The Effect of a Ketogenic Diet on Mitochondria Function in Human Skeletal Muscle During

Home , Exogenous ketone, Ketosis, Starvation response

The effect of a ketogenic diet on mitochondria function in human skeletal muscle during

adaptation to chronic exercise training and the potential involvement of metabolic

dysregulation

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy

in the Graduate School of The Ohio State University

Vincent J. Miller, MS

Ohio State University Nutrition Program

The Ohio State University

2019

Dissertation Committee

Jeff S. Volek, PhD, RD, Advisor

William J. Kraemer, PhD

W. David Arnold, MD

Frederick A. Villamena, PhD

Copyrighted by

Vincent J. Miller

2019

Abstract

Objective: The prominent influence of skeletal muscle mitochondria on health and physical capacity can be enhanced through diet and exercise. Ketogenic diets have great potential to drive this enhancement, but prior research is limited, particularly in humans. Therefore, the objective of the present research was to characterize changes in skeletal muscle mitochondria function induced by a ketogenic diet during adaptation to chronic exercise. Methods: Twenty-nine participants completed a 12-week supervised exercise program while following a ketogenic diet

(KD, n=15, males=13) or their habitual mixed diet (MD, n=14, males=12). Body composition was measured using dual-energy x-ray absorptiometry (iDXA, GE Healthcare, Chicago, IL).

Blood was drawn in a fasted and resting state and serum insulin and glucose were measured using an enzyme-linked immunosorbent assay kit (Calbiotech, El Cajon, CA) and a hexokinase reagent set (Pointe Scientific, Canton, MI), respectively. Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated based on insulin and glucose values. Respiratory quotient (RQ) was measured through gas exchange (TruOne 2400, Parvo Medics, Sandy, UT).

Muscle biopsies were collected from the Vastus lateralis, from which mitochondria were isolated. O2 consumption and membrane potential were measured with a Clark-type electrode fitted with a tetraphenylphosphonium electrode. H2O2 and ATP production were measured using fluorescence (Amplex Ultra Red) and luminescence (luciferase) assays, respectively. Each test was repeated with a carbohydrate- (pyruvate), fat- (palmitoyl-L-carnitine), and ketone-based (β-

ii hydroxybutyrate+acetoacetate) substrate. Results: Participants were matched by age, gender, and body fat (KD vs MD: 27.4±1.8 vs 24.6±2.4 yrs, 25.6±1.3% vs 22.0±2.3%). At baseline, HOMA-

IR was greater in KD (2.1±0.3 vs 1.5±0.2, p=0.056) and decreased during the intervention

(2.11±0.3 to 1.11±0.1, p=0.008). Weight loss was greater for KD (-6.9±0.9 vs 0.7±0.4 kg, p<0.001), as was decrease in body fat percentage (-5.4±0.7 vs -0.7±0.5 %, p<0.001). Mean daily blood β-hydroxybutyrate concentration for KD was 1.2±0.2 mM and RQ decreased only in KD

(0.82 to 0.75, p=0.001), all indicating the ketogenic diet induced a profound shift in energy metabolism towards reliance on fat oxidation. An effect of time was observed for increases in mitochondrial protein (p=0.019) and respiratory control ratio (RCR, p=0.003). Time by diet interactions indicate a lesser increase in H2O2 (p=0.098) and a relative increase in ATP production (p=0.003) and efficiency (based on ATP/O2 and ATP/H2O2, p<0.005) for KD. With the fat-based substrate, RCR and ATP production increased only for KD (4.7±0.3 to 5.6±0.2, p=0.009; 20.9±4.2 to 28.4±4.6 nmol/mg/min, p=0.028). ATP production with the ketone-based substrate was 4 to 8 times lower than with other substrates, indicating that ketones are minimally oxidized in human skeletal muscle. Conclusions: While the effects of time for mitochondrial protein and RCR indicate exercise-induced enhancement of mitochondria function, the time by diet interactions for ATP, ATP/O2, and ATP/H2O2 indicate augmentation of this enhancement by the ketogenic diet, particularly in relation to fat oxidation. The improvement in HOMA-IR for

KD suggests that these improvements may have partly been related to rescue of metabolic impairment. Further research is strongly encouraged for determination of mitochondria function as a target through which ketogenic diets improve metabolic health.

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Dedication

Dad, you always wanted me to be a doctor, and even though you meant the other kind of doctor, I know without a doubt there is nobody who would be more proud. I will be forever grateful for everything you have done for me and your memory will forever inspire me to live my life in a way that would make you proud and to help others avoid what stole your brilliant mind. I miss you dearly. 49!

Acknowledgments

A variety of people have made the research in this dissertation possible by providing critical contributions, directly supporting my work, or by influencing my path towards pursuing a

PhD. I would like to acknowledge my sincere appreciation to each of these individuals.

Thank you, Mr. Mandracchia, for inspiring me to care about education. Your interest in my ability was the turning point that converted me from an apathetical high-school student to an avid, life-long learner.

Thank you, Mom, for always believing in me, for pushing me to be the best I can be, and for giving me the gift of persistence.

Thank you, Kayla, for understanding and gracefully accepting the sacrifices involved in this process. As much as I have tried to teach and demonstrate to you the meaning of good character, you continue to teach me instead.

Thank you, Kim, for standing by me for the past two decades. Actions speak louder than words and the sacrifices you have made on my behalf speak volumes. Even when you think my ideas are extreme or obsessive, which they often are, you still support me.

Thank you, Dr. Sarah Everman, for helping me through the decision to pursue a PhD, and thank you, Dr. Paul Arciero, for welcoming me into your lab to gain research experience. I consider you both friends as much as mentors. Each of you were instrumental in helping me get to this point and I will always be grateful.

Thank you, Dr. Jon Parquette, for allowing me to use your freeze dryer, and thank you,

Cassidy Creemer, for your assistance.

Thank you, Jacquie Stewart and Dr. Eric England, for providing me with the animal tissue needed to develop the mitochondria assays that are the foundation of this research.

Thank you to my labmates for your support and assistance, especially Rich LaFountain for co-leading this study with me and Emily Barnhart for supervising the exercise training.

Thank you to our study participants who graciously subjected themselves to the muscle biopsies that made this research possible.

Finally, thank you to my committee members, each of whom supported me throughout this project and helped to make it the best it could be.

Dr. Villamena, thank you for sharing your expertise on oxidative stress. The knowledge I gained from your outstanding course greatly enhanced this project and your support exemplifies the benefit of the interdisciplinary nature of the OSUN program.

Dr. Arnold, thank you for enabling this research to be a reality! The possibility of not finding a physician to perform muscle biopsies was close to becoming a very disappointing reality until you saved the day and managed to fit us into your busy schedule. My involvement with the biopsy procedure was an unexpected experience that turned out to be one of my best memories from this project. Thank you also for your support with other areas of the project and for the opportunities you have given me to expand my research exposure. Although I was unable to follow through with our plans for the motor neuron study, I enjoyed collaborating with you and very much appreciate the opportunities to analyze spinal cord mitochondria and gain exposure to animal research.

Dr. Kraemer, thank you for inspiring me to better understand the implications of mitochondria localization in the myocyte and for expanding my knowledge of endocrinology. I came to Ohio State excited about the prospect of working with you and maintaining kinesiology as part of my focus on nutrition. Having the opportunity to interact with you and your team has been both a pleasure and a privilege. I am grateful for all of your support and honored to have you as a member of my committee.

Dr. Volek, you have been the driving force behind this life-changing process and I will forever be grateful for your wise guidance and generous support. You invited me into your lab despite my limited research experience, and you fully supported my interest in mitochondria research despite it being a complex and completely new area of focus within our team. The sacrifices I made and imposed on my family to pursue a PhD came with tremendous pressure, but the work that you enabled me to do confirms that I made the right choice. Your appreciation for genuine academic pursuit is highly admirable. I feel privileged to have benefited from it and am very proud of having contributed to your mission of advancing scientific understanding of nutritional ketosis and its potential to benefit public health (and service).

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Vita

1993 ……………….. Carmel High School

1997 ……………….. BS Mechanical Engineering, Rensselaer Polytechnic Institute

2012 ……………….. MS Human Nutrition, University of Bridgeport

2014 ……………….. MS Human Movement/Kinesiology, A. T. Still University

2015 to present ……. PhD Ohio State University Nutrition, The Ohio State University

Publications

LaFoundtain, R. A., Miller, V. J., Barnhart, E. C., Hyde, P. N., Crabtree, C. D., McSwiney, F. T., . . . Volek, J. S. Extended ketogenic diet and physical training intervention in military personnel. Submitted to Military Medicine, 2019.

Hyde, P. N., Sapper, T. N., Crabtree, C. D., LaFoutain, R. A., Bowling, M. L., Buga, A., . . . Miller, V. J., . . . Volek, J. S. You are not what you eat: Dietary carbohydrate restriction improves metabolic syndrome independent of weight loss. Submitted to JCI Insight, 2019.

Short, J. A., Bowling, M. L., Hyde, P. N., LaFountain, R. A., Miller, V. J., Dickerson, R. M., . . . Volek, J. S. Effects of a ketone-caffeine supplement on high-intensity exercise performance. Submitted to the Journal of the American College of Nutrition, 2018.

Miller, V. J., Villamena, F. A., & Volek, J. S. (2018). Nutritional ketosis and mitohormesis: Potential implications for mitochondrial function and human health. Journal of Nutrition and Metabolism, 2018, 5157645.

Hyde, P. N., Lustberg, M. B., Miller, V. J., LaFountain, R. A., Volek, J. S. (2017). Pleiotropic effects of nutritional ketosis: Conceptual framework for keto-adaptation as a breast cancer therapy. Cancer Treatment and Research Communications, 12(2017), 32- 39.

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Hyde, P., Miller, V. J., & Volek, J. S. (2016). Keto-adaptation in health & fitness. In Boison, D., D’Agostino, D. P., Kossoff, E. H., & Rho, J. M. (Eds.), Ketogenic diet and metabolic therapies: Expanded roles in health and disease.

Ives, J. I., Norton, C., Miller, V., Minicucci, O., Robinson, J., O’Brien, G., . . . Arciero, P. J. (2016). Multi-modal exercise training and protein-pacing enhances physical performance adaptations independent of growth hormone and BDNF but may be dependent on IGF-1 in exercise-trained men. Growth Hormone & IGF Research, 32, 60- 70

Arciero, P. J., Ives, S. J., Norton, C., Escudero, D., Minicucci, O., O’Brien, G., Paul, M., Ormsbee, M. J., Miller, V., . . . He, F. (2016). Protein-pacing and multi-component exercise training improves physical performance outcomes in exercise-trained women: The PRISE 3 Study. Nutrients, 8(6), E332.

Arciero, P. J., Miller, V. J., & Ward, E. (2015). Performance enhancing diets and the PRISE Protocol to optimize athletic performance. Journal of Nutrition and Metabolism, 2015, 715159.

Fields of Study

Major Field: Ohio State University Nutrition Program

Minor Field: Statistical Data Analysis

Table of Contents

Abstract ...... ii Dedication ...... iv Acknowledgments ...... v Vita ...... viii List of Tables...... xii List of Figures ...... xiii Chapter 1. Introduction ...... 1 Chapter 2. Literature Review: Effects of Ketogenic Diets on Mitochondria Function and the Potential Involvement of Mitohormesis ...... 4 Introduction ...... 4 Nutritional Ketosis ...... 5 Formation of mtROS and Associated Antioxidant Defense ...... 7 Mitohormesis ...... 9 Ketones as Antioxidants and Signaling Molecules ...... 14 Mitochondrial Uncoupling ...... 19 Oxidative Capacity ...... 22 Bioenergetic Signal Transduction ...... 25 AMPK ...... 25 SIRT1 and SIRT3 ...... 29 Direct Involvement of AMPK and Sirtuins in Redox Balance ...... 31 Downstream Bioenergetic and Antioxidant Signaling ...... 31 PGC-1α...... 32 FOXO3a ...... 34 NRF-1, NRF-2, and TFAM ...... 36 NFE2L2 ...... 36 x

Overlap Between Bioenergetic and Antioxidant Signal Transduction ...... 38 Exercise as an Adjunct to Nutritional Ketosis ...... 40 Conclusion...... 43 Chapter 3. Methods ...... 46 Chapter 4. Results ...... 60 Chapter 5. Discussion ...... 77 Bibliography ...... 90

List of Tables

Table 1. Bioenergetic proteins upregulated by ketogenic or low-carbohydrate diets (by species)...... 25 Table 2. Tests with limited sample sizes ...... 57 Table 3. Pre-intervention participant characteristics ...... 60 Table 4. Metabolic markers before and after 12 weeks of a ketogenic diet combined with exercise training ...... 62 Table 5 Comparison of pre-intervention mitochondria function (with fat substrate only) between the highest and lowest tertiles for markers of metabolic dysregulation ...... 73

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List of Figures

Figure 1. β-hydroxybutyrate, and in some cases acetoacetate, contribute to protection against oxidative stress...... 19 Figure 2. Nutritional ketosis may initiate bioenergetic and mitohormetic signaling through multiple mechanisms...... 40 Figure 3. Protein concentration of isolated mitochondria before and after 12 weeks of a ketogenic diet combined with exercise training ...... 63 Figure 4. General function (by substrate) of isolated mitochondria before and after 12 weeks of a ketogenic diet combined with exercise training ...... 64 Figure 5. Capacity and efficiency (by substrate) of isolated mitochondria before and after 12 weeks of a ketogenic diet combined with exercise training ...... 66 Figure 6. Ketone metabolism in isolated mitochondria before and after 12 weeks of a ketogenic diet combined with exercise training ...... 68 Figure 7. Shift in macronutrient metabolism in response to 12 weeks of a ketogenic diet combined with exercise training ...... 70 Figure 8. ATP production in isolated muscle mitochondria in response to 12 weeks of a ketogenic diet combined with exercise...... 74 Figure 9. Metabolic damage before and after 12 weeks of a ketogenic diet combined with exercise training ...... 76

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Chapter 1. Introduction

Although it is commonly understood that mitochondria are the “power house” of the cell, it is easy to take their importance for granted. In skeletal muscle, contraction is a prominent and obvious example of the need for ATP production, which is consistent with the majority of mitochondria in muscle being located near myofibrils [1-6]. Other critical, but less obvious requirements for mitochondrial ATP production include gene expression, cell signaling (protein phosphorylation), and active transport, all of which apply to nearly all human cells. Impaired mitochondria function and the associated decline of ATP availability can therefore lead to cellular dysfunction and, in turn, promote or exacerbate chronic disease [7-9]. Such impairment is often associated with increased mitochondrial production of reactive oxygen species (ROS) and further mitochondrial and cellular dysfunction caused by oxidative damage [8-10].

Mitochondria are therefore more than an energy source, they are foundational to human health.

In skeletal muscle, impaired mitochondria function can decrease physical capacity and likely contributes to the pathogenesis of sarcopenia [10-12]. Mitochondria dysfunction in skeletal muscle is also associated with type 2 diabetes and neuromuscular conditions [13]. In healthy humans, it has been estimated that skeletal muscle accounts for approximately 90% of insulin- mediated glucose uptake [14]. Although it is debatable whether mitochondria dysfunction is a cause or effect of insulin resistance and type 2 diabetes, a decrease in capacity for glucose uptake clearly predisposes to worsening of either condition. Furthermore, insulin resistance is believed

1 to have an important role in the contribution of mitochondria dysfunction to the pathogenesis of sarcopenia [15] and is relevant to a wide variety of degenerative disease beyond the scope of skeletal muscle [16-21].

Mitochondria function can be enhanced by calorie restriction and other nutritional interventions [22]. In particular, dietary carbohydrate restriction is expected to increase mitochondria capacity based on the increased demand for fat oxidation associated with limited carbohydrate availability. Ketogenic diets, which increase the demand for fat oxidation to the extent of inducing ketogenesis, can enhance mitochondria function in a variety of ways

(described extensively in Chapter 2). Exercise also enhances mitochondria function [13, 23], and it is possible that the combination of diet and exercise may have a synergistic effect in this regard

(also described in Chapter 2). A ketogenic diet, coupled with exercise, is therefore a promising approach for the enhancement of mitochondria function and prevention and treatment of chronic health conditions associated with mitochondria dysfunction. This is especially the case for mitochondria dysfunction associated with insulin resistance, as indicated by the impressive efficacy of ketogenic diets for treating conditions closely associated with insulin resistance, including metabolic syndrome [24-26] and type 2 diabetes [27, 28].

Mitochondria function was a secondary focus of the present investigation. The primary aim was to assess the potential for a ketogenic diet to enhance physical and cognitive resilience in military personnel during adaptation to chronic exercise training. A ketogenic diet has particular relevance to this population based on the potential for better maintenance of physical and cognitive performance during limited food availability and faster recovery from strenuous physical exertion [29]. In addition, the prevalence of overweight and obesity in the military is a

2 major concern, affecting two thirds of active-duty military personnel [30, 31], and the high prevalence of overweight and obesity among the general population is also concerning based on the limitations it imposes on military recruitment [32]. Since ketogenic diets are particularly effective for decreasing body fat while maintaining physical capacity [33-39], they may be a valuable solution to this concern. Given the ubiquitous requirement for ATP in human physiology, mitochondria are critical to the resilience of nearly all tissue, including skeletal muscle, which is highly relevant to the general physical resilience required for military duty.

Regardless of military affiliation, prior research on the effects of a ketogenic diet on mitochondria function is limited, particularly in humans. The purpose of the present research is to help fill this gap.

There are several major limitations of previous research that the design of the present investigation was intended to address. As discussed in Chapter 2, a large majority of the research on the influence of dietary carbohydrate restriction on mitochondria function has been done in rodents. In addition, much of the research on ketogenic diets in general has been based on processed chow for animals or extreme ketogenic diets intended for therapeutic purposes in humans. And finally, many prior investigations on ketogenic diets have neglected to account for the time required for full metabolic adaptation, which is at least four weeks [40], if not longer

[41]. By addressing each of these limitations in a single investigation, the present research should make a considerable contribution to advancing the understanding the physiological effects of ketogenic diets.

Chapter 2. Literature Review: Effects of Ketogenic Diets on Mitochondria Function and the Potential Involvement of Mitohormesis

Introduction

All cells of the human body require ATP as the fundamental energy source to support life. Because mitochondria produce the majority of ATP, impaired mitochondrial function is implicated in the majority of today’s most concerning chronic and degenerative health conditions including obesity, cardiovascular disease, cancer, diabetes, sarcopenia, and neurodegenerative diseases [42]. Much of this association between mitochondrial function and disease can be attributed to excessive mitochondrial production of ROS [10].

Although mitochondrial ROS (mtROS) are generally considered harmful, which is certainly the case at high concentrations, modest levels stimulate necessary biological processes such as proliferation, differentiation, and immunity [43]. Adaptations that enhance resistance to oxidative stress are also induced by mtROS [43], possibly decreasing net ROS production during basal metabolism. This adaptive response is called mitohormesis [44-46] and is a promising mechanism through which lifestyle interventions that enhance mitochondrial function may, in turn, enhance resistance to chronic and degenerative diseases.

By dramatically shifting energy metabolism towards ketogenesis and fatty acid oxidation, ketogenic diets are likely to have a profound effect on mitochondrial function. However, despite the rapidly growing amount of research on ketogenic diets and their effects on various disease

4 states, only a small amount of this research has focused on mitochondrial function or oxidative stress. The well-established increase in fat oxidation induced by a ketogenic diet [26, 41] clearly indicates prominent connection with mitochondrial function and, in turn, oxidative stress and mitohormesis [45-47]. Therefore, the purpose of this review is to describe the current, but limited understanding of how ketogenic diets may affect mitochondrial function and resistance to oxidative stress, particularly within the context of extending human healthspan.

Nutritional Ketosis

The use of lifestyle interventions to treat and prevent chronic disease is attractive because of their potential to lower medical costs and produce more robust and holistic improvements in health. Ketogenic diets have been studied sporadically for more than 100 years, but over the last

15 years, a growing number of researchers have contributed to what is now a critical mass of discoveries that link the process of keto-adaptation to a broad range of health benefits [28, 48-

70]. Early clinical research focused on the use of “extreme” versions of ketogenic diets to treat seizures, but recent research indicates that benefits related to the management of epilepsy, weight loss, metabolic syndrome, and type 2 diabetes can be achieved with an approach that is less restrictive in carbohydrate and protein, and therefore more satisfying, sustainable, and feasible for the general population. A “well formulated” ketogenic diet is generally characterized by a total carbohydrate intake of less than 50 g/d and a moderate protein intake of approximately 1.5 g/d per kg of body weight [71]. This typically increases circulating β-hydroxybutyrate (BHB) and acetoacetate (ACA) from concentrations that are typically less than 0.3 mM into the range of nutritional ketosis, which for BHB, we define as 0.5-3 mM [72]. This range is below the typical

5-10 mM range for BHB that occurs during prolonged fasting, and well below concentrations 5 characteristic of ketoacidosis [71, 72]. From the perspective of meeting energy demands, the reduced carbohydrate and moderate protein intakes necessarily make ketogenic diets high in fat.

Despite this contradiction with mainstream dietary guidelines, ketogenic diets may be beneficial for many health conditions, particularly the previously mentioned conditions related to mitochondrial impairment, which includes obesity [48, 49], diabetes [28, 50, 51], cardiovascular disease [52-54], cancer [52, 55-63], neurodegenerative diseases [56, 57, 64-67], and even aging

[68-70, 73, 74].

In both the nutrition literature and public dietary guidelines, non-starchy vegetables are one of the few dietary components nearly unanimously agreed upon as healthful. Given their health-supporting characteristics and low carbohydrate content, they should be a prominent component of any ketogenic diet. Beyond the primary features of a well-formulated ketogenic diet, such as macronutrient proportions, adequate mineral intake, and appropriate selection of fat sources, which have been discussed more thoroughly elsewhere [71, 72], inclusion of non- starchy vegetables is an important consideration given that reports in the literature of adverse effects resulting from ketogenic diets are often associated with extreme implementations that typically lack plant matter. In fact, for this reason, it has recently been recommended to increase the non-starchy vegetable content of ketogenic diets used to treat epilepsy [75]. Beyond adding variety to the diet, benefits of non-starchy vegetables that may be particularly relevant to nutritional ketosis include the maintenance of adequate micronutrient status and the presence of prebiotic fiber as substrate for the gut microbiota. In addition to the importance of prebiotic fiber for basic health, the short-chain fatty acids produced by the gut microbiota from this dietary fiber support ketogenesis [76] and metabolic signaling related to mitochondrial function and

6 antioxidant defense [77]. Furthermore, non-starchy vegetables are a source of the many micronutrients needed to support energy metabolism. As such, there is more to a ketogenic diet than simply restricting carbohydrate. Selection of a variety of nutrient-dense foods is an important component of nutritional ketosis that should be given consideration in any clinical or academic implementation.

Formation of mtROS and Associated Antioxidant Defense

As with other sources of oxidative stress, mtROS can damage enzymes and cell membranes and subsequently facilitate the pathogenesis of chronic disease [78]. Oxidative damage to mitochondrial DNA (mtDNA) is of particular concern because of its proximity to the electron transport chain (mtETC). Furthermore, compared to nuclear DNA, mtDNA is more prone to oxidative damage and is not repaired as effectively [79-82], although this has been debated based on more recent evidence [83-87]. Unrepaired mtDNA damage leads to mitochondrial dysfunction, which is implicated in the pathogenesis of a variety of chronic diseases [88] and associated with shorter lifespan [89]. Therefore, while moderate levels of mtROS have important roles in signaling and health, protection against excessive levels is also important.

Although there are numerous sites of mtROS formation, the most prominent are those in

•- the mtETC, where superoxide (O2 ) is formed through reduction of O2 by leaked electrons,

•- particularly at complexes I and III [78, 90-92]. Production of O2 at complex I is particularly high during reverse electron transport (RET), which occurs when a high protonmotive force (Δp) develops across the inner mitochondrial membrane in conjunction with the pool of coenzyme Q

(CoQ) being in a highly reduced state (i.e. mostly present as ubiquinol) as a result of electron 7 transfer through complex II and electron transfer flavoprotein:ubiquinone oxidoreductase (ETF-

QO) [93-99]. This dependence of mtROS production on Δp during RET is mainly influenced by proton gradient (ΔpH) [96].

•- Formation of O2 at complexes I and III primarily occurs in the mitochondrial matrix, but

•- some of the O2 produced at complex III is produced in the intermembrane space [100].

•- Within the matrix, O2 is rapidly dismutated into hydrogen peroxide (H2O2) by manganese

•- superoxide dismutase (SOD2) [78, 90]. Some O2 may escape into the mitochondrial intermembrane space [101] and cytosol [102], where copper/zinc superoxide dismutase (SOD1) can dismutate it into H2O2 [78]. The large majority of mitochondrial H2O2 is removed by peroxiredoxin (Prx) 3, followed by much smaller contributions from Prx5 and glutathione peroxidases (GPx) 1 and 4 [103]. GPx also removes other peroxides, including lipid hydroperoxides [78]. Catalase is another antioxidant enzyme capable of removing H2O2, but is primarily located in peroxisomes and is therefore unlikely to directly remove mitochondrial H2O2

[78, 103]. However, H2O2 can be transported out of mitochondria [104], and it is possible that the majority of mitochondrial H2O2 is removed in the cytosol. Since Prxs and GPxs rely on NADPH for recycling of their cofactors (thioredoxins and glutathione, respectively) [78], and since

NADH is required for recycling of NADPH [105], activity of these enzymes would decrease availability of NADH for oxidative phosphorylation. Therefore, transport of H2O2 out of mitochondria for removal in the cytosol may be a more likely defense mechanism [104], implying a more important role of catalase and other antioxidant enzymes outside of mitochondria. Despite the lower reactivity of H2O2, it is still reactive and can oxidize metal ions, particularly iron, to form the hydroxyl radical (•OH), which readily damages DNA, lipids, and

8 proteins [78]. •OH is scavenged by metallothionein I and II [106, 107] and glutatathione [108], indicating that these antioxidant proteins may be important defenses against byproducts of unaddressed mtROS. Other important antioxidant enzymes include glutamate-cysteine ligase

(GCL), which is the rate limiting step in glutathione synthesis, and glutathione reductase (GSR) and thioredoxin reductase (TRXR), which recycle glutathione and thioredoxin, respectively, to their reduced forms [78].

Mitohormesis

Increased reliance on mitochondrial respiration will increase the flow of electrons through the mtETC and, in turn, increase potential for mtROS formation. Although oxidative stress is traditionally viewed as harmful, a modest increase in ROS is now established as a signaling stimulus that induces hormetic adaptation [43]. In regard to mitohormesis and mtROS, such adaptation is largely centered around antioxidant defense [44-46], making mitohormesis an attractive target for the prevention and treatment of chronic disease.

Although mitohormesis has not been studied comprehensively in higher-level organisms, its occurrence is supported by compelling evidence in lower-level organisms. For example, inhibition of glycolysis in C. elegans increased fat oxidation (based on nematode triglyceride content) and mitochondrial O2 consumption, which was followed by increases in ROS production at day 2 and catalase activity at day 6 [109]. The increase in catalase activity occurred in conjunction with increases in lifespan and resistance to the mitochondrial stressors sodium azide and paraquat. However, antioxidant treatment (N-acetylcysteine) decreased the elevation of

ROS at day 2 and eliminated the resistance to sodium azide and paraquat treatments, indicating a requirement of ROS as a stimulus [109]. 9

In a subsequent series of experiments, glucose metabolism in C. elegans was inhibited by knockout of the insulin receptor, insulin-like growth factor 1 (IGF-1) receptor, and insulin receptor substrate 1 (IRS-1) [110]. Consistent with the previous study [109], inhibition of glucose metabolism increased mitochondrial respiration concomitant with ROS-dependent increases in lifespan, stress resistance, and antioxidant enzyme activity. However, in this case, detection of ROS was mitochondria-specific, and repeated measures allowed for changes in antioxidant enzyme activities to be evaluated more closely in relation to the timing of changes in mtROS. Compared to controls, inhibition of glucose metabolism resulted in higher mitochondrial

O2 consumption at 12 h, higher mtROS production at 24 h, and higher activities of SOD and catalase at 48 h, suggesting a dependence of antioxidant activity on mtROS and a dependence of mtROS on mitochondrial respiration. The most striking result is the lower mtROS at 120 h, indicating that the initial increase in mtROS and subsequent increase in antioxidant enzyme activity ultimately lowered net mtROS production to a level lower than controls, which is the proposed explanation for the more than 2-fold increase in lifespan. As with the previous study, this demonstration of mitohormesis is further supported by the changes in ROS production, antioxidant enzyme activity, and lifespan having been prevented with antioxidant treatment.

The occurrence of mitohormesis is further supported by the potential for mtROS to simultaneously induce bioenergetic and antioxidant adaptations through a single signaling mediator. As discussed later in this review, this mediator is the transcription factor peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), and its role in simultaneously inducing bioenergetic and antioxidant adaptations has been demonstrated in several experimental models of mtROS production, including treatments with paraquat and H2O2. Paraquat is an

10 herbicide that is reduced by the mtETC and subsequently initiates mtROS production by reacting

•- with O2 to produce O2 [111, 112], and H2O2 is a common form of mtROS. Treatment of a fibroblast cell line (10T1/2) with H2O2 has induced expression of both antioxidant enzymes

(SOD1, SOD2, and GPx1) and proteins involved in mitochondrial oxidative phosphorylation, all in a manner largely dependent on PGC-1α [113]. Demonstrating the hormetic benefit of this response in a variety of brain cells, overexpression of PGC-1α protected against cell death induced by H2O2 or paraquat treatment, and this occurred in conjunction with changes in gene expression similar to those observed with the 10T1/2 cells [113]. Although the central role of

PGC-1α in linking mitochondrial bioenergetics with antioxidant defense appears to not have been thoroughly investigated in vivo, some suggestive evidence does exist. In skeletal muscle of mice treated with paraquat, content of proteins involved in mitochondrial oxidative phosphorylation and uncoupling have been found to increase in conjunction with greater nuclear localization of PGC-1α [114]. Traditional antioxidant proteins were not measured, but as will be discussed later, the increase in uncoupling proteins can be regarded as an indication of enhanced antioxidant defense based on the potential of these proteins to decrease mtROS production.

Ketogenic and low-carbohydrate diets greatly increase reliance on fat oxidation [115-

126], which would logically be expected to increase mitochondrial respiration and mtROS production and, in turn, induce mitohormesis. Furthermore, mtROS produced through RET appears to have particular relevance to hormetic adaptation, including increased lifespan [127].

Nutritional ketosis is likely to increase RET by altering the FADH2 to NADH ratio. As the primary source of acetyl CoA shifts from glycolysis to β-oxidation and ketolysis, this ratio increases, more than doubling for β-oxidation of longer-chain fatty acids. Electrons from FADH2

11 reduce the CoQ pool through complex II and ETF-QO, thereby increasing RET [128, 129]. This induction of RET by alteration of substrate availability can also be influenced by configuration of mtETC complexes into supercomplexes [127]. The greater potential for mtROS production through RET is consistent with evidence of mitochondria producing more H2O2 during oxidation of palmitoyl carnitine versus pyruvate [130, 131]. Furthermore, succinate is generated during ketolysis by succinyl-CoA:3-oxoacid CoA-transferase (SCOT), which also promotes RET by reducing the CoQ pool through complex II. Demonstrating the likely role of RET in mitohormesis, particularly within the context of nutritional ketosis, extension of lifespan in C. elegans through BHB treatment is dependent on both complex I function and expression of bioenergetic and antioxidant proteins [132].

A study on hippocampal mitochondrial function in rats more directly supports the induction of mitohormesis by a ketogenic diet. After the first day of the diet (Bio-Serv F3666),

H2O2 production by isolated mitochondria was increased [133]. After the third day, mitochondrial levels of oxidized glutathione (GSSG) and hippocampal levels of 4-hydroxy-2- nonenal (4-HNE), both indicators of oxidative stress, were also increased. However, at completion of the first week, upregulation of antioxidant signaling occurred, indicated by increased nuclear content and transcriptional activity of nuclear factor erythroid-derived 2-like 2

(NFE2L2), which persisted through the remainder of the study. By the third week, mitochondrial

H2O2 production decreased to below baseline [133]. In the liver, content of reduced acetyl CoA, which is indicative of mitochondrial redox status, decreased after three days of the ketogenic diet, but increased relative to the control diet after three weeks, indicating an initial increase in oxidative stress followed by a decrease [133]. This was in conjunction with changes in NFE2L2

12 nuclear content and transcriptional activity similar to those observed in the hippocampus. As with the previously described C. elegans experiments, the time course of these observations is a strong indication of mitohormesis, and the similarity in results between the liver and hippocampus suggests that a ketogenic diet can induce mitohormesis in a variety of tissues.

Several other rodent studies provide additional evidence of ketogenic diets upregulating antioxidant defense, but without enough data to convincingly attribute the results to mitohormesis. Content of SOD2 has increased in the livers of mice fed a ketogenic diet (% energy: 89 fat, < 1 carbohydrate, 10 protein), which occurred in conjunction with increased median lifespan and decreases in tumors and age-associated losses of physical and cognitive performance [73]. In addition, activity of GCL and the protein content of its two subunits increased in the hippocampal homogenate of rats fed a ketogenic diet (Bio-Serv F3666) for 3 weeks [134]. This was in conjunction with higher levels of reduced glutathione (GSH) and lower

ROS production in hippocampal mitochondria. The ketogenic diet also increased resistance to mtDNA damage in hippocampal mitochondria exposed to H2O2 [134]. Consistent with these results, total antioxidant capacity and activities of GPx and catalase were increased in hippocampal homogenate of rats fed a ketogenic diet (% energy: 86 fat, <1 carbohydrate, 13 protein) for 8 weeks [135]. In cortical homogenate of rats induced with traumatic brain injury, a ketogenic diet increased cytosolic and mitochondrial protein contents of NAD(P)H:quinone oxidoreductase 1 (NQO1) and SOD1, as well as mitochondrial protein content of SOD2, and also prevented mitochondrial oxidative damage (indicated by 4-HNE) [136].

Additional evidence, although disparate and primarily based on neuronal mitochondrial function related to epileptic seizures, further supports the potential for nutritional ketosis to

13 induce mitohormesis [47]. Much of this is based on signal transduction, antioxidant defense, and oxidative capacity, all of which will be discussed in proceeding sections.

Ketones as Antioxidants and Signaling Molecules

Although ketones may not induce mitohormesis directly, they do influence antioxidant defense (Figure 1). Furthermore, ketone metabolism is highly relevant to mitochondrial adaptation since the ketogenic and ketolytic enzymes needed to support nutritional ketosis are located in mitochondria.

BHB, in addition to being an important energy substrate, is also a signaling molecule

[137-139]. Although not induced through mtROS, BHB inhibits class I and II histone deacetylases (HDACs) in a dose-dependent manner, resulting in greater histone acetylation regardless of whether BHB is elevated through fasting, caloric restriction, or infusion [140]. This inhibition is associated with increased expression of forkhead box O (FOXO) 3a and metallothionein II and increased protein content of FOXO3a, SOD2, and catalase [140].

Consistent with these changes, the kidneys of mice with elevated blood BHB concentrations

(~1.2 mM) were protected from paraquat-induced (50 mg/kg) oxidative damage to proteins and lipids, which was indicated by lower levels of protein carbonyls, 4-HNE, and lipid peroxides

[140]. Upregulation of antioxidant defense by BHB-induced HDAC inhibition also appears to be the mechanism through which exogenous BHB extends lifespan in C. elegans [132]. The dependence of this response on FOXO3a, NFE2L2, and several bioenergetic signaling proteins that influence the activities of these two transcription factors [132] is indicative of the overlap between bioenergetics and antioxidant defense that is characteristic of mitohormesis.

Additional indications of exogenous BHB upregulating antioxidant defense have been observed, although without consideration of HDAC inhibition. In rats, injection of BHB has increased activities of SOD and catalase and prevented the increase in lipid peroxidation and decreases in SOD, catalase, and GSH induced by paraquat injection, all of which was observed in kidney homogenate [141]. Furthermore, BHB also prevented the paraquat-induced decrease in nuclear NFE2L2, indicating involvement of antioxidant signaling [141]. Similarly, BHB treatment has increased FOXO3a, SOD2, and catalase content in cardiomyocytes [142], indicating that BHB may also influence antioxidant defense in the heart. In this study, BHB also prevented the decrease of FOXO3a, SOD2, and catalase content that resulted from H2O2 treatment [142]. Despite the amount of research that has been done on the antiseizure mechanisms of ketogenic diets, the influence of BHB on HDAC inhibition and related antioxidant defense appears to have not yet been investigated in brain tissue. However, BHB appears to inhibit HDAC2 in microvascular and neuronal brain cells [143], and BHB-induced

HDAC inhibition is thought to have a role in the antiseizure effects of ketogenic diets [144].

In addition to BHB inducing upregulation of antioxidant defense, ketones have direct antioxidant capacity. BHB scavenges •OH, as does ACA, although to a lesser extent [145]. The applicability of this antioxidant capacity has been investigated in vitro and in vivo in the context of hypoglycemia. In cultured hippocampal neurons, treatment with BHB or ACA decreased ROS during hypoglycemia induced through inhibition of glycolysis, and in hypoglycemic rats, infusion of BHB decreased hippocampal lipid peroxidation [145].

More specific to mitochondrial function, treatment with BHB+ACA (1 mM each) has

•- decreased O2 production in isolated rat neuronal mitochondria following glutamate exposure

[146]. This occurred in conjunction with decreased NADH levels, suggesting that ketones may additionally decrease mtROS production by enhancing NADH oxidation and, in turn, decreasing

•- •- mitochondrial Δp and associated O2 production. The observed decrease in mitochondrial O2 production occurred independently of glutathione [146], but in isolated and stunned hearts from guinea pigs, treatment with 5 mM ACA increased GSH and the NADPH/NADP+ ratio [147], suggesting that glutathione may be involved to some extent.

Ketones also influence mtROS production through alteration of electron transport.

Treatment of rat hippocampal slices with BHB+ACA (1 mM each) prevented the increase in mtROS and mitigated the decrease in ATP production that otherwise result from inhibition of mtETC complex I with rotenone [148]. In mitochondria isolated from the brains of mice injected with BHB, although inhibition of complex I with rotenone and 1-methyl-4-phenylpyridinium increased rather than decreased mtROS production, the BHB treatment prevented the decrease in

O2 consumption caused by inhibition of complex I, and this occurred independently of uncoupling [149]. Consistent with the results from hippocampal brain slices, the BHB treatment also mitigated the decrease in ATP production caused by complex I inhibition [149]. These effects were prevented by inhibition of complex II with 3-nitropropionic acid or malonate, indicating that BHB primarily influences mitochondrial respiration at complex II [149], which is consistent with ketolysis increasing formation of succinate and FADH2. However, in mutated cells prone to complex I disassembly and an associated severe decrease in complex I activity, treatment with BHB+ACA (5 mM each) increased both the assembly and activity of complex I

[150], indicating that ketones somehow promote repair of complex I damage and may therefore influence mitochondrial respiration at more than one site.

Another aspect of mitochondrial function influenced by ketones is the mitochondrial permeability transition pore (mPTP). Prolonged opening of the mPTP is one of the mechanisms through which mtROS can induce cellular injury and promote disease [151]. In neurons isolated from rat brain slices, treatment with BHB+ACA has decreased the mtROS production, mPTP opening, and cell death induced by H2O2 [152]. This protective effect was duplicated with catalase, even in conjunction with diamide-induced opening of the mPTP, indicating that the protective effect of BHB and ACA is at least partly due to defense against ROS [152]. In a mouse model of epilepsy, this decrease in mPTP opening was found to be induced exclusively by

BHB, and in a manner dependent on the cyclophilin D subunit of the mPTP [153]. BHB in combination with ACA also appears to promote opening of mitochondrial ATP-sensitive K+

+ (mtKATP) channels [154], which in heart mitochondria is known to protect against Ca overload

[155] and dissipate ΔΨ [156]. Since high ΔΨ promotes mtROS production, dissipation of ΔΨ through mtKATP channels may partly explain the potential for ketones to decrease mtROS production. However, opening of mtKATP channels by pinacidil decreases mitochondrial ATP production [156], which is consistent with dissipation of ΔΨ and suggests a compromise between

ATP and mtROS production.

Ketones may also be important, or even necessary, for the bioenergetic signaling associated with mitohormesis. As will be discussed later, peroxisome proliferator-activated receptor α (PPARα) is a nuclear receptor that is responsible for many of the bioenergetic adaptations associated with nutritional ketosis and mitohormesis [157]. In mice, a ketogenic diet

(90% fat, 0% carbohydrate, 10% protein) increased blood BHB concentration to 1-2 mM and upregulated expression of numerous PPARα targets in the liver [74]. However, in mice fed a

17 non-ketogenic low-carbohydrate diet (% energy: 75 fat, 15 carbohydrate, 10 protein), which did not raise blood concentration of BHB, the increased expression of PPARα targets did not occur

[74], implying that induction of PPARα signaling by a ketogenic diet is dependent on ketones.

This response may be, at least in part, a result of the epigenetic effects of BHB. In addition to

HDAC inhibition, BHB also influences gene expression through β-hydroxybutyrylation of histone lysine residues [158]. In the livers of mice subjected to prolonged fasting, this β- hydroxybutyrylation has been associated with upregulation of PPAR signaling, oxidative phosphorylation, fatty acid metabolism, the proteasome, and amino acid metabolism related to redox balance [158]. Upregulation of these pathways is largely influenced by β- hydroxybutyrylation of histone residue H3K9 [158], which is also involved in the upregulation of antioxidant defense through BHB-induced HDAC inhibition [140]. This potential for BHB to influence expression of both mitochondrial and antioxidant genes through a common histone residue is further indication of the overlap between bioenergetics and antioxidant defense and suggests that if mitohormesis is indeed induced during nutritional ketosis, induction may be dependent on ketones and may therefore not occur during a low-carbohydrate diet that is not ketogenic.

In regard to the practicality of BHB signaling, many of the outcomes described above, including HDAC inhibition, were achieved with BHB concentrations within the range of 0.6-2 mM [74, 140, 142, 145, 146, 148, 149, 153], which is well within the physiological range of nutritional ketosis for humans and even suggests potential benefit at low to moderate levels.

Figure 1. β-hydroxybutyrate, and in some cases acetoacetate, contribute to protection against oxidative stress by decreasing production of mitochondrial reactive oxygen species (mtROS), increasing expression or protein content of antioxidant enzymes through histone deacetylase (HDAC) inhibition, and directly scavenging ·OH. Upregulation of antioxidant enzymes through HDAC inhibition includes manganese superoxide dismutase (SOD2), catalase, and metallothionein II, and is likely mediated by the transcription factor forkhead box O 3a (FOXO3a).

Mitochondrial Uncoupling

As previously discussed, RET is a prominent source of mtROS and is dependent on a high Δp across the inner mitochondrial membrane. During ATP production, Δp is dissipated as

H+ enters the mitochondrial matrix through ATP synthase. Mitochondrial uncoupling also dissipates Δp, but by allowing translocation of H+ into the matrix independent of ATP synthase.

Uncoupling is therefore regarded as an antioxidant defense in that it can mitigate mtROS production [159-163]. In fact, only a small dissipation of ΔΨ or ΔpH (components of Δp) is needed for a large decrease in mtROS production [94-97, 164].

Mitochondrial uncoupling is primarily facilitated by uncoupling proteins (UCPs) and adenine nucleotide translocase (ANT) [161, 165, 166]. Although UCP1 is primarily expressed in brown adipose, UCP2 is expressed across a wide variety of tissues, and expression of UCP3 appears to be limited to skeletal muscle and the heart [167]. Knockout of UCP2 [168] or UCP3

[131, 169] increases mtROS production, and both proteins are inactivated through glutathionylation by GSH [170], further establishing their involvement in antioxidant defense.

UCP2 and UCP3 may also be activated by products of lipid peroxidation induced by mtROS

[159]. However, the potential for UCP2 and UCP3 to reduce mtROS through uncoupling is not fully agreed upon [165]; UCPs may alternatively protect against oxidative damage merely by exporting lipid hydroperoxides [165]. Furthermore, UCP3 is less abundant in type I and type IIa muscle fibers [171], which are more oxidative, and its expression and content are further decreased by endurance exercise training [172, 173], suggesting that UCP3 may not be a primary defense against mtROS.

Although the primary purpose of ANT is to exchange newly synthesized ATP in the mitochondrial matrix for cytosolic ADP [166], it shares a common feature with UCPs and other inner membrane proteins in that they translocate anions, including fatty acids. The uncoupling mechanism of ANT, along with UCP2 and UCP3, may be the exchange of protonated fatty acids from the mitochondrial intermembrane space for fatty acid anions in the matrix, thereby dissipating Δp [160, 174-176]. Inhibition studies indicate that ANT may contribute more to uncoupling than UCPs [177, 178].

Independent of nutritional ketosis, increased dietary fat intake increases expression of

UCP2 and UCP3 in muscle [179], and fatty acids facilitate uncoupling through UCP2 [180, 181],

UCP3 [131, 180, 181], and ANT [182]. Given the high fat intake that is characteristic of a ketogenic diet, it is logical to expect nutritional ketosis to increase mitochondrial uncoupling.

Certain ionophores are capable of completely uncoupling mitochondria by transporting

H+ across the inner membrane. Such ionophores are therefore commonly used to measure maximal mitochondrial respiration. In mice fed a ketogenic diet (Bio-Serv F3666, ~6:1 ratio of fat to carbohydrate + protein) for 6 days, respiration of hippocampal mitochondria was fully uncoupled with the ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP)

[183]. The ratio of respiration during oxidation of palmitic acid to maximally uncoupled respiration induced by FCCP was greater in response to the ketogenic diet, indicating increased uncoupling [183]. Although this interpretation relies on the assumption that ATP production was not changed by diet, it is further supported by the higher levels of UCP2, UCP4, and UCP5 detected in mitochondria after the ketogenic diet. Furthermore, mtROS production was lower in the ketogenic diet group [183], supporting the role of uncoupling as an antioxidant defense.

Although not based on direct measurement of mitochondrial function, in rats fed a ketogenic diet

(% energy: 89.5 fat, 0.1 carbohydrate, 10.4 protein), increased uncoupling in response to nutritional ketosis is further indicated by increases in fat oxidation and overall O2 consumption occurring in conjunction with decreases in CO2 production and energy expenditure [126].

However, based on observations of greater palmitate-induced uncoupling (determined by measurement of ΔΨ) during state 4 respiration in rats fed a high-fat, low carbohydrate diet (% energy: 50 fat, 21 carbohydrate, 29 protein) [184] that was likely too high in carbohydrate and protein to induce nutritional ketosis, it is possible that moderate carbohydrate restriction may increase mitochondrial uncoupling independently of ketones.

Several additional rodent studies have shown ketogenic diets to increase protein content of UCPs. However, since mitochondrial function was not measured in these studies, it is not known if uncoupling was affected by these changes in UCP content. In obese mice fed a ketogenic diet (0.4% of energy as carbohydrate), expression of UCP1 and UCP2 increased in adipose and the liver, respectively [185]. Similarly, expression of UCP1 has increased in brown adipose of mice fed a low-carbohydrate diet (18.5% of energy) supplemented with ketone esters

(6% w/v) [186]. The increase in hepatic UCP2 expression during a ketogenic diet has been demonstrated by other studies as well [74, 187, 188]. Ketogenic diets also induce expression of

UCP3 in skeletal muscle. In rats fed a ketogenic diet (% energy: 78.1 fat, 0 carbohydrate, 21.9 protein) for 8 weeks, UCP3 expression increased in the soleus but not the extensor digitorum longus, which is consistent with the soleus containing mostly oxidative, type I muscle fibers

[189]. In humans, glycogen depleting exercise followed by two days of a low-carbohydrate diet

(0.7 g/kg body mass) increased UCP3 expression in the vastus lateralus [190].

Oxidative Capacity

As the rate of oxidative phosphorylation approaches the capacity of the mtETC, Δp will increase and facilitate mtROS production [90]. Higher oxidative capacity should therefore decrease the potential for mtROS production and subsequent oxidative damage. Furthermore, greater oxidative capacity may compensate for the resulting decrease in efficiency of ATP production associated with increased mitochondrial uncoupling. Since oxidative phosphorylation occurs exclusively in mitochondria, mitochondrial density is a key determinant of oxidative capacity [191].

As previously mentioned, numerous studies have demonstrated a profound increase in fat oxidation in response to ketogenic and low-carbohydrate diets. Some studies have even shown an increase in O2 consumption [185, 192-195]. However, fats contain fewer O2 molecules per carbon than carbohydrates, thereby necessitating greater O2 intake to produce the same amount of energy [196]. Furthermore, since β-oxidation and ketolysis produce a greater proportion of

FADH2 to NADH, the resulting decrease in passage of electrons through complex I decreases potential for ATP production per unit of O2 consumption [197]. Increased O2 consumption in response to a ketogenic diet may therefore merely be an effect of the differences in the metabolism and molecular structures of fat and carbohydrate rather than a true indication of increased capacity for oxidative phosphorylation. However, in rat hearts perfused with glucose, the addition of ketones has decreased O2 consumption [198]. This discrepancy may be related to variations in mitochondrial uncoupling. Either way, several studies have shown ketogenic diets to increase mitochondrial content, and numerous studies have shown these diets to increase expression, content, or activity of mitochondrial proteins involved in oxidative phosphorylation and fat oxidation. Compared to O2 consumption alone, these findings provide more conclusive support for an increase in oxidative capacity in response to nutritional ketosis.

In rats fed a ketogenic diet (Bio-Serv F3666) for 22 days, mitochondrial density

(determined by electron microscopy) in the hippocampus increased in conjunction with increased transcription of 39 of the 42 mitochondrial proteins analyzed [199]. Similarly, mitochondrial content (mtDNA copy number) increased in skeletal muscle of mice fed a ketogenic diet

(Research Diets D05052004, % energy: 89.5 fat, 0.1 carbohydrate, 10.4 protein) for 10 months

[200]. Higher mtDNA copy number was also observed in skeletal muscle of rats fed a high-fat,

23 low-carbohydrate diet (% energy: 60 fat, 20 carbohydrate, 20 protein) for 4 weeks in conjunction with daily injections of heparin (0.5 U/g) heparin to increase circulation of fatty acids [124]. In humans, after just 3 days of a low-carbohydrate, high-fat diet (50% fat, 34% carbohydrate, 16% protein), fat oxidation significantly increased and 49% of the variance was explained by mtDNA content [116]. Despite this, the content of mtDNA did not change significantly, but this was expected given the brief duration of the diet.

As will be discussed in proceeding sections, many of the signaling proteins involved in regulating antioxidant defense also regulate oxidative phosphorylation and fat oxidation. There is abundant evidence (Table 1) showing ketogenic and low-carbohydrate diets to increase expression, content, or activity of many targets of these signaling proteins, further indicating increased oxidative capacity. It is particularly striking that ketogenic or low-carbohydrate diets upregulate expression of proteins associated with each of the five mtETC complexes.

Table 1. Bioenergetic proteins upregulated by ketogenic or low-carbohydrate diets (by species) Oxidative Phosphorylation NADH dehydrogenase (complex I) Rat [199] Succinate dehydrogenase (complex II) Mouse [186], Rat [124, 199, 201, 202] Cytochrome c reductase (complex III) Rat [199] Cytochrome c oxidase (complex IV) Mouse [186], Rat [124, 199] ATP synthase (complex V) Rat [124, 199] Cytochrome c Mouse [186], Rat [199] Citric Acid Cycle Citrate synthase Rat [193, 203] Isocitrate dehydrogenase Rat [199] Succinate dehydrogenase (complex II) Mouse [186], Rat [124, 199, 201, 202] Malate dehydrogenase Rat [199, 202] Fatty Acid Oxidation Carnitine palmitoyltransferase Human [204, 205], Mouse [73, 74], Rat [124, 189, 206] Medium-chain acyl-CoA dehydrogenase (MCAD) Mouse [73, 185, 207], Rat [124] Long-chain acyl-CoA dehydrogenase (LCAD) Mouse [185, 188], Rat [124] Very-long-chain acyl-CoA dehydrogenase (VLCAD) Rat [124] β-hydroxyacyl-CoA dehydrogenase (β-HAD) Human [205, 208, 209], Mouse [185, 187, 188] , Rat [189, 193, 202, 203, 206, 210] Ketolysis β-hydroxybutyrate dehydrogenase Mouse [185, 187, 188]

Bioenergetic Signal Transduction

Perturbations in bioenergetic homeostasis induce signal transduction that leads to upregulation of mitochondrial capacity and antioxidant defense. Three key enzymes involved in the sensing of these perturbations and the subsequent induction of signal transduction are AMP- activated protein kinase (AMPK) and silent mating type information regulation 2 homologues 1 and 3 (SIRT1 and SIRT3).

AMPK

AMPK is activated through phosphorylation of the Thr172 residue of the AMPK α catalytic subunit [211-213], and this phosphorylation is largely regulated by molecules related to

25 bioenergetic homeostasis including AMP, ADP, catecholamines, adiponectin, glycogen, and insulin. In general, AMPK is activated by energy deficit and induces signaling that upregulates energy production. AMP and ADP are direct byproducts of energy depletion while adiponectin and catecholamines serve as endocrine signals to increase energy production, often in response to energy depletion. In contrast, indications of energy surplus, such as glycogen and insulin, inhibit activation of AMPK. Nutritional ketosis increases the aforementioned factors that activate

AMPK and decreases those that inhibit AMPK, suggesting that nutritional ketosis is similar to caloric restriction in inducing a signal of energy depletion.

AMP competes with ATP for binding to the γ regulatory subunit of AMPK [214, 215], and by doing so, greatly increases AMPK activity, but only in the presence of an upstream kinase such as liver kinase B1 (LKB1) [216]. This binding of AMP to the γ subunit appears to promote

AMPK activity through at least two mechanisms: facilitated phosphorylation of the α subunit

[217-220] and inhibition of dephosphorylation by protein phosphatases 2Cα and 2Ac [216, 218,

220, 221]. ADP also binds to the γ subunit of AMPK to inhibit dephosphorylation [220, 222,

223] and possibly facilitate phosphorylation [222]. This is important to the energy sensing sensitivity of AMPK based on the much higher physiological concentration of ADP compared to

AMP [223]. Data on changes in AMP and ADP levels in response to a ketogenic diet are lacking.

However, the decreased availability of carbohydrate and increased mitochondrial uncoupling

(previously described) during nutritional ketosis suggest a decline in ATP production, at least until compensatory adaptations occur. A decline in ATP implies a relative increase in AMP and

ADP, which would facilitate AMPK phosphorylation and activation. In addition, ketogenic diets

26 are commonly reported to have a satiating effect [224], which may further increase the AMP and

ADP to ATP ratios through spontaneous caloric restriction.

Adiponectin increases AMPK activity in skeletal muscle [225, 226] and the liver [226] by promoting Thr172 phosphorylation, likely in response to an increase in the AMP to ATP ratio

[226]. Similarly, α-adrenergic signaling increases AMPK activity in skeletal [227] and cardiac muscle [228], and β-adrenergic signaling increases AMPK activity in adipose [229, 230], all through promotion of Thr172 phosphorylation. While activation through β-adrenergic signaling appears to involve the AMP to ATP ratio [229], α-adrenergic signaling appears to work independently of AMP and ATP [227]. Increases in adiponectin have been observed during ketogenic or low-carbohydrate diets, although primarily in obese individuals [231-233]. BHB induces adiponectin secretion in adipocytes [234], indicating that the level of nutritional ketosis may be an important determinant of the extent to which ketogenic diets influence AMPK activity through adiponectin. In regard to catecholamines, epinephrine increases during fasting, and this appears to be dependent on carbohydrate restriction [235], implying that epinephrine is likely to be elevated during nutritional ketosis. Consistent with this, dietary carbohydrate restriction increases catecholamines at rest [192, 236] and in response to exercise [192, 236-239]. This may be, at least in part, a result of glycogen depletion [237, 240], suggesting both a direct and indirect effect of glycogen on AMPK activity. The potential for nutritional ketosis to increase catecholamines is further supported by the dependency of the antiseizure effects of ketogenic diets on norepinephrine [241].

Glycogen influences AMPK activity by binding to a glycogen binding domain on the β regulatory subunit of AMPK [242, 243]. In human and rodent skeletal muscle, AMPK activity is

27 lower when glycogen is bound to this domain [244, 245] and higher when muscle is depleted of glycogen [246-249]. In direct contrast to the effect of AMP and ADP, glycogen inhibits the phosphorylation of AMPK by upstream kinases such as LKB1 [250]. Although muscle glycogen concentration has recently been demonstrated to be similar in ultra-endurance athletes regardless of a ketogenic or high-carbohydrate diet [41], concentrations generally decrease in response to dietary carbohydrate restriction [40, 193, 203, 210, 251-257]. Furthermore, the long-term adaptations to nutritional ketosis that may enable some athletes to replenish glycogen at a normal rate may not apply to less physically active individuals.

Insulin inhibits AMPK activity by stimulating protein kinase B (PKB) to phosphorylate the Ser485 residue of the α subunit, thereby inhibiting phosphorylation at Thr172 [258]. One of the most prominent features of nutritional ketosis is that due to restricted carbohydrate intake, postprandial insulin is dramatically decreased. Furthermore, numerous studies have shown ketogenic or low-carbohydrate diets to decrease fasting insulin [192, 232, 259-261], particularly in the presence of metabolic dysregulation associated with hyperinsulinemia [25, 121, 262-264].

Consistent with the mechanisms described above, changes in AMPK in response to a ketogenic or low-carbohydrate diet have been reported in several studies. In rodents, a ketogenic diet (Bio-Serv F3666) has increased AMPK activity in skeletal muscle [187] and AMPK phosphorylation in the liver [265], and a low-carbohydrate diet (18.5% of energy) supplemented with ketone esters (6% w/v) increased AMPK content in brown adipose [186]. In humans, a non- ketogenic low-carbohydrate diet (50% fat, 30% carbohydrate, 20% protein) has increased AMPK phosphorylation in skeletal muscle [266].

SIRT1 and SIRT3

The sirtuin isoforms SIRT1 [267, 268] and SIRT3 [269-271] are nicotinamide adenine dinucleotide (NAD+)-dependent deacetylases associated with longevity. Many reactions are regulated by the redox state of NAD+ and its phosphorylated form, NADP+. Among these reactions, a prominent role of reduced NADP+ (i.e. NADPH) is to support reductive biosynthesis and antioxidant defense, requiring the NADP+/NADPH ratio to be kept low [272]. In contrast, the NAD+/NADH ratio is kept high to support energy metabolism [272], thereby linking sirtuin function to bioenergetic status [273]. Although sirtuins are inhibited by high concentrations of

NADH, their activity is influenced more by absolute NAD+ concentration than the NAD+/NADH ratio [273].

SIRT1 is present in the cytosol and nucleus [274], while SIRT3 is primarily located in mitochondria where it regulates bioenergetics and ROS production [274-276]. The sirtuins, particularly SIRT1, appear to participate in a feed-forward cycle of reciprocal activation with

AMPK. In skeletal muscle, AMPK indirectly activates SIRT1 by increasing NAD+ through increased mitochondrial β-oxidation [277] and increased expression of nicotinamide phosphoribosyltransferase (NAMPT) [278], which is the rate-limiting enzyme in NAD+ synthesis

[279]. Completing the cycle, SIRT1 and SIRT3 can deacetylate and activate LKB1, thereby promoting further activation of AMPK. LKB1 is known to be activated by SIRT1 in adipose and liver [280] and by SIRT3 in cardiac muscle [281].

Based on the reciprocal activation described above, nutritional ketosis is likely to activate

SIRT1 and SIRT3 indirectly through activation of AMPK. However, more direct activation of sirtuins by nutritional ketosis is possible. Since reduction of NAD+ to NADH occurs outside of

29 mitochondria only during glycolysis, which is less active during nutritional ketosis, more cytosolic NAD+ remains oxidized, further facilitating activation of SIRT1 [282]. In addition to the decrease in glucose availability during nutritional ketosis, glycolysis may be further inhibited through activation of pyruvate dehydrogenase kinase and subsequent inhibition of pyruvate dehydrogenase (PDH), which occurs in response to dietary carbohydrate restriction [283-286] or infusion of BHB, ACA, or fatty acids [287]. Consistent with the relevance of these inducers to nutritional ketosis, a ketogenic diet (% energy: 89 fat, < 1 carbohydrate, 10 protein) has decreased expression of PDH in mouse liver [73]. More importantly, there is direct evidence of nutritional ketosis promoting an increase in NAD+ concentration. Treatment with BHB+ACA (1 mM each) has increased NADH oxidation in rat neocortical mitochondria [146], and a ketogenic diet (Bio-Serv F3666) has increased NAD+ concentration in rat hippocampus [288]. There is also evidence of nutritional ketosis regulating sirtuin expression. A low-carbohydrate (20% of energy) diet combined with ketone esters (6% w/v) has increased SIRT1 protein content in brown adipose of mice [186], and a ketogenic diet (% energy: 90 fat, 0 carbohydrate, 10 protein) has increased SIRT3 expression in mouse liver [74].

In addition to the downstream bioenergetic and antioxidant signaling induced by sirtuins, they directly facilitate ketogenesis and β-oxidation. SIRT1 [289] and SIRT3 [290] deacetylate 3- hydroxy-3-methylglutaryl CoA (HMG CoA) synthase, which is the rate limiting enzyme for ketogenesis [291], resulting in increased levels of β-hydroxybutyrate [290]. In addition, SIRT3 deacetylates and increases activity of long-chain acyl-CoA dehydrogenase (LCAD) [292], which participates in β-oxidation and therefore supports ketogenesis. SIRT3 has a similar influence on medium-chain acyl-CoA dehydrogenase (MCAD) as well [293]. Since sirtuins facilitate

30 ketogenesis, which then facilitates sirtuin activation, nutritional ketosis may promote, to some extent, a feed-forward cycle of sirtuin activity.

Direct Involvement of AMPK and Sirtuins in Redox Balance

Although the majority of links between energy sensing and antioxidant defense are manifested further downstream, there is some direct influence at the level of AMPK and sirtuins.

AMPK is activated by oxidative stress [294, 295], likely through ATP depletion and a subsequent increase in the AMP to ATP ratio, or facilitation of tyrosine phosphorylation, which occurs independently of AMP and ATP concentrations [294]. SIRT3 contributes more directly to antioxidant defense by deacetylating and activating SOD2 [296-298]. The overlapping effect of

SIRT3 on antioxidant defense and bioenergetics is further supported by SIRT3 knockout increasing lipid peroxidation in conjunction with decreased O2 consumption in mouse skeletal muscle, and also by SIRT3 knockdown increasing H2O2 production and decreasing O2 consumption in myoblasts [299].

Downstream Bioenergetic and Antioxidant Signaling

AMPK and sirtuins are the interface between the metabolic stimuli of nutritional ketosis and the downstream signaling that influences expression of proteins related to bioenergetics and antioxidant defense. Some of the primary downstream signaling molecules involved include

PGC-1α, FOXO3a, nuclear respiratory factors 1 and 2 (NRF-1, NRF-2), mitochondrial transcription factor A (TFAM), and NFE2L2.

PGC-1α

The coordinated effects of AMPK, SIRT1, and SIRT3 are primarily mediated through

PGC-1α, which is activated through phosphorylation by AMPK [277, 300] and deacetylation by

SIRT1 [114, 277, 301-304]. SIRT3 also increases PGC-1α activity [305], possibly through cAMP response element binding protein (CREB) [306, 307], but the exact mechanism has not been elucidated. In addition to phosphorylating PGC-1α, activated AMPK also increases PGC-

1α expression [295, 308-311]. Activation of β2-adrenergic receptors [312-315] and the adiponectin AdipoR1 receptor [316] also increase PGC-1α expression, independently of AMPK activation [313, 316]. PGC-1α activity is increased by oxidative stress [113, 114, 317-319], possibly through activation of AMPK [294, 295] or p38 mitogen-activated protein kinase

(MAPK) [318, 319], or inhibition of glycogen synthase kinase 3β, which inhibits PGC-1α through phosphorylation [114, 318]. In contrast, insulin decreases PGC-1α activity through phosphorylation by PKB [320]. Once activated, PGC-1α interacts with the PPAR family of nuclear receptors [321] and the FOXO family of transcription factors [322] to influence expression of a variety of bioenergetic and antioxidant proteins. PGC-1α most notably increases transcription of proteins involved in mitochondrial biogenesis and respiration [113, 277, 300,

302, 304, 309, 314, 317, 320, 323-328], but also increases transcription of antioxidant proteins including SOD1 [113], SOD2 [113, 317, 324, 327-329], catalase [317], GPx [113, 329], thioredoxins [317, 318, 327], TRXR [317, 327], Prx3 [317, 327], and Prx5 [317, 327], as well as the mitochondrial uncoupling proteins UCP2 [113, 300, 317, 323, 329], UCP3 [113, 300, 329], and ANT [113, 330].

PGC-1α coactivates all three known PPAR isoforms (PPARα, PPARδ, and PPARγ)

[321]. Although each isoform is expressed in a variety of tissues, PPARα is prominently expressed in the liver, PPARδ in skeletal muscle, the heart, and the pancreas, and PPARγ in adipose [321, 331]. PGC-1α was discovered and named based on its promotion of brown adipose differentiation through coactivation of PPARγ and subsequent induction of mitochondrial biogenesis and UCP1 expression [332]. However, it is the PGC-1α coactivation of PPARα that is responsible for the upregulated transcription of many of the enzymes responsible for increased ketogenesis and fatty acid metabolism in response to a ketogenic diet [157]. Consistent with the role of PGC-1α in inducing mitochondrial biogenesis, it also shifts skeletal muscle fiber composition towards type I [333, 334] and type IIa [334], which are more oxidative. AMPK also contributes to fiber type changes and is required for the transition of highly-glycolytic, type IIb fibers to more oxidative, type IIa fibers [311]. Although PGC-1α is primarily known for inducing transcription of nuclear DNA, it may also, in conjunction with SIRT1, induce expression of mtDNA [335].

PGC-1α is also influenced by p38 MAPK, which is well known for being involved in development [336] and adaptation [337] in skeletal muscle. PGC-1α is activated by p38 MAPK

[318, 338] through phosphorylation [339], which prevents repression [338] by blocking interaction with the p160 myb binding protein [339]. In addition, expression of PGC-1α is increased by p38 MAPK [340, 341], and the overlap in bioenergetic and antioxidant signaling is further indicated based on p38 MAPK activation by AMPK [342-344], oxidative stress [345-

349], and β-adrenergic signaling [315, 350, 351].

Nutritional ketosis may facilitate PGC-1α activity through multiple mechanisms. Since

PGC-1α is activated by AMPK and SIRT1, nutritional ketosis may initiate PGC-1α activity through these enzymes. As previously mentioned, catecholamines and adiponectin facilitate

PGC-1α activity by promoting its expression, and insulin inhibits PGC-1α through downstream phosphorylation, all independent of AMPK. As previously discussed, a ketogenic diet may increase catecholamines and adiponectin and is well known to decrease insulin, indicating that nutritional ketosis may directly facilitate PGC-1α activity through these hormones. Supporting these potential mechanisms, a ketogenic or low-carbohydrate diet has increased expression, protein content, and activation of PGC-1α [186, 266, 352], as well as expression of its target

PPARα [124, 185]. Furthermore, in skeletal muscle of mice following a ketogenic diet, the resulting increases in O2 consumption and expression of genes related to fat oxidation appear to be dependent on PGC-1α [194]. Ketones likely contribute to this signaling as well based on the recent observation that the increased hepatic expression of PPARα targets induced by a ketogenic diet did not occur with a non-ketogenic low-carbohydrate diet [74].

FOXO3a

The FOXO family of transcription factors is highly conserved and promotes longevity and resistance to cellular stress. Although there are a variety of FOXO isoforms with varying tissue distribution [353-355], FOXO3a has been the most thoroughly studied in relation to energy sensing, mitochondrial function, and antioxidant defense. Similar to PGC-1α, FOXO3a is activated through phosphorylation by AMPK [356-358] and deacetylation by SIRT1 [359, 360] and SIRT3 [361-364], and its transcriptional activity is at least partly dependent on AMPK [357] and SIRT1 [360]. In a variety of organisms, tissues, and cell types, FOXO3a increases 34 mitochondrial biogenesis and expression of mitochondrial transcription factor A (TFAM) [364], but is more known for increasing expression of antioxidant and repair proteins, including SOD2

[322, 365, 366], catalase [322, 365, 367, 368], glutathione S-transferse (GST) [357], thioredoxins

[322, 358], Prx3 [322, 369], Prx5 [322], and metallothionein I and II [357], as well as UCP2

[322, 357] and the DNA repair enzyme growth arrest and DNA damage-inducible 45 (GADD45)

[357, 359, 370, 371]. FOXO3a is also activated by oxidative stress [359, 366, 368], possibly in a

SIRT1-dependent manner [359], and likely mediated through c-Jun N-terminal protein kinase

(JNK), which allows FOXOs to translocate to the nucleus by promoting dissociation of 14-3-3

[372, 373]. Furthermore, FOXO3a and SIRT3 interact in mitochondria to induce mitochondrial gene expression in an AMPK dependent manner [374]. FOXO3a also induces expression of

LKB1 [375] and NAMPT [376], indicating a feed-forward cycle of activation with AMPK and sirtuins. Like PGC-1α, FOXO3a transcriptional activity is inhibited by insulin through PKB

[366].

As with PGC-1α, nutritional ketosis may activate FOXO3a by increasing activity of

AMPK and sirtuins or by decreasing insulin. Expression of FOXO3a is increased by fasting, caloric restriction, and BHB [140, 142], all of which are or can be components of a ketogenic diet. Furthermore, BHB treatment has extended lifespan in C. elegans in a manner dependent on

FOXO3a [132], and a ketogenic diet (% energy: 89 fat, < 1 carbohydrate, 10 protein) has increased median lifespan and decreased tumors and age-associated losses of physical and cognitive performance, all in conjunction with increased hepatic content of FOXO3a [73].

NRF-1, NRF-2, and TFAM

Nuclear respiratory factors 1 and 2 (NRF-1, NRF-2) are transcription factors that increase expression of TFAM [377], which is required for full initiation of mtDNA transcription [378-

380], and hence mitochondrial biogenesis. PGC-1α induces expression of NRF-1 and NRF-2 and facilitates TFAM expression by coactivating NRF-1 [323]. Oxidative stress increases this signaling [381, 382] in conjunction with increased mitochondrial biogenesis [381]. AMPK also contributes to mitochondrial biogenesis, but by inducing mitochondrial fission through phosphorylation of mitochondrial fission factor (MFF) [383], which is in addition to and independent of AMPK’s role in activating PGC-1α.

NFE2L2

Nuclear factor erythroid-derived 2-like 2 (NFE2L2 or NRF2) is a transcription factor that has a prominent role in antioxidant signaling and also influences mitochondrial bioenergetics.

The NFE2L2 abbreviation is used in this review to avoid confusion with nuclear respiratory factor 2, which despite being a different protein, has overlapping function with NFE2L2 and shares the same NRF2 abbreviation [384]. Although the mechanisms of NFE2L2 signaling are not fully elucidated [385], oxidative stress has a clear role in interacting with cysteine residues of

Kelch-like ECH-associated protein 1 (Keap1), which decreases proteasomal degradation of

NFE2L2 and thereby allows entry of NFE2L2 into the nucleus to induce transcription [386-390].

Although the influence of PGC-1α on antioxidant enzyme expression is not dependent on

NFE2L2 [113, 391], PGC-1α increases NFE2L2 expression [392], indicating that NFE2L2 activity is influenced by perturbations in both energy and redox homeostasis. NFE2L2 primarily

36 increases expression of antioxidant enzymes, including SOD1 [393], SOD2 [393], catalase [393-

396], GPx [395], NQO1 [389, 394-397], GCL [394-396], GST [397], GSR [394-396], and PrxI

[387], but also increases expression of proteins involved in mitochondrial biogenesis and bioenergetics including NRF-1, NRF-2, TFAM, cytochrome c oxidase, and citrate synthase

[393].

In the previously described C. elegans experiments demonstrating mitohormesis, knockout of the NFE2L2 homolog SKN-1 attenuated the increases in antioxidant enzyme activity and lifespan [110], indicating mitohormesis may, at least in part, be dependent on

NFE2L2 signaling. Similarly, a ketogenic diet (Bio-Serv F3666) increased nuclear content of

NFE2L2 and expression of its target NQO1 in the hippocampi of rats, all of which occurred after an initial increase in mtROS [133]. This increase in NFE2L2 content appears to have mediated the subsequent decrease in mtROS to a level below baseline [133], thereby further indicating a likely role of NFE2L2 in the induction of mitohormesis during a ketogenic diet.

Additional evidence, although independent of mitohormesis, further supports the induction of NFE2L2 activity by nutritional ketosis. Succinate is a byproduct of ketolysis and is oxidized to fumarate by succinate dehydrogenase. Therefore, the increased presence of ketones and increased rate of ketolysis during nutritional ketosis is likely to increase fumarate, which can succinylate cysteine residues of proteins [398]. In particular, fumarate can succinylate Keap1, thereby allowing NFE2L2 to enter the nucleus to induce transcription [399, 400]. In the retinas of rats injected with BHB, nuclear content of NFE2L2 and total homogenate content of SOD2 and GCL increased in conjunction with increased fumarate concentration [401]. BHB injection also decreased retinal ROS production and degeneration following induction of ischemia, and

37 this protection was dependent on NFE2L2 [401]. These effects were observed at blood concentrations of BHB between 1-2 mM, which is consistent with nutritional ketosis.

Overlap Between Bioenergetic and Antioxidant Signal Transduction

As described throughout preceding sections, there are many instances of co-dependencies and feed-forward loops in bioenergetic and antioxidant signal transduction, which supports the well-known potential for metabolic stimuli, such as diet or exercise, to have a profound physiological influence. Given the central role of mitochondria in oxidative phosphorylation and

ROS production, the overlap between bioenergetic and antioxidant signaling is not surprising and is possibly an outcome of evolution favoring efficiency. PGC-1α is at the center of this overlapping and complex network of co-dependencies. The likely role of PGC-1α as a coactivator of FOXO3a indicates a possible dependence of FOXO3a transcriptional activity on

PGC-1α [322], indicating FOXO3a as a central mediator as well. Furthermore, FOXO3a induces transcription of PGC-1α [322, 357, 402], and formation and antioxidant transcriptional activity of the PGC-1α-FOXO3a complex is partly dependent on interaction with SIRT1 [360]. In muscle, expression of many of the bioenergetic and antioxidant proteins previously discussed is dependent on PGC-1α [300]. Upstream, activation of PGC-1α is dependent on AMPK [277] and

SIRT1 [277, 304] and partly dependent on SIRT3 [305]. Furthermore, activation of SIRT1 is dependent on AMPK [277], which may also be the case for SIRT3. AMPK and PGC-1α are therefore two key factors, with critical supporting roles of the sirtuins, in the signal transduction linking bioenergetics to antioxidant defense. Further supporting the relevance of this linkage to nutritional ketosis, expression of AMPK, SIRT1, FOXO3a, and NFE2L2 is required for extension of lifespan in C. elegans by exogenous BHB [132], and expression of AMPK, p38 38

MAPK, and NFE2L2 are required for the extension of lifespan, also in C. elegans, by mitohormesis induced through inhibition of glucose metabolism [110]. The induction of AMPK

[294, 295], SIRT3 [298, 364], p38 MAPK [345-348], PGC-1α [113, 114, 295, 317, 318],

FOXO3a [359, 366, 368], and NFE2L2 [393-395] activity by oxidative stress also makes this signaling highly relevant to mitohormesis [298, 317, 395], especially given that activation of these proteins have been shown to decrease mitochondrial or cellular ROS [113, 298, 317, 324,

358, 367, 369, 391, 394, 402]. Furthermore, mitochondrial biogenesis [381] and the activities of

AMPK [294, 295], SIRT3 [364], p38 MAPK [346, 347], PGC-1α [113, 114, 295, 318], FOXO3a

[359], and NFE2L2 [403] are increased by H2O2, more specifically associating this signaling with mitohormesis. Given that AMPK and sirtuins are upstream of the majority of this signaling, and that AMPK and sirtuin activities are stimulated by both bioenergetic and oxidative stressors, these stressors are likely the primary signals through which nutritional ketosis may induce the mitochondrial and antioxidant adaptations characteristic of mitohormesis (Figure 2).

Figure 2. Nutritional ketosis may initiate bioenergetic and mitohormetic signaling through an increase in catecholamines or adiponectin, a decrease in insulin or glycogen, or an increase in β-oxidation that leads to an increase in mitochondrial reactive oxygen species (mtROS) or NAD+. This leads to further signaling involving AMP-activated protein kinase (AMPK), silent mating type information regulation 2 homologue 1 (SIRT1), peroxisome proliferator-activated receptor γ co-activator 1α (PGC-1α), forkhead box O 3a (FOXO3a), and nuclear factor erythroid-derived 2-like 2 (NFE2L2), ultimately leading to transcription of genes related to oxidative capacity, mitochondrial uncoupling, and antioxidant defense. These adaptations collectively contribute to resistance against oxidative stress. Other proteins involved include liver kinase B1 (LKB1), which activates AMPK; nicotinamide phosphoribosyltransferase (NAMPT), which facilitates SIRT1 activation through NAD+ synthesis; and nuclear respiratory factors 1 and 2 (NRF1, NRF2) and mitochondrial transcription factor A (TFAM), which promote mitochondrial biogenesis.

Exercise as an Adjunct to Nutritional Ketosis

Although resting skeletal muscle is less metabolically active than the heart, kidneys, brain, or liver, it rivals even the brain in being the body’s most metabolically demanding tissue

40 when considered relative to total tissue mass [404]. Physical activity can greatly increase this demand, making exercise a practical and powerful way to induce bioenergetic adaptations.

In skeletal muscle, impaired mitochondrial function contributes to age-associated atrophy, impaired contraction, and insulin resistance [10]. While exercise provides a direct stimulus for mitochondrial adaptation in muscle, with great potential to prevent or treat the aforementioned conditions, the global effects of exercise on bioenergetic homeostasis may lead to mitochondrial adaptations in other tissues as well. Based on this, exercise has the potential to influence any condition for which impairments in global energy metabolism or local mitochondrial function are a contributing factor, which is arguably the case for a majority of chronic diseases. Exercise is therefore an excellent adjunct to nutritional ketosis because it facilitates β-oxidation and ketogenesis by increasing energy demand and depleting glycogen storage, which is likely to augment the signaling induced by nutritional ketosis.

In skeletal muscle, oxidative capacity and mitochondrial content is related to fiber type.

Compared to type II fibers, type I fibers have larger mitochondria [405] with greater oxidative enzyme content [406]. While fiber type is plastic, particularly in response to endurance exercise, transformation from oxidative, slow-twitch fibers (type I) to glycolytic, fast-twitch fibers (type

II) is unlikely to occur [407, 408]. Type II fibers, however, can shift in humans from highly glycolytic (type IIx) to more oxidative (type IIa) [408]. Compared to type IIx fibers, type IIa fibers have greater citrate synthase activity, indicating greater mitochondrial content [409]. The relevance of oxidative capacity and fiber type to oxidative stress has been demonstrated by greater mitochondrial respiration with less H2O2 production in permeabilized fibers from rat muscle consisting primarily of type I or IIa fibers versus type IIb fibers [410]. Although muscle

41 fiber type transformation has been well characterized in response to exercise, this appears to not be the case for ketogenic diets. However, in rats, β-hydroxyacyl-CoA dehydrogenase (β-HAD) has been shown to increase most prominently in glycolytic, type IIb fibers following 4 weeks of a ketogenic diet (% energy: 70 fat, 6 carbohydrate, 24 protein) [202], suggesting transition of these fibers towards type IIa fibers and, in turn, indicating potential for nutritional ketosis to promote a more oxidative muscle fiber composition.

Bioenergetic and oxidative stressors may be largely responsible for inducing many of the beneficial adaptations to exercise, and for this reason, exercise research provides much of the basis for mitohormesis [44-46], As previously discussed, an increase in fat oxidation appears to be a prerequisite for increasing mtROS and, in turn, inducing mitohormesis. Given that ketogenic diets prominently increase fat oxidation during submaximal exercise [40, 41, 125, 251, 252, 254,

255, 411-416], the combination of the two interventions may induce mitohormetic adaptations to a greater extent. Furthermore, much of the signaling that is relevant to mitohormesis, and likely induced by nutritional ketosis, is also induced by exercise, further suggesting the possibility of an additive or even synergistic effect. Demonstrating this, exercise or muscle contraction increases activity, activation, or expression of AMPK [246-248, 310, 319, 417-421], SIRT1 [419-424],

SIRT3 [307, 425, 426], NFE2L2 [393, 395, 427], p38 MAPK [319, 340, 348-350, 428-430],

PGC-1α [310-314, 319, 340, 349, 420-424, 431-435], NRF-1 [393], and TFAM [393, 423, 424].

Exercise also increases expression or activity of antioxidant enzymes [348, 393, 395, 431, 432,

436], uncoupling proteins [131], and bioenergetic proteins involved in oxidative phosphorylation

[431, 432, 435] and the citric acid cycle [431], all of which appears to be at least partly mediated

42 by ROS-induced activity of p38 MAPK [319, 345, 348, 349], PGC-1α [319, 345, 432, 436],

TFAM [345, 349, 393, 432], NRF-1 [345, 393, 432], NRF-2 [393, 395], and NFE2L2 [393].

In addition to increased mitochondrial demand and mtROS production, there are several other commonalities in the mechanisms through which exercise and nutritional ketosis induce adaptive signaling. Exercise-induced activation of AMPK is greater when the exercise is performed in a glycogen depleted state [246-248, 417, 418], and exercise-induced activation of p38 MAPK [350] and PGC-1α [312-314] occurs at least partly through β-adrenergic signaling.

Although changes in NAD+ and NADH are difficult to measure and are complicated by conflicting results, exercise is also likely to increase sirtuin activation by increasing the NAD+ to

NADH ratio [437].

In controlled studies on exercise-trained humans and animals, ketogenic diets have been shown to increase fat oxidation [41, 204] and expression or activity of carnitine palmitoyltransferase (CPT) [204, 206] and β-HAD [206, 209], demonstrating that nutritional ketosis induces adaptation beyond exercise. Similarly, in a study comparing the independent and combined effects of exercise and a ketogenic diet on rats, the combination resulted in greater β-

HAD and citrate synthase activities in skeletal muscle and higher maximal O2 consumption than either intervention alone, further indicating the potential for exercise to magnify adaptations induced by nutritional ketosis [193].

Conclusion

Among the chronic and degenerative diseases in which impaired mitochondrial function is a contributing factor, many respond favorably to lifestyle interventions focused on diet and exercise. The therapeutic potential of nutritional ketosis stands out in this regard. For example, in 43 just the first 10 weeks of an ongoing clinical trial with hundreds of type 2 diabetics following a ketogenic diet, glycated hemoglobin (HbA1c) decreased to below the diagnostic threshold in more than a third of patients, and prescription medication was reduced or eliminated for more than half of patients [50]. Convincing arguments for a ketogenic diet to be the default treatment for diabetes are a decade old [51] and have continued to gain support since then [28]. Similar arguments are developing for obesity [48, 49], neurodegenerative diseases [56, 57, 64-67], cardiovascular disease [52-54], cancer [55-63], and even aging [68, 69]. Although the mechanisms through which a ketogenic diet may improve these conditions expand beyond mitochondrial function, the great extent to which nutritional ketosis increases reliance on mitochondrial metabolism strongly suggests that mitochondrial adaptation is a central factor.

The clinical relevance of nutritional ketosis to mitochondrial function is further indicated by promotion of ketogenic diets for treatment of mitochondrial disorders [56, 57, 63, 67, 282,

438]. The most prominent example is the study of mitochondrial adaptations as a mechanism for the well-known antiseizure effect of ketogenic diets [56, 66, 70, 199, 282, 438-440]. As previously discussed, the dramatic shift in energy metabolism and subsequent increase in circulating ketones induced by a ketogenic diet can enhance mitochondrial function and endogenous antioxidant defense. The primary mechanism behind these adaptations appears to be the increased demand for fat oxidation resulting from carbohydrate restriction. However, ketones themselves have important metabolic and signaling effects that enhance mitochondrial function and endogenous antioxidant defense, implying that a well-formulated ketogenic diet should have greater benefit than a non-ketogenic low-carbohydrate diet. Regardless of the mechanism(s), the

44 potential outcomes imply protection against chronic disease through improved mitochondrial function and, in turn, decreased potential for oxidative stress and subsequent pathology.

Chapter 3. Methods

Experimental Design

The present investigation on mitochondria function was part of a larger 12-week intervention study designed to assess the effect of a ketogenic diet on the physical and mental performance of military personnel during adaptation to chronic exercise training. All participants followed the same exercise training intervention and chose to continue their habitual mixed diet

(MD) or follow a ketogenic diet (KD). Self-selection was used for group assignments instead of randomization to maximize compliance and applicability. This is based on the dedication required to maintain long-term dietary changes and the freedom that most military personnel have to make dietary choices. A unique feature of this design is the use of daily monitoring of capillary BHB levels to track compliance and provide personalized dietary coaching to each participant.

Participants

All participants were affiliated with a military organization and included officers and cadets from the Army Reserve Officer Training Corps (ROTC), Marine Corps veterans, and

National Guard reservists. Exclusion criteria included prior experience with dietary carbohydrate restriction; health concerns including injuries, smoking, cardiovascular disease, endocrine dysfunction, allergies, and use of medication; age greater or equal to 50 years; and failure to meet the safety criteria defined by the American College of Sports Medicine [441] for maximal exercise testing. All study procedures were approved by The Ohio State University Institutional

Review Board. Written informed consent was provided by all participants prior to participation.

A total of 34 participants began the intervention, 5 of whom withdrew (3 from the MD group, 2 from the KD group) for personal reasons unrelated to the study. Among the 29 participants who completed the study, 15 were in the KD group and 14 in the MD group. Participant were initially matched between groups based on age, gender, height, body mass, body mass index (BMI), and body fat percentage. However, participant withdrawal resulted in significant between-group differences at baseline for some of these variables.

Exercise Intervention

Exercise training consisted of two supervised sessions plus one unsupervised session each week. The supervised sessions were focused on enhancement of strength, power, and metabolic conditioning and consisted of traditional strength training exercises with free weights,

Olympic weightlifting, and high-intensity interval training. Unsupervised sessions were focused on aerobic exercise and intended for maintenance of cardiorespiratory fitness.

Diet Intervention

The primary intent of the KD intervention was for participants to maintain a consistent and robust state of nutritional ketosis characterized by daily capillary BHB readings of 1 mM or greater. Participants in the KD group attended an information session at which they were provided a hand-held glucose and ketone meter (Precision Xtra, Abbott, Chicago, IL), trained on how to use the meter, and educated on implementation of the ketogenic diet. In addition, KD participants were instructed to immediately report each meter reading by sending a clearly legible picture of the meter, including date, to a member of the research team. To minimize the time required to achieve the target capillary BHB concentration, participants were encouraged to limit carbohydrate intake to 25 g/d or less and protein intake to 90 g/d or less. Once daily BHB

47 readings stabilized above 1 mM, participants were encouraged to gradually increase carbohydrate and protein intakes to the greatest amounts that would still allow maintenance of the target BHB concentration.

During the initial stages of the diet and anytime capillary BHB readings were less than 1 mM, participants were encouraged to use their smartphone app of choice to track food intake.

The resulting data was used by the research team to provide personalized recommendations to help each participant stay as consistently close to the target BHB concentration as possible.

Participants were provided with frozen meals donated by Quest Nutrition (El Segundo,

CA) and food prepared in our research kitchen. However, participants were free to consume food from other sources. Consistent with the general guidelines of a “well-formulated” ketogenic diet

[71], participants were encouraged to prioritize whole foods, consume the majority of their carbohydrates from non-starchy vegetables and berries, avoid oils high in polyunsaturated fats, and increase sodium intake to compensate for the natriuretic effect of dietary carbohydrate restriction.

Regardless of group assignment, all participants were encouraged to consult with one of the research team’s Registered Dietitians to discuss any questions or concerns related to their diet. Both groups were encouraged to eat ad libitum. However, weight loss was a preconceived dependent variable, and as such, no attempts were made to discourage participants in either group from losing weight.

Pre-Testing Procedures

Prior to each testing session, participants were instructed to adequately hydrate

(consumption of 16 oz of water prior to sleep and 8 oz upon waking), avoid strenuous activity for

48 hrs, avoid alcohol and caffeine for 24 hrs, get 6-8 hrs of sleep, and fast for 8-12 hrs immediately prior to the time of testing. Urine specific gravity was measured to verify adequate hydration prior to any testing or specimen collection procedures. Participants with a urine specific gravity greater than 1.025 were retested following water consumption or rescheduled for a different testing day.

Anthropometric Measurements

Body mass and composition were measured using a standard digital scale (Seca, Chino,

CA) and dual-energy x-ray absorptiometry (DXA; iDXA, GE Healthcare, Chicago, IL), respectively. Visceral fat was measured using magnetic resonance imaging (MRI; MAGNETOM

Avanto 1.5T, Siemens Healthcare, Germany). Fat percentage and water percentage image maps were created using variable projection [442], and custom software was developed to process the resulting images. DXA values for visceral fat strongly correlate with MRI values (r=0.81, p<0.001), justifying generation and use of a single linear regression equation (r2=0.66, p<0.001) to provide estimations for four missing values in the MRI data. The MRI data set, supplemented with these four estimates, was used for visceral fat analysis.

Gas Exchange and Whole-Body Substrate Oxidation

Prior to measurement of gas exchange, the equipment (TruOne 2400, Parvo Medics,

Sandy, UT) was calibrated using standard procedures and the participant was instructed to rest quietly in a supine position. After allowing the participant to rest for approximately 30 min, the mixing chamber pump was started and a plastic canopy was placed over the participant’s head and adjusted to prevent exchange with outside air. Following stabilization of gas exchange rates, data was collected every 30 sec for approximately 20 min. The testing room was kept dark and at

49 a temperature of approximately 23 C throughout the testing session. Substrate oxidation rates were calculated based on the formulas derived by Peronnet and Massicotte [443, 444].

During a separate testing visit, aerobic capacity (VO2max) was measured according to the

Bruce protocol [445], using gas exchange (TruOne 2400, Parvo Medics, Sandy, UT) and a hydraulic treadmill (EXCMR, Columbus, OH) designed for concurrent use with MRI.

Blood Collection and Processing

Blood was drawn from each participant through venipuncture pre- and post- intervention.

All vacutainers were centrifuged at 1,200 g for 10 min at 4 C. Prior to centrifugation, serum and serum-separating vacutainers were left at room temperature for 30 min to allow clotting. During this time, plasma vacutainers were chilled in ice. After centrifugation, one serum-separating tube was sent out for a comprehensive metabolic profile and standard lipid profile (Quest Diagnostics,

Wood Dale, IL). All remaining serum and plasma was aliquoted into microcentrifuge tubes, frozen in liquid nitrogen, and stored at -80 C for future analysis.

Blood Analysis

All samples were thawed only one time, which was immediately prior to analysis.

Enzyme-linked immunosorbent assay (ELISA) kits were used to measure plasma c-reactive protein (CRP; Cayman Chemical, Ann Arbor, MI), serum insulin (Calbiotech, El Cajon, CA), and serum myoglobin (Calbiotech, El Cajon, CA). Respective intra-assay coefficients of variation (CV) were 10.8, 2.1, and 4.1%. Plasma cortisol was measured using a competitive

ELISA kit and had an intra-assay CV of 2.9%. Serum glucose was measured enzymatically using a hexokinase reagent set (Pointe Scientific, Canton, MI) and had an intra-assay CV of 4.1%.

Insulin Resistance and Metabolic Dysregulation

Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as

(fasting insulin (µU/mL) x glucose (mM) / 22.5) [446]. Based on the diagnostic criteria for metabolic syndrome from the National Cholesterol Education Program’s Adult Treatment Panel

III, HOMA-IR threshold values of 2.27 for men and 2.12 for women were used to classify participants as insulin resistant [447].

To help identify aspects of metabolic dysregulation that may inhibit mitochondria function, participants were divided into tertiles based on HOMA-IR, CRP, and visceral fat and comparisons were made for mitochondrial ATP production, state 3 O2 consumption, and membrane potential (each with a fat-derived substrate) between highest and lowest tertiles.

Based on the results, further comparisons were made within the KD group for mitochondria ATP production between participants with the highest versus lowest HOMA-IR values. High values were defined as 2.27 or above, based on the threshold for insulin resistance, and low values were defined as 1.6 or lower, which is the lowest threshold allowing a nearly equal number of participants per comparison group.

Muscle Biopsy

Muscle biopsies were taken from the vastus lateralis by a trained physician in a sterile environment. To minimize variation in muscle fiber type composition, pre- and post-intervention biopsies were taken in close proximity to each other, each from the right leg, and at similar depths. For each biopsy, approximately 10 cc of 1% lidocaine was injected to the biopsy site, a 1 to 2 cm incision was created, and a Bergstrom biopsy needle was inserted into the muscle through the incision. Suction was applied with a 60 mL syringe to draw muscle into the needle for cutting. The needle was rotated after each cut to allow up to 3 separate cuts of tissue per

51 needle insertion. Following withdrawal of the needle from the muscle and removal of the tissue samples, the needle was reinserted into the incision to repeat the process a second time, leading to a total yield of approximately 300 mg of tissue. The sample was immediately placed in ice- cold phosphate buffered saline (PBS, free of Ca and Mg). Visible blood, fat, and connective tissue were removed and 100-140 mg of the remaining sample was used for mitochondria isolation. A remaining portion of the sample was placed in a microcentrifuge tube, frozen in liquid nitrogen, and stored at -80 C for future analysis, including determination of glycogen and triglyceride content. Stored samples were partially thawed for further aliquoting and fully thawed an additional time immediately prior to analysis.

Muscle Glycogen

As previously described [41], muscle samples (~10 mg) were boiled for 2 hr in 500 µL of

2 M HCl to hydrolyze glycogen content. After boiling, each sample was weighed and water was added in an amount to compensate for the loss of solution during boiling. Each sample was then neutralized with 500 µL 2 M NaOH and 50 µL of 2 M Tris. Sample (5 µL) was combined with

100 µL of Glucose Hexokinase Reagent (TR15421, ThermoFisher Scientific, Waltham, MA) in a

96-well microplate. Absorbance was read at 350 nm (Synergy H1, BioTek, Winooski, VT) and compared to a standard curve for glucose determination. The final glucose concentration was normalized to the wet weight of each sample. Intra-assay CV was 4.3%.

Muscle Triglyceride

Samples were freeze dried overnight and 0.5-2.5 mg of dried sample was manually homogenized with a disposable pestle in 0.5 mL of methanol [448]. Chloroform (500 µL) was added to the homogenate and the resulting solution was incubated overnight at 4 C for lipid

52 extraction. MgCl2 (1 mL, 400 µM) was added to the solution, which was then centrifuged at

3,500 rpm for 30 min at 4 C. The aqueous phase of the centrifuged solution was discarded and

250 µL of the chloroform phase was transferred to a new tube and heated at 70 C until complete evaporation occurred (20-30 min). The remaining residue was resuspended in 100 µL of 250 mM

KOH dissolved in ethanol and incubated at room temperature for 30 min. Triglyceride content was hydrolyzed with addition of 25 µL of 1 M HCl and the absorbance of the resulting glycerol was enzymatically enhanced by adding 1 mL of Free Glycerol Reagent (F6428, Sigma Aldrich,

St. Louis, MO). Absorbance was read at 540 nm (Synergy H1, BioTek, Winooski, VT) from a

96-well microplate and compared to a standard curve derived from tripalmitin for glycerol determination. The final glycerol concentration was normalized to the dry weight of each sample. Intra-assay CV was 2.1%.

Mitochondria Isolation

Immediately after biopsy, each sample of muscle tissue designated for mitochondria isolation was minced with scissors for 90 sec in 990 μL of ice-cold isolation buffer consisting of:

100 mM sucrose, 100 mM KCl, 50 mM Tris, 1 mM KH2PO4, 0.1 mM EGTA, 0.2% BSA, ph 7.4

[449]. Protease from Bacillus licheniformis (Sigma-Aldrich, St. Lous, MO, P5380) was added to the minced tissue, resulting in a final concentration of 0.2 mg/mL, and the solution was incubated for 2 min. The minced tissue was then transferred to a 15 mL PTFE/glass tissue grinder (VWR, Radnor, PA) and homogenized, while on ice, at 610 rpm for three 15 sec intervals separated by 5 sec each. Immediately following homogenization, 3 mL of isolation buffer was added to the homogenization vessel and the homogenate was divided into three 1.5 mL microcentrifuge tubes and centrifuged at 700 g for 10 min at 4 C. Supernatant from each tube

53 was transferred to a new 1.5 mL microcentrifuge tube and centrifuged at 10,000 g for 10 min at 4

C. Supernatant from each tube was discarded and the pellets were resuspended into a single microcentrifuge tube with 1 mL of ice-cold isolation buffer. The resuspension was then centrifuged at 7,000 g for 10 min at 4 C, the supernatant discarded, and the final pellet resuspended with a solution (0.6 μL/mg muscle) consisting of: 225 mM mannitol, 75 mM sucrose, 10 mM Tris, 0.1 mM EDTA, 0.2% BSA, pH 7.4. A 5 μL aliquot of the final suspension was stored at -80 C for measurement of protein content (Pierce BCA Protein Assay Kit, Thermo

Scientific, Waltham, MA). The final suspension was incubated on ice for 30-40 minutes prior to further analysis.

Mitochondria Substrates

Three substrate combinations were used to evaluate potential differences in mitochondria function relative to carbohydrate, fat, and ketone metabolism. For each assay, the final concentration of the three respective substrate combinations were: 5 mM pyruvate + 2 mM malate, 0.01 mM L-palmitoylcarnitine chloride + 2 mM malate, and 1 mM lithium acetoacetate +

1 mM sodium-R-3-hydroxybutyrate. The concentrations for the carbohydrate- and fat-derived substrates have been reported to maximize mitochondria respiration [450]. Ketone concentrations were based on available evidence, which was limited to brain mitochondria [146].

Mitochondrial Respiration and Membrane Potential

Mitochondrial O2 consumption and membrane potential were simultaneously measured using a Clark-type electrode (Oxygraph Plus, Hansatech, King’s Lynn, UK) fitted with a tetraphenylphosphonium (TPP) selective electrode (KWIKTPP-2, World Precision Instruments,

Sarasota, FL) and reference electrode (DRIREF-2, World Precision Instruments, Sarasota, FL).

Heated water was circulated through the water jacket surrounding the reaction chamber of the electrode to maintain the temperature of the mitochondria suspension at 37 C. The reaction chamber was filled with 0.5 mL of reaction buffer consisting of: 225 mM mannitol, 75 mM sucrose, 10 mM KCl, 10 mM Tris, 10 mM K2HPO4, 0.08 mM MgCl2, 0.1 mM EDTA, 0.2%

BSA, pH 7.2 [449, 451]. The reaction solution was supplemented with 10 μL of 0.1 mM tetraphenylphosphonium chloride + 1 mM KCl, which was added in three successive volumes (5,

2.5, and 2.5 μL) to establish a calibration curve for estimation of TPP concentration [452]. Based on the ability of TPP to diffuse across the inner mitochondria membrane, changes in TPP concentration can be used to estimate membrane potential [453, 454]. Contents of the reaction chamber were stirred with a magnetic stir bar at 60% of max speed. State 4 mitochondria respiration was initiated with the addition of 8 μL of mitochondria suspension to the reaction chamber. State 3 respiration was initiated with the addition of 10 μL of 10 mM of K-ADP, resulting in a final ADP concentration of approximately 0.2 mM. Measurement of respiration and changes in membrane potential were repeated for each of the three substrate solutions. State

3 and 4 respiration values were normalized to the protein concentrations of the final suspension of isolated mitochondria. Respiratory control ratio (RCR) was calculated as the quotient of state

3 and state 4 O2 consumption rates. Membrane potential was calculated using the Nernst equation with the observed changes in TPP concentration [452, 453].

Mitochondrial H2O2 Production

The rate of mitochondrial H2O2 production during state 4 respiration was measured using

Amplex UltraRed (Invitrogen, Carlsbad, CA), which reacts with horseradish peroxidase (HRP) in the presence of H2O2 to form a fluorescent byproduct [455, 456]. A 10 μL volume of 10X

55 diluted mitochondria suspension was incubated at 37 C in a total well volume of 200 μL consisting of the same reaction buffer used for respiration measurement, 15 U/mL horseradish peroxidiase, and 50 mM Amplex UltraRed Also included was 90 U/mL SOD to maximize

- conversion of O2 to H2O2. A separate reaction was prepared for each of the three substrate combinations. Fluorescence was measured from a 96-well microplate for 10 min at 50 sec intervals with excitation and emission wavelengths of 530 and 590 nm, respectively (Synergy

H1, BioTek, Winooski, VT). H2O2 production rate was calculated based on the average rate of change throughout all reads. Intra-assay CV was 7.2%.

Mitochondrial ATP Production

The rate of mitochondrial ATP production during state 3 respiration was measured using a luciferase-based assay (ATP Bioluminescence Assay Kit CLS II, Roche, Basel, Switzerland).

In the presence of ATP, luciferase converts luciferin into a detectable bioluminescent byproduct

[457, 458]. A 5 μL volume of 40X diluted mitochondria suspension was incubated at 37 C in a total well volume of 100 μL consisting of the same reaction buffer used for respiration measurement, 50 μL of luciferase reagent, and 0.5 mM K-ADP. A separate reaction was prepared for each of the three substrate combinations. Bioluminescence was measured from a 96- well microplate for 8 min at 123 sec intervals (Synergy H1, BioTek, Winooski, VT) and ATP production rate was calculated based on the average rate of change throughout the first three reads. Intra-assay CV was 5.5%.

Oxidative Damage

The remaining suspension of isolated mitochondria leftover after completion of testing was stored at -80 C and later thawed for measurement of protein carbonyls and nitrotyrosine.

Both markers were measured using OxiSelect ELISA kits (Cell Biolabs, San Diego, CA). Intra- assay CVs were 5.2% for protein carbonyl and 10.1% for nitrotyrosine. Plasma thiobarbituric acid reactive substances (TBARS), which is a marker of lipid peroxidation, was measured with a colorimetric assay kit (Cayman Chemical, Ann Arbor, MI). Intra-assay CV was 4.6%.

Sample Sizes

Due to technical complications and limited sample availability, sample sizes differed across tests. The sample sizes of tests for which it was not possible to include all participants are listed in Table 2.

Table 2. Tests with limited sample sizes Mixed Diet Ketogenic Diet Mitochondria O2 consumption (states 3 and 4) * Fat and carbohydrate substrates 12 14 Ketone substrate 11 13 Mitochondria respiratory control ratio * Fat and carbohydrate substrates 12 14 Ketone substrate 11 13 Mitochondria membrane potential † 10 13 Mitochondria ATP production ‡ 14 13 Mitochondria ATP/O2 Fat and carbohydrate substrates *‡ 12 12 Ketone substrate 11 11 Mitochondria ATP/H2O2 ‡ 14 13 Muscle triglyderide §¶ 13 13 Plasma c-reactive protein § 14 14 Plasma cortisol § 14 14 Serum glucose § 14 14 Serum insulin § 14 14 Serum myoglobin § 14 14 * 3 participants (all substrates) excluded due to equipment failure, 2 participants (ketone substrate only) excluded due to abnormally high values not consistent with ATP data † 6 participants excluded due to equipment failure ‡ 2 participants excluded due to abnormally low values not consistent with O2 consumption data § 1 participant excluded due to limited sample availability ¶ 2 participants excluded due to suspected contamination with extramyocellular lipid

Statistical Analysis

Data are reported as means with standard error. Independent t-tests or Wilcoxon rank sum tests, depending on normality of the data, were used for pre-intervention participant characteristics and pairwise comparisons associated with repeated measures analyses. Linear mixed-effects models were used for two- and three-factor repeated measures analyses. Random intercepts were used in these models to account for the non-independence of repeated measures among participants. Body mass, HOMA-IR, CRP, and visceral fat were included as covariates in all models to account for pre-intervention between group differences, except where doing so would be redundant. Normality and variance of model residuals and random intercepts were analyzed for validation of model assumptions. Where appropriate, transformation based on Box-

Cox log-likelihood or trial and error was used to improve model diagnostics. Bidirectional stepwise linear regression was used to identify potential influence of anthropometric, demographic, and metabolic variables on key mitochondria variables. Spearman’s correlations were used in place of Pearson’s correlations where appropriate based on lack of a linear relationship. All analyses were completed using R 3.4.0 [459]. A significance level of α=0.05 was used for all tests.

P-values associated with multiple comparisons and correlations were not adjusted for family-wise error. Such adjustment is intended to minimize the occurrence of Type I errors, but at the cost of an increase in Type II errors. Therefore, use of adjustment should correspond to the severity of the respective consequences associated with Type I and Type II errors [460-464].

Given the exploratory nature of this investigation and the intent for it to support hypothesis generation, adjustment for family-wise error was avoided to facilitate identification of reasonable patterns and relationships that might otherwise go unnoticed and be neglected for consideration in future investigations.

Chapter 4. Results

Participants

Pre-intervention participant characteristics are shown in Table 3. Although participants were originally matched as best as possible with no significant differences between groups, participant dropouts resulted in significant between-group differences for body mass and BMI.

Table 3. Pre-intervention participant characteristics Mixed Diet (n=14) Ketogenic Diet (n=15) Mean±SE Range Mean±SE Range P-value Age (years) 24.6±2.4 18-50 27.4±1.8 20-37 0.350 Gender (F,M) 2,12 NA 2,13 NA NA Height (cm) 179.4±1.4 167.6-190.5 175.5±1.5 165.1-182.9 0.060† Body mass (kg) 79.7±1.5 70.3-87.1 85.6±2.0 71.7-93.0 0.026* BMI (kg/m2) 24.8±0.6 21.0-27.8 27.8±0.7 24.0-33.7 0.005* Body fat (%) 21.2±2.2 11.8-40.5 24.5±1.2 14.7-35.1 0.211 F, female; M, male; NA, not applicable HOMA-IR, homeostatic model assessment of insulin resistance; CRP, c-reactive protein * p<0.05 † p<0.10

Dietary Compliance

KD participants provided capillary glucose and BHB meter readings for 97% of intervention days with a mean BHB concentration of 1.2±0.2 mM. Daily BHB concentration was at or above the target of 1 mM for more than half (52%) of readings, and at or above the commonly used 0.5 mM threshold for nutritional ketosis for 79% of readings. In addition, only one participant had a mean BHB concentration (0.9 mM) below the 1 mM target. These data

60 indicate remarkable compliance throughout the intervention. However, during the last 7 days of each participant’s readings, which roughly corresponds to the post-intervention testing period,

BHB concentrations decreased despite encouragement to maintain the target concentration until post-testing was complete. During this 7-day period, 10 of the 15 (67%) KD participants had a mean BHB concentration below the 1 mM target, 5 of which had concentrations below 0.5 mM.

This suggests a decline in compliance, although further adaptation to the diet, possibly including increased ketone utilization, decreased ketogenesis, or a combination of both, may have contributed as well. This is supported by unpublished observations from another investigation in our lab in which BHB concentration steadily decreased during weeks 2 through 6 of a consistent and tightly controlled ketogenic diet.

Body Composition and Metabolic Markers

At completion of the intervention, the KD group lost more weight (-6.9±0.9 vs 0.7±0.4 kg, p<0.001) and had a greater decrease in body fat percentage (-5.1±0.7 vs -0.7±0.5 %, p<0.001). The KD group also had improvements in nearly all other body composition and metabolic markers including fat mass, visceral fat mass, insulin, HOMA-IR, and VO2max (Table

4). Although all participants were generally healthy upon entry to the study, the pre-intervention differences in visceral fat, insulin, HOMA-IR, and CRP imply early-stage metabolic dysregulation in the KD group. Based on the significant improvement and time by diet interaction for each of these markers (except CRP), the ketogenic diet appears to have resolved this dysregulation.

Table 4. Metabolic markers before and after 12 weeks of a ketogenic diet combined with exercise training Mixed Diet Ketogenic Diet Time by (Mean±SE) (Mean±SE) Diet Pre Post Pre Post P-value VO2max (mL/kg/min) 45.9±2.2 45.8±1.8 44.9±1.5 48.5±1.6† 0.049§ Body Mass (kg) 79.7±1.5* 80.4±1.3 85.6±2.0 78.7±1.6† <0.001§ Fat Mass (kg) 17.3±2.1 16.7±2.0 21.1±1.2 15.2±0.9† <0.001§ Body Fat (%) 22.0±2.3 21.3±2.2 25.6±1.3 20.3±1.3† <0.001§ Visceral Fat (kg) 0.8±0.2# 0.8±0.2 1.3±0.2 0.7±0.1† <0.001§ Insulin (mIU/L) 8.0±1.2* 7.3±0.8 10.5±1.3 5.9±0.7† 0.019§ Glucose (mg/dL) 75.8±1.6 74.8±2.3 79.1±2.8 74.8±1.6 0.391 HOMA-IR 1.5±0.2# 1.4±0.1 2.1±0.3 1.1±0.1† 0.022§ CRP (µg/mL) 0.5±0.1* 0.7±0.2# 1.2±0.3 1.2±0.2 0.630 Pre, pre-intervention; Post, post-intervention HOMA-IR, homeostatic model assessment of insulin resistance * p<0.05, # p<0.10 for between-group difference at associated timepoint; † p<0.05 for within-group difference between timepoints for associated diet group; § p<0.05 for time by diet interaction

Mitochondria Content

Since the volume of the final suspension of isolated mitochondria was based on the mass of muscle tissue used for isolation, the concentration of protein in the final suspension is proportional to mitochondria content per unit mass of muscle. There were significant effects of time and CRP for mitochondria protein content, but no other significant effects or interactions, and no significant between- or within-group differences (Figure 3), indicating that the ketogenic diet did not influence mitochondria content beyond exercise.

Figure 3. Protein concentration of isolated mitochondria before and after 12 weeks of a ketogenic diet combined with exercise training – effect of time, p=0.016; effect of c-reactive protein, p=0.027 MD, mixed diet; KD, ketogenic diet; Pre, pre-intervention; Post, post-intervention

General Mitochondria Function

State 3 and 4 O2 consumption rates and RCR are presented in Figure 4. RCR with the fat substrate increased in the KD group, but not the MD group, suggesting increased capacity for fat oxidation Although there were not any other clear or consistent patterns of change in these markers, state 3 O2 consumption was much lower with the ketone substrate than the carbohydrate and fat substrates. In contrast, mitochondrial H2O2 production increased for all substrates in the

MD group, leading to a nearly significant time by diet interaction (Figure 4D). Time by diet interactions were also nearly significant for state 3 and 4 O2 consumption (p=0.110 and p=0.091, respectively), which appears to have been primarily driven by the carbohydrate and ketone substrates.

Figure 4. General function (by substrate) of isolated mitochondria before and after 12 weeks of a ketogenic diet combined with exercise training A. State 3 O2 consumption - effect of diet, p=0.048; effect of substrate, p<0.001; time by diet by substrate interaction, p=0.050; effect of homeostatic model assessment of insulin resistance (HOMA-IR), p=0.001 B. State 4 O2 consumption – effect of time, p=0.010; effect of diet, p=0.090; effect of substrate, p<0.001; time by diet interaction, p=0.091; effect of HOMA-IR, p=0.003 C. Respiratory control ratio - effect of time, p=0.003, effect of substrate, p<0.001; time by diet by substrate interaction, p=0.038 D. H2O2 production - effect of time, p=0.001; effect of substrate, p<0.001; time by diet interaction, p=0.098; effect of c- reactive protein, p=0.001; effect of body mass, p=0.091 Carbohydrate, pyruvate + malate; Fat, palmitoylcarnitine + malate; Ketone, β-hydroxybutyrate + acetoacetate; MD, mixed diet; KD, ketogenic diet; Pre, pre-intervention; Post, post-intervention * p<0.05, #p<0.10 for between-group difference at associated timepoint; † p<0.05, ‡ p<0.10 for within-group difference between timepoints for associated diet group

Mitochondria Capacity and Efficiency

Mitochondria ATP production, membrane potential, and the ratios of ATP production to state 3 O2 consumption and H2O2 production are presented in Figure 5. For all variables, there was a clear between-group contrast in the direction of change indicating increased capacity and

64 efficiency for the KD group relative to the MD group. This is further indicated by a highly significant time by diet interaction (p<0.005) for most variables. Similar to O2 consumption,

ATP production with the ketone substrate was much less compared to the fat and carbohydrate substrates. These differences with the ketone substrate can be seen more clearly in Figure 6 and occur regardless of diet and time.

Several modifications to the ketone substrate solution were tested to rule out the composition of the substrate as the reason for the differences compared to the carbohydrate and fat substrates. The carbohydrate and fat substrates included malate, but the ketone substrate did not. On four occasions immediately following measurement of O2 consumption with the ketone substrate, measurement was repeated with the addition of the same amount of malate included in the carbohydrate and fat substrates. Although this led to a 26% increase in O2 consumption, the increase is inconsequential relative to the 400-800% greater rates of O2 consumption with the other two substrates. O2 consumption was also measured using a greater concentration of the ketone substrate and with ketone substrate constituted with a different BHB reagent (H6501 in place of 54965, Sigma-Aldrich, St. Louis, MO). Neither variation in the ketone substrate increased O2 consumption. The low rates of ketone oxidation observed are therefore unlikely to be a result of the substrate itself.

Figure 5. Capacity and efficiency (by substrate) of isolated mitochondria before and after 12 weeks of a ketogenic diet combined with exercise training A. ATP production - effect of diet, p=0.015; effect of substrate, p<0.001; time by diet interaction, p=0.003; effect of c-reactive protein (CRP), p=0.055 B. Membrane potential - effect of time, p=0.019; effect of diet, p=0.090; effect of substrate, p<0.001; effect of homeostatic model assessment of insulin resistance (HOMA-IR, p=0.001; effect of visceral fat, p=0.033; effect of body mass, p=0.061 C. ATP production relative to state 3 O2 consumption - effect of diet, p=0.049; effect of substrate, p=0.015; time by diet interaction, p=0.002; effect of HOMA-IR, p=0.020 D. ATP production relative to H2O2 production – effect of diet, p=0.048; effect of substrate, p<0.001; time by diet interaction, p=0.003; effect of CRP, p=0.001; effect of HOMA-IR, p=0.056 Carbohydrate, pyruvate + malate; Fat, palmitoylcarnitine + malate; Ketone, β-hydroxybutyrate + acetoacetate; MD, mixed diet; KD, ketogenic diet; Pre, pre-intervention; Post, post-intervention * p<0.05, # p<0.10 for between-group difference at associated timepoint; † p<0.05, ‡ p<0.10 for within-group difference between timepoints for associated diet group

VO2max strongly correlates with mitochondria function [449] and can therefore provide indication of the extent to which the exercise intervention in the present investigation may have influenced the observed improvements in mitochondria capacity in the KD group. VO2max increased in the KD group (Table 4), but only when normalized to body mass, indicating that the

66 increase is attributable to weight loss rather than increased O2 intake. There are no significant between-group differences or within-group changes for absolute VO2max (not normalized to body mass). Furthermore, normalizing mitochondria ATP production to absolute VO2max does not change the significant time by diet interaction or the significant increase in the KD group with the fat substrate. When normalized to body mass, post-intervention VO2max correlated with

ATP/O2 consumption (r=-0.39, p=0.043), but only with the carbohydrate substrate, and the correlation is not significant when evaluated in the KD group alone. Without normalization to body mass, VO2max did not correlate with any markers of mitochondria function. Collectively, these data indicate that cardiorespiratory fitness did not influence the changes observed in mitochondria function.

Figure 6. Ketone metabolism in isolated mitochondria before and after 12 weeks of a ketogenic diet combined with exercise training A. State 3 O2 consumption by substrate and time - State 3 O2 consumption with the ketone substrate (β-hydroxybutyrate + acetoacetate) was approximately 6 to 8 times lower compared to the carbohydrate (pyruvate + malate) and fat (palmitoylcarnitine + malate) substrates regardless of diet or time B. ATP production by substrate and time - ATP production with the ketone substrate was approximately 4 to 8 times lower compared to the carbohydrate and fat substrates regardless of diet or time C. ATP production with the ketone substrate relative to the carbohydrate and fat substrates – effect of diet, p=0.067; effect of substrate, p<0.001; time by diet interaction, p<0.001; effect of homeostatic model assessment of insulin resistance, p=0.075 MD, mixed diet; KD, ketogenic diet; Pre, pre-intervention; Post, post-intervention * p<0.05 for between-group difference at associated timepoint; † p<0.05, ‡ p<0.10 for within-group difference between timepoints for associated diet group

Shift in Macronutrient Metabolism

The ratio of mitochondrial ATP production with the fat substrate versus the carbohydrate substrate reveals an increase in relative mitochondria capacity for fat oxidation in the KD group, 68 but not the MD group (Figure 7A). Similarly, the ratio of whole-body fat oxidation to carbohydrate oxidation indicates increased reliance on fat oxidation for whole-body resting energy expenditure in the KD group, but not the MD group (Figure 7B). More specifically, baseline reliance on fat and carbohydrate oxidation for whole-body resting energy expenditure was similar between groups, but after the intervention, reliance on fat oxidation in the KD group was nearly three times greater than carbohydrate oxidation, clearly indicating a prominent metabolic shift. Despite similar shifts occurring both globally and in muscle mitochondria, whole-body fat oxidation did not correlate with mitochondrial ATP production with the fat substrate, which is reasonable given that the former is a measure of capacity while the latter represents actual utilization during submaximal demand. Changes in muscle contents of glycogen (Figure 7C) and triglyceride (Figure 7D) are consistent with the shift towards fat oxidation, as indicated by the post-intervention ratio of whole-body fat oxidation to carbohydrate oxidation inversely correlating with muscle glycogen (ρ=-0.37, p=0.046) and directly correlating with muscle triglyceride (r=0.53, p=0.004). Within the KD group, post-intervention muscle glycogen also inversely correlated with average daily BHB concentration (r=-0.56, p=0.032).

A shift in ketone metabolism is also apparent despite the small magnitude of ketone oxidation. The significant time by diet interaction for the ratio of mitochondrial ATP production with the ketone substrate relative to the carbohydrate and fat substrates (Figure 6C) indicates that relative capacity to oxidize ketones increased in the KD group and decreased in the MD group.

Figure 7. Shift in macronutrient metabolism in response to 12 weeks of a ketogenic diet combined with exercise training A. Ratio of mitochondria ATP production from fat substrate (palmitoylcarnitine + malate) to that with carbohydrate substrate (pyruvate + malate) – effect of time, p=0.019; time by diet interaction, p=0.001 B. Ratio of whole-body fat oxidation to carbohydrate oxidation – effect of time, p=0.052; effect of diet, p=0.003; time by diet interaction, p=0.001; effect of homeostatic model assessment of insulin resistance, p=0.057 C. Glycogen content (measured as glucosyl units) per wet weight of muscle tissue – effect of diet, p=0.081; time by diet interaction, p=0.099; effect of visceral fat, p=0.066 D. Triglyceride content (measured as glycerol) per dry weight of muscle tissue – effect of time, p=0.060; time by diet interaction, p<0.001 MD, mixed diet; KD, ketogenic diet; Pre, pre-intervention; Post, post-intervention * p<0.05, # p<0.10 for between-group difference at associated timepoint; † p<0.05, ‡ p<0.10 for within-group difference between timepoints for associated diet group

Potential Influence of Metabolic Dysregulation on Mitochondria Function

To identify explanations for the pre-intervention between-group differences in mitochondria ATP production, O2 consumption, and membrane potential, stepwise multiple linear regression was run with nearly 30 possible predictors representing anthropometric,

70 demographic, and metabolic data. Separate models were created with each aforementioned marker of mitochondria function as the dependent variable. None of the possible predictor variables were consistently selected by the stepwise regression as a significant predictor for each model. This analysis therefore failed to provide any clear explanations for the pre-intervention differences.

Despite not being consistently selected as a significant predictor by stepwise regression,

CRP was significantly higher in the KD group at baseline (Table 4) and consistently inversely correlated with pre-intervention values for each of the three mitochondria markers (with the fat substrate): ATP production (ρ=-0.42, p=0.032), state 3 O2 consumption (ρ=-0.36, p=0.069), and membrane potential (ρ=-0.45, p=0.021). Inspired by the association between CRP, inflammation, and metabolic dysregulation [465, 466], additional indicators of metabolic dysregulation

(HOMA-IR and visceral fat) were analyzed for relevance to mitochondria function. To gain a general understanding of how these indicators related to pre-intervention mitochondria function, all participants (regardless of group assignment) were divided into tertiles for each indicator.

Comparisons between the highest and lowest tertiles are presented in Table 5.

For each of the three markers of metabolic dysregulation considered (CRP, HOMA-IR, and visceral fat), at least 7 KD participants were in the highest tertile and at least 7 MD participants in the lowest tertile. This is consistent with greater pre-intervention values in the KD group for each marker (Table 4), collectively indicating greater metabolic dysregulation in this group. Although the tertile analysis is weakly powered, mitochondrial capacity is consistently lower in the tertile with the highest values for each metabolic marker (Table 5). This is particularly the case for ATP production and O2 consumption. Among the three metabolic

71 markers assessed, HOMA-IR appears to be the strongest predictor of mitochondrial impairment and visceral fat appears to be the weakest predictor. However, pre-intervention visceral fat correlates with HOMA-IR (r=0.44, p=0.019) and CRP (r=0.35, p=0.075), suggesting that it may influence muscle mitochondria function indirectly. Based on these analyses, metabolic dysregulation is the most likely explanation for the pre-intervention between group differences in mitochondria ATP production, O2 consumption, and membrane potential. To account for this as best as possible, CRP, HOMA-IR, and visceral fat were each included as covariates in repeated- measures analysis of all variables except the metabolic health markers listed in Table 4.

Metabolic function clearly improved in the KD group, largely based on the significant decreases in fasting insulin, HOMA-IR, and visceral fat (Table 4). Based on HOMA-IR scores, 6 of 15 (40%) of KD participants were insulin resistant at baseline compared to 2 of 14 (14%) in the MD group. No participants in either group were insulin resistant after the intervention.

Table 5 Comparison of pre-intervention mitochondria function (with fat substrate only) between the highest and lowest tertiles for markers of metabolic dysregulation Highest Tertile Lowest Tertile KD/MD Mean±SE KD/MD Mean±SE Diff (%) P-value CRP (µg/mL) 7/3 1.7±0.3 3/7 0.3±0.0 480.1 0.001* ATP production 22.0±4.9 34.4±7.6 -35.9 0.191 O2 consumption 107.3±12.2 126.6±14.6 -15.3 0.324 Membrane potential 171.0±3.6 180.4±4.6 -5.2 0.125 HOMA-IR 8/2 2.9±0.3 2/8 1.0±0.1 201.7 <0.001* ATP production 20.5±4.2 32.7±4.9 -37.4 0.075† O2 consumption 98.8±13.5 136.2±10.6 -27.4 0.045* Membrane potential 173.3±4.7 179.3±3.8 -3.4 0.133 Visceral fat (kg) 7/3 1.9±0.2 2/8 0.3±0.0 551.8 <0.001* ATP production 30.8±4.1 38.1±8.2 -19.2 0.440 O2 consumption 116.3±14.2 129.8±10.7 -10.4 0.460 Membrane potential 177.2±2.6 181.0±4.4 -2.1 0.467 KD, ketogenic diet; MD, mixed diet CRP, c-reactive protein; HOMA-IR, homeostatic model assessment of insulin resistance Units for ATP production and O2 consumption, nmol/mg/min; units for membrane potential, mv * p<0.05, † p<0.10

To gain further insight on the extent to which the improvement in mitochondria function in the KD group was true enhancement versus rescue of metabolic impairment, the KD group was split according to pre-intervention HOMA-IR values, and mitochondrial ATP production in the two resulting groups was compared (Figure 8). Although there is not a significant interaction of time by HOMA-IR group, which is at least partly due to the small sample size, there is a pattern of greater relative increase in individuals starting the intervention with insulin resistance, indicating that the improvements in mitochondria function observed in the KD group are likely partially, but not completely attributable to rescue of impairment associated with metabolic dysregulation.

Among the 8 KD participants in the highest HOMA-IR tertile, pre- to post-intervention improvement in HOMA-IR coincided with improvement in mitochondria function. In these

73 participants, HOMA-IR decreased by 55% (2.8±0.4 to 1.2±0.2), indicating reversal of insulin resistance, and mitochondria O2 consumption and ATP production (each with the fat substrate) increased by 42% and 30%, respectively. All three post-intervention means are statistically similar to the pre-intervention means for the lowest HOMA-IR tertile (primarily including MD participants), indicating that the impaired mitochondria function associated with insulin resistance at baseline was resolved by the ketogenic diet and exercise intervention. In contrast,

H2O2 production in these 8 participants only changed by 6%, suggesting that impaired fat oxidation was more relevant to insulin resistance than oxidative stress.

Figure 8. ATP production in isolated muscle mitochondria in response to 12 weeks of a ketogenic diet combined with exercise. HOMA-IR, homeostatic model assessment of insulin resistance; Low (n=4), pre-intervention HOMA-IR ≤ 1.6; High (n=5), pre-intervention HOMA-IR ≥ 2.27

Metabolic Damage

Despite increases in mitochondrial H2O2 production, mitochondrial nitrotyrosine decreased in the MD group (Figure 9B), and there were no other significant effects or differences observed for mitochondrial protein carbonyls (Figure 9A) or nitrotyrosine. Likewise, there were no significant effects or differences observed for plasma TBARS (Figure 9D). Interestingly, post-intervention H2O2 production inversely correlated with protein carbonyls for each substrate

(carbohydrate, r=-0.46, p=0.022; fat, r=-0.46, p=0.022; ketone, r=-0.48, p=0.014). In addition, post-intervention protein carbonyls inversely correlated with whole-body fat oxidation (r=-0.45, p=0.023), and post-intervention nitrotyrosine correlated with muscle glycogen content (r=0.63, p=0.029), each suggesting greater oxidative damage in conjunction with increased carbohydrate availability, which is consistent with the relatively greater H2O2 production in the MD group, but not consistent with the inverse correlation between H2O2 production and protein carbonyls.

Although myoglobin and cortisol are not closely related based on function, they are each used as markers of physiological exercise burden [467]. Serum myoglobin decreased in the KD group, resulting in a lower post-intervention concentration compared to the MD group (Figure

9C), suggesting better tolerance of the exercise training. No significant effects or differences were observed for plasma cortisol.

Figure 9. Metabolic damage before and after 12 weeks of a ketogenic diet combined with exercise training A. Mitochondria content of protein carbonyls - effect of diet, p=0.030; effect of homeostatic model assessment of insulin resistance, p=0.004 B. Mitochondria content of nitrotyrosine – effect of c-reactive protein, p=0.015 C. Serum myoglobin – effect of diet, p=0.019; time by diet interaction, p=0.005 D. Plasma thiobarbituric acid reactive substances (TBARS) – no significant effects or interactions MD, mixed diet; KD, ketogenic diet; Pre, pre-intervention; Post, post-intervention * p<0.05, # p<0.10 for between-group difference at associated timepoint; † p<0.05 for within-group difference between timepoints for associated diet group

Chapter 5. Discussion

In addition to this being the first investigation to assess a closely monitored and robust level of nutritional ketosis (1 mM) achieved through a ketogenic diet, it is also the first to thoroughly evaluate the effects of a ketogenic diet on mitochondria function in human skeletal muscle. In addition, the well-formulated ketogenic diet used for the intervention is more relevant to human health than the processed chow used in animal studies or the extreme implementations used for therapeutic purposes in clinical studies. Furthermore, the 12-week intervention duration provided additional time for metabolic adaptation, which is a consideration that has often been neglected in prior investigations. The results show favorable changes in mitochondrial capacity and efficiency associated with the ketogenic diet, particularly in relation to fat oxidation and consistent with the prominent shift in whole-body substrate oxidation towards fat, all of which appears to be related to reversal of mild to moderate metabolic dysregulation.

Ketogenic diets are highly efficacious for weight loss [49], even in the absence of prescribed caloric restriction [224]. Although weight loss was a planned dependent variable for this investigation, it potentially confounds the changes in mitochondria function despite inclusion of body mass as a covariate in statistical analysis. However, the lack of consistent post- intervention correlation between body mass, fat mass, or fat percentage with any of the primary mitochondria markers measured (O2 consumption, membrane potential, ATP production, H2O2 consumption) suggests that weight loss had minimal influence on mitochondria function.

Furthermore, based on single linear regression, none of these body composition markers are a significant predictor for any of the primary mitochondria markers. These results are consistent with previous investigations. Weight loss combined with exercise has been shown to increase muscle mitochondria capacity [468], but in a subsequent investigation by the same lab, diet- induced weight loss without exercise failed to produce similar results [469], indicating it was exercise rather than weight loss that affected mitochondria capacity. This is also supported by similar findings based on activities of oxidative enzymes [470].

Despite this influence of exercise on mitochondria function, the ketogenic diet was likely a stronger factor in the improvements observed in the KD group given that both groups followed the same exercise program. This is indicated by the numerous time by diet interactions showing more favorable changes in mitochondria function in the KD group. Although VO2max increased in the KD group, this can be attributed to weight loss, and the fact that the increase in mitochondrial ATP production is still significant after normalization to absolute VO2max indicates that the influence of cardiorespiratory fitness was partial at most. Consistent with these results, prior investigations have shown ketogenic diets to increase whole-body fat oxidation [41,

204] and activity of mitochondrial enzymes [193, 204, 206, 209] in muscle, all beyond the effects of exercise.

The conclusion that exercise played a relatively small role in the observed changes in mitochondria function is further supported by the lack of post-intervention correlation between

VO2max and markers of mitochondria function. This lack of correlation, however, is surprising given that mitochondrial respiration represents the primary requirement for O2 intake, but is consistent with the observed changes in mitochondria function having occurred despite minimal

78 change in mitochondrial O2 consumption. Furthermore, even if the increase in VO2max in the

KD group was not completely attributable to weight loss, it could also be a result of the greater amount of oxygen required for fat oxidation [196] and greater reliance on FADH2 relative to

NADH for ATP production [197] rather than improvement in cardiorespiratory fitness, which is supported by the lack of change in VO2max in the MD group, with or without normalization to body mass. This is consistent with the exercise intervention having been primarily focused on strength and power development, which is not expected to have notable influence on cardiorespiratory fitness (or mitochondria function) [471-474].

Despite the seemingly small contribution of exercise to the observed changes in mitochondria function, the effect of time for mitochondria protein content (Figure 3) and the lack of a time by diet interaction indicate that the similar increases in mitochondria content between groups is primarily attributable to exercise, which also suggests that the changes observed in mitochondria function occurred independently of mitochondria content. Prior investigation has shown that a ketogenic diet increased mitochondria content in rodent skeletal muscle [200, 475], but this has not been previously studied in human muscle. However, after just three days of restricted carbohydrate intake in humans, mitochondria content was determined to be the primary predictor of the observed increase in resting fat oxidation, explaining 49% of the variance [116].

Despite these indications that ketogenic diets increase mitochondria content, the results of the present investigation suggest that the influence of a ketogenic diet on mitochondria biogenesis is masked by exercise and not strong enough to induce additive benefit.

As expected, the ketogenic diet resulted in a profound and global shift toward reliance on fat oxidation, indicated by post-intervention fat oxidation in the KD group contributing nearly

79 three-fold more to whole-body resting energy expenditure than carbohydrate oxidation (Figure

7B). In contrast, carbohydrate oxidation was the primary contributor to energy expenditure in the

MD group, indicating that this metabolic shift was primarily an effect of the ketogenic diet rather than the exercise training. This shift appears to have occurred in muscle mitochondria as well given that capacity for ATP production with the fat substrate relative to the carbohydrate substrate increased in the KD group but not the MD group (Figure 7A).

Changes in muscle content of glycogen (Figure 7C) and triglyceride (Figure 7D) correspond to the global shift towards fat oxidation, as indicated by the inverse correlation of glycogen and the direct correlation of triglyceride with the ratio of whole-body fat oxidation to carbohydrate oxidation. The inverse correlation between average daily BHB concentration and muscle glycogen reinforces the expectation that the dietary carbohydrate restriction associated with the ketogenic diet caused the decrease in muscle glycogen in the KD group. Given that glycogen inhibits AMPK activity [242-249], and that AMPK signaling regulates mitochondria function, including fat oxidation [476, 477], partial depletion of muscle glycogen may have been a contributing factor, if not the primary mechanism through which mitochondrial function improved in the KD group. Since muscle represents a large proportion of total energy expenditure [478], glycogen-related induction of AMPK signaling may also partly explain the dramatic whole-body shift towards fat oxidation. Similar to glycogen, AMPK is also inhibited by insulin [258]. Therefore, the decrease in insulin in the KD group may have additionally contributed to the enhancement of mitochondrial function and the whole-body shift towards fat oxidation. However, unlike glycogen, post-intervention insulin does not correlate with the ratio

80 of whole-body fat oxidation to carbohydrate oxidation, suggesting that any influence of insulin was weaker or less direct.

With at least 20 proteins involved [479], there are multiple steps at which fat oxidation can be regulated, including β-oxidation and transport of fatty acids through the cellular and mitochondrial membranes [480-482]. Carnitine palmitoyl transferase 1 (CPT1) regulates entrance of long-chain fatty acids into mitochondria such that inhibition of CPT1 inhibits β- oxidation [480]. CPT1 is inhibited by malonyl-CoA, which in muscle is created by acetyl-CoA carboxylase 2 (ACC2) as the first metabolite in fatty acid synthesis from acetyl-CoA during energy abundance [483]. In contrast, malonyl-CoA decarboxylase (MCD) reverses this reaction, resulting in disinhibition of CPT1 and β-oxidation [484]. Further supporting the role of glycogen-related activation of AMPK in increase in fat oxidation observed in the present investigation, AMPK inhibits ACC2 and activates MCD [483, 484], thereby facilitating β- oxidation. In addition, insulin and glucose increase expression of ACC2 and therefore further inhibit β-oxidation during insulin resistance [483, 485], indicating that the improvement in

HOMA-IR in the KD group may have also contributed to the increase in fat oxidation.

Although the decrease in muscle glycogen is statistically significant, it is moderate (14%) in magnitude. Post-intervention glycogen content in the present investigation is similar to that in trained cyclists after four weeks of a ketogenic diet [40], but more than three-fold greater than levels observed after intense exercise [486]. Furthermore, in elite ultra-endurance runners who followed a ketogenic diet for at least 6 months, muscle glycogen levels were similar to those in controls who were following a high-carbohydrate diet [41]. It is therefore likely that adaptation to a ketogenic diet includes enhanced capacity to replenish glycogen from alternative sources

81 during limited dietary carbohydrate intake. Lactate and glycerol have been proposed as likely sources for such replenishment [41].

As metabolism shifts towards greater reliance on fat oxidation, the ratio of FADH2 to

NADH available for oxidative phosphorylation can more than double [128]. Under typical circumstances, characterized by abundant glucose availability, complexes I and III of the electron transport chain tend to be associated in supercomplexes, favoring oxidation of NADH.

As the ratio of FADH2 to NADH increases during greater reliance on fat oxidation, complex I is dissociated from complex III, facilitating greater oxidation of FADH2 [487-490]. Therefore, it appears that oxidative phosphorylation can be optimized according to dietary macronutrient ratios, but at the cost of decreased efficiency for the less abundant macronutrient. This may further explain why mitochondrial state 3 O2 consumption and membrane potential in the KD group decreased with the carbohydrate substrate, but increased with the fat substrate (Figure 4A and Figure 5B, respectively). In addition, dietary carbohydrate restriction, BHB, and fatty acids, all of which are highly relevant to ketogenic diets, promote inhibition of pyruvate dehydrogenase

[283-287], which may further explain the decreases in O2 consumption and membrane potential with the carbohydrate substrate.

The minimal oxidation of ketones by muscle mitochondria (Figure 6) was initially surprising, but is logical given that SCOT, an enzyme critical for ketolysis, is minimally expressed in human skeletal muscle relative to the heart, kidneys, and brain [491] and has an activity level in rat muscle that is only 6% of the activity in the kidneys [492]. However, consistent with the shift towards fat oxidation in the KD group, mitochondrial capacity to produce ATP from ketone oxidation increased relative to capacities from carbohydrate and fat

82 oxidation (Figure 6C), which warrants future investigation of changes in SCOT in skeletal muscle during adaptation to a ketogenic diet. Previous investigation [493], including from our lab [494], indicates that exogenous ketone supplementation can enhance physical performance.

However, the minimal rate of ketone oxidation by muscle mitochondria observed in the present investigation, combined with the relatively low levels of SCOT in human muscle, indicate that the mechanism of enhancement is not related to ketones acting as an energy substrate in skeletal muscle.

Another surprising outcome was the relative decline in mitochondria function in the MD group, which is clearly shown by the significant decrease in the ratio of mitochondria ATP production to H2O2 production (Figure 5D), indicating decreased efficiency of energy production relative to oxidative burden. The increase in H2O2 production (with each substrate) in the MD group, but not the KD group, possibly suggests upregulation of antioxidant defense through mitohormesis in the KD group. However, given the lack of increase in mitochondrial protein carbonyls (Figure 9A) and nitrotyrosine (Figure 9B), the changes in mitochondria ROS production were likely not enough to alter levels of oxidative damage within mitochondria.

Although the inverse correlation between protein carbonyls and whole-body fat oxidation and the direct correlation between nitrotyrosine and muscle glycogen are consistent with the increase in

H2O2 production in the MD group, these results are contradicted by the inverse correlations between H2O2 and protein carbonyls. This inconsistency is further indication that the KD intervention did not have meaningful influence on oxidative burden.

The time by diet interaction for myoglobin (Figure 9C) may be more relevant to the relative decline in mitochondria function in the MD group. The presence of myoglobin in blood

83 is indicative of muscle damage and is therefore commonly used to assess the physiological burden of exercise training. The significant decrease (38%) in serum myoglobin in the KD group and the non-significant 22% increase in the MD group imply that the ketogenic diet enhanced capacity to recover from exercise, which is consistent with anecdotal reports from elite endurance athletes [29]. Such enhancement may have enabled the KD group to more readily tolerate the exercise training, and the lack of such tolerance in the MD group may explain, at least partly, the seemingly unfavorable changes in mitochondria function. However, based on the oxidative damage markers evaluated, any such differences in recovery capacity appear to not be directly related to antioxidant defense, which is further supported by the lack of post-intervention correlation between myoglobin and H2O2 production or any markers for oxidative stress or damage.

Acute exercise-induced elevations in myoglobin, including elevations resulting from resistance training, generally resolve within 24 hours [495-497]. Given that participants were instructed to avoid intense activity for 48 hours prior to testing, and that post-exercise levels of myoglobin (unreported) were approximately two-fold greater than resting values, the changes in resting myoglobin may be more indicative of an adaptive response in sarcolemmal permeability than unresolved muscle damage. Lipid peroxidation induced by mitochondrial ROS production can attack the fatty acids and proteins comprising cell membranes, thereby compromising membrane integrity [78]. The similar patterns in serum myoglobin and mitochondria H2O2 production support this possibility, but the lack of correlation between these two markers and the lack of change in plasma TBARS and mitochondria protein carbonyls suggest otherwise.

Alternatively, ketogenic diets consistently result in higher phospholipid levels of arachidonic

84 acid and lower levels of its precursor fatty acid, dihomo-γ-linolenic acid [498, 499], suggesting less demand for arachidonic acid synthesis. Ketogenic diets also tend to decrease inflammation

[34, 498, 500-502], leading to the hypothesis that less membrane arachidonic acid is utilized for prostaglandin synthesis [498]. In cultured cardiomyocytes, release of arachidonic acid from the sarcolemma during inhibition of ATP production coincides with release of creatine kinase, indicating sarcolemmal damage and increased permeability [503]. Therefore, less use of sarcolemmal arachidonic acid for prostaglandin synthesis in the KD group may have resulted in greater sarcolemmal integrity.

Exhaustive exercise is known to impair skeletal muscle mitochondria, including abnormal structural morphology, decreased respiration and ATP production, and increased H2O2 production [504]. Increased mitochondrial Ca2+ content [505] and faster opening of the mPTP

[506] may also occur, which can lead to further impairment and even cellular apoptosis. Given the potential of these effects to persist for two to three days following an exhaustive bout of activity [507, 508], frequent bouts of intense exercise may result in accumulation of mitochondria impairment that may take longer to resolve. Elevated myoglobin has been observed in conjunction with exercise-induced mitochondria impairment [506, 509], suggesting a link between the two. Furthermore, impairment in resting muscle mitochondria function appears to be a strong predictor of myoglobin elevation in blood following exhaustive exercise [509], suggesting that muscle resilience is dependent on mitochondria function, which is consistent with the changes observed in myoglobin and mitochondria function in the present investigation.

This consistency implies the expectation of an inverse correlation between myoglobin and mitochondria markers. Post-intervention myoglobin inversely correlates with mitochondria

85 membrane potential for the carbohydrate and ketone substrates (ρ=-0.61, p=0.003; ρ=-0.57, p=0.005; respectively). Although myoglobin does not correlate with any other mitochondria markers, membrane potential is a telling predictor. Establishment of membrane potential not only dictates capacity to produce ATP, but is also dependent on the integrity of the inner mitochondrial membrane. Similar to the sarcolemma, the phospholipids comprising this membrane are vulnerable to oxidative damage. Therefore, the indication of enhanced muscle resilience in the KD group based on decreased blood concentration of myoglobin appears to be related to the improvement in mitochondria function.

Metabolic dysregulation, largely represented by insulin resistance, is clearly associated with mitochondrial impairment [15, 510]. This is consistent with the lower pre-intervention levels of mitochondrial O2 consumption, membrane potential, and ATP production in the highest tertiles for CRP, HOMA-IR, and visceral fat (Table 5). The greater pre-intervention frequency of insulin resistance and levels of CRP, HOMA-IR, and visceral fat in the KD group strongly suggests greater metabolic dysregulation in this group and is a likely explanation for the several pre-intervention between-group differences observed for mitochondria function.

It is not clear if insulin resistance is a cause or effect of mitochondria impairment. Insulin function supports mitochondria function, and based on this, it has been proposed that insulin resistance causes mitochondrial impairment [15]. However, evidence of this causal relationship occurring in the opposite direction is more convincing. Oxidative stress and impaired fat oxidation are both mechanisms through which insulin signaling is inhibited. Both mechanisms are highly relevant to mitochondria and are therefore probable explanations for induction of

86 insulin resistance through mitochondria dysfunction. Although both mechanisms are plausible, mitochondrial ROS production appears to be more likely [15, 510].

In the present investigation, participants in the highest tertile for pre-intervention HOMA-

IR had lower mitochondria O2 consumption and ATP production with the fat substrate than the lowest tertile, but H2O2 production did not differ, suggesting that in this population, impairment of fat oxidation was a more important factor for insulin resistance than oxidative stress. This is further supported by the changes observed in the 8 KD participants who started the intervention in the highest tertile for HOMA-IR. In these participants, a decrease in HOMA-IR, indicating resolution of insulin resistance, coincided with increases in mitochondria O2 consumption and

ATP production with the fat substrate, but change in H2O2 production was minimal.

The highly significant improvements in HOMA-IR and visceral fat in the KD group make it unclear if the concurrent improvements in mitochondria function observed in response to the ketogenic diet were a true enhancement, a rescue of impairment, or a combination of both.

The significant time by diet interactions for multiple mitochondria markers despite inclusion of

HOMA-IR, CRP, and visceral fat as covariates and the greater, but not significantly different improvement in mitochondria ATP production in KD participants with a high HOMA-IR (insulin resistance) compared to those with a lower HOMA-IR suggests that it may be a combination.

Further research is needed and strongly encouraged to more thoroughly answer this question.

More than half of Americans are estimated to be diabetic or pre-diabetic [511], which implies that an even a greater proportion is insulin resistant. Given the involvement of both insulin resistance and mitochondria impairment in a wide variety of degenerative diseases, if rescue of impaired mitochondria function is indeed a mechanism through which ketogenic diets

87 resolve insulin resistance, further research in this area could reinforce and expand the already impressive potential for ketogenic diets to greatly benefit public health.

Although the mitochondrial analysis included in the present investigation would not have been possible without several compromises, these compromises impose limitations. The pre- intervention between-group differences in several mitochondria markers lead to a compelling case for further research on the influence of ketogenic diets on mitochondria function in the context of metabolic dysregulation, but complicate the interpretation of the original research objective to identify this influence under normal circumstances and independent of weight loss.

Likewise, the inclusion of an exercise intervention was necessary to support the primary aim of the investigation, but makes it difficult to definitively distinguish the effects of the diet intervention. Finally, although body mass was used as a covariate in statistical analyses to account for the pre-intervention between-group difference that resulted from participant withdrawal, this difference may have introduced more confounding than can be corrected by statistical adjustment.

Conclusion

This investigation is the first to provide empirical evidence of how a closely monitored state of nutritional ketosis achieved through a practical and well-formulated ketogenic diet influences mitochondria function in human skeletal muscle. During adaptation to 12 weeks of exercise training, the ketogenic diet resulted in enhanced mitochondrial function and efficiency, particularly in the context of fat oxidation. This is primarily indicated by an increase in total ATP production as well as ATP production relative to O2 consumption and H2O2 production. Pre- intervention between group differences in these markers appear to be related to metabolic

88 dysregulation, particularly insulin resistance, implying that the ketogenic diet may have enhanced mitochondria function through improvement of metabolic health. This possibility warrants further research, which may provide new insights on addressing insulin resistance and associated conditions.

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