Mobilization of fatty acids from stored triacylglycerol (TG) in adipose tissue and skeletal muscle [intramyocellular triacylglycerol (IMTG)] requires activity of lipases. Although exercise training increases the lipolytic capacity of skeletal muscle, the expression of hormone-sensitive lipase (HSL) is not changed. Recently, adipose triglyceride lipase (ATGL) was identified as a TG-specific lipase in various rodent tissues. To investigate whether human skeletal muscle ATGL protein is regulated by endurance exercise training, 10 healthy young men completed 8 wk of supervised endurance exercise training. Western blotting analysis on lysates of skeletal muscle biopsy samples revealed that exercise training induced a twofold increase in skeletal muscle ATGL protein content. In contrast to ATGL, expression of comparative gene identification 58 (CGI-58), the activating protein of ATGL, and HSL protein was not significantly changed after the training period. The IMTG concentration was significantly decreased by 28% at termination of the training program compared with before. HSL-phoshorylation at Ser 660 was increased, HSL-Ser 659 phosporylation was unchanged, and HSL-phoshorylation at Ser 565 was decreased altogether, indicating an enhanced basal activity of this lipase. No change was found in the expression of diacylglycerol acyl transferase 1 (DGAT1) after training. Inhibition of HSL with a monospecific, small molecule inhibitor (76-0079) and stimulation of ATGL with CGI-58 revealed that significant ATGL activity is present in human skeletal muscle. These results suggest that ATGL in addition to HSL may be important for human skeletal muscle lipolysis.comparative gene identification 58; hormone sensitive lipase; diacylglycerol acyl transferase 1; intramyocellular triacylglycerol; lipolysis HORMONE-SENSITIVE LIPASE (HSL) has generally been accepted to be the primary lipase responsible for hydrolysis of intramyocellular triacylglycerol (IMTG). This notion is supported by findings in both rat and human skeletal muscle demonstrating that immunoinhibition of HSL with an anti-HSL antibody completely abolished contraction-induced increase in triacylglycerol (TG)-lipase activity (29,40,52). On the other hand, dissociations between HSL activity and net change of IMTG content during skeletal muscle contractions in humans have been observed (40,50,51). Also, in resting human skeletal muscle, it was shown that 40 -80% TG-hydrolase activity was still remaining after immunoinhibition of HSL (40,52). In line with this, recent studies (17) revealed that basal TG-hydrolase (lipolytic) activity was not reduced in the skeletal muscle of HSL knockout mice compared with wild-type controls and that, in the wild-type mice, diacylglycerol (DAG) rather than TG was found to accumulate in skeletal muscle and in adipose tissue. These findings together indicate that TG lipases other than HSL may be of importance in skeletal muscle TG hydrolysis. Recently, a previously unknown TG lipase, named adipose triglyceride lipase (ATGL), was identified (2...

Circulating interleukin (IL)-18 is elevated in obesity, but paradoxically causes hypophagia. We hypothesized that IL-18 may attenuate high-fat diet (HFD)-induced insulin resistance by activating AMP-activated protein kinase (AMPK). We studied mice with a global deletion of the α-isoform of the IL-18 receptor (IL-18R−/−) fed a standard chow or HFD. We next performed gain-of-function experiments in skeletal muscle, in vitro, ex vivo, and in vivo. We show that IL-18 is implicated in metabolic homeostasis, inflammation, and insulin resistance via mechanisms involving the activation of AMPK in skeletal muscle. IL-18R−/− mice display increased weight gain, ectopic lipid deposition, inflammation, and reduced AMPK signaling in skeletal muscle. Treating myotubes or skeletal muscle strips with IL-18 activated AMPK and increased fat oxidation. Moreover, in vivo electroporation of IL-18 into skeletal muscle activated AMPK and concomitantly inhibited HFD-induced weight gain. In summary, IL-18 enhances AMPK signaling and lipid oxidation in skeletal muscle implicating IL-18 in metabolic homeostasis.

This article is available online at http://www.jlr.orgCarbohydrates and FAs are the major energy substrates in the working muscle. The use of FAs for energy conversion depends, among other things, on exercise intensity and duration. During prolonged exercise, when carbohydrate reserves get depleted, oxidation of FAs becomes increasingly important ( 1, 2 ). Under these conditions, FAs are imported from plasma sources or mobilized from intramyocellular stores. Most of the body's energy reserves are stored in white adipose tissue (WAT) implicating that the supply of the muscle with energy during prolonged exercise is largely dependent on adipose lipolysis. The mobilization of FAs in adipose tissue is tightly controlled by hormones. Catecholamines and other effectors activate lip ases resulting in increased FA release into the circulation ( 3, 4 ). Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major triglyceride (TG) lipases in this process. HSL most effi ciently hydrolyses diglycerides (DGs), but is also capable of degrading several other lipid substrates including TGs, monoglycerides, cholesteryl ester, and retinyl ester ( 5 ). In HSL-deficient WAT, TG hydrolysis results in the accumulation of DG, suggesting that HSL is a major DG hydrolase in vivo ( 6 ). ATGL specifi cally performs the fi rst step in lipolysis, generating DGs and FAs ( 7 ). ATGL-defi ciency in mice results in obesity caused by severely reduced TG hydrolysis in adipose tissue. Increased deposition of TG is also observed in many other tissues ( 8 ). In the absence of both enzymes, hormone-induced FA release in WAT is decreased by more than 95% ( 9 ). Together, these observations suggest that effi cient lipolysis is dependent on the coordinate action of Abstract FAs are mobilized from triglyceride (TG) stores during exercise to supply the working muscle with energy. Mice defi cient for adipose triglyceride lipase (ATGL-ko) exhi bit defective lipolysis and accumulate TG in adipose tissue and muscle, suggesting that ATGL defi ciency affects energy availability and substrate utilization in working muscle. In this study, we investigated the effect of moderate treadmill exercise on blood energy metabolites and liver glycogen stores in mice lacking ATGL. Because ATGL-ko mice exhibit massive accumulation of TG in the heart and cardiomyopathy, we also investigated a mouse model lacking ATGL in all tissues except cardiac muscle (ATGL-ko/CM). In contrast to ATGL-ko mice, these mice did not accumulate TG in the heart and had normal life expectancy. Exercise experiments revealed that ATGL-ko and ATGL-ko/CM mice are unable to increase circulating FA levels during exercise. The reduced availability of FA for energy conversion led to rapid depletion of liver glycogen stores and hypoglycemia. Together, our studies suggest that ATGL-ko mice cannot adjust circulating FA levels to the increased energy requirements of the working muscle, resulting in an increased use of carbohydrates for energy conversion. Thus, ATGL activity is required...

We evaluated the response of various muscle and bone adaptation parameters with 24 wk of strength training in healthy, early postmenopausal women when a nutrient supplement (protein, carbohydrate, calcium, and vitamin D) or a placebo supplement (a minimum of energy) was ingested immediately following each training session. At inclusion, each woman was randomly and double-blindedly assigned to a nutrient group or a placebo (control) group. Muscle hypertrophy was evaluated from biopsies, MRI, and dual-energy X-ray absorptiometry (DEXA) scans, and muscle strength was determined in a dynamometer. Bone mineral density (BMD) was measured using DEXA scans, and bone turnover was determined from serum osteocalcin and collagen type I cross-linked carboxyl terminal peptide. The nutrient group improved concentric and isokinetic (60 degrees /s) muscle strength from 6 to 24 wk by 9 +/- 3% (P < 0.01), whereas controls showed no change (1 +/- 2%, P > 0.05). Only the nutrient group improved lean body mass (P < 0.05) over the 24 wk. BMD responded similarly at the lumbar spine but changed differently in the two groups at the femoral neck (P < 0.05) [control: 0.943 +/- 0.028 to 0.930 +/- 0.024 g/mm(3) (-1.0 +/- 1.4%); nutrient group: 0.953 +/- 0.051 to 0.978 +/- 0.043 g/mm(3) (3.8 +/- 3.4%)] when adjusted for age, body mass index, and BMD at inclusion. Bone formation displayed an interaction (P < 0.05), mainly caused by increased osteocalcin at 24 wk in the nutrient group. In conclusion, we report that nutrient supplementation results in superior improvements in muscle mass, muscle strength, femoral neck BMD, and bone formation during 24 wk of strength training. The observed differences following such a short intervention emphasize the significance of postexercise nutrient supply on musculoskeletal maintenance.

Skeletal muscle protein synthesis rate decreases during contractions but the underlying regulatory mechanisms are poorly understood. It was hypothesized that there would be a coordinated regulation of eukaryotic elongation factor 2 (eEF2) and eukaryotic initiation factor 4E-binding protein 1 (4EBP1) phosphorylation by signalling cascades downstream of rises in intracellular [Ca 2+ ] and decreased energy charge via AMP-activated protein kinase (AMPK) in contracting skeletal muscle. When fast-twitch skeletal muscles were contracted ex vivo using different protocols, the suppression of protein synthesis correlated more closely with changes in eEF2 than 4EBP1 phosphorylation. Using a combination of Ca 2+ release agents and ATPase inhibitors it was shown that the 60-70% suppression of fast-twitch skeletal muscle protein synthesis during contraction was equally distributed between Ca 2+ and energy turnover-related mechanisms. Furthermore, eEF2 kinase (eEF2K) inhibition completely blunted increases in eEF2 phosphorylation and partially blunted (i.e. 30-40%) the suppression of protein synthesis during contractions. The 3-to 5-fold increase in skeletal muscle eEF2 phosphorylation during contractions in situ was rapid and sustained and restricted to working muscle. The increase in eEF2 phosphorylation and eEF2K activation were downstream of Ca 2+ -calmodulin (CaM) but not other putative activating factors such as a fall in intracellular pH or phosphorylation by protein kinases. Furthermore, blunted protein synthesis and 4EBP1 dephosphorylation were unrelated to AMPK activity during contractions, which was exemplified by normal blunting of protein synthesis during contractions in muscles overexpressing kinase-dead AMPK. In summary, in fast-twitch skeletal muscle, the inhibition of eEF2 activity by phosphorylation downstream of Ca 2+ -CaM-eEF2K signalling partially contributes to the suppression of protein synthesis during exercise/contractions.

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 ϭ 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 Ͻ 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. physical inactivity; insulin resistance; prediabetic subjects PHYSICAL ACTIVITY IS AN IMPORTANT environmental moderator of metabolism, and sedentary lifestyle has been identified as a major risk factor for the metabolic syndrome, including type 2 diabetes mellitus (T2D), hypertension, and dyslipidemia (7,9,12,18,41). Thus there is an urgent need to gain more insight into the impact of physical inactivity on physiological mechanisms involved in the development of T2D and metabolic syndrome (23)(24)(25). Previous studies (28, 29) of physical inactivity in healthy individuals showed that 7 days bed rest diminished whole body glucose uptake as a result of decreased insulin action in inactive muscles. The Dallas Bed Rest and Training Study showed that 3 wk of bed rest caused a decrease in maximal exercise capacity (V O 2 max ) comparable to 30 yr of aging (26,27).An association between low birth weight (LBW) and impairment of glucose homeostasis was first proposed by Hales and Barker in 1991 (17). Since then, several studies have confirmed and elaborated on these findings, thereby highlighting the importance of the intrauterine environment in the development of diseases in adulthood, including hypertension, cardiovascular disease (16), and abnormal glucose tolerance (35,37,43). Accordingly, studies from the UK (2) as well as from our group (19, 20, 30, 31, 34 -36, 39) have provided evidence in favor of an important additional role of an adverse intrauterine environment associated with LBW in the development of insulin resistance,...

The activation and function of Ca 2+ -calmodulin-dependent kinase II (CaMKII) in contracting rat skeletal muscle was examined. The increase in autonomous activity and phosphorylation at Thr 287 of CaMKII of gastrocnemius muscle in response to contractions in situ was rapid and transient, peaking at 1-3 min, but reversed after 30 min of contractions. There was a positive correlation between CaMKII phosphorylation at Thr 287 and autonomous CaMKII activity. In contrast to the rapid and transient increase in autonomous CaMKII activity, the phosphorylation of the putative CaMKII substrate trisk95/triadin was rapid and sustained during contractions. There were no changes in CaMKII activity and phosphorylation or trisk95 phosphorylation in the resting contralateral muscles during stimulation. When fast-twitch muscles were contracted ex vivo, CaMKII inhibition resulted in a greater magnitude of fatigue as well as blunted CaMKII and trisk95 phosphorylation, identifying trisk95 as a physiological CaMKII substrate. In summary, skeletal muscle CaMKII activation was rapid and sustained during exercise/contraction and is mediated by factors within the contracting muscle, probably through allosteric activation via Ca 2+ -CaM. CaMKII may signal through trisk95 to modulate Ca 2+ release in fast-twitch rat skeletal muscle during exercise/contraction.

Key points• In skeletal muscle hormone-sensitive lipase (HSL) is considered the only enzyme responsible for breakdown of intramyocellular triacylglycerol (IMTG) during contractions. This notion is based on indirect measures in which important cellular events are not taken into account.• Using two histochemical techniques to measure breakdown of IMTG during contractions in isolated skeletal muscles we found that IMTG was decreased (1) in rat muscles during acute pharmacological blockade of HSL, and (2) in muscles of HSL knockout mice.• We demonstrated that adipose triglyceride lipase (ATGL) and HSL collectively account for at least 98% of the total TG lipase activity in mouse muscle, and other TG lipases accordingly seem of negligible importance for breakdown of IMTG.• In conclusion, breakdown of IMTG occurs in the contracting muscle in the absence of HSL activity. Our data suggest that ATGL is activated during contractions and plays a major role in breakdown of IMTG.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.