In experiments with PKC and MEK inhibitors or phorbol-12-myristate-13-acetate (PMA) (see below), control soleus muscles were incubated with the same concentration of DMSO as used in media with inhibitors or PMA. In experiments with muscle contractions and corresponding controls (Figs 1, ?,2,2, ?,33 and ?and7)7) the muscles were fixed Rabbit Polyclonal to IL11RA in the vertical position at resting length by small clips attached to the tendons. activity. Activated Picoprazole ERK increased Picoprazole HSL activity in supernatant from basal but not from electrically stimulated muscle. In conclusion, in muscle, PKC can stimulate HSL through ERK. Contractions and adrenaline enhance muscle HSL activity by different signalling mechanisms. The effect of contractions is mediated by PKC, at least partly via the ERK pathway. Triacylglycerol contained in lipid droplets in the cytoplasm of skeletal muscle cells represents an important energy store, which can be mobilized by catecholamines and exercise (Carlson 1971; Reitman 1973; Froberg 1975; Abumrad 1980; Oscai 1990; Schick 1993; Van der Vusse & Reneman, 1996). However, the enzymatic regulation of muscle triacylglycerol breakdown has until recently been poorly understood. Then the presence of the neutral lipase hormone-sensitive lipase (HSL), which controls triacylglycerol breakdown in adipose tissue, was demonstrated by Western blotting in all muscle fibre types (Langfort 1999). Furthermore, it was shown that analysed under conditions optimal for HSL, neutral lipase activity in muscle can be increased by adrenaline as well as by muscle contractions, and these increases were abolished by the presence of anti-HSL anti-body during analysis (Langfort 1999, 2000). Moreover, immunoprecipitation with affinity-purified anti-HSL antibody caused similar reductions in muscle HSL protein concentration and in measured neutral lipase responses to contractions (Langfort 2000). Adrenaline has been shown to stimulate Picoprazole HSL in muscle via -adrenergic activation of PKA (Langfort 1999). From findings in adipocytes it is likely that PKA phosphorylates HSL at residues Ser563, Ser659 and Ser660, the latter two being the major activity controlling sites (Anthonsen 1998). Contractions probably also enhance muscle-HSL activity by phosphorylation, because the contraction-induced increase in HSL activity is increased by the protein phosphatase inhibitor okadaic acid and reversed by alkaline phosphatase (Langfort 2000). However, the signalling mechanisms mediating such an effect of contractions are not known. The fact that the effects of adrenaline and contractions on HSL activity in muscle are partially additive (Langfort 2003) may suggest that the two stimuli activate different kinases, which, in turn, phosphorylate HSL at different sites. Because an increase in intracellular Ca2+ is characteristic of contractions, and because HSL activity increases at the onset of contractions (Langfort 2000), it is tempting to speculate that contraction-induced HSL activation is mediated by a Ca2+-dependent protein kinase. Ca2+/calmodulin-dependent protein kinase II has been shown to inhibit rather than enhance HSL from adipose tissue (Garton 1989), a fact that makes Ca2+-activated protein kinase C (PKC) a more likely mediator of contraction-induced HSL activation. Because the regulation of HSL in skeletal muscle is probably of physiological as well as pathophysiological importance, we wanted to unravel the signalling mechanisms involved in contraction-induced HSL activation. While we were doing this by applying PKC inhibitors in incubated, electrically stimulated rat muscle, it was reported that, in adipocytes, PKC can stimulate lipolysis through a mitogen-activated protein kinase (MAPK)/ERK-mediated phosphorylation of HSL (Greenberg 2001). Interestingly, this novel mechanism involves a new phosphorylation site, Ser600, located in close proximity to the already known phosphorylation sites within the regulatory module of HSL (Anthonsen 1998; Greenberg 2001). In the present study, we found that in muscle also, PKC can stimulate HSL through ERK. Furthermore, the contraction-induced HSL activation is mediated by PKC, at least partly via the ERK pathway. Finally, in adipocytes, -adrenergic activation of HSL is in part mediated via ERK (Greenberg 2001), while in muscle, activation of ERK does not contribute to adrenaline-induced stimulation of HSL. METHODS Muscle incubation and stimulation The experiments were approved by the Animal Research Committee of the Danish Ministry of Justice. Male Wistar rats weighing about 70 g were obtained from Charles River Laboratories (Sulzfeld, Germany). Young rats were used to avoid adipocytes interlaced between muscle.