mM βHB resulted in a 28 % increasein hydraulic work in the working perfused rat heart and asignificant decrease in oxygen consumption [28, 41, 42].Ketone bodies have been shown to increase cerebral bloodflow and perfusion [95]. Also, ketone bodies have beenshown to increase ATP synthesis and enhance the effi-ciency of ATP production [14, 28, 40]. It is possible thatsustained ketosis results in enhanced cardiac efficiencyand O2 consumption. Even though the size of theheart did not change for any of the ketone supple-ments, further analysis of tissues harvested from theketone-supplemented rats will be needed to determineany morphological changes and to understand changes inorgan size. It should be noted that the Harlan standardrodent chow 2018 is nutritionally complete and formu-lated with high-quality ingredients to optimize gestation,lactation, growth, and overall health of the animals. Thesame cannot be said for the standard American diet(SAD). Therefore, we plan to investigate the effects ofketone supplements administered with the SAD to deter-mine if similar effects will be seen when the micronutrientdeficiencies and macronutrient profile mimics what mostAmericans consume.MCT oil has recently been used to induce nutritional
ketosis although it produces dose-dependent gastrointes-tinal (GI) side effects in humans that limit the potentialfor its use to significantly elevate ketones (>0.5 mM).Despite these limitations, Azzam and colleagues pub-lished a case report in which a 43-year-old-man had asignificant decrease in seizure frequency after supple-menting his diet with 4 tablespoons of MCT oil twicedaily [96]. An attempt to increase his dosage to 5 table-spoons twice daily was halted by severe GI intolerance.Henderson et al. observed that 20 % of patients re-ported GI side effects with a 20 g dose of ketogenicagent AC-1202 in a double blind trial in mild to moderateAlzheimer’s patients [24]. We visually observed similargastrointestinal side effects (loose stools) in the ratstreated with MCT oil in our study. Rats were closely mon-itored to avoid dehydration, and gastric motility returnedto normal between 12–24 h. Interestingly, the BMS +MCT supplement elevated βHB similarly to MCT oilalone, without causing the adverse gastrointestinal effectsseen in MCT-supplemented rats. However, this could bedue to the fact in a 10 g/kg dose of BMS +MCT, only 5 g/kg is MCT alone, which is less than the 10 g/kg dose thatelicits the GI side effects. This suggests that this novelcombination may provide a more useful therapeuticoption than MCT oil alone, which is limited in its abilityto elevate ketones in humans.Exogenously delivered ketone supplements signifi-
cantly altered rat weight gain for the duration of thestudy (Fig. 6). However, rats did not lose weight andmaintained a healthy range for their age. Rats have been
shown to effectively balance their caloric intake to preventweight loss/gain [97–99]. Due to the caloric density of theexogenous ketone supplements (Table 1) it is possible forthe rats to eat less of the standard rodent chow and there-fore less carbohydrates while maintaining their caloricintake. Food intake was not measured for this study.However, if there was a significant carbohydrate restric-tion there would be a signifcant change in basal blood ke-tone and blood glucose levels. As the hallmark to the KD,carbohydrate restriction increases blood ketone levels andreduces blood glucose levels. Neither an increase in basalblood ketone levels nor a decrease in basal blood glucoselevels was observed in this study (Fig. 7). Additionally, ifthere were an overall blood glucose decrease due to achange in food intake, this would not explain therapid reduction (within 30 min) in blood glucose cor-related with an elevation of blood ketone levels afteran intragastric bolus of ketone supplement (Figs. 2, 3and 4).
ConclusionsSeveral studies have investigated the safety and efficacyof ketone supplements for disease states such as AD andParkinson’s disease, and well as for parenteral nutrition[40, 48–50, 100–103]. Our research demonstrates thatseveral forms of dietary ketone supplementation can ef-fectively elevate blood ketone levels and achieve deleted:therapeutic nutritional ketosis without the need for diet-ary carbohydrate restriction. We also demonstrated thatketosis achieved with exogenous ketone supplementationcan reduce blood glucose, and this is inversely associatedwith the blood ketone levels. Although preliminary resultsare encouraging, further studies are needed to determineif oral ketone supplementation can produce the sametherapeutic benefits as the classic KD in the broad-spectrum of KD-responsive disease states . Additionally,further experiments need to be conducted to see if the ex-ogenous ketone supplementation affects the same physio-logical features as the KD (i.e. ROS, inflammation, ATPproduction). Ketone supplementation could be used as analternative method for inducing ketosis in patientsuninterested in attempting the KD or those who have pre-viously had difficulty implementing the KD because ofpalatability issues, gall bladder removal, liver abnormal-ities, or intolerance to fat. Additional experiments shouldbe conducted to see if ketone supplementation could beused in conjunction with the KD to assist and ease thetransition to nutrition ketosis and enhance the speed ofketo-adaptation. In this study we have demonstrated theability of several ketone supplements to elevate bloodketone levels, providing multiple options to induce thera-peutic ketosis based on patient need. Though additionalstudies are needed to determine the therapeutic potentialof ketone supplementation, many patients that previously
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were unable to benefit from the KD may now have analternate method of achieving therapeutic ketosis. Ketonesupplementation may also represent a means to furtheraugment ketonemia in those responsive to therapeuticketosis, especially in those individuals where maintaininglow glucose is important.
AbbreviationsAcAc: acetoacetate; ALS: amyotrophic lateral sclerosis; AD: Alzheimer’s;βHB: beta-hydroxybutyrate; BMS: sodium/potassium βHB mineral salt; BMS +MCT: BMS + MCT 1:1 mixture; BD: 1,3-butanediol; CNS: central nervoussystem; HDL: high density lipoprotein; HDACs: histone deacetylases; LDL: lowdensity lipoprotein; KD: ketogenic diet; KE: 1, 3-butanediol acetoacetate dies-ter/ketone ester; MCT: medium chain triglyceride oil; PCOS: polycystic ovarysyndrome; ROS: reactive oxygen species; SAD: standard American diet;TBI: traumatic brain injury; T2D: type-2 diabetes.
Competing interestsInternational Patent # PCT/US2014/031237, University of South Florida, D.P.D’Agostino, S. Kesl, P. Arnold, “Compositions and Methods for ProducingElevated and Sustained Ketosis”. P. Arnold (Savind) has received financialsupport (ONR N000140610105 and N000140910244) from D.P. D’Agostino(USF) to synthesize ketone esters. The remaining authors have no conflicts ofinterest.
Authors’ contributionsConceived and designed the experiments: SK, AP, NW, TF, DP. Performed theexperiments: SK, AP, NW, TF, CA, JS, AVP. Analyzed the data: SK, AP, DP.Contributed reagents/materials/analysis tools: PA. Helped draft themanuscript: SK, AP, NW, CA, DP. All authors read and approved the finalmanuscript.
AcknowledgementsThe authors would like to thank Savind Inc for manufacturing some of theketone supplements, Jay Dean for the use of his Hyperbaric ResearchLaboratory, and the ONR and Scivation Inc. for funding the project.
GrantsThis study was supported by the Office of Naval Research (ONR) GrantN000140610105 (DPD); and a Morsani College of Medicine Department ofMolecular Pharmacology and Physiology departmental grant.
Author details1Department of Molecular Pharmacology and Physiology, Morsani College ofMedicine, University of South Florida, 12901 Bruce B. Downs Blvd. MDC8,Tampa, FL 33612, USA. 2Savind Inc, 205 South Main Street, Seymore, IL61875, USA.
Received: 10 September 2015 Accepted: 28 January 2016
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Kesl et al. Nutrition & Metabolism (2016) 13:9 Page 15 of 15
ABOUT THE AUTHOR
Shannon Kesl was born and raised in Springfield, Ohio. She attended Wittenberg
University in Springfield, Ohio until Spring 2007. In 2007, she moved to Tampa, FL to continue
her studies in Biomedical Sciences at the University of South Florida; she received her BS in fall
of 2008. In 2010, Shannon began her graduate training as a student in the Ph.D. Program in
Integrated Biomedical Sciences at the Morsani College of Medicine, University of South Florida.
In 2014, she married the love of her life and currently shares her heart with her two dogs Bailey
and Isa. Shannon has performed her dissertation research in the Laboratory of Nutritional and
Metabolic Medicine under the co-mentorship of Dr. Dominic D’Agostino and Dr. Mack Wu.
During her time as a graduate student, she presented her research at many national conferences,
won numerous travel awards, and was a member of ASN, AAA, WHS, and APS societies. She
received her Masters of Science in Medical Science in 2013, and successfully defended her