Contraction-induced lipolysis is not impaired by inhibition of hormone-sensitive lipase in skeletal muscle

Abstract

In skeletal muscle hormone-sensitive lipase (HSL) has long been accepted to be the principal enzyme responsible for lipolysis of intramyocellular triacylglycerol (IMTG) during contractions. However, this notion is based on in vitro lipase activity data, which may not reflect the in vivo lipolytic activity. We investigated lipolysis of IMTG in soleus muscles electrically stimulated to contract ex vivo during acute pharmacological inhibition of HSL in rat muscles and in muscles from HSL knockout (HSL-KO) mice. Measurements of IMTG are complicated by the presence of adipocytes located between the muscle fibres. To circumvent the problem with this contamination we analysed intramyocellular lipid droplet content histochemically. At maximal inhibition of HSL in rat muscles, contraction-induced breakdown of IMTG was identical to that seen in control muscles (P < 0.001). In response to contractions IMTG staining decreased significantly in both HSL-KO and WT muscles (P < 0.05). In vitro TG hydrolase activity data revealed that adipose triglyceride lipase (ATGL) and HSL collectively account for ∼98% of the TG hydrolase activity in mouse skeletal muscle, other TG lipases accordingly being of negligible importance for lipolysis of IMTG. The present study is the first to demonstrate that contraction-induced lipolysis of IMTG occurs in the absence of HSL activity in rat and mouse skeletal muscle. Furthermore, the results suggest that ATGL is activated and plays a major role in lipolysis of IMTG during muscle contractions.

Figure 1. Effects of contraction time on IMTG degradation in rat soleus muscles

A, representative images of muscles in the basal state and electrically stimulated (ES) for 5 and 20 min, respectively. Results are expressed as total lipid doplet (LD) staining per area (B), number of LDs per 200 μm² muscle fibre area (C) and average LD size (nm²) (D) (n= 8); data are mean ± SEM. *P < 0.05 compared with corresponding basal; #P < 0.05 compared with ES 5 min; §P < 0.05 interaction between time and ES.

Figure 2. Effects of HSL inhibition in vitro and ex vivo

A, TG hydrolase activity in lysates of quadriceps muscles from HSL-KO and wild type (WT) mice incubated with vehicle (VEH) or increasing doses of a mono-specific, small molecule HSL inhibitor (76-0079). Data are expressed as mean ± SEM. B, effect of HSL inhibition on muscle DAG content in electrically stimulated rat soleus muscles (n= 6). Data are expressed as mean percentage of the basal control muscle ± SEM. *P < 0.001 compared with VEH within WT; #P < 0.05 compared with HSL-KO at the corresponding dose; ‡P < 0.05 compared with ES 0 and VEH.

Figure 3. Acute inhibition of HSL does not impair contraction-induced IMTG breakdown, glycogen breakdown or force production

A–C, rat soleus muscles were stimulated electrically to contract (ES) for 20 min or remained in the basal state in the presence of the specific HSL inhibitor 76-0079 (2 mm) or vehicle (VEH). Results are expressed as total LD staining per area (A), number of LDs per 200 μm² muscle fibre area (B) and average LD size (nm²) (C) (n= 9). D and E, glycogen content before and after ES (n= 9) (D) and force production during ES (n= 18) (E) in rat soleus muscles in the presence of the specific HSL-inhibitor 76-0079 (2 mm) or VEH. Data are expressed as mean ± SEM. *P < 0.001 compared with corresponding basal.

Figure 4. Contraction-induced IMTG breakdown is slightly impaired while glycogen breakdown and muscle contractile properties are not altered in HSL-KO mice

A, representative images of LDs stained in cross sections from basal and electrically stimulated (ES) muscles of HSL-KO mice and WT littermates. Results are expressed as total LD staining per area (B), average LD size (nm²) (C) and number of LDs per 200 μm² muscle fibre area (D) (n= 8). E and F, glycogen content (n= 8) before and after ES (E) and force production (n= 16) during ES (F) in soleus muscles of HSL-KO and WT littermates. Data are expressed as mean ± SEM. *P < 0.05 compared with corresponding basal; #P < 0.05 compared with HSL-KO, same condition; §P < 0.001 interaction between genotype and ES; ‡P < 0.001 main effect of ES.

Figure 5. Effects of contractions on muscle signalling proteins

Immunoblotting of rat soleus muscles before and after 20 min of electrically stimulated (ES) contractions in the presence of the specific HSL inhibitor 76-0079 (2 mm) or VEH (A–D) and mouse soleus muscles of HSL-KO and WT (E–H) before and after ES. Values for protein phosphorylation are corrected for the corresponding total protein. Data are expressed as mean ± SEM, n= 8–9 per group. ‡P < 0.01 main effect of ES. AU, arbitrary units.

Figure 6. TG hydrolase activities and protein expressions

A, TG hydrolase activity in lysates of quadriceps muscles from ATGL-KO and wild-type (WT) littermates incubated with the specific HSL inhibitor 76-0079 or vehicle (VEH). B–E, protein expression of ATGL (C) and ATGL activity regulating proteins CGI-58 (D) and G0S2 (E) in mouse soleus muscles of HSL-KO and WT mice before and after electrically stimulated (ES) contractions. No HSL protein was detected in muscles of HSL-KO mice (B). For protein expression, values are normalized to actin to correct for potential unequal protein loading. Data are expressed as mean ± SEM, n= 8 per group. *P < 0.001 compared with VEH, same genotype; #P < 0.001 compared with WT at the corresponding dose. AU, arbitrary units.