Hormone-sensitive Lipase Protein Levels Research Articles

Effect of Resistance Exercise on the Lipolysis Pathway in Obese Pre- and Postmenopausal Women.

Physical exercise may stimulate lipolytic activity within adipose tissue. Furthermore, resistance exercise may contribute to the more efficient reduction in adipose tissue mass and prevent the accumulation thereof in obese women. The purpose of this study was to examine the effects of regular resistance exercise for 12 weeks on the lipolysis pathway in women with obesity. Twenty-three pre- and postmenopausal women with body fat percentages of 30% or more were divided into the premenopausal group (n = 9) and the postmenopausal group (n = 14). All subjects participated in resistance exercise training for 12 weeks. Anthropometric and physical fitness tests were performed on all participants. Protein analyses were performed on extracted subcutaneous fatty tissue, and changes in the relevant protein levels in the samples were analyzed by Western blotting. All serum samples were submitted for enzyme-linked immunosorbent assay measurements of adipocyte factors. After 12 weeks, the adipose triglyceride lipase, monoacylglycerol lipase, and perilipin1 protein levels were significantly lower in the postmenopausal group than in the premenopausal group. The hormone-sensitive lipase protein levels were significantly higher in the postmenopausal group than in the premenopausal group. In addition, leptin concentrations were significantly decreased after resistance exercise in the postmenopausal group. Adiponectin concentrations were significantly increased after resistance exercise in both groups. These findings indicate that regular resistance exercise is effective in reducing the weight and body fat of obese premenopausal women, and in the secretion of adiponectin. On the other hand, postmenopausal women were found to have redeced weight and body fat, and were found to be positive for the secretion of adipokine factors. In addition, positive changes in lipolysis pathway factors in adipose tissue promote lipid degradation and reduce fat mass. Thus, regular resistance exercise shows positive changes in the lipolysis pathway more effectively in weight and body fat reduction in postmenopausal women than in premenopausal women.

Feeding influences adipose tissue responses to exercise in overweight men.

Feeding profoundly affects metabolic responses to exercise in various tissues, but the effect of feeding status on human adipose tissue responses to exercise has never been studied. Ten healthy overweight men aged 26 ± 5 yr (mean ± SD) with a waist circumference of 105 ± 10 cm walked at 60% of maximum oxygen uptake under either fasted or fed conditions in a randomized, counterbalanced design. Feeding comprised 648 ± 115 kcal 2 h before exercise. Blood samples were collected at regular intervals to examine changes in metabolic parameters and adipokine concentrations. Adipose tissue samples were obtained at baseline and 1 h after exercise to examine changes in adipose tissue mRNA expression and secretion of selected adipokines ex vivo. Adipose tissue mRNA expression of pyruvate dehydrogenase kinase isozyme 4 (PDK4), adipose triglyceride lipase, hormone-sensitive lipase (HSL), fatty acid translocase/CD36, glucose transporter type 4 (GLUT4), and insulin receptor substrate 2 (IRS2) in response to exercise were lower in fed compared with fasted conditions (all P ≤ 0.05). Postexercise adipose IRS2 protein was affected by feeding (P ≤ 0.05), but Akt2, AMPK, IRS1, GLUT4, PDK4, and HSL protein levels were not different. Feeding status did not impact serum and ex vivo adipose secretion of IL-6, leptin, or adiponectin in response to exercise. This is the first study to show that feeding before acute exercise affects postexercise adipose tissue gene expression, and we propose that feeding is likely to blunt long-term adipose tissue adaptation to regular exercise.

Specific visible radiation facilitates lipolysis in mature 3T3-L1 adipocytes via rhodopsin-dependent β3-adrenergic signaling

The regulation of fat metabolism is important for maintaining functional and structural tissue homeostasis in biological systems. Reducing excessive lipids has been an important concern due to the concomitant health risks caused by metabolic disorders such as obesity, adiposity and dyslipidemia. A recent study revealed that unlike conventional care regimens (e.g., diet or medicine), low-energy visible radiation (VR) regulates lipid levels via autophagy-dependent hormone-sensitive lipase (HSL) phosphorylation in differentiated human adipose-derived stem cells. To clarify the underlying cellular and molecular mechanisms, we first verified the photoreceptor and photoreceptor-dependent signal cascade in nonvisual 3T3-L1 adipocytes. For a better understanding of the concomitant phenomena that result from VR exposure, mature 3T3-L1 adipocytes were exposed to four different wavelengths of VR (410, 505, 590 and 660nm) in this study. The results confirmed that specific VR wavelengths, especially 505nm than 590nm, increase intracellular cyclic adenosine monophosphate (cAMP) levels and decrease lipid droplets. Interestingly, the mRNA and protein levels of the Opn2 (rhodopsin) photoreceptor increased after VR exposure in mature 3T3-L1 adipocytes. Subsequent treatment of mature 3T3-L1 adipocytes at a specific VR wavelength induced rhodopsin- and β3-adrenergic receptor (AR)-dependent lipolytic responses that consequently led to increases in intracellular cAMP and phosphorylated HSL protein levels. Our study indicates that photoreceptors are expressed and exert individual functions in nonvisual cells, such as adipocytes. We suggest that the VR-induced photoreceptor system could be a potential therapeutic target for the regulation of lipid homeostasis in a non-invasive manner.

Curcumin induces brown fat-like phenotype in 3T3-L1 and primary white adipocytes

Recent advances have been made in the understanding of pharmacological and dietary agents that contribute to browning of white adipose tissue in order to combat obesity by promoting energy expenditure. Here, we show that curcumin induces browning of 3T3-L1 and primary white adipocytes via enhanced expression of brown fat-specific genes. Curcumin-induced browning in white adipocytes was investigated by determining expression levels of brown adipocyte-specific genes/proteins by real-time reverse transcriptase polymerase chain reaction, immunoblot analysis and immunocytochemical staining. Curcumin increased mitochondrial biogenesis, as evidenced by transmission electronic microscopic detection and enhanced expression of proteins involved in fat oxidation. Cucurmin also increased protein levels of hormone-sensitive lipase and p-acyl-CoA carboxylase, suggesting its possible role in augmentation of lipolysis and suppression of lipogenesis. Increased expression of UCP1 and other brown adipocyte-specific markers was possibly mediated by curcumin-induced activation of AMP-activated protein kinase (AMPK) based on the fact that inhibition of AMPK by dorsomorphin abolished expression of PRDM16, UCP1 and peroxisome proliferator-activated receptor gamma co-activator 1-alpha while the activator 5-Aminoimidazole-4-carboxamide ribonucleotide elevated expression of these brown marker proteins. Our findings suggest that curcumin plays a dual modulatory role in inhibition of adipogenesis as well as induction of the brown fat-like phenotype and thus may have potential therapeutic implications for treatment of obesity.

Green tea catechins enhance norepinephrine-induced lipolysis via a protein kinase A-dependent pathway in adipocytes

Green tea catechins have been shown to attenuate obesity in animals and humans. The catechins activate adenosine monophosphate-activated protein kinase (AMPK), and thereby increase fatty acid oxidation in liver and skeletal muscles. Green tea catechins have also been shown to reduce body fat in humans. However, the effect of the catechins on lipolysis in adipose tissue has not been fully understood. The aim of this study was to clarify the effect of green tea catechins on lipolysis in adipocytes and to elucidate the underlying mechanism. Differentiated mouse adipocyte cell line (3T3-L1) was stimulated with green tea catechins in the presence or absence of norepinephrine. Glycerol and free fatty acids in the media were measured. Phosphorylation of hormone-sensitive lipase (HSL) was determined by Western blotting, and the mRNA expression levels of HSL, adipose triglyceride lipase (ATGL), and perilipin were determined by quantitative RT-PCR. The cells were treated with inhibitors of protein kinase A (PKA), protein kinase C (PKC), protein kinase G (PKG), or mitogen-activated protein kinase (MAPK) to determine the responsible pathway. Treatment of 3T3-L1 adipocytes with green tea catechins increased the level of glycerol and free fatty acids released into the media in the presence, but not absence, of norepinephrine, and increased the level of phosphorylated HSL in the cells. The catechins also increased mRNA and protein levels of HSL and ATGL. PKA inhibitor (H89) attenuated the catechin-induced increase in glycerol release and HSL phosphorylation. The results demonstrate that green tea catechins enhance lipolysis in the presence of norepinephrine via a PKA-dependent pathway in 3T3-L1 adipocytes, providing a potential mechanism by which green tea catechins could reduce body fat.

Lipolytic Effect of Eumicrotremus orbis Water Extracts in Differentiated 3T3-L1 Adipocytes

In this study, we investigated the effects of Eumicrotremus orbis water extracts (EOWE) on lipolysis in differentiatied 3T3-L1 adipocytes and its mechanism. It was observed that there was no cytotoxicity for adipocytes of EOWE. The content of glycerol released in cell culture medium increased dependent on sample concentration. In differentiated 3T3-L1 adipocytes treated EOWE, hormone sensitive lipase (HSL) and phosphorylated HSL (p-HSL) protein level increased by approximately 3.43 and 1.77 times as compared to the controls. But, adipose triglyceride lipase (ATGL) protein level did not effect as compared to the controls. Treated EOWE inhibited the protein expression of perilipin A. In other word, it is judged to be due to, a content of glycerol released in cell culture medium increased through not only increase of HSL, p-HSL and p-PKA protein level, but inhibit of perilipin A. Therefore, our results suggest that EOWE may induce lipoylsis by increasing the protein levels of HSL, p-HSL and p-PKA.

Fasting, But Not Exercise, Increases Adipose Triglyceride Lipase (ATGL) Protein and Reduces G(0)/G(1) Switch Gene 2 (G0S2) Protein and mRNA Content in Human Adipose Tissue

Fasting and exercise are characterized by increased lipolysis, but the underlying mechanisms are not fully understood. The study was designed to test whether fasting and exercise affect mRNA and protein levels of adipose triglyceride lipase (ATGL) and G(0)/G(1) switch gene 2 (G0S2), a recently discovered ATGL inhibitor, in humans. We studied eight healthy men (age, 25.5 ± 4.3 yr) for 6 h (a 4-h basal and a 2-h clamp period) on three occasions in a randomized crossover design: 1) in the basal state and after; 2) 72-h fasting; and 3) 1-h exercise (65% VO(2max)). Subcutaneous abdominal adipose tissue (AT) biopsies were taken at t = 30 and 270 min. The study was conducted at a university hospital research unit. Circulating free fatty acids and GH were increased, and C-peptide was decreased by both fasting and exercise. During fasting, insulin failed to suppress free fatty acid levels, suggesting AT insulin resistance. ATGL protein was increased 44% (P < 0.001), and G0S2 mRNA and protein were decreased 56% (P = 0.02) and 54% (P = 0.01), respectively, after fasting, but both ATGL and G0S2 were unaffected by exercise. Protein levels of hormone-sensitive lipase and comparative gene identification-58 were unaffected throughout. We found increased AT content of ATGL and decreased protein and mRNA content of the ATGL inhibitor G0S2, suggesting increased ATGL activity during fasting, but not after short-term exercise. These findings are compatible with the notion that the ATGL-G0S2 complex is an important long-term regulator of lipolysis under physiological conditions such as fasting in humans.

B56α/Protein Phosphatase 2A Inhibits Adipose Lipolysis in High-Fat Diet-Induced Obese Mice

Lipolysis and lipogenesis are two opposite processes that control lipid storage in adipocytes. Impaired adipose lipolysis has been observed in both obese human subjects and animal models. This study investigated the mechanisms underlying impaired adipose lipolysis in a high-fat diet-induced obese (DIO) mouse model. DIO models were created using male C57BL/6 mice. Our results show that beta3 adrenergic receptor-specific agonist BRL37344 induced adipose lipolysis was significantly blunted in DIO mice. The levels of Ser660 phosphorylation of hormone-sensitive lipase (HSL) were significantly decreased in the epididymal fat of DIO mice. However, protein levels of HSL, adipose triglyceride lipase and its coactivator comparative gene identification-58 were similar between DIO and control mice. It is known that upon lipolytic hormone stimulation, protein kinase A phosphorylates HSL Ser660 and activates HSL, whereas protein phosphatase 2A (PP2A) dephosphorylates and inactivates HSL. Interestingly, our study shows that high-fat feeding did not alter epididymal fat cAMP and protein kinase A protein levels but significantly increased the expression of the alpha-isoform of PP2A regulatory subunit B' (B56alpha). To study the role of B56alpha in obesity-associated lipolytic defect, B56alpha was overexpressed or knocked down by adenovirus-mediated gene transduction in cultured 3T3-L1CARDelta1 adipocytes. Overexpression of B56alpha significantly decreased HSL Ser660 phosphorylation. In contrast, knocking down B56alpha increased hormone-stimulated HSL activation and lipolysis in mature 3T3-L1CARDelta1 adipocytes. These results strongly suggest that elevated B56alpha/PP2A inhibits HSL and lipolysis in white adipose tissue of DIO mice.

Differential effects of high MUFA with high or low P/S ratio (polyunsaturated to saturated fatty acids) on improving hepatic lipolytic enzymes and mediating PPARγ related with lipoprotein lipase and hormone-sensitive lipase of white adipose tissue in diet-induced obese hamster

The aim of this study was to assess the relationship between high monounsaturated fatty acids (MUFAs) with different levels of polyunsaturated-to-saturated fatty acid (P/S) ratios and body fat loss in diet-induced obesity (DIO) models. Male Golden Syrian hamsters were randomly assigned to the control group (n=12) and obesity group (n=24) for 4 weeks of the high-fat DIO period; afterward, six hamsters from each group were killed. The remaining control hamsters were still fed a low-fat diet. For an additional 8 weeks, the remaining obesity hamsters were switched to a low-fat diet and subdivided into three subgroups (n=6/group): the obesity-control (ObC) group, high MUFA with high P/S ratio oil (HMHR) group and olive oil (OO) group. Serum insulin and leptin concentrations were measured, and hepatic fatty acid metabolic enzymes and adipose differentiation markers were determined using enzyme activities analysis, western blot and semiquantification reverse-transcription PCR. No difference was observed in the mean energy intake through all study periods. After the DIO period, the obesity group increased in weight gain and epididymal fat weight compared with the control group. DIO hamsters in the HMLR group had significant reductions in white adipose tissue deposition and plasma leptin levels, suppression in adipose peroxisome proliferator-activated receptor-γ (PPARγ) and lipoprotein lipase (LPL) mRNA expressions and increases in hepatic acyl-CoA oxidase and carnitine palmitoyltransferase-I activities and mRNA levels compared with those in the ObC group. The HMHR group had upregulated phosphorylation of hormone-sensitive lipase (HSL) relative to total HSL protein levels compared with the OO group. However, the OO group had significantly elevated hepatic de novo lipogenesis compared with the HMHR group. HMHR seemed to be beneficial in depleting white adipose tissue accumulation by decreasing adipose PPARγ and LPL mRNA expressions and mediating phosphorylation of HSL, and by improving hepatic lipolytic enzyme activities and mRNA expressions involved in β-oxidation in DIO hamsters.

Direct Effect of Glucocorticoids on Lipolysis in Adipocytes

Hypercortisolemia and glucocorticoid treatment cause elevated level of circulating free fatty acids (FFAs). The basis of this phenomenon has long been linked to the effect of glucocorticoids permitting and enhancing the adipose lipolysis response to various hormones. In this study, we demonstrate that glucocorticoids directly stimulate lipolysis in rat primary adipocytes in a dose- and time-responsive manner; this lipolytic action was attenuated by treatment with the glucocorticoid antagonist RU486. Dexamethasone down-regulates mRNA and protein levels of cyclic-nucleotide phosphodiesterase 3B, thereby elevating cellular cAMP production and activating protein kinase A (PKA). On inhibition of PKA but not other kinases, the lipolysis response ceases. Furthermore, dexamethasone induces phosphorylation and down-regulation of perilipin, a lipid droplet-associating protein that modulates lipolysis; this effect is restored by RU486 or PKA inhibitor H89. Dexamethasone up-regulates mRNA and protein levels of hormone-sensitive lipase (HSL) and adipose triglyceride lipase; these effects, parallel to increased lipolysis, are attenuated by RU486 or actinomycin D. Phosphorylation at Ser-563 and Ser-660 residues of HSL and activity of cellular lipases are elevated on dexamethasone stimulation but abrogated by the coaddition of H89. However, dexamethasone does not induce HSL translocation to the lipid droplet surface in differentiated adipocytes. We show that elevated FFA concentration in plasma is associated with increased lipase activity and lipolysis in vivo in adipose tissues of dexamethasone-treated rats. Therefore, the lipolytic action of glucocorticoids liberates FFA efflux from adipocytes to the bloodstream, which could be a cellular basis of systemic FFA elevation in response to glucocorticoid challenge.

Direct effect of glucocorticoids on lipolysis in adipocytes.

Hypercortisolemia and glucocorticoid treatment cause elevated level of circulating free fatty acids (FFAs). The basis of this phenomenon has long been linked to the effect of glucocorticoids permitting and enhancing the adipose lipolysis response to various hormones. In this study, we demonstrate that glucocorticoids directly stimulate lipolysis in rat primary adipocytes in a dose- and time-responsive manner; this lipolytic action was attenuated by treatment with the glucocorticoid antagonist RU486. Dexamethasone down-regulates mRNA and protein levels of cyclic-nucleotide phosphodiesterase 3B, thereby elevating cellular cAMP production and activating protein kinase A (PKA). On inhibition of PKA but not other kinases, the lipolysis response ceases. Furthermore, dexamethasone induces phosphorylation and down-regulation of perilipin, a lipid droplet-associating protein that modulates lipolysis; this effect is restored by RU486 or PKA inhibitor H89. Dexamethasone up-regulates mRNA and protein levels of hormone-sensitive lipase (HSL) and adipose triglyceride lipase; these effects, parallel to increased lipolysis, are attenuated by RU486 or actinomycin D. Phosphorylation at Ser-563 and Ser-660 residues of HSL and activity of cellular lipases are elevated on dexamethasone stimulation but abrogated by the coaddition of H89. However, dexamethasone does not induce HSL translocation to the lipid droplet surface in differentiated adipocytes. We show that elevated FFA concentration in plasma is associated with increased lipase activity and lipolysis in vivo in adipose tissues of dexamethasone-treated rats. Therefore, the lipolytic action of glucocorticoids liberates FFA efflux from adipocytes to the bloodstream, which could be a cellular basis of systemic FFA elevation in response to glucocorticoid challenge.

Omental adipose tissue overexpression of fatty acid transporter CD36 and decreased expression of hormone-sensitive lipase in insulin-resistant women with polycystic ovary syndrome

Elevated free fatty acids (FFAs) are involved in insulin resistance in polycystic ovary syndrome (PCOS). We investigated the role of fatty acid transporter CD36, hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) in regulation of lipolysis in insulin-resistant women with PCOS. CD36, HSL and ATGL proteins were analyzed in omental adipose tissue from 10 women with PCOS and 10 healthy, BMI- and age-matched controls by western blotting. Women with PCOS had higher fasting and 2 h insulin levels (P < 0.002, P < 0.029, respectively) and a higher homeostasis model insulin resistance index (P < HOMA(IR), 0.005) and a lower fasting glucose-to-insulin ratio (G(0)/I(0)) (P < 0.001) than controls. CD36 protein levels in the PCOS women were higher (268% of control levels, P < 0.05) and HSL protein levels were lower (43% of control levels, P < 0.05). However, ATGL protein levels were not different in the two groups. Fasting serum insulin levels showed a positive correlation with CD36 levels (P = 0.01, r = 0.875) and a negative correlation with HSL levels (P = 0.045, r = -0.73). Furthermore, a positive correlation was found between serum testosterone levels and CD 36 protein levels (P = 0.025, r = 0.817) but the correlation did not reach significance after controlling for HOMA(IR). After adjusting insulin resistance index of HOMA(IR) by analysis of covariance, only CD36 differed between PCOS and control (P = 0.026). Our results suggest that, in insulin-resistant women with PCOS, changes in CD36 and HSL expression may result in altered FFA uptake.

Bacterial Endotoxin Stimulates Adipose Lipolysis via Toll-Like Receptor 4 and Extracellular Signal-regulated Kinase Pathway

Bacterial endotoxin/lipopolysaccharide elicits inflammatory responses and also elevates circulating levels of free fatty acids (FFAs) and impairs insulin sensitivity. Serum FFA elevation in acute endotoxemia has long been thought to be due to endotoxin dysregulating lipid disposal and counterregulatory hormones and cytokines. Here, we investigated the direct lipolysis effect of endotoxin in rodents and in isolated primary adipocytes. Endotoxin increases lipolysis in vivo in adipose tissues, elevates circulating FFA level, induces insulin resistance in rats, and directly stimulates chronic lipolysis in vitro in adipocytes. The lipolytic action of endotoxin is mediated via its lipid A moiety and is blocked by anti-endotoxin peptides. Neither adipocytokine secretion nor nuclear factor-kappaB activation is involved in endotoxin-induced lipolysis. Different from catecholamine, endotoxin stimulates lipolysis without elevating cAMP production and activating protein kinase A and protein kinase C. Instead, endotoxin induces phosphorylation of Raf-1, MEK1/2, and ERK1/2. Upon inhibition of ERK1/2 but not JNK and p38 MAPK, endotoxin-stimulated lipolysis ceases. Endotoxin causes perilipin down-regulation and phosphorylation and increases the activity and protein levels of hormone-sensitive lipase and adipose triglyceride lipase but does not induce hormone-sensitive lipase translocation to intracellular lipid droplets. In TLR4 (Toll-like receptor 4)-deficient mice and adipocytes, endotoxin fails to increase in vivo and in vitro lipolysis. These findings suggest that endotoxin stimulates lipolysis via TLR4 and ERK1/2 signaling in adipocytes. The lipolytic action of endotoxin liberates FFA efflux from adipocytes to the bloodstream, which is a possible basis for systemic FFA elevation and insulin resistance in endotoxemia or Gram-negative bacterial infection.

Salicylate Blocks Lipolytic Actions of Tumor Necrosis Factor-α in Primary Rat Adipocytes

Increased systemic free fatty acids (FFA) impair insulin sensitivity. In obese and diabetic subjects, production of tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, is elevated. TNF-alpha has a variety of effects by inducing inflammation, decreasing glucose utilization, and stimulating adipocyte lipolysis to release FFA to plasma. High doses of nonsteroidal anti-inflammatory drug salicylates have long been recognized to lower blood FFA and glucose in humans, although the mechanisms are not fully understood. In this report, we show that sodium salicylate at therapeutic concentrations directly blocks TNF-alpha-stimulated lipolysis and therefore inhibits FFA release from primary rat adipocytes. To elucidate the cellular basis of this action, we show that salicylate suppresses TNF-alpha-induced extracellular signal-related kinase activation and intracellular cAMP elevation, two early events during the lipolysis response to TNF-alpha. Furthermore, salicylate prevents the down-regulation of cyclic-nucleotide phosphodiesterase 3B, an enzyme responsible for cAMP hydrolysis. Perilipins coat intracellular lipid droplet surface by restricting lipase access to the triacylglycerol substrates. TNF-alpha down-regulates perilipin but promotes its phosphorylation during lipolysis stimulation; these actions are efficiently reversed by salicylate. Salicylate slightly reduces basal but completely inhibits TNF-alpha-liberated lipase activity. In contrast, neither salicylate nor TNF-alpha alters the protein levels of hormone-sensitive lipase and adipose triglyceride lipase. In addition, sodium salicylate restricts basal lipolysis simulated by a high concentration of glucose and significantly diminishes the high glucose-enhanced lipolysis response to TNF-alpha. These results provide novel evidence that salicylate directly blocks TNF-alpha-mediated FFA efflux from adipocytes, hence reducing plasma FFA levels and increasing insulin sensitivity.

Comparative studies of the role of hormone-sensitive lipase and adipose triglyceride lipase in human fat cell lipolysis

Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) regulate adipocyte lipolysis in rodents. The purpose of this study was to compare the roles of these lipases for lipolysis in human adipocytes. Subcutaneous adipose tissue was investigated. HSL and ATGL protein expression were related to lipolysis in isolated mature fat cells. ATGL or HSL were knocked down by RNA interference (RNAi) or selectively inhibited, and effects on lipolysis were studied in differentiated preadipocytes or adipocytes derived from human mesenchymal stem cells (hMSC). Subjects were all women. There were 12 lean controls, 8 lean with polycystic ovary syndrome (PCOS), and 27 otherwise healthy obese subjects. We found that norepinephrine-induced lipolysis was positively correlated with HSL protein levels (P < 0.0001) but not with ATGL protein. Women with PCOS or obesity had significantly decreased norepinephrine-induced lipolysis and HSL protein expression but no change in ATGL protein expression. HSL knock down by RNAi reduced basal and catecholamine-induced lipolysis. Knock down of ATGL decreased basal lipolysis but did not change catecholamine-stimulated lipolysis. Treatment of hMSC with a selective HSL inhibitor during and/or after differentiation in adipocytes reduced basal lipolysis by 50%, but stimulated lipolysis was inhibited completely. In contrast to findings in rodents, ATGL is of less importance than HSL in regulating catecholamine-induced lipolysis and cannot replace HSL when this enzyme is continuously inhibited. However, both lipases regulate basal lipolysis in human adipocytes. ATGL expression, unlike HSL, is not influenced by obesity or PCOS.

Depot- and Gender-related Differences in the Lipolytic Pathway of Adipose Tissue from Severely Obese Patients

The present study was performed to analyze in detailgender- and site-related alterations in the adrenergicsignal transduction pathway of lipolysis in fat cellsisolated from subcutaneous abdominal and visceralfat depots from severely obese patients. The studygroup consisted of 30 morbidly obese subjects (9 menand 21 women) aged 41.1±1.9 years, with a bodymass index (BMI) of 54.7±1.7 kg/m2, who hadundergone abdominal surgery. Protein levels ofhormone-sensitive lipase (HSL) and adrenergicreceptors (AR), as well as HSL activity and the lipolyticresponse to adrenergic agents were analyzed. Bothfat depots had similar basal lipolysis, but the capacityof catecholamines to activate lipolysis was greater invisceral fat, both at AR and postreceptor levels. Basallipolysis and lipolytic activity induced by dibutyryl cyclicAMP were higher in men than in women. However,the visceral depot of women showed a higher maximalstimulation by noradrenaline than that of men, inaccordance with higher ß1- and ß3-AR protein levels.In conclusion, the main gender-related differenceswere located in the visceral depot, with womenexhibiting a higher sensitivity to catecholaminesassociated with an increased provision of ß-AR, whilemen showed an enhanced lipolytic capacity at thepostreceptor level.

Relationship of the hormone-sensitive lipase-mediated modulation of cholesterol metabolism in individual compartments of the testis to serum pituitary hormone and testosterone concentrations in a seasonal breeder, the mink (Mustela vison).

The role of cholesterol differs in the two compartments of the testis. In the interstitial tissue, cholesterol is necessary for the synthesis of testosterone, whereas in the seminiferous tubules, membrane cholesterol content in developing germ cells will influence the gametes' fertility. Here we evaluate the hormone-sensitive lipase (HSL) modulation of the cholesterol metabolism in each compartment of the testis. Two HSL immunoreactive bands of 104- and 108-kDa were detected in Western blots performed with polyclonal anti-human HSL antibodies in the interstitial tissue (ITf)- and seminiferous tubule (STf)-enriched fractions generated from testes harvested at 30-day intervals during puberty and, in the adult mink, during the annual seasonal reproductive cycle. Epididymal spermatozoa expressed a 104-kDa HSL isoform, and HSL was active in these cells. Immunolabeling localized HSL to interstitial macrophages; Sertoli cells, where its distribution was stage specific; spermatids; and the equatorial segment of spermatozoa. Total HSL protein levels, specific enzymatic activity, and free cholesterol (FC):esterified cholesterol (EC) ratios varied concomitantly in STf and ITf and reached maximal values in the adult during the period of maximal spermatogenic activity. In STf, HSL-specific activity correlated with FC:EC ratios but not with triglyceride levels. In STf, high HSL-specific activity occurred concomitantly with high FSH serum levels. In ITf, HSL-specific activity was high during periods of low serum prolactin levels and high serum testosterone levels. The results suggest that 1) modulation of cholesterol metabolism in individual testicular compartments may be regulated by HSL isoforms expressed by distinct cells; 2) interstitial macrophages may be part of a system involved in the synthesis of steroid hormones and in the recycling of sterols in the interstitium, whereas in the tubules, recycling could be ensured by Sertoli cells; 3) there is distinctive substrate preference for testicular HSL; and 4) HSL may be the only cholesterol esterase in this location.

The effect of exercise training on hormone-sensitive lipase in rat intra-abdominal adipose tissue and muscle.

1. Adrenaline-stimulated lipolysis in adipose tissue may increase with training. The rate-limiting step in adipose tissue lipolysis is catalysed by the enzyme hormone-sensitive lipase (HSL). We studied the effect of exercise training on the activity of the total and the activated form of HSL, referred to as HSL (DG) and HSL (TG), respectively, and on the concentration of HSL protein in retroperitoneal (RE) and mesenteric (ME) adipose tissue, and in the extensor digitorum longus (EDL) and soleus muscles in rats. 2. Rats (weighing 96 +/- 1 g, mean +/- S.E.M.) were either swim trained (T, 18 weeks, n = 12) or sedentary (S, n = 12). Then RE and ME adipose tissue and the EDL and soleus muscles were incubated for 20 min with 4.4 microM adrenaline. 3. HSL enzyme activities in adipose tissue were higher in T compared with S rats. Furthermore, in RE adipose tissue, training also doubled HSL protein concentration (P < 0.05). In ME adipose tissue, the HSL protein levels did not differ significantly between T and S rats. In muscle, HSL (TG) activity as well as HSL (TG)/HSL (DG) were lower in T rats, whereas HSL (DG) activity did not differ between groups. Furthermore, HSL protein concentration in muscle did not differ between T and S rats (P > 0.05). 4. In conclusion, training increased the amount of HSL and the sensitivity of HSL to stimulation by adrenaline in intra-abdominal adipose tissue, the extent of the change differing between anatomical locations. In contrast, in skeletal muscle the amount of HSL was unchanged and its sensitivity to stimulation by adrenaline reduced after training.

The expression of hormone-sensitive lipase in clonal beta-cells and rat islets is induced by long-term exposure to high glucose.

Hormone-sensitive lipase (HSL) is expressed and enzymatically active in beta-cells and has been proposed to be involved in the generation of the lipid-derived signal that seems to be necessary for glucose-stimulated insulin secretion. In this study, we investigated whether the expression of HSL in INS-1 cells and in rat islets is affected by exposure to high glucose concentrations. Incubation of INS-1 cells in 25 mmol/l glucose for 16 and 32 h induced HSL protein expression twofold, whereas no effect was observed after 4 and 8 h of incubation. The HSL activity, defined as the diglyceride lipase activity inhibited by anti-rat HSL antibodies, constituted approximately 25% of total diglyceride lipase activity and was induced to a similar extent as HSL protein levels. The glucose effect at 16 h on HSL protein expression level was confirmed in freshly isolated rat islets. Exposure of INS-1 cells to different glucose concentrations for 16 h showed that the inductive effect on HSL protein levels was maximum at 20 mmol/l glucose (2- to 2.5-fold). Northern blot analysis demonstrated a more than threefold elevation of HSL mRNA levels. The induction was blocked by actinomycin D, and the half-life of the transcript seemed to be unchanged by high glucose, suggesting a transcriptional nature of the glucose effect on HSL gene expression. The nonmetabolizable glucose analog 2-deoxyglucose, which has no mitogenic effect, induced HSL approximately 1.3-fold, whereas mannose was similar to glucose, stimulating HSL expression 1.7- to 2-fold. The results suggest that HSL is involved in the beta-cell responses to hyperglycemia and also in generating the lipid signal that is needed in stimulus-secretion coupling.

Expression, activity, and subcellular localization of testicular hormone-sensitive lipase during postnatal development in the guinea pig.

The present work reports on testicular hormone-sensitive lipase (HSL), the biological significance of which has been documented in male fertility. The HSL protein levels and enzymatic activity were measured, respectively, by densitometry of immunoreactive bands in Western blots, performed with antibodies against recombinant rat HSL, and by spectrophotometry in seminiferous tubules (STf) and interstitial tissue (ITf) enriched fractions generated from neonatal, pubertal, and adult guinea pig testes. In addition, HSL was studied in subcellular fractions obtained from STf isolated from adult testes and in epididymal spermatozoa (Spz). A 104-kDa HSL protein was detected in STf and ITf, the expression and activity of which increased with testicular development. Three immunoreactive bands of 104, 110, and 120 kDa were detected in the lysosomal subfraction, and two bands of 104 and 120 kDa were detected in Spz. The HSL activity was positively correlated with free (FC) and esterified (EC) cholesterol ratios in STf and ITf, but not with triglyceride (TG) levels, during testicular development. Immunolabeling localized HSL to elongated spermatids and Sertoli cells, where its distribution was stage-dependent, and within the cells lining the excurrent ducts of the testis. The findings of the 104- and 120-kDa HSL immunoreactive bands and of HSL activity in Spz as well, as the detection of the 104-, 110-, and 120-kDa immunoreactive bands in lysosomes, suggest that part of HSL may originate from germ cells and be imported in Sertoli cells. The HSL protein levels and enzymatic activity in ITf and STf were positively correlated with serum testosterone levels during development. To the best of our knowledge, this study is the first to contribute insights regarding the impact of HSL on FC:EC cholesterol ratios and TG levels in the interstitial tissue and tubules in relation to serum testosterone levels during postnatal development, and regarding the immunolocalization of the enzyme in regions of the male gamete consistent with spermatozoa-oocyte interaction.