Adipose triglyceride lipase and hormone-sensitive lipase are the major enzymes in adipose tissue triacylglycerol catabolism

The mobilization of free fatty acids from adipose triacylglycerol (TG) stores requires the activities of triacylglycerol lipases. In this study, we demonstrate that adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are the major enzymes contributing to TG breakdown in in vitro assays and in organ cultures of murine white adipose tissue (WAT). To differentiate between ATGL- and HSL-specific activities in cytosolic preparations of WAT and to determine the relative contribution of these TG hydrolases to the lipolytic catabolism of fat, mutant mouse models lacking ATGL or HSL and a mono-specific, small molecule inhibitor for HSL (76-0079) were used. We show that 76-0079 had no effect on TG catabolism in HSL-deficient WAT but, in contrast, essentially abolished free fatty acid mobilization in ATGL-deficient fat. CGI-58, a recently identified coactivator of ATGL, stimulates TG hydrolase activity in wild-type and HSL-deficient WAT but not in ATGL-deficient WAT, suggesting that ATGL is the sole target for CGI-58-mediated activation of adipose lipolysis. Together, ATGL and HSL are responsible for more than 95% of the TG hydrolase activity present in murine WAT. Additional known or unknown lipases appear to play only a quantitatively minor role in fat cell lipolysis.     

Adipose triglyceride lipase activity is inhibited by long-chain acyl-coenzyme A

Adipose triglyceride lipase (ATGL) is required for efficient mobilization of triglyceride (TG) stores in adipose tissue and non-adipose tissues. Therefore, ATGL strongly determines the availability of fatty acids for metabolic reactions. ATGL activity is regulated by a complex network of lipolytic and anti-lipolytic hormones. These signals control enzyme expression and the interaction of ATGL with the regulatory proteins CGI-58 and G0S2. Up to date, it was unknown whether ATGL activity is also controlled by lipid intermediates generated during lipolysis. Here we show that ATGL activity is inhibited by long-chain acyl-CoAs in a non-competitive manner, similar as previously shown for hormone-sensitive lipase (HSL), the rate-limiting enzyme for diglyceride breakdown in adipose tissue. ATGL activity is only marginally inhibited by medium-chain acyl-CoAs, diglycerides, monoglycerides, and free fatty acids. Immunoprecipitation assays revealed that acyl-CoAs do not disrupt the protein–protein interaction of ATGL and its co-activator CGI-58. Furthermore, inhibition of ATGL is independent of the presence of CGI-58 and occurs directly at the N-terminal patatin-like phospholipase domain of the enzyme. In conclusion, our results suggest that inhibition of the major lipolytic enzymes ATGL and HSL by long-chain acyl-CoAs could represent an effective feedback mechanism controlling lipolysis and protecting cells from lipotoxic concentrations of fatty acids and fatty acid-derived lipid metabolites.     

脂肪组织甘油三酯水解酶参与脂肪分解调控

循环中游离脂肪酸增高与肥胖、胰岛素抵抗和2型糖尿病密切相关,其主要来源于脂肪细胞内甘油三酯水解.调控脂肪分解的脂肪酶主要包括激素敏感脂肪酶(hormone-sensitive lipase,HSL)和最近发现的脂肪组织甘油三酯水解酶(adipose triglyceride lipase,ATGL),后者主要分布在脂肪组织,特异水解甘油三酯为甘油二酯,其转录水平受多种因素调控.CGI-58(属于α/β水解酶家族蛋白),可以活化ATGL,基础条件下该蛋白和脂滴包被蛋白(perilipin)紧密结合于脂滴表面,蛋白激酶A激活刺激脂肪分解时,CGI-58与perilipin分离,进而活化ATGL.     

Adipose triglyceride lipase plays a key role in the supply of the working muscle with fatty acids

FAs are mobilized from triglyceride (TG) stores during exercise to supply the working muscle with energy. Mice deficient for adipose triglyceride lipase (ATGL-ko) exhibit defective lipolysis and accumulate TG in adipose tissue and muscle, suggesting that ATGL deficiency 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 for proper energy supply of the skeletal muscle during exercise.     

Fate of fat: The role of adipose triglyceride lipase in lipolysis

Lipolysis, the coordinated catabolism of triacylglycerol (TG) stored in cellular lipid droplets, provides fatty acids, di-, and monoglycerides. These products are important energy substrates, precursors for other lipids, or lipid signaling molecules. Following their discovery by Hollenberg, C.H., Raben, M.S., and Astwood, E.B.(1961) and Vaughan, M., Berger, J.E., and Steinberg, D. (1964), hormone-sensitive lipase (HSL) and monoacylglycerol lipase stayed in the focus of research for three decades. Within the last decade, however, it became evident that the lipolytic pathway is incompletely understood. Studies on the regulation of lipolysis and the characterization of HSL-deficient mice indicated that additional previously unrecognized factors that contribute to fat catabolism must exist. This led to the discovery of the perilipin, adipophilin, Tip47 (PAT) family of lipid droplet binding proteins and the identification of a novel TG hydrolase named adipose triglyceride lipase (ATGL). This review focuses on the importance of ATGL as TG lipase within the “lipolytic machinery” and the current knowledge of molecular mechanisms that regulate ATGL activity.     

ATGL and HSL are not coordinately regulated in response to fuel partitioning in fasted rats

Prolonged fasting is characterized by lipid mobilization (Phase 2), followed by protein breakdown (Phase 3). Knowing that body lipids are not exhausted in Phase 3, we investigated whether changes in the metabolic status of prolonged fasted rats are associated with differences in the expression of epididymal adipose tissue proteins involved in lipid mobilization. The final body mass, body lipid content, locomotor activity and metabolite and hormone plasma levels differed between groups. Compared with fed rats, adiposity and epididymal fat mass decreased in Phase 2 (approximately two- to threefold) and Phase 3 (∼4.5-14-fold). Plasma nonesterified fatty acids (NEFA) concentrations were increased in Phase 2 (approximately twofold) and decreased in Phase 3 (approximately twofold). Daily locomotor activity was markedly increased in Phase 3 (∼11-fold). Compared with the fed state, expressions of adipose triglyceride lipase (ATGL; mRNA and protein), hormone-sensitive lipase (HSL; mRNA) and phosphorylated HSL at residue Ser660 (HSL Ser⁶⁶⁰) were increased during Phase 2 (∼1.5-2-fold). HSL (mRNA and protein) and HSL Ser⁶⁶⁰ levels were lowered during Phase 3 (∼3-12-fold). Unlike HSL and HSL Ser⁶⁶⁰, ATGL expression did not correlate with circulating NEFA, mostly due to data from animals in Phase 3. At this stage, ATGL could play an essential role for maintaining a low mobilization rate of NEFA, possibly to sustain muscle performance and hence increased locomotor activity. We conclude that ATGL and HSL are not coordinately regulated in response to changes in fuel partitioning during prolonged food deprivation, ATGL appearing as the major lipase in late fasting.     

Adipocyte triglyceride lipase expression in human obesity

We have investigated the gene and protein expression of adipose triglyceride lipase (ATGL) and triglyceride (TG) lipase activity from subcutaneous and visceral adipose tissue of lean and obese subjects. Visceral and subcutaneous adipose tissue was obtained from 16 age-matched lean and obese subjects during abdominal surgery. Tissues were analyzed for mRNA expression of lipolytic enzymes by real-time quantitative PCR. ATGL protein content was assessed by Western blot and TG lipase activity by radiometric assessment. Subcutaneous and visceral adipose tissue of obese subjects had elevated mRNA expression of PNPLA2 (ATGL) and other lipases including PNPLA3, PNPLA4, CES1, and LYPLAL1 (P < 0.05). Surprisingly, ATGL protein expression and TG lipase activity were reduced in subcutaneous adipose tissue of obese subjects. Immunoprecipitation of ATGL reduced total TG lipase activity in adipose lysates by 70% in obese and 83% in lean subjects. No significant differences in the ATGL activator CGI-58 mRNA levels (ABHD5) were associated with obesity. These data demonstrate that ATGL is important for efficient TG lipase activity in humans. They also demonstrate reduced ATGL protein expression and TG lipase activity despite increased mRNA expression of ATGL and other novel lipolytic enzymes in obesity. The lack of correlation between ATGL protein content and in vitro TG lipase activity indicates that small decrements in ATGL protein expression are not responsible for the reduction in TG lipase activity observed here in obesity, and that posttranslational modifications may be important.     

Several agents and pathways regulate lipolysis in adipocytes

Adipose tissue is the only tissue capable of hydrolyzing its stores of triacylglycerol (TAG) and of mobilizing fatty acids and glycerol in the bloodstream so that they can be used by other tissues. The full hydrolysis of TAG depends on the activity of three enzymes, adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and monoacylglycerol lipase, each of which possesses a distinct regulatory mechanism. Although more is known about HSL than about the other two enzymes, it has recently been shown that HLS and ATGL can be activated simultaneously, such that the mechanism that enables HSL to access the surface of lipid droplets also permits the stimulation of ATGL. The classical pathway of lipolysis activation in adipocytes is cAMP-dependent. The production of cAMP is modulated by G-protein-coupled receptors of the Gs/Gi family and cAMP degradation is regulated by phosphodiesterase. However, other pathways that activate TAG hydrolysis are currently under investigation. Lipolysis can also be started by G-protein-coupled receptors of the Gq family, through molecular mechanisms that involve phospholipase C, calmodulin and protein kinase C. There is also evidence that increased lipolytic activity in adipocytes occurs after stimulation of the mitogen-activated protein kinase pathway or after cGMP accumulation and activation of protein kinase G. Several agents contribute to the control of lipolysis in adipocytes by modulating the activity of HSL and ATGL. In this review, we have summarized the signalling pathways activated by several agents involved in the regulation of TAG hydrolysis in adipocytes.     

Lipolysis - a highly regulated multi-enzyme complex mediates the catabolism of cellular fat stores

Lipolysis is the biochemical pathway responsible for the catabolism of triacylglycerol (TAG) stored in cellular lipid droplets. The hydrolytic cleavage of TAG generates non-esterified fatty acids, which are subsequently used as energy substrates, essential precursors for lipid and membrane synthesis, or mediators in cell signaling processes. Consistent with its central importance in lipid and energy homeostasis, lipolysis occurs in essentially all tissues and cell types, it is most abundant, however, in white and brown adipose tissue. Over the last 5years, important enzymes and regulatory protein factors involved in lipolysis have been identified. These include an essential TAG hydrolase named adipose triglyceride lipase (ATGL) [annotated as patatin-like phospholipase domain-containing protein A2], the ATGL activator comparative gene identification-58 [annotated as α/β hydrolase containing protein 5], and the ATGL inhibitor G0/G1 switch gene 2. Together with the established hormone-sensitive lipase [annotated as lipase E] and monoglyceride lipase, these proteins constitute the basic "lipolytic machinery". Additionally, a large number of hormonal signaling pathways and lipid droplet-associated protein factors regulate substrate access and the activity of the "lipolysome". This review summarizes the current knowledge concerning the enzymes and regulatory processes governing lipolysis of fat stores in adipose and non-adipose tissues. Special emphasis will be given to ATGL, its regulation, and physiological function.     

Adipose triglyceride lipase regulation of skeletal muscle lipid metabolism and insulin responsiveness

Adipose triglyceride lipase (ATGL) is important for triglyceride (TG) metabolism in adipose tissue, and ATGL-null mice show increased adiposity. Given the apparent importance of ATGL in TG metabolism and the association of lipid deposition with insulin resistance, we examined the role of ATGL in regulating skeletal muscle lipid metabolism and insulin-stimulated glucose disposal. ATGL expression in myotubes was reduced by small interfering RNA and increased with a retrovirus encoding GFP-HA-ATGL. ATGL was also overexpressed in rats by in vivo electrotransfer. ATGL was down-regulated in skeletal muscle of obese, insulin-resistant mice and negatively correlated with intramyocellular TG levels. ATGL small interfering RNA in myotubes reduced TG hydrolase activity and increased TG content, whereas ATGL overexpression induced the reciprocal response, indicating that ATGL is an essential TG lipase in skeletal muscle. ATGL overexpression in myotubes increased the oxidation of fatty acid liberated from TG and diglyceride and ceramide contents. These responses in cells were largely recapitulated in rats overexpressing ATGL. When ATGL protein expression and TG hydrolase activity in obese, insulin-resistant rats were restored to levels observed in lean rats, TG content was reduced; however, the insulin resistance induced by the high-fat diet persisted. In conclusion, ATGL TG hydrolysis in skeletal muscle is a critical determinant of lipid metabolism and storage. Although ATGL content and TG hydrolase activity are decreased in obese, insulin-resistant phenotypes, overexpression does not rescue the condition, indicating reduced ATGL is unlikely to be a primary cause of obesity-associated insulin resistance.     

Hepatic overexpression of hormone-sensitive lipase and adipose triglyceride lipase promotes fatty acid oxidation, stimulates direct release of free fatty acids, and ameliorates steatosis

Hepatic steatosis is often associated with insulin resistance and obesity and can lead to steatohepatitis and cirrhosis. In this study, we have demonstrated that hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), two enzymes critical for lipolysis in adipose tissues, also contribute to lipolysis in the liver and can mobilize hepatic triglycerides in vivo and in vitro. Adenoviral overexpression of HSL and/or ATGL reduced liver triglycerides by 40-60% in both ob/ob mice and mice with high fat diet-induced obesity. However, these enzymes did not affect fasting plasma triglyceride and free fatty acid levels or triglyceride and apolipoprotein B secretion rates. Plasma 3-beta-hydroxybutyrate levels were increased 3-5 days after infection in both HSL- and ATGL-overexpressing male mice, suggesting an increase in beta-oxidation. Expression of genes involved in fatty acid transport and synthesis, lipid storage, and mitochondrial bioenergetics was unchanged. Mechanistic studies in oleate-supplemented McA-RH7777 cells with adenoviral overexpression of HSL or ATGL showed that reduced cellular triglycerides could be attributed to increases in beta-oxidation as well as direct release of free fatty acids into the medium. In summary, hepatic overexpression of HSL or ATGL can promote fatty acid oxidation, stimulate direct release of free fatty acid, and ameliorate hepatic steatosis. This study suggests a direct functional role for both HSL and ATGL in hepatic lipid homeostasis and identifies these enzymes as potential therapeutic targets for ameliorating hepatic steatosis associated with insulin resistance and obesity.     

启动脂肪细胞脂动员过程的新成员ATGL

 过去近20年里,激素敏感脂酶（HSL）一直被认为是脂肪细胞脂动员过程中唯一的脂肪水解限速酶,但随着HSL基因敲除鼠的出现,其限速作用受到了质疑.脂肪甘油三酯脂酶（adipose triglyceride lipase,ATGL)是随后发现的启动脂动员的又一个脂肪分解酶.本文就ATGL基因的结构和功能特征、表达及其调控途径和影响因素等方面的研究进展进行了综述,并对今后的研究方向和应用做了展望.     

Chronic TNFα and cAMP pre-treatment of human adipocytes alter HSL, ATGL and perilipin to regulate basal and stimulated lipolysis

We examined the effects of chronic TNFα and dibutyryl-cAMP (Db-cAMP) pre-treatment on the lipolytic machinery of human hMADS adipocytes. TNFα decreased adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) protein content and triglycerides (TG)-hydrolase activity but increased basal lipolysis due to a marked reduction in perilipin (PLIN) protein content. Conversely, Db-cAMP increased ATGL and HSL protein content but prevented PLIN phosphorylation, the net result being accentuated basal lipolysis. In forskolin-stimulated conditions, TNFα and Db-cAMP pre-treatment decreased stimulated TG-hydrolase activity and impaired PLIN phosphorylation. Together, this resulted in a severely attenuated response to forskolin-stimulated lipolysis.     

Adipose triglyceride lipase activity regulates cancer cell proliferation via AMP-kinase and mTOR signaling

Aberrant fatty acid (FA) metabolism is a hallmark of proliferating cells, including untransformed fibroblasts or cancer cells. Lipolysis of intracellular triglyceride (TG) stores by adipose triglyceride lipase (ATGL) provides an important source of FAs serving as energy substrates, signaling molecules, and precursors for membrane lipids. To investigate if ATGL-mediated lipolysis impacts cell proliferation, we modified ATGL activity in murine embryonic fibroblasts (MEFs) and in five different cancer cell lines to determine the consequences on cell growth and metabolism. Genetic or pharmacological inhibition of ATGL in MEFs causes impaired FA oxidation, decreased ROS production, and a substrate switch from FA to glucose leading to decreased AMPK-mTOR signaling and higher cell proliferation rates. ATGL expression in these cancer cells is low when compared to MEFs. Additional ATGL knockdown in cancer cells did not significantly affect cellular lipid metabolism or cell proliferation whereas the ectopic overexpression of ATGL increased lipolysis and reduced proliferation. In contrast to ATGL silencing, pharmacological inhibition of ATGL by Atglistatin© impeded the proliferation of diverse cancer cell lines, which points at an ATGL-independent effect. Our data indicate a crucial role of ATGL-mediated lipolysis in the regulation of cell proliferation. The observed low ATGL activity in cancer cells may represent an evolutionary selection process and mechanism to sustain high cell proliferation rates. As the increasing ATGL activity decelerates proliferation of five different cancer cell lines this may represent a novel therapeutic strategy to counteract uncontrolled cell growth.     

Adipose triglyceride lipase expression and fasting regulation are differently affected by cold exposure in adipose tissues of lean and obese Zucker rats

Adipose triglyceride lipase (ATGL) hydrolyzes triacylglycerols to diacylglycerols in the first step of lipolysis, providing substrates for hormone-sensitive lipase (HSL). Here we studied whether ATGL messenger RNA (mRNA) and protein levels were affected by 24-h cold exposure in different white adipose tissue depots and in interscapular brown adipose tissue of lean and obese Zucker rats submitted to feeding and 14-h fasting conditions. HSL mRNA expression was also studied in selected depots. In both lean and obese rats, as a general trend, cold exposure increased ATGL mRNA and protein levels in the different adipose depots, except in the brown adipose tissue of lean animals, where a decrease was observed. In lean rats, cold exposure strongly improved fasting up-regulation of ATGL expression in all the adipose depots. Moreover, in response to fasting, in cold-exposed lean rats, there was a stronger positive correlation between circulating nonesterified fatty acids (NEFA) and ATGL mRNA levels in the adipose depots and a higher percentage increase of circulating NEFA in comparison with control animals not exposed to cold. In obese rats, fasting-induced up-regulation of ATGL was impaired and was not improved by cold. The effects of obesity and cold exposure on HSL mRNA expression were similar to those observed for ATGL, suggesting common regulatory mechanisms for both proteins. Thus, cold exposure increases ATGL expression and improves its fasting-up-regulation in adipose tissue of lean rats. In obese rats, cold exposure also increases ATGL expression but fails to improve its regulation by fasting, which could contribute to the increased difficulty for mobilizing lipids in these animals.     

Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes

Phosphorylation of the lipid droplet-associated protein perilipin A (Peri A) mediates the actions of cyclic AMP-dependent protein kinase A (PKA) to stimulate triglyceride hydrolysis (lipolysis) in adipocytes. Studies addressing how Peri A PKA sites regulate adipocyte lipolysis have relied on non-adipocyte cell models, which express neither adipose triglyceride lipase (ATGL), the rate-limiting enzyme for triglyceride catabolism in mice, nor the "downstream" lipase, hormone-sensitive lipase (HSL). ATGL and HSL are robustly expressed by adipocytes that we generated from murine embryonic fibroblasts of perilipin knock-out mice. Adenoviral expression of Peri A PKA site mutants in these cells reveals that mutation of serine 517 alone is sufficient to abrogate 95% of PKA (forskolin)-stimulated fatty acid (FA) and glycerol release. Moreover, a "phosphomimetic" (aspartic acid) substitution at serine 517 enhances PKA-stimulated FA release over levels obtained with wild type Peri A. Studies with ATGL-and HSL-directed small hairpin RNAs demonstrate that 1) ATGL activity is required for all PKA-stimulated FA and glycerol release in murine embryonic fibroblast adipocytes and 2) all PKA-stimulated FA release in the absence of HSL activity requires serine 517 phosphorylation. These results provide the first demonstration that Peri A regulates ATGL-dependent lipolysis and identify serine 517 as the Peri A PKA site essential for this regulation. The contributions of other PKA sites to PKA-stimulated lipolysis are manifested only in the presence of phosphorylated or phosphomimetic serine 517. Thus, serine 517 is a novel "master regulator" of PKA-stimulated adipocyte lipolysis.