HSL serine phosphorylation and glycerol exchange across skeletal muscle in lean and obese subjects : effect of beta-adrenergic stimulation

Objective: Increased intramuscular triacylglycerol (IMTG) storage is a characteristic of the obese insulin resistant state. We aimed to investigate whether a blunted fasting or beta-adrenergically mediated lipolysis contributes to this increased IMTG storage in obesity. Research design and Methods: Forearm skeletal muscle (SM) lipolysis was investigated in thirteen lean and ten obese men using [H5]-glycerol combined with the measurement of arteriovenous differences before and during beta-adrenergic stimulation using the non-selective betaagonist isoprenaline (ISO). Muscle biopsies were taken from the vastus lateralis muscle before and during ISO to investigate hormone-sensitive lipase (HSL) protein expression and serine phosphorylation. Results: Baseline total glycerol release across the forearm was significantly blunted in obese compared with lean subjects (P=0.045). This was accompanied by lower HSL protein expression (P=0.004), and HSL phosphorylation on PKA sites Ser (P=0.041) and Ser (P=0.09) and on the AMPK site Ser (P=0.007), suggesting a blunted skeletal muscle lipolysis in obesity. Total forearm glycerol uptake during baseline did not differ significantly between groups while higher net fatty acid uptake across the forearm was observed in the obese (P=0.064). ISO induced an increase in total glycerol release from SM, which was not significantly different between groups. Interestingly, this was accompanied by an increase in HSL Ser phosphorylation in obese subjects during ISO compared with baseline (P=0.008). Conclusions: Obesity is accompanied by impaired fasting glycerol release, lower HSL protein expression and serine phosphorylation. It remains to be determined whether this is a primary factor or an adaptation to the obese insulin resistant state. he obese insulin resistant state is characterized by increased triacylglycerol (TAG) storage in adipose and non-adipose tissues (ectopic fat), such as skeletal muscle (1). A strong link between increased intramuscular triacylglycerol (IMTG) stores and skeletal muscle insulin resistance has been shown in lean and obese subjects (2; 3). Recent studies have, however, indicated that intramuscular accumulation of lipid intermediates rather than TAG per se may be the direct cause of skeletal muscle insulin resistance, through interference with insulin signaling (4). Impaired uptake and a reduced oxidation of fatty acids has been reported in skeletal muscle under post-absorptive conditions, during beta-adrenergic stimulation and moderate-intensity exercise in obese subjects with type 2 diabetes (5-7). Besides impaired fatty acid handling, disturbances in the regulation of skeletal muscle lipolysis may contribute to the increased storage of IMTG and lipid metabolites. So far, little is known on the in vivo regulation of skeletal muscle lipolysis in obesity. Data from our laboratory indicate that the catecholamine-induced increase in interstitial glycerol concentration as well as local blood flow is blunted in obese subjects (8), a factor that may contribute to an increase in content of muscle TAG and diacylglycerol (DAG). Although the molecular mechanisms that underlie muscle lipolysis are not known into detail, it has been shown that hormonesensitive lipase (HSL) is expressed in skeletal muscle of rodents (9; 10) and humans (11). HSL activity appears to be regulated by sitespecific phosphorylation on several serine residues. It has been demonstrated that skeletal muscle HSL can be phosphorylated on at least five serine residues (Ser, Ser, Ser, Ser and Ser) (11-13). Catecholamines increase intracellular cyclic AMP concentration resulting in the activation of protein kinase A (PKA). HSL Ser, Ser and Ser are major PKA phosphorylation sites, although Ser may not affect HSL activity directly (14). It is still unclear which of the PKA phosphorylation sites on HSL are important in mediating the effect of catecholamines on in vivo muscle HSL activity. Ser appears to be a likely candidate, since HSL Ser phosphorylation and HSL activity show a similar response to exercise with concomitant increase in circulating epinephrine (13). In vitro studies on purified bovine adipocyte HSL have shown that AMP-activated protein kinase (AMPK) phosphorylates HSL on Ser thereby abolishing PKA induced HSL activation (15). In human skeletal muscle, changes in AMPK activity during exercise were also associated with an increased HSL Ser phosphorylation but this was not accompanied by an increased HSL activity, suggesting that AMPK can phosphorylate HSL on Ser but that AMPK is of minor importance as a regulator of HSL activity in human skeletal muscle during exercise (11). So far, limited data are available on the differences in in vivo regulation of skeletal muscle lipolysis between lean and obese subjects. The aim of the present study was to investigate whether in vivo baseline and/or catecholamine-induced lipolysis is impaired in skeletal muscle of obese compared with lean subjects. For this reason [H5]-glycerol tracer methodology was used to investigate in vivo whole-body and regional forearm skeletal muscle lipolysis in lean and obese subjects after an overnight fast and during beta-adrenergic stimulation, using the nonselective beta-adrenergic agonist isoprenaline. To obtain more information on the underlying mechanism at the molecular level we measured skeletal muscle HSL protein expression and serine phosphorylation on Ser, Ser and Ser. T