Figure 1.
(A,B) Human PGC-1α mRNA levels were determined relative to B2M using RT and real-time PCR. (A) Six samples were analyzed, three from cultured skm cells and three from skm biopsies. Data are expressed as mean values ± SEM of 2−ΔCt. The significance of the difference is *p<0.05. (B) Cultured skm cells were transduced with Ad-GFP or Ad-PGC-1α. Data are expressed as mean values ± SEM of 2−ΔCt for three experiments performed in triplicate. The significance of the difference is *p<0.001. (C) Cultured skm cells were transduced with Ad-GFP or Ad-PGC-1α. Immunoblot analyses were performed on nuclear cell extracts (30 µg protein) and membranes were hybridized with antibodies against PGC-1α and α-actin. A representative image is shown. Bands were quantified with ImageQuant LAS 4000. Data are means ± SEM from two experiments performed in triplicate. Significance of differences versus cells treated with Ad-GFP: *p<0.05.
Table 1.
Differentially expressed genes in response to PGC-1α-overexpression in cultured muscle cells.
Table 2.
Gene ontology annotation of upregulated genes in response to PGC-1α-overexpression in cultured muscle cells.
Figure 2.
Genes regulated by both PGC-1α-overexpression in cultured skm cells and skm cultures in comparison to skm tissue.
Heat map including the genes (Gene symbol/Gene ID for Entrez Gene) in Table 1 (PGC-1α- versus control-skm cells) that were also found to be differentially expressed after human skm culture according to Table S2 from [25] (cultured skm cells versus skm tissue). Each cell displays the absolute fold change in the corresponding comparison and is filled according to the color gradient shown at the top (green and red for down and upregulated genes, respectively).
Table 3.
Genes validated for differential expression in PGC-1α-overexpressing cultured muscle cells.
Figure 3.
Upregulation of IL-8 and FITM1 protein content by PGC-1α-overexpression in cultured skm cells.
Cultured skm cells were transduced with Ad-GFP or Ad-PGC-1α. In cell extracts, (A) ELISA assay for IL-8 and (B) immunoblot analysis of FITM1 were performed. (B) For FITM1, α-actinin was used as loading control; a representative image is shown and bands were quantified. (A and B) Data are means ± SEM from three experiments performed in triplicate. Significance of differences versus cells treated with Ad-GFP: *p<0.05.
Figure 4.
Effect of PGC-1α-overexpression on mitochondrial COX activity, mitochondrial to nuclear DNA ratio and oxidation of metabolic substrates in cultured muscle cells.
Cultured muscle cells were transduced with Ad-GFP or Ad-PGC-1α adenoviruses. (A) Cytochemical staining of COX activity was performed in cultured skm cells that had been transduced with Ad-GFP (a, b and c) or Ad-PGC-1α (d, e and f). Images were obtained with a camera (a,b,d,e) or with an inverted microscope at 800× magnification (c,f). Images from two independent experiments performed in duplicate were quantitatively analyzed and the data expressed as percentages of the values in the controls: means ± SEM are shown on the graph. The significance of the difference is *p<0.005. (B) Cells were harvested and total DNA isolated to measure the ratio of mitochondrial DNA (mtDNA) content to nuclear DNA (nDNA) content. Data are expressed as a percentage of control. Data are means ± SEM from two experiments performed in triplicate. (C–E) Cells were then incubated for 4 h with: (C) 0.5 mM [1-14C]-palmitate (2.8 µCi/µmol), (D) 10 mM [U-14C]-glucose (0.21 µCi/µmol) and (E) 2 mM [U-14C]-lactate (0.6 µCi/µmol). Production of 14CO2 was subsequently quantified. Data are expressed as means ± SEM from two experiments performed in sextuplicate. The significance of the difference is *p<0.005 and **p<0.001.
Figure 5.
Effect of PGC-1α-overexpression on lipid droplets and triglyceride accumulation in cultured muscle cells.
Cultured skm cells were transduced with Ad-GFP or Ad-PGC-1α and incubated with 0.5 mM oleate for 16 h. (A,B,C) Cells were fixed and then stained with Nile Red. (A) Representative lipid droplet micrographs obtained via confocal microscopy are shown. White bars represent 5 µm. (B) The number of lipid droplets per cell area and (C) the lipid droplet mean area values were quantified. Data are means ± SEM of (B) four cells or (C) at least 414 droplets. (D) Triglyceride content was measured. Data are means ± SEM of three experiments performed in quadruplicate. (B,C,D) The significance of the differences versus cells treated with Ad-GFP is *p<0.05 and **p<0.01.
Figure 6.
Effect of PGC-1α-overexpression on glycogen metabolism and glucose uptake in cultured muscle cells.
Cultured skm cells were transduced with Ad-GFP or Ad-PGC-1α. (A) Cells were incubated with 10 mM [U-14C]-glucose (0.10 µCi/µmol) for 18 h and then harvested to asses glucose incorporation into glycogen. Data are means ± SEM from two experiments performed in triplicate. (B) Glucose uptake was measured using 0.5 mM 2-deoxy-D-[3H]glucose (0.5 µCi/well). Data are means ± SEM from two experiments performed in triplicate. (C,D) Cells were harvested to measure (C) glycogen synthase and (D) glycogen phosphorylase activity. Data are means ± SEM from three experiments performed in quadruplicate. The significance of differences versus cells treated with control adenoviruses is indicated as follows: *p<0.05 and **p<0.01.