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PREX protein family (PREX1 and PREX2) guanine-nucleotide exchange factors (GEFs) activate the small G protein Rac. They are important in many physiological and pathophysiological processes, including inflammation, neuronal plasticity, cancer progression and metastasis, with an exciting new role in metabolic processes currently emerging. PREX1 was shown to regulate the thermogenic capacity and insulin-stimulated glucose uptake in adipocytes, whereas PREX2 was reported to mediate glucose tolerance, possibly through its adaptor function as an inhibitor of the tumour suppressor PTEN. The aims of this project were to explore the importance of PREX proteins in glucose homeostasis and insulin signalling, and to assess the significance of PREX Rac-GEF activity in these processes. Western blotting showed that both Prex proteins are expressed in brain, liver and brown adipose tissue, whereas Prex1 is also expressed white adipose tissue. Prex1 deficient mice had small livers, with reduced glycogen storage, eosinophilia in brown adipose tissue and reduced fasting plasma levels of adiponectin. Surprisingly, Prex1 deficiency lowered fasting blood glucose levels and protected mice from age-related glucose intolerance, in a diet- and sex-dependent manner. Bone marrow transplantation revealed that hematopoietic Prex1 deficiency was sufficient for the improved glucose tolerance. In contrast, Prex2 deficiency only affected glucose homeostasis in mice on high-fat diet, causing glucose intolerance and insulin resistance in old age. The phenotype of Prex1/Prex2 double-deficient mice suggested limited sex- and diet-dependent redundancy between both Prex proteins, as well as compensation by other regulators of glucose homeostasis. Overexpression of wild type or catalytically-inactive PREX1 and PREX2 in HepG2 cells caused increased insulin-stimulated Akt activity, suggesting that both PREX proteins can mediate insulin signalling at least in part through adaptor functions. Therefore, catalytically-inactive Prex1 and Prex2 mouse strains were generated and the metabolic phenotype of the catalytically-inactive Prex2 strain was assessed. In contrast to the Prex2 KO mice, catalytically-inactive Prex2 mice had reduced fasting blood glucose levels and improved glucose tolerance, both on chow and high fat diet. Taken together, these finding indicate that Prex1 and Prex2 have unexpected, important and divergent physiological roles in the regulation of glucose homeostasis, and that some of these roles are through adaptor functions. My work suggests that the development of therapeutics to target PREX activity may be a new avenue for the treatment of metabolic syndrome.

Obesity is widely considered a primary risk factor for type 2 diabetes (T2D). However, less is known about the early adaptive responses to short-term periods of high-fat energy excess (HFEE). Previous reports detailing whole-body adaptation to fat and energy oversupply are equivocal, perhaps, in part, owing to use of different experimental protocols, varying durations of dietary manipulation and participant cohorts with individuals of varying characteristics. In addition to use of different dietary protocols between studies, alterations in functional end-point measures due to the type of dietary fat consumed warrants consideration. Daily n-3 PUFA intake, commonly obtained from pelagic fish oil (FO) consumption, has been shown to positively associate with insulin sensitivity in epidemiological studies and thus may be a useful dietary strategy for slowing insulin resistance development. Chapter 2 of this thesis extends previous literature by demonstrating that 6 d HFEE (150 % habitual energy intake; 60 % of energy from fat) does not clearly alter whole- body insulin sensitivity, irrespective of FO consumption. However, investigation of metabolism at the tissue level, as presented in Chapter 3 of this thesis, offers insight into a potential tissue-specific level of regulation that precedes whole-body regulation. Skeletal muscle insulin signalling protein (e.g. protein kinase B (PKB)) activity, levels of certain ceramide species, and AMPK α2 activity were altered following HFEE and may explain the early maladaptive responses to short-term HFEE. Moreover, FO intake as 10 % of total fats mediated some of these molecular responses, including PKB and AMPK α2 activity, reflecting possible functional effects of FO at the subcellular level. Regulation of these metabolic / molecular responses at both the tissue and whole- body level can be explained, in part, by genetic predisposition, environmental influence and more recently epigenetics, including microRNAs (miRNAs). In Chapter 4, we characterised the plasma and skeletal muscle miRNA responses to HFEE and oral glucose ingestion. We demonstrate transient changes in levels of certain miRNAs following oral glucose ingestion in both tissue types and in response to HFEE in skeletal muscle. However, no significant correlations between basal plasma and skeletal muscle miRNA levels were observed, suggesting that our candidate plasma miRNAs may be co-ordinating functional changes in other tissue types. Plasma miR- 145-5p and skeletal muscle miR-204-5p predicted a significant proportion of the variance in mean whole-body insulin sensitivity change in response to HFEE. These data indicate that these miRNAs may be useful biomarkers of insulin resistance development following HFEE. A constraint of this thesis is that all conclusions are made within the context of statistically unaltered insulin sensitivity. Therefore, future investigations of diet-induced maladaptation should consider establishing a time course of insulin resistance development in response to HFEE, or use different study populations. Populations that are more susceptible to T2D development, e.g., overweight, sedentary individuals would be of particular interest. These data would aid development of a working model of diet-induced insulin resistance that has more direct application to T2D progression and extends the data presented herein.

Alzheimer’s disease (AD) is the most common cause of dementia, accounting for around 60-70% of cases. AD encompasses large-scale neuronal loss, resulting in progressive memory and other cognitive decline. Presently, there is no cure for dementia and in light of the ageing population demographic, this represents a clear unmet medical and socioeconomic challenge Worldwide. Much of the current AD research focuses on studying the brain once hallmark amyloid plaque and neurofibrillary tangle pathologies have presented. However their appearance is extremely end stage and to date, any therapeutic interventions aimed at alleviating them having failed to halt symptoms progression. It may therefore be beneficial to look for earlier changes, with metabolic and oxidative stress events as well as reduced cerebral metabolism thought to occur early on in disease progression. Evidence from rare, familial AD cases suggests a causative role for A in AD pathogenesis. For this reason, the enzyme beta-site amyloid precursor protein (APP) cleaving enzyme 1 (BACE1), the rate-limiting step in A production is currently of great therapeutic interest. With the prevailing view being that reducing BACE1 levels will be beneficial in AD, there remains a need to better understand the physiological roles of BACE1 to avoid potential side effects of BACE1 inhibition. Herein is presented data showing that, in agreement with the previous literature, BACE1 is fundamentally regulated by cell stress. Notably, both acute and prolonged bouts of oxidative and metabolic stress result in significant increases in BACE1 and APP protein expression. These changes also result in a shift in APP metabolism, with amyloidogenic processing of APP predominating during times of stress. It has also been shown that chronic elevation of BACE1 and/or manipulation of APP processing can alter cellular glucose uptake and use. These changes were determined through the use of radiolabelled substrate uptake and oxidation as well as extracellular flux assays. These data highlighted a fundamental shift in cellular metabolism, with aerobic glycolysis being utilised over oxidative metabolism of glucose. These changes were later shown to come as a result of metabolic lesions, which acted to impair substrate delivery to the electron transport chain of the mitochondria. Taken together, these data show that overexpression of the AD-associated protein BACE1 phenocopies a number of the earliest detectable changes observed in the brains of people who later develop AD. Finally, these data highlighted the potential importance of a number of novel pathways (Sirtuin, AMP-activated protein kinase, and peroxisome proliferator-activated receptor- coactivator signalling) that may underlie these changes and offer therapeutic avenues for earlier and more targeted treatment to halt AD progression.

Stress is defined as a challenge to homeostatic equilibrium by physical or psychological events, generating a coping response consisting of central and peripheral changes, with the aim of exerting control over the threatening events. Corticotropin-releasing factor (CRF) is well known as a hypothalamic factor which controls the hypothalamic-pituitary-adrenocortical (HPA) axis during basal activity and stress. CRF also serves a neurotransmitter function in the brain, where it is implicated in a range of stress-related behaviours. The measurement of local cerebral glucose utilisation (LCGU) using radiolabelled 2-deoxy-D-glucose (2DG) provides an estimate of cellular activity in the brain. 2DG competes with glucose in its metabolic pathway, but is not fully metabolised, instead accumulating within astrocytes where it can be quantified. After consideration of available modifications to the LCGU technique, the effect of manipulating the CRF system on LCGU was studied, in order to test the hypothesis that CRF and other endogenously expressed CRF-related peptides would induce different patterns of LCGU, and to examine the involvement of CRF receptors in any response. The CRF1 receptor has been implicated in the mediation of stress- and anxiety-related behaviour, while recent evidence has suggested a role for CRF2 in mediating the delayed effects of stress, although it has previously been postulated that CRF2 may be involved in the attenuation of stress-related behaviour. CRF and the endogenous CRF-related peptide Urocortin 1 both induced increases in LCGU in a number of brain regions associated with the CRF system, with concomitant activation of the HPA axis. CRF induced increases in LCGU in the dissected hypothalamus, thalamus, cerebellum and hippocampus, while Urocortin 1 induced a significant increase in LCGU in a dissected hindbrain region, with trend-like effects in frontal cortex and hippocampus. These regions contain components of the CRF system, or receive projections from regions involved in the CRF system, and have been implicated in stress-related function. The effects of CRF on LCGU appear to be mediated by the CRF2 receptor, as they were abolished by the selective CRF2 antagonist antisauvagine-30, but persisted in mice lacking CRF1 and were unaffected by a selective CRF1 antagonist. However, neither of the endogenous CRF-related peptides selective for CRF2, Urocortin 2 and Stresscopin, affected LCGU, which may indicate ligand-specific effects within the CRF system. In contrast to the effects of CRF, restraint stress reduced LCGU, while activating the HPA axis, and this response was unaffected by a selective CRF1 antagonist. This data suggests that the role of CRF receptors in restraint-induced LCGU changes may be overshadowed by effects on other neurotransmitter systems. These studies support the hypothesis that CRF and other endogenously expressed CRF-related peptides would induce different patterns of LCGU, and highlight the involvement of particular brain regions in the response to CRF receptor stimulation. Furthermore, these studies provide evidence suggesting ligand-specific effects within the CRF system.

Alzheimer's disease is the most common form of dementia, and its prevalence is increasing worldwide. Therefore, understanding the disease's pathogenesis is of utmost importance. To this end, we performed untargeted metabolomic assays and targeted qPCR on postmortem visual cortex tissue from subjects diagnosed as cognitively normal control (Ctrl), mildly cognitively impaired (MCI), or with Alzheimer’s disease (AD). The subjects were closely split by sex, had comparable post-mortem intervals, were all Caucasian, and none were diagnosed with metabolic disease. The metabolomic assay revealed an upregulation in multiple metabolites in the glycogen synthesis and glycolysis pathways in the AD subjects. To further probe these findings, we generated heat maps and found a reduction in acetyl-CoA in the AD group. When evaluating the mRNA expression of key GLUTs in the CNS, we found a decrease in the AD group compared to the others. The AD groups also had a reduction in mRNA expression of key rate-limiting enzymes in the glycolytic pathway. Furthermore, we compared key energy metabolites and found that NADH, NAD+, and FAD were all reduced in the AD group compared to the MCI group. Lastly, we evaluated the mRNA expression of key proteins regulating insulin signaling. There was an increase in the mRNA expression of protein tyrosine phosphatase 1B and Src homology 2 domain-containing protein tyrosine phosphatases, and a decrease in the insulin degradation enzyme. In conclusion, the visual cortex tissue of the AD subjects demonstrates a shift in the metabolites involved in glucose metabolism and energy availability, a reduction in the mRNA expression of enzymes that break down glucose, and a shift in mRNA expression of enzymes that regulate insulin signaling in a way that promotes insulin resistance. Our results corroborate other laboratory’s findings and suggest that improving metabolic status may be a promising therapeutic strategy for treating AD.

Bariatric surgery has become a recognized and effective procedure for treating obesity and type 2 diabetes (T2D). Our objective was to directly compare the caloric intake-independent effects of sleeve gastrectomy (SG) and single anastomosis duodenoileal bypass with SG (SADI-S) on glucose tolerance in rats with diet-induced obesity (DIO) and to elucidate the differences between bariatric surgery and caloric restriction.A total of 120 adult male Wistar rats with DIO and insulin resistance were randomly assigned to surgical (sham operation, SG, and SADI-S) and dietary (pair-feeding the amount of food eaten by animals undergoing the SG or SADI-S surgeries) interventions. Body weight and food intake were weekly monitored, and 6 weeks after interventions, fasting plasma glucose, oral glucose and insulin tolerance tests, plasma insulin, adiponectin, GIP, GLP-1, and ghrelin levels were determined.The body weight of SADI-S rats was significantly (p < 0.001) lower as compared to the sham-operated, SG, and pair-fed groups. Furthermore, SADI-S rats exhibited decreased whole body fat mass (p < 0.001), lower food efficiency rates (p < 0.001), and increased insulin sensitivity, as well as improved glucose and lipid metabolism compared to that of the SG and pair-fed rats.SADI-S was more effective than SG, or caloric restriction, in improving glycemic control and metabolic profile, with a higher remission of insulin resistance as well as long-term weight loss.

TXNIP Mediated Crosstalk Between Oxidative Stress and Glucose Metabolism Thioredoxin-interacting protein (TXNIP) has emerged as a key player in cancer and diabetes since it targets thioredoxin (TRX)-mediated redox regulation and glucose transporter (GLUT)-mediated metabolism. Here, we report that TXNIP shuffles between TRX and GLUTs to regulate homeostasis of intracellular oxidative stress and glucose metabolism. TXNIP interacted with GLUTs through three essential amino acid residues (K212, R238, and K287) located within the C- terminal arrestin (ARR) region. Through its di-leucine motif, TXNIP downregulated the surface expression of class I GLUTs, triggered their lysosomal degradation, and attenuated glucose uptake. When intracellular ROS levels increased, TXNIP showed enhanced interactions with GLUT1 and a robust downregulation in glucose uptake. As a consequence, TXNIP significantly limited glucose uptake, leading to the suppression of glycolysis, hexosamine biosynthesis, and pentose phosphate pathway. Our findings establish a fundamental link between ROS and glucose metabolism through TXNIP, and provide a promising target for drug development against GLUT-related metabolic disorders. KSHV-K1 Enhances Fc Binding to Low Affinity Fc Gamma Receptors The Kaposi's sarcoma-associated herpesvirus (KSHV) K1 glycoprotein plays a pivotal role in driving the development of Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD). The primary aim of this project was to screen for K1’s ligand by utilizing K1-Fc, a fusion protein combining K1's extracellular domain with the Fc domain of human IgG1. Serendipitously, we discovered that the extracellular domain of K1 drives K1-Fc oligomerization, leading to Fc clustering and heightened binding to low affinity Fc gamma receptors (FcγRs). This enhanced binding, dependent on K1 oligomerization, involves multivalent interactions between Fc and low affinity FcγRs. Remarkably, commercial Fc blockers prove ineffective in blocking K1-Fc binding to low affinity FcγRs, demonstrating its superior binding capacity. Ig-Fc, a truncation mutant of K1 with the Ig-like region fused to Fc, exhibits the most pronounced oligomerization potential and activates FcγRIIa signaling. This finding highlights the potential of Ig-Fc as a powerful molecular tool for investigating the physicochemical properties and potential clinical applications of Fc-fusion proteins.

Elevated circulating erythritol is a predictive biomarker of weight gain, type 2 diabetes mellitus, and cardiovascular disease. In individuals with existing cardiometabolic disorders, elevated erythritol is also associated with comorbid conditions. Erythritol is a sugar alcohol that is found naturally in fermented foods and is regularly used as a nonnutritive sweetener. In addition to the diet, erythritol can be derived endogenously in mammals from glucose through the pentose phosphate pathway. This dissertation explores the role of both dietary and endogenous erythritol in the progression of cardiometabolic diseases. We observed that chronic exposure to dietary erythritol had no impact on weight and adiposity or glucose tolerance in mice. This was true in mice exposed to low-fat diet or an obesogenic high-fat diet. We then focused on the dietary and intracellular factors that regulate the endogenous synthesis of erythritol. We identified that glucose availability and reactive oxygen species both promote endogenous erythritol synthesis in cell culture models. In addition, erythritol synthesis from glucose is regulated by non-oxidative pentose phosphate enzymes in cultured cells. We observed that the enzymes sorbitol dehydrogenase (SORD) and alcohol dehydrogenase 1 (ADH1), both of which catalyze the final step in the conversion of glucose to erythritol, were not essential for erythritol synthesis in mice. Finally, we found that diet-induced obesity and glucose intolerance did not impact endogenous erythritol synthesis in mice. High sucrose intake, however, elevated circulating erythritol levels and kidney and skeletal muscle erythritol content. We concluded that erythritol synthesis and excretion may be an additional mechanism to dispose of glucose when it is consumed in excess.

Neddylation is a post-translational mechanism that adds a ubiquitin-like protein, namely neural precursor cell expressed developmentally downregulated protein 8 (NEDD8). Here, we show that neddylation in mouse liver is modulated by nutrient availability. Inhibition of neddylation in mouse liver reduces gluconeogenic capacity and the hyperglycemic actions of counter-regulatory hormones. Furthermore, people with type 2 diabetes display elevated hepatic neddylation levels. Mechanistically, fasting or caloric restriction of mice leads to neddylation of phosphoenolpyruvate carboxykinase 1 (PCK1) at three lysine residues—K278, K342, and K387. We find that mutating the three PCK1 lysines that are neddylated reduces their gluconeogenic activity rate. Molecular dynamics simulations show that neddylation of PCK1 could re-position two loops surrounding the catalytic center into an open configuration, rendering the catalytic center more accessible. Our study reveals that neddylation of PCK1 provides a finely tuned mechanism of controlling glucose metabolism by linking whole nutrient availability to metabolic homeostasis.

Despite the growing obesity epidemic and its adverse health effects, such as diabetes and cardiovascular diseases, effective treatments are still missing. This is partly due to our insufficient understanding of the regulatory mechanisms underlying energy homeostasis. This work used transgenic and knockout mouse models combined with neuronal manipulation and pathway tracing to investigate the role of the Neuropeptide FF (NPFF) and Neuropeptide VF (NPVF) in the regulation of energy balance and glucose homeostasis. This study reveals that NPFF signalling plays important roles in fluid and glucose homeostasis, since Npff-/- mice exhibit an increased water intake and improved glucose tolerance, the latter being due to higher insulin responsiveness. Coherently, chemogenetic activation of brainstem NPFF neurons via DREADD technology impaired glucose tolerance, whereas inhibition improved it. In addition, the lack of NPFF signalling leads to a rise in energy expenditure and brown adipose tissue thermogenesis, however, shows impaired high fat diet-induced thermogenesis at thermoneutrality. Together this indicates a critical role of NPFF signalling in the thermos-sensory and dietary regulation of thermogenesis. Consistent with a role of NPFF in the control of energy metabolism, targeted stimulation of NPFF neurons induces a torpor-like state, characterized by strong hypothermia and hypometabolic reactions. Lack of NPVF shows strong gender-specific effects on glucose homeostasis and energy metabolism, such as providing protection against HFD-induced glucose intolerance in male mice. An exacerbated diet-induced obesity in Npvf-/- mice, attributable to reduced energy expenditure observed in females only, and/or higher food intake observed in both genders, suggests a role of NPVF signalling in the regulation of feeding and energy metabolism under positive energy balance. Furthermore, this work reveals important roles of both NPFF and NPVF signalling in the regulation of anxiety-related behaviours, since both Npff-/- and Npvf-/- mice exhibit reduced anxiety and more explorative behaviours in the open field and elevated plus maze test. Together, these findings reveal the NPFF and NPVF systems as potential targets for treating metabolic disorders and anxiety, with NPFF being particularly important for a possible induction of hypometablic and hypothermic states during surgical (brain) recovery or space engineering

Apolipoprotein E (APOE) is encoded by the APOE gene, present in humans as three main isoforms (E2, E3, and E4). E4 carriers face up to a 15-fold increased risk for developing late-onset Alzheimer’s disease (AD), while E2 carriers are protected. Understanding the risk conferred by E4 has been an extensive research focus for nearly three decades, but the exact mechanism has yet to be proven. Many studies have demonstrated attenuated roles of E4 in classical hallmarks of AD, notably amyloid processing and neurofibrillary formation, which normally present later in disease progression. How APOE influences hallmarks that present much earlier are not as well- characterized and may indicate how E4 confers greater risk with an earlier age of disease onset. One such hallmark of AD is cerebral glucose hypometabolism, which happens to also be present in young cognitively normal E4 carriers. The specific cell types responsible for this reduction are unknown; however, an important role of astrocytes, which are the main producers of APOE in the brain, is to facilitate glucose uptake from the vasculature to provide metabolic support for neurons. Therefore, I hypothesize astrocytes are primarily responsible for the reduction in cerebral glucose uptake associated with E4, specifically due to impairments in glucose metabolism. In vivo brain metabolism in human APOE mice (E2, E3, or E4) was examined using an oral gavage of [U-13C] glucose. Glucose metabolism in immortalized astrocytes expressing human APOE E2, E3, or E4 was measured using stable isotope resolved metabolomics via a [U-13C] glucose tracer supplemented growth media. Cell culture and brain metabolite profiles were analyzed using mass spectrometry to determine glucose utilization by tracing the enrichment of 13C atoms in central carbon metabolism pathways. Lastly, a combined multi-omics approach was utilized to determine the metabolic and transcriptomic changes associated with inflammation in astrocytes. Immortalized astrocytes (E3 and E4) were subjected to an acute inflammatory challenge consisting of pro-inflammatory mediators (TNFα, IL-1α, and C1q). Metabolic phenotyping of E3 and E4 astrocytes was conducted by Seahorse mitochondrial and glycolytic rate assays, steady state metabolomics, and further examined using stable isotope resolved metabolomics with a [U-13C] glucose tracer in primary mixed glial cultures. Transcriptional changes in E3 and E4 astrocytes after inflammatory challenge were determined using Nanostring neuroinflammatory gene expression arrays. E4 mice exhibited alterations in glucose flux through central carbon metabolism, specifically in glycolysis and the TCA cycle. In vitro findings revealed E4 astrocytes redirected glucose flux through glycolysis into the non-oxidative pentose phosphate pathway. Glutathione, phospholipid species, NADH, and nucleotide biosynthesis from glucose was also increased in E4 astrocytes, suggesting an inflammatory or oxidative stress component. Following acute inflammatory stimulation, E4 astrocytes exhibited altered glycolytic function, disrupted metabolic responses, and blunted inflammatory gene expression compared to E3 astrocytes. These findings begin to shed light on the cell type specific effects of APOE by which immunometabolism, especially in E4 carriers, may lead to progression of AD and may direct future development of therapeutics.

Throughout the last decade, interest has intensified in intermittent fasting, ketogenic diets, and exogenous ketone therapies as prospective health-promoting, therapeutic, and performance-enhancing agents. However, the regulatory roles of ketogenesis and ketone metabolism on liver homeostasis remain unclear. This thesis seeks to develop a better understanding of the metabolic consequences of hepatic ketone body metabolism by focusing on the redox-dependent interconversion of acetoacetate (AcAc) and D-β-hydroxybutyrate (D-βOHB). Using targeted and isotope tracing high-resolution liquid chromatography-mass spectrometry, dual stable isotope tracer nuclear magnetic resonance spectroscopy-based metabolic flux modeling, dietary-induced mouse models of nonalcoholic fatty liver disease (NAFLD), and complementary physiological approaches in novel cell type-specific knockout mice, the roles of hepatocyte D-β-hydroxybutyrate dehydrogenase (BDH1), a mitochondrial enzyme required for NAD+/NADH-dependent oxidation/reduction of ketone bodies, are quantified. Exogenously administered AcAc is reduced to D-βOHB, and increases hepatic NAD+/NADH ratio, reflecting hepatic BDH1 activity. Livers of hepatocyte-specific BDH1 deficient mice produced no D-βOHB, but due to extrahepatic BDH1, these mice nonetheless remained capable of AcAc/D-βOHB interconversion. Compared to littermate controls, hepatocyte specific BDH1 deficient mice, maintained on either a chow or NAFLD-inducing western-style diet, showed diminished liver tricarboxylic acid (TCA) cycle flux and impaired gluconeogenesis but normal overall hepatic energy charge. Furthermore, the livers of knockout mice maintained on a 60% high fat diet were less fibrotic, with reduced markers of oxidative stress, than littermate controls. Collectively, this thesis illustrates how ketone bodies and BDH1 activity influence liver homeostasis and health. While liver BDH1 is not required for whole body equilibration of AcAc and D-βOHB, loss of the ability to interconvert these ketone bodies in hepatocytes results in impaired TCA cycle flux and glucose production, with a beneficial effect on liver fibrosis. Therefore, BDH1 is a significant contributor to hepatic mitochondrial redox, liver physiology, and organism-wide ketone body homeostasis, and augmentation of hepatic BDH1 activity could prove beneficial in the treatment of NAFLD.

Akt is a signalling protein that regulates many processes in the cell, such as cell growth, anti-apoptosis, and glucose metabolism. The abnormal regulation of Akt is implicated in the development of a number of diseases, including cancer, cardiovascular disease, and type two diabetes. Initially synthesized on the endoplasmic reticulum, Akt moves to the plasma membrane in response to insulin, where it is activated by phosphorylation. Phosphorylated Akt is found in many cellular locations, and the spatial distribution of Akt is thought to be an important determinant of downstream regulation. However, this aspect of Akt signalling has received scant treatment in the mathematical modelling literature to date. This thesis consists of a number of mathematical models of Akt translocation and phosphorylation. The Akt Switch model is a simple, linear, ordinary differential equation (ODE) model of Akt activation that tracks both the biochemical state and cellular location of Akt. Whilst elucidating some of the apparent anomalies of Akt signalling, it enables the differential regulation of downstream substrates via the two branches of Akt signalling (plasma membrane-bound and cytosolic), without recourse to complex feedback mechanisms. However, the Akt Switch model has some limitations, including a noticeable discrepancy between the model output and the experimental data in the early stages of simulation. As a result, the Akt Translocation model was developed to further investigate the translocation of Akt in response to insulin in vitro. The Akt Translocation model is a three-compartment ODE model that reproduces the salient features of Akt translocation. Analysis of the model shows that it behaves as a heavily damped harmonic oscillator with solution curves that either increase monotonically or overshoot. Optimisation of the model to TIRF microscopy data quantified a time delay of approximately 0.4 min between the application of insulin and the Akt translocation response. In addition, the optimisation revealed that the processes regulating the size of the plasma membrane bound pool vary with the insulin level. For physiological insulin, the rate limiting step is the release of Akt to the plasma membrane. At high insulin levels, however, the down-regulation of Akt movement away from the plasma membrane is also necessary to explain the data. The models developed in this thesis provide a framework for understanding the dynamics of this vital signalling node.

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes mellitus (DM). Its pathology is consisted of the breakdown of the blood-retinal barrier (BRB), particularly the inner blood-retinal barrier (iBRB). The exact mechanism that leads to the iBRB impairment and the development of DR has not been elucidated yet. However, chronic inflammation that is hallmark of the type I DM has recently become a key focus in DR study. Hyperglycemia causes dysregulation of nuclear factor-kappa B (NF-kB); and its constant activation increases the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and intercellular adhesion molecule-1 (ICAM-1) in the retina and vitreous of DR patients and animal models. This increase in inflammatory mediators leads to the degradation of junctional molecules, occludin, connexin-43, death of retinal cells via pyroptosis, and thus disruption of the iBRB. Derailment of the iBRB induces fluid accumulation into the retina of DR patients, which eventually progresses into vision impairment. The purpose of this study is to investigate whether chronic inflammation in DM induces disruption of iBRB; and whether inhibition of the expression and activity of NF-kB by Methyl-N1-(3-phenyl propyl) benzene-1, 2-diamine (JSH-23) would mitigate this iBRB disruption. Methods: Type 1 diabetic mouse model (Akita) on a C57BL/6J background along with non-diabetic littermate were used in the experiments. All mice were 22-24 weeks of age at the start of the experiments. The experimental and the control animals were treated for four weeks on alternate days with JSH-23 @5mg/Kg body weight. The intraocular pressure (IOP), blood pressure, blood glucose, and body weights of all animals were measured before and after treatment. Fluorescence angiography (FA), optical coherence tomography (OCT), and dark-adapted electroretinogram were performed before and after the treatment. Also, intraperitoneal glucose tolerance test (GTT) were performed in the animals before and after the treatment. Then, the retinas and plasma were collected. Western blotting and real-time polymerase chain reaction were performed to analyze protein expression and the mRNA levels of NF-kB p65, iNOS, COX-2, ICAM-1, occludin, connexin-43, NLRP3, caspase-1, and IL-1b. Additionally, plasma insulin levels were also analyzed via ELISA kit. Results: The body weight of Akita mice was significantly lower than the C57BL/6J mice. The treatment with JSH-23 did not have any effect on body weight. The blood glucose of Akita mice was significantly higher than the blood glucose of non-diabetic littermate control mice. The treatment substantially decreased blood glucose in Akita mice. Also, the plasma insulin level for the Akita mice was significantly lower compared to non-diabetic littermate control mice. The intraperitoneal GTT showed that the Akita mice were unable to lower their blood glucose levels. The total and phosphorylated protein expression levels of NF-kB p65 were significantly decreased after treatment with JSH-23 in Akita mice, and the gene expression of NF-kB p65 also reduced. The protein and gene expression levels of iNOS, COX-2, and ICAM-1 were reduced after treatment in Akita mice, and the treatment significantly lowered the expression of pyroptosis marker NLRP3. The gene expression of other proteins in the pyroptosis pathway were also reduced, including caspase-1 and IL-1b. On the other hand, the protein and gene expression levels of junctional molecules occludin, connexin-43 were significantly increased in Akita mice after treatment with JSH-23. The blood pressure of the animals was normal and the treatment did not have any effect on the blood pressure. FA depicted an increase in retinal capillary permeability in Akita mice, and the treatment significantly reduced that permeability. The OCT showed retinal layers thickening in the retina of Akita mice, and treatment increased the retinal layers thickness. Electroretinogram (ERG) showed an increase in the amplitude of the A-wave and the B-wave in Akita mice. Conclusions: The results showed that inhibitor (JSH-23) improved visual function by decreasing the retinal vascular permeability, IOP, and the retinal inflammation in Akita mice.

Our ability to taste sugar evolved to tell us that certain foods are good to eat because they provide us with energy. However, in the modern food environment of ubiquitous “added sugars”, this good taste can be hijacked in ways that cause overeating. This dissertation details the discovery of a means by which dietary sugar causes overeating in the fruit fly Drosophila melanogaster, via impairment of peripheral sweet taste sensitivity through increased intracellular glucose metabolic signaling by a conserved protein modifier. This in turn affects central value/motivational processing of the sweet sensation to alter feeding. We also present a new fly feeding assay, the optoFLIC, that is optimized for the monitoring and the closed-loop, optogenetic manipulation of long-term, steady-state feeding behaviors. Together, these findings depict a theory overeating that links excess dietary sugar with peripheral sensation and central reward, and they guide future research into the etiology and treatment of diet-induced obesity.

Proliferative cells, including many types of cancer and pluripotent stem cells, rely primarily on glycolysis and lactate metabolism for energy, regardless of oxygen availability. This metabolic phenotype – referred to as the Warburg effect – results in wasteful lactate accumulation. Although cancer cells and pluripotent stem cells share this central metabolic characteristic, the sensitivities of each of these cell types to lactate stress appear contradictory. While lactate accumulation is thought to adversely impact pluripotent stem cell proliferation and differentiation capacities, cancer cells have been shown to possess bioenergetic plasticity to utilize lactate catabolism for fuel. As a result, lactate buildup within the hypoxic tumor microenvironment has been hypothesized to promote cancer progression and malignancy, in part by selecting for cancer populations capable of catabolizing lactate. Moreover, lactate has been shown to promote "stemness" gene expression in cancer, suggesting that lactate plays a functional role in regulating pluripotency gene expression. However, an incomplete understanding of the impact of lactate on intracellular metabolism for proliferative cells remains. In this work, 13C-metabolic flux analysis was used to quantify the intracellular metabolic responses of breast cancer cells and induced pluripotent stem cells (iPSCs) to high extracellular lactate; in particular, to determine the role of lactate as a metabolic substrate. In this research, it was demonstrated that both iPSCs and breast cancer cells employ dual consumption of glucose and lactate to support growth. In addition, this is the first study to determine and quantify intracellular contribution of lactate in proliferative iPSC metabolism. These results provide insight into the metabolic flexibility of highly proliferative cells with respect to lactate metabolism and suggest that, much like many types of cancer cells, iPSCs possess the capacity to metabolize lactate to promote exponential growth and maintain pluripotency.

Abstract: Background: Chronic diseases related to the metabolic syndrome, such as type 2 diabetes and hypertension lead to complications when not treated properly. Natural approaches able to tackle multiple aspects of the metabolic syndrome and cause minimal side effects are desired. Bioactive peptides are short amino acid sequences that exert benefits beyond their nutritional value, improving health. Egg white is a rich source of bioactive peptides that are promising candidates as functional foods. Egg white peptides and hydrolysates (EWH) have been shown to reduce blood pressure, inflammation and fat mass, all related to the metabolic syndrome. Previously, feeding EWH for 6 weeks improved glucose and insulin tolerance in insulin resistant (IR) rats. Here, I aimed to identify EWH mechanism(s) of action related to glucose homeostasis. In this work I hypothesized that EWH supplementation affects multiple signaling pathways in insulin sensitive tissues to improve glucose tolerance in high fat diet (HFD)-induced insulin resistant rats.Methods: Sprague-Dawley rats were fed HFD for 6 weeks and then divided into 2 groups, HFD (n=7) and HFD+4% EWH (n=8) for another 6 weeks. 10 min prior to euthanization insulin was injected in half of the animals in each group. Insulin sensitive tissues (skeletal muscle, liver and white adipose tissue (WAT)) were tested, when applicable, for changes in insulin signalling, renin-angiotensin system (RAS) components, gluconeogenesis and lipolysis enzymes by western blot. In addition, WAT inflammatory markers and adipokines were measured by ELISA, PPARγ activation by DNA binding activity and adipocyte size by histological analysis. Results: EWH enhanced Akt phosphorylation in muscle and WAT of EWH-treated animals. RAS components abundance presented minimal changes, but notably there was increased angiotensin type 2 receptor (AT2R) in liver and WAT after EWH supplementation. The abundance of liver gluconeogenesis enzymes did not change. PPARγ DNA binding activity was enhanced and lipolytic capacity reduced by EWH in WAT. Moreover, the treatment reduced adipocyte size in epidydimal and retroperitoneal WAT, despite no changes in tissue inflammatory cytokines, resistin or adiponectin levels.Conclusion: In this study WAT was more responsive to EWH effects than other insulin sensitive tissues. In summary, the main findings of this study were that EWH changed adipocyte morphology, reduced adipocyte lipolytic capacity, enhanced insulin sensitivity in peripheral tissues, had little effect on local inflammation and induced only AT2R of the RAS components examined. The results are in accordance with our hypothesis showing that EWH is affecting insulin sensitive tissue to improve glucose tolerance through a variety of mechanisms and acts independently of changes in local inflammatory markers or adipokines. In WAT, a model linking EWH to AT2R activation, with downstream effects on PPARγ and Akt activation is proposed for further investigation. This research contributes to evidence for EWH as a potential product in the functional food market.

Pancreatic cancer ranks as third deadliest cancer worldwide in both men and women. The lack of diagnostic methods, chemo-resistance and invasiveness of the disease diminishes the overall survival rate in patients. Dysregulated glucose metabolism and several molecular determinants including oncogenic signaling pathways play pivotal roles in the development, progression and metastasis of pancreatic cancer. The aberrant glucose metabolism is the most prevalent key factor that influences pancreatic tumorigenesis. The cancer cells of pancreas utilize oxygen for the breakdown of glucose to lactate, which is known as Warburg effect. This phenotype of pancreatic cancer encourages proliferation, growth, migration and invasive phenotypes of cancer cells that worsens the disease. The upstream regulatory molecules responsible for deviant activity of glucose transporters during aberrant glucose metabolism was not very well defined. Thus, identification of new molecular determinants that regulate glucose metabolic pathway would provide promising therapeutic outcomes. Our lab has established the role of MUC13 protein in pancreatic cancer development. Another interesting molecule, Protein Kinase D1 has been reported to play essential role in hypoxic metabolism in squamous cell carcinoma, and glucose uptake in cardiac hypertrophy. Also, it has been extensively studied in our research group, and has been suggested by some other groups to have an essential role in pancreatic cancer. However, its role in regulating glucose metabolism in pancreatic cancer remains elusive. In this dissertation, we investigated that MUC13 and PKD1 proteins are involved in abrupt glucose metabolism in pancreatic cancer cells. We have demonstrated that targeted overexpression of these oncogenes upregulates key oncogenic signaling components involved in aberrant glucose metabolism in pancreatic cancer cells. We also investigated a natural agent Steviol that restored glucose metabolism in pancreatic cancer cells via repression of MUC13 and PKD1 expression. MUC13 is a transmembrane glycoprotein which has an elevated expression in pancreatic cancer. Due to the presence of a cytoplasmic domain, it confers kinase activity and regulates many signaling processes. Therefore, we investigated the effect of MUC13 in metabolic reconditioning of pancreatic cancer. As a result, it was observed that presence of MUC13 in pancreatic cancer upregulated glucose uptake and lactate secretion. This in turn led to more aggressive behavior of the tumor, as evidenced by enhanced proliferation, migration and invasive characteristics of the cells as compared to MUC13 null cells. Interestingly, we observed the interaction of MUC13 with Glut-1 protein, which influences glucose uptake in cancer cells. This was confirmed through various assays such as: immunoprecipitation, immunofluorescence, co-capping and proximity ligation assay. The interpretation of inhibitor results envisaged the role of NFҡB pathway during the molecular interactions of MUC13 and Glut-1. To understand the clinical importance of this mechanism, we evaluated this interaction in tumor samples from the patients with pancreatic disease in advanced stages. Our findings fortified the mechanistic role of MUC13 in rewiring of aberrant glucose metabolism. Altogether, we believed that MUC13 has clinical implication as a key molecule that might be responsible for dysregulated glucose metabolism. As discussed earlier about the paucity of associated proteins related to dysregulated glucose metabolism, we further investigated the possible key players with kinase domain for association and activation with other signaling molecules.
Protein Kinase D1 (PKD1), a Ser/Thr kinase has been shown to be involved in progression of pancreatic cancer. In this study, we established PKD1 as a novel molecular target and its involvement in regulating aberrant glucose metabolism in pancreatic cancer. We observed that PKD1 was overexpressed in pancreatic cancer tissues compared to normal human tissues. We also found constitutive expression of PKD1 in various pancreatic cancer cells as compared to normal pancreatic ductal epithelial cells. The overexpression of PKD1 in low PKD1 expressing pancreatic cancer cells enhanced tumorigenic characteristics. We observed that specific knockdown of PKD1 inhibited key oncogenic signaling components in pancreatic cancer cells suggesting its oncogenic role in pancreatic cancer. Additionally, during the PKD1 overexpression, cells displayed increased glucose consumption and lactate production. This suggested role of PKD1 in metabolic reprogramming in pancreatic cancer. Also, we observed that PKD1 stimulates the glucose uptake through mTORC1, a component of mTOR signaling pathway. Additionally, the knockdown of PKD1 promoted the chemosensitivity of cells towards gemcitabine and 2DG (2-deoxyglucose). Overall, these results indicated a significant function of PKD1 in rewiring of glucose metabolism in pancreatic cancer. It was mandating to target and counteract the aberrant glucose metabolism along with the proteins identified in our previous results. In this perspective herein, we proposed steviol, a natural sweetener from plant origin to assess its inhibitory action towards dysregulated glucose metabolism and its associated proteins (MUC13 and PKD1). The reason behind steviol selection for this approach includes that it mimics the glucose molecule that facilitates enhanced uptake of drug (steviol) within the cells and on the other side, it provides glucose homeostasis to adjacent normal cells. These features made steviol as dual functional drug by overtaking other natural drugs for promising results. The exposure of steviol inhibited the proliferative (IC50: 10mM) and clonogenic abilities of pancreatic cancer cells. It also suppressed the cell migration and cell invasion capabilities of pancreatic cancer cells. Further, steviol caused the selective inhibition of intracellular glucose intake and lactate accumulation in a concentration dependent manner. The selective inhibition of MUC13 and PKD1 (upstream key proteins of glucose metabolism) that caused the disruption of glucose intake in cancer cells, was observed during steviol treatment. Secondly, steviol interfered with translation-initiation machinery, causing destabilization of the cellular functionality. The impairment of cellular translation process had promoted G1 cell cycle arrest in cells due to the lack of G1-S transition proteins during drug exposure. Collectively, all these events engendered the cellular integrity and directed towards activation of apoptosis. In conclusion, we evaluated the role of MUC13 and PKD1 in aberrant glucose metabolism leading to growth and aggressive metastatic phenotypes of pancreatic cancer. We also demonstrated efficacy of steviol to repress aberrant glucose metabolism via suppression of MUC13 and PKD1 proteins.

Diabetes is currently affecting 30 million Americans. To combat disease onset, lifestyle interventions have been focused on. Low water intake, alongside elevated concentrations of circulating copeptin, has been linked to increased risk for. However, it is unknown whether this effect on glucose regulation is acute or chronic. Furthermore, no study to date assessed the effects of copeptin independent of the renin-angiotensin-aldosterone system (RAAS). Purpose: The purpose of these studies was therefore two-fold: 1) to establish high endogenous copeptin concentrations independent of RAAS, and 2) to assess glucose metabolism following a standardized oral glucose tolerance test (OGTT) during high concentrations of c copeptin. Methods: 60 subjects participated in this randomized, crossover design study. On two trial days, subjects were infused for 120-min with either 0.9 % sodium chloride (NaCl, ISO) or 3.0 % NaCl (HYPER) (Study 1). Post infusion, a 240-min oral glucose tolerance test (OGTT, 75 g) was administered (Study 2). Changes in plasma volume (ΔPV), plasma osmolality (POsm), electrolyte, copeptin, glucose, insulin, glucagon, crh, acth and cortisol concentrations were measured every 30 min. Results: During HYPER, plasma osmolality (POsm) and copeptin increased (P0.05) at baseline and during the 120-min of infusion. During the OGTT the positive integer of the area under the curve (AUC) for glucose was greater during HYPER (401.5±190.5 mmol·L-1·min) vs. the ISO trial (354.0±205.8 mmol·L-1·min, P< 0.05). The positive integer of the AUC for insulin during OGTT did not differ between trials (HYPER: 55,850±36,488 vs. ISO: 57,205±31,119 pmol·L-1·min). Baseline values of serum glucagon were not different between the two trials, however, the AUC of glucagon during the OGTT was also significantly greater in HYPER (19,303±3,939 ng·L-1·min) vs. the ISO trial (18,600±3,755 ng·L-1·min; P < 0.05). Conclusion: The present data indicate that acute osmotic stimulation of copeptin induced greater hyperglycemic responses during the oral glucose challenge possibly due to greater glucagon levels.

The neonatal period in mammals is characterized by high growth rates and is dominated by skeletal muscle hypertrophy. Low birth weight (LBWT) neonates experience restricted growth and development of skeletal muscle, leading to metabolic perturbations later in life. The overall hypothesis of this dissertation was that in utero disturbances in glucose metabolism and increased energy requirements predisposes LBWT neonatal pigs to metabolic disturbances after birth. We sought to increase growth of skeletal muscle and improve glucose production through increasing dietary energy and to determine the changes in glucose catabolism and metabolic flexibility in different skeletal muscle fiber types in LBWT neonates. Piglets were considered normal birth weight (NBWT) and LBWT when birth weight was within 0.5 SD and below 2 SD of the litter average, respectively. Increasing dietary energy increased lean deposition in the longissimus dorsi (LD) in both NBWT and LBWT neonates. Although glucose rate of appearance was greater in LBWT compared to their NBWT sibling, glucose concentrations were reduced in LBWT compared to NBWT pigs, regardless of diet fed. Postprandial glucose concentrations were lower in LBWT compared to NBWT pigs, regardless of diet fed, although rate of appearance did not differ between them. This would suggest that glucose is being absorbed in the peripheral tissues to be utilized. However, expression of enzymes related to glycolysis were downregulated in both the soleus and LD of LBWT compared to NBWT neonatal pigs. In addition, expression of enzymes related to the catabolism of glucose in the serine biosynthetic pathway were decreased in both the soleus and LD muscles of LBWT compared to NBWT neonatal pigs. Expression of the pentose phosphate pathway was slightly increased in LBWT compared to NBWT siblings in both muscle types. Increased expression of pyruvate dehydrogenase 4 was exhibited in both the soleus and LD of LBWT pigs compared to NBWT siblings. This would indicate a switch in fuel utilization to more fatty acid oxidation. By contrast, CO2 production from the oxidation of palmitate was reduced in LBWT compared with NBWT pigs along with reduced oxidation of glucose and pyruvate. In conclusion, lipid supplementation increased growth at the expense of fat deposition in the liver of NBWT and LBWT pigs. However, supplementing with fat did not increase glucose production due to the contribution of glycerol remaining constant. Hypoglycemia cannot be attributed to greater catabolism in skeletal muscle due to decreased expression of glycolytic genes and the addition of fatty acids did not spare glucose oxidation in skeletal muscle of LBWT pigs.

In the late 90’s, emerging evidence revealed that the brain is insulin-sensitive, highlighted by broad expression of brain-specific insulin receptors and reports of circulating brain insulin. Contemporary literature robustly supports the role of insulin signaling in normal brain function and suggests that insulin-related processes diminish with aging, evidenced by decreased signaling markers, reduced insulin receptor density, and lower levels of insulin transport across the blood-brain barrier. In the context of pathological cognitive decline, clinical trials using intranasal insulin delivery have reported positive outcomes on memory and learning in patients with mild cognitive decline or early-stage Alzheimer’s disease. However, while the importance of insulin and its related actions in the brain are robustly supported, the distinct mechanisms and pathways that mediate these effects remain unclear. To address this, I conducted a series of experiments exploring the impact of insulin on memory and learning in two models: primary hippocampal cell cultures and the Fisher 344 animal model of aging. These studies attempted to identify relationships between insulin receptor signaling, neuronal gene expression, glucose metabolism, and calcium homeostasis in the hippocampus using either expression of a constitutively active human insulin receptor or administration of intranasal insulin. The following dissertation summarizes this work and provides valuable insights into the potential pathways mediating these relationships. Of note, intranasal studies reported that insulin is able to significantly alter gene expression patterns in the hippocampus of both young and aged rats following chronic, repeated exposure to the ligand. In cell culture, constitutive insulin signaling correlated with significantly elevated neuronal glucose uptake and utilization, as well as with significant alterations in the overall expression and localization of the neuron-specific glucose transporter 3. Interestingly, continued activity of the insulin receptor did not appear to alter voltage-gated calcium channels in hippocampal neurons despite prior evidence of the ligand’s role in other calcium-related processes. The results reported in this manuscript suggest that in the brain, insulin may be involved in a myriad of complex and dynamic events dependent on numerous variables, such as age, length of the exposure, and/or the insulin formulation used. Nevertheless, this work highlights the validity of using insulin to ameliorate age-related cognitive decline and supports the need for further studies exploring alternative approaches to enhance insulin receptor signaling in the brain.

Among the main complications associated with obesity are insulin resistance and altered glucose and lipid metabolism within the liver. It has previously been described that insulin receptor isoform A (IRA) favors glucose uptake and glycogen storage in hepatocytes compared with isoform B (IRB), improving glucose homeostasis in mice lacking liver insulin receptor. Thus, we hypothesized that IRA could also improve glucose and lipid metabolism in a mouse model of high-fatdiet-induced obesity. We addressed the role of insulin receptor isoforms in glucose and lipid metabolism in vivo. We expressed IRA or IRB specifically in the liver by using adeno-associated viruses (AAVs) in a mouse model of diet-induced insulin resistance and obesity. IRA, but not IRB, expression induced increased glucose uptake in the liver and muscle, improving insulin tolerance. Regarding lipid metabolism, we found that AAV-mediated IRA expression also ameliorated hepatic steatosis by decreasing the expression of Fasn, Pgc1a, Acaca and Dgat2 and increasing Scd-1 expression. Taken together, our results further unravel the role of insulin receptor isoforms in hepatic glucose and lipid metabolism in an insulin-resistant scenario. Our data strongly suggest that IRA is more efficient than IRB at favoring hepatic glucose uptake, improving insulin tolerance and ameliorating hepatic steatosis. Therefore, we conclude that a gene therapy approach for hepatic IRA expression could be a safe and promising tool for the regulation of hepatic glucose consumption and lipid metabolism, two key processes in the development of non-alcoholic fatty liver disease associated with obesity.

Many studies investigating mechanisms of diet-induced insulin resistance impose controlled experimental conditions (constant insulin stimulation, constant glucose and/or fatty acid availability) designed to make interpretation of results simpler. These models may alter important parameters such as insulin or glucose to levels that are not ever experienced by the animal during normal feeding or fasting. The aim of this thesis was to investigate mechanisms of insulin resistance in the muscle of rodents fed a high- fat, high-sucrose diet (HFHS) under physiologically relevant conditions. We examined glucose uptake and markers of insulin action in the muscle of chow and HFHS rats at four time-points during the normal diurnal cycle. Chow and four week HFHS-fed rats were implanted with chronic venous catheters and were infused with a bolus of glucose tracer in the conscious state to determine glucose dynamics. HFHS animals displayed hyperinsulinemia but had reduced systemic glucose disposal and impaired muscle glucose uptake at the time-points studied across the feeding period. There were no differences in the phosphorylation of the canonical insulin signalling protein Akt or its downstream targets, AS160 and GSK3B in the muscle of chow and HFHS rats. Transfection of a constitutively active Akt into the tibialis muscle of HFHS rats did not rescue diet-induced reductions in insulin-stimulated glucose uptake. Phosphoproteomic analysis of the red quadriceps muscle comparing chow and HFHS identified differentially regulated phosphopeptides that tended to be from proteins involved directly in energy metabolism rather than protein kinases. Proteins involved in the regulation of pyruvate dehydrogenase (PDH) were altered in both the phospho, and total, proteomic analysis. In order to investigate the role of PDH in insulin-stimulated glucose uptake, we utilised the pyruvate dehydrogenase kinase inhibitor dichloroacetate (DCA). Both acute and chronic administration of DCA in rodents did not affect insulin sensitivity systemically or in muscle, measured by hyperinsulinemic-euglycemic clamp. Overall, these studies suggest that insulin resistance in muscle induced by a relatively short-term of HFHS-feeding is not associated with defective canonical insulin signalling. Additionally, although PDH activity in muscle is reduced by HFHS-feeding, activation of PDH does not rescue lipid-induced insulin resistance. The work in this thesis highlights the advantage of conducting investigations under a variety of experimental conditions (in vivo, ex vivo, free-living) to determine the relevance of molecular processes to whole body metabolism particularly in the context of insulin sensitivity.

The epidemicity of metabolic diseases such as obesity and diabetes globally has become a public health concern. Therefore, understanding interactions of macronutrients such as glucose and micronutrients such as vitamin A (VA) has become urgent. Both skeletal muscle and adipose tissues are critical for the regulation of glucose homeostasis. Retinoid acid (RA) is an active metabolite of retinol which mediates the major functions of VA. RA and insulin signals have been shown to regulate glucose metabolism. However, their roles in glucose metabolism in skeletal muscle and adipocytes remain to be revealed. We postulate that insulin and RA signals may work together to regulate glucose metabolism in skeletal muscle cells and adipocytes. In the first study, the glucose transporter 4 (GLUT4) expression were measured in L6 myocyes treated with increasing doses of RA in the absence or presence of insulin for 6 days. RA and insulin were found to inhibit the expression levels of GLUT4 in L6 cells after 4 and 6 days of treatment. The synergy inhibition effects of RA and insulin on GLUT4 can be observed at Day 4 with RA at 1 μM RA. To study the RA effects on adipocytes, recombinant adenoviruses were used to overexpress retinoid X receptor α (RXRα) and chicken ovalbumin upstream transcription factor II (COUP-TFII), two transcription factors with the potential to mediate RA signaling. 3T3-L1 CARΔ1 cells were transfected with ad-β-gal, ad-RXRα or ad-COUPTFII and treated with increasing RA doses during differentiation. Lipid accumulation and GLUT4 protein level were examined. Interestingly, overexpression of RXRα enhanced the inhibitory effects of RA on lipid accumulation. Moreover, the inhibitory effect of RA on GLUT4 expression can be attenuated in the presence of RXRα overexpression. The overexpression of COUPTFII may enhance the inhibitory effects of RA on the expression of GLUT4. The fact that RA and insulin synergized to inhibit the GLUT4 expression in L6 cells seems to indicate that cautions should be taken when VA supplement is recommended for patients with diabetes or obesity.

Myeloid derived suppressor cells (MDSCs) are a heterogeneous group of immature myeloid cells that are significantly increased in cancer patients and correlate with higher stage and poor prognosis. MDSCs negatively modulate anti-tumor immunity, suppress T cell activity, promote angiogenesis and increase the risk of metastasis. In this study, we report that monocytic-MDSCs (M-MDSCs) but polymorphonuclear-MDSCs (PMN-MDSCs) over-express 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatases 3 (PFKFB3), an important regulator of glycolysis. Furthermore in the melanoma model, M-MDSCs but not PMN-MDSCs suppressed T cell function which correlated with PFKFB3 over-expression and increased rate of glycolysis. PFKFB3 inhibition with the first-in-class small molecule inhibitor, PFK-158 reversed M-MDSC mediated T cell suppression and decreased the expression of arginase 1 and inducible nitric oxide synthase (iNOS) both in vitro and in vivo. In addition, both in B16-F10 tumor-bearing mice and patients enrolled in Phase 1 clinical trial, PFKFB3 inhibition resulted in decrease in M-MDSC frequency and increase in effector T cell populations. In this first-of-a-kind study, we present strong evidence for targeting metabolic profile of M-MDSCs to modulate their immune suppressive phenotype. This study provides the basis to study PFK-158 as an immunomodulatory agent in combination with immune checkpoint blockade therapies to improve T cell activity and anti-tumor responses.

The in vitro leishmanicidal activities of a series of 48 recently synthesized selenium derivatives against Leishmania infantum and Leishmania braziliensis parasites were tested using promastigotes and intracellular amastigote forms. The cytotoxicity of the tested compounds for J774.2 macrophage cells was also measured in order to establish their selectivity. Six of the tested compounds (compounds 8, 10, 11, 15, 45, and 48) showed selectivity indexes higher than those of the reference drug, meglumine antimonate (Glucantime), for both Leishmania species; in the case of L. braziliensis, compound 20 was also remarkably selective. Moreover, data on infection rates and amastigote numbers per macrophage showed that compounds 8, 10, 11, 15, 45, and 48 were the most active against both Leishmania species studied. The observed changes in the excretion product profile of parasites treated with these six compounds were also consistent with substantial cytoplasmic alterations. On the other hand, the most active compounds were potent inhibitors of Fe superoxide dismutase (Fe-SOD) in the two parasite species considered, whereas their impact on human CuZn-SOD was low. The high activity, low toxicity, stability, low cost of the starting materials, and straightforward synthesis make these compounds appropriate molecules for the development of affordable antileishmanicidal agents.

Physical inactivity and obesity are associated with elevated systemic oxidative stress and the activation of JNK, p38 MAPK, NF-κB, and protein kinase C (PKC) signalling pathways in skeletal muscle. Sustained activation of these stress and mitogen activated protein kinase (SAPK) pathways are associated with impaired glycaemic control, and the development and progression of cardiometabolic disease. Paradoxically, acute exercise also increases oxidative stress and SAPK signalling, yet glycaemic control and skeletal muscle function are enhanced. Research now supports a role for the transient induction of oxidative stress and associated activation of SAPK signalling in the physiological response and adaptation to acute exercise and exercise training. High-intensity interval exercise (HIIE) is a potent exercise stimulus for the improvement of metabolic health and skeletal muscle adaption, however the effect of HIIE on oxidative stress and SAPK signalling is unclear. The aims of this thesis were to explore the effect of HIIE on glycaemic control, exercise-induced oxidative stress, and skeletal muscle SAPK signalling, in a series of independent but related studies.

The most successful approaches for improving glucose metabolism in obese individuals are reducing energy intake (dieting) and increasing energy expenditure (exercise). Approaches aimed at pharmacologically or genetically increasing or inhibiting fatty acid metabolism to increase glucose utilisation have been only partially successful. This is because without an increase in energy expenditure, the breakdown products of increased fatty acid or glucose metabolism will eventually lead to a build-up of alternative storage products. In this thesis we examined the concept of promoting a futile cycle in both fatty acid and glucose metabolic pathways, by increasing the activity of a specific enzyme and altering metabolism in a way that might also alter energy demand. Acyl CoA synthase enzymes (ACSL1) and phosphofructokinase (PFK1) are metabolic enzymes in the muscle with high basal activities. It was proposed that overexpressing the opposing enzymes thioesterase (Acot7) or fructose bis-phosphatase (FBP2) respectively, would promote futile cycling between Acot7 and ACSL or FBP2 and PFK leading to energy wastage, an increased energy demand, and thereby an increase in substrate utilisation. This was hypothesised to have beneficial effect on insulin action in the muscle. Overexpressing Acot7 altered fatty acid oxidation in an ex vivo muscle system, but there was no effect of increasing thioesterase activity on insulin-stimulated glucose uptake in muscle in vivo. One explanation for this lack of effect is that the subcellular localisation or the level of overexpression of Acot7 may not have been ideal for promoting futile cycling at this step of fatty acid metabolism. Overexpressing FBP2 in the muscle increased glucose uptake under basal conditions ex vivo and under insulin-stimulated state in vivo indicating that altering glycolysis in this way was successful in increasing glucose uptake in muscle. However it was not clear if these effects were accompanied by an increase in energy expenditure. The results demonstrate the potential for creating futile cycles of metabolism in muscle and suggest that creating a futile cycle in glycolysis can have a beneficial impact on glucose metabolism in vivo.

Membrane transporters are widely expressed throughout the body, transporting nutrients, metabolites, toxins, and drugs across cellular membranes. In drug development, transporters are clinically important for drug absorption and disposition, and play significant roles in pharmacokinetics and treatment response. In addition, there is a growing interest in physiologic roles of transporters as polymorphisms in many drug transporters have been significantly associated with human diseases. Metformin is widely used as first-line treatment of type 2 diabetes. It exists primarily as a hydrophilic cation at physiological pHs, and therefore, membrane transporters are involved in its intestinal absorption, tissues distribution, and renal elimination. Multiple organic cation transporters play roles in the pharmacokinetics of metformin, and many of them play important roles in its pharmacological response, as mediators of metformin entry into target tissues. Here, we investigated transporters of metformin, with an emphasis on the pharmacological and biological roles of transporters in the intestine and liver. We characterized the human thiamine transporter (THTR-2; SLC19A3), which plays a major role in the intestinal absorption of thiamine (Vitamin B1). We found that human THTR-2 is capable of transporting other compounds including the drugs metformin and famotidine, and the neurotoxin MPP+. Other chemical agents including phenformin, chloroquine, verapamil, famotidine, amprolium, and pyrithiamine were inhibitors of hTHTR-2 mediated uptake of both thiamine and metformin. Interestingly, previous studies from our laboratory and others identified thiamine as an endogenous substrate for several organic cation transporters including OCT1, OCT2, and MATE1. In our second study, we focused on the endogenous role of OCT1, which is the major hepatic transporter for metformin. Through extensive experiments in Oct1 knockout mice, our data support the notion that hepatic thiamine deficiency is the underlying mechanism for the clinical phenotypes associated with reduced OCT1 function, particularly elevated LDL- cholesterol and total cholesterol levels. Our data suggest that reduced OCT1-mediated thiamine uptake in the liver leads to reduced levels of thiamine pyrophosphate, TPP, and a decreased activity of key TPP-dependent enzymes. As a result, there is a shift from glucose to fatty acid oxidation in the liver, which disrupts key metabolic flux pathways. In parallel, through analysis of published genomewide association studies, we show that reduced function OCT1 polymorphisms are associated with several metabolic traits in humans. Taken together, our studies suggest that changes in OCT1 activity modulate the disposition of thiamine and its active metabolite, TPP, which results in alterations in hepatic energy metabolism, ultimately affecting total body energy homeostasis. Finally, we developed a comparative structural model of OCT1 that discriminates ligands from non-ligands and is the model together with an in vitro high-throughput screening (HTS) assay to screen a drug library to explore the molecular properties of drugs interacting with OCT1. To extend the in silico methods in predicting endogenous metabolites that interact with OCT1, we conducted a virtual screen of 29,332 human metabolites against the structure-based model and an SAR model generated from the HTS data. In conclusion, our findings have important implications for understanding metformin absorption and disposition, and have expanded our understanding of the physiologic and pathophysiologic roles of membrane transporters.

Evidence from animal models and clinical data suggest that maternal obesity programs a range of detrimental effects in offspring. Limiting gestational weight gain may be a useful strategy to reduce these effects. This thesis investigated the impact of voluntary exercise during pregnancy on health outcomes in offspring of obese and lean mothers. Female Sprague-Dawley rats were fed chow (C n=28) or high fat (F n=32) diet for 6 weeks. Ten days before mating, half of each group were offered voluntary exercise with a running wheel (CE/FE); the others remained sedentary (CS/FS). Wheel access continued until parturition. Pup body weight (BW) was monitored and two pups from each mother were killed at postnatal day (PND)19. Thereafter siblings were fed chow. At 5-6 weeks, male pups were tested for anxiety on the Elevated Plus Maze (EPM); all offspring were killed at 13 weeks (adult) for metabolic assessment. Lean and obese mothers showed similar modest levels of exercise with no significant effect on maternal BW. At PND1, lean exercised mothers had lighter male and female pups with no effect in those from obese mothers. At PND19, maternal obesity increased offspring BW and adiposity with no effect of exercise. Exercise reduced insulin concentrations in males, with reduced glucose in male FE pups. In males, maternal obesity significantly decreased muscle MYOD1 and GLUT4 mRNA expression and these were normalised by exercise. Maternal exercise upregulated adipose GLUT4, IL6, TNFα, and PGC1α mRNA expression in CE offspring. Maternal obesity had no effect on anxiety-like behavior while CE offspring showed increased anxiety-like behavior. Adult FS males had higher plasma insulin, leptin and triglyceride concentrations, which were normalised by maternal exercise, while in females only the insulin concentration was raised. In males, hippocampus TLR4, FTO and IL-1β mRNA expression were upregulated by maternal obesity, and normalised by exercise. Taken together, maternal obesity increased the risk of metabolic disease in offspring, with higher BW, adiposity, hyperleptinemia, hyperinsulinemia and increased brain inflammatory markers. Modest levels of maternal exercise appeared to decrease the metabolic risk induced by maternal obesity. Exercise had sex-specific benefits that were more marked in male offspring.

The circadian system plays an essential role in regulation of glucose homeostasis, and the disruption of this system leads to deleterious health consequences, such as Type 2 Diabetes Mellitus (T2DM). Disruption of circadian organization in humans is strongly associated with development of metabolic dysfunction and increases the propensity for development of T2DM. Importantly, the correlation between circadian disruption and T2DM is partly attributed to loss of beta-cell function and mass, called beta-cell failure. To address this association, we first hypothesized that exposure to environmental conditions associated with disruption of circadian rhythms and susceptibility to T2DM in humans disrupts islet clock and beta-cell function. We reported that prolonged exposure to LL disrupts islet circadian clock function through impairment in the amplitude, phase, and interislet synchrony of clock transcriptional oscillations. We also reported that exposure to LL leads to diminished glucose-stimulated insulin secretion due to a decrease in insulin secretory pulse mass. Second, obesity-mediated insulin resistance is an important contributory factor to induction and progression of T2DM. We, therefore, hypothesized that disruption of circadian rhythms compromises pancreatic beta cell functional and morphological adaption to diet-induced obesity leading to development of T2DM. We found that concomitant exposure to LL and HFD resulted in development of hyperglycemia characterized by loss of circadian rhythms in insulin secretion, compromised beta cell function, and induction of beta cell apoptosis, suggesting that circadian disruption and diet-induced obesity synergize to promote development of beta cell failure, likely mediated as a consequence of impaired beta cell clock function. Third, as accumulating evidence pointed out that metabolic signals can feed back into the circadian system, we tested whether the impaired glucose homeostasis disturbs the circadian system in return. We examined the expression of circadian rhythms in the human islet amyloid polypeptide transgenic (HIP) rats: a validated non-obese, beta-cell dysfuntion model of T2DM. Diurnal and circadian rhythms of locomotor activity, circadian entrainment, and clock gene oscillation in SCN, pancreatic islets and aorta all remains intact in the HIP rats. In summary, these studies examined mechanisms by which circadian disruption predisposes to beta-cell failure in the development of T2DM. We also showed that glucose intolerance caused by beta-cell dysfunction might not be sufficient to cause circadian deficits, which emphasized the causal role of circadian disruption in the development of T2DM.

The increased number of individuals with metabolic diseases has become a public health concern. Vitamin A (VA, retinol) is required to maintain the general health of an individual. How VA contributes to the regulation of glucose and lipid homeostasis in normal and metabolic disease states is unclear. VA’s physiological activities are mainly mediated by its metabolite, retinoic acid (RA), which activates several transcriptional factors in the nuclear receptor super family and in turn, regulates the expression of numerous genes for macronutrient metabolism. For the RA production, retinol is first oxidized into retinal and then from retinal to RA. We hypothesize that VA status and dynamic production of RA play a role in the control of glucose and fatty acid metabolism. This dissertation summarized my investigation of VA’s role in metabolism: (1) We compared the expression levels of RA-producing enzymes in the liver of Zucker lean (ZL) and fatty (ZF) rats. We found that the hepatic expression levels of retinaldehyde dehydrogenase family 1 gene (Raldh1) mRNA and RALDH1 protein were higher in ZF rats than that in ZL rats. This elevated RALDH1 expression may cause the excessive RA production, which may enhance the hepatic lipogenesis and lead to the hepatic insulin resistance; (2) We compared the expression levels of insulin-regulated genes in the liver of VA deficient (VAD) and VA sufficient (VAS) ZL rats in response to the cycle of fasting and refeeding, a normal physiological condition. We observed that VA status and its dietary availability play a role in the expression levels of insulin-regulated gene in the rat liver; (3) We further used streptozotocin (STZ) to induce insulin-dependent diabetes mellitus in both VAD and VAS rats, a disease condition. The hepatic expression levels of insulin-regulated genes in these animals treated with control, RA, insulin and insulin + RA were investigated. The effects of VA status and RA production on the regulation of the hepatic gene expression were also observed. We conclude that VA status and dynamic RA production contribute to the expression of hepatic genes, which in turn regulate glucose and fatty acid homeostasis.

Obese individuals present with an increased inflammatory tone as compared to healthy, normal-weight individuals, which is associated with insulin resistance. One factor hypothesized to contribute to increased inflammation in obese and diabetic states is elevated blood endotoxin levels, also known as metabolic endotoxemia. In healthy rodents (non-obese and insulin sensitive), there is evidence that blood endotoxin levels fluctuate over the course of the day with elevations in the post-prandial state that return to baseline levels in the post-absorptive state. High-fat feeding in these animals altered these fluctuations causing endotoxin levels to remain high throughout the day. The effects of alterations in endotoxin levels on glucose metabolism are not understood. The goal of this study was to determine the effects of short-term and long-term increases in endotoxin of a low magnitude on insulin signaling in a human primary cell line as well as the effects of short-term endotoxin treatments on glucose homeostasis in a C57/Bl6 mouse model. First, we tested the hypothesis in cell culture that short-term low-dose endotoxin treatments would enhance insulin-signaling and glycogen synthesis while long-term treatments would have inhibitory effects. Under our second hypothesis, we examined whether short-term low-dose treatments of endotoxin would contribute to improvements in glucose tolerance in a mouse model. In contrast to our first hypothesis, short-term endotoxin treatments did not improve insulin signaling or glycogen synthesis although long-term treatments did contribute to decreases in glycogen synthesis. Interestingly, short-term endotoxin treatments resulted in significant improvements in glucose clearance in the mouse model; this is believed to be partly attributed to LPS inhibiting gluconeogenesis. Future studies are necessary to understand the mechanisms responsible for altered glucose metabolism in response to low magnitude changes in LPS levels.

Hormonal and nutritional stimuli coordinately regulate the glucose and lipid metabolism in the liver. Dysregulated hormonal balance and nutrient metabolism not only disrupt the energy homeostasis, but also predispose individuals to the development of obesity and its related metabolic diseases. Research presented in this dissertation investigated the effects of a nutritional factor, vitamin A (VA), and a potential component of insulin signaling cascade, atypical protein kinase C (aPKC), on the glucose and lipid metabolic genes transcription in primary hepatocytes from Zucker lean (ZL) and fatty (ZF) rats. To examine VA effects, we put ZL and ZF rats with different VA statuses on various feeding conditions. After the isolation of their primary hepatocytes, we analyzed the expressions of hepatic model genes in response to the treatments of insulin and retinoic acid (RA). We report that the insulin- and RA-regulated glucokinase (Gck), sterol regulatory element-binding protein-1c (Srebp-1c) and cytosolic form of phosphoenolpyruvate carboxykinase (Pck1) expressions are impaired in hepatocytes of ZF rats fed chow or a VA sufficient (VAS) diet ad libitum. The impairment is partially corrected when ZF rats are fed a VA deficient (VAD) diet ad libitum or pair-fed a VAS diet to the intake of their VAD counterparts in non-fasting conditions. Interestingly, in the pair-fed ZL and ZF rats, transient overeating on the last day of pair-feeding regimen changes the expression levels of some VA catabolic genes, and impairs the insulin- and RA-regulated gene expression in hepatocytes. To investigate the roles of aPKC, PKCζ(zeta) and PKCι/λ(iota/lambda), they were overexpressed in primary hepatocytes from ZL rats using recombinant adenoviruses. aPKC overexpression in primary hepatocytes significantly attenuated the insulin- regulated expressions of Gck, Srebp-1c and Pck1. Additionally, aPKC overexpression increased their basal transcript levels. The impairment is associated with reduced protein level of insulin receptor substrate 1, and the phosphorylation of protein kinase B in response to insulin treatment. Taken together, the data demonstrate that feeding state, VA status, and aPKC expression regulate the hepatic glucose and lipid metabolism at the gene expression level, and provide interesting insights into the development of metabolic diseases.

Faced with an environment of constantly changing nutrient availability, mammals have adapted complex homeostatic mechanisms to maintain energy balance. Deviations from this balance are largely corrected through a concerted, multi-organ effort that integrates hormonal signals with transcriptional regulatory networks. When these relationships are altered, as with over-nutrition and insulin resistance, metabolic disease ensues. Here, I present data concerning two distinct transcriptional pathways--one for thyroid hormone (TH) and one for insulin--that confer hormone responsiveness on metabolic gene programs that preserve energy homeostasis.

Mammals have to adapt quickly to the changes of nutrition availability. The liver is the central organ that coordinates the responses to food deprivation upon fasting and nutrient overload during feeding. In liver, hormonal and nutrient pathways converge into the regulation of transcriptional programs that are involved in maintaining energy homeostasis. When these fine-tuned regulations in liver are altered due to constant surplus of nutrients or insufficient hormonal actions, multiple metabolic diseases including type II diabetes can occur, followed by severe complications. As a part of those regulatory programs, PGC-1alpha (peroxisome proliferator-activated receptor gamma coactivator-1alpha) links hormonal signaling to the expression of glucose and lipid metabolic genes. Its transcriptional co-activator activity is tightly controlled via post-translational modification; GCN5 (histone acetyltransferase KAT2A) acetylates PGC-1alpha and suppresses its transcriptional activity, whereas Sirt1 deacetylates and activates PGC-1alpha. Herein, cyclin D1-CDK4 (cyclin-dependent kinase 4) kinase is identified as a new regulator of glucose metabolism in liver that modulates PGC-1alpha's transcriptional activity. Through a cell-based high throughput chemical screen, a CDK4 inhibitor was discovered to potently decrease PGC-1alpha acetylation. Cyclin D1-CDK4 kinase phosphorylates and activates GCN5, which then acetylates and inhibits PGC-1alpha activity on hepatic gluconeogenic genes. Feeding activates cyclin D1-CDK4 kinase in liver, which, in turn, suppresses glucose production independently of cell cycle progression. As part of the feeding response, insulin/GSK3beta (glycogen synthase kinase 3beta) signaling stabilizes cyclin D1 protein via sequestering cyclin D1 in the nucleus. In parallel, dietary amino acids increase hepatic cyclin D1 mRNA transcripts. Loss of hepatic cyclin D1 in mice leads to mild diabetic phenotypes. In diabetic models, cyclin D1-CDK4 is chronically elevated and refractory to fasting/feeding transitions; nevertheless further activation of this kinase normalizes glycemia. Thus, these findings show that hormonal and nutrient pathways utilize components of the cell cycle machinery in post-mitotic cells to control glucose homeostasis independently of cell cycle progression.

Exercise has benefits on body and fat mass, glucose metabolism, and lipid metabolism. Likewise, exercise is an effective therapy against the metabolic syndrome. Yet, the molecular pathways underlying the advantageous effects of exercise are imperfectly understood. Emerging evidence suggests that the melanocortin system and the fibroblast growth factor 21 (FGF21) signaling axis, two endocrine pathways involved in regulation of energy homeostasis, are mediators of exercise benefits.

Objective: To examine whether serum vitamin D levels are related to markers of glucose metabolism as measured by A1C, fasting glucose, and fasting insulin, Design: Cross-sectional design using data from the National Health and Nutrition Examination Survey (NHANES), 2005 – 2006.Subjects: Non-diabetic adults, ages 20 years and older, with measures of serum vitamin D, fasting glucose, fasting insulin, and A1C were included in the study. Race/ethnicity, as reported by the subject, was categorized as Mexican American (N = 254), other Hispanic (N = 46), non-Hispanic White (N = 579), African American (N = 278), & multi-racial (N = 57)Methods: Data collected from 1214 adult participants in the National Health and Nutrition Examination Survey (NHANES) 2005 – 2006 was accessed via the public domain and analyzed using SAS Enterprise software, version 6.1. Pearson correlation, odds ratio, and chi-squared tests were performed to determine the strength and significance of the relationship between levels of serum vitamin D and A1C, fasting glucose, and fasting insulin status. Results: Serum vitamin D was inversely correlated with A1C (r = -0.12; p = < 0.0001), fasting glucose (r = -0.08; p = 0.005), and fasting insulin (r = -0.18; p = < 0.0001), across the study population. For the total study population, the likelihood of having an abnormal marker of glucose metabolism with adequate serum vitamin D (= 30 ng/mL) was reduced for fasting insulin, (OR = 0.40; 95% CI = 0.29 – 0.54) and A1C (OR = 0.67; 95% CI = 0.44 – 0.99) but not significant for glucose (OR = 0.78; 95% CI = 0.57 – 1.06). When racial and ethnic groups were subdivided, the only marker that remained significant was fasting insulin in the Caucasian (OR = 0.50; 95% CI = 0.34 – 0.73) and Mexican-American (OR = 0.16; 95% CI = 0.07 – 0.39) groups.Conclusion: The results of this study show an inverse association between serum vitamin D and A1C, fasting glucose, and fasting insulin. Adequate levels of serum vitamin D (= 30 ng/mL) was also found to be associated with reduced risk of impaired fasting insulin and A1C in the total population. In subgroups, the odds of having an abnormal insulin level was only significant in Mexican-American and non-Hispanic white participants. This data extends previous research and provides new evidence that serum vitamin D may play a role in glucose metabolism, which may have implications for the pathogenesis of type 2 diabetes.

The past decade has witnessed remarkable progress toward understanding the reprogrammed metabolism in cancer, an emerging hallmark as well as an active area of basic, translational, and clinical research. About ninety years ago, Otto Warburg pioneered quantitative investigations of cancer metabolism and discovered that cancer cells exhibit a phenotype of increased glycolysis even under aerobic conditions, known as the Warburg effect. Warburg speculated that the reason for the upregulated glycolysis was for compensating the ATP shortage due to dysfunctions of mitochondria in cancer cells. Despite subsequent progresses, the biological reasons for ATP synthesis by aerobic glycolysis in cancer cells are only partially understood. Intriguingly, intratumoral (extracellular) ATP levels are 103 to 104 times higher than those in normal tissues. We showed that although extracellular ATP is not known to cross the plasma membrane by itself, extracellular ATP in the range of the intratumoral ATP levels induced large intracellular ATP concentration increase in A549 human lung cancer cells and promoted cancer cell survival. More importantly, we reported that a nonhydrolyzable fluorescent ATP was internalized by A549 cells through macropinocytosis as visualized by fluorescence microscopy. The induced ATP increase was reduced by the macropinocytosis inhibitor EIPA but persisted even when mitochondrial oxidative phosphorylation and glycolysis were inhibited, without involving transcription or translation. The increases were also observed in several other cancer cell lines, but not in noncancerous cells. Furthermore, extracellular ATP enhanced cancer cell survival under various stress conditions and promoted drug resistance to tyrosine kinase inhibitors that compete with ATP for their anticancer action. Collectively, these results provide the first piece of evidence that extracellular ATP is internalized by cancer cells via macropinocytosis and potentially other endocytic process, which significantly contribute to their growth and to drug resistance. These findings potentially change our understanding of ATP supply and sharing among cancer cells, enhance our understanding of the Warburg effect, and highlight a novel anticancer target. Current understanding of glucose transport and metabolism in cancer, anticancer therapeutics targeting glycolysis and glucose transporters, and daunting challenges are also summarized. Based on the previous discovery on WZB-117, the first generation of glucose transport inhibitor, we screened a library of its derivatives and identified WZB-173 and DRB-18 with higher stabilities and potencies as lead compounds in new generations of novel glucose transport inhibitors. Both compounds can serve as models for further development of glucose transport inhibitors.

Irisin is assumed to be a relevant link between muscle and weight maintenance as well as to mediate exercise benefits on health. The aim of this study was to assess the possible associations between irisin levels and glucose homeostasis in obese subjects with metabolic syndrome (MetS) following an energy-restricted treatment. Ninety-six adults with excessive body weight and MetS features underwent a hypocaloric dietary pattern for 8 weeks, within the RESMENA randomized controlled trial (www.clinicaltrials.gov; NCT01087086). After the intervention, dietary restriction significantly reduced body weight and evidenced a dietary-induced decrease in circulating levels of irisin in parallel with improvements on glucose homeostasis markers. Interestingly, participants with higher irisin values at baseline (above the median) showed a greater reduction on glucose (P=0.022) and insulin (P=0.021) concentrations as well as on the homeostasis model assessment index (P=0.008) and triglycerides (P=0.006) after the dietary intervention, compared with those presenting low-irisin baseline values (below the median). Interestingly, a positive correlation between irisin and carbohydrate intake was found at the end of the experimental period. In conclusion, irisin appears to be involved in glucose metabolism regulation after a dietary-induced weight loss.

The liver is a vital organ for the homeostatic control of both carbohydrate and lipid metabolism, functioning as a major regulatory site of energy storage, processing, and transformation. This project focused on liver lipid metabolism--it's production and trafficking of very low density density lipoproteins in particular. Specifically, we investigated whether VLDL trafficking is affected by 1) glucose and/or insulin, and 2) source of fatty acid (de novo or exogenous). As these were two distinct questions, each required its own sub-study, both of which are outlined separately below. Although it is well known that glucose and insulin influence the metabolism of fatty acids (FA) and the secretion of very low-density lipoprotein (VLDL), little is understood about how they regulate the partitioning of FA hydrolyzed from hepatic stored triacylglycerol (TAG) to different metabolic pathways. The aims of this study were to investigate the effects of differing concentrations of glucose and insulin on TAG hydrolysis and the subsequent partitioning of FA to pathways of secretion and oxidation in primary hepatocytes. We utilized primary mouse hepatocytes for pulse-chase experiments using [1-14C] oleate to examine the turnover and partitioning of the labeled FA. In addition to looking at the effects of differing glucose and insulin concentrations, we wanted to investigate whether any effects differed following acute (6 h) vs. chronic (24 h) treatment. Although we did not observe any acute effects of either glucose or insulin on TAG hydrolysis or partitioning of hydrolyzed FA, chronic insulin exposure decreased both FA oxidation and TAG secretion. These results suggest that insulin and glucose do not acutely influence TAG hydrolysis or channeling of hydrolyzed FA, but longer exposure to insulin reduces FA oxidation and secretion. There is a great deal of evidence suggesting that before being incorporated into VLDL for secretion, hepatic FA are first directed to storage in the cytosolic lipid droplet pool, and must subsequently be mobilized and channeled to assembly into VLDL. It remains unclear, however, whether the liver stores FA from different sources in distinct lipid droplet pools. Furthermore, should distinct storage pools exist, it is unknown whether they are preferentially channeled to different pathways, such as VLDL assembly. We performed experiments utilizing [1-14C] acetate and [1-14C] palmitate to represent FA derived from de novo lipogenesis and exogenous uptake, respectively. There was no difference between percentage of cellular acetate (via conversion to long chain FA) or palmitate secreted into the media at 4, 8, and 16 h in primary hepatocytes. However, we consistently saw a greater percentage of the label from acetate (de novo FA) secreted into the media after 2 h. Following the same 2 h labeling period in primary hepatocytes, we observed no difference between the partitioning of [1-14C] acetate and [1-14C] palmitate to the microsomal (ER) and lipid droplet fractions. Finally, to examine in vivo partitioning of the different FA, we injected mice with labeled FA and isolated their livers to quantify the contribution of the respective FA to cellular TAG and phospholipids. There was no difference in the incorporation of differentially sourced FA in liver TAG or phospholipid fractions. The results of these studies suggest that given the choice between FA taken up from exogenous sources and FA synthesized de novo, the liver may acutely preferentially secrete FA from de novo synthesis, however more work is needed to confirm this finding.

PURPOSE: Dietary food composition influences postprandial glucose homeostasis. Thus, the objective was to investigate the effects of an acute intake of three different types of strawberry jam, differing in carbohydrate and antioxidants content, on postprandial glucose metabolism, lipid profile, antioxidant status, and satiety. METHODS: Sixteen healthy adults participated in a randomized, crossover, double-blind study with three arms, receiving 60 g of three different strawberry jams. Blood samples were collected at fasting and at 30, 60, 90, and 120 min after its intake. Blood analyses were performed with validated procedures and satiety was estimated with visual analogue scale (VAS). RESULTS: Blood glucose concentrations were maintained at normal values and without peaks within the 2 h after consumption of low-sugar jams. However, blood glucose and insulin were significantly higher at 30 and 60 min after high-sugar (HS) jam intake versus both low-sugar jams. Furthermore, HS jam produced more satisfaction at short time, but decreased as soon as blood glucose concentration began to decrease. Moreover, HS ingestion produced lower free fatty acid levels (p < 0.05) throughout the trial with respect both the low-sugar jams. However, no additional benefits on oxidative status (malondialdehyde, glutathione peroxidase, total antioxidant capacity, and uric acid), glucose, lipid, and satiety variables were observed due to the inclusion of an antioxidant to low-sugar jam. CONCLUSIONS: This study reinforces the idea that products without added sugars are appropriate for the management of glycemic alterations and provides further insight into the effect of natural antioxidants as a functional ingredient on oxidative status and related metabolic disturbances.

The studies described in this dissertation focus on a novel mechanism of post-translational control as well as a novel pro-survival role for a canonical tumor-suppressor BH3-only protein, Noxa, in human hematopoietic cells and cancers. An investigation into regulatory mechanisms underlying the constitutive expression of this pro-apoptotic Bcl-2 family member in leukemias, led to the identification of a phosphorylated serine on the protein. The phospho-modification suppressed its pro-apoptotic function and was regulated by glucose levels. Glucose deprivation promoted Noxa dephosphorylation and activated its apoptotic function. The atypical cyclin dependent kinase, Cdk5, was identified as the glucose-sensitive Noxa kinase. Further investigation pointed to a role for phospho-modified Noxa in glucose metabolism; cells over-expressing Noxa increased their uptake of glucose and promoted its utilization in anabolic metabolic pathways that are favored in proliferating cells. Finally, studies showed Noxa and its anti-apoptotic binding partner Mcl-1 associated within cytosolic multi-protein complexes in proliferating T-leukemia and activated primary T-cells. Early characterization and identification of the components point to a novel glucose-responsive signaling role for these complexes. Taken together, these studies offer fresh insights into cancer cell metabolism and the regulation of Bcl-2 proteins and are expected to contribute to novel therapeutic strategies aimed at activating Noxa's pro-apoptotic function in leukemias.

As the importance of glucose metabolism in tumorigenesis is increasingly recognized, glycolysis has been emerging as a general and promising target of cancer therapeutics. Cancer cells upregulate their basal glucose transporter (Glut1) and glycolysis rate to generate energy, obtain biomass and maintain redox balance. This property is known as the Warburg effect. Based on the fundamental characteristic of cancer cells and considered it as a potential anticancer target, I screened a small library of natural derived small molecule inhibitors and identified novel basal glucose transport inhibitors. Three generations of these inhibitors were identified and studied here: WZB27 and 115; WZB117; and WZB131, 133 and 134, classified based on their improved anticancer activities and chemical stability. WZB27 and WZB115 were the first identified basal glucose transport inhibitors capable of selectively inhibiting cancer cell proliferation in multiple cancer cells lines without harming normal cells. The action mechanism of WZB27 and WZB115 was similar to that of glucose deprivation. WZB117 was selected among the second generation of the inhibitors and its action mechanisms were further investigated. Daily intraperitoneal (ip) injection of WZB117 at 10mg/kg resulted in about 70% reduction in tumor size in A549 cells xenografted tumor mice models. Target identification study showed that WZB117 was able to inhibit glucose transport in human red blood cells that express Glut1 as their sole glucose transporter. A following computer docking study revealed that WZB117 had high binding affinity to the central channel of Glut1. Cancer cells treated with WZB117 were found to downregulate Glut1 protein, intracellular ATP, and glycolytic enzymes. All these changes were followed by activation of energy-sensing enzyme AMPK and declines in growth signaling. Cell cycle G0/G1 arrest was identified as the major mechanism leading to cancer cell proliferation inhibition, and decreased levels of cyclin E2 and Rb phosphorylation further confirmed the observation. About 10% of the WZB117 treated cancer cells underwent necrosis, not apoptosis. In addition, senescence was found for the first time in Glut1 inhibited cancer cells. Therefore, cell cycle arrest, necrosis and senescence together contributed to the irreversible growth inhibition in WZB117 treated cancer cells. The third generation inhibitors showed significant improvement in compound stability. Thus, these glucose transport inhibitors can serve as prototypes for further development of anticancer therapeutics targeting Glut1-mediated glucose transport and glucose metabolism.

Selenium (Se) is an essential trace element used for biosynthesis of selenoproteins, acquired either through the diet or cellular recycling mechanisms involving the enzyme selenocysteine lyase (Scly). This dissertation characterizes the Sedependent regulation of Scly gene expression in in vitro and in vivo models. Scly mRNA negatively correlates with Se levels in HepG2 cells, liver and brain tissues, but not in Ht22 cells. Scly is also downregulated in the liver of the Selenoprotein P (Sepp1) knockout (KO) mouse, a model lacking the Se transporter protein with elevated hepatic Se content. Additionally, this dissertation reports the development and characterization of a Scly KO mouse. This mouse model presents diminished Se content and increased expression of selenoprotein mRNAs in the liver compared with wild type mice. Scly KO mice maintain circulating Se levels and most selenoprotein mRNA levels in Se target tissues, such as brain and testis. Disruption of the Scly mechanism also affects hepatic energy metabolism. Scly KO mice raised on a Se adequate diet exhibit hyperinsulinemia and mild hepatic steatosis, with attenuated insulin signaling as measured by Akt phosphorylation and increased oxidative stress, without changes in phosphorylation or protein levels of AMP-activated Protein Kinase alpha, a regulator of cholesterol biosynthesis. On Se restriction, Scly KO mice develop characteristics of metabolic syndrome, such as obesity, fatty liver, hypercholesterolemia and insulin resistance. Moreover, hepatic glutathione peroxidase 1 and circulating Sepp1 levels are also diminished, but mRNA levels of Acetyl-CoA carboxylase are increased. When exposed to a high fat, Se adequate diet, Scly KO mice become more vulnerable to obesity. The findings of this dissertation unveil a strong connection between Se recycling and glucose and lipid metabolism, and demonstrate for the first time in an animal model the unique physiological role of the Se recycling pathway in lipid and glucose metabolism.

The ER-localized DnaJ homologue 4 (ERdj4) is a soluble ER chaperone induced by the unfolded protein response (UPR) to assist in the removal of unfolded/misfolded proteins from the ER lumen for proteasomal degradation. To elucidate the function of ERdj4 in vivo, ERdj4 gene trap mice were generated from embryonic stem cells harboring a gene trap cassette inserted into the ERdj4 locus. ERdj4 gene trap mice expressed hypomorphic levels of ERdj4 with a 10-100 fold reduction in all tissues and cell types examined. Approximately 30-50% of ERdj4 gene trap mice died perinatally in association with growth retardation and hypoglycemia. ERdj4 gene trap neonates exhibited signs of delayed pancreatic development, including abnormal distribution of pancreatic α- and β-cells and reduced insulin and glucagon in the pancreas. Surviving adult ERdj4 gene trap mice were glucose intolerant, resulting from hypoinsulinemia rather than insulin resistance. Pancreatic β-cells exhibited increased ER stress, including ER dilation and upregulation of the UPR. Proinsulin accumulated in the ER of β-cells from ERdj4 gene trap mice consistent with our previous finding that proinsulin is a substrate for ERdj4. The insulin processing enzymes, including Pcsk1, Pcsk2 and CPE, also associated with ERdj4, contributing to defects in proinsulin biosynthesis in ERdj4 gene trap mice. ERdj4 deficiency also resulted in pancreatic a-cell hyperplasia in association with increased ER stress. Since previous studies had demonstrated that the UPR is required for both early and late B lymphopoiesis, we hypothesized that ERdj4 deficiency would affect B cell development. Pro-B, pre-B, immature and mature B cell populations were significantly reduced in the bone marrow of ERdj4 gene trap mice in association with increased pro-B cell death. Further, mature B cell populations were reduced in the spleen and peritoneal cavity. ERdj4 gene trap donor cells transplanted into wildtype recipients rescued all stages of B cell development, confirming a defect in the microenvironment. Osteogenic cells support B cell development and the UPR is required for osteoblast differentiation and function. Bone-lining cells, which differentiate from osteoblasts, were reduced in ERdj4 gene trap mice in association with increased ER stress. Further, primary bone cells from ERdj4 gene trap mice had reduced expression of osteogenic-specific markers. These data suggest that the loss of ERdj4 affects the maturation/survival of osteogenic cells, which in turn leads to a defect in B lymphopoiesis. Collectively, this work reveals an important role for ERdj4 in survival, glucose metabolism and B cell development.