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

Cultured human myotubes offer a unique model to distinguish between primary and environmental factors in the aetiology of insulin resistance in human skeletal muscle. The objective of this review was to summarize our and other group studies on insulin resistance in human myotubes established from lean, obese and type 2 diabetes (T2D) subjects. Overall, studies of human myotubes established from lean, obese and T2D subjects clearly show that part of the diabetic phenotype observed in vivo is preserved in diabetic myotubes.

Carnitine acetyltransferase: A new player in skeletal muscle insulin resistance?

Carnitine acetyltransferase (CRAT) deficiency has previously been shown to result in muscle insulin resistance due to accumulation of long-chain acylcarnitines. However, differences in the acylcarnitine profile and/or changes in gene expression and protein abundance of CRAT in myotubes obtained from obese patients with type 2 diabetes mellitus (T2DM) and glucose-tolerant obese and lean controls remain unclear. The objective of the study was to examine whether myotubes from obese patients with T2DM express differences in gene expression and protein abundance of CRAT and in acylcarnitine species pre-cultured under glucose and insulin concentrations similar to those observed in healthy individuals in the over-night fasted, resting state.

Primary defects in lipolysis and insulin action in skeletal muscle cells from type 2 diabetic individuals.

A decrease in skeletal muscle lipolysis and hormone sensitive-lipase (HSL) expression has been linked to insulin resistance in obesity. The purpose of this study was to identify potential intrinsic defects in lipid turnover and lipolysis in myotubes established from obese and type 2 diabetic subjects. Lipid trafficking and lipolysis were measured by pulse-chase assay with radiolabeled substrates in myotubes from non-obese/non-diabetic (lean), obese/non-diabetic (obese) and obese/diabetic (T2D) subjects.

Testosterone treatment increases androgen receptor and aromatase gene expression in myotubes from patients with PCOS and controls, but does not induce insulin resistance.

Polycystic ovary syndrome (PCOS) is associated with insulin resistance and increased risk of type 2 diabetes. Skeletal muscle is the major site of insulin mediated glucose disposal and the skeletal muscle tissue is capable to synthesize, convert and degrade androgens. Insulin sensitivity is conserved in cultured myotubes (in vitro) from patients with PCOS, but the effect of testosterone on this insulin sensitivity is unknown.

Genetic alterations within the DENND1A gene in patients with polycystic ovary syndrome (PCOS).

Polycystic ovary syndrome (PCOS), the most common endocrine disease among premenopausal women, is caused by both genes and environment. We and others previously reported association between single nucleotide polymorphisms (SNPs) in the DENND1A gene and PCOS. We therefore sequenced the DENND1A gene in white patients with PCOS to identify possible alterations that may be implicated in the PCOS pathogenesis.

FA1 Induces Pro-Inflammatory and Anti-Adipogenic Pathways/Markers in Human Myotubes Established from Lean, Obese, and Type 2 Diabetic Subjects but Not Insulin Resistance.

Aims: Delta like 1/fetal antigen 1 (Dlk1/FA1) is a protein secreted by hormone producing cells in adult human and mice that is known to inhibit adipogenesis. Recent studies demonstrated the role of Dlk1/FA1 in inducing insulin resistance in mice. To investigate the involvement of circulating Dlk1/FA1 in insulin resistance and type 2 diabetes in human subjects, we studied the effects of chronic FA1 on the intermediary metabolism in myotubes established from lean, obese, and type 2 diabetic (T2D) subjects.

Free carnitine and acylcarnitines in obese patients with polycystic ovary syndrome and effects of pioglitazone treatment.

Objective: To determine fasting and insulin-stimulated levels of carnitine precursors, total and free carnitine, and acylcarnitines, and evaluate the impact of pioglitazone treatment in obese patients with polycystic ovary syndrome (PCOS).

Design: The present study is a secondary analysis of a previously published case-control study, followed by a double-blind randomized clinical trial.

Setting: Academic tertiary care medical center.

A primary reduced TCA flux governs substrate oxidation in T2D skeletal muscle.

Our current knowledge on substrate oxidation in skeletal muscle in relation to insulin resistance and type 2 diabetes (T2D) originate mainly from in vivo studies. The oxidative capacity of skeletal muscle is highly influenced by physical activity, ageing, hormonal status, and fiber type composition, rendering it difficult to determine the contribution of heritable factors to the alteration in oxidative metabolism. Cultured human myotubes offer a unique model to distinguish between primary and environmental factors in the etiology of insulin resistance.

Palmitic acid follows a different metabolic pathway than oleic acid in human skeletal muscle cells; lower lipolysis rate despite an increased level of adipose triglyceride lipase.

Development of insulin resistance is positively associated with dietary saturated fatty acids and negatively associated with monounsaturated fatty acids. To clarify aspects of this difference we have compared the metabolism of oleic (OA, monounsaturated) and palmitic acids (PA, saturated) in human myotubes. Human myotubes were treated with 100μM OA or PA and the metabolism of [(14)C]-labeled fatty acid was studied.

Mesenchymal stromal cell phenotype is not influenced by confluence during culture expansion.

Background: Accumulating preclinical and clinical evidence indicates that human mesenchymal stromal cells (MSCs) are good candidates for cell therapy. For clinical applications of MSCs extensive in vitro expansion is required to obtain an adequate number of cells. It is evident that the pursuit for cell quantity must not affect quality, but it is also a fact that in vitro culture conditions affect MSC phenotype.

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

The diabetic phenotype is complex, requiring elucidation of key initiating defects. Diabetic myotubes express a primary reduced tricarboxylic acid (TCA) cycle flux but at present it is unclear in which part of the TCA cycle the defect is localised. In order to localise the defect we studied ATP production in isolated mitochondria from substrates entering the TCA cycle at various points.

Association of polycystic ovary syndrome susceptibility single nucleotide polymorphism rs2479106 and PCOS in Caucasian patients with PCOS or hirsutism as referral diagnosis.

Context: Polycystic ovary syndrome (PCOS) is the most common endocrine disease among premenopausal women. A recent study found association between three single nucleotide polymorphisms (SNPs) and PCOS in a cohort of Han Chinese women.

Objective: To investigate the association between rs13405728 (LHCGR gene), rs13429458 (THADA gene) and rs2479106 (DENND1A gene), PCOS, hirsutism and metabolic and hormonal parameters in a well characterized cohort of Caucasian patients of Danish descendant with PCOS or hirsutism.

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.

Uncoupling of sarcoplasmic reticulum Ca²⁺-ATPase by N-arachidonoyl dopamine. Members of the endocannabinoid family as thermogenic drugs.

Background And Purpose: The sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) plays a role in thermogenesis. The exogenous compound capsaicin increased SERCA-mediated ATP hydrolysis not coupled to Ca²⁺ transport. Here, we have sought to identify endogenous compounds that may function as SERCA uncoupling agents.

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.

Expression of perilipins in human skeletal muscle in vitro and in vivo in relation to diet, exercise and energy balance.

The perilipin proteins enclose intracellular lipid droplets. We describe the mRNA expression of the five perilipins in human skeletal muscle in relation to fatty acid supply, exercise and energy balance. We observed that all perilipins were expressed in skeletal muscle biopsies with the highest mRNA levels of perilipin 2, 4 and 5.

Hydrogen peroxide production is not primarily increased in human myotubes established from type 2 diabetic subjects.

Context: Increased oxidative stress and mitochondrial dysfunction have been implicated in the development of insulin resistance in type 2 diabetes. To date, it is unknown whether increased mitochondrial reactive oxygen species (ROS) production in skeletal muscle from patients with type 2 diabetes is primarily increased or a secondary adaptation to environmental, lifestyle, and hormonal factors.

Objective: This study investigates whether ROS production is primarily increased in isolated diabetic myotubes.

Characterization of human myotubes from type 2 diabetic and nondiabetic subjects using complementary quantitative mass spectrometric methods.

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.

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.

Human myotubes from myoblast cultures undergoing senescence exhibit defects in glucose and lipid metabolism.

Adult stem cells are known to have a finite replication potential. Muscle biopsy-derived human satellite cells (SCs) were grown at different passages and differentiated to human myotubes in culture to analyze the functional state of various carbohydrate and lipid metabolic pathways. As the proliferative potential of myoblasts decreased dramatically with passage number, a number of cellular functions were altered: the capacity of myoblasts to fuse and differentiate into myotubes was reduced, and metabolic processes in myotubes such as glucose uptake, glycogen synthesis, glucose oxidation and fatty acid β-oxidation became gradually impaired.

Insulin resistance is not conserved in myotubes established from women with PCOS.

Background: Polycystic ovary syndrome (PCOS) is the most common endocrine disorder among premenopausal women, who often develop insulin resistance. We tested the hypothesis that insulin resistance in skeletal muscle of patients with polycystic ovary syndrome (PCOS) is an intrinsic defect, by investigating the metabolic characteristics and gene expression of in vitro differentiated myotubes established from well characterized PCOS subjects.

Methods: Using radiotracer techniques, RT-PCR and enzyme kinetic analysis we examined myotubes established from PCOS subjects with or without pioglitazone treatment, versus healthy control subjects who had been extensively metabolically characterized in vivo.