Diabetes Genome Anatomy Project

Projects > Project 2

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)

Specific Aims

1. To create a map of gene expression across the diabetes metabolic spectrum by quantitating gene expression in the fasting state in skeletal muscle biopsy specimens from human subjects across a broad range of insulin sensitivity (Si), as classified by intravenous glucose tolerance testing (IVGTT) and minimal model analysis.
2. To identify genes whose expression is altered in response to acute in vivo insulin stimulation (euglycemic clamp) in human skeletal muscle, and to compare the type and magnitude of insulin-mediated changes in subjects chosen from across the metabolic spectrum (as defined in Specific Aim 1) in order to define: (a) insulin-regulated genes in insulin sensitive subjects and (b) defects in the ability of insulin to modulate expression in insulin resistant and diabetic subjects.
3. To assess time stability of gene expression by analyzing repeated biopsy samples of skeletal muscle in subjects with varying degrees of insulin resistance 2-3 years after initial biopsy and correlate with changes in metabolic control over time, in order to identify genes which predict declining metabolic control and development of diabetes.

Summary

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.

We hypothesize that each of these risk factors alters gene expression, likely in a unique but partially overlapping way, and that the superimposition of multiple risk-related changes in gene expression are likely to be the final common pathway by which both variations in primary gene sequence and environmental factors mediate diabetes risk. Since the earliest detectable abnormality in subjects at risk for type 2 diabetes is insulin resistance in skeletal muscle, we are using oligonucleotide arrays to analyze expression of both known and potentially novel genes and EST in skeletal muscle biopsy specimens from metabolically characterized human subjects across the spectrum of metabolic stages of diabetes, ranging from normal insulin sensitivity to insulin resistance to overt diabetes.

Primary Investigator

The long-range goal of our laboratory investigation is to define molecular mechanisms by which changes in gene expression resulting from primary gene sequence or the metabolic/nutritional environment mediate risk for type 2 diabetes in humans. We hypothesize that diabetes risk factors, including family history, low birth weight, and obesity, alters gene expression, likely in a unique but partially overlapping way, and that the superimposition of multiple risk-related changes in gene expression are likely to be the final common pathway by which both variations in primary gene sequence and environmental factors mediate diabetes risk. (See project 2 summary.) Therefore, our laboratory is utilizing genomics approaches, including high-density oligonucleotide arrays and restriction-based differential display, to identify alterations in gene expression which confer diabetes susceptibility.

Since insulin resistance is the earliest observable metabolic defect in the majority of prediabetic subjects, we have focused our efforts primarily in skeletal muscle and adipose tissue from metabolically characterized human subjects at risk for diabetes on the basis of family history, obesity, and low birth weight to identify primary, potentially pathogenic changes in gene expression. While we are using human tissues in metabolically characterized individuals whenever possible for primary data, refinement and confirmation of hypotheses are performed in cell culture and animal models. Clearly, validation and functional characterization of diabetes risk genes identified from differential gene expression studies will be a major endeavor over the next few years.

Co-Investigator

Dr. Goldfine is the clinically focused co-investigator for project 2. Research investigations in Dr. Goldfine's lab focus on clinical and molecular translational projects related to insulin action and development of type 2 diabetes and molecular mechanisms of drug action. Current work for the DGAP project involves careful phenotyping of insulin sensitivity and beta-cell function of subjects with risk factors for type 2 diabetes to allow characterization of physiologic determinants of disease and to obtain tissue for genomic analysis. Dr. Goldfine also has ongoing projects to explore mechanisms of cardiovascular complications of diabetes through evaluation of endothelial dependent and endothelial independent vasodilation as influenced by hyperglycemia or hyperinsulinemia, the effects of antioxidants on restoring endothelial function, and the role of hormone replacement therapy on vascular function. Additional clinical trials are in progress to evaluate clinical and molecular mechanisms of drug targets in key pathways regulating insulin sensitivity, including peroxisome proliferator activated receptor-? (PPAR?) and insulin resistant kinase IRK (also known as IKK) and NFkB pathways.

Protocols

• RNA Isolation from Tissue Culture Cells w/ Trizol
• Procedure for RNA isolation from human muscle or fat
• cDNA & Biotinylated cRNA Synthesis Reactions for GeneChips
• Real-time quantitative RT-PCR ( Taqman )

Microarray Data

• Human skeletal muscle - type 2 diabetes and family history positive individuals - Mexican American