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Project 1: The Normal and Morbid Anatomy of Gene Expression in Mice
Primary Investigator: C. Ronald Kahn, M.D. (Joslin Diabetes Center)
This project focuses on determining the normal and morbid anatomy of gene expression in the mouse, and takes advantage of a large number of animal models in which insulin action is altered genetically in one tissue at a time, but tissue specific knockout or the insulin receptor, as well as other models of both type 1 and type 2 diabetes, insulin resistance and the metabolic syndrome. The overall goals of this project are to define set of insulin responsive genes define the set of insulin responsive genes in classic insulin sensitive tissues (liver, muscle, fat) in mice and the time course of the acute insulin response using euglycemic hyperinsulinemic clamps; determine the impact of diabetes on gene expression in these tissues; and differentiate insulin-regulated versus diabetes-regulated genes by comparing gene expression in tissue specific knockout animals models, such as the muscle insulin receptor knockout (MIRKO), fat insulin receptor knockout (FIRKO) and liver insulin receptor knockout (LIRKO) mice, with streptozotocin diabetic mice. These patterns of alterations in gene expression can then be compared and contrasted to the changes that are observed in human tissues from normal, insulin resistant and diabetic patients (Dr. Patti, Project 2) to provide sets of regulated genes which are common to both the human and rodent, and thus high priority for further genetic analysis in Project 5. In addition, we will define the set of differentially regulated genes in fat of FIRKO mice and correlate this with a proteomic analysis of the same tissue in collaboration with Dr. Covera and Project 4; and compare patterns of gene expression and insulin regulation in mice with specific components of the insulin signaling network inactivated to define the genes downstream of each signaling molecule. Finally, cellular derived from knockout mice with specific defects in the insulin signaling pathway will be used for phosphoproteomic and small molecule analysis in Projects 7 and 8.
Project 2: Identifying the underlying alterations in gene expression which result in type 2 diabetes
Primary Investigator: Mary-Elizabeth Patti, M.D. (Joslin Diabetes Center)
Co-Investigator: Allison B. Goldfine, M.D. (Joslin Diabetes Center)
The overall goal of this project is to identify the underlying alterations in gene expression which result in type 2 diabetes. Though details of disease pathogenesis are increasingly complex, epidemiologic studies in humans have clearly defined risk factors for the development of and/or progression of diabetes, including: (1) genetics/family history, resulting in alterations in primary gene sequence, and (2) both prenatal and postnatal environmental factors, including suboptimal intrauterine environment and low birth weight, obesity, nutrient excess (even in the absence of obesity), inactivity, gestational diabetes, and advancing age (Figure). Each of these risk factors can, via largely undefined mechanisms, lead to skeletal muscle, adipose, and hepatic insulin resistance, ?-cell dysfunction, and overt diabetes. In turn, diabetes-related hyperglycemia and associated metabolic abnormalities can further alter signal transduction and gene expression, thus contributing to a vicious cycle.
Project 3: The Anatomy of Gene Expression in Insulin Resistant States
Primary Investigator: Michael Czech, Ph.D. (U. Mass Medical Center)
Genomics is a particularly powerful approach to the problem of identifying genes involved in insulin signaling to glucose transport since this insulin effect is restricted to muscle and fat. Heterologous expression of insulin receptors and the insulin-regulated transporter GLUT4 in other cell types fails to restore insulin regulation of glucose transport to these cells, strongly indicating that additional fat- and muscle-specific gene products are involved. Furthermore, adipocytes during differentiation, maturation and enlargement undergo a dramatic conversion from an insulin-unresponsive, fibroblastic state to a highly insulin-sensitive state, and then to an insulin resistant state in which they are virtually unresponsive to insulin. Thus, identifying subsets of genes selectively expressed in adipocytes and muscle, as well as genes differentially expressed between insulin-sensitive and insulin-resistant states, should include potential insulin signaling components or modifiers of this signaling pathway. Such genes may in turn be candidates for susceptibility to type 2 diabetes because insulin resistance appears to be a key primary aspect of the etiology of the disease.
Primary Investigator: Silvia Corvera, M.D. (U. Mass Medical Center)
Co-Investigator: John Leszyk, Ph.D. (U. Mass Medical Center)
The role of project 4 and the proteomics core facility is to complement the DGAP with information regarding the levels, modification states and subcellular distribution of proteins in cells and tissues that are being analyzed by gene chip methodologies.
The discovery aspect of this project focuses on the identification of novel targets or molecular processes that are rapidly induced by insulin in adipose cells. The underlying hypothesis for this project is that insulin action will cause rapid changes in the subcellular localization and/or oligomerization state of key regulatory proteins and protein complexes, and that such changes can be detected using a comprehensive approach that combines subcellular fractionation, detergent solubilization, velocity gradient centrifugation and Mass spectroscopy.
Project 5A: Genetic Variability of Highly Regulated Diabetogenes
Primary Investigator: David Altshuler, M.D., Ph.D. (Broad Institute of Harvard and MIT)
The Diabetes Genome Anatomy Project (DGAP) is using expression profiling to identify genes with putative roles in insulin action and the pathophysiology of diabetes. Genetic variability at these loci may play a causal role in the development of insulin-resistance and type 2 diabetes. One powerful way to test such hypothesis is to study whether inherited sequence differences are associated with diabetes in human populations. With the advent of the human genome sequence, improved understanding of genetic variation, and high-throughput technologies it is now possible to comprehensively test allelic variation at candidate loci.
Project 5B: Genetics of Adipose-Regulated Genes in Insulin-Resistance and Type 2 Diabetes
Primary Investigator: Alessandro Doria, M.D., Ph.D. (Joslin Diabetes Center)
The Diabetes Genome Anatomy Project (DGAP) is using expression profiling to identify genes with putative roles in insulin action and the pathophysiology of diabetes. Genetic variability at these loci may play a causal role in the development of insulin-resistance and type 2 diabetes. One powerful way to test such hypothesis is to study whether inherited sequence differences are associated with diabetes in human populations. With the advent of the human genome sequence, improved understanding of genetic variation, and high-throughput technologies it is now possible to comprehensively test allelic variation at candidate loci. In this project, we propose to generate data on the association between polymorphisms and type 2 diabetes or other metabolic traits for a carefully selected set of 100 genes that are identified by the DGAP as being highly adipose-regulated, insulin-regulated, and/or abnormally expressed in type 2 diabetes. Since molecules secreted by the adipose tissue are emerging as major modulators of insulin-sensitivity and the risk of type 2 diabetes, special emphasis is placed on genes that reside in the cellular pathways regulating the release of adipokines and FFA from adipocytes or their action on peripheral tissues.
Project 6: Transcriptional Profiling of PGC-1 Family in Liver
Primary Investigator: Bruce Spiegelman, Ph.D. (Dana-Farber Cancer Institute)
PGC-1a is a transcriptional coactivator of nuclear receptors and other transcription factors. It is a dominant regulator of mitochondrial biogenesis in many cell types and regulates a program of thermogenesis in brown fat, where it is inducible in the cold. PGC-1a is also involved in several other important tissue-specific metabolic programs. This coactivator is preferentially expressed in type 1 muscle fibers; when expressed transgenically in muscle beds that contain predominantly type 2 fibers, it induces a program of fiber-type switching including expression of type 1 myofibrillar proteins.
Project 7: Interfacing Small Molecules with the Diabetes Genome Anatomy Project
Primary Investigator: Stuart L. Schreiber, Ph.D. (Harvard University)
Chemical genomics aims to use small molecules to probe genetic variation. Specifically, experimental design in chemical genomic screening takes into account genetic and genomic differences in identifying cellular reactions to small molecules. Further, this approach provides a temporal control over cell states not normally achieved by molecular biology methods. We are using chemical genomic methods in order to probe the effects of small molecules within the cellular context of type 2 diabetes.
Project 8: Signal-dependent Interactions in Insulin Action
Primary Investigator: Matthias Mann, Ph.D. (University of Southern Denmark)
Insulin binding to its receptor initiates a complex network of events, starting with a tyrosine phosphorylation cascade that branches out to affect multiple endpoints. It is likely that many steps of the insulin pathway are adversely affected in various forms of type II diabetes. However, the polygenic nature of the disease makes it difficult to understand its molecular level. While gene expression methods are very well developed, they do not address many of the changes that may occur in cell signaling. To study changes in protein abundance, we are using a combination of nano-flow high performance liquid chromatography, sensitive mass spectrometers (LTQ-FTMS), and the SILAC method (Stable Isotope Labeling by Amino acids in Cell culture). These techniques will allow us to define changes in the insulin-dependent tyrosine phosphorylation on a proteomic scale.