Regulation of hormone-sensitive lipase activity and Ser563 and Ser565 phosphorylation in human skeletal muscle during exercise

Abstract

Hormone-sensitive lipase (HSL) catalyses the hydrolysis of myocellular triacylglycerol (MCTG), which is a potential energy source during exercise. Therefore, it is important to elucidate the regulation of HSL activity in human skeletal muscle during exercise. The main purpose of the present study was to investigate the role of 5'AMP-activated protein kinase (AMPK) in the regulation of muscle HSL activity and Ser565 phosphorylation (the presumed AMPK target site) in healthy, moderately trained men during 60 min bicycling (65%). Alpha2AMPK activity during exercise was manipulated by studying subjects with either low (LG) or high (HG) muscle glycogen content. HSL activity was distinguished from the activity of other neutral lipases by immunoinhibition of HSL using an anti-HSL antibody. During exercise a 62% higher (P < 0.01) alpha2AMPK activity in LG than in HG was paralleled by a similar difference (61%, P < 0.01) in HSL Ser565 phosphorylation but without any difference between trials in HSL activity or MCTG hydrolysis. HSL activity was increased (117%, P < 0.05) at 30 min of exercise but not at 60 min of exercise. In both trials, HSL phosphorylation on Ser563 (a presumed PKA target site) was not increased by exercise despite a fourfold increase (P < 0.001) in plasma adrenaline. ERK1/2 phosphorylation was increased by exercise in both trials (P < 0.001) and was higher in LG than in HG both at rest and during exercise (P = 0.06). In conclusion, the present study suggests that AMPK phosphorylates HSL on Ser565 in human skeletal muscle during exercise with reduced muscle glycogen. Apparently, HSL Ser565 phosphorylation by AMPK during exercise had no effect on HSL activity. Alternatively, other factors including ERK may have counterbalanced any effect of AMPK on HSL activity.

Figure 1. Testing of HSL antibodies

A, representative Western blots of HSL total protein and of HSL phosphorylated on Ser⁵⁶³ or Ser⁵⁶⁵ in human skeletal muscle and in human paraumbilical adipose tissue sampled at rest. SM, skeletal muscle; AT, adipose tissue. B, specificity of anti-HSL Ser⁵⁶³-phos and anti-HSL Ser⁵⁶⁵-phos tested using recombinant rat HSL. HSL was incubated with MgATP either without added kinase, with AMPK, with PKA, with AMPK followed by PKA, or with PKA followed by AMPK. The protein was analysed by SDS-PAGE and blots were probed with anti-HSL Ser⁵⁶³-phos, anti-HSL Ser⁵⁶⁵-phos, or a non-phosphospecific anti-HSL antibody.

Figure 2. AMPK phosphorylation and activity in the vastus lateralis muscle at rest and after 60 min bicycling at 65% V̇_o2peak with low (LG) or high (HG) muscle glycogen

A, αAMPK Thr¹⁷² phosphorylation. B, α₁AMPK activity. C, α₂AMPK activity. *Different from HG, P < 0.01; # different from rest, P < 0.01.

Figure 3. HSL total protein, activity and phosphorylation in the vastus lateralis muscle at rest and after 60 min bicycling at 65% V̇_o2peak with low (LG) or high (HG) muscle glycogen

A, HSL protein. B, HSL activity calculated as neutral lipase activity measured in the absence of anti-HSL antibody minus neutral lipase activity measured in the presence of anti-HSL antibody. C, HSL Ser⁵⁶⁵ phosphorylation. D, HSL Ser⁵⁶³ phosphorylation. *Different from HG, P < 0.01; # different from rest, P < 0.01; † different from rest and 60 min exercise, P < 0.05.

Figure 4. ERK1/2 phosphorylation in the vastus lateralis muscle at rest and after 60 min bicycling at 65% with low (LG) or high (HG) muscle glycogen

A, ERK1 Thr²⁰²/Tyr²⁰⁴ phosphorylation. B, ERK2 Thr¹⁸⁵/Tyr¹⁸⁷ phosphorylation. ^#Different from rest, P < 0.01.