Fig 2: G0S2 knockdown reduces glucose metabolism and enhances mitochondrial function. (A) Glucose oxidation and (B) glycogen synthesis were measured in control myotubes (shNT) and myotubes knocked down for G0S2 (shG0S2) using [U–14C] glucose. (C) PDK4 gene expression was measured in control myotubes (shNT) and myotubes knocked down for G0S2 (shG0S2) in absence or presence of a selective PPARd antagonist GSK0660 500 nM PCG1a gene expression (D), mitochondrial mass (E), and mitochondrial membrane potential (F) were measured in control myotubes (shNT) and myotubes knocked down for G0S2 (shG0S2) (n = 9). *p < 0.05, **p < 0.01, ***p < 0.001 versus shNT.

Fig 3: PDK4 knockdown prevents induction of fatty acid oxidation in brite adipocytes. Britening-associated changes were explored in 18 day-differentiated hMADS cells transfected with either a control-or PDK4-targeting siRNA and treated or not with rosiglitazone or GW7647 for the last 4 days. (A) PDK4 mRNA and protein levels. (B) Gene expression levels of UCP1, CIDEA and CITED1. (C) Ser293-phosphorylated PDHE1a protein levels. (D) Pyruvate release into the medium. (E) Glyceroneogenesis assessed by incorporation of pyruvic acid into the glycerol moiety of neutral lipids. (F) Pyruvate oxidation. (G) Fatty acid (oleate) oxidation. Data represent mean ± SEM expressed as percentage of control (n = 6–11). Open bars: siCtrl-treated cells, full bars: siPDK4-treated cells. *: p < 0.05 for R or GW vs. C; **: p < 0.01; ***: p < 0.001. $: p < 0.05 for siPDK4 vs. siCtrl; $$: p < 0.01; $$$: p < 0.001. #: p < 0.05 for R vs. GW; ###: p < 0.001.

Fig 4: Effect of chronic galactose treatment on gene and protein expressions.Myotubes were grown in DMEM-media with 5.5 mM glucose or 5.5 mM galactose. Total RNA was isolated from the cells and analyzed by qPCR, while protein samples were harvested analyzed as described in Materials and Methods. Gene expressions were normalized to 36B4 and protein expressions to ß-actin, except phosphorylated AMP-activated protein kinase (p-AMPK), which were normalized to total AMPK. Values in A and B represent fold change of genes/proteins in galactose-treated myotubes relative to glucose-treated myotubes, given as means ± SEM (n = 5). (A) Genes analyzed; CPT1b, carnitine palmitoyltransferase-1b; CYC1, cytochrome C; MCAD, acyl-coenzyme A-dehydrogenase; MYH2, myosin heavy chain 2, SLC2A1 and SLC2A4; glucose transporter 1 and 4, HKII; hexokinase II, GALK1 and 2; galactokinase 1 and 2, GALT; galactose-1-phosphate uridylyltransferase, PDK4; pyruvate dehydrogenase kinase 4. (B) Protein expression of myosin, ATP synthase subunit, slow muscle, complex II subunit, complex IV subunit II, complex I subunit NDUFB8, PDK4; pyruvate dehydrogenase 4, P-AMPK and AMPK. (C) Representative corresponding Western blots.

Fig 5: PPARa deficiency disrupts WAT britening-induced PDK4 expression in vivo. (A, C, E, G and I) WT and PPARa-null male mice housed at thermoneutrality were treated with CL316,243 (CL, 0.1 mg/kg/d) or vehicle (DMSO) for 10 days. (B, D, F and H) WT and PPARa-null male mice housed at standard temperature were treated with CL316,243 (CL, 1 mg/kg/d) or vehicle (DMSO) for 7 days. Analyses were performed on the inguinal white adipose tissue depot. (A, B) UCP1 mRNA and protein levels. (C–F) Gene expression levels of Cidea, Cpt1b, Cited1 and Tmem26. (G, H) PDK4 mRNA and protein levels. (I) Spearman correlation between UCP1 and PDK4 protein content (n = 23). Data represent mean ± SEM expressed as percentage of vehicle-treated WT mice (n = 7–9 mice per group). Open bars: wild-type mice, hatched bars: PPARa-null mice. NA: not available. *: p < 0.05 for CL vs. DMSO; **: p < 0.01; ***: p < 0.001. $: p < 0.05 for PPARa-null vs. wild type; $$: p < 0.01; $$$: p < 0.001.