Diabetes Genome Anatomy Project

Projects > Project 1

Project 1:
The Normal and Morbid Anatomy of Gene Expression in Mice

Primary Investigator: C. Ronald Kahn, M.D. (Joslin Diabetes Center)

Specific Aims

1. 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.
2. Determine the impact of diabetes on gene expression in these tissues, and differentiate insulin-regulated versus diabetes-regulated genes using 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.
3. Define the set of differentially regulated genes in fat of FIRKO mice and correlate this with a proteomic analysis of the same tissue.
4. 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.

Summary

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.

Protocols

• Brown Fat Cell Isolation
• Genotyping of IRlox mice by PCR
• Genotyping of IR-heterozygous mice
• IRS-1 GENOTYPING BY PCR
• Genotyping of MCK-Cre mice by PCR
• Myocyte Isolation
• Measurement of Liver Triglyceride Content

Microarray Data

• Mouse skeletal muscle - controls, streptozotocin diabetes and insulin treated
• Control Insulin Receptor (IR) and IRS-1 Single and Double Heterozygous (DH) Knockouts - Comparison of Age (6 weeks vs 6 months) and Genetic Background (B6 vs. 129) - Liver
• Control Insulin Receptor (IR) and IRS-1 Single and Double Heterozygous (DH) Knockouts - Comparison of Age (6 weeks vs 6 months) and Genetic Background (B6 vs. 129) - Skeletal Muscle
• Comparison of Low Fat and High Fat Diet on Mice of Two Genetic Backgrounds (B6 vs. 129) - Fat
• Comparison of Low Fat and High Fat Diet on Mice of Two Genetic Backgrounds (B6 vs. 129) - Liver
• Comparison of Low Fat and High Fat Diet on Mice of Two Genetic Backgrounds (B6 vs. 129) - Skeletal Muscle
• Control Insulin Receptor (IR) and IRS-1 Single and Double Heterozygous (DH) Knockouts - Comparison of Age (6 weeks vs 6 months) and Genetic Background (B6 vs. 129) - Epididymal White Fat
• Brown preadipocyte IRS knockout profiling
• Liver - ob/ob mice
• Isolated adipocytes from normal and fat insulin receptor knockout (FIRKO) mice sorted into small and large cells
• Effect of insulin infusion on skeletal muscle
• Skeletal Muscle - Muscle Insulin Receptor Knockout and Control Mice - Control, Streptozotocin Diabetic and Insulin Treated
• Human pancreatic islets from normal and Type 2 diabetic subjects