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Awarded for 2004:
Awarded for 2003:
PI: Sally C. Kent, Ph.D.
Project Title: Pancreatic Lymph Node Derived T Cell Autoantigen Recognition in Human Type 1a Diabetes
Abstract: A central issue in autoimmunity is the recognition of cognate self-target(s) by pathogenic T lymphocytes. In patients with Type 1 diabetes, studies have been limited to peripheral blood. We have developed techniques to examine T cells from pancreatic lymph nodes (PLN, site of autoreactive T cell triggering) from patients with diabetes and control subjects using single cell cloning. Our aims are to determine if there is oligoclonal expansion of PLN T cells in patients with diabetes and to determine antigen-specificity of clonally expanded T cell clones isolated from patients by screening a human islet-specific cDNA library. This library will be used to transfect autologous EBV transformed B cells and these cells will be used as antigen presenting cells to hybridomas transfected with the TCRVa and Vb chains and an IL-2 responsive reporter gene. A rare sample is from a new onset diabetic in which we have observed lymphocytic infiltrate containing CD4+ T cells in pancreatic islets. We will laser microcapture individual T cells from islets and identify TCRVa and Vb chain usage and use these TCR chains to transfect hybridomas and identify islet cell antigens. This will give us the first direct evidence of antigen reactivity of an islet-infiltrating T cell in human Type 1 diabetes. Thus, our goal is to identify the self-antigen driving the clonal expansion of T cells in the PLN of patients with autoimmune diabetes.
PI: Andreas Lechner, M.D.
Project Title: Lineage-tracing analysis of pancreatic beta-cell turnover and renewal in
adult mice
Abstract: Adequate pancreatic beta-cell mass is important to ensure physiologic glucose control and to prevent diabetes mellitus. Since beta-cells undergo apoptosis at a steady rate, constant formation of new beta-cells is necessary to maintain an equilibrium. However, the mechanisms by which new beta-cells are generated in the adult pancreas are unclear. While many scientists believe that the life-long replication of mature beta-cells is the mainstay of this process others argue that beta-cells are post-mitotic and new beta-cells are primarily formed by differentiation from self-renewing progenitor cells (neogenesis).
We hypothesize that neogenesis from progenitor cells is indeed a major component of beta-cell turnover, particularly under conditions of increased insulin demand or islet injury. To test this hypothesis we propose a lineage-tracing study that permits the discrimination between beta-cell proliferation and neogenesis in the adult mouse pancreas. As a basis for this study we generated transgenic mice that express a tamoxifen-inducible Cre-recombinase under the control of the insulin-promoter. These mice will be crossed with a Cre-Lox reporter line, thus creating a mouse model that allows the irreversible genetic marking of beta-cells and their daughter cells in vivo at the time of tamoxifen-injection. Using this model we plan to analyze beta-cell turnover under the following conditions: Steady state beta-cell renewal, the adaptation of beta-cell mass to increased insulin demand, and the regeneration of beta-cells after islet injury. The lineage-tracing approach will be complemented by immunostaining and detection of dividing cells through BrdU-labeling.
The proposed project should facilitate a better understanding of the replicative capacity of mature beta-cells, the mechanisms of beta-cell renewal, and the role of adult pancreatic stem/progenitor cells.
PI: Anthony Rosenzweig, M.D.
Project Title: Role of Akt signaling in diabetic cardiomyopathy
Abstract: Diabetic cardiomyopathy is an important complication of diabetes that is characterized by cardiomyocyte apoptosis, systolic and diastolic cardiac dysfunction, and abnormalities of cardiac metabolism. The serine-threonine kinase, Akt, is a critical intracellular mediator of the actions of insulin and IGF-I. In other settings, Akt activation in the heart counteracts the defects common to diabetic cardiomyopathy by promoting cardiomyocyte survival, improving cellular calcium handling and contractile function, and enhancing glucose uptake. The goal of the current proposal is to understand the role of Akt signaling in diabetic cardiomyopathy.
This proposal is based on the hypothesis that Akt activation in the heart will inhibit diabetes-induced cardiomyocyte apoptosis while mediating significant functional and metabolic benefits in diabetic cardiomyopathy. To test this hypothesis, activation and/or inhibition of Akt in the heart will be achieved through somatic and germline gene transfer in models of chemically induced diabetes. In Specific Aim 1, we will define the intracellular pathways controlling hyperglycemia-induced cardiomyocyte apoptosis and dysfunction in vitro. In Specific Aim 2, we will evaluate the effects of transgenic cardiac Akt activation or inhibition on streptozotocyn (STZ)-induced cardiac dysfunction and apoptosis. If successful, additional studies in the second year will seek to define the downstream mechanisms responsible for Akt-mediated protection against diabetic cardiomyopathy.
Understanding the role of specific signaling pathways in diabetic cardiomyopathy and developing approaches to local modulation of these pathways, may provide novel therapeutic approaches for this important condition.
PI: Lisa Getty
Project Title: Mechanism and Role of Oscillatory Lipolysis in Adipocytes and Islets
Abstract: It has been shown that lipolysis oscillates in plasma, isolated adipocytes, and isolated islets. In adipocytes, lipolytic oscillations are dependent on glucose. It is hypothesized that lipolytic oscillations are driven by oscillatory glucose metabolism which leads to oscillatory inhibition of HSL by LC-CoA. Both lipolysis and glycolysis generate important signals in the b-cell necessary for normal oscillatory insulin release. Therefore, we hypothesize that oscillatory FFA may play a central role in modulating and synchronizing oscillatory insulin release. Aim 1 will test the hypothesis that oscillatory FFA release from fat cells is due to inhibition of HSL by oscillatory changes in LC-CoA. Adipocytes will be perifused with exogenous FFA to determine the effect of lipolytic oscillations and the time course of changes in LC-CoA will be determined by direct measurement. Adipocytes from HSL deficient mice will be used to further investigate this hypothesis. Aim 2 will examine the hypothesis that lipolytic oscillations are dependent on glucose oscillations. Adipocytes will be perfused with insulin at different glucose concentrations to determine the effect on lipolytic oscillations. Also lipolytic oscillations from adipocytes of PFK-M deficient mice (the putative glycolytic oscillator) will be examined. Aim 3 will test the hypothesis that oscillatory FFA release from fat cells cont4ributes to synchronize insulin secretion from islets. Sequential co-perifusion of isolated adipocytes and islets will be undertaken and secretion and lipolysis measured and temporally related in response to stimulation. Adipocytes and islets from HSL and PFK-M deficient mice will be used to further probe adipocyte/islet interactions.
PI: Maria Koulmanda
Project Title: The effect of streptozotocin on the autoimmune disease of the nonobese diabetic (NOD) mouse.
Abstract: NOD mice develop autoimmunity that destroys their native beta cells causing diabetes. Recurrent autoimmunity also destroys syngeneic islet transplants into these mice after they have become diabetic. We have generated preliminary data showing that: a) treatment of NOD mice with streptozotocin (STZ) prevents the onset of autoimmunity, b) STZ treatment reverses autoimmuninty in these mice after they have developed diabetes, and c) depletion of CD8+ cells prevents the beneficial effects of streptozotocin treatment. On the basis of these data, we hypothesize that streptozotocin therapy modulates the autoimmune response by generating regulatory T cells. In order to obtain sufficient preliminary data to allow us to submit a full RO1 application to the NIH based on this hypothisis, we would like to perform four experiments. 1) Determine whether spleen cells from diabetic NOD mice can cause recurrent autoimmunity in the STZ-treated recipients. 2) Determine whether depletion of specific T cell subsets, at different times, prevents the beneficial effect of STZ-treatment. 3) Determine whether regulatory T cells exist in the STZ-treated animals by co-transferring cells from these mice with spleen cells of diabetic animals into NOD/scid recipients. 4) Determine whether infusion of islets treated ex vivo with streptozotocin reverses autoimmunity. These studies may suggest new strategies to reverse autoimmunity in patients with type one diabetes
PI: Evan D. Rosen
Project Title: Novel Transcriptional Pathways in Adipogenesis
Abstract: The dramatic rise in the incidence of obesity in the developed world has focused attention on the biology of the adipocyte. The experiments in this proposal are designed to address crucial questions in the area of adipogenesis, or fat cell differentiation. Specifically, these studies will focus on the control of terminal gene expression in adipocytes. We have generated fibroblasts that lack the critical adipogenic transcription factor PPARgamma. These cells are utterly unable to undergo differentiation unless ectopic PPARgamma is added. These cells are a unique model system for identifying factors that promote adipose conversion downstream of PPARgamma. Using retroviral vectors expressing either candidate transcription factors or an adipocyte cDNA library, we will screen for proteins responsible for various aspects of the adipocytic program, including lipid accumulation, insulin sensitivity, and marker expression. It is hoped that these studies will expand our understanding of gene expression in adipocytes, which may ultimately be manipulated the benefit of those with obesity, type 2 diabetes, or lipodystrophy.
PI: Denise Faustman, MD, Ph.D.
Project Title: Development of a quantitative flow cytometric assay for TNF-a sensitivity in diabetic lymphocytes
Abstract: In NOD mice, a model of autoimmune diabetes, a proteasome defect in select lymphoid cells has been identified. This impairs the presentation of self antigens in MHC class I structures and increases the susceptibility of these autoimmune disease causing cells to TNF-a induced apoptosis by defective proteasome mediated NF-kB activation. A novel treatment regimen that targets these defective cells permanently eliminates hyperglycemia in diabetic NOD mice by eliminating autoimmunity and islets regrow in the pancreas.
In order to logically move this data forward to diabetic humans, standardized assays similarly detecting heightened TNF-a sensitivity in diabetes is essential. The development of these assays will potentially establish a similar genetic basis to disease and provide the research community with an assay that someday could be advanced to in vitro monitoring of diabetic patients receiving TNF-a. Preliminary human data suggests most diabetic patients have subpopulations of cells with TNF-a induced death but these cell death assays were performed with in vitro TNF-a exposure and trypan-blue exclusion, a non-quantitative method.
The goal of this 3-year pilot grant is to develop a quantitative flow cytometric assay of TNF-a sensitivity of diabetic lymphocytes.
- AIM1. Demonstrate the prevalence of TNF-a sensitivity of diabetic PBL in 50 random diabetic patients.
- AIM2. Correlate the clinical characteristics of TNF-a sensitivity compared to the magnitude of the apoptotic defect.
- AIM3. Compare the stability and the magnitude of the TNF-a sensitivity in the same diabetic with the passage of time.
- AIM4. Determine the optimal blood drawing conditions for TNF-a induced cell death.
- AIM5. In control individuals, optimize the conditions to keep non-specific cell death to a minimum to magnify specific diabetic induced TNF-a apoptosis.
This pilot grant could provide both valuable information on underlying apoptotic defects in human diabetes and the development and standardization of a human assay for future in vivo human studies
PI: Shane T Grey, Ph.D.
Project Title: The Role of A20 in Protection from Non Insulin Dependent Diabetes mellitus in the Zucker Diabetic Fatty Rat
Abstract: During the pre-diabetic phase of non insulin dependent diabetes mellitus (NIDDM), normoglycemia is maintained through compensatory hyperinsulinemia. However in certain individuals the increased demand for insulin is unable to be met and diabetes ensues. The exact mechanism leading to the failure of compensation and onset of diabetes is not known, though studies in humans indicate that the b-cell mass in diabetic (NIDDM) patients is reduced by 50%. Like humans with NIDDM, Zucker diabetic fatty (ZDF) fa/fa rats have a pre-diabetic phase with insulin resistance which is compensated by increased preproinsulin production and insulin secretion. However in contrast to both wild type and non diabetic ZF rats who maintain euglycemia, ZDF rats become diabetic (~14 weeks) and at this time exhibit a 50-75% reduction in their b-cell mass. This reduction is due to b-cell death by apoptosis, despite active proliferation of the remaining b-cells. High intraislet FFA and TG levels are thought to be the principal causative agents in the loss of b-cell volume (lipotoxicity), driving the progression to NIDDM in ZDF fafa rats. The loss of b-cell mass is the result of profound apoptosis of the b-cells induced by over production of NO. The "natural" sensitivity of islets to free radical damage due to low expression of cyto-protective genes may be further compounded in NIDDM by high FFA levels which down regulate Bcl-2 expression. We have shown that A20 is part of the natural physiological defence of islets as demonstrated by its rapid induction by cytokines in human and rodent islets (manuscript submitted). A20, a cytoplasmic zinc finger protein, was originally described as an anti-apoptotic TNF-a inducible gene in endothelial cells. Besides protection from apoptosis, we have previously demonstrated that A20 also inhibits pro-inflammatory responses in endothelial cells through inhibition of NF-kB activation. These functions of A20 are preserved in islets as A20 can protect islets from cytokine induced apoptosis and block IL-1b induced iNOS protein induction and NO generation (manuscript submitted, see below) . This suppresive effect of A20 on cytokine induced expression of iNOS is mediated by inhibiting NF-kB activation. This proposal aims to evaluate whether the natural cyto-protective response (ie. A20 expression) of islets is perturbed during NIDDM. Additionally we will examine whether over expression of A20 by recombinant adenovirus mediated gene transfer can protect ZDF fafa islets from lipotoxicity. We also will determine whether other anti-apoptotic molecules, namely members of the bcl family (e.g. A1, bcl-xl), are involved in NIDDM development. These studies may be of relevance to our understanding of the pathogenesis of human NIDDM and lead to the development of novel therapeutic strategies for the treatment of NIDDM.
This pilot grant could provide both valuable information on underlying apoptotic defects in human diabetes and the development and standardization of a human assay for future in vivo human studiesPI: Lynne L. Levitsky, M.D.
Project Title: Islet function in mice with selective loss of HNF1b in b-cells
Abstract: Objective: Our objective in this project is to examine the role of the hepatocyte nuclear factor (HNF)1b transcription factor in b cell development and glucose sensing. Understanding the transcriptional mechanisms underlying b cell differentiation and the regulation of insulin secretion will lead to improved strategies to restore b cell function, to engineer non-b cells to perform b cell functions, and to promote the complete differentiation of stem cells into functioning b cells.
Background and rationale: Rare monogenic forms of diabetes (maturity onset diabetes of the young, MODY) have provided insight into the role of transcription factors in b cell differentiation and function. MODY5 is caused by mutations in the gene for HNF1b, a member of the transcription factor network expressed in b cells. Availability of a mouse model for MODY5 has been hindered because HNF1b null mice die before birth. To overcome this obstacle, we generated viable mice with selective loss of HNF1b in b cells (bbKO mice) using Cre-LoxP technology. bbKO mice have a selective defect in glucose-stimulated insulin secretion in pancreatic b cells, demonstrating that HNF1b is necessary for b cell glucose sensing or glycolytic signaling. bbKO mice are an excellent model system to identify the functions of HNF1b in b cells.
Description of project: The hypothesis to be tested in this pilot project is that b cell function depends on HNF1b activity. Glucose-stimulated insulin release and gene expression will be examined in impaired bbKO islets vs. normal islets engineered with different HNF1b levels by transient expression of wild type vs. mutant HNF1b alleles. Anticipated outcome: We expect to identify important downstream HNF1b target genes in b cells and to define the functions of HNF1b in the regulation of insulin secretion.
PI: Melissa K. Thomas, M.D., Ph.D.
Project Title: Activation of SMAD transcription factors in pancreatic neogenesis
Abstract: In diabetes mellitus relative or absolute insulin deficiency results from a decrease in the functioning mass of insulin-producing pancreatic b-cells. The signaling pathways that promote the generation of new pancreatic b-cells in the adult pancreas are largely undefined. Elucidation of mechanisms to produce new insulin-producing cells may lead to novel therapeutic approaches to restore insulin production in patients with diabetes. The proposed pilot and feasibility studies address the hypothesis that glucagon-like-peptide-1 (GLP-1) and TGF-b signal transduction pathways converge at the transcriptional level to initiate a pancreatic b-cell differentiation program. Signaling via the TGF-b and activin pathway activates transcriptional regulatory proteins in the SMAD family. TGF-b and activin signals regulate embryonic pancreas development and the induction of b-cell differentiation. In cellular models of b-cell neogenesis (the production of new pancreatic b-cells from precursor cells), activin potentiates b-cell differentiation as assessed by induction of insulin production. The incretin hormone GLP-1 and its analog exendin-4 induce similar patterns of b-cell differentiation in vitro and promote b-cell neogenesis in vivo. Induction of b-cell differentiation from precursor cells correlates with the induction of expression of the insulin gene and of the pancreas-specific homeoprotein IDX-1, a critical regulator of both pancreas development and b-cell-specific gene transcription. The proposed pilot and feasibility studies will address the following specific aims: I) to determine whether GLP-1 and TGF-b signals converge to activate nuclear SMAD transcription factors in b-cell neogenesis and II) to characterize the regulation of IDX-1 function by activation of nuclear SMAD transcription factors
PI: Jonathan S. Bogan, M.D.
Project title: Role of ARF proteins in GLUT4 trafficking
Abstract: The GLUT4 glucose transporter recycles between the plasma membrane and intracellular sites of sequestration in adipose and muscle tissue. Insulin stimulates GLUT4 exocytosis, such that the net distribution of cellular GLUT4 is shifted to the plasma membrane. Insulin likely regulates GLUT4 exocytosis both at the level of membrane fusion involving the plasma membrane, and at the level of budding from an intracellular compartment. The identification of 3-phosphoinositide-regulated ARF GTP exchange factors as targets for insulin signaling further supports the notion that insulin regulates membrane budding, since ARF GTPases are known to initiate bud formation. Neither the membranes at which insulin might stimulate budding of GLUT4-containing vesicles, nor the specific ARF GTPases that might be involved in this process are known. We therefore propose experiments to test the potential roles of known ARF proteins in GLUT4 recycling.
We developed a novel assay to measure changes in the proportion of GLUT4 distributed to the plasma membrane of cultured adipocytes, as well as a set of bicistronic retroviral vectors that allow us to test many proteins rapidly in our assay. By overexpressing wildtype and mutant ARF proteins and measuring alterations in GLUT4 recycling, we have already implicated ARF5 and ARF6 in GLUT4 this process. To determine where these ARF proteins act, we plan kinetic, microscopic, and biochemical analyses. Kinetic studies will determine the effects of ARF overexpression on GLUT4 externalization after insulin addition and reinternalization after insulin removal, as well as on the steady-state rate of GLUT4 endocytosis in the presence or absence of insulin. Control experiments will study the effects of these proteins on trafficking of transferrin receptor and GLUT1. We will use immunofluorescence microscopy and biochemical fractionation experiments to determine the subcellular distribution of ARF proteins we implicate in GLUT4 trafficking, particularly ARF5, and will assess how overexpression of these proteins alters intracellular GLUT4 distribution. Finally, we plan a yeast two-hybrid screen to identify proteins that interact with ARFs that control GLUT4 trafficking, particularly ARF5.
Our present data have convinced us that our assay and vectors are powerful tools to dissect the regulation of GLUT4 trafficking. Funding of this Pilot and Feasibility application will allow us to further establish the usefulness of these tools, as well as providing insight into the regulation of GLUT4 trafficking through membrane budding.
PI: Chiara Gerhardinger
Project Title: Glial cell abnormalities in background diabetic retinopathy
Abstract: The long-term goal of my investigation is to identify the molecular events responsible for increased vascular permeability in diabetic retinopathy. In this pilot project, I intend to test the novel hypothesis that glucose-induced retinal glial cell abnormalities mediate or contribute to altered expression of tight junction proteins in capillary endothelial cells. I have observed that diabetes induces increased levels of glial fibrillar acidic protein (GFAP) in retinal Müller glial cells, and that this increase in GFAP is accompanied by increased levels of occludin, one of the integral transmembrane protein of vascular endothelial tight junctions. Because glial cells contribute to the formation of tight junctions, and GFAP itself appears to be involved in regulating barrier properties, I propose to investigate the hypothesis that high glucose induces overexpression of GFAP, which in turn modulates the expression of occludin.
The specific aims of this research proposal are: 1) to determine the role of high glucose levels in causing GFAP overexpression, by studying the synthesis (mRNA levels) of GFAP and occludin in the retinas of diabetic donors, streptozotocin-diabetic rats, and in culture of Müller and retinal endothelial cells exposed to high glucose; and 2) to determine the role of GFAP overexpression or other glial cell abnormalities in the increased levels of occludin by investigating the expression of occludin in GFAP-transgenic and GFAP-knockout mice, and in coculture of retinal endothelial and GFAP-overexpressing Müller cells. Studies proposed in this application will help elucidate whether Müller cells are a target of glucose-induced abnormalities, and the potential mechanisms for the occludin overexpression observed in diabetic retinas. This in turn will help identify novel targets and strategies for pharmacological interventions to prevent this complication of diabetes.
PI: Colin A. Leech, PhD
Project Title: Regulation of non-selective cation channels by IRS-2
Abstract: The pancreatic islets of type 2 diabetics characteristically show an impairment of glucose-induced insulin secretion that can be normalized by treatment with agents that stimulate electrical activity andan influx of calcium ions in b cells. This stimulation of electrical activity can be induced therapeutically by sulfonylureas, that act through their receptors to close ATP-sensitive K+ (K+ATP)-channels, or by hormones such as glucagon-like peptide-1 (GLP-1) that stimulate cAMP production. GLP-1 regulates both K+ATP-channel activity and maitotoxin (MTX)-sensitive Ca2+-dependent non-selective cation (Ca-NS) channels, in addition to a direct stimulation of exocytosis. Our hypothesis is that increased blood glucose initiates bursting activity in b cells by decreasing K+ATP-channel activity relative to a fixed, or increasing, depolarizing conductance formed by Ca-NS channels. The specific aim of this proposal is to test the hypothesis that Ca-NS channels are regulated by insulin receptor substrate-2 (IRS-2). This hypothesis is suggested by our new preliminary data suggesting that MTX induces tyrosine phosphorylation of IRS-2 and is consistent with our previous observation that tyrosine kinase inhibitors reducesMTX-sensitive current amplitudes. The first specific aim of this proposal is to conclusively determine whether the MTX-sensitive phosphoprotein is IRS-2 by immunoprecipitation and sequencing of the precipitated peptide. Our second aim is to test a hypothesis for the mechanism by which IRS-2 might regulate Ca-NS channels. The mechanism to be tested involves a pathway whereby IRS-2 activates phosphoinositide 3-kinase and protein kinase Cz leading to the activation of Ca-NS channels. Our final aim is to test whether MTX, whose receptor is unknown, acts through the insulin receptor-related receptor, whose ligand is unknown. These proposed studies will increase our understanding of the mechanisms regulating b cell electrical activity and insulin secretion.
PI: Gordon Yancy, Ph.D.
Project Title: Adipocyte lipolysis and b-cell stimulation
Abstract: Glucose-induced insulin secretion appears to involve a metabolically generated lipid signal, long chain acyl CoA (LC-CoA), which directly or indirectly modulates cellular effectors including protein kinase C (PKC). Exogenous free fatty acids (FFA) also increase intracellular LC-CoA and strongly potentiate glucose-stimulated secretion with certain FFA mixtures being more effective than others. Stimulation of adipocyte-specific b-3 adrenergic receptors rapidly increases serum FFA and dramatically raises insulin levels in mice. Preliminary work shows that 1) genetic knockout of the adipocyte-specific fatty acid binding protein, aP2, blocks the b3-stimulated rise in insulin, but not in fatty acids levels in the serum and that 2) serum from b3-stimulated wild type mice, but not aP2 (-/-) mice directly stimulates insulin secretion in the b-cell . In addition, aP2 (-/-) mice fail to produce the cytokine TNF-alpha following b-3 adrenergic stimulation.
Based on these observations we propose: 1) to determine whether the FFA composition of the sera differs between wild type and knockout mice or the action of TNF-alpha in concert with FFA accounts for the increased insulin secretion, 2) to determine if the increased secretion involves changes in the intracellular distribution or activity of PKC isoforms found in the b-cell, and 3) whether b-cell signalling involving Ca2+ or cAMP is altered and 4) whether FFA metabolism in the b-cell is altered following exposure to the secretory products of b-3 adrenergic adipocytes.
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