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.

Obesity has reached epidemic proportions worldwide, with some of the greatest severity in the United States. The most recent data reports ~70% of the American population is overweight (BMI ⥠25 kg/m2) and ~35% obese (BMI â¥30 kg/m2). Obesity is a metabolic disorder leading to increased risk for cardiovascular disease, type 2 diabetes, asthma, certain cancers, and various other diseases. A hallmark of obesity is adipose tissue (AT) inflammation and AT dysfunction. It is important to understand how immune cells accumulate in AT and regulate inflammation. We used CCR2-/- mice to study macrophage chemotaxis to AT, and also discovered CCR2-/- regulates chemotactic factors that upregulate AT eosinophil accumulation. Previous studies suggested that directly manipulating eosinophils (particularly in AT) could impart beneficial effects in obese subjects. Thus we developed an interventional treatment model of restoring obese AT eosinophils to higher levels of lean AT by injection of rIL5. AT eosinophils were successfully increased with rIL5, but there was no reduction in obesity and its comorbidities. Lastly, we discovered that repeated exposure to a foreign substance such as bovine serum albumin (BSA) could greatly increase AT eosinophils. While there were no metabolic improvements in mice chronically exposed to BSA, we have evidence to believe AT is capable of mounting a type 2 allergic response to antigens similar to the lung of an asthmatic, resulting in this large increase in AT eosinophils. Future studies will determine whether the AT eosinophilia following BSA exposure feeds back to the lung in allergic models, increasing both incidence and severity. Such studies will help in explaining the clinical link between obesity and allergic conditions such as asthma. In conclusion, we have found that restoring AT eosinophils to either physiological levels or super-physiological levels during obesity is not able to improve metabolic fitness (e.g. weight gain, glucose intolerance). Furthermore, we may have discovered a novel site of allergy that could offer insights and treatment opportunities for obese subjects that have increased difficulty with allergic disease.

Haploinsufficiency of the melanocortin-4 receptor (MC4R) is the most common monogenetic obesity syndrome in humans. This syndrome is associated with a reduction in autonomic tone, bradycardia, hyperinsulinemia and a reduced prevalence of obesity-associated hypertension. Thus, it has been assumed that melanocortin obesity syndrome may be protective with respect to obesity-associated cardiovascular disease. We show here that deletion of the Mc4r in mice causes a dilated cardiomyopathy characterized by reduced contractility and increased left ventricular diameter. This cardiomyopathy is independent of obesity as weight matched wild type mice are spared from systolic dysfunction. Mc4r-/- heart tissue further displays ultrastructural abnormalities in mitochondrial morphology and cardiomyocyte organization. Remarkably, mitochondrial function testing of myocardial tissue from Mc4r-/- mice revealed increased ADP stimulated respiration. This is in contrast to the reduction in O2 consumption seen in other models of cardiomyopathy, as well as the reduction in whole animal energy expenditure detected in MC4R knockout animals by indirect calorimetry. However, we show that this increase in respiration correlates with increased reactive oxygen species production â a canonical mediator of tissue damage. In keeping with this hypothesis, Mc4r-/- heart tissue displays a similar transcriptional profile to that of doxorubicin treatment â a free radical generating chemotherapy. Furthermore, Mc4r+/- mice are hypersensitive to both the cachexigenic and cardiac suppressive side effects of doxorubicin treatment. Together this study identifies MC4R deletion as a novel and potentially clinically important cause of heart failure.

The two-pore domain K⁺ (K2P) channel TALK-1 is highly expressed in the pancreatic islet and is linked to type 2 diabetes mellitus (T2DM) risk through a non-synonymous polymorphism (rs1535500). Here, we established that TALK-1 channels are functionally expressed in mouse and human β-cells where they modulate insulin secretion by limiting electrical excitability and cytosolic Ca²⁺ influx. We found that the rs1535500 polymorphism (encoding TALK-1 A277E) increases TALK-1 channel activity. When placed on a high-fat diet, mice lacking TALK-1 channels were protected from elevations in fasting glycemia. Therefore, rs1535500 may contribute to T2DM etiology by exacerbating hyperglycemia under diabetogenic conditions. We next determined that endoplasmic reticulum (ER)-localized TALK-1 channels conduct ER K⁺ countercurrents, facilitating β-cell and δ-cell ER Ca²⁺ leak. In β-cells, TALK-1 regulation of ER Ca²⁺ handling influences activation of K_slow, a Ca²⁺-dependent K⁺ current which repolarizes the plasma membrane potential, terminating each Ca²⁺ oscillation. K_slow is significantly reduced in KO β-cells, contributing to an elevated frequency of Ca²⁺ oscillations in TALK-1 KO islets. Furthermore, we determined that islets from mice lacking TALK-1 channels were resistant to ER stress induced by chronic exposure to a high-fat diet. Finally, we showed that TALK-1 channel regulation of δ-cell ER Ca²⁺ handling impacts δ-cell function. Somatostatin secretion is amplified by Ca²⁺-induced Ca²⁺ release (CICR) from the ER, and we found that TALK-1 regulates δ-cell Ca²⁺ handling and somatostatin secretion by modulating the ER Ca²⁺ stores which underlie CICR. Our data establish TALK-1 channels as key determinants of islet cell Ca²⁺ handling, and suggest that TALK-1 channels may be a therapeutic target to reduce islet cell ER Ca²⁺ defects during the pathogenesis of diabetes.

Type 2 diabetes is a major healthcare concern and is characterized by chronic hyperglycemia and low-grade inflammation. Hyperglycemia and systemic inflammation can induce the production of Prostaglandin E2 (PGE2) in islets. PGE2 signals through its four receptors, termed E-Prostanoid (EP) 1-4, to modulate many physiological functions, including insulin secretion and systemic inflammation. EP3 and EP4 often play opposing roles due to signaling through different G proteins, resulting in Gi inhibition (EP3) or Gs stimulation (EP4) of adenylyl cyclase. Previous work from our group revealed that Ptger4 (EP4) is increased while Ptger3 (EP3) is decreased in a mouse model of enhanced beta-cell proliferation and survival. Additional evidence from our lab suggests that EP3 inhibits mouse beta-cell proliferation in the setting of insulin resistance. Using ex vivo assays, we have determined that EP3 and EP4 play opposing roles in regulating beta-cell proliferation and survival in mouse and human islets: EP3 inhibits beta-cell proliferation via inhibition of PLC-gamma1 and enhances beta-cell death whereas EP4 activates beta-cell proliferation and promotes beta-cell survival in a PKA-dependent mechanism.