Epigenetic modification through DNA methylation is implicated in metabolic disease. Using whole-genome promoter methylation analysis of skeletal muscle from normal glucose-tolerant and type 2 diabetic subjects, we identified cytosine hypermethylation of peroxisome proliferator-activated receptor gamma (PPARgamma) coactivator-1 alpha (PGC-1alpha) in diabetic subjects. Methylation levels were negatively correlated with PGC-1alpha mRNA and mitochondrial DNA (mtDNA). Bisulfite sequencing revealed that the highest proportion of cytosine methylation within PGC-1alpha was found within non-CpG nucleotides. Non-CpG methylation was acutely increased in human myotubes by exposure to tumor necrosis factor-alpha (TNF-alpha) or free fatty acids, but not insulin or glucose. Selective silencing of the DNA methyltransferase 3B (DNMT3B), but not DNMT1 or DNMT3A, prevented palmitate-induced non-CpG methylation of PGC-1alpha and decreased mtDNA and PGC-1alpha mRNA. We provide evidence for PGC-1alpha hypermethylation, concomitant with reduced mitochondrial content in type 2 diabetic patients, and link DNMT3B to the acute fatty-acid-induced non-CpG methylation of PGC-1alpha promoter.

Simulated shift work uncovers potential tissue mechanisms for metabolic effects attributed to sleep loss and circadian disruption.

AMP-activated protein kinase (AMPK) is a heterotrimeric complex, composed of a catalytic subunit (␣) and two regulatory subunits (␤ and ␥), that works as a cellular energy sensor. The existence of multiple heterotrimeric complexes provides a molecular basis for the multiple roles of this highly conserved signaling system. The AMPK ␥3 subunit is predominantly expressed in skeletal muscle, mostly in type II glycolytic fiber types. We determined whether the AMPK ␥3 subunit has a role in signaling pathways that mediate mitochondrial biogenesis in skeletal muscle. We provide evidence that overexpression or ablation of the AMPK ␥3 subunit does not appear to play a critical role in defining mitochondrial content in resting skeletal muscle. However, overexpression of a mutant form (R225Q) of the AMPK ␥3 subunit (Tg-AMPK␥3 225Q ) increases mitochondrial biogenesis in glycolytic skeletal muscle. These adaptations are associated with an increase in expression of the co-activator PGC-1␣ and several transcription factors that regulate mitochondrial biogenesis, including NRF-1, NRF-2, and TFAM. Succinate dehydrogenase staining, a marker of the oxidative profile of individual fibers, was also increased in transversal skeletal muscle sections of white gastrocnemius muscle from Tg-AMPK␥3 225Q mice, independent of changes in fiber type composition. In conclusion, a single nucleotide mutation (R225Q) in the AMPK ␥3 subunit is associated with mitochondrial biogenesis in glycolytic skeletal muscle, concomitant with increased expression of the co-activator PGC-1␣ and several transcription factors that regulate mitochondrial proteins, without altering fiber type composition.Skeletal muscle is a heterogeneous tissue that is composed of different fiber types, which are characterized by distinct contractile and metabolic properties. Skeletal muscle adapts to different conditions that require quick or long term alterations in power, force production, or substrate availability and utilization. Regular endurance exercise training promotes mitochondrial biogenesis (1) through an orchestrated change in the expression of genes that encode mitochondrial proteins (2). The AMPK 2 pathway plays a major role in the physiological regulation of mitochondrial function and biogenesis (3-8) and has been suggested to prevent or reverse peripheral insulin resistance associated with Type 2 diabetes (9 -11).AMPK is a cellular energy sensor that is activated in response to an increase in the AMP/ATP ratio. Once activated, AMPK coordinates signaling events to initiate catabolic processes that increase energy production and terminate anabolic processes, such as protein synthesis. AMPK activation modulates transcription of specific genes involved in energy metabolism, thereby exerting long term metabolic control. AMPK exists as a heterotrimeric complex composed of a catalytic (␣) and two regulatory (␤ and ␥) subunits. The ␣-and ␤-subunits are each encoded by two genes (␣1, ␣2, ␤1, and ␤2), and the ␥ subunit is encoded by three genes (␥1, ␥2, and ␥3), yielding 1...

Abstract-Recent work has demonstrated the importance of the epicardium in the development of the heart. During embryogenesis, these epithelial cells provide the progenitors for the epicardium, coronary smooth muscle, endothelium, and cardiac fibroblasts. The epicardium sends important signals to the developing myocardium. Still, analysis of these epithelial cells has lagged behind that of other cardiac cell types largely because of the lack of a defined experimental cell system in which epicardial cell differentiation can be studied. The present report examines the developmental potential of a cell line derived from rat epicardial mesothelial cells. These analyses demonstrate that the cell line retains many characteristics of the intact epithelium, including the ability to form a polarized epithelium and express many epicardial genes. Our data show for the first time that these cells retain the ability to produce mesenchyme in response to specific growth factors and, importantly, to generate smooth muscle cells. Thus, this study provides evidence that these cells can serve as an important model system for the analysis of the cellular and molecular mechanisms that govern epicardial development and function.