In vitro myogenesis in skeletal muscle cells from type 2 diabetic and non-diabetic subjects : Comparison of metabolic properties, mitochondrial content and mass, ATP concentration and differentiation capacity

Abstract

Diabetes mellitus is a metabolic disorder resulting from defective insulin secretion, impaired insulin action or both. The consequence of this is chronic hyperglycemia with disturbance of carbohydrate, fat and protein metabolism. The prevalence of diabetes, especially type 2 diabetes (T2D), is rapidly increasing throughout the world, parallel to this; we also see an increase in the obesity epidemic. The mechanisms underlying the metabolic defects of T2D and obesity are still largely unknown, but there are several studies indicating a strong association between insulin resistance, increased lipid accumulation and impaired fat oxidation in insulin sensitive tissues. Muscular mitochondrial dysfunction has been proposed as an underlying cause to the metabolic disorders, due to observations of reduced mitochondrial oxidative capacity and number in skeletal muscle of insulin resistant and T2D individuals.

The purpose of this study was to determine possible differences in metabolic properties between skeletal muscle cells from T2D and non-diabetic (N-D) subjects. Secondly, it was of interest to assess mitochondrial measures (i.e. mitochondrial content and mass, ATP concentration and OXPHOS content) and MHC content (differentiation marker) in muscle cells from the two donor groups. This was done in order to test the hypothesis of whether a possible impairment in metabolic properties in skeletal muscle of T2D individuals, could be caused by a lower mitochondrial activity/number or by an impaired differentiation capacity of myoblasts.

Muscle biopsies from 10 obese, T2D patients and 10 lean, healthy control subjects were obtained at baseline (pre-intervention). Metabolic properties of myoblasts and myotubes, from the two donor groups were evaluated by measuring rates of oxygen consumption (OCR) and extracellular acidification (ECAR) in an XF24 Analyzer. Mitochondrial content and mass were measured by rt-PCR and mitotracker, respectively. ATP concentration was determined by the EnzyLight ATP assay kit, while OXPHOS and MHC content were determined by Western immunoblotting.

Results showed that the skeletal muscle cells, established from obese, T2D subjects, had a substantially reduction in OCR and ECAR compared with cells from the control group. Measurements of mitochondrial mass and content, ATP concentration and OXPHOS content were not different between cells from the two groups. The MHC content was significantly lower in myotubes from T2D individuals compared to cells from the control group. These results may indicate an impaired mitochondrial function in T2D cells, rather than a lower mitochondrial number. However, the reduction in OCR and ECAR seen in T2D subjects could simply be caused by less differentiated myotubes in this group.