FIGURE 4.

Rapamycin inhibits mitochondrial transcription factors, but has no major effect on mitochondrial protein levels or PKCθ phosphorylation. (A) Forty-eight hours of rapamycin treatment decreases expression of PGC-1α and TFAM, two of the major transcription factors involved in mitochondrial biogenesis, as well as the level of MT-CO1, an mtDNA-encoded transcript. (B) Rapamycin mildly increases intracellular triglyceride levels in myotubes. The result show is representative of three separate experiments. (C) Forty-eight hours of rapamycin treatment has no effect on mitochondrial DNA copy number, as reflected by the ratio between MT-CO1 (mitochondrial DNA-encoded) and Ndufv1 (nuclear-encoded) DNA. (D) Preincubation of myotubes with rapamycin for 24 h or more induces insulin resistance irrespective of the presence of palmitate. Rapamycin-induced insulin resistance is not associated with major increases in serine phosphorylation of IRS1 or phosphorylation of PKCθ, two hallmarks of insulin resistance caused by insufficient oxidation of fatty acids. Moreover, there is no change in the expression of several proteins involved in electron transport complexes or in the level of VDAC, a structural protein in the mitochondria. *p < 0.05. Error bars show SEM.