Table 1.
Pathway analysis of PFI-induced gene activation in human primary myotubes.
Figure 1.
Cultured myotubes were incubated with 100 µM of [1-14C] oleate (A) or [1-14C] palmitate (B) for 3 h, and radiolabel incorporation into CO2 was determined as a measure of complete fat oxidation (FAO). Radiolabel incorporation into acid soluble molecules (ASM) was measured to assess incomplete FAO. The ratio of CO2 to ASM was calculated for oleate (C) and palmitate (D). Data are expressed as mean ± SEM of 4 separate experiments. ** p<0.01, *** p<0.001 versus control.
Figure 2.
Mitochondrial content and enzymes activity.
(A) Mitochondrial content determined by mtDNA copy number. (B) Mitochondrial content determined by Mitotracker Green FM. (C) β-hydroxyacyl-CoA dehydrogenase (β-HAD) activity adjusted for protein content. (D) Citrate synthase (CS) activity adjusted for protein content. Data are expressed as mean ± SEM of 4 separate experiments. ** p<0.01, *** p<0.001 versus control.
Table 2.
Treatment of human primary myotubes with PFI increases expression of several genes related to mitochondrial biogenesis, lipolysis and lipid storage.
Figure 3.
Confocal microscopy of lipid droplets.
Lipid droplets were stained with Bodipy 493/503 (green) and nuclei were stained with DAPI (blue). Note the increase in the organization of lipid droplets along the myofibrillar apparatus in panels B (20×) and D (40×) upon PFI treatment.
Figure 4.
Lipid storage and lipolytic capacity.
(A) Cultured myotubes were incubated with 100 µM [1-14C] oleate for 3 h, then the cells were harvested and total lipid extracted. Total lipid synthesis was measured as incorporation of radiolabel into total cellular lipids. (B) Lipid intermediates species such as phospholipids (PL), diacylglycerol (DAG), and triacylglycerol (TAG) rates of synthesis were measured by thin-layer chromatography. (C) Lipolysis measured as glycerol release was determined by subtracting baseline values to values during the stimulation with 1 µM of isoproterenol (a non-selective β-adrenergic agonist). The graph represents the changes in lipolysis at baseline in response to isoproterenol in control and PFI conditions. Data are expressed as mean ± SEM of 4 separate experiments. * p<0.05, ** p<0.01 versus control.
Figure 5.
Glucose metabolism and insulin sensitivity.
(A) Incorporation of [U-14C] glucose into glycogen was measured in either the absence (open bar) or the presence (black bar) of 100 nM of insulin. ** p<0.01 versus baseline, † p<0.05 versus control. (B) Insulin-stimulated glycogen storage was measured by the change in glycogen storage at baseline in response to insulin and used as a marker of insulin responsiveness in this model. Data are expressed as mean ± SEM of 4 separate experiments. ** p<0.01 versus control.
Table 3.
Correlations between changes in components of lipid metabolism and changes in insulin responsiveness during activation of cAMP/PKA and calcium signaling pathways in cultured human myotubes.