MiP2005: Session 9

Mitochondrial Physiology Network 10.9: 104-105 (2005) - download pdf

 

Proteomic and biochemical analyses of diabetic human and obese rodent skeletal muscle mitochondria reveals altered concentration, oxidation, and activity of respiratory chain proteins.

Douglas R Moellering1, HS Vestri1, E Ulasova2, CL Krumdieck1, P Wallace1, AL Landar2, VM Darley-Usmar2, WT Garvey1

1Dept. Nutrition Sciences; 2Dept. Pathology, University of Alabama at Birmingham, Birmingham, AL. 35294. - dmoellering@uab.edu

    Altered mitochondria-related gene expression in skeletal muscle has been observed in Type 2 Diabetes (T2DM) patients compared to Insulin Sensitive (IS) individuals, implicating mitochondrial dysfunction in the development of insulin resistance (IR). Obesity-related and diabetes-related IR in skeletal muscle has been linked to intramyocellular lipid (IMCL) accumulation and impaired mitochondrial oxidative phosphorylation [1,2]. It is hypothesized that specific mitochondrial defects in skeletal muscle, including altered expression of individual respiratory chain complex proteins and increased reactive oxygen species (ROS) generation, are directly related to increased IMCL and insulin resistance. 

    Using proteomic and biochemical analyses, we have assessed whether there are altered expression, oxidative modification, and/or activity of respiratory chain protein complexes in 6 pairs of IS and T2DM patients matched for age, race, gender, and BMI and in an animal model of obesity using Zucker (fa/Br) obese rats.  Mitochondrial preparations were subjected to 2-Dimension Blue-Native gel electrophoresis for optimal separation of membrane proteins [3,4]. Multiple proteins associated with respiratory complexes I, III, IV, and V were found to be altered in T2DM compared to IS individuals and expression of complexes I and IV were decreased in the obese rat compared to lean littermates.  Alterations in the ETC protein subunits could also increase ROS formation. Western blot analysis confirmed increased levels of oxidized mitochondrial thiols and increased protein carbonyl adducts in T2DM individuals compared to insulin sensitive controls and in the obese rodents compared to their controls which could further compromise function [5-7].

    These abnormalities in respiratory chain protein expression and oxidation could affect function of respiratory complexes resulting in defects in substrate oxidation and accumulation of IMCL. The dramatic increases in respiratory protein oxidation in diabetes implicate increased ROS generation as a potential key mechanism for impaired insulin action and metabolism in insulin resistant humans.

1.  Petersen KF, Dufour S, Befroy D, Garcia R, Shulman GI (2004) Impaired mitochondrial activity in the insulin-resistant offspring of patients with type 2 diabetes. N. Engl. J. Med. 350: 664-671.

2.  Machann J, Haring H, Schick F, Stumvoll M (2004) Intramyocellular lipids and insulin resistance.  Diabetes Obes. Metab. 6: 239-248.

3.  Schagger H, Cramer WA, von Jagow G (1994) Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis. Anal. Biochem. 217: 220-230.

4.  Brookes PS, Pinner A, Ramachandran A, Coward L, Barnes S, Kim H, Darley-Usmar VM (2002) High throughput two-dimensional blue-native electrophoresis: a tool for functional proteomics of mitochondria and signaling complexes. Proteomics 2: 969-977.

5.  Kim JR, Yoon HW, Kwon KS, Lee SR, Rhee SG (2000) Identification of proteins containing cysteine residues that are sensitive to oxidation by hydrogen peroxide at neutral pH. Anal. Biochem. 283: 214-221.

6.  Ramachandran A, Ceaser E, Darley-Usmar VM (2004) Chronic exposure to nitric oxide alters the free iron pool in endothelial cells: role of mitochondrial respiratory complexes and heat shock proteins. Proc. Natl. Acad. Sci. 101: 384-389.

7.  Conrad CC, Choi J, Malakowsky CA, Talent JM, Dai R, Marshall P, Gracy RW (2001) Identification of protein carbonyls after two-dimensional electrophoresis. Proteomics 1: 829-834.


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Mitochondrial Physiology