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. Results Myotubes established from PCOS and matched control subjects comprehensively expressed all insulin-sensitive biomarkers; glucose uptake and oxidation, glycogen synthesis and lipid uptake. There were no significant differences between groups either at baseline or during acute insulin stimulation, although in vivo skeletal muscle was insulin resistant. In particular, we found no evidence for defects in insulin-stimulated glycogen synthase activity between groups. Myotubes established from PCOS patients with or without pioglitazone treatment also showed no significant differences between groups, neither at baseline nor during acute insulin stimulation, although in vivo pioglitazone treatment significantly improved insulin sensitivity. Consistently, the myotube cultures failed to show differences in mRNA levels of genes previously demonstrated to differ in PCOS patients with or without pioglitazone treatment (PLEK, SLC22A16, and TTBK).

Conclusion

These results suggest that the mechanisms governing insulin resistance in skeletal muscle of PCOS patients in vivo are not primary, but rather adaptive.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00145340","term_id":"NCT00145340"}}NCT00145340     

Insulin-sensitizing effects of thiazolidinediones are not linked to adiponectin receptor expression in human fat or muscle

Circulating adiponectin levels are increased by the thiazolidinedione (TZD) class of PPARgamma agonists in concert with their insulin-sensitizing effects. Two receptors for adiponectin (AdipoR1 and AdipoR2) are widely expressed in many tissues, but their physiological significance to human insulin resistance remains to be fully elucidated. We examined the expression patterns of AdipoR1 and AdipoR2 in fat and skeletal muscle of human subjects, their relationship to insulin action, and whether they are regulated by TZDs. Expression patterns of both AdipoRs were similar in subcutaneous and omental fat depots, with higher expression in adipocytes than in stromal cells and macrophages. To determine the effects of TZDs on AdipoR expression, subcutaneous fat and quadriceps muscle were biopsied in 14 insulin-resistant subjects with type 2 diabetes mellitus after 45 mg pioglitazone or placebo for 21 days. This duration of pioglitazone improved insulin's suppression of glucose production by 41% and enhanced stimulation of glucose uptake by 27% in concert with increased gene expression and plasma levels of adiponectin. Pioglitazone did not affect AdipoR expression in muscle, whole fat, or cellular adipose fractions, and receptor expression did not correlate with baseline or TZD-enhanced insulin action. In summary, both adiponectin receptors are expressed in cellular fractions of human fat, particularly adipocytes. TZD administration for sufficient duration to improve insulin action and increase adiponectin levels did not affect expression of AdipoR1 or AdipoR2. Although TZDs probably exert many of their effects via adiponectin, changes in these receptors do not appear to be necessary for their insulin-sensitizing effects.     

Glutathione peroxidase 3 mediates the antioxidant effect of peroxisome proliferator-activated receptor gamma in human skeletal muscle cells

Oxidative stress plays an important role in the pathogenesis of insulin resistance and type 2 diabetes mellitus and in diabetic vascular complications. Thiazolidinediones (TZDs), a class of peroxisome proliferator-activated receptor gamma (PPARgamma) agonists, improve insulin sensitivity and are currently used for the treatment of type 2 diabetes mellitus. Here, we show that TZD prevents oxidative stress-induced insulin resistance in human skeletal muscle cells, as indicated by the increase in insulin-stimulated glucose uptake and insulin signaling. Importantly, TZD-mediated activation of PPARgamma induces gene expression of glutathione peroxidase 3 (GPx3), which reduces extracellular H(2)O(2) levels causing insulin resistance in skeletal muscle cells. Inhibition of GPx3 expression prevents the antioxidant effects of TZDs on insulin action in oxidative stress-induced insulin-resistant cells, suggesting that GPx3 is required for the regulation of PPARgamma-mediated antioxidant effects. Furthermore, reduced plasma GPx3 levels were found in patients with type 2 diabetes mellitus and in db/db/DIO mice. Collectively, these results suggest that the antioxidant effect of PPARgamma is exclusively mediated by GPx3 and further imply that GPx3 may be a therapeutic target for insulin resistance and diabetes mellitus.     

Effects of pioglitazone and metformin on beta-cell function in nondiabetic subjects at high risk for type 2 diabetes

Thiazolidinediones (TZDs) and metformin decreased the incidence of diabetes in subjects at risk for developing diabetes and improved peripheral or hepatic insulin sensitivity, respectively. Whether they also directly improved beta-cell function is not clear. In vitro studies showed improved beta-cell function in response to TZDs and metformin; however, the effects of TZDs or metformin on beta-cell function in humans are still uncertain. We hypothesized that both TZDs and metformin directly affect beta-cell function. We evaluated beta-cell function and insulin sensitivity (S(I)) in subjects with impaired glucose tolerance or a history of gestational diabetes using oral and intravenous glucose tolerance tests in addition to the glucose-potentiated arginine stimulation test. In contrast to metformin, pioglitazone improved S(I), glucose tolerance, and insulin-independent glucose disposal [glucose effectiveness (S(G))]. Neither pioglitazone nor metformin significantly improved beta-cell compensation for insulin resistance [disposition index (DI)], but the change in DI significantly correlated with baseline S(I). Insulin secretion in response to arginine at maximally potentiating glucose levels (AIR(max)) tended to increase after metformin and to decrease after pioglitazone; however, when adjusted for S(I), the changes were not significant. Our results demonstrate that, in nondiabetic subjects at risk for diabetes, pioglitazone, but not metformin, significantly improved glucose tolerance by improving S(I) and S(G). We did not find any evidence that either pioglitazone or metformin improved beta-cell function. Improved beta-cell compensation was observed primarily in the subgroup of subjects that had the lowest S(I) at baseline.     

Enhanced liver but not muscle OXPHOS in diabetes and reduced glucose output by complex I inhibition

Mitochondrial function is critical in energy metabolism. To fully capture how the mitochondrial function changes in metabolic disorders, we investigated mitochondrial function in liver and muscle of animal models mimicking different types and stages of diabetes. Type 1 diabetic mice were induced by streptozotocin (STZ) injection. The db/db mice were used as type 2 diabetic model. High-fat diet-induced obese mice represented pre-diabetic stage of type 2 diabetes. Oxidative phosphorylation (OXPHOS) of isolated mitochondria was measured with Clark-type oxygen electrode. Both in early and late stages of type 1 diabetes, liver mitochondrial OXPHOS increased markedly with complex IV-dependent OXPHOS being the most prominent. However, ATP, ADP and AMP contents in the tissue did not change. In pre-diabetes and early stage of type 2 diabetes, liver mitochondrial complex I and II-dependent OXPHOS increased greatly then declined to almost normal at late stage of type 2 diabetes, among which alteration of complex I-dependent OXPHOS was the most significant. In contrast, muscle mitochondrial OXPHOS in HFD, early-stage type 1 and 2 diabetic mice, did not change. In vitro, among inhibitors to each complex, only complex I inhibitor rotenone decreased glucose output in primary hepatocytes without cytotoxicity both in the absence and presence of oleic acid (OA). Rotenone affected cellular energy state and had no effects on cellular and mitochondrial reactive oxygen species production. Taken together, the mitochondrial OXPHOS of liver but not muscle increased in obesity and diabetes, and only complex I inhibition may ameliorate hyperglycaemia via lowering hepatic glucose production.     

Not only insulin stimulates mitochondriogenesis in muscle cells, but mitochondria are also essential for insulin-mediated myogenesis

Viability and myogenesis from C2C12 muscle cells and L6 rat myoblasts were dose-dependently stimulated by insulin. The metabolic inhibitors of phosphatidyl-inositol-3-kinase (PI-3K, LY294002) and of MAPKK/ERK kinase (MEK, PD98059) differently affected insulin-stimulated myogenesis of the cells. After LY294002 and PD98059 treatment, viability deteriorated and apparently an additive effect of both metabolic inhibitors was observed, irrespective of the method of measurement (neutral red or MTT assay). These inhibitors were antagonistic in myogenesis. Our results confirm that insulin regulates cell viability by at least two distinct pathways, namely by PI-3K- and MEK-dependent signalling cascades. Both pathways are agonistic in cell viability, whereas PI-3K rather than MEK supports insulin-mediated myogenicity. Accordingly, inhibition of insulin action by LY294002, but not PD98059, was accompanied with a reduced level of Ser473-phosphorylated Akt with additional loss of myogenin protein. Besides, repression of insulin signalling by either PI-3K or MEK inhibitor diminished expression of selected subunits of the mitochondrial oxidative phosphorylation enzymes (OXPHOS). In turn, insulin raised and accelerated protein expression of subunits I and IV of mitochondrial cytochrome-c oxidase (COX). In addition, the level of myogenin, the molecular marker of terminal and general muscle differentiation indices decreased if selected OXPHOS enzymes were individually blocked by rotenone, myxothiazol or oligomycin. Summing up, our results pointed to mitochondria as an essential organelle for insulin-dependent myogenesis. Insulin positively affects mitochondrial function by induction of OXPHOS enzymes, which provide energy indispensable for the anabolic effect of insulin.     

Genetic Factors Modulate the Impact of Pubertal Androgen Excess on Insulin Sensitivity and Fertility

Polycystic ovary syndrome (PCOS) is the most common endocrine disorder of reproductive age women. The syndrome is caused by a combination of environmental influences and genetic predisposition. Despite extensive efforts, the heritable factors contributing to PCOS development are not fully understood. The objective of this study was to test the hypothesis that genetic background contributes to the development of a PCOS-like reproductive and metabolic phenotype in mice exposed to excess DHEA during the pubertal transition. We tested whether the PCOS phenotype would be more pronounced on the diabetes-prone C57BL/6 background than the previously used strain, BALB/cByJ. In addition, we examined strain-dependent upregulation of the expression of ovarian and extra-ovarian candidate genes implicated in human PCOS, genes containing known strain variants, and genes involved with steroidogenesis or insulin sensitivity. These studies show that there are significant strain-related differences in metabolic response to excess androgen exposure during puberty. Additionally, our results suggest the C57BL/6J strain provides a more robust and uniform experimental platform for PCOS research than the BALB/cByJ strain.     

Retinoblastoma protein promotes oxidative phosphorylation through upregulation of glycolytic genes in oncogene‐induced senescent cells

Metabolism is closely linked with cellular state and biological processes, but the mechanisms controlling metabolic properties in different contexts remain unclear. Cellular senescence is an irreversible growth arrest induced by various stresses, which exhibits active secretory and metabolic phenotypes. Here, we show that retinoblastoma protein (RB) plays a critical role in promoting the metabolic flow by activating both glycolysis and mitochondrial oxidative phosphorylation (OXPHOS) in cells that have undergone oncogene‐induced senescence (OIS). A combination of real‐time metabolic monitoring, and metabolome and gene expression analyses showed that OIS‐induced fibroblasts developed an accelerated metabolic flow. The loss of RB downregulated a series of glycolytic genes and simultaneously reduced metabolites produced from the glycolytic pathway, indicating that RB upregulates glycolytic genes in OIS cells. Importantly, both mitochondrial OXPHOS and glycolytic activities were abolished in RB‐depleted or downstream glycolytic enzyme‐depleted OIS cells, suggesting that RB‐mediated glycolytic activation induces a metabolic flux into the OXPHOS pathway. Collectively, our findings reveal that RB essentially functions in metabolic remodeling and the maintenance of the active energy production in OIS cells.     

Effects of high-fat overfeeding on mitochondrial function, glucose and fat metabolism, and adipokine levels in low-birth-weight subjects

Low birth weight (LBW) is associated with an increased risk of insulin resistance and downregulation of oxidative phosphorylation (OXPHOS) genes when exposed to a metabolic challenge of high-fat overfeeding (HFO). To elaborate further on the differential effects of HFO in LBW subjects, we measured in vivo mitochondrial function, insulin secretion, hepatic glucose production, and plasma levels of key regulatory hormones before and after 5 days of HFO in 20 young LBW and 26 normal-birth-weight (NBW) men. The LBW subjects developed peripheral insulin resistance after HFO due to impaired endogenous glucose storage (9.42 ± 4.19 vs. 5.91 ± 4.42 mg·kg FFM(-1)·min(-1), P = 0.01). Resting muscle phosphorcreatine and total ATP in muscle increased significantly after HFO in LBW subjects only, whereas additional measurements of mitochondrial function remained unaffected. Despite similar plasma FFA levels, LBW subjects displayed increased fat oxidation during insulin infusion compared with normal-birth-weight (NBW) subjects after HFO (0.37 ± 0.35 vs. 0.17 ± 0.33 mg·kg FFM(-1)·min(-1), P = 0.02). In contrast to NBW subjects, the plasma leptin levels of LBW subjects did not increase, and the plasma gastric inhibitory polypeptide (GIP) as well as pancreatic polypeptide (PP) levels increased less in LBW compared with NBW subjects during HFO. In conclusion, HFO unmasks dissociation between insulin resistance and mitochondrial dysfunction in LBW subjects, suggesting that insulin resistance may be a cause, rather than an effect, of impaired muscle OXPHOS gene expression and mitochondrial dysfunction. Reduced increments in response to HFO of fasting plasma leptin, PP, and GIP levels may contribute to insulin resistance, lower satiety, and impaired insulin secretion in LBW subjects.     

Mitochondrial adaptation in obesity is a ClpPicated business

Quality control systems that maintain mitochondrial oxidative phosphorylation (OXPHOS) include rescue by mitochondrial fusion, elimination of dysfunctional mitochondria by mitophagy, and degradation of damaged proteins by proteases. ClpP is an ATP‐dependent protease located in the mitochondrial matrix and mutated in Perrault syndrome, causing gonadal atrophy and hearing loss. Given that hearing loss is common in mitochondrial diseases caused by mtDNA mutations, ClpP was proposed to be part of the quality control system to maintain proper mitochondrial OXPHOS function. Two recent studies independently report that deletion of ClpP in mice protects from insulin resistance and obesity by increasing mitochondrial OXPHOS capacity and browning in gonadal white adipose tissue and mitochondrial coupling in brown adipose tissue 1 , 2 . Furthermore, liver‐ and muscle‐specific deletion of ClpP has no major effects on insulin resistance. These studies reveal that ClpP might be involved in tissue‐specific mitochondrial remodeling in response to metabolic demands, rather than exclusively removing damaged proteins to maintain OXPHOS capacity.