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Context: Insulin resistance in skeletal muscle is a major risk factor for the development of type 2 diabetes in women with polycystic ovary syndrome (PCOS). Despite this, the mechanisms underlying insulin resistance in PCOS are largely unknown. Objective: To investigate the genome-wide DNA methylation and gene expression patterns in skeletal muscle from women with PCOS and controls and relate them to phenotypic variations. Design/Participants: In a case-control study, skeletal muscle biopsies from women with PCOS (n = 17) and age-, weight-, and body mass index‒matched controls (n = 14) were analyzed by array-based DNA methylation and mRNA expression profiling. Results: Eighty-five unique transcripts were differentially expressed in muscle from women with PCOS vs controls, including DYRK1A, SYNPO2, SCP2, and NAMPT. Furthermore, women with PCOS had reduced expression of genes involved in immune system pathways. Two CpG sites showed differential DNA methylation after correction for multiple testing. However, an mRNA expression of ∼30% of the differentially expressed genes correlated with DNA methylation levels of CpG sites in or near the gene. Functional follow-up studies demonstrated that KLF10 is under transcriptional control of insulin, where insulin promotes glycogen accumulation in myotubes of human muscle cells. Testosterone downregulates the expression levels of COL1A1 and MAP2K6. Conclusion: PCOS is associated with aberrant skeletal muscle gene expression with dysregulated pathways. Furthermore, we identified specific changes in muscle DNA methylation that may affect gene expression. This study showed that women with PCOS have epigenetic and transcriptional changes in skeletal muscle that, in part, can explain the metabolic abnormalities seen in these women.

A single nucleotide substitution in the third intron of insulin-like growth factor 2 (IGF2) is associated with increased muscle mass and reduced subcutaneous fat in domestic pigs. This mutation disrupts the binding of the ZBED6 transcription factor and leads to a threefold up-regulation of IGF2 expression in pig skeletal muscle. Here, we investigated the biological significance of ZBED6-IGF2 interaction in the growth of placental mammals using two mouse models, ZBED6 knock-out (Zbed6(-/-)) and Igf2 knock-in mice that carry the pig IGF2 mutation. These transgenic mice exhibit markedly higher serum IGF2 concentrations, higher growth rate, increased lean mass, and larger heart, kidney, and liver; no significant changes were observed for white adipose tissues. The changes in body and lean mass were most pronounced in female mice. The phenotypic changes were concomitant with a remarkable up-regulation of Igf2 expression in adult tissues. Transcriptome analysis of skeletal muscle identified differential expression of genes belonging to the extracellular region category. Expression analysis using fetal muscles indicated a minor role of ZBED6 in regulating Igf2 expression prenatally. Furthermore, transcriptome analysis of the adult skeletal muscle revealed that this elevated expression of Igf2 was derived from the P1 and P2 promoters. The results revealed very similar phenotypic effects in the Zbed6 knock-out mouse and in the Igf2 knock-in mouse, showing that the effect of ZBED6 on growth of muscle and internal organs is mediated through the binding site in the Igf2 gene. The results explain why this ZBED6 binding site is extremely well conserved among placental mammals.

AIMS/HYPOTHESIS: Insulin-mediated signals and AMP-activated protein kinase (AMPK)-mediated signals are activated in response to physiological conditions that represent energy abundance and shortage, respectively. Focal adhesion kinase (FAK) is implicated in insulin signalling and cancer progression in various non-muscle cell types and plays a regulatory role during skeletal muscle differentiation. The role of FAK in skeletal muscle in relation to insulin stimulation or AMPK activation is unknown. We examined the effects of insulin or AMPK activation on FAK phosphorylation in human skeletal muscle and the direct role of FAK on glucose and lipid metabolism. We hypothesised that insulin treatment and AMPK activation would have opposing effects on FAK phosphorylation and that gene silencing of FAK would alter metabolism.METHODS: Human muscle was treated with insulin or the AMPK-activating compound 5-aminoimadazole-4-carboxamide ribonucleotide (AICAR) to determine FAK phosphorylation and glucose transport. Primary human skeletal muscle cells were used to study the effects of insulin or AICAR treatment on FAK signalling during serum starvation, as well as to determine the metabolic consequences of silencing the FAK gene, PTK2.RESULTS: AMPK activation reduced tyrosine phosphorylation of FAK in skeletal muscle. AICAR reduced p-FAKY397in isolated human skeletal muscle and cultured myotubes. Insulin stimulation did not alter FAK phosphorylation. Serum starvation increased AMPK activation, as demonstrated by increased p-ACCS222, concomitant with reduced p-FAKY397. FAK signalling was reduced owing to serum starvation and AICAR treatment as demonstrated by reduced p-paxillinY118. Silencing PTK2 in primary human skeletal muscle cells increased palmitate oxidation and reduced glycogen synthesis.CONCLUSIONS/INTERPRETATION: AMPK regulates FAK signalling in skeletal muscle. Moreover, siRNA-mediated FAK knockdown enhances lipid oxidation while impairing glycoge

OBJECTIVE: Parental nutrition and lifestyle impact the metabolic phenotype of the offspring. We have reported that grandpaternal chronic high-fat diet (HFD) transgenerationally impairs glucose metabolism in subsequent generations. Here we determined whether grandpaternal diet transgenerationally impacts the transcriptome and lipidome in skeletal muscle. Our aim was to identify tissue-specific pathways involved in transgenerational inheritance of environmental-induced phenotypes.METHODS: F0 male Sprague-Dawley rats were fed a HFD or chow for 12 weeks before breeding with chow-fed females to generate the F1 generation. F2 offspring were generated by mating F1 males fed a chow diet with an independent line of chow-fed females. F1 and F2 offspring were fed chow or HFD for 12 weeks. Transcriptomic and LC-MS lipidomic analyses were performed in extensor digitorum longus muscle from F2-females rats. Gene set enrichment analysis (GSEA) was performed to determine pathways reprogrammed by grandpaternal diet.RESULTS: GSEA revealed an enrichment of the unfolded protein response pathway in skeletal muscle of grand-offspring from HFD-fed grandfathers compared to grand-offspring of chow-fed males. Activation of the stress sensor (ATF6α), may be a pivotal point whereby this pathway is activated. Interestingly, skeletal muscle from F1-offspring was not affected in a similar manner. No major changes were observed in the skeletal muscle lipidome profile due to grandpaternal diet.CONCLUSIONS: Grandpaternal HFD-induced obesity transgenerationally affected the skeletal muscle transcriptome. This finding further highlights the impact of parental exposure to environmental factors on offspring's development and health.

DNA methylation is altered by environmental factors. We hypothesized DNA methylation is altered in skeletal muscle in response to either insulin or glucose exposure. We performed a genome-wide DNA methylation analysis in muscle from healthy men before and after insulin exposure. DNA methylation of selected genes was determined in muscle from healthy and type 2 diabetic men before and after a glucose tolerance test. Insulin altered DNA methylation in the 3'UTR of the calcium pump ATP2A3 gene. Insulin increased DNA methylation in the gene body of DAPK3, a gene involved in cell proliferation, apoptosis and autophagy. DAPK3 methylation was reduced in type 2 diabetic patients. Carbohydrate ingestion reduced DAPK3 DNA methylation in healthy and type 2 diabetic men, suggesting glucose may play a role. Supporting this, DAPK3 DNA methylation was inversely correlated with the 2 hr glucose concentration. While glucose incorporation to glycogen was unaltered by siRNA against DAPK3, palmitate oxidation was increased. In conclusion, insulin and glucose exposure acutely alter the DNA methylation profile of skeletal muscle, indicating DNA methylation constitutes a rapidly and adaptive epigenetic mark. Furthermore, insulin and glucose modulate DAPK3 DNA methylation in a reciprocal manner suggesting a feedback loop in the control of the epigenome.