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.
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.
|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|
|Chao, Alex; Zhang, Xiangmin; Ma, Danjun et al. (2012) Site-specific phosphorylation of protein phosphatase 1 regulatory subunit 12A stimulated or suppressed by insulin. J Proteomics 75:3342-50|
|Langlais, P; Yi, Z; Finlayson, J et al. (2011) Global IRS-1 phosphorylation analysis in insulin resistance. Diabetologia 54:2878-89|
|Geetha, Thangiah; Langlais, Paul; Luo, Moulun et al. (2011) Label-free proteomic identification of endogenous, insulin-stimulated interaction partners of insulin receptor substrate-1. J Am Soc Mass Spectrom 22:457-66|
|Holmes, R M; Yi, Z; De Filippis, E et al. (2011) Increased abundance of the adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain and leucine zipper motif (APPL1) in patients with obesity and type 2 diabetes: evidence for altered adiponectin signalling. Diabetologia 54:2122-31|
|Xie, Xitao; Yi, Zhengping; Bowen, Benjamin et al. (2010) Characterization of the Human Adipocyte Proteome and Reproducibility of Protein Abundance by One-Dimensional Gel Electrophoresis and HPLC-ESI-MS/MS. J Proteome Res 9:4521-34|
|Hwang, Hyonson; Bowen, Benjamin P; Lefort, Natalie et al. (2010) Proteomics analysis of human skeletal muscle reveals novel abnormalities in obesity and type 2 diabetes. Diabetes 59:33-42|
|Lefort, Natalie; Glancy, Brian; Bowen, Benjamin et al. (2010) Increased reactive oxygen species production and lower abundance of complex I subunits and carnitine palmitoyltransferase 1B protein despite normal mitochondrial respiration in insulin-resistant human skeletal muscle. Diabetes 59:2444-52|
|Langlais, Paul; Mandarino, Lawrence J; Yi, Zhengping (2010) Label-free relative quantification of co-eluting isobaric phosphopeptides of insulin receptor substrate-1 by HPLC-ESI-MS/MS. J Am Soc Mass Spectrom 21:1490-9|
|Hojlund, Kurt; Bowen, Benjamin P; Hwang, Hyonson et al. (2009) In vivo phosphoproteome of human skeletal muscle revealed by phosphopeptide enrichment and HPLC-ESI-MS/MS. J Proteome Res 8:4954-65|