Insulin resistance in skeletal muscle is a condition that is common to obesity, type 2 diabetes, and cardiovascular disease. These diseases affect millions of individuals worldwide. The insulin signaling pathway is crucial to a wide variety of biological processes in skeletal muscle such as glycogen synthesis and glucose transport. It involves precise, controlled protein-protein interactions as well as protein phosphorylation to relay the insulin signal. Defects in the insulin signaling pathway have been implicated in the development of skeletal muscle insulin resistance but the precise abnormalities in protein-protein interactions and protein phosphorylation are largely unclear. The present project will analyze proteins isolated from muscle biopsies of lean healthy, obese nondiabetic and type 2 diabetic volunteers utilizing state-of-the-art HPLC-nanospray- tandem mass spectrometry (HPLC-ESI-MS/MS) to assess interacting partners of insulin receptor substrate-1 (IRS-1) and to discover and quantify novel phosphorylation of proteins in the insulin signaling pathway. The central hypothesis of this investigation is that there are differences in protein-protein interactions and protein phosphorylation in the insulin signaling pathway in human skeletal muscle in obesity and type 2 diabetes as compared to lean and healthy conditions. The overall goal of our research is to identify molecular mechanisms responsible for insulin resistance in human skeletal muscle and to provide novel targets for prevention and treatment of type 2 diabetes. The outcome of this proposed work will be the discovery of novel protein complexes and phosphorylation sites associated with insulin resistance and regulated by insulin that will expedite the generation of hypotheses to tackle the challenging issues of type 2 diabetes. Specifically, we propose to: 1. Determine how protein-protein interactions involving IRS-1 in human skeletal muscle are altered in obesity and type 2 diabetes and are regulated by insulin. We will test the hypotheses that abnormal protein-protein complexes in human skeletal muscle are associated with insulin resistance that may impede signaling pathways involved in insulin action, and that these protein-protein interactions are regulated by insulin. We will perform co-immunoprecipitation experiments using a specific antibody against IRS-1, a major player in insulin signaling, followed by identification and quantification of IRS-1 associated proteins by HPLC-ESI-MS/MS. 2. Determine how phosphorylation of proteins involved in the insulin signaling pathway in skeletal muscle are dysfunctional in obesity and type 2 diabetes. We will test the hypothesis that abnormal phosphorylation patterns of proteins in the insulin signaling pathway in human skeletal muscle are associated with obesity and type 2 diabetes. We will employ the mass spectrometry based phosphorylation identification and quantification approach developed in our laboratory to target proteins of interest in the PI-3 kinase pathway, such as PI-3 kinase, PDK1, AKT and AS160. New and known phosphorylation sites will be quantified under both basal conditions and upon insulin infusion in vivo.

Public Health Relevance

We propose to utilize innovative mass spectrometry based proteomic technology to study differences in protein-protein interactions and protein phosphorylation in the insulin signaling pathway in skeletal muscle from lean healthy, obese nondiabetic and type 2 diabetic volunteers. The overall goal of our research is to identify molecular mechanisms responsible for insulin resistance in human skeletal muscle and to provide novel targets for prevention and treatment of type 2 diabetes.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
7R01DK081750-03
Application #
8012803
Study Section
Clinical and Integrative Diabetes and Obesity Study Section (CIDO)
Program Officer
Sechi, Salvatore
Project Start
2009-02-01
Project End
2013-01-31
Budget Start
2011-02-01
Budget End
2012-01-31
Support Year
3
Fiscal Year
2011
Total Cost
$324,021
Indirect Cost
Name
Wayne State University
Department
Radiation-Diagnostic/Oncology
Type
Schools of Pharmacy
DUNS #
001962224
City
Detroit
State
MI
Country
United States
Zip Code
48202
Zhang, Xiangmin; Damacharla, Divyasri; Ma, Danjun et al. (2016) Quantitative proteomics reveals novel protein interaction partners of PP2A catalytic subunit in pancreatic β-cells. Mol Cell Endocrinol 424:1-11
Xie, Xitao; Yi, Zhengping; Sinha, Sandeep et al. (2016) Proteomics analyses of subcutaneous adipocytes reveal novel abnormalities in human insulin resistance. Obesity (Silver Spring) 24:1506-14
Wu, Kai; Li, Lingzhi; Thakur, Chitra et al. (2016) Proteomic Characterization of the World Trade Center dust-activated mdig and c-myc signaling circuit linked to multiple myeloma. Sci Rep 6:36305
Caruso, Michael; Zhang, Xiangmin; Ma, Danjun et al. (2015) Novel Endogenous, Insulin-Stimulated Akt2 Protein Interaction Partners in L6 Myoblasts. PLoS One 10:e0140255
Tu, Chengjian; Sheng, Quanhu; Li, Jun et al. (2015) Optimization of Search Engines and Postprocessing Approaches to Maximize Peptide and Protein Identification for High-Resolution Mass Data. J Proteome Res 14:4662-73
Berhane, Feven; Fite, Alemu; Daboul, Nour et al. (2015) Plasma Lactate Levels Increase during Hyperinsulinemic Euglycemic Clamp and Oral Glucose Tolerance Test. J Diabetes Res 2015:102054
Wang, Wei; Lu, Yongju; Stemmer, Paul M et al. (2015) The proteomic investigation reveals interaction of mdig protein with the machinery of DNA double-strand break repair. Oncotarget 6:28269-81
Caruso, Michael; Ma, Danjun; Msallaty, Zaher et al. (2014) Increased interaction with insulin receptor substrate 1, a novel abnormality in insulin resistance and type 2 diabetes. Diabetes 63:1933-47
Zhang, Xiangmin; Ma, Danjun; Caruso, Michael et al. (2014) Quantitative phosphoproteomics reveals novel phosphorylation events in insulin signaling regulated by protein phosphatase 1 regulatory subunit 12A. J Proteomics 109:63-75
Geetha, Thangiah; Langlais, Paul; Caruso, Michael et al. (2012) Protein phosphatase 1 regulatory subunit 12A and catalytic subunit δ, new members in the phosphatidylinositide 3 kinase insulin-signaling pathway. J Endocrinol 214:437-43

Showing the most recent 10 out of 20 publications