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

Abstract

Abstract. 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 lowmolecular-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. IntroductionThe application of gel electrophoresis-based proteomics in diabetes research is a fast growing field (1), including the proteomic analysis of diabetic skeletal muscle tissues (2-7). Since diabetes is increasingly prevalent in the general population, research into the complex pathophysiological mechanisms that underlie abnormal signaling in crucial target organs, such as skeletal muscle, is of central importance (8-10). It is now clear that type-2 diabetes mellitus represents a group of heterogeneous disorders with abnormal expression patterns in various genes and protein products (11-13). Peripheral insulin resistance in the liver, adipose tissue and muscles, as well as impaired pancreatic β-cell functioning, are the principal features of type-2 diabetes (11). The worldwide incidence of type-2 diabetes is dramatically increasing (14) and it has been estimated that the incidence of diabetes will rise to a staggering 4.4% by the year 2030, with 366 million affected patients (15). Importantly, type-2 diabetes is associated with a loss of skeletal muscle mass and contractile strength (16-19) warranting detailed investigations into diabetes-related muscle weakness (20). In this respect, large-scale biochemical approaches, such as gel electrophoresis-based proteomics, are ideal analytical tools for an unbiased identification of novel protein factors that are associated with abnormal functioning in diabetic fibres.High-resolution two-dimensional gel electrophoresis has long been established as one of the most powerful biochemical techniques for the comparative analysis of large protein complements (21-23). The more recent combination of advanced gel electrophoretic methods with mass spectrometry has further reinforced the central importance of gel electrophoretic techniques for analytical protein chemistry (24-26). The unprecedented advancements of mass spectrome...

2D-DIGE proteomic analysis of vastus lateralis from COPD patients with low and normal fat free mass index and healthy controls

“…HSPB1 participates in the regulation of apoptosis, protects the cell against oxidative stress, and is involved in the regulation of the cytoskeleton [49]. In turn HSPB1 has been related to better fatigue resistance [50] and has been shown to be up-regulated in animal models of diabetes-related muscle weakness [51]. Moreover, it has been reported that the chaperone effect of CRYAB on the cytoskeleton in relation to tubulin/microtubule, is a key mechanism for muscle adaptation, muscle differentiation and protection from atrophy [52].…”

Proteomics of Skeletal Muscle: Focus on Insulin Resistance and Exercise Biology

“…Over the last decades, several research groups have performed skeletal muscle proteome analysis from diabetic patients and rodent models of diabetes [68,69,70,71]. These pioneering studies have already begun to advance our understanding of skeletal muscle insulin resistance.…”

“…Another study that involved proteome analysis of lean, obese and Type 2 diabetic skeletal muscles revealed that the insulin-resistance muscle bears reduced expression of mitochondrial and structural proteins [72]. These abnormalities were also observed in skeletal muscle from rodent model of Type 2 diabetes [71]. Using two-dimensional electrophoresis combined with mass spectrometry, Choi et al demonstrated gender specific differences in rodent model for Type 2 diabetes [73].…”

Pathobiochemical Changes in Diabetic Skeletal Muscle as Revealed by Mass-Spectrometry-Based Proteomics

“…The proteomic profiling of crude muscle extracts and subcelluar fractions from the Goto-Kakizaki rat has employed different protein staining methods for the visualization of protein spots in two-dimensional gels, that is, colloidal Coomassie Blue [82], fluorescent RuBPs [82, 83], and fluorescent CyDyes for difference in-gel electrophoretic analysis [83, 84]. The different labeling techniques clearly showed variations in their dynamic visualization range, so proteomic findings from the 3 dyes used for densitometric scanning could be combined for a more complete coverage of changes within the diabetic muscle proteome.…”

“…The different labeling techniques clearly showed variations in their dynamic visualization range, so proteomic findings from the 3 dyes used for densitometric scanning could be combined for a more complete coverage of changes within the diabetic muscle proteome. Colloidal Coomassie staining, routinely used for reliable protein detection following gel electrophoresis [114, 115], and fluorescence difference in-gel electrophoresis, which represents one of the most powerful biochemical tools for the comparative analysis of protein complements [116–118], resulted in the detection of 929 [82] and 1734 [83] protein spots, respectively. The silver staining technique and protein labeling with the fluorescent RuBPs dye (ruthenium bathophenanthroline disulfonate) [119] were employed as independent visualization methods for the verification of key findings in altered protein expression patterns [82, 83].…”