Morten Donsmark

Decrease in intramuscular lipid droplets and translocation of HSL in response to muscle contraction and epinephrine

A better understanding of skeletal muscle lipid metabolism is needed to identify the molecular mechanisms relating intramuscular triglyceride (IMTG) to muscle metabolism and insulin sensitivity. An increasing number of proteins have been reported to be associated with intracellular triglyceride (TG), among them the PAT family members: perilipin, ADRP (for adipocyte differentiation-related protein), and TIP47 (for tail-interacting protein of 47 kDa). Hormone-sensitive lipase (HSL) is thought to be the major enzyme responsible for IMTG hydrolysis in skeletal muscle. In adipocytes, regulation of HSL by intracellular redistribution has been demonstrated. The existence of such regulatory mechanisms in skeletal muscle has long been hypothesized but has never been demonstrated. The aim of this study was to characterize the PAT family proteins associated with IMTG and to investigate the effect of epinephrine stimulation or muscle contraction on skeletal muscle TG content and HSL intracellular distribution. Rat soleus muscles were either incubated with epinephrine or electrically stimulated for 15 min. Single muscle fibers were used for morphological analysis by confocal and transmission electron microscopy. We show a decrease in IMTG in response to both lipolytic stimuli. Furthermore, we identify two PAT family proteins, ADRP and TIP47, associated with IMTG. Finally, we demonstrate HSL translocation to IMTG and ADRP after stimulation with epinephrine or contraction.

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

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%). α2AMPK 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)α2AMPK 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.

Denervation and High-Fat Diet Reduce Insulin Signaling in T-Tubules in Skeletal Muscle of Living Mice

OBJECTIVE—Insulin stimulates muscle glucose transport by translocation of GLUT4 to sarcolemma and T-tubules. Despite muscle glucose uptake playing a major role in insulin resistance and type 2 diabetes, the temporal and spatial changes in insulin signaling and GLUT4 translocation during these conditions are not well described. RESEARCH DESIGN AND METHODS—We used time-lapse confocal imaging of green fluorescent protein (GFP) ADP-ribosylation factor nucleotide-binding site opener (ARNO) (evaluation of phosphatidylinositide 3-kinase activation) and GLUT4-GFP–transfected quadriceps muscle in living, anesthetized mice either muscle denervated or high-fat fed. T-tubules were visualized with sulforhodamine B dye. In incubated muscle, glucose transport was measured by 2-deoxy-d-[3H]-glucose uptake, and functional detubulation was carried out by osmotic shock. Muscle fibers were immunostained for insulin receptors. RESULTS—Denervation and high-fat diet reduced insulin-mediated glucose transport. In denervated muscle, insulin-stimulated phosphatidylinositol 3,4,5 P3 (PIP3) production was abolished in T-tubules, while PIP3 production at sarcolemma was increased 2.6-fold. Correspondingly, GLUT4-GFP translocation to T-tubules was abolished, while translocation to sarcolemma was increased 2.3-fold. In high fat–fed mice, a ∼65% reduction in both insulin-induced T-tubular PIP3 production and GLUT4-GFP translocation was seen. Sarcolemma was less affected, with reductions of ∼40% in PIP3 production and ∼15% in GLUT4-GFP translocation. Access to T-tubules was not compromised, and insulin receptor distribution in sarcolemma and T-tubules was unaffected by denervation or high-fat feeding. Detubulation of normal muscle reduced basal and abolished insulin-induced glucose transport. CONCLUSIONS—Our findings demonstrate, for the first time, that impaired insulin signaling and GLUT4 translocation is compartmentalized in muscle and primarily localized to T-tubules and not sarcolemma during insulin resistance.

Regulation and role of hormone-sensitive lipase in rat skeletal muscle

Intramyocellular triacylglycerol (TG) is an important energy store, and the energy content of this depot is higher than the energy content of the muscle glycogen depot. It has recently been shown that the mobilization of fatty acids from this TG pool may be regulated by the neutral lipase hormone-sensitive lipase (HSL). This enzyme is known to be rate limiting for intracellular TG hydrolysis in adipose tissue. The presence of HSL has been demonstrated in all muscle fibre types by Western blotting of muscle fibres isolated by collagenase treatment or after freeze-drying. The content of HSL varies between fibre types, being higher in oxidative fibres than in glycolytic fibres. When analysed under conditions optimal for HSL, neutral lipase activity in muscle can be stimulated by adrenaline as well as by contractions. These increases are abolished by the presence of anti-HSL antibody during analysis. Moreover, immunoprecipitation with affinity-purified anti-HSL antibody causes similar reductions in muscle HSL protein concentration and in measured neutral lipase responses to contractions. The immunoreactive HSL in muscle is stimulated by adrenaline via beta-adrenergic activation of cAMP-dependent protein kinase (PKA). From findings in adipocytes it is likely that PKA phosphorylates HSL at residues Ser(563), Ser(659) and Ser(660). Contraction probably also enhances muscle HSL activity by phosphorylation, because the contraction-induced increase in HSL activity is elevated by the protein phosphatase inhibitor okadaic acid and reversed by alkaline phosphatase. A novel signalling pathway in muscle by which HSL activity may be stimulated by protein kinase C (PKC) via extracellular signal-regulated kinase (ERK) has been demonstrated. In contrast to previous findings in adipocytes, in muscle the activation of ERK is not necessary for stimulation of HSL by adrenaline. However, contraction-induced HSL activation is mediated by PKC, at least partly via the ERK pathway. In fat cells ERK is known to phosphorylate HSL at Ser(600). Hence, phosphorylation of different sites may explain the finding that in muscle the effects of contractions and adrenaline on HSL activity are partially additive. In line with the view that the two stimuli act by different mechanisms, training increases contraction-mediated HSL activation but diminishes adrenaline-mediated HSL activation in muscle. In conclusion, HSL is present in skeletal muscle and can be activated by phosphorylation in response to both adrenaline and muscle contractions. Training increases contraction-mediated HSL activation, but decreases adrenaline-mediated HSL activation in muscle.

Hormone-sensitive lipase as mediator of lipolysis in contracting skeletal muscle.

The authors propose that the enzyme hormone-sensitive lipase (HSL), which is the rate-limiting enzyme for hydrolysis of triacylglycerol in adipocytes, also regulates the intramyocellular triacylglycerol mobilization and is controlled by mechanisms similar to those regulating glycogen phosphorylase. From an exercise perspective, it is fascinating that the primary enzymatic setting allows simultaneous mobilization of all major extramuscular and intramuscular energy stores.

Hormone-sensitive Lipase (HSL) Expression and Regulation By Epinephrine and Exercise in Skeletal Muscle

Triacylglycerol (TG) is stored in lipid droplets in the cytoplasm of skeletal muscle. The energy content of the TG depot is higher than the energy content of the muscle glycogen depot. The enzymatic regulation of intracellular TG hydrolysis in skeletal muscle has not been elucidated. Therefore, we investigated the expression and the regulation of hormone-sensitive lipase (HSL) in skeletal muscle. This enzyme is a neutral lipase and known as the rate-limiting enzyme of intracellular TG hydrolysis in adipose tissue. The total and the activated form of the neutral lipase are referred to as MOME and TO, respectively. In isolated rat skeletal muscle fibers, the presence of HSL was demonstrated by Western blotting. The expression of HSL was correlated to fiber type, being higher in oxidative than in glycolytic fibers. In incubated soleus and extensor digitorum longus (EDL) muscles stimulation with epinephrine or electrically induced contractions increased neutral lipase activity against triolein (TO), but not against a diacylglycerol analogue (MOME). Glycogen phosphorylase activity increased in parallel with TO activity. No measurable increase in muscle homogenate TO activity existed in the presence of an anti-HSL antibody. The effect of epinephrine could be blocked by propanolol and mimicked by incubation of a crude supernatant from control muscle with the catalytic subunit of cAMP-dependent protein kinase. The effect of contractions was transient as TO activity declined to basal levels after 10 min of electrical stimulation. Indicating involvement of protein kinase C the effect of contractions was abolished by Calphostin C. Okadaic acid doubled the contraction-mediated increase in TO activity, whereas the increase was reversed by phosphatase treatment. The effects of epinephrine and contractions were partially additive. In rats training increased epinephrine-stimulated TO activity and HSL concentration in adipose tissue but not in muscle. In humans, at the end of 60 min of exercise muscle, TO activity was increased in healthy, but not in adrenalectomized, subjects. In conclusion, HSL is present in skeletal muscle and can be activated by phosphorylation by both epinephrine and muscle contractions. In addition, HSL and glycogen phosphorylase are stimulated in parallel in muscle indicating simultaneous activation of triacylglycerol and glycogen breakdown.