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Lab Research Projects

These research projects are what is called "bench science" or "wet lab" projects in that they take place in the lab and do not involve human subjects. These lab research projects are an important precursor to the clinical studies done with human subjects. In the lab, our researchers can test their theories until the results provide evidence that is later translated into clinical studies, then hopefully treatments — and, ultimately, prevention, reversal, and cures!

 

Peptide Hormone Sorting to the Secretory/Storage Granule

Molecular Mechanisms of Oxidative Stress and Cardiac Neuropathy in Metabolic Syndrome

Bypass Angioplasty Revascularization Investigation Diabetes 2 (BARI 2D)

The Epidemiology of Diabetes Interventions and Complications - Neuropathy ( NEUROEDIC)

Leptin Receptor Action in Obesity and Diabetes

Genomics and Lipomics in a Model of the Metabolic Syndrome

Regulation and Function of Leptin Receptor-Expressing LHA Neurons

Improving Prediction of IDDM

Insulin Resistance and Hepatic Steatosis in Hepatitis C

Biology of Aerobic Capacity and Insulin Resistance

Using Systems Biology to Understand Islet Adaptation and Failure Diabetes

Epidemiology of Heterogenity in Type 2 Diabetes

Proteomics of Autoimmune Type 1 Diabetes

Mechanisms of Leptin Receptors Signal Attenuation in vivo

Role of the Lateral Hypothalamic Area in Leptin Action

Molecular Mechanisms of Leptin Receptor/Jak2 Action

 

 

Peptide Hormone Sorting to the Secretory/Storage Granule

Investigator:                  Peter Arvan, M.D., Ph.D.
Sponsor:                        National Institute of Diabetes and Digestive and Kidney Diseases

Purpose of Study:  The biology of the secretory pathway is of central importance to physiological insulin production:

I) Pancreatic beta-cells normally synthesize and export massive quantities of proinsulin, and these quantities are further increased upon elevation of blood glucose. This poses a challenge to the secretory pathway protein folding system and exposes the cells to risk of proteotoxicity. One mechanism that limits the translation of islet proinsulin, especially after elevation of extracellular glucose, involves signaling at the endoplasmic reticulum (ER) by the PERK transmembrane protein kinase that phosphorylates eukaryotic translational initiation factor eIF2alpha on Ser51, which diminishes translation of secretory proteins while allowing the continued translation of a subset of mRNAs including those for ER chaperones. Deficient signaling via this pathway results in a failure to normally downregulate secretory protein translation, and PERK-/- mice exhibit phenotypes that include apoptotic beta-cell failure. Similarly, expression of the Akita [Cys(A7)Tyr] mutant proinsulin that has improper disulfide bonding reportedly also induces apoptosis of beta-cells, even in the absence of genetic defects in ER signaling.

II) It is generally believed that the fidelity of sorting-packaging functions within the TGN and immature secretory granules is supported by active recycling of membrane proteins between the biosynthetic and endocytic pathways.

Research Aims:

1. In Aim #1, we propose to pursue the hypothesis of disulfide mispairing for a fraction of wild-type proinsulin, and the possibility that this misfolding can lead to beta-cell toxicity - even more so in cells rendered susceptible because of insufficient ER stress response.

2. In Aim #2, we wish to pursue established and new models of misfolded, disulfide-mispaired mutant proinsulin, as well as the possibility of molecular/genetic rescue of cells from death due to proteotoxicity.

3. In Aim #3, we now propose to extend a two-pronged approach for studies in beta-cells involving molecular perturbation of candidate gene products relevant to protein egress from the biosynthetic pathway, as well as endosomal return, in order to examine the relationship of these steps to insulin secretory granule biogenesis.

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Molecular Mechanisms of Oxidative Stress and Cardiac Neuropathy in Metabolic Syndrome

Investigator:            Rodica Pop-Busui, M.D., Ph.D.
Sponsor:                 American Diabetes Association

Purpose of Study: This study explores the relationship between components of the metabolic syndrome, in the presence of prediabetic range glucose elevations, and oxidative stress in the development of cardiac autonomic neuropathy (CAN, a failure of the nerves surrounding the heart) and the effects of a lifestyle intervention comprised of a Mediterranean style diet and exercise in subjects with metabolic syndrome (MS). We also investigate the salutary effects of an intensive lifestyle intervention in MS on a clustering of major cardiovascular disease (CVD) risk factors, which include elevated fasting glucose (IFG) or impaired glucose tolerance (IGT), low high density lipoprotein (HDL) cholesterol, elevated triglycerides, elevated blood pressure and central adiposity. These components associate with chronic inflammation, oxidative stress and increased CVD risk. CAN also strongly associates with increased CVD risk and mortality.

Several lines of evidence point towards a central role for oxidative stress in the pathogenesis of insulin resistance, CVD and the development of diabetic complications  including CAN. However, the mechanisms underlying CAN development and its links to CVD risk are poorly understood. We hypothesize that components of the metabolic syndrome in the presence of mild glycemic elevations induce activation of specific oxidative stress and inflammatory pathways which in turn lead to impaired sympathetic activity, CAN and increased CVD risk.

The Met Fit Program at the University of Michigan includes supervised exercise program and evidence-based dietary counseling to improve metabolic health in patients with MS. Using highly sensitive and specific gas chromatography (GC) mass spectrometry we have characterized and validated sensitive markers of oxidative stress in plasma and HDL of humans with CVD and diabete.  These markers serve as molecular fingerprints for specific oxidation pathways. We have also developed positron emission tomography (PET) imaging with norepinephrine radioanalogs to study regional cardiac sympathetic innervation.

With this study, we are performing a detailed analysis of specific oxidative fingerprints and study their relationship with functional measurements of early CAN- left ventricle (LV) PET with [11C]meta-hydroxyephedrine ([11C]HED)  and quantitative heart rate (HR) variability testing in subjects with MS. We are following these subjects prospectively during a 6-month period of supervised lifestyle modifications comprised of a structured exercise and Mediterranean diet program.

Research Aims:
1. To evaluate the relationship between MS, CAN and oxidative stress in subjects with MS.
2. To prospectively investigate the effects of an intensive lifestyle intervention comprised of a structured exercise–diet program on biomarkers of oxidative stress and CAN in subjects with MS.

Combining quantitative oxidative biomarker measurements with functional analysis of CAN will provide insights into the pathogenesis of CAN and help define the salutary effects of an intensive lifestyle intervention in this high-risk population. Collectively, these studies using novel techniques and a systematic approach will be pivotal in defining novel sensitive biomarkers to identify persons at risk for the development of CAN and targeted for preventive measures in future larger trials.

                     

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Bypass Angioplasty Revascularization Investigation Diabetes 2 (BARI 2D)

Investigator:            Rodica Pop-Busui, M.D., Ph.D.
Sponsor(s):              National Institutes of Health / National Heart, Lung, and Blood Institute

Purpose of Study: The primary aim of the BARI 2D Trial is to test the coronary revascularization vs. intensive glycemic control hypothesis of treatment efficacy in 2,800 patients with type 2 diabetes mellitus and documented stable coronary artery disease, in the setting of uniform glycemic control and intensive management of all other risk factors including dyslipidemia, hypertension, smoking, and obesity. 

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The Epidemiology of Diabetes Interventions and Complications - Neuropathy  (NEUROEDIC)

Investigator:            William Herman, M.D., M.P.H.; Rodica Pop-Busui, M.D., Ph.D.
Sponsor(s):              National Institutes of Health / National Institute of Diabetes and Digestive and Kidney Diseases

Purpose of Study: The primary aim is to evaluate the development and progression of distal symmetrical peripheral neuropathy and autonomic neuropathy as part of a multi-center longitudinal observational study of the Diabetes control and Complications Trial ( DCCT) cohort who are participating in the Epidemiology of Diabetes Interventions and Complications (EDIC) study.  

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Leptin Receptor Action in Obesity and Diabetes

Investigator:            Martin G. Myers, Jr., M.D., Ph.D.
Sponsor(s):              American Diabetes Association

Purpose of Study: This study explores the role of obesity to the pathogenesis of type 2 diabetes.  Development of disease modifying treatments has been deterred by poor understanding of the links between weight regulation and disease development. One contributing factor is loss of leptin signaling, demonstrated by the obese phenotypes of mice deficient in either leptin (ob/ob) or the long form leptin receptor (LRb, db/db). These mice develop type 2 diabetes, suggesting that loss of the leptin/LRb signaling system promotes disease development. There are numerous populations of LRb-expressing neurons in the brain, but outside of LRb-expressing neurons in the acuate nucleus, little is known about the contributions of other populations of LRb-expressing neurons in energy homeostasis. This research will characterize a novel population of LHA LRb-expressing neurons and will collectively advance understanding of LRb-mediated signaling in neurons and in weight-regulation circuits. Increased understanding of how LRb-expressing neurons signal and contribute to weight regulation circuits is critical for identifying potential sites of therapeutic intervention for type 2 diabetes.

Research Aims:
1. Understand the contribution of a large group of LRb-expressing neurons in the lateral hypothalamus, a known "feeding center"
2. Define the molecular profile of LHA LRb neurons via microdissection and microarray technology.
3. Investigate the connectivity of LHA LRb neurons via novel tract tracing techniques.
4. Explore the physiological contribution of LHA LRb neurons in energy homeostasis by utilizing mouse models allowing for LHA-specific deletion of LRb expression.   

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Genomics and Lipomics in a Model of the Metabolic Syndrome

Investigator:            Charles F. Burant, M.D., Ph.D.
Sponsor(s):              American Diabetes Association

Purpose of Study: The metabolic syndrome is a constellation of risk factors for diabetes and cardiovascular disease. Understanding the underlying cause of the metabolic syndrome will help in the prevention of type 2 diabetes and the prevention of cardiovascular disease. The animal model of the metabolic syndrome used in this study was developed over the last 10 years by breeding rats for their intrinsic ability to run. The animals that run more poorly (Low Capacity Runners or LCR) have all the traits of someone with “prediabetes” including insulin resistance with higher glucose and insulin levels, elevated blood pressure, high blood lipid levels and obesity. They are genetically more susceptible to high fat diet while the High Capacity Runners (HCR) is resistant to high fat diets.

Research Aims:
1. This study will determine whether it is possible to exploit this genetic heterogeneity to determine which genes are responsible for the poor running capacity, the elevated blood pressure, the altered lipid levels and the increased insulin levels.   To do this, the expression of genes in skeletal muscle and liver will be examined.
2. In addition, because changes in the concentration and 'flux' of fatty acids in the blood are thought to contribute to many of the abnormalities in the metabolic syndrome, the profile of fatty acids in different kinds of lipid in the blood and in the tissues will be determined as well .
3. A systems biology analysis of the data using a variety of computational approaches will be performed to make predictions as to which genes and lipids contribute to the different phenotypes. First, this will provide new insight into the development of insulin resistance and, second, may allow the ability to predict what types of diets or exercise might be beneficial to the prevention or treatment of diabetes.   

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Regulation and Function of Leptin Receptor-Expressing LHA Neurons

Investigator:               Martin Myers, Jr., M.D., Ph.D.
Sponsor:                     American Diabetes Association

Purpose of Study:  This project deals with the basic mechanisms by which signals from the brain control eating and glucose homeostasis- important for obesity and type 2 diabetes (“diabesity”). The central hypothesis is that the appetite-suppressing hormone, leptin, acts in part via a novel and important set of neurons in the 'feeding center' of the lateral hypothalamus. If the data that we generate in this project support this theory, then we will have taken several steps toward characterizing potential therapeutic targets for the treatment of “diabesity.”    

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Improving Prediction of IDDM

Investigator:           Massimo Pietropaolo, M.D.
Sponsor:                 National Institute of Diabetes and Digestive and Kidney Diseases

Purpose of Study: The primary aim of this research is to investigate the role of new islet autoantibodies, HLADQ high risk haplotypes and a novel potential T1DM susceptibility genetic locus (polymorphism of the gene ICA1) in risk of conversion to T1DM in the largest population based cohort of first degree relatives of T1DM patients, with and without T1DM, from the same geographical area. Small sample sizes, limited follow-up and the lack of advanced technology have limited previous studies in this field. It is now possible to determine the risk of conversion to T1DM utilizing new islet biochemical islet autoantibody assays in this specific population. Recent data indicate that the most popular screening assays for detecting islet autoantibodies are not sufficient for predicting T1DM. Over the past few years, molecular cloning techniques have been applied to identify T1DM-related targets of islet autoimmunity. We now provide preliminary data suggesting that diabetes the progression rate can be increased to nearly 100% with the addition of two novel assays detecting autoantibodies to IA-2 Fragment 1 (aa residues 761- 964) and IA-2ic (aa residues 601-979).

To increase sensitivity, autoantibodies will be applied that recognize specific epitopes of the antigen GAD65 in combination with the conventional islet autoantibody markers. Two HLA-DQ high-risk haplotypes and SNPs within the ICA1 locus (encoding the antigen ICA69) will also be evaluated as risk factors for insulin-requiring diabetes. This research sample includes 123 first degree relatives, who converted to insulin-requiring diabetes during follow-up (converters), from a pool of over 7,000 relatives of T1DM probands.  Also, DNA has been collected from relatives of T1DM patients, and specimens on complete singleton and multiplex families in our repository with affected and unaffected siblings are available. This is a unique set of serum and DNA samples for the testing of immunologic and genetic hypotheses. The outcome of the proposed investigation should facilitate the stage for the application of a new screening strategy based upon the use of IA-2 and GAD65 epitope markers in combination with conventional islet autoantibody testing that could be of benefit in major clinical trials aimed at evaluating new approaches for understanding, preventing, and treating Type 1 diabetes. 

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Insulin Resistance and Hepatic Steatosis in Hepatitis C

Investigator:          Charles F. Burant, M.D., Ph.D.
Sponsor:                National Institute of Diabetes and Digestive and Kidney Diseases

Purpose of Study: Approximately 1.8% of the population in the United States has antibody to HCV and the infection rate is increasing. Multiple retrospective, case-control and population-based studies have demonstrated that infection with the hepatitis C virus (HCV) is a significant risk factor for the development of insulin resistance, hepatic steatosis and type 2 diabetes mellitus which confer an increased risk for hepatic fibrosis, cirrhosis and hepatocellular carcinoma. The site (hepatic vs. peripheral) and severity of insulin resistance in HCV infection has never been systematically examined in humans. In addition, the cause-effect relationship between insulin resistance and hepatic steatosis in HCV infection has not been clarified.

In this study, the hypothesis to be tested is that human HCV infection results in specific changes in gene expression that leads to significant hepatic insulin resistance which provides a 'fertile ground' for the development of hepatic steatosis.

Research Aims:
1. In Aim 1, the site and severity of insulin resistance (IR) in patients with hepatitis C infection will be assessed by stepped insulin-euglycemic clamp before and following anti-viral therapy. The results will be compared with a group of weight matched normal controls and individuals with non-alcoholic fatty liver disease.
2. In Aim 2, directed and global gene profiling will be performed on biopsy samples of subjects studied in Aim 1 to test the hypothesis that HCV infection results in a distinct pattern of gene expression that is responsible for the induction of hepatic insulin resistance.
3. The output of the gene arrays will be analyzed by different but complimentary methods: i) Gene Set Enrichment Analysis, a robust method for determining coordinated changes in gene expression of a priori defined sets of functionally related genes, ii) identification of networks of related genes populated with genes which have significant differences in expression, iii) performing a novel 'index gene network enrichment analysis' using index genes to generate networks which can be statistically compared between conditions.

The results of these studies will provide the first data on the physiological mechanisms of insulin resistance and the relationship to the development of steatosis and will provide the first insight into the molecular mechanism mechanisms underlying HCV-induced insulin resistance.

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Biology of Aerobic Capacity and Insulin Resistance

Investigator:              Charles Burant, M.D., Ph.D.
Sponsor:                     National Institute of Diabetes and Digestive and Kidney Diseases

Purpose of Study: Recent studies have show a decrease in oxidative capacity is associated with obesity, insulin resistance, hypertension and in some individuals, the development of type 2 diabetes. Indeed, oxidative capacity is an excellent predictor of life expectancy. Decreases in skeletal muscle mitochondrial oxidative phosphorylation can be found in individuals who are prone to the development of type 2 diabetes. While these alterations can be found in relatively young individuals at high risk for diabetes, it is not clear whether this alteration is due to an inherent defect in skeletal muscle oxidative capacity or is due to acquired changes. In addition, it remains to be determined whether interventions at an early age can alter the metabolic profile of those at risk for type 2 diabetes. An outbred, genetically heterogeneous colony of rats with inherited differences in oxidative capacity based on low (LCR) and high (HCR) running capacity show differences in the propensity to develop a phenotype consistent with the “metabolic syndrome”. The LCR colony has many of the same alteration in mitochondrial-related function as has been described in humans and thus provides a model to investigate the molecular causes and treatment of the metabolic syndrome. Interestingly, the HCR colony was found to have gene expression profiles suggesting that they perceive a caloric restriction, despite an increase in food intake. The studies will use a systems approach, combining metabolomics, gene expression and bioinformatics to define the underlying molecular changes in metabolism that lead to alterations in oxidative capacity.

Research Aims:

1. In Specific Aim 1, detailed metabolic phenotyping in LCR and HCR animals with a range of oxidative capacity will be performed at baseline and following exercise training. Physiologic, morphometric and metabolomic data will be evaluated.
2. In Specific Aim 2, a series of bioinformatic studies will be performed, including Bayesian analysis connecting gene expression, metabolome differences with phenotype to identify pathways and genes. Detailed promoter analysis will be performed to identify upstream factors that may underly differences in oxidative capacity in the LCR and HCR rats.
3. In Aim 3, temporal caloric restriction will be used to test the potential for “metabolic imprinting” to alter the physiologic and biochemical profiles of the HCR and LCR animals in adulthood. These studies will provide a mechanistic understanding of the contributions of “nature” and “nurture” in the development of insulin resistance and type 2 diabetes.

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Using Systems Biology to Understand Islet Adaptation and Failure Diabetes

Investigator:               Charles Burant, M.D., Ph.D.
Sponsor:                     National Institute of Diabetes and Digestive and Kidney Diseases

Purpose of Study: The pancreatic islet is a dynamic tissue which can increase insulin secretion in response to increased secretory demand. The adaptation is due to both increased intrisic islet secretory capacity and expansion of beta-cell mass in response to excess nutrition. The development of type 2 diabetes is due to the inability of beta-cells to maintain insulin secretion in the face of prevailing insulin resistance. The complexy underlying biology of islet adaptation and failure is still incompletely understood but lends itself to a global, intradisciplinary approach. In this application we will use both an animal model and human tissue to test our hypothesis that increases in glucose and fatty acid flux to the islet results in anaplerosis which will augment the secretion of insulin in the short term and generate signals that result in longer term adaptation, including expansion of the beta-cell mass. In addition, we propose that in a genetically susceptible islet, specific changes in the metabolome occur, when exposed to a specific nutrient mix, that results in islet failure. The results of these investigations will lead to an integrated understanding of how nutrients interact with genetically resistant and susceptible islets to increase secretion an how adaptation fails leading to diabetes. The studies will also provide the first insights into the ways in which human islets react to specific nutrient challenges.

Research Aims:
1. In Specific Aim 1 we will examine the temporal effect of a control, diabetogenic and non- diabetogenic high fat/high sucrose diets on physiological parameters of islet function in vivo and in vitro. Initial studies will use islets from female control, Zucker Fatty Rats and Zucker Diabetic Fatty Rats exposed to diabetogenic and non-diabetogenic diets for varying periods of time.
2. In Aim 2, relevant islet metabolites, induding anaplerotic and cataplerotic intermediates, lipid species and oxidative stress markers will be measured under different in vitro conditions to examine the adaptation/maladaptation of the islet following different dietary interventions.
3. In Aim 3, the temporal changes in gene expression profiles under errant dietary conditions will be evaluated and integrated with the metabolomic, lipomic and functional data to using new computational methods to provide a systems overview of the effects of the diets on islet function.
4. In Aim 4, we will translate the techniques and tools that we develop to evaluate the function and metabolism of human islets isolated from living donors with known physiological status.

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Epidemiology of Heterogenity in Type 2 Diabetes

Investigator:               Massimo T. Pietropaolo, M.D.
Sponsor:                     National Institute of Diabetes and Digestive and Kidney Diseases

Purpose of Study: Several lines of evidence indicate that Type 2 (non-insulin-dependent) diabetes mellitus is a heterogeneous disease that results from a combination of abnormalities in both insulin secretion and insulin action. The causes of decreased insulin secretion in Type 2 diabetes are still not completely understood, but in a subgroup of Type 2 diabetic patients they may be related to an autoimmune destruction of the pancreatic beta-cells. We showed that in Type 2 diabetic patients the presence of GAD65 antibodies is strongly related to the use of insulin therapy. We now propose that GAD65 antibody-positive diabetes that we are observing is a unique subclass of Type 2 diabetes, which is associated with relatively severe insulin deficiency. It is important to note that these cases are all reasonably well documented as Type 2 diabetes rather than Type 1 and those they are older individuals and, clearly, are different than traditional Type 1 diabetes or young adults in regard to their clinical characteristics and needs for insulin.

We hypothesize that this subset of older T2DM patients carrying GAD65 antibodies is unique in that is associated with severe insulin deficiency, with HLADQ susceptibility and with the development micro vascular disease. In contrast, diabetic patients who are not GAD65 antibody positive are probably primarily insulin resistant, have elevated blood insulin levels and are at primary risk for macro vascular disease. As a model for understanding the causes of insulin deficiency in GAD65 antibody positive older patients (>50 yr of age), we plan to evaluate the metabolic and genetic abnormalities in GAD65 autoantibody positive T2DM patients and compare these abnormalities with those of a subgroup of GAD65 antibody negative T2DM patients. The characterization of individuals at risk of developing this unique form of Type 2 diabetes is of public health interest because therapeutic strategies could potentially be instituted early enough to prevent the complications related with hyperglycemia and, possibly, the time of onset of insulin requirement. A more appropriate characterization of this subgroup of older Type 2 diabetic patients, presumably of autoimmune pathogenesis, will be of benefit to future research into the etiology, natural history as well as treatment of Type 2 diabetes mellitus.

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Proteomics of Autoimmune Type 1 Diabetes

Investigator:              Massimo T. Pietropaolo, M.D.
Sponsor:                    National Institute of Diabetes and Digestive and Kidney Diseases

Purpose of Study: A growing number of pilot trials, such as TrialNet (http://www.niddk.nih.gov/welcome/releases/06-05-04.htm), are now being carried out nationwide and worldwide in an effort to find the cure for Type 1 diabetes (T1DM). All of these trials are based on the understanding that only 50% of "high risk" first-degree relatives of T1DM probands enrolled in these trials develop insulin requirement at 5 year follow-up. Endless discussions have taken place on how to develop new strategies to enhance sensitivity of multiple markers and in turn effectively enroll first-degree relatives into such trials prior to T1 DM onset. As of today, based on conventional autoantibody markers alone, the number of these relatives nationwide would be insufficient to complete all the proposed clinical trials. In the Preliminary Studies we demonstrate our expertise in applying a proteomic-based technology to identify pancreatic islet proteins reactive with antibodies in the sera of islet cell antibody (ICA) positive T1 DM patients but not in the sera of controls. To date, we have identified several candidate proteins by proteomic technology deemed worthy of investigation as new candidate autoantigens in T1DM.

Our data also provide indirect evidence for the presence of an important subset of ICA that likely reacts with unidentified islet autoantigens. These data suggest that a novel subset of ICA is present in GAD65/IA-2 AA negative newly diagnosed T1DM patients and that a subset of ICA might also be related with rapid progression to insulin-requiring diabetes. A further characterization of this ICA response should facilitate a rational approach to ultimately discover a novel biochemical islet autoantibody marker(s) associated with rapid progression to T1DM. This objective will be initially exploited using proteomic-based technology and this approach will subsequently be coupled with our longstanding expertise in developing biochemical islet autoantibody assays. Novel surrogate markers that will be identified by this approach might ultimately aid in monitoring the response to therapy aimed at delaying or reversing the disease process.

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Mechanisms of Leptin Receptors Signal Attenuation in vivo

Investigator:               Martin Myers, Jr., M.D., Ph.D.
Sponsor:                     National Institute of Diabetes and Digestive and Kidney Diseases

Purpose of Study: The long (or LRb) isoform of the leptin receptor mediates signaling and the physiologic action of leptin to regulate energy balance (decreasing feeding and increasing energy expenditure) and neuroendocrine function. It is not clear why leptin fails to adequately protect from obesity and the predisposition to Type 2 diabetes in humans; it is thus critical to understand the molecular details of LRb signaling and especially mechanisms by which LRb signaling is attenuated in order to understand potential mechanisms of leptin resistance and/or to identify potential targets for the therapy of obesity. The long-term outlook of our previous and future studies is to understand the regulation and mechanisms of signaling by LRb and their role in physiologic leptin action. LRb is a type 1 cytokine receptor that, although devoid of enzymatic activity, mediates phosphotyrosine-dependent signaling by means of an associated Jak2 tyrosine kinase and two phosphorylation sites on the intracellular LRb. Tyr1138 activates STAT3, which is responsible for important positive leptin signals as well as for the induction of SOCS3 by leptin.

In addition to its role in SHP- 2/ERK signaling in cultured cells, Tyr985 binds SOCS3 to mediate some elements of LRb signal attenuation in vivo as well as in cultured cells; SOCS3 also binds to the LRb-associated Jak2 to directly inhibit Jak2 signaling. We propose to study the function of SOCS3 binding to Jak2 and Tyr985 in signal attenuation in cultured cells and in vivo. In addition to shedding light upon the biology of LRb specifically, our analysis will illuminate basic mechanisms of cytokine receptor/tyrosine kinase signaling.

Research Aims:

1. Determine the molecular mechanisms of LRb signal attenuation in cultured cells.
2. Examine the role of LRb phosphorylation sites in leptin sensitivity in vivo.
3. Address the mechanism(s) of SOCS3-mediated inhibition of LRb action in vivo. 

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Role of the Lateral Hypothalamic Area in Leptin Action

Investigator:               Martin Myers, Jr., M.D., Ph.D.
Sponsor:                     National Institute of Diabetes and Digestive and Kidney Diseases

Purpose of Study:   The ongoing epidemic of obesity in the United States represents a public health emergency that remains unchecked and without specific therapy. To design specific treatments to prevent and treat obesity, we must first understand the mechanisms that regulate feeding and energy expenditure in order to identify potential therapeutic targets. In this proposal, entitled, Role of the lateral hypothalamic area in leptin action, we will analyze novel leptin-regulated neural pathways in the lateral hypothalamic area (LHA) that likely contribute to body energy homeostasis.

We have defined the existence of a novel population of LRb-expressing, leptin-responsive LHA neurons that project locally as well as densely innervating the ventral tegmental area (VTA). We hypothesize that LRb-mediated signaling in the LHA is crucial to the regulation of the VTA and activity of the mesolimbic dopamine system, and thereby for the regulation of feeding and energy balance. We will thus study the regulation of LHA LRb neurons and their role in the regulation of physiology by leptin.

Research Aims:
1. Understand the regulation of LHA LRb neurons. Analyze the regulation of gene expression and activity of LHA LRb neurons, and define the mechanisms by which leptin and other factors mediate this regulation.
2. Define the action of LHA LRb neurons on downstream neurons. Define the downstream target neurons of LHA LRb neurons and the role of LHA LRb neurons in the regulation of these downstream neurons, including the mesolimbic dopamine system.
3. Determine the function of LHA LRb neurons in physiologic leptin action. Examine the physiologic function of LHA LRb neurons by examining energy balance and behavior in animals following a variety of genetic and pharmacologic manipulations directed at these neurons.

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Molecular Mechanisms of Leptin Receptor/Jak2 Action

Investigator:               Martin Myers, Jr., M.D., Ph.D.
Sponsor:                     National Institute of Diabetes and Digestive and Kidney Diseases

Purpose of Study:       The long-term outlook of our previous and future studies is to understand mechanisms of LepRb signaling and to determine how LepRb signals contribute to the regulation of neural function and thence to the control of energy balance, glucose homeostasis, and neuroendocrine function. LepRb mediates tyrosine phosphorylation (Tyr(P))-dependent signaling by means of an associated Jak2 tyrosine kinase. Leptin binding stimulates the Tyr(P) of Jak2 and tyrosine residues on LepRb; each Tyr(P) site mediates a unique complement of intracellular signals. To this point, we have defined the mechanisms of LRb/Jak2 interaction, defined the function of Jak2 Tyr(P) sites, and examined the biology of LepRb Tyr1138AESTAT3 signaling and LepRb Tyr985AESHP2/SOCS3 signaling. Recently, we defined a third Tyr(P) site on LepRb (Tyr1077), which regulates STAT5 signaling and potentially other LepRb signals, and have generated novel mouse models to probe the function of Jak2 and Tyr1077 in LepRb action in vivo.

Preliminary data suggest that contributions from LepRb phosphorylation sites are required for most known leptin effects, including some actions that are independent of Tyr1138 and Tyr985. In contrast, Tyr1077 is crucial to the regulation of glycemic control and potentially other physiologic leptin effects. In the context of a variety of data that suggest the importance of the acute (non-transcriptional) effects of leptin in the short-term regulation of energy balance and glycemic control, these data suggest the hypothesis that LepRb Tyr1077 mediates cellular signals required for the acute effects of leptin. In addition to testing this core hypothesis, the proposed research will define the signals and mechanisms by which leptin mediates a variety of physiologic effects and by which leptin modulates blood glucose levels.

Research Aims:
1. Understand the roles for LepRb signals in the regulation of physiology, focusing on Jak2 and Tyr1077.
2. Define the leptin-mediated regulation of neural function in mouse models of altered LepRb signaling.
3. Determine the signaling mechanisms by which LepRb Tyr1077 and/or other Jak2 and LepRb Tyr(P) sites control physiology and neural function.  

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