Muscle Hormone-sensitive Lipase Research Articles

Effects of fasting on the activities and mRNA expression levels of lipoprotein lipase (LPL), hormone-sensitive lipase (HSL) and fatty acid synthetase (FAS) in spotted seabass Lateolabrax maculatus.

To investigate the effects of fasting on lipid metabolism in spotted seabass muscle and liver tissues, we analyzed mRNA levels and enzyme activities of lipoprotein lipase (LPL), hormone-sensitive lipase (HSL) and fatty acid synthetase (FAS), and the relationship among fat content, mRNA level, and enzyme activity during fasting of 35days. The results showed that expressions of all the three genes were ubiquitous. During the fasting experiment, the hepatosomatic index (HSI) and fat content of muscle and liver tissues significantly decreased before 5days of fasting (P < 0.05). mRNA levels of LPL increased significantly after 5days of fasting in liver and 7days in muscle. Abundance of HSL transcripts increased significantly after 14days of fasting in both muscle and liver. The activities of LPL and HSL presented a trend that increased firstly, decreased subsequently, and then raised again with the prolonged fasting experiment (P < 0.05). However, activities and mRNA levels of FAS decreased significantly after 1day of fasting in both muscle and liver. Moreover, activities and mRNA levels of FAS showed a moderate correlation in muscle. These results suggested that FAS had a sooner response to fasting than LPL and HSL in both muscle and liver tissues. LPL and HSL played important roles in lipolysis mainly by increasing enzyme activities in the early stage of fasting and mRNA levels in the later stage of fasting in both muscle and liver. Our results also provided useful information on regulating muscle fat content by fasting.

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

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.

Altered skeletal muscle lipase expression and activity contribute to insulin resistance in humans.

OBJECTIVEInsulin resistance is associated with elevated content of skeletal muscle lipids, including triacylglycerols (TAGs) and diacylglycerols (DAGs). DAGs are by-products of lipolysis consecutive to TAG hydrolysis by adipose triglyceride lipase (ATGL) and are subsequently hydrolyzed by hormone-sensitive lipase (HSL). We hypothesized that an imbalance of ATGL relative to HSL (expression or activity) may contribute to DAG accumulation and insulin resistance.RESEARCH DESIGN AND METHODSWe first measured lipase expression in vastus lateralis biopsies of young lean (n = 9), young obese (n = 9), and obese-matched type 2 diabetic (n = 8) subjects. We next investigated in vitro in human primary myotubes the impact of altered lipase expression/activity on lipid content and insulin signaling.RESULTSMuscle ATGL protein was negatively associated with whole-body insulin sensitivity in our population (r = −0.55, P = 0.005), whereas muscle HSL protein was reduced in obese subjects. We next showed that adenovirus-mediated ATGL overexpression in human primary myotubes induced DAG and ceramide accumulation. ATGL overexpression reduced insulin-stimulated glycogen synthesis (−30%, P < 0.05) and disrupted insulin signaling at Ser1101 of the insulin receptor substrate-1 and downstream Akt activation at Ser473. These defects were fully rescued by nonselective protein kinase C inhibition or concomitant HSL overexpression to restore a proper lipolytic balance. We show that selective HSL inhibition induces DAG accumulation and insulin resistance.CONCLUSIONSAltogether, the data indicate that altered ATGL and HSL expression in skeletal muscle could promote DAG accumulation and disrupt insulin signaling and action. Targeting skeletal muscle lipases may constitute an interesting strategy to improve insulin sensitivity in obesity and type 2 diabetes.

Skeletal Muscle Lipase Content and Activity in Obesity and Type 2 Diabetes

The obese insulin-resistant state is characterized by elevated lipid storage in skeletal muscle tissue. We tested whether differences in muscle triacylglycerol (TAG) and diacylglycerol (DAG) lipase content and activity are associated with incomplete in vivo lipolysis and lipid accumulation. Two case-control studies were conducted on skeletal muscle biopsies from lean (n=13) and obese (n=10) men (study 1) and from 11 nonobese type 2 diabetic (T2D), obese T2D, and healthy normoglycemic men (study 2). Skeletal muscle lipase protein content and activity and muscle lipid content (TAG and DAG) were determined. Skeletal muscle hormone-sensitive lipase protein content was lower (0.39±0.07 vs. 1.00±0.19 arbitrary units; P=0.004) and adipose triglyceride lipase protein content was higher in obese men compared with lean controls (2.17±0.40 vs. 0.42±0.23 arbitrary units; P=0.008). This apparent difference in lipase content was accompanied by a 60% lower ratio of DAG to TAG hydrolase activity in the obese men (11.4±2.3 vs. 26.5±7.3 nmol/h·mg; P=0.045), implying incomplete lipolysis. Lower hormone-sensitive lipase and higher adipose triglyceride lipase content was confined to obesity per se, because it was observed solely in obese T2D men but not in healthy normoglycemic controls and nonobese T2D men. Muscle total DAG content was not higher in obese men but was even lower (6.2±0.7 vs. 9.4±0.9 μmol/mg dry weight; P=0.017). TAG content did not differ between groups (84.7±18.9 vs. 70.4±12.4 μmol/mg dry weight; P=0.543). Our data do not support an important role of total muscle DAG content in the development of insulin resistance in obese men.

Effect of starvation on activities and mRNA expression of lipoprotein lipase and hormone-sensitive lipase in tilapia (Oreochromis niloticus × O. areus)

A 4-week study was conducted to determine the effect of starvation on activities and mRNA expression of lipoprotein lipase (LPL) and hormone-sensitive lipase (HSL) in hybrid tilapia (Oreochromis niloticus x O. areus). The tissue samples were sampled once a week. Results showed that body weight (BW) and hepatosomatic index (HSI) were decreased significantly (P < 0.05) during starvation. The percentages of crude fat and crude protein in the whole body and the fat content in muscle decreased significantly (P < 0.05), while the rate of moisture and crude ash increased significantly (P < 0.05). The response of LPL, HSL activities and mRNA expression in tissues was tissue dependent. The activities of LPL and HSL in muscle at day 7 were elevated by 2.5 times (P < 0.05) and 11.8 times (P < 0.05) of the value at day 0, respectively, and both then decreased to pre-starvation levels at day 14 and finally stabilized at a certain level afterward. LPL and HSL mRNA abundance in muscle remained relatively stable between 0 and 14 day; then, a significant increase was seen after 14 days. In the liver, LPL activity maintained a significantly increasing trend during starvation, while HSL activity rose dramatically at day 7 of starvation by 2.35 times (P < 0.05) and finally stabilized at a certain level. The mRNA abundance of liver LPL increased significantly during the whole process of starvation (P < 0.05), whereas the mRNA abundance of liver HSL decreased significantly at day 7 of starvation, elevating significantly afterward (P < 0.05).

Increased rate of whole body lipolysis before and after 9 days of bed rest in healthy young men born with low birth weight

Individuals born with low birth weight (LBW) are at risk of developing type 2 diabetes mellitus (T2D), which may be precipitated by physical inactivity. Twenty-two LBW subjects and twenty-three controls were studied before and after bed rest by the hyperinsulinemic euglycemic clamp combined with indirect calorimetry and infusion of stable isotope tracers and preceded by an intravenous glucose tolerance test. LBW subjects had a similar body mass index but elevated abdominal obesity compared with controls. The basal rate of whole body lipolysis (WBL) was elevated in LBW subjects with and without correction for abdominal obesity before and after bed rest (all P = 0.01). Skeletal muscle hormone-sensitive lipase (HSL) protein expression and phosphorylation at Ser565 were similar in the two groups. Bed rest resulted in a decrease in WBL and an increased skeletal muscle HSL Ser565 phosphorylation indicating a decreased HSL activity in both groups. All subjects developed peripheral insulin resistance in response to bed rest (all P < 0.0001) with no differences between groups. LBW subjects developed hepatic insulin resistance in response to bed rest. In conclusion, increased WBL may contribute to the development of hepatic insulin resistance when exposed to bed rest in LBW subjects. Nine days of bed rest causes severe peripheral insulin resistance and reduced WBL and skeletal muscle HSL activity, as well as a compensatory increased insulin secretion, with no differences in LBW subjects and controls.

Low‐intensity training dissociates metabolic from aerobic fitness

This study investigated the effect of prolonged whole-body low-intensity exercise on blood lipids, skeletal muscle adaptations and aerobic fitness. Seven male subjects completed a 32-day crossing of the Greenland icecap on cross-country skies and before and after this arm or leg cranking was performed on two separate days and biopsies were obtained from arm and leg muscle, and venous blood was sampled. During the crossing, subjects skied for 342+/-42 min/day and body mass was decreased by 7.1+/-0.7 kg. Peak leg oxygen uptake (4.6+/-0.2 L/min) was decreased (P<0.05) by 7% whereas peak arm oxygen uptake (3.0+/-0.2 L/min) remained unchanged. Total and low-density lipoprotein cholesterol (5.0+/-0.2 and 3.20.2 mmol/L) were decreased by 8% and 20%, respectively. Muscle beta-hydroxy-acyl-CoA dehydrogenase activity was increased with 22% in arm (P=0.08) and remained unchanged in leg muscle. Hormone sensitive lipase activity was similar in arm and leg muscle prior to the expedition and was not significantly affected by the crossing. In conclusion, an improved blood lipid profile and thus metabolic fitness was present after prolonged low-intensity training and this occurred in spite of a decreased aerobic fitness and an unchanged arm and leg muscle hormone-sensitive lipase activity.

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.

Sex differences in hormone-sensitive lipase expression, activity, and phosphorylation in skeletal muscle at rest and during exercise

Women have been shown to use more intramuscular triacylglycerol (IMTG) during exercise than men. To investigate whether this could be due to sex-specific regulation of hormone-sensitive lipase (HSL) and to use sex comparison as a model to gain further insight into HSL regulation, nine women and eight men performed bicycle exercise (90 min, 60% Vo(2peak)), and skeletal muscle HSL expression, phosphorylation, and activity were determined. Supporting previous findings, basal IMTG content (P < 0.001) and net IMTG decrease during exercise (P < 0.01) were higher in women than in men and correlated significantly (r = 0.72, P = 0.001). Muscle HSL mRNA (80%, P = 0.11) and protein content (50%, P < 0.05) were higher in women than in men. HSL total activity increased during exercise (47%, P < 0.05) but did not differ between sexes. Accordingly, HSL specific activity (HSL activity per HSL protein content) increased during exercise (62%, P < 0.05) and was generally higher in men than in women (82%, P < 0.05). A similar pattern was observed for HSL Ser(659) phosphorylation, suggesting a role in regulation of HSL activity. Likewise, plasma epinephrine increased during exercise (P < 0.05) and was higher in men than in women during the end of the exercise bout (P < 0.05). We conclude that, although HSL expression and Ser(659) phosphorylation in skeletal muscle during exercise is sex specific, total muscle HSL activity measured in vitro was similar between sexes. The higher basal IMTG content in women compared with men is therefore the best candidate to explain the higher IMTG net hydrolysis during exercise in women.

Muscle triacylglycerol and hormone-sensitive lipase activity in untrained and trained human muscles

During exercise, triacylglycerol (TG) is recruited in skeletal muscles. We hypothesized that both muscle hormone-sensitive lipase (HSL) activity and TG recruitment would be higher in trained than in untrained subjects in response to prolonged exercise. Healthy male subjects (26 +/- 1 years, body moss index 23.3 +/- 0.5 kg m(-2)), either untrained (N = 8, VO(2max) 3.8 +/- 0.2 l min(-1)) or trained (N = 8, VO(2max) 5.1 +/- 0.1 l min(-1)), were studied. Before and after 3-h exercise (58 +/- 1% VO(2max)), a biopsy was taken. Muscle citrate synthase (32 +/- 2 vs. 47 +/- 6 mumol g(-1) min(-1) d.w.) and beta-hydroxy-acyl-CoA-dehydrogenase (38 +/- 3 vs. 52 +/- 5 mumol g(-1) min(-1) d.w.) activities were lower in untrained than in trained subjects (p < 0.05). Throughout the exercise, fat oxidation was higher in trained than in untrained subjects (p < 0.05). Muscle HSL activity was similar at rest (0.72 +/- 0.08 and 0.74 +/- 0.03 mU mg(-1) protein) and after exercise (0.71 +/- 0.1 and 0.68 +/- 0.03 mU mg(-1) protein) in untrained and trained subjects. At rest, the chemically determined muscle TG content (37 +/- 8 and 26 +/- 5 mmol g(-1) d.w.) was similar (p > 0.05), and after exercise it was unchanged in untrained and lower (p < 0.05) in trained subjects (41 +/- 9 and 10 +/- 2 mmol g((1) d.w.). Determined histochemically, TG was decreased (p < 0.05) after exercise in type I and II fibres. Depletion of TG was not different between fibre types in untrained, but tended to be higher (p = 0.07) in type I compared with type II fibres in trained muscles. In conclusion, HSL activity is similar in untrained and trained skeletal muscles both before and after prolonged exercise. However, the tendency to higher muscle TG recruitment during exercise in the trained subjects suggests a difference in the regulation of HSL or other lipases during exercise in trained compared with untrained subjects.

Adrenergic regulation of HSL serine phosphorylation and activity in human skeletal muscle during the onset of exercise

Skeletal muscle hormone-sensitive lipase (HSL) activity is increased by contractions and increases in blood epinephrine (EPI) concentrations and cyclic AMP activation of the adrenergic pathway during prolonged exercise. To determine the importance of hormonal stimulation of HSL activity during the onset of moderate- and high-intensity exercise, nine men [age 24.3 +/- 1.2 yr, 80.8 +/- 5.0 kg, peak oxygen consumption (VO2 peak) 43.9 +/- 3.6 ml x kg(-1) x min(-1)] cycled for 1 min at approximately 65% VO2 peak, rested for 60 min, and cycled at approximately 90% VO2 peak for 1 min. Skeletal muscle biopsies were taken pre- and postexercise, and arterial blood was sampled throughout exercise. Arterial EPI increased (P < 0.05) postexercise at 65% (0.45 +/- 0.10 to 0.78 +/- 0.27 nM) and 90% VO2 peak (0.57 +/- 0.34 to 1.09 +/- 0.50 nM). HSL activity increased (P < 0.05) following 1 min of exercise at 65% VO2 peak [1.05 +/- 0.39 to 1.78 +/- 0.54 mmol x min(-1) x kg dry muscle (dm)(-1)] and 90% VO2 peak (1.07 +/- 0.24 to 1.91 +/- 0.62 mmol x min(-1) x kg dm(-1)). Cyclic AMP content also increased (P < 0.05) at both exercise intensities (65%: 1.52 +/- 0.67 to 2.75 +/- 1.12, 90%: 1.85 +/- 0.65 to 2.64 +/- 0.93 micromol/kg dm). HSL Ser660 phosphorylation (approximately 55% increase) and ERK1/2 phosphorylation ( approximately 33% increase) were augmented following exercise at both intensities, whereas HSL Ser563 and Ser565 phosphorylation were not different from rest. The results indicate that increases in arterial EPI concentration during the onset of moderate- and high-intensity exercise increase cyclic AMP content, which results in the phosphorylation of HSL Ser660. This adrenergic stimulation contributes to the increase in HSL activity that occurs in human skeletal muscle in the first minute of exercise at 65% and 90% VO2 peak.

Transcriptome and proteome analysis of soleus muscle of hormone-sensitive lipase-null mice

Hormone-sensitive lipase (HSL), a key enzyme in fatty acid mobilization in adipocytes, has been demonstrated also in skeletal muscle. To gain further insight into the role and importance of HSL in skeletal muscle, a transcriptome analysis of soleus muscle of HSL-null mice was performed. A total of 161 transcripts were found to be differentially expressed. Increased mRNA levels of fructose-1,6-bisphosphatase, fructose-2,6-bisphosphatase, and phosphorylase kinase gamma1A suggest a higher glycogen flux in soleus muscle of HSL-null mice. An observed increase in the utilization of glycogen stores supports this finding. Moreover, an increased amount of intramyocellular lipid droplets, observed by transmission electron microscopy, suggests decreased mobilization of lipid stores in HSL-null mice. To complement the transcriptome data, protein expression analysis was performed. Five spots were found to be differentially expressed: pyruvate dehydrogenase E1alpha, creatine kinase (CK), ankyrin-repeat domain 2, glyceraldehyde-3-phosphate dehydrogenase, and one protein yet to be identified. The increased protein level of CK indicates creatine phosphate degradation to be of increased importance in HSL-null mice. The results of this study suggest that in the absence of HSL, a metabolic switch from reliance on lipid to carbohydrate energy substrates takes place, supporting an important role of HSL in soleus muscle lipid metabolism.

Regulation of HSL serine phosphorylation in skeletal muscle and adipose tissue

Hormone-sensitive lipase (HSL) is important for the degradation of triacylglycerol in adipose and muscle tissue, but the tissue-specific regulation of this enzyme is not fully understood. We investigated the effects of adrenergic stimulation and AMPK activation in vitro and in circumstances where AMPK activity and catecholamines are physiologically elevated in humans in vivo (during physical exercise) on HSL activity and phosphorylation at Ser(563) and Ser(660), the PKA regulatory sites, and Ser(565), the AMPK regulatory site. In human experiments, skeletal muscle, subcutaneous adipose and venous blood samples were obtained before, at 15 and 90 min during, and 120 min after exercise. Skeletal muscle HSL activity was increased by approximately 80% at 15 min compared with rest and returned to resting rates at the cessation of and 120 min after exercise. Consistent with changes in plasma epinephrine, skeletal muscle HSL Ser(563) and Ser(660) phosphorylation were increased by 27% at 15 min (P < 0.05), remained elevated at 90 min, and returned to preexercise values postexercise. Skeletal muscle HSL Ser(565) phosphorylation and AMPK signaling were increased at 90 min during, and after, exercise. Phosphorylation of adipose tissue HSL paralleled changes in skeletal muscle in vivo, except HSL Ser(660) was elevated 80% in adipose compared with 35% in skeletal muscle during exercise. Studies in L6 myotubes and 3T3-L1 adipocytes revealed important tissue differences in the regulation of HSL. AMPK inhibited epinephrine-induced HSL activity in L6 myotubes and was associated with reduced HSL Ser(660) but not Ser(563) phosphorylation. HSL activity was reduced in L6 myotubes expressing constitutively active AMPK, confirming the inhibitory effects of AMPK on HSL activity. Conversely, in 3T3-L1 adipocytes, AMPK activation after epinephrine stimulation did not prevent HSL activity or glycerol release, which coincided with maintenance of HSL Ser(660) phosphorylation. Taken together, these data indicate that HSL activity is maintained in the face of AMPK activation as a result of elevated HSL Ser(660) phosphorylation in adipose tissue but not skeletal muscle.

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%). 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.

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.

Contractions induce phosphorylation of the AMPK site Ser 565 in hormone-sensitive lipase in muscle

Intramyocellular triglyceride is an important energy store which is related to insulin resistance. Mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by epinephrine via PKA and by contractions via PKC and ERK. 5 ′ AMP-activated protein kinase (AMPK) is an intracellular fuel gauge which regulates metabolism. In this study we incubated rat soleus muscle to investigate if AMPK influences HSL during 5 min of repeated tetanic contractions. An eightfold increase in AMPK activity was accompanied by a 2.5-fold increase in phosphorylation of the AMPK-site Ser 565 in HSL ( p<0.05). Inhibition of PKC by Calphostin C abolished the contraction-mediated HSL activation while HSL-Ser 565 phosphorylation was not reduced. The study indicates that during contractions AMPK phosphorylates HSL in Ser 565, but this phosphorylation is not directly responsible for the contraction-induced activation of HSL.

Contractions activate hormone-sensitive lipase in rat muscle by protein kinase C and mitogen-activated protein kinase.

Intramuscular triacylglycerol is an important energy store and is also related to insulin resistance. The mobilization of fatty acids from this pool is probably regulated by hormone-sensitive lipase (HSL), which has recently been shown to exist in muscle and to be activated by both adrenaline and contractions. Adrenaline acts via cAMP-dependent protein kinase (PKA). The signalling mediating the effect of contractions is unknown and was explored in this study. Incubated soleus muscles from 70 g male rats were electrically stimulated to perform repeated tetanic contractions for 5 min. The contraction-induced activation of HSL was abolished by the protein kinase C (PKC) inhibitors bisindolylmaleimide I and calphostin C and reduced 50% by the mitogen-activated protein kinase kinase (MEK) inhibitor U0126, which also completely blocked extracellular signal-regulated kinase (ERK) 1 and 2 phosphorylation. None of the inhibitors reduced adrenaline-induced HSL activation in soleus muscle. Both phorbol-12-myristate-13-acetate (PMA), which activates PKC and, in turn, ERK, and caffeine, which increases intracellular Ca2+ without eliciting contraction, increased HSL activity. Activated ERK increased 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.

Hormone-sensitive lipase in skeletal muscle: regulatory mechanisms.

The enzymatic regulation of intramuscular triacylglycerol (TG) breakdown has until recently not been well understood. Our aim was to elucidate the role of hormone-sensitive lipase (HSL), which controls TG breakdown in adipose tissue. Isolated rat muscle as well as exercising humans were studied. The presence of HSL was 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 than in glycolytic fibres. 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 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 protein kinase A (PKA). From findings in adipocytes it is likely that PKA phosphorylates HSL at residues Ser563, Ser659 and Ser660. Contraction probably also enhances 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. 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 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 Ser600. So, phosphorylation of different sites may explain 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 the contraction-mediated, but diminishes the adrenaline mediated HSL activation in muscle. The existence and regulation of HSL in skeletal muscle indicate a role of HSL in muscle TG metabolism.

HORMONE SENSITIVE LIPASE ACTIVITY AND FATTY ACYL-COA CONTENT IN HUMAN SKELETAL MUSCLE DURING PROLONGED EXERCISE

Hormone sensitive lipase (HSL) catalyzes the hydrolysis of intramuscular triacylglycerol (IMTG) but HSL regulation is poorly understood in skeletal muscle. PURPOSE To measure human skeletal muscle HSL activity and several putative regulators of HSL at rest and during prolonged moderate intensity exercise. METHODS Healthy male volunteers (n = 7; 21 ± years, 77 ± kg) cycled for 120 min at 60% peak pulmonary oxygen uptake. Muscle, venous blood and respiratory gas samples were obtained at rest and 10, 60 and 120 min of exercise. RESULTS HSL activity increased from resting levels by 10 min of exercise (2.09 ± 0.19 to 2.56 ± 0.22 mmol/min/kg dm, P < 0.05), increased further by 60 min (3.1227 ± 0. mmol/min/kg dm, P < 0.05) and decreased to near-resting rates after 120 min of cycling. Skeletal muscle LCFA CoA increased (P < 0.05) above rest by 60 min (15.9 ± 3.0 to 50.4 ± 7.9 mmol/kg dm) and increased further by 120 min. Estimated free AMP increased (P < 0.05) from rest to 60 min, and was ∼20-fold greater than rest by 120 min. Epinephrine was increased above rest (P < 0.05) at 60 (1.47 ± 0.15 nM) and 120 min (4.87 ± 0.76 nM) of exercise. Insulin concentrations decreased rapidly and were lower than rest by 10 min and continued to decrease throughout exercise. CONCLUSION HSL activity was increased above rest by 10 min, increased further by 60 min and decreased to near-resting values by 120 min. The increased HSL activity from 10–60 min was associated with the stimulating effect of increased epinephrine and decreased insulin levels. After 120 min, the decreased HSL activity was associated with the proposed inhibitory effects of increased free AMP. The LCFA CoA accumulation in the second hour of exercise may have reduced flux through HSL and accounted for the reduction in IMTG utilization previously observed late in prolonged exercise. Supported by NSERC and CIHR, Canada.

Additivity of adrenaline and contractions on hormone-sensitive lipase, but not on glycogen phosphorylase, in rat muscle.

Hormone-sensitive lipase (HSL) has been proposed to regulate triacylglycerol (TG) breakdown in skeletal muscle. In muscles with different fibre type compositions the influence on HSL of two major stimuli causing TG mobilization was studied. Incubated soleus and extensor digitorum longus (EDL) muscles from 70 g rats were stimulated by adrenaline (5.5 microm, 6 min) or contractions (200 ms tetani, 1 Hz, 1 min) in maximally effective doses or by both adrenaline and contractions. Hormone-sensitive lipase activity was increased significantly by adrenaline as well as contractions, and the highest activity (P < 0.05) was seen with combined stimulation [Soleus: 0.40 +/- 0.03 (SE) m-unit mg protein(-1) (basal), 0.65 +/- 0.02 (adrenaline), 0.65 +/- 0.03 (contractions), 0.78 +/- 0.03 (adrenaline and contractions); EDL: 0.18 +/- 0.01, 0.30 +/- 0.02, 0.26 +/- 0.02, 0.32 +/- 0.01]. Glycogen phosphorylase activity was always increased more by adrenaline compared with contractions [Soleus: 60 +/- 4 (a/a + b)% vs. 46 +/- 3 (P < 0.05); EDL: 60 +/- 5 vs. 39 +/- 6 (P < 0.05)]. After combined stimulation glycogen phosphorylase activity in soleus [59 +/- 3 (a/a + b)%] was identical to and in EDL [45 +/- 4 (a/a + b)%] smaller (P < 0.05) than the activity after adrenaline only. In slow-twitch oxidative as well as in fast-twitch glycolytic muscle HSL is activated by both adrenaline and contractions. These stimuli are partially additive indicating at least partly different mechanisms of action. Contractions may impair the enhancing effect of adrenaline on glycogen phosphorylase activity in muscle.