Thesis advisor: Thomas N. Seyfried / Dietary therapy has been used since ancient times to treat the symptoms of disease and disorder. Dietary therapy has long captured the interest of the public in modern times, dating back to the mid-nineteenth century with Englishman William Banting's "Letter on Corpulence, Addressed to the Public", which addressed Banting's anecdotal use of a high-fat diet to treat obesity. High-fat diets became popular in the United States in the early twentieth-century to treat epilepsy. The utility of dietary therapy to treat diseases and disorder has not been embraced widely, as there is a paucity of standardized clinical trials that demonstrate robust safety and therapeutic efficacy for specific diseases and disorders. Additionally, preclinical studies of dietary therapy do not adhere to standardized guidelines, which can hinder cross-study interpretation and reproducibility. To that end, my dissertation updates diet implementation guidelines for preclinical studies that adhere to standardized experimental design and biomarker monitoring in mouse models in order to maximize therapeutic efficacy, diet regimen safety, and cross-study interpretability. With these guidelines, I explored the effect of various diets on circulating glucose and ketone bodies in mice, a measure of glycolytic flux, along with biomarkers of health. I found that calorie-restricted diets, regardless of macronutrient composition, lowers circulating glucose and increases circulating ketone levels, along with improving biomarkers of health, including lowering circulating triglyceride levels. In demonstrating the utility of dietary therapy to treat disease, I also explored the mechanisms on how dietary therapy can be used to treat epilepsy in a preclinical mouse model. I showed that reduced glucose utilization underlies the seizure-protective effects of dietary therapy in EL mice, a mouse model of idiopathic epilepsy. Lastly, I developed a novel tool, the Glucose Ketone Index Calculator, to track the progress of dietary therapy in brain cancer patients through a ratio of circulating glucose to circulating ketone bodies. Evidence is presented that demonstrates a low ratio of glucose to ketone bodies is associated with improved prognosis of brain cancer management in humans and mice. Overall, this dissertation demonstrates the utility of dietary therapy in treating disease using standardized guidelines, and suggests the use of a novel tool to apply and track the progress of dietary therapy in the clinical brain cancer population. / Thesis (PhD) — Boston College, 2014. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Dietary manipulations and their pharmacological outcomes have been increasingly studied in neurodegenerative diseases. However, a systematic comparison among different methods in validated animal models of Alzheimer's disease is made necessary due to several different approaches applied in recent studies. Moreover, despite the large body of evidence on the effects of calorie restriction (CR) and ketogenic diets (KDs) on amyloid pathology, no consistent data is available on the effects of calorie restriction, ketogenic diet or ketone supplements on tau pathology in transgenic models of AD. Moreover, the ketogenic diet used in our studies was custom made with low carbohydrate content and rich in medium chain triglyceride (MCT) oils, known to be rapidly metabolized in the liver, resulting in sustained peripheral ketosis. Chapter 1 tested the ability of KD to induce significant ketosis in a mouse model of amyloid deposition. We showed that, despite the mild ketosis induced, KD fed APP mice presented subtle behavioral improvement shown as faster learning in the radial arm water maze, making less errors than APP mice kept on a control diet. Additionally, we observed decreased Aβ immunoreactivity in the anterior cortex of KD fed versus control fed APP mice, despite the lack of changes in congophilic deposits. Due to the mild ketosis induced, a modified ketogenic diet was devised with decreased maltodextrin content and showed greater peripheral levels of β-hydroxybutyrate. Chapter 2 investigated the effects of a ketogenic diet in two transgenic mouse models of Alzheimer's pathology. Interestingly, we found that both transgenic lines, regardless of diet, weighed less than nontransgenic mice, despite their elevated food intake. The reduced body weight may, in part, be explained by the increased locomotor activity shown by both transgenic lines in both the open field and y-maze. Moreover, KD fed mice performed significantly better on the rotarod compared to mice on the control diet independent of genotype. We did not observed KD-induced changes in spatial or associative memory in the radial arm water maze or contextual fear conditioning, respectively. Furthermore, immunohistochemical levels of amyloid, tau, astrocytic and microglial markers showed no differences between animals fed KD or the control diet. Chapter 3 studied the effects of calorie restriction on a mouse model of tau deposition. We show here that 35% body weight reduction in Tg4510 mice did not prevent increased locomotor activity in the open field, previously reported in chapter 2. Similarly, CR did not affect motor performance or spatial memory assessed by the rotarod and radial arm water maze, respectively. Interestingly, CR Tg4510 mice showed improved short-term memory tested by the novel object recognition despite spending a minimal percentage of the trial time interacting with the objects presented. However, this improvement was not observed when the test was modified to replace the objects with mice. In this case, we noticed that nontransgenic mice spent most of the trial time interacting with the novel mouse whereas Tg4510 mice spent roughly the same amount of time at any of the areas in the test chamber. Moreover, no changes in histopathological or biochemical levels of tau, astrocytic, microglial or synaptic markers were observed. Chapter 4 sought to investigate alternative approaches to inducing ketosis in the brain by either administering BHB intracerebroventricularly (i.c.v.) or by using the acetoacetate (AcAc) diester as a dietary supplement in mice. We observed that i.c.v administration of BHB in 20 months old APP mice did not affect body weight or food intake. Consistent with the lack of effects on behavioral performance, amyloid and congophilic load were not different between APP mice infused with either saline or BHB. We also found that enteral administration of AcAc diester was well tolerated and induced peripheral ketosis for at least 3 hours. Acute ketosis, however, was not sufficient to attenuate behavioral deficits in old APP mice. Chronic dietary supplementation with AcAc was tested in control tet mice and was shown to effectively induce ketosis in mice fed a diet with normal contents of carbohydrates. Nonetheless, we observed that AcAc-induced ketosis was not significantly greater than levels induced by the ketogenic diet tested in our lab. Considering that KD did not rescue behavioral or histopathological features of either amyloid or tau depositing mouse models, we anticipated that dietary supplementation with AcAc would not likely modify the phenotype of the same mouse models tested previously. Taken together, our findings show that our custom made ketogenic diet was effective in inducing and sustaining ketosis and may play an important role in enhancing motor performance in mice. However, the lack of changes on the cognitive and histopathological phenotype of the models studied suggests that KD may not be a disease modifying therapeutic approach to AD. Moreover, calorie restriction showed inconsistent effects on behavioral and histopathological outcomes of a mouse model of tauopathies. Furthermore, dietary supplementation with acetoacetate diester was successful in inducing peripheral ketosis to the same extend as a ketogenic diet even in the context of normal carbohydrate intake, suggesting that it may be of therapeutic interest for diseases of hypometabolism but not a disease modifying therapy in mouse models of Alzheimer's pathology.

Obesity is one the few modifiable risk factors for breast cancer. Hence, an evaluation of the metabolic and cancer inhibitory effects of the obesity reversing strategies, calorie restriction (CR) and exercise, is important for breast cancer prevention. Additionally, a better understanding of the mechanisms underlying the effects of these interventions on cancer will provide scientific basis for therapeutic recommendations, and facilitate the identification of therapeutic agents for breast cancer treatment in obese patients. We found that CR is more effective than exercise in reversing the metabolic and cancer enhancing effects of obesity. Even at comparable levels of adiposity, CR effects on insulin resistance, energy balance related hormones levels, and metabolic genes expression in adipose tissue were more profound than those of exercise. The mechanism by which CR influences tumor progression is thought to involve molecules that respond to energy balance changes and control cell growth, such as the insulin-like growth factor-1 (IGF-1) and the mammalian target of rapamycin (mTOR). The mTOR inhibitor rapamycin decreased mammary tumor burden to levels comparable to CR. While established tumors did not display decreased mTOR activity, constitutively active mTOR was capable of overcoming some of the inhibitory effects of CR on tumor cells invasion and migration. Effects of increasing levels of CR on gene expression indicate that 30% and 40% CR, but not 20% CR, induce beneficial metabolic changes in the liver. However, 40% CR also increases apoptosis of hepatic cells which appears to be detrimental for the liver. IGF-1 infusion partially overcame the beneficial effects of CR on expression of tumor-related genes in the mammary fat pad and on mammary tumor growth. Taken together, our data show that CR, but not exercise, is able to reverse the metabolic and tumorigenic effects of obesity. Furthermore, the IGF-1 and mTOR pathways may mediate, at least in part, many of the beneficial effects of CR on metabolism and tumor progression. / text

Obesity is an established risk and progression factor for many types of cancer, including pancreatic and colon cancer, and is characterized by abnormal metabolic hormone production and a chronic low-grade state of inflammation. However, the links between obesity, hormones, inflammation and tumorigenesis in colon and pancreatic tissue are poorly understood. Calorie restriction (CR), an anti-obesity dietary regimen with potent anticancer effects, reduces serum metabolic hormones and protumorigenic cytokines. Insulin-like growth factor (IGF)-1 is a metabolic hormone that activates NF-[kappa]B, a key regulator of inflammation. NF-[kappa]B is a transcription factor that mediates transcription of many cancer- and inflammation-related genes and is upregulated in both colon and pancreatic cancer. We hypothesized that CR inhibits colon and pancreatic tumor cell growth through modulation of hormone-stimulated NF-[kappa]B activation and protumorigenic gene expression. To test this hypothesis, we used CR and ad libitum feeding to generate a lean and overweight (control) phenotype, respectively; in C57BL/6 mice transplanted with MC38 colon cancer cells or Panc 02 pancreatic cancer cells, and analyzed the effect of diet on circulating hormone levels, markers of inflammation, and tumor growth. We also investigated the in vitro effects of IGF-1 on NF-[kappa]B activation and downstream protumorigenic gene expression in MC38 and Panc 02 cells. CR, relative to control diet, reduced body weight, circulating IGF-1 levels, and transplanted MC38 and Panc 02 tumor growth, as well as protumorigenic gene expression in the MC38 and Panc 02 tumor microenvironment. IGF-1 increased cell viability, NF-[kappa]B nuclear translocation and DNA binding, transcriptional activation, and downstream gene expression of inflammation and other protumorigenic genes in MC38 colon cancer cells and Panc 02 pancreatic cancer cells in vitro. Knockdown studies of NF-[kappa]B in Panc 02 cells using si-RNA established that the IGF-1-induced increase in protumorigenic gene expression is mediated, at least partially, through an NF-[kappa]B-dependent mechanism. In conclusion, these findings in models of pancreatic and colon cancer help clarify the links between obesity, IGF-1, NF-[kappa]B-mediated inflammation, and cancer. This work provides the underpinnings for several new molecular targets and strategies to test in model systems and translational studies for preventing or controlling obesity-related cancer. / text

Calorie restriction (CR) is one of the few treatments that has been observed to significantly extend life in a wide variety of species. While its life-extending properties are still being investigated in primates, there is general agreement that it reduces oxidative stress and inflammation. Interestingly, there is some evidence that it may ameliorate or delay the onset of a number of neurodegenerative diseases, including age-related white matter degeneration. The processes underlying its neuroprotective effects in non-human primates are unknown, but oxidative stress and inflammation are potential contributors to age-related white matter pathologies that characterize aging in the monkey brain and correlate with cognitive decline. To determine if CR reduces damage due to oxidative stress and inflammation in the monkey brain, brains from four calorie restricted monkeys and four matched controls brains were processed for immunohistochemical analysis using an antibody against the pro-inflammatory protein S100b. S100b is a widely expressed calcium-binding cytoplasmic protein associated with neurological insults like ischemia, atrophy, and neurofibrillary tangles and plaques in Alzheimer's disease. It is primarily expressed in astrocytes, but is also expressed to a lesser extent in microglia, oligodendrocytes, and some neuronal populations. Stereology was used to estimate density of S100b labeling in the cingulum, corpus callosum and visual cortex. No significant difference between calorie restricted animals and controls was found. More specific markers of oxidative stress and inflammation may be more effective in revealing any significant differences between CR and control brains. Potential alternatives include antibodies against 4-hydroxynonenal, a lipid peroxidation product, and encephalitogenic peptides of myelin basic protein, which are only exposed to the extracellular environment when myelin is damaged.

Recently, Magnetic Resonance Imaging (MRI) has found great traction in monitoring the effects of Caloric Restriction (CR) on the brain, specifically gray matter. However, there are no streamlined, simple pipelines in existence to analyze data generated from these kinds of MRI studies. Therefore, my hypothesis is two-fold: the first part being the development of a dynamic and straightforward image processing pipeline, which I have tailored to fit the unique needs of the CR data involved in this study. This data brings me to the second part of my hypothesis, which is to use that pipeline to highlight the decreased attenuation in grey matter induced by long-term CR. In order to test the second portion of my hypothesis, T1/MPRAGE scans were collected from 17 male Rhesus Macaques, half of which were maintained on a 30% reduced calorie diet for an average of 22 years, starting around age 3. Using this basis, the inherent properties of the MR images were exploited by the novel pipeline, and used to analyze whether or not CR reduces the attenuation of grey matter atrophy, with regards to aging

Obesity is considered to be a worldwide epidemic disease characterized by an accumulation of AT. Increased adiposity can perturb normal metabolic functions and lead to the development of diseases like insulin resistance and other metabolic disorders. A large amount of clinical studies have been shown that changes in inflammatory signaling in adipose tissue cells, increased infiltration of immune cells into AT as well as stress of endoplasmic reticulum belong to the key molecular steps leading to the development of metabolic disturbances associated with this disease. Adverse metabolic effects of AT accumulation can be diminished by calorie restriction resulting in weight loss. In addition, stress of endoplasmic reticulum could be alleviated by chemical chaperones including bile acids. These two approaches for the treatment of obesity or the obesity-associated disturbances were basis for this PhD thesis. In the first part of this work, we studied inflammation status of gluteal in comparison with abdominal AT and differentiation and secretory capacity of adipocytes after weight loss in obese patients. We revealed that inflammatory profile of gluteal AT, estimated by mRNA level of macrophages and cytokines as markers of inflammatory status of the body, did not explain the different clinical impact of...

Sarcopenia, loss of muscle mass and function, is a common feature of aging. Oxidative damage and apoptosis are likely underlying factors. Autophagy, a process for the degradation of cellular constituents, may be a mechanism to combat cell damage and death. We investigated the effect of age on autophagy and apoptosis in plantaris muscle of male Fischer 344 rats that were either fed ad libitum, or mild, life-long calorie restricted (CR) alone or combined with life-long voluntary exercise. Upstream autophagy-regulatory proteins were either upregulated with age (Beclin-1) or unchanged (Atg7 and 9). LC3 gene and protein expression pattern as well as LAMP-2 gene expression, both downstream regulators of autophagy, however, suggested an age-related decline in autophagic degradation. Atg protein expression and LC3 and LAMP-2 gene expression were improved in CR rats with or without exercise. The age-related increase in oxidative damage and apoptosis were attenuated by the treatments. Both, oxidative damage and apoptosis correlated negatively with autophagy. We conclude that mild CR attenuates the age-related impairment of autophagy in rodent skeletal muscle, which might be one of the mechanisms by which CR attenuates age-related cellular damage and cell death in skeletal muscle in vivo.

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