Skeletal muscle tissue from the Goto-Kakizaki rat model of type-2 diabetes exhibits increased levels of the small heat shock protein Hsp27.

In order to increase our understanding of diabetes-related muscle weakness, we carried out a mass spectrometry-based proteomic analysis of skeletal muscle preparations from the Goto-Kakizaki rat model of type-2 diabetes. Fluorescence difference in-gel electrophoresis was performed to determine potential differences in the global protein expression profile of muscle extracts. Besides changes in contractile proteins and metabolic enzymes, the abundance of the small stress proteins αB-crystallin and Hsp27 was significantly increased. The up-regulation of the low-molecular-mass heat shock protein Hsp27 was confirmed by an alternative fluorescent staining method of two-dimensional gels and immunoblotting. The observed protein alterations in the cellular stress response, distinct metabolic pathways, regulatory mechanisms and the contractile apparatus might be directly or indirectly associated with peripheral resistance to insulin signalling, making these newly identified muscle proteins potential biomarkers of type-2 diabetes. Increased levels of molecular chaperones suggest considerably enhanced cellular stress levels in diabetic muscle fibres.

Proteomic profiling of non-obese type 2 diabetic skeletal muscle.

Abnormal glucose handling has emerged as a major clinical problem in millions of diabetic patients worldwide. Insulin resistance affects especially one of the main target organs of this hormone, the skeletal musculature, making impaired glucose metabolism in contractile fibres a major feature of type 2 diabetes. High levels of circulating free fatty acids, an increased intramyocellular lipid content, impaired insulin-mediated glucose uptake, diminished mitochondrial functioning and an overall weakened metabolic flexibility are pathobiochemical hallmarks of diabetic skeletal muscles. In order to increase our cellular understanding of the molecular mechanisms that underlie this complex diabetes-associated skeletal muscle pathology, we initiated herein a mass spectrometry-based proteomic analysis of skeletal muscle preparations from the non-obese Goto-Kakizaki rat model of type 2 diabetes. Following staining of high-resolution two-dimensional gels with colloidal Coomassie Blue, 929 protein spots were detected, whereby 21 proteins showed a moderate differential expression pattern. Decreased proteins included carbonic anhydrase, 3-hydroxyisobutyrate dehydrogenase and enolase. Increased proteins were identified as monoglyceride lipase, adenylate kinase, Cu/Zn superoxide dismutase, phosphoglucomutase, aldolase, isocitrate dehydrogenase, cytochrome c oxidase, small heat shock Hsp27/B1, actin and 3-mercaptopyruvate sulfurtransferase. These proteomic findings suggest that the diabetic phenotype is associated with a generally perturbed protein expression pattern, affecting especially glucose, fatty acid, nucleotide and amino acid metabolism, as well as the contractile apparatus, the cellular stress response, the anti-oxidant defense system and detoxification mechanisms. The altered expression levels of distinct skeletal muscle proteins, as documented in this study, might be helpful for the future establishment of a comprehensive biomarker signature of type 2 diabetes. Reliable markers could be used for improving diagnostics, monitoring of disease progression and therapeutic evaluations.

DIGE analysis of rat skeletal muscle proteins using nonionic detergent phase extraction of young adult versus aged gastrocnemius tissue.

Contractile weakness and loss of muscle mass are critical features of the aging process in mammalians. Age-related fibre wasting has a profound effect on muscle metabolism, fibre type distribution and the overall physiological integrity of the neuromuscular system. This study has used mass spectrometry-based proteomics to investigate the fate of the aging rat muscle proteome. Using nonionic detergent phase extraction, this report shows that the aged gastrocnemius muscle exhibits a generally perturbed protein expression pattern in both the detergent-extracted fraction and the aqueous protein complement from senescent muscle tissue. In the detergent-extracted fraction, the expression of ATP synthase, isocitrate dehydrogenase, enolase, tropomyosin and beta-actin was increased. Different isoforms of creatine kinase and prohibitin showed differential changes. In the aqueous fraction, malate dehydrogenase, sulfotransferase, triosephosphate isomerase, aldolase, cofilin-2 and lactate dehydrogenase showed increased levels. Interestingly, differential effects on dissimilar 2-D spots of the same protein species were shown for Cu/Zn superoxide dismutase, albumin, annexin A4 and phosphoglycolate phosphatase. Mitochondrial Hsp60, Hsp71 and nucleoside diphosphate kinase B exhibited a reduced abundance in aged muscle. The majority of altered proteins were found to be involved in mitochondrial metabolism, glycolysis, metabolic transportation, regulatory processes, the cellular stress response, detoxification mechanisms and muscle contraction.

The pathobiochemical role of the dystrophin-dystroglycan complex and the Ca2+-handling apparatus in diabetes-related muscle weakness (Review).

Serious diabetic complications affect millions of patients worldwide. Skeletal muscle represents the largest insulin-regulated glucose sink in the body, making insulin resistance and abnormal glucose disposal in muscle fibres a critical aspect of diabetes mellitus. Advances in the biomedical analysis of the molecular mechanisms underlying diabetic complications rely heavily on the study of suitable disease models. The Goto-Kakizaki (GK) rat is an established animal model of non-obese type 2 diabetes. This review discusses the recent finding that expression of the dystrophin-dystroglycan complex is drastically altered in diabetic GK skeletal muscle fibres. In normal muscle, the dystrophin-glycoprotein complex provides a stabilizing connection between the actin membrane cytoskeleton and the extracellular matrix component laminin. A reduction in dystrophin-associated proteins may be associated with a weakening of the fibre periphery, abnormal sarcolemmal signaling and/or a decreased cytoprotective mechanism in diabetic skeletal muscle. Stimulation by insulin might be altered due to impaired linkage between the dystrophin-anchored actin cytoskeleton and the intracellular pool of essential glucose transporters. The diminished recruitment of GLUT4 transporter molecules to the sarcolemma may be a key step in the development of insulin resistance in diabetic skeletal muscles. Thus, analogous to certain forms of muscular dystrophy, altered dystrophin levels may have pathological effects in type 2 diabetes. In contrast, the dystrophin-glycoprotein complex does not appear to be altered in diabetic cardiac muscle. However, reduced expression of the sarcoplasmic reticulum Ca2+-ATPase isoform SERCA2 is characteristic of cardiac abnormalities in type 2 diabetes. Reduced Ca2+ removal from the sarcoplasm may be associated with impaired relaxation kinetics, and could therefore play a pathophysiological role in diabetic cardiomyopathy. Here, the potential impact of these molecular alterations in diabetic muscle tissues is discussed and critically examined with respect to the future design of alternative treatment strategies to counteract diabetes-associated muscle weakness.