PI: Elena Arystarkova, PhD
Abstract: Role of FXYD2 in regulation of proliferation of beta cells in pancreas
Restoration of functional potency of pancreatic islets either through enhanced proliferation (hyperplasia) or increased in size (hypertrophy) of beta cells is a major objective for diabetes. We have obtained experimental evidence that global knockout of a small, single-span membrane protein, FXYD2, interferes with glucose metabolism. Adult mice showed significantly lower blood glucose level and much faster rate of glucose clearance compared to their littermate controls. Strikingly, there was a substantial hyperplasia in pancreatic beta cells from the FXYD2-/-deficient mice compared to the wild type littermates. No changes were seen in the exocrine compartment of pancreas. The hypothesis to be tested is that reduction of FXYD2 is beneficial for selective proliferation of functionally active beta cells. First, we will establish whether the hyperplasia seen in FXYD2-/- mice is a developmental phenomenon, or it is ongoing in adults. We will perform a systematic analysis of pancreata from the knockout and wild type mice taken at different stages of development. If the rate of proliferation is elevated in adult mice, this would make the FXYD2 protein an attractive therapeutic target for restoration of beta cell mass in diabetic pancreas. Second, we will test the hypothesis that hyperplasia observed in the knockout mice is a direct effect of selective deletion of FXYD2 from beta cells in pancreas. We will perform knockdown of FXYD2 in vivo by injecting specific siRNA(s) followed by BrdU incorporation assay in order to exclude potential developmental effects of the knockout mouse. Finally we will cross a NOD mouse with FXYD2-/- in order to test whether reduction of FXYD2 in pancreatic beta cells is beneficial in diabetic mice To complement, we will also attempt to knockdown FXYD2 in NOD mouse with specific siRNA(s). We anticipate that these initial steps of characterization of FXYD2 as a potential target for intervention in replication of beta cells will set the stage for future development of novel therapeutics in treatment of insulin dependent diabetes.
PI: Harald von Boehmer, MD, Ph.D
Abstract: Prevention of T1D by Treg-vaccination with an insulin-mimetope
Type 1 diabetes (T1D) results from the destruction of pancreatic β cells by the immune system. Especially, peptide epitopes of insulin appear to be targeted since changes in antigenic epitopes of insulin that do not affect metabolic function prevent the spontaneous development of T1D in NOD mice, a rodent model of human disease.
In the proposed project we aim at inducing Treg-mediated dominant tolerance to β-cell derived antigens by converting naïve and also activated insulin-specific T cells into regulatory T cells (Treg). Attempts to do this by using natural insulin peptides (epitopes) have failed. Recent evidence suggests that the natural epitopes recognized by diabetogenic T cells have low agonist activity. We therefore aimed at using mimetopes with high agonist activity and thereby could completely prevent the development of disease in NOD mice. To this end the arginine residue in position 22 of the essential 9-23 epitope of the insulin ? chain was mutated to glutamic acid to improve binding to the positively charged p9 pocket of I-Ag7 in the relevant third binding register.
Based on these preliminary findings we aim to define whether Treg-mediated dominant tolerance is responsible for the observed protection from disease development. Moreover we are seeking to determine whether conversion of already activated T cells into Foxp3 Tregs can be achieved by a pharmacological approach using suitable drugs plus mimetope. This aim has the goal to allow not only for prevention of disease but possibly also for treatment of recent onset disease.
To conclude, the proposed studies will be helpful in order to design suitable strategies for the future translation of the antigen-specific Treg-vaccination approach to clinical protocols in order to prevent T1D in humans at risks.
PI: José, M Cacicedo,
Abstract: The Role of AMP-Activated Protein Kinase (AMPK) in the Prevention of Diabetic Retinopathy
Diabetic retinopathy (DR), a leading cause of blindness in adults worldwide, has generally been considered a consequence of hyperglycemia. The recent ACCORD Eye Trial and the FIELD study both demonstrated that in patients with diabetes use of the lipid lowering agent fenofibrate reduced the progression to retinopathy necessitating laser treatment by 30%. These results suggest (1) that lipids could play a significant role in the etiology and progression of diabetic retinopathy and (2) that perhaps AMPK activation in the retina could account for these findings since fenofibrate is an AMPK activator. In studies carried out over the past few years, I have shown that the saturated fatty acid palmitate, but not the monounsaturated fatty acid oleate, at concentrations present in the plasma of patients with diabetes causes apoptosis of retinal pericytes (PCs) and endothelial cells (ECs). I also found that activation of AMPK prevents both the apoptosis and the pro-apoptotic pathways induced by palmitate including NF-kB activation and ceramide synthesis (IOVS under review). The proposed studies will have the following specific aims. Aim 1: To characterize further the mechanism by which palmitate induces apoptosis in cultured primary retinal PCs and ECs, and to assess whether hyperglycemia potentiates the effects of the fatty acid. A special focus will be on measurements by western blot and RT-PCR of markers for endoplasmic reticulum (ER) stress, such as the chaperone protein BiP and the death effector CHOP, since two recent studies in animal models have shown that ER stress occurs early in the diabetic retina and contributes to the progression of DR. Aim 2: To assess whether AMPK activators such as AICAR, metformin, statins, and fenofibrate administered locally by eye drops (to deliver the drug at the site of injury and avoid confounding systemic effects) or systemically via drinking water activate AMPK in the rat retina and in which cells. To address this question immunohistochemistry (IHC) for “activated” phospho-AMPK of the whole retina will be employed, as will trypsin digestion of the retinas followed by IHC analysis to determine whether AMPK is activated specifically in the ECs and PCs of the retina. These studies will use a methodology I developed to demonstrate AMPK activation in aortic endothelium after exercise in the mouse. Aim 3: Once an effective AMPK activator and route of delivery in vascular cells is found, to determine whether AMPK activation prevents or diminishes diabetes-induced death of retinal ECs and PCs (i.e. DR), and the signals related to cell death (from Aim 1) in mouse models of type 1 (streptozotocin-induced) and type 2 (diet-induced) diabetes. This line of research represents a new way of thinking about DR, one in which diabetes-induced hyperlipidemia and ER stress is part of its etiology and AMPK activators may be useful for its prevention.
PI: Paola Divieti Pajevic MD, PhD.
Abstract: Loss of Gsα Signaling in Osteocytes Decreases Peripheral Fat Accumulation
Traditionally bone has been viewed as a structural support organ that helps in locomotion and protection of other organs. Recent studies have identified novel endocrine functions of bone that include a role of bone cells in pancreatic insulin secretion, glucose homeostasis, and peripheral fat accumulation via osteocalcin actions. We hypothesized that the GPCR signaling in osteocytes, the most abundant cells of the bone, might play an important role in glucose homeostasis through direct or indirect actions of osteocalcin. To test this hypothesis, mice lacking the stimulatory G-protein alpha subunit, Gsα, specifically in osteocytes in homozygous (DMP1-GsαKO) or heterozygous (DMP1-GsαHet) manner were generated. Dual energy X-ray absorptiometry (DXA) and microcomputed tomography (micro-CT) analysis of 21-week old DMP1-GsαKO mice showed dramatic osteopenia with 86% decrease in trabecular bone volume in DMP1-GsαKO mice compared to controls. The osteopenia was due to a dramatic decrease in osteoblast number and functions as revealed by histomorphometric analysis. Interestingly, osteocyte density in DMP1-GsαKO mice was doubled as compared to controls, but resulted in dramatically disorganized canaliculi network as assessed by SEM. Neither micro-CT nor DXA bone parameters differed between control and DMP1-GsαHet. Interestingly, both DMP1-GsαKO and DMP1-GsαHet mice showed a significant decrease (>40%) in peripheral fat at 21-weeks of age as analyzed by DXA. These mice also showed random fed hypoglycemia. The decrease in fat in both the KO and Het mice appears to be due to decreased gene expression makers of adipocyte differentiation (PPARg), fat storage (PLIN) and fat uptake (LPL) as revealed by real time PCR on gonadal fat. Adiponectin and leptin gene expression in fat tissue in both DMP1-GsαKO and DMP1-GsαHet mice were also significantly decreased as compared to controls. These results suggest a possible direct role of Gsα signaling in osteocytes in regulating not only bone metabolism, but also peripheral adiposity. Surprisingly, the serum osteocalcin levels were decreased in both the KO and Het mice where we expected to see an increase. Based on this preliminary data, we hypothesize that Gsα signaling in osteocytes regulate fat accumulation and glucose homeostasis through other mechanisms independent of osteocalcin. We propose two specific aims: 1) Identify the metabolic defect in DMP1-GsαKO and DMP1-GsαHet mice leading to the lean phenotype, 2) Identify cellular signals generated by Gsα-null osteocytes that affect glucose homeostasis and body fat accumulation.
Taken together, our preliminary data suggests a possible direct role of Gsα signaling in osteocytes in regulating peripheral adiposity and glucose homeostasis.
PI: Dong Kong, Ph.D.
Abstract: Blood Glucose Control by Hypothalamic Glucose-sensing Neurons
Hypothalamus functions as a key monitor in the brain on nutrient status and regulates whole body energy and glucose homeostasis. Discrete groups of neurons in hypothalamus were found to be responsible to extracellular glucose change, providing a premise for central mechanism in maintaining euglycemic state. Glucose can inhibit certain neurons (glucose-inhibited neurons, such as AgRP neurons in the arcuate nucleus and orexin neurons in the lateral hypothalamus) and stimulate some others (glucose-excited neurons, such as POMC neurons in the arcuate nucleus, MCH neurons in the lateral hypothalamus, and some chemically undefined neurons in the ventral medial hypothalamus). While the mechanism for glucose-inhibited neurons is still unclear, a well-defined pancreatic β-cell-like mechanism has been proposed for most cases of glucose excitation of neurons (glucose®ATP ®closure of KATP channels®Firing rate).
By neuron-specific mouse genetic tools, we recently perturbed glucose excitation in both POMC and MCH neurons (summarized in the left figure). Expressing a mutant KATP channel subunit (Kir6.2[K185Q,ΔN30]) in these neurons, which blocks glucose sensing, impairs whole body glucose tolerance. Specific deletion of Ucp2, a mitochondrial uncoupling protein, which negatively regulates ATP production and is up-regulated in diabetic hypothalamus, augments glucose sensing and greatly improves global glucose homeostasis. Of great note, the mice without Ucp2 in POMC or MCH neurons displayed greatly reduced hyperglycemia when treated with either high-fat diet (type 2 diabetes model) or streptozotocin (type 1 diabetes model). These findings strongly suggest that impaired glucose sensing of MCH and POMC neurons, likely via increased Ucp2 expression, plays important roles in pathogenesis of Diabetes.
While much has been learned about the mechanism and physiologic effects of glucose-excited POMC/MCH neurons, comparatively less is known about how these neurons achieve the effect on blood glucose. This presents the unique opportunity to identify novel mechanisms controlling glucose homeostasis and could reveal innovative strategies to treat Diabetes. In this grant, I propose to determine the following: 1) the “glucose-lowering” factor released from POMC/MCH neurons (an important role of CART peptide has been proposed) (Aim 1); 2) the neurocircuitry by which POMC/MCH neurons employ to control blood glucose (we propose liver to be the major downstream effecting organ and autonomic efferents bridge between central and peripheral) (Aim 2). To accomplish these Aims, we will utilize the following innovative technologies: 1) neuron-specific and neural projection-specific gene manipulation to test molecular mechanisms in an in vivo context (in both Aims), 2) optogenetics to identify functionally relevant, monosynaptic, downstream targets of POMC/MCH neurons (Aim 2), and 3) Acute manipulation of specific groups of neurons (both activation and inhibition) for in vivo functional test by DREADD system (Aim 2).