Unique regulation of adipose triglyceride lipase (ATGL) by perilipin 5, a lipid droplet-associated protein

Lipolysis is a critical metabolic pathway contributing to energy homeostasis through degradation of triacylglycerides stored in lipid droplets (LDs), releasing fatty acids. Neutral lipid lipases act at the oil/water interface. In mammalian cells, LD surfaces are coated with one or more members of the perilipin protein family, which serve important functions in regulating lipolysis. We investigated mechanisms by which three perilipin proteins control lipolysis by adipocyte triglyceride lipase (ATGL), a key lipase in adipocytes and non-adipose cells. Using a cell culture model, we examined interactions of ATGL and its co-lipase CGI-58 with perilipin 1 (perilipin A), perilipin 2 (adipose differentiation-related protein), and perilipin 5 (LSDP5) using multiple techniques as follows: anisotropy Forster resonance energy transfer, co-immunoprecipitation, [(32)P]orthophosphate radiolabeling, and measurement of lipolysis. The results show that ATGL interacts with CGI-58 and perilipin 5; the latter is selectively expressed in oxidative tissues. Both proteins independently recruited ATGL to the LD surface, but with opposite effects; interaction of ATGL with CGI-58 increased lipolysis, whereas interaction of ATGL with perilipin 5 decreased lipolysis. In contrast, neither perilipin 1 nor 2 interacted directly with ATGL. Activation of protein kinase A (PKA) increased [(32)P]orthophosphate incorporation into perilipin 5 by 2-fold, whereas neither ATGL nor CGI-58 was labeled under the incubation conditions. Cells expressing both ectopic perilipin 5 and ATGL showed a 3-fold increase in lipolysis following activation of PKA. Our studies establish perilipin 5 as a novel ATGL partner and provide evidence that the protein composition of perilipins at the LD surface regulates lipolytic activity of ATGL.     

Human frame shift mutations affecting the carboxyl terminus of perilipin increase lipolysis by failing to sequester the adipose triglyceride lipase (ATGL) coactivator AB-hydrolase-containing 5 (ABHD5)

Perilipin (PLIN1) is a constitutive adipocyte lipid droplet coat protein. N-terminal amphipathic helices and central hydrophobic stretches are thought to anchor it on the lipid droplet, where it appears to function as a scaffold protein regulating lipase activity. We recently identified two different C-terminal PLIN1 frame shift mutations (Leu-404fs and Val-398fs) in patients with a novel subtype of partial lipodystrophy, hypertriglyceridemia, severe insulin resistance, and type 2 diabetes (Gandotra, S., Le Dour, C., Bottomley, W., Cervera, P., Giral, P., Reznik, Y., Charpentier, G., Auclair, M., Delépine, M., Barroso, I., Semple, R. K., Lathrop, M., Lascols, O., Capeau, J., O'Rahilly, S., Magré, J., Savage, D. B., and Vigouroux, C. (2011) N. Engl. J. Med. 364, 740-748.) When overexpressed in preadipocytes, both mutants fail to inhibit basal lipolysis. Here we used bimolecular fluorescence complementation assays to show that the mutants fail to bind ABHD5, permitting its constitutive coactivation of ATGL, resulting in increased basal lipolysis. siRNA-mediated knockdown of either ABHD5 or ATGL expression in the stably transfected cells expressing mutant PLIN1 reduced basal lipolysis. These insights from naturally occurring human variants suggest that the C terminus sequesters ABHD5 and thus inhibits basal ATGL activity. The data also suggest that pharmacological inhibition of ATGL could have therapeutic potential in patients with this rare but metabolically serious disorder.     

Identification and characterization of adipose triglyceride lipase (ATGL) gene in birds

Adipose triglyceride lipase (ATGL) is a triglyceride hydrolysis lipase and is generally related to lipid metabolism in animals. The ATGL gene was well studied in mammals, however very less was known in birds that differed significantly with mammals for lipid metabolism. In this study, cloning, mRNA real time and association analysis was performed to characterize the ATGL gene in birds. Results showed that the obtained ATGL gene cDNA of parrot, quail, duck were 1,651 bp (NCBI accession number: GQ221784), 1,557 bp (NCBI accession number: GQ221783) and 1,440 bp each, encoded 481-, 482- and 279-amino acid (AA) peptide, respectively. The parrot ATGL (pATGL) gene was found to predominantly express in breast muscle and leg muscle, and very higher ATGL mRNA level was also found in heart, abdominal fat and subcutaneous fat. The quail ATGL (qATGL) gene was also predominantly expressed in breast muscle and leg muscle, and then to a much lesser degree in heart. The duck ATGL (dATGL) gene was found to predominantly express in subcutaneous fat and abdominal fat, quite higher ATGL mRNA was also found in heart, spleen, breast muscle and leg muscle. Blast analyses indicated the high homology of ATGL and its patatin region, and moreover, and the active serine hydrolase motif (“GASAG” for “GXSXG”) and the glycine rich motif (“GCGFLG” for “GXGXXG”) were completely conservative among 14 species. Association analyses showed that c.950+24C>A, c.950+45C>G, c.950+73G>A, c.950+83C>T and c.950+128delA of chicken ATGL gene (cATGL) were all significantly or highly significantly with cingulated fat width (CFW) (P < 0.05 or P < 0.01), and c.777−26C>A, c.950+45C>G, c.950+73G>A and c.950+118C>T were all significantly or highly significantly with pH value of breast muscle (BMPH) (P < 0.05).     

cDNA cloning, characterization, and variation analysis of chicken adipose triglyceride lipase (ATGL) gene

Adipose triglyceride lipase (ATGL) is an important triglyceride-specific lipase that catalyzes the initial step in triglyceride hydrolysis. In this study, cloning, sequencing, and mRNA real-time analyses were employed to characterize the chicken ATGL gene. We obtained a total of 1,528-bp long chicken ATGL cDNA fragment including 51-bp 5'UTR, 1,452-bp open reading frame (ORF), and 25-bp 3'UTR. The predicted chicken ATGL had 483 amino acids and a molecular weight of 53.5 kDa, giving rise to identities of 66.5%, 67.3%, 68.2%, 64.8%, and 66.5% with that of human, mouse, rat, pig, and cattle, respectively. The chicken ATGL gene spanned over 30,197 bp and comprised of nine exons and eight introns, in which the intron 1 (21,146 bp) was far longer than others. It predominantly expressed in subcutaneous fat and abdominal fat and then in kidney and lung. Very low but detectable mRNA level was also observed in other 15 tissues. However, no mRNA was detected in spleen. A total of 15 single nucleotide polymorphisms (SNPs) were identified in its complete cDNA sequences with an average of one SNP in every 102 bp and a summarized nucleotide diversity of 3.02 x 10(-3). Seven of the 15 SNPs were non-synonymous. All SNPs had allelic frequencies over 5% and could be considered as candidate markers for future marker-trait association analysis.     

Structure-activity relationship studies for the development of inhibitors of murine adipose triglyceride lipase (ATGL)

High serum fatty acid (FA) levels are causally linked to the development of insulin resistance, which eventually progresses to type 2 diabetes and non-alcoholic fatty liver disease (NAFLD) generalized in the term metabolic syndrome. Adipose triglyceride lipase (ATGL) is the initial enzyme in the hydrolysis of intracellular triacylglycerol (TG) stores, liberating fatty acids that are released from adipocytes into the circulation. Hence, ATGL-specific inhibitors have the potential to lower circulating FA concentrations, and counteract the development of insulin resistance and NAFLD. In this article, we report about structure–activity relationship (SAR) studies of small molecule inhibitors of murine ATGL which led to the development of Atglistatin. Atglistatin is a specific inhibitor of murine ATGL, which has proven useful for the validation of ATGL as a potential drug target.     

Interactions of perilipin-5 (Plin5) with adipose triglyceride lipase

Members of the perilipin family of lipid droplet scaffold proteins are thought to play important roles in tissue-specific regulation of triglyceride metabolism, but the mechanisms involved are not fully understood. Present results indicate that adipose triglyceride lipase (Atgl) interacts with perilipin-5 (Plin5) but not perilipin-1 (Plin1). Protein interaction assays in live cells and in situ binding experiments showed that Atgl and its protein activator, α-β-hydrolase domain-containing 5 (Abhd5), each bind Plin5. Surprisingly, competition experiments indicated that individual Plin5 molecules bind Atgl or Abhd5 but not both simultaneously. Thus, the ability of Plin5 to concentrate these proteins at droplet surfaces involves binding to different Plin5 molecules, possibly in an oligomeric complex. The association of Plin5-Abhd5 complexes on lipid droplet surfaces was more stable than Plin5-Atgl complexes, and oleic acid treatment selectively promoted the interaction of Plin5 and Abhd5. Analysis of chimeric and mutant perilipin proteins demonstrated that amino acids 200-463 are necessary and sufficient to bind both Atgl and Abhd5 and that the C-terminal 64 amino acids of Plin5 are critical for the differential binding of Atgl to Plin5 and Plin1. Mutant Plin5 that binds Abhd5 but not Atgl was defective in preventing neutral lipid accumulation compared with wild type Plin5, indicating that the ability of Plin5 to concentrate these proteins on lipid droplets is critical to functional Atgl activity in cells.     

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.     

Application of a sensitive collection heuristic for very large protein families: Evolutionary relationship between adipose triglyceride lipase (ATGL) and classic mammalian lipases

Background  

Manually finding subtle yet statistically significant links to distantly related homologues becomes practically impossible for very populated protein families due to the sheer number of similarity searches to be invoked and analyzed. The unclear evolutionary relationship between classical mammalian lipases and the recently discovered human adipose triglyceride lipase (ATGL; a patatin family member) is an exemplary case for such a problem.     

Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover

Although neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRP-expressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells.     

Perilipin 5, a lipid droplet-associated protein, provides physical and metabolic linkage to mitochondria

Maintaining cellular lipid homeostasis is crucial to oxidative tissues, and it becomes compromised in obesity. Lipid droplets (LD) play a central role in lipid homeostasis by mediating fatty acid (FA) storage in the form of triglyceride, thereby lowering intracellular levels of lipids that mediate cellular lipotoxicity. LDs and mitochondria have interconnected functions, and anecdotal evidence suggests they physically interact. However, the mechanisms of interaction have not been identified. Perilipins are LD-scaffolding proteins and potential candidates to play a role in their interaction with mitochondria. We examined the contribution of LD perilipin composition to the physical and metabolic interactions between LD and mitochondria using multiple techniques: confocal imaging, electron microscopy (EM), and lipid storage and utilization measurements. Using neonatal cardiomyocytes, reconstituted cell culture models, and rodent heart tissues, we found that perilipin 5 (Plin5) recruits mitochondria to the LD surface through a C-terminal region. Compared with control cells, Plin5-expressing cells show decreased LD hydrolysis, decreased palmitate β-oxidation, and increased palmitate incorporation into triglycerides in basal conditions, whereas in stimulated conditions, LD hydrolysis inhibition is lifted and FA released for β-oxidation. These results suggest that Plin5 regulates oxidative LD hydrolysis and controls local FA flux to protect mitochondria against excessive exposure to FA during physiological stress.     

Adipose triglyceride lipase (ATGL) expression in human skeletal muscle is type I (oxidative) fiber specific

Accumulation of triacylglycerol (TAG) and lipid intermediates in skeletal muscle plays an important role in the etiology of insulin resistance and type 2 diabetes mellitus. Disturbances in skeletal muscle lipid turnover and lipolysis may contribute significantly to this. So far, knowledge on the regulation of muscle lipolysis is limited. Recently the identification of a new lipase was reported: adipose triglyceride lipase (ATGL). ATGL deficient animals show significant lipid accumulation in skeletal muscle, which may indicate that ATGL plays a pivotal role in skeletal muscle lipolysis. However, until now, it is still unknown whether ATGL protein is expressed in human skeletal muscle. Therefore, the aim of the present study was to investigate whether ATGL is expressed at the protein level in human skeletal muscle, and to examine whether its expression is fiber-type specific. To accomplish this, we established an imunohistochemical and immunofluorescent staining procedure to study ATGL protein expression in relation to fiber type in human vastus lateralis muscle of eight male subjects (BMI range: 21.0–34.5 kg/m² and age: 38–59 years). In the present paper we report for the first time that ATGL protein is indeed expressed in human skeletal muscle. Moreover, ATGL is exclusively expressed in type I (oxidative) muscle fibers, suggesting a pivotal role for ATGL in intramuscular fatty acid handling, lipid storage and breakdown.     

Breed difference and regulation of the porcine adipose triglyceride lipase and hormone sensitive lipase by TNFα

Adipose triglyceride lipase (ATGL) and hormone sensitive lipase (HSL) are major novel triglyceride lipases in animals. The aim of this study was to determine if there are differences in the porcine ATGL ( pATGL ) and HSL genes between Jinhua pigs (a fatty breed) and Landrace pigs (a leaner breed). In addition, the effect of TNFα and pATGL-specific siRNA ( pATGL-siRNA ) on the expression of pATGL and HSL in porcine adipocytes was also examined. Compared with Landrace pigs, the body weight ( BW ) of Jinhua pigs was lower ( P <  0.01), while intramuscular fat content (in the longissimus dorsi muscle), as well as the back fat thickness and body fat content were higher ( P <  0.01). The expression of pATGL and HSL mRNA in Jinhua pigs was lower ( P <  0.01) in subcutaneous adipose tissue, and greater ( P <  0.01) in longissimus dorsi muscle compared with Landrace pigs. In vitro treatment of porcine adipocytes with TNFα decreased ( P <  0.01) the glycerol release and the gene expression of pATGL , HSL and PPARγ in porcine adipocytes. Furthermore, transfection with pATGL-siRNA significantly decreased ( P <  0.01) the expression of pATGL , while it had no effect on the expression of HSL . Treatment with 25 ng/ml TNFα in conjunction with pATGL-siRNA significantly decreased ( P <  0.01) the expression of pATGL and HSL in cultured porcine adipocytes. These results provide useful information to further the understanding of the function of pATGL and HSL in porcine lipid metabolism, which should be applicable to the regulation of fat deposition and improvement of meat quality.     

Insulin increases macrophage triglyceride accumulation under diabetic conditions through the down regulation of hormone sensitive lipase and adipose triglyceride lipase

Diabetes mellitus (DM) is a major risk factor for the development of atherosclerosis, and high-serum levels of insulin are strongly associated with type 2 DM. Atherosclerosis is characterized by lipid-laden macrophage foam cell formations, which contain substantial amount of cholesterol and triglycerides (TG). This study analyzed for the first time, the effects of insulin on TG metabolism in macrophages under normal and diabetic conditions. Mouse peritoneal macrophages from C57BL6 mice were cultured under normal (5 mM) or high (diabetic condition, 25 mM) glucose concentration, with or without insulin, followed by the assessment of TGs metabolism in these cells. Under diabetic condition, insulin increased TG accumulation in macrophages by 100%, decreased cellular TG degradation by 21%, and increased C-reactive protein levels in macrophages by 83%. Insulin decreased hormone-sensitive lipase mRNA and protein expression by 28 and 60%, respectively, and adipose TG lipase (ATGL) protein expression by 36%, with no significant reduction in ATGL mRNA levels. The inhibition of insulin-mediated phosphorylation, and the addition of cyclic adenosine 3'5'-monoposphate, abolished the insulin-mediated inhibition of TGs degradation in cells. Insulin increases macrophage TGs accumulation only under diabetic conditions, suggesting that impaired glycemic control in diabetic patients treated with insulin may contribute to foam cell formations and enhanced inflammation in macrophages.     

The minimal domain of adipose triglyceride lipase (ATGL) ranges until leucine 254 and can be activated and inhibited by CGI-58 and G0S2, respectively

Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme of lipolysis. ATGL specifically hydrolyzes triacylglycerols (TGs), thereby generating diacylglycerols and free fatty acids. ATGL's enzymatic activity is co-activated by the protein comparative gene identification-58 (CGI-58) and inhibited by the protein G0/G1 switch gene 2 (G0S2). The enzyme is predicted to act through a catalytic dyad (Ser47, Asp166) located within the conserved patatin domain (Ile10-Leu178). Yet, neither an experimentally determined 3D structure nor a model of ATGL is currently available, which would help to understand how CGI-58 and G0S2 modulate ATGL's activity. In this study we determined the minimal active domain of ATGL. This minimal fragment of ATGL could still be activated and inhibited by CGI-58 and G0S2, respectively. Furthermore, we show that this minimal domain is sufficient for protein-protein interaction of ATGL with its regulatory proteins. Based on these data, we generated a 3D homology model for the minimal domain. It strengthens our experimental finding that amino acids between Leu178 and Leu254 are essential for the formation of a stable protein domain related to the patatin fold. Our data provide insights into the structure-function relationship of ATGL and indicate higher structural similarities in the N-terminal halves of mammalian patatin-like phospholipase domain containing proteins, (PNPLA1, -2,- 3 and -5) than originally anticipated.     

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.     

Partial purification and characterization of a triglyceride lipase from pig adipose tissue.

A triglyceride lipase was extracted from defatted pig adipose tissue powder with dilute ammonia and purified about 230-fold by a combination of ammonium sulfate fractionation, heparin-Sepharose 4B, DEAE-cellulose, and Sephadex G-150 column chromatographies and isoelectrofocusing electrophoresis. The enzyme was distinguishable in physical and kinetic properties from the two previously defined lipases in adipose tissue, lipoprotein lipase, and hormone-sensitive lipase. The purified enzyme was fully active in the absence of serum lipoprotein and was not stimulated by adenosine 3':5'-monophosphate-dependent protein kinase. In marked contrast to the already defined lipases, the enzyme was strongly inhibited by serum albumin. The enzyme had a molecular weigt of about 43,000, a pI of 5.2, and pH optimum of 7.0. The enzyme hydrolyzed triolein to oleic acid and glycerol, and did not exhibit esterase activity. The apparent Km for triolein was 0.05 mM. Physiological roles of this new species of lipase remained to be explored.     

The C-terminal region of human adipose triglyceride lipase affects enzyme activity and lipid droplet binding

Adipose triglyceride lipase (ATGL) catalyzes the first step in the hydrolysis of triacylglycerol (TG) generating diacylglycerol and free fatty acids. The enzyme requires the activator protein CGI-58 (or ABHD5) for full enzymatic activity. Defective ATGL function causes a recessively inherited disorder named neutral lipid storage disease that is characterized by systemic TG accumulation and myopathy. In this study, we investigated the functional defects associated with mutations in the ATGL gene that cause neutral lipid storage disease. We show that these mutations lead to the expression of either inactive enzymes localizing to lipid droplets (LDs) or enzymatically active lipases with defective LD binding. Additionally, our studies assign important regulatory functions to the C-terminal part of ATGL. Truncated mutant ATGL variants lacking approximately 220 amino acids of the C-terminal protein region do not localize to LDs. Interestingly, however, these mutants exhibit substantially increased TG hydrolase activity in vitro (up to 20-fold) compared with the wild-type enzyme, indicating that the C-terminal region suppresses enzyme activity. Protein-protein interaction studies revealed an increased binding of truncated ATGL to CGI-58, suggesting that the C-terminal part interferes with CGI-58 interaction and enzyme activation. Compared with the human enzyme, the C-terminal region of mouse ATGL is much less effective in suppressing enzyme activity, implicating species-dependent differences in enzyme regulation. Together, our results demonstrate that the C-terminal region of ATGL is essential for proper localization of the enzyme and suppresses enzyme activity.