Primary RC dysfunction transcriptionally and post-transcriptionally dysregulates the integrated nutrient-sensing signaling network.

<p>(<b>A</b>) <b>Integrated overview modeling general interactions between central nutrient-sensing signaling pathways.</b> Arrows and bars convey activating and inhibiting effects, respectively. TFs, physiologic signals, and drugs known to modulate this pathway are indicated in green, blue, and purple font, respectively. “P” indicates pathway components whose activity is modulated by phosphorylation. Red boxes detail physiologic effects. (<b>B</b>) <b>Oligomycin-based pharmacologic RC inhibition in human FCLs alters mTORC1 and AMPK pathway activities.</b> To confirm primary mitochondrial RC dysfunction was sufficient to alter mTORC1 signaling, FCLs from a healthy individual were treated in DMEM medium containing 20% fetal bovine serum for 24 hours and either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose, with either the complex V inhibitor, Oligomycin, (“O”, 5 uMol), an AMPK activator, AICAR (“A”, 2 mMol), or the mTORC1 inhibitor, rapamycin (“R”, 100 nMol). Regardless of glucose concentration, expression of a standard readout of mTORC1 pathway activity, phospho-S6 protein level, was reduced by oligomycin treatment, while increased phospho-AMPK expression was evident in high glucose media. (<b>C</b>) <b>Oligomycin-based alteration of mTORC1 and AMPK pathway activities is dose- and nutrient-dependent.</b> The same control FCLs as in (B) were treated for 24 hours in either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose media with oligomycin in doses ranging from 0.25 uMol to 5 uMol. A general trend is evident of overall reduced phospho-S6 and phospho-AMPK at increasing doses of oligomycin. See also <b>Fig. S13 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069282#pone.0069282.s003" target="_blank">File S3</a></b>. (<b>D</b>) <b>Pharmacologic RC inhibition increases </b><b><i>FOXO1</i></b><b> expression in high-glucose conditions.</b> FCLs were treated for 24 hours in either low (1 g/L or 5 mMol) or high (4.5 g/L or 25 mMol) glucose media with a RC inhibitor targeting either complex I (rotenone, 0.1 uMol), III (antimycin A, 0.5 uMol), or V (oligomycin, 1 uMol). As expected, <i>FOXO1</i> expression was inhibited by high-glucose media. All three RC inhibitors increased <i>FOXO1</i> relative expression in high-glucose conditions, whereas <i>FOXO1</i> expression was decreased by oligomycin in low-glucose media. (<b>E–F</b>) <b>Integrated view of detailed gene and sub-gene level transcriptional alterations in RC disease among central nutrient-sensing signaling network modulators.</b> Their major downstream targets and pathways were presented in separated plots. An overall inverse expression pattern is evident in RC disease between (<b>E</b>) skeletal muscle and (<b>F</b>) FCL.