No abstract

The skeletal muscle of obese individuals exhibits an impaired ability to increase the expression of genes linked with fatty acid oxidation (FAO) upon lipid exposure. The present study determined if this response could be attributed to differential DNA methylation signatures. RNA and DNA were isolated from primary human skeletal muscle cells (HSkMC) from lean and severely obese women following lipid incubation. mRNA expression and DNA methylation were quantified for genes that globally regulate FAO [PPARγ coactivator (PGC-1α), peroxisome proliferator-activated receptors (PPARs), nuclear respiratory factors (NRFs)]. With lipid oversupply, increases in NRF-1, NRF-2, PPARα, and PPARδ expression were dampened in skeletal muscle from severely obese compared with lean women. The expression of genes downstream of the PPARs and NRFs also exhibited a pattern of not increasing as robustly upon lipid exposure with obesity. Increases in CpG methylation near the transcription start site with lipid oversupply were positively related to PPARδ expression; increases in methylation with lipid were depressed in HSkMC from severely obese women. With severe obesity, there is an impaired ability to upregulate global transcriptional regulators of FAO in response to lipid exposure. Transient changes in DNA methylation patterns and differences in the methylation signature with severe obesity may play a role in the transcriptional regulation of PPARδ in response to lipid. The persistence of differential responses to lipid in HSkMC derived from lean and obese subjects supports the possibility of stable epigenetic programming of skeletal muscle cells by the respective environments.

Phosphorylase kinase (PhK), the key enzyme that regulates glycogenolysis, has traditionally been thought to be expressed predominantly in muscle and liver. In this study, we show by two different database searches (Expressed Sequence Tag and UniGene) that PhK gene expression occurs in at least 28 -36 different tissues, and that the genes encoding the α, β and γ subunits of PhK undergo extensive transcriptional processing. In particular, we have identified exon 6 of PHKG1 as a 3′ composite terminal exon due to the presence of a weak polyadenylation and cleavage site in intron 6. We have verified biochemically that transcriptional processing of PHKG1 does occur in vivo; mRNA corresponding to the alternate variant is expressed in skeletal muscle, brain, heart, and tongue. In silico translation of this mRNA yields a PhK γ subunit that contains the first 181 residues of the protein, followed by an additional 21 amino acids. The implication of this alternate processing is discussed within the context of γ catalysis and regulation.

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Quantification of nitric oxide (NO) from cultured cells is a valuable tool for studying cell signaling. Detection of NO in biological fluids can be difficult however, due to its transient half-life and low physiological concentrations. In this study, we have refined an existing amperometric method to determine relative levels of accumulated nitrogen oxides (NO X ) in cell culture and have used this method to reproducibly quantify NO from cultured pulmonary myofibroblasts. Basal levels of NO produced by pulmonary myofibroblasts ranged from 0.6 nM to 20 nM and varied due to the growth conditions of the cells, i.e. higher NO concentrations were observed in differentiated cells. The constitutive eNOS isoform is primarily responsible for the observed NO accumulation in these cells since transcript levels of eNOS are 10-fold higher than the inducible iNOS form while nNOS was undetectable. Treatment of myofibroblasts with the inhibitors L-NNA and L-NAME resulted in a concentration dependent decrease in measured NOx. Overall, the improved assay presented here should be applicable to measuring NOX levels from many different cell types and under a wide variety of conditions.