Insulin resistance in skeletal muscle tissues and diabetes-related muscle weakness are serious pathophysiological problems of increasing medical importance. In order to determine global changes in the protein complement of contractile tissues due to diabetes mellitus, mass-spectrometry-based proteomics has been applied to the investigation of diabetic muscle. This review summarizes the findings from recent proteomic surveys of muscle preparations from patients and established animal models of type 2 diabetes. The potential impact of novel biomarkers of diabetes, such as metabolic enzymes and molecular chaperones, is critically examined. Disease-specific signature molecules may be useful for increasing our understanding of the molecular and cellular mechanisms of insulin resistance and possibly identify new therapeutic options that counteract diabetic abnormalities in peripheral organ systems. Importantly, the biomedical establishment of biomarkers promises to accelerate the development of improved diagnostic procedures for characterizing individual stages of diabetic disease progression, including the early detection of prediabetic complications.
The incidence of diabetes mellitus has reached epidemic proportions and is one of the great biomedical challenges of the 21st century [
With respect to muscle tissues, diabetes affects primarily cardiac cells [
The fact that drastic lifestyle modifications, such as an altered diet and increased physical activity [
Modern proteomics attempts to identify, catalogue, and characterize the entire protein constellation of specific cell types, tissues, or biological fluids using large-scale separation methods, such as two-dimensional gel electrophoresis or liquid chromatography, and high-throughput identification approaches focusing mostly on mass spectrometric techniques [
However, aside from these technical problems, several physiological, cell biological, and biochemical parameters make the proteomic analysis of skeletal muscle an especially difficult task. In order to cover as many proteins as possible in proteomic studies, the initial extraction process of tissue samples is crucial. Since muscle fibres are rigid structures that are embedded in a complex system of connective tissue, these biological properties make it an extremely tough tissue to homogenize. Approximately half of the muscle protein content consists of the contractile apparatus with its various isoforms of myosin light chains, myosin heavy chains, troponins, tropomyosins, and associated proteins. Due to the high density of these proteins, a certain degree of cross-contamination of protein spots cannot be avoided during two-dimensional gel electrophoresis. In addition, a large number of very high-molecular-mass proteins exist in skeletal muscle fibres, with some of them being susceptible to proteolytic degradation [
With respect to the under-representation of integral proteins in most proteomic investigations of crude tissue extracts, it is important to stress that skeletal muscles exhibit a highly elaborate membrane system, consisting of the sarcolemma, transverse tubules, triad junctions, the sarcoplasmic reticulum, and abundant organelles such as mitochondria. Since mature skeletal muscle tissues are heterogeneous in composition, it has to be taken into account that total extracts contain muscle fibre proteins and components derived from nerves, capillaries, basal lamina, and satellite cells [
The cellular and functional integrity of muscles depends heavily on constant loading and proper activity levels. On the one hand, chronic electrostimulation or physical training triggers profound changes in the protein expression pattern of muscles [
Proteomic profiling strategy to evaluate the effect of type 2 diabetes on skeletal muscle tissues. Shown is a flowchart of the proteomic workflow to identify new protein factors involved in the molecular pathogenesis of insulin resistance, abnormal cellular signaling, and contractile weakness in diabetic skeletal muscle tissues. Listed are the various analytical steps of mass-spectrometry-based proteomic surveys of muscle samples, such as sample preparation, protein extraction, gel electrophoretic separation, densitometric scanning, mass spectrometric identification of novel biomarkers, and the independent validation of proteomic data by immunoblotting, enzyme assays, and immunofluorescence microscopy.
Over the last few years, mass-spectrometry-based proteomics has been established as a crucial analytical tool for biomarker discovery in the heterogeneous pathology of diabetes [
List of major proteomic profiling studies of skeletal muscle tissues from prediabetic and diabetic patients or animal models of type 2 diabetes.
Proteomic study | New potential biomarkers | References |
---|---|---|
Proteomic analysis of human vastus lateralis muscle from type 2 diabetic subjects | Select number of potential markers of diabetes; abnormal phosphorylation of ATP synthase; elevated levels of stress proteins | [ |
Proteomic profiling of skeletal muscle biopsy material from patients suffering from type 2 diabetes | Establishment of a large number of muscle-associated signature molecules of type 2 diabetes; changed abundance in various mitochondrial proteins; abnormal phosphorylation of ATP synthase beta-subunit | [ |
Proteomic profiling of rectus abdominus tissue from obese and morbidly obese women with potential prediabetic side effects | Increased levels of key glycolytic enzymes suggests an obesity-related compensatory glycolytic shift in muscle metabolism | [ |
Proteomic analysis of gastrocnemius muscle from the nonobese Goto-Kakizaki rat model of type 2 diabetes | Changes in muscle proteins associated with the contractile apparatus, the antioxidant defense system, detoxification mechanisms, the cellular stress response, glucose metabolism, fatty acid utilization, nucleotide metabolism, and amino acid metabolism | [ |
Subproteomic survey of the muscle mitochondria-enriched fraction from the non-obese Goto-Kakizaki rat model of type 2 diabetes | Differential expression of various mitochondrial marker proteins agrees with the idea that mitochondrial dysregulation plays a role in type 2 diabetes | [ |
Comparative proteomic study of fenofibrate-dependent protein expression in skeletal muscle from type 2 diabetic OLETF rats | Increased levels of the functionally unknown muscle protein C11orf59 in a fenofibrate-dependent manner in diabetic rat muscle | [ |
Proteomic analysis of obese and potentially prediabetic animal models | Generally perturbed protein expression levels in obese muscles, affecting especially metabolic enzymes | [ |
The occurrence of diabetes and cardiovascular disease has reached epidemic rates and there is a clear association of these disorders with obesity-related complications. Impaired insulin-mediated glucose uptake in skeletal muscle fibres is related to high levels of circulating free fatty acids, an increased intramyocellular lipid content, diminished mitochondrial functioning and an overall weakened metabolic flexibility [
Two-dimensional gel electrophoresis in combination with MALDI-ToF MS analysis identified a small number of potential muscle-associated biomarkers for type 2 diabetes [
Recently, a more comprehensive proteomic study of diabetic muscle was carried out by one-dimensional gel electrophoresis and HPLC-ESI-MS/MS analysis. Hwang et al. [
Since abnormal skeletal muscle functioning is a major feature of type 2 diabetes, it can be expected that distinct changes occur in the muscle proteome during the development of insulin resistance and diabetes. Established animal models with key symptoms of diabetes are, therefore, an ideal experimental tool to study global alterations in the abundance, isoform expression pattern, and/or posttranslational modifications of muscle proteins.
It is clearly evident from the large number of publications on animal model research covering diabetes mellitus that studying laboratory animals had a significant impact on understanding diabetic pathogenesis, testing novel drugs, and evaluating toxic side effects [
With the emergence of genomics and proteomics, the search for novel genes and protein factors involved in the pathophysiology of diabetes has been decisively improved. The proteomic profiling of animal disease models is highly suitable for the identification of new biomarker signatures, for the development of superior diagnostics, and for the evaluation of novel treatment options to counteract diabetes-related side effects in skeletal muscle tissues. As reviewed by Resjö et al. [
As discussed in general by Doran et al. [
Since mass-spectrometry-based proteomic studies of potential changes in the skeletal muscle proteome have mostly focused on the Goto-Kakizaki rat [
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 [
The gel electrophoresis-based proteomic survey of normal versus nonobese diabetic rats showed an altered expression profile for muscle-associated proteins that are involved in lipolytic catabolism, the citric acid cycle, oxidative phosphorylation, glycolysis, nucleotide metabolism, carbon dioxide removal, oxygen transportation, amino acid catabolism, and cellular detoxification mechanisms [
The proteomic analysis of nonobese diabetic muscle revealed a differential effect on glycolytic enzymes. The concentration of enolase appears to be decreased, while aldolase and phosphoglucomutase showed elevated levels in muscle preparations from the Goto-Kakizaki rat [
Interestingly, increased levels of adenylate kinase isoform AK1 and monoglyceride lipase were shown to exist in diabetic gastrocnemius muscle from the Goto-Kakizaki rat [
In addition to newly detected changes in metabolic enzymes, the fluorescence difference in-gel electrophoretic analysis of diabetic muscle revealed significantly increased levels of the small stress proteins
Since mitochondrial dysfunction in skeletal muscle has been implicated in the progression of diabetic pathology [
Besides proteomic surveys of skeletal muscles from nonobese type 2 diabetic animal models, mass spectrometric methodology was also applied to the evaluation of fenofibrate-fed type 2 diabetic OLETF rats [
The main findings of proteomic surveys of diabetic muscle preparations are summarized in Figure
Overview of changes in diabetic skeletal muscle as revealed by mass-spectrometry-based proteomics. Shown is a diagram of a skeletal muscle fibre outlining sarcolemmal proteins involved in insulin signaling and glucose uptake, as well as major cellular mechanisms affected in diabetic muscle tissues. Listed are established diabetes-related impairments of insulin receptor (IR) phosphorylation and abnormal signaling and transporter recruitment involving IRS1, p85, p110, Akt, PI-3-kinase, and glucose transporter isoform GLUT4. Proteomic profiling of muscle tissues from patients and established animal models of type 2 diabetes have revealed changes in components downstream from these plasmalemmal signaling cascades, including proteins involved in mitochondrial metabolism, glycolysis, contractile apparatus, detoxification mechanisms, cellular stress response, glucose metabolism, fatty acid utilization, nucleotide metabolism, and amino acid metabolism.
Cardiovascular diseases, obesity-related disorders affecting multiple organ systems, the metabolic syndrome, and diabetes mellitus affect hundreds of millions of patients worldwide. These epidemiological facts warrant detailed biomedical studies into the molecular and cellular mechanisms of these crippling diseases. It is hoped that a better understanding of the molecular pathogenesis of common human disorders will help in the identification of novel therapeutic targets. In the long term, this should translate into the development of superior pharmacological approaches and better clinical treatment options. The etiology of type 2 diabetes appears to be influenced by both genetic and environmental factors, making it a great challenge to determine the causative interplay of genetic susceptibilities, pathophysiological aspects, and external features. A change in lifestyle, such as increased physical activity and improved nutritional regimes, can reverse some of the diabetic symptoms such as insulin resistance. Diabetes is clearly a heterogeneous group of disorders and can cause extremely serious side effects, such as cardiomyopathy, stroke, lower limb amputation, kidney failure, blindness, and skeletal muscular weakness. In the older population, type 2 diabetes can result in significantly decreased muscle strength and may thus contribute to the development of physical disability in the elderly. Sarcopenia of old age, the natural age-related decline in skeletal muscle mass and strength, can be exacerbated by the negative influence of the diabetic phenotype on muscle metabolism. It is, therefore, of the uttermost urgency to devise novel approaches to counteract diabetes-related muscle weakness and abnormal hormone signaling in of the most crucial insulin-dependent organs for glucose disposal. Over the last few years, mass-spectrometry-based proteomics has been applied to diabetes research as an unbiased analytical tool for the global determination of abnormal protein expression patterns in diabetic muscle tissues. The proteomic profiling of both biopsy samples from diabetic subjects and muscle extracts from established animal models has resulted in the identification of a variety of new potential markers of diabetes. The biomarker signature of muscle-related changes due to diabetes includes components that are associated with the actomyosin apparatus, the cellular stress response, glucose utilization, fatty acid oxidation, and other metabolic pathways. Interestingly, a very recent proteomic study of human diabetic muscle clearly supports the idea of oxidative-to-glycolytic shifts in energy metabolism of the diabetic phenotype [
This work was supported by a project grant from the Irish Health Research Board (HRB-RP/2005/3) and equipment grants from the Higher Education Authority and Science Foundation Ireland.