Regulation of skeletal muscle lipolysis and oxidative metabolism by the co-lipase CGI-58

Abstract

We investigated here the specific role of CGI-58 in the regulation of energy metabolism in skeletal muscle. We first examined CGI-58 protein expression in various muscle types in mice, and next modulated CGI-58 expression during overexpression and knockdown studies in human primary myotubes and evaluated the consequences on oxidative metabolism. We observed a preferential expression of CGI-58 in oxidative muscles in mice consistent with triacylglycerol hydrolase activity. We next showed by pulse-chase that CGI-58 overexpression increased by more than 2-fold the rate of triacylglycerol (TAG) hydrolysis, as well as TAG-derived fatty acid (FA) release and oxidation. Oppositely, CGI-58 silencing reduced TAG hydrolysis and TAG-derived FA release and oxidation (-77%, P < 0.001), whereas it increased glucose oxidation and glycogen synthesis. Interestingly, modulations of CGI-58 expression and FA release are reflected by changes in pyruvate dehydrogenase kinase 4 gene expression. This regulation involves the activation of the peroxisome proliferator activating receptor-δ (PPARδ) by lipolysis products. Altogether, these data reveal that CGI-58 plays a limiting role in the control of oxidative metabolism by modulating FA availability and the expression of PPARδ-target genes, and highlight an important metabolic function of CGI-58 in skeletal muscle.

Fig. 1.

Distribution and function of CGI-58 in skeletal muscle. A: Representative blot of CGI-58 protein expression in different types of muscle and EWAT [whole gastrocnemius (gastroc), soleus, quadriceps (quad) and heart, as well as epididimal white adipose tissue] in 20-week-old mice. Forty micrograms of total protein were loaded for the different muscles and 10 µg for EWAT (n = 3). B: Triacylglycerol hydrolase activity (TAGH) was measured in different types of muscle and EWAT in 20-week-old mice as previously (n = 6), One-way ANOVA * P < 0.05. C: TAGH was measured in human adipose tissue (hAT) and skeletal muscle (hSkM) in absence (control) or presence of recombinant human CGI-58 (rhCGI-58) (n = 6). A positive control experiment was performed in presence of COS7 cell extracts overexpressing human ATGL (COS7/ATGL), paired Student's t-test ** P < 0.01 when compared with control.

Fig. 2.

CGI-58 overexpression promotes TAG breakdown. A: CGI-58 and (B) ATGL protein content were measured in control myotubes (Ad-GFP) and myotubes overexpressing CGI-58 (Ad-CGI58) (n = 5). Insets are showing representative blots and loading control of each protein, * P < 0.05 versus Ad-GFP. C: TAGH activity and (D) total TAG content were measured in control myotubes (Ad-GFP) and myotubes overexpressing CGI-58 (Ad-CGI58) (n = 5), paired Student's t-test * P < 0.05, ** P < 0.01 versus Ad-GFP.

Fig. 3.

CGI-58 increases TAG-derived FA release and oxidation. Pulse-chase experiments were performed to determine the time-course over 6 h of (A) TAG hydrolysis and (B) FA release into the culture medium, in control myotubes (Ad-GFP) and myotubes overexpressing CGI-58 (Ad-CGI58) (n = 6). TAG content was expressed as a function of the value at the 0 time point (% of T0). All parameters were measured at the end of the Pulse (0 time point) and during the Chase period (1, 3, and 6 h time points). Two-way ANOVA * P < 0.05, ** P < 0.01 when compared with Ad-GFP. C: Endogenous oxidation, i.e., TAG-derived FA oxidation was measured in absence of triacsin C after 3 h of Chase in the same conditions. Paired Student's t-test ** P < 0.01 when compared with Ad-GFP.

Fig. 4.

CGI-58 silencing reduces lipolysis and TAG-derived FA oxidation. A: Representative blot of CGI-58 protein expression in control myotubes and myotubes knocked down for CGI-58 (siCGI58) (n = 2 lane per condition). We next measured by pulse-chase the time-course over 6 h of (B) TAG hydrolysis, (C) FA release into the culture medium in control myotubes and myotubes knocked down for CGI-58 (siCGI58) (n = 6). All parameters were measured at the end of the Pulse (0 time point) and during the Chase period (1, 3, and 6 h time points). TAG content was expressed as a function of the value at the 0 time point (% of T0). Two-way ANOVA ** P < 0.01, *** P < 0.0001 when compared with control. D: Endogenous oxidation, i.e., TAG-derived FA oxidation, was measured in absence of triacsin C after 3 h of Chase in the same conditions. Paired Student's t-test *** P < 0.0001 when compared with control.

Fig. 5.

CGI-58 silencing induces a cellular energy deficit. A: Mitochondrial mass was determined in control myotubes (left panel) and myotubes knocked down for CGI-58 (siCGI58) (middle panel) using the Mitotracker Green FM (20×). Quantitative bar graph of the fluorescence intensity signal (right panel) (n = 4). B: Mitochondrial membrane potential was determined in control myotubes (left panel) and myotubes knocked down for CGI-58 (siCGI58) (middle panel) using the Mitotracker Red CMX-Ros (10×). Quantitative bar graph of the fluorescence intensity signal (right panel) (n = 4). C: Representative blot (left panel) and quantitative bar graph (right panel) of phosphoAMPK to AMPK ratio in control myotubes (left panel) and myotubes knocked down for CGI-58 (siCGI58) (n = 5). Paired Student's t-test * P < 0.05, ** P < 0.01 versus control.

Fig. 6.

CGI-58 silencing favors glucose metabolism. A: Basal glucose oxidation was measured in control myotubes and myotubes knocked down for CGI-58 (siCGI58). B: Glycogen synthesis was measured in absence (open bars) or presence (black bars) of 100 nM insulin. * P < 0.05, ** P < 0.01 versus basal; ^## P < 0.01, ^### P < 0.001 versus respective control (n = 6). C: Representative blot (left panel) and quantitative bar graph (right panel) of PDK4 protein in control myotubes and myotubes knocked down for CGI-58 (siCGI58) (n = 6), paired Student's t-test *** P < 0.001 versus control.

Fig. 7.

CGI-58-mediated lipolysis specifically modulates PPARδ-target gene expression. A: PDK4 relative gene expression in myotubes treated for 24 h in absence (control) or presence of the selective PPARδ agonist GW0742 1 nM and the selective PPARδ antagonist GSK0660 500 nM (n = 6); One-way ANOVA *** P < 0.001 versus control. B: PDK4 relative gene expression in myotubes treated for 24 h in absence (control) or presence of 500 µM of oleate alone or in combination with the selective PPARδ antagonist GSK0660 2 µM, and with the selective PPARα agonist GW7647 100 nM (n = 8); One-way ANOVA ***P < 0.001 versus control. C: PDK4 relative gene expression in control myotubes (Ad-GFP) or overexpressing CGI-58 (Ad-CGI58) in absence or presence of the selective PPARδ antagonist GSK0660 500 nM; One-way ANOVA ** P < 0.01, *** P < 0.001 (n = 6). D: PDK4 relative gene expression in control myotubes (Ad-GFP) and myotubes knocked down for CGI-58 (siCGI58) in absence or presence of the selective PPARδ agonist GW0742 1 nM (n = 6), One-way ANOVA, *** P < 0.001 versus GW0742, ^## P < 0.01 versus control.