PPARα, δ and FOXO1 Gene Silencing Overturns Palmitate-Induced Inhibition of Pyruvate Oxidation Differentially in C2C12 Myotubes

SIMPLE SUMMARY: Frequent high-dietary fat intake increases muscle lipid use and reduces muscle carbohydrate use, thereby inducing metabolic inflexibility. The latter term can be described as a poor muscle biochemical and molecular response to increased availability of insulin, which in the long term results in chronically excessive-high glucose levels in blood. Chronic hyperglycaemia is associated with many pathological conditions, including type 2 diabetes mellitus, which can cause severe health damages in humans. Here, we attempt to unravel the underlying mechanism and its associated factors behind the inhibition of muscle glucose use by a high-fat diet, thereby providing evidence for appropriate therapeutic intervention. ABSTRACT: The molecular mechanisms by which free fatty acids (FFA) inhibit muscle glucose oxidation is still elusive. We recently showed that C2C12 myotubes treated with palmitate (PAL) presented with greater protein expression levels of PDK4 and transcription factors PPARα and PPARδ and lower p-FOXO/t-FOXO protein ratios when compared to control. This was complemented with the hallmarks of metabolic inflexibility (MI), i.e., reduced rates of glucose uptake, PDC activity and maximal pyruvate-derived ATP production rates (MAPR). However, the relative contribution of these transcription factors to the increase in PDK4 and reduced glucose oxidation could not be established. Therefore, by using a similar myotube model, a series of individual siRNA gene silencing experiments, validated at transcriptional and translation levels, were performed in conjunction with measurements of glucose uptake, PDC activity, MAPR and concentrations of metabolites reflecting PDC flux (lactate and acetylcarnitine). Gene silencing of PPARα, δ and FOXO1 individually reduced PAL-mediated inhibition of PDC activity and increased glucose uptake, albeit by different mechanisms as only PPARδ and FOXO1 silencing markedly reduced PDK4 protein content. Additionally, PPARα and FOXO1 silencing, but not PPARδ, increased MAPR with PAL. PPARδ silencing also decreased FOXO1 protein. Since FOXO1 silencing did not alter PPARδ protein, this suggests that FOXO1 might be a PPARδ downstream target. In summary, this study suggests that the molecular mechanisms by which PAL reduces PDC-mediated glucose-derived pyruvate oxidation in muscle occur primarily through increased PPARδ and FOXO1 mediated increases in PDK4 protein expression and secondarily through PPARα mediated allosteric inhibition of PDC flux. Furthermore, since PPARδ seems to control FOXO1 expression, this may reflect an important role for PPARδ in preventing glucose oxidation under conditions of increased lipid availability.