The heart must function properly to perform its essential role in supplying the body with the oxygen and nutrients required for survival. Over the course of a lifetime the heart will eventually be exposed to conditions of oxygen depletion or obstructed flow. At such times it is essential that the heart maintain its function and adapt to these conditions by altering its metabolism in response to the decrease in oxygen. Glucose, fatty acids, lactate and most other substrates require oxygen for full energy yield, making them unsuitable during an anoxic or ischemic period. Amino acids have a wide array of uses in the body, including as metabolic substrates. In particular, glutamine and glutamate can be easily converted to α-ketoglutarate and shuttled into the TCA cycle as metabolic substrates. For this reason we hypothesized that it may be possible to prolong cardiac function during and improve recovery after oxygen depletion by supplying the heart with excess glutamine and glutamate. Using action potential duration (APD) as a readout for the electrical function of the heart, we exposed rabbit hearts to anoxic and ischemic challenges and monitored their behavior. We show that elevated levels of glutamate and glutamine increased APD90 in anoxic hearts by 11% over controls. In ischemic hearts, however, the effects were even greater, as the enriched hearts had a 29% increase in APD90 and a 38% increase in APD50 compared to controls. We also demonstrate that blockage of amino acid transamination eliminates this effect and show that metabolism of these amino acids through the TCA cycle is the primary mechanism. These results are significant and conserved across both anoxia and ischemia, indicating that this could be a reliable and effective intervention for extending APD and possibly improving cardiac function.

Diabetes is characterized by excess levels of glucose and lipid in the blood, which have been proposed to directly and negatively impact islet β cells, exacerbating symptoms and promoting disease progression. Our understanding of glucotoxicity and lipotoxicity in human type 2 diabetes is limited, because most investigation has used either rodent islets or human islets in vitro. Based on these studies, numerous mechanisms have been proposed, but not tested, in human islets in vivo. To investigate the molecular mechanisms of excess glucose or lipid, we developed and employed three complementary models of nutrient excess, hyperglycemia (excess glucose), insulin resistance (excess lipid), or both, and examined direct effects on human islets in vivo. In response to hyperglycemia or insulin resistance, stimulated insulin secretion, the main indicator of β cell function, is severely reduced from human islet grafts. Surprisingly, in contrast to rodent islets, neither excess of lipid or glucose stimulated β cell proliferation or caused β cell apoptosis. In human islets in vivo, insulin resistance (1) blunted the unfolded protein response (UPR), (2) increased reactive oxygen species production and oxidative stress, (3) increased lipid droplets and amyloid deposits, and (4) reduced the key β cell transcription factors MAFB and NKX6.1. In contrast, hyperglycemia only blunted the UPR and reduced expression MAFB. Importantly, we found fundamental differences between human and mouse islets in response to identical metabolic conditions, such as stimulated insulin secretion, proliferative response, UPR induction, and transcription factor expression. To complement these studies, we performed a comprehensive, systematic, post-hoc analysis of variation of attributes and function across human islet preparations. Based on our data, we propose a model of glucotoxic and liptoxic consequences for human islets in vivo in which insulin resistance induces significantly more detrimental effects for human β cells in vivo than does hyperglycemia alone, but both conditions mechanistically converge to impair stimulated insulin secretion from human β cells in vivo. This work is the first to demonstrate and mechanistically investigate glucotoxic and lipotoxic consequences for human β cells in vivo, and the models developed will be useful to better understand and test interventions for gluco- and lipotoxicity.

Reduced pancreatic β cell mass is a hallmark of diabetes, which makes the ability to increase or restore β cell mass a major therapeutic goal. While testing the hypothesis that increased endothelial cell signaling would increase β cell mass using a model of inducible vascular endothelial growth factor-A (VEGF-A) overexpression in β cells (βVEGF-A mouse), we found that increased VEGF-A leads to reduced, not increased, β cell mass. Surprisingly, withdrawal of the VEGF-A stimulus is followed by robust β cell proliferation, leading to islet regeneration, normalization of β cell mass, and reestablishment of the intra-islet capillary network. Using islet and bone marrow transplantation approaches we found that β cell proliferation is dependent on the local microenvironment of endothelial cells, β cells, and bone marrow-derived macrophages recruited to the islets upon VEGF-A induction. Depleting macrophages greatly reduced β cell proliferation, indicating that these macrophages are required for the β cell proliferative response in regenerating islets. Based on these data, in addition to transcriptome analysis of FACS-sorted islet β cell, and islet-derived endothelial cell and macrophage populations during VEGF-A induction and normalization, we propose a new paradigm for β cell regeneration where β cell self-renewal is mediated by coordinated interactions between macrophages recruited to the site of β cell injury, intra-islet endothelial cells, and β cells. In this model, (1) increased growth factors produced by β cells, endothelial cells, macrophages, (2) increased production of growth factor receptors and integrins on β cells, and (3) increased integrin activation by the extracellular matrix cause simultaneous, and potentially synergistic, activation of the PI3K/Akt and MAPK signaling pathways, leading to β cell proliferation.

Loss-of-function mutations causing haploinsufficiency of the MC4R are the most common monogenic cause of severe early-onset obesity in humans. Research has primarily focused on how central MC4R contributes to energy homeostasis with the eventual goal of developing anti-obesity therapeutics. MC4R expression has been found in vago-vagal circuitry, as well as in enteroendocrine cell populations in the gastrointestinal tract. Multi-choice feeding studies in MC4R deficient mice revealed a surprising reduction in preference for palatable foods high in either fats or carbohydrates. This loss of macronutrient preference was also coupled with a homeostatic drive for hyperphagia. These complex defects in feeding behaviors were suggestive of defects in the gut-brain axis, a cascade of neural and hormonal signals which bi-directionally control feeding behaviors. Gastrointestinal expression of MC4R in L cells was shown to be functionally significant as MC4R agonism stimulated peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) secretion in vivo. These findings highlighted a complementary role for peripheral MC4R in the gut-brain axis that has implications for energy and glucose homeostasis. Furthermore, pharmacological action of melanocortin compounds at peripheral sites must now be considered when examining effects of the receptor on whole-animal physiology.

In the past decade, evidence has emerged that obesity induces a neuroinflammatory response in the hypothalamus, a part of the brain that contains neuronal circuitry controlling feeding and metabolism. The potential contribution of non-neuronal central nervous system (CNS) cells, including glia, to the regulation of energy homeostasis has only recently begun to be appreciated. Here we report on the role of two glial cell types, astrocytes and microglia, which act in concert as mediators of the neuroinflammatory response of the CNS. Neuroinflammation is thought to take place in two phases: an early acute phase necessary for homeostatic and defense mechanisms and a self-perpetuating long-term chronic phase associated with neurologic disease. The results herein describe evidence for opposing roles of this biphasic pattern of neuroinflammation in the regulation of energy homeostasis and the pathophysiology of obesity. Using mice with green-fluorescent protein (GFP)-labeled immune cells in peripheral circulation, we show that chronic high-fat diet (HFD) intake increases recruitment of monocytes in to the brain. Histological examination showed that these cells acquire morphology similar to activated phagocytic microglia, suggesting that recruitment of peripheral immune cells into the CNS may contribute to the neuroinflammatory response to obesity. Further studies were then conducted to describe the localization and activation of astrocytes in obesity. Increased expression of the astrocyte activation marker GFAP was found within several nuclei of the hypothalamus following chronic exposure to HFD. We also found that astrocytes in the hypothalamus were activated acutely after high-fat feeding. To begin to address the physiological significance of astrocyte activation to the regulation of energy homeostasis we examined the effect of inactivation of astroglial NF-κB, an essential component of astrocyte activation, on food intake. Suppression of NF-κB signaling in astrocytes in a tetracycline-inducible transgenic mouse model led to increased food intake following acute exposure to a HFD. This study provides novel evidence that astrocytes have a regulatory role in the regulation of feeding behavior in response to HFD.

Diabetes mellitus has been a disease of increasing prevalence for nearly a century and is attributed to a dysregulation of hormones secreted from the pancreatic Islets of Langerhans; insulin from β-cells and glucagon from α-cells. Dysregulated α-cell glucagon secretion is responsible for the chronic hyperglycemia that accompanies diabetes and may be an important therapeutic avenue. Despite its importance, the normal molecular regulation of glucagon secretion is poorly understood. This work characterized a novel mechanism by which somatostatin and insulin coordinate to lower cAMP and phosphorylated PKA in the α-cells with rising glucose to suppress glucagon secretion in a Ca2+-independent manner. This decrease in cAMP/PKA normally arises from somatostatin preventing cAMP production by adenylyl cyclases via the Gαi subunit of the SSTR2 and from insulin receptor activation of phosphodiesterase 3B to drive degradation of cAMP in a glucose-dependent manner. Our data indicate that both somatostatin and insulin signaling is required to decrease cAMP and PKA sufficiently to inhibit glucagon secretion from islets and isolated α-cells. We conclude that somatostatin and insulin together are critical paracrine mediators of glucose-inhibited glucagon secretion and function by lowering cAMP/PKA signaling with increasing glucose. The complex inter-relationships in these results demonstrate the need for simultaneous measurements of multiple signaling pathways. For example, the roles of Ca2+ and cAMP in regulating glucose-stimulated insulin secretion from β-cells have been long known. However, it has been challenging to study temporal relationships between these signaling molecules due to the spectral overlap of most [Ca2+]i and cAMP biosensors. We have developed a hyperspectral image mapping spectrometry technique for simultaneously monitoring these biosensors in real time. Using the IMS, we can resolve the effects of glucose and known stimulating drugs on these signaling molecules simultaneously and show that their glucose-induced oscillations are anti-correlated. We have also recently demonstrated the capability of this method for monitoring two Forster resonance energy transfer based biosensors simultaneously, a feat rarely attempted by other spectral imaging systems. This was used to study the relationship between cAMP signaling and caspase-3 mediated β-cell apoptosis, a critical event in developing diabetes.

Obesity is an increasingly prevalent condition that increases risk factors for type-2 diabetes and heart disease. Weight loss reverses the complications of obesity. Long-term maintenance of weight loss, however, is difficult. Mechanisms that improve metabolic health in obese people are therefore attractive targets for study. In my dissertation work, I have identified a novel role for Cholesteryl Ester Transfer Protein (CETP) to protect female mice against insulin resistance and exercise intolerance caused by obesity. CETP is a lipid transfer protein that shuttles lipids between lipoproteins, culminating in delivery of cholesterol esters to the liver for secretion as bile. Bile acids are known to have insulin-sensitizing effects. Mice naturally lack CETP expression. I discovered that female mice transgenic for CETP were protected from high fat diet-induced insulin resistance. This effect was modest in males. In female mice I found activation of bile acid signaling pathways in liver and muscle as well as increased glucose rate of disappearance and increased muscle glycolysis. These results suggest that CETP can ameliorate insulin resistance associated with obesity in female mice by promoting muscle glucose utilization. Based on the observations of improved muscle function in the CETP mice, I hypothesized that CETP could improve exercise capacity by increasing muscle oxidative metabolism. While there is no difference in exercise capacity between lean, chow fed CETP-expressing mice and their non-transgenic littermates, CETP-expressing female mice are protected against the decline in exercise capacity caused by obesity. This improvement in exercise capacity corresponded with increased mitochondrial oxidative capacity. My dissertation work has demonstrated a novel role for CETP to promote metabolic health in obese animals potentially through its effect on bile acid signaling to muscle. I propose that targeting bile acid signaling pathways could promote metabolic health in obese people. The sexual dimorphism observed adds to the growing body of evidence that CETP likely has a positive impact on metabolism in females. Further understanding the role of CETP and bile acid signaling will help to provide new strategies for promoting metabolic health in obese people.

Vitamin C (VC) concentration in the brain is crucial for neuronal defense against oxidative stress and proper function. VC transport is a balance between regulated uptake mechanisms that include the Sodium-dependent Vitamin C Transporter, Type 2 (SVCT2) and efflux mechanisms that may involve several types of trans-membrane proteins. Neuronal function, and ultimately neurobehavioral defects, may result from dysregulation in acute uptake or efflux processes. However, the mechanisms that explain the relationships between these phenomena are yet to be understood. Thus, our overarching hypothesis is that vitamin C transport is tightly regulated in functional areas of the neuron, specifically, the nerve terminal. First, we sought to determine whether oxidative stress could contribute to neurobehavioral defects in mice unable to synthesize VC or partially lacking the SVCT2. Our results showed that combined dietary VC and vitamin E (VE) deprivation only minimally increased neuronal oxidative stress markers compared to single deficiencies. Yet, with combined VC and VE deficiency in addition to decreased cellular uptake of VC (SVCT2+/-), deficits in motor and coordination skills became evident. Whereas this effect may be an early manifestation of scurvy, its mechanism does not appear to be due to an increase in neuronal lipid peroxidation and remains to be determined. It is clear, however, that SVCT2 function contributes to the neurobehavioral phenotype in mice. Next, we investigated the regulation mechanisms involved in acute VC transport at the cortical nerve terminal. Using synaptosomes as a model, our immunoblot studies demonstrate that SVCT2 protein is expressed predominately at the pre-synaptic terminal while our transport studies conclude that it functions to mediate VC uptake at the synapse. While the mechanisms for VC efflux are not completely clear, our studies suggest that Volume-Regulated Anion Channels (VRACs) are likely to have a substantial role in mediating glutamate-induced VC efflux at the cortical nerve terminal. These data may extend the current hetero-exchange theory, which was established in astrocytes, to include neurons as well. Altogether, this body of work has implications in understanding the mechanisms involved in VC regulation and how they contribute to proper neuronal function.

Inappropriate glucagon secretion contributes to hyperglycemia in inflammatory disease. Previous work implicates the pro-inflammatory cytokine, interleukin-6 (IL-6), in glucagon secretion. IL-6 knock-out mice have a blunted glucagon response to lipopolysaccharide (LPS) that is restored by intravenous replacement of IL-6. Given that IL-6 has previously been demonstrated to have a transcriptional (i.e. slow) effect on glucagon secretion from islets, I hypothesized that the rapid increase in glucagon following LPS occurred by a faster mechanism such as by action within the brain. Using chronically catheterized, conscious mice, it was found that central IL-6 stimulates glucagon secretion uniquely in the presence of an accompanying stressor (hypoglycemia or LPS). Contrary to the original hypothesis, however, IL-6 was found to amplify glucagon secretion in two ways: IL-6 not only stimulates glucagon secretion via the brain but also by direct action on islets. Interestingly, IL-6 augments glucagon secretion from both sites only in the presence of an accompanying stressor (such as epinephrine). Given that both adrenergic tone and plasma IL-6 are elevated in multiple inflammatory diseases, the interactions of the IL-6 and catecholaminergic signaling pathways in regulating GCG secretion may contribute to our present understanding of these diseases.

Regeneration of endogenous β-cells is a promising therapy to treat diabetes, but there are considerable gaps in our understanding of the microenvironmental signals necessary to stimulate β-cell proliferation and the unique ways human β-cells differ from rodents. Our group previously modulated the islet microenvironment using a mouse model in which vascular endothelial growth factor A (VEGF-A) overexpression causes β-cell loss and endothelial cell (EC) expansion, followed by β-cell proliferation and regeneration that requires infiltrating macrophages. To determine the role of proliferative and quiescent ECs, we conditionally inactivated the key receptor mediating VEGF-A signaling, VEGFR2, in ECs and found that EC signaling was necessary for maximal macrophage recruitment and phenotype activation. We also showed that ablation of VEGFR2 in quiescent ECs during the β-cell regenerative phase induced rapid vessel regression that promoted β-cell proliferation, possibly mediated by growth factor release from the extracellular matrix. Extending these findings to human pancreas development, we determined that intra-islet EC area was greatest during the first year of postnatal life and coincided with the peak of β-cell proliferation, suggesting that vascular arrangement or EC-derived signals may impact human β-cell proliferation. Next, to advance the methodologies for studying human islets, we identified two molecular markers of developing and mature human β-cells. Secretory granule membrane major glycoprotein 2 (GP2) marks a population of multipotent pancreatic progenitor cells in the neonatal human pancreas, and can be utilized to improve efficiency of generating β-like cells from stem cells. Nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) is a cell surface marker of adult human β-cells, and is a unique tool for isolating live β-cells by flow cytometry and performing in vivo β-cell imaging. These two markers will further our knowledge of islet development and allow us to assess β-cell gene expression and mass during the disease process, which we demonstrated by utilizing our islet cell isolation strategy to reveal transcriptional dysregulation in α-cells from donors with type 1 diabetes. Together, this work provides a framework for future efforts aimed at promoting β-cell regeneration and increasing functional β-cell mass.