Characterization of Human Myotubes From Type 2 Diabetic and Nondiabetic Subjects Using Complementary Quantitative Mass Spectrometric Methods

Abstract

Skeletal muscle is a key tissue site of insulin resistance in type 2 diabetes. Human myotubes are primary skeletal muscle cells displaying both morphological and biochemical characteristics of mature skeletal muscle and the diabetic phenotype is conserved in myotubes derived from subjects with type 2 diabetes. Several abnormalities have been identified in skeletal muscle from type 2 diabetic subjects, however, the exact molecular mechanisms leading to the diabetic phenotype has still not been found. Here we present a large-scale study in which we combine a quantitative proteomic discovery strategy using isobaric peptide tags for relative and absolute quantification (iTRAQ) and a label-free study with a targeted quantitative proteomic approach using selected reaction monitoring to identify, quantify, and validate changes in protein abundance among human myotubes obtained from nondiabetic lean, nondiabetic obese, and type 2 diabetic subjects, respectively. Using an optimized protein precipitation protocol, a total of 2832 unique proteins were identified and quantified using the iTRAQ strategy.Despite a clear diabetic phenotype in diabetic myotubes, the majority of the proteins identified in this study did not exhibit significant abundance changes across the patient groups. Proteins from all major pathways known to be important in type 2 diabetic subjects were wellcharacterized in this study. This included pathways like the trichloroacetic acid (TCA) cycle, lipid oxidation, oxidative phosphorylation, the glycolytic pathway, and glycogen metabolism from which all but two enzymes were found in the present study. None of these enzymes were found to be regulated at the level of protein expression or degradation supporting the hypothesis that these pathways are regulated at the level of post-translational modification. Twelve proteins were, however, differentially expressed among the three different groups. Thirty-six proteins were chosen for further analysis and validation using selected reaction monitoring based on the regulation identified in the iTRAQ discovery study. The abundance of adenosine deaminase was considerably downregulated in diabetic myotubes and as the protein binds propyl dipeptidase (DPP-IV), we speculate whether the reduced binding of adenosine deaminase to DPP-IV may contribute to the diabetic phenotype in vivo by leading to a higher level of free DPP-IV to bind and inactivate the anti-diabetic hormones, glucagon-like peptide-1 and glu-

The diabetic phenotype is preserved in myotubes established from type 2 diabetic subjects: a critical appraisal

“…Slow fibres are more oxidative and insulin sensitive than fast fibres . Our proteomic‐based observation that fast myosin (MHCIIa) expression is upregulated in diabetic myotubes is consistent with the idea that primary insulin resistance in myotubes may also be associated with loss of slow fibres, although we did not confirm a concomitant increase in slow myosin expression in lean myotubes. Currently, it is unknown to what extent fibres co‐expressing developmental and neonatal myosin represent more than a transitional stage in the development of mature muscle fibres .…”

“…. Utilizing day 8 cultures, we showed that basal glucose uptake is increased in diabetic myotubes compared to age‐matched lean myotubes ; however, GLUT1 expression did not differ between the diabetic and control myotubes .…”

Reduced TCA Flux in Diabetic Myotubes: Determined by Single Defects?

“…Augmented phosphorylation of mitochondrial aconitase was associated with a reduced TCA cycle flux, based on an increased reverse activity, while the forward reaction was normal. That PTM could be part of the explanation for a reduced TCA cycle flux in diabetic mitochondria is supported by our previous finding of a 14% reduced citrate synthase activity in diabetic myotubes [12] which cannot be explained by changes in protein expression [15]. Additional phosphoproteomic studies are required to test whether the impaired TCA cycle flux, in diabetic mitochondria, is based on posttranscriptional modifications of TCA cycle enzymes.…”

The proteomic signature of insulin-resistant human skeletal muscle reveals increased glycolytic and decreased mitochondrial enzymes

“…There have been several previous approaches to the study of the human skeletal muscle proteome [13,14] and its alterations in insulin-resistant muscle [15][16][17][18]. Two studies have used the two-dimensional gel approach [15,16].…”