Impaired TCA cycle flux in mitochondria in skeletal muscle from type 2 diabetic subjects: marker or maker of the diabetic phenotype?

The diabetic phenotype is complex, requiring elucidation of key initiating defects. Recent research has shown that diabetic myotubes express a primary reduced tricarboxylic acid (TCA) cycle flux. A reduced TCA cycle flux has also been shown both in insulin resistant offspring of T2D patients and exercising T2D patients in vivo.

Cultured senescent myoblasts derived from human vastus lateralis exhibit normal mitochondrial ATP synthesis capacities with correlating concomitant ROS production while whole cell ATP production is decreased.

The free radical theory of aging says that increased oxidative stress and mitochondrial dysfunction are associated with old age. In the present study we have investigated the effects of cellular senescence on muscle energetic by comparing mitochondrial content and function in cultured muscle satellite cells at early and late passage numbers. We show that cultured muscle satellite cells undergoing senescence express a reduced mitochondrial mass, decreased whole cell ATP level, normal to increased mitochondrial ATP production under ATP utilization, increased mitochondrial membrane potential and increased superoxide/mitochondrial mass and hydrogen peroxide/mitochondrial mass ratios.

Intact primary mitochondrial function in myotubes established from women with PCOS.

Context: Polycystic ovary syndrome (PCOS) affects 5-8% of fertile women and is often accompanied by insulin resistance, leading to increased risk of developing type 2 diabetes. Skeletal muscle from insulin-resistant PCOS subjects display reduced expression of nuclear encoded genes involved in mitochondrial oxidative metabolism.

Objective: We aimed to investigate whether there was a primary mitochondrial dysfunction or difference in mitochondria content that might contribute to the in vivo detected insulin resistance.

The dynamic equilibrium between ATP synthesis and ATP consumption is lower in isolated mitochondria from myotubes established from type 2 diabetic subjects compared to lean control.

Although, most studies of human skeletal muscle in vivo have reported the co-existence of impaired insulin sensitivity and reduced expression of oxidative phosphorylation genes, there is so far no clear evidence for whether the intrinsic ATP synthesis is primarily decreased or not in the mitochondria of diabetic skeletal muscle from subjects with type 2 diabetes. ATP synthesis was measured on mitochondria isolated from cultured myotubes established from lean (11/9), obese (9/11) and subjects with type 2 diabetes (9/11) (female/male, n=20 in each group), precultured under normophysiological conditions in order to verify intrinsic impairments. To resemble dynamic equilibrium present in whole cells between ATP synthesis and utilization, ATP was measured in the presence of an ATP consuming enzyme, hexokinase, under steady state.

ATP synthesis is impaired in isolated mitochondria from myotubes established from type 2 diabetic subjects.

To date, it is unknown whether mitochondrial dysfunction in skeletal muscle from subjects with type 2 diabetes is based on primarily reduced mitochondrial mass and/or a primarily decreased mitochondrial ATP synthesis. Mitochondrial mass were determined in myotubes established from eight lean, eight obese and eight subjects with type 2 diabetes precultured under normophysiological conditions. Furthermore, mitochondria were isolated and ATP production was measured by luminescence at baseline and during acute insulin stimulation with or without concomitant ATP utilization by hexokinase.

Pyruvate carboxylase is expressed in human skeletal muscle.

Pyruvate carboxylase (PC) is a mitochondrial enzyme that catalyses the carboxylation of pyruvate to oxaloacetate thereby allowing supplementation of citric acid cycle intermediates. The presence of PC in skeletal muscle is controversial. We report here, that PC protein is easily detectable by streptavidin blot and describe the presence of considerable amounts of PC in cultured human myotubes and in human muscle tissue.

The intracellular domain of teneurin-1 interacts with MBD1 and CAP/ponsin resulting in subcellular codistribution and translocation to the nuclear matrix.

Teneurin-1 is a type II transmembrane protein expressed in neurons of the developing and adult central nervous system. To investigate the intracellular signaling of teneurin-1, we searched for proteins interacting with its intracellular domain. One of the proteins identified is the c-Cbl-associated protein CAP/ponsin, an adaptor protein containing SH3 domains.