Molecular evidence supporting the portal theory: a causative link between visceral adiposity and hepatic insulin resistance
Morvarid KabirKaryn J. CatalanoSuchitra AnanthnarayanStella P. KimG. W. van CittersMelvin K. DeaRichard N. Bergman
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The mechanism by which increased central adiposity causes hepatic insulin resistance is unclear. The "portal hypothesis" implicates increased lipolytic activity in the visceral fat and therefore increased delivery of free fatty acids (FFA) to the liver, ultimately leading to liver insulin resistance. To test the portal hypothesis at the transcriptional level, we studied expression of several genes involved in glucose and lipid metabolism in the fat-fed dog model with visceral adiposity vs. controls (n = 6). Tissue samples were obtained from dogs after 12 wk of either moderate fat (42% calories from fat; n = 6) or control diet (35% calories from fat). Northern blot analysis revealed an increase in the ratio of visceral to subcutaneous (v/s ratio) mRNA expression of both lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor-gamma (PPARgamma). In addition, the ratio for sterol regulatory element-binding transcription factor-1 (SREBP-1) tended to be higher in fat-fed dogs, suggesting enhanced lipid accumulation in the visceral fat depot. The v/s ratio of hormone-sensitive lipase (HSL) increased significantly, implicating a higher rate of lipolysis in visceral adipose despite hyperinsulinemia in obese dogs. In fat-fed dogs, liver SREBP-1 expression was increased significantly, with a tendency for increased fatty acid-binding protein (FABP) expression. In addition, glucose-6-phosphatase (G-6-Pase) and phosphoenolpyruvate carboxykinase (PEPCK) increased significantly, consistent with enhanced gluconeogenesis. Liver triglyceride content was elevated 45% in fat-fed animals vs. controls. Moreover, insulin receptor binding was 50% lower in fat-fed dogs. Increased gene expression promoting lipid accumulation and lipolysis in visceral fat, as well as elevated rate-limiting gluconeogenic enzyme expression in the liver, is consistent with the portal theory. Further studies will need to be performed to determine whether FFA are involved directly in this pathway and whether other signals (either humoral and/or neural) may contribute to the development of hepatic insulin resistance observed with visceral obesity.Keywords:
Hyperinsulinemia
Adipose triglyceride lipase
Gluconeogenesis
Hormone-sensitive lipase
Hepatic lipase
Key points Hormone‐sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) are the key enzymes involved in intramuscular triglyceride (IMTG) lipolysis. In isolated rat skeletal muscle, HSL translocates to IMTG‐containing lipid droplets (LDs) following electrical stimulation, but whether HSL translocation occurs in human skeletal muscle during moderate‐intensity exercise is currently unknown. Perilipin‐2 (PLIN2) and perilipin‐5 (PLIN5) proteins have been implicated in regulating IMTG lipolysis by interacting with HSL and ATGL in cell culture and rat skeletal muscle studies. This study investigated the hypothesis that HSL (but not ATGL) redistributes to LDs during moderate‐intensity exercise in human skeletal muscle, and whether the localisation of these lipases with LDs was affected by the presence of PLIN proteins on the LDs. HSL preferentially redistributed to PLIN5‐associated LDs whereas ATGL distribution was not altered with exercise; this is the first study to illustrate the pivotal step of HSL redistribution to PLIN5‐associated LDs following moderate‐intensity exercise in human skeletal muscle. Abstract Hormone‐sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) control skeletal muscle lipolysis. ATGL is present on the surface of lipid droplets (LDs) containing intramuscular triglyceride (IMTG) in both the basal state and during exercise. HSL translocates to LD in ex vivo electrically stimulated rat skeletal muscle. Perilipin‐2‐ and perilipin‐5‐associated lipid droplets (PLIN2+ and PLIN5+ LDs) are preferentially depleted during exercise in humans, indicating that these PLINs may control muscle lipolysis. We aimed to test the hypothesis that in human skeletal muscle in vivo HSL (but not ATGL) is redistributed to PLIN2+ and PLIN5+ LDs during moderate‐intensity exercise. Muscle biopsies from 8 lean trained males (age 21 ± 1 years, BMI 22.6 ± 1.2 kg m −2 and 48.2 ± 5.0 ml min −1 kg −1 ) were obtained before and immediately following 60 min of cycling exercise at ∼59% . Cryosections were stained using antibodies targeting ATGL, HSL, PLIN2 and PLIN5. LDs were stained using BODIPY 493/503. Images were obtained using confocal immunofluorescence microscopy and object‐based colocalisation analyses were performed. Following exercise, HSL colocalisation to LDs increased ( P < 0.05), and was significantly greater to PLIN5+ LDs (+53%) than to PLIN5− LDs (+34%) ( P < 0.05), while the increases in HSL colocalisation to PLIN2+ LDs (+16%) and PLIN2− LDs (+28%) were not significantly different. Following exercise, the fraction of LDs colocalised with ATGL (0.53 ± 0.04) did not significantly change ( P < 0.05) and was not affected by PLIN association to the LDs. This study presents the first evidence of exercise‐induced HSL redistribution to LDs in human skeletal muscle and identifies PLIN5 as a facilitator of this mechanism.
Perilipin
Adipose triglyceride lipase
Hormone-sensitive lipase
Lipid droplet
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Adipose triglyceride lipase
Hormone-sensitive lipase
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Significance Fat mass is controlled by the balance of triacylglycerol (TAG) degradation and synthesis. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are key players in TAG catabolism providing fatty acids (FAs) as energy substrates and metabolic intermediates. Here, we show that ATGL and HSL metabolize TAGs containing antidiabetic lipid mediators (FA esters of hydroxy FAs), distinctly controlling the release of bioactive lipids. Our paper connects lipolysis-mediated TAG metabolism with the regulation of antidiabetic signaling lipids.
Adipose triglyceride lipase
Catabolism
Hormone-sensitive lipase
Triglyceride lipase
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Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are key enzymes involved in intracellular degradation of triacylglycerols. It was the aim of this study to elucidate how the deficiency in one of these proteins affects the residual lipolytic proteome in adipose tissue. For this purpose, we compared the lipase patters of brown and white adipose tissue from ATGL (−/−) and HSL (−/−) mice using differential activity-based gel electrophoresis. This method is based on activity-recognition probes possessing the same substrate analogous structure but carrying different fluorophores for specific detection of the enzyme patterns of two different tissues in one electrophoresis gel. We found that ATGL-deficiency in brown adipose tissue had a profound effect on the expression levels of other lipolytic and esterolytic enzymes in this tissue, whereas HSL-deficiency hardly showed any effect in brown adipose tissue. Neither ATGL- nor HSL-deficiency greatly influenced the lipase patterns in white adipose tissue. Enzyme activities of mouse tissues on acylglycerol substrates were analyzed as well, showing that ATGL-and HSL-deficiencies can be compensated for at least in part by other enzymes. The proteins that responded to ATGL-deficiency in brown adipose tissue were overexpressed and their activities on acylglycerols were analyzed. Among these enzymes, Es1, Es10, and Es31-like represent lipase candidates as they catalyze the hydrolysis of long-chain acylglycerols. Adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) are key enzymes involved in intracellular degradation of triacylglycerols. It was the aim of this study to elucidate how the deficiency in one of these proteins affects the residual lipolytic proteome in adipose tissue. For this purpose, we compared the lipase patters of brown and white adipose tissue from ATGL (−/−) and HSL (−/−) mice using differential activity-based gel electrophoresis. This method is based on activity-recognition probes possessing the same substrate analogous structure but carrying different fluorophores for specific detection of the enzyme patterns of two different tissues in one electrophoresis gel. We found that ATGL-deficiency in brown adipose tissue had a profound effect on the expression levels of other lipolytic and esterolytic enzymes in this tissue, whereas HSL-deficiency hardly showed any effect in brown adipose tissue. Neither ATGL- nor HSL-deficiency greatly influenced the lipase patterns in white adipose tissue. Enzyme activities of mouse tissues on acylglycerol substrates were analyzed as well, showing that ATGL-and HSL-deficiencies can be compensated for at least in part by other enzymes. The proteins that responded to ATGL-deficiency in brown adipose tissue were overexpressed and their activities on acylglycerols were analyzed. Among these enzymes, Es1, Es10, and Es31-like represent lipase candidates as they catalyze the hydrolysis of long-chain acylglycerols. Excess lipids are stored as intracellular triacylglycerol and steryl ester deposits in animals, plant seeds, and fungi. In mammals adipose tissue is the body's largest storage organ for triacylglycerols (TAG) 1The abbreviations used are:TAGTriacylglycerolApo AIVApolipoprotein A-IVATGLAdipose triglyceride lipaseBATBrown adipose tissueDABGEDifferential activity-based gel electrophoresisDAGDiacylglycerolFAFatty acidHSLHormone-sensitive lipasekoKnock-outLacZβ-galactosidaseLyPLLysophospholipaseMAGMonoacylglycerolMGLMonoglycerid lipasePAF-AHPlatelet-activating factor acetylhydrolasepNPp-NitrophenolTGHTriacylglycerol hydrolaseWATWhite adipose tissuewtWild-typeARPactivity recognition probe. 1The abbreviations used are:TAGTriacylglycerolApo AIVApolipoprotein A-IVATGLAdipose triglyceride lipaseBATBrown adipose tissueDABGEDifferential activity-based gel electrophoresisDAGDiacylglycerolFAFatty acidHSLHormone-sensitive lipasekoKnock-outLacZβ-galactosidaseLyPLLysophospholipaseMAGMonoacylglycerolMGLMonoglycerid lipasePAF-AHPlatelet-activating factor acetylhydrolasepNPp-NitrophenolTGHTriacylglycerol hydrolaseWATWhite adipose tissuewtWild-typeARPactivity recognition probe. as the primary source of energy during periods of starvation and increased energy demand. Two types of adipose tissue, namely brown (BAT) and white (WAT) adipose tissue exist in mammals, localizing to anatomically distinct areas. BAT and WAT differ in almost all their structural and functional features. Whereas BAT develops prenatally, WAT is subject to maturation postnatally. The different appearance of brown and white adipose tissue is caused by differences in lipid content and the abundance of mitochondria in the constituent adipocytes. Brown fat cells contain several small multilocular lipid droplets and a high number of large mitochondria with numerous cristae. In addition, BAT is highly vascularized and highly innervated by the sympathetic nervous system. In contrast, white adipocytes, usually contain one major unilocular lipid droplet that fills most of the cytoplasm leaving space for only few mitochondria (1Cinti S. The adipose organ: morphological perspectives of adipose tissues.Proc. Nutr. Soc. 2001; 60: 319-328Crossref PubMed Google Scholar, 2Cinti S. 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In vivo regulation of lipolysis in humans.J. Lipid Res. 1994; 35: 177-193Abstract Full Text PDF PubMed Google Scholar, 5Zechner R. Strauss J.G. Haemmerle G. Lass A. Zimmermann R. Lipolysis: pathway under construction.Curr. Opin. Lipidol. 2005; 16: 333-340Crossref PubMed Scopus (222) Google Scholar). The balance of lipid storage and mobilization is tightly regulated to ensure whole body energy homeostasis. The mobilization of triacylglycerol stores by activation of lipolytic enzymes is specifically stimulated by hormones and chemical agents. In addition, a number of specific physiological conditions owing to exercise, aging, and nutritional status (feeding, fasting) also regulate degradation of TAGs (6Large V. Arner P. Regulation of lipolysis in humans. Pathophysiological modulation in obesity, diabetes, and hyperlipidaemia.Diabetes Metab. 1998; 24: 409-418PubMed Google Scholar). 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Investig. 2002; 32: 14-23Crossref PubMed Scopus (1001) Google Scholar). The sequential hydrolysis of triacylglycerols in adipocytes producing FFAs is catalyzed by a cascade of lipolytic enzymes, with different substrate preferences (11Watt M.J. Steinberg G.R. Regulation and function of triacylglycerol lipases in cellular metabolism.Biochem. J. 2008; 414: 313-325Crossref PubMed Scopus (126) Google Scholar). The committed enzyme catalyzing the first step of TAG hydrolysis is ATGL, which was identified in three different laboratories in 2004 (12Jenkins C.M. Mancuso D.J. Yan W. Sims H.F. Gibson B. Gross R.W. Identification, cloning, expression, and purification of three novel human calcium-independent phospholipase A2 family members possessing triacylglycerol lipase and acylglycerol transacylase activities.J. Biol. Chem. 2004; 279: 48968-48975Abstract Full Text Full Text PDF PubMed Scopus (678) Google Scholar, 13Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. Desnutrin, an adipocyte gene encoding a novel patatin domain-containing protein, is induced by fasting and glucocorticoids: Ectopic expression of desnutrin increases triglyceride hydrolysis.J. Biol. Chem. 2004; 279: 47066-47075Abstract Full Text Full Text PDF PubMed Scopus (506) Google Scholar, 14Zimmermann R. Strauss J.G. Haemmerle G. Schoiswohl G. Birner-Gruenberger R. Riederer M. Lass A. Neuberger G. Eisenhaber F. Hermetter A. Zechner R. Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.Science. 2004; 306: 1383-1386Crossref PubMed Scopus (1505) Google Scholar). Its activity appears to be largely dependent on association with CGI-58 (14Zimmermann R. Strauss J.G. Haemmerle G. Schoiswohl G. Birner-Gruenberger R. Riederer M. Lass A. Neuberger G. Eisenhaber F. Hermetter A. Zechner R. Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.Science. 2004; 306: 1383-1386Crossref PubMed Scopus (1505) Google Scholar, 15Lass A. Zimmermann R. Haemmerle G. Riederer M. Schoiswohl G. Schweiger M. Kienesberger P. Strauss J.G. Gorkiewicz G. Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman Syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). HSL exhibits a much broader substrate spectrum, with preference for diacylglycerols as well as cholesteryl and retinyl esters (16Doolittle M. Reue K. Holm C. Østerlund T. Hormone-sensitive lipase and neutral cholesteryl ester lipase.in: Lipase Phospholipase Protocols. Vol. 109. Humana Press, 1999: 109-121Google Scholar, 17Kraemer F.B. Shen W.J. Hormone-sensitive lipase.J. Lipid Res. 2002; 43: 1585-1594Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). In the final step of lipolysis, monoacylglycerol lipase (MGL) degrades MAG thereby generating free fatty acid and glycerol (18Tornqvist Hans Belfrage Per Lowenstein J.M. [75] Monoacylglycerol lipase from rat adipose tissue: EC 3.1.1.23 Glycerol-monoester acylhydrolase.Methods Enzymol. 1981; 71: 646-652Crossref PubMed Scopus (11) Google Scholar). ATGL is the major TAG lipase in adipose tissue. Expression in other tissues is rather low. Currently it cannot be excluded, that other lipases also exist that are important for lipid catabolism (19Birner-Gruenberger R. Susani-Etzerodt H. Waldhuber M. Riesenhuber G. Schmidinger H. Rechberger G. Kollroser M. Strauss J.G. Lass A. Zimmermann R. Haemmerle G. Zechner R. Hermetter A. The lipolytic proteome of mouse adipose tissue.Mol. Cell. Proteomics. 2005; 4: 1710-1717Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Recent functional proteomic screens in various mouse tissues led to the identification of enzyme candidates that are currently subject to functional characterization (unpublished data). The intracellular degradation of triacylglycerols is catalyzed by a cascade of lipolytic enzymes. There appears to be an overlap of substrate preferences as well as a redundancy of lipases to ensure a proper function of this important catabolic process if individual lipase activities are reduced or entirely absent. This study aimed at identifying the effects of ATGL and HSL-deficiency on the expression of other lipolytic enzymes in adipose tissue. For this purpose, we compared the lipolytic proteomes of BAT and WAT from ATGL (−/−) and HSL (−/−) mice with the enzyme patterns of wt tissues using differential activity-based gel electrophoresis (DABGE) (20Morak M. Schmidinger H. Krempl P. Rechberger G. Kollroser M. Birner-Gruenberger R. Hermetter A. Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities.J. Lipid Res. 2009; 50: 1281-1292Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). This method is based on activity-recognition probes containing same substrate analogous structures but carrying different fluorophores for specific detection of the individual enzyme patterns of two different tissues. These inhibitors react with the nucleophilic serine in the active center of lipolytic enzymes thereby generating covalent bound lipid-protein complexes, which can be separated by gel electrophoresis. We found, that ATGL-deficiency in BAT had a profound effect on the expression levels of other lipolytic and esterolytic enzymes in this tissue, whereas HSL-deficiency hardly showed any effect in BAT. Neither ATGL- nor HSL-deficiency greatly influenced the lipase patterns in WAT. ATGL-deficiency led to a significant but not total reduction in the TAG hydrolyzing activity of adipose tissues. Obviously, there must be (an)other enzyme(s) compensating for the hydrolytic capacity of ATGL. Three proteins that responded to ATGL-deficiency in BAT were overexpressed and their activities on acylglycerols were analyzed. Among these proteins, Es1, Es10, and Es31-like emerged as novel lipase candidates in these studies. ATGL-deficient (ATGL-ko) and HSL-deficient (HSL-ko) mice were generated by targeted homologous recombination (21Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. Kratky D. Wagner E.F. Klingenspor M. Hoefler G. Zechner R. defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase.Science. 2006; 312: 734-737Crossref PubMed Scopus (1011) Google Scholar, 22Haemmerle G. Zimmermann R. Hayn M. Theussl C. Waeg G. Wagner E.F. Sattler W. Margin T.M. Zechner R. Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.J. Biol. Chem. 2002; 277: 4806-4815Abstract Full Text Full Text PDF PubMed Scopus (486) Google Scholar). Nontransgenic littermates expressing two intact alleles of mouse HSL were used as wild-type (wt) control. All animals were maintained on a regular light-dark cycle (14 h light, 10 h dark) and kept on a standard laboratory chow diet containing 4.5% fat and 21% protein (SSNIFF, Germany) with free access to water. Fat pads were collected from fed (free access to food over night) or fasted (food was removed for 20 h) animals aged between 3–6 months between 9:00 and 10:00 a.m. All procedures in this study were in conformity with the Public Health Service Policy on the use of Laboratory Animals and were approved by local ethical committees. Gonadal fat pads (white adipose tissue) and brown adipose tissue of fed and fasted mice were surgically removed and washed in phosphate buffered saline (PBS). Homogenization was performed on ice in lysis buffer (0.25 m sucrose, 1 mm EDTA, 1 mm dithioerythritol, 20 μg/ml leupeptin, 2 μg/ml antipain, 1 μg/ml pepstatin) using a motor-driven Teflon-glass homogenizer (8 strokes, at 1500 rpm, Schuett Labortechnik, Germany). Cell debris was removed by centrifugation at 1000 × g for 15 min to obtain cytoplasmic extracts. Protein concentration was determined using the BIORAD Protein Assay based on the method of Bradford (23Bradford M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215653) Google Scholar). Unless otherwise indicated, incubations of proteomes with activity tags were conducted as follows: For a sample containing 50 μg of protein, the following reagent was prepared. 5 μl of a 10 mm solution of Triton X-100 in CHCl3 (final sample concentration 1 mm) and 5 μl of activity recognition probe dissolved in CHCl3 (1 nmol/10 μl, final sample concentration 10 μm) were mixed and the organic solvent was removed under a stream of argon. Fifty microliters of homogenate (1.0 mg/ml protein) were added and the resulting mixture was incubated at 37 °C under light protection for 2 h. Wild-type and knock-out were labeled with Cy3- and Cy5- tagged ARPs, respectively, and vice versa in a dye-swap experiment. 1:1 (protein amount) mixtures of wild-type and knock-out tissue homogenates were labeled with Cy2b-tagged inhibitor as an internal standard. The samples were mixed, proteins were precipitated in 10% ice-cold trichloroacetic acid on ice for 1 h and collected by centrifugation at 4 °C at 14000 g for 15 min. The pellet was washed once with ice-cold acetone and resuspended in sample buffer for 1D SDS-PAGE (20 mm KH2PO4, 6 mm EDTA, 60 mg/ml SDS, 100 mg/ml glycerol, 0.5 mg/ml bromophenol blue, 20 μl/ml mercaptoethanol, pH 6.8) or sample buffer for 2D PAGE (7 m urea, 2 m thiourea, 4% 3-[(3-cholamidopropyl)dimethylammonio]propanesulfonate, 2% Pharmalyte 3–10). Before loading onto the gel, the samples for 1D SDS-PAGE were heated to 95 °C for 5 min. SDS-PAGE was performed essentially according to the method of Fling and Gregerson (24Fling S.P. Gregerson D.S. Peptide and protein molecular weight determination by electrophoresis using a high-molarity tris buffer system without urea.Anal. Biochem. 1986; 155: 83-88Crossref PubMed Scopus (782) Google Scholar) using a Tris/glycine buffer system. Proteins (15 μg protein/lane) were applied onto a 5% stacking gel and separated in a 10% resolving gel at 20 mA constant current (BIORAD Mini PROTEAN 3), respectively. 2D-gelelectrophoresis was performed as described by Görg et al. (25Görg A. Postel W. Günther S. Two-dimensional electrophoresis. The current state of two-dimensional electrophoresis with immobilized pH gradients.Electrophoresis. 1988; 9: 531-546Crossref PubMed Scopus (865) Google Scholar, 26Görg A. Postel W. Günther S. Weser J. Improved horizontal two-dimensional electrophoresis with hybrid isoelectric focusing in immobilized pH gradients in the first dimension and laying-on transfer to the second dimension.Electrophoresis. 1985; 6: 599-604Crossref Scopus (100) Google Scholar, 27Görg A. Weiss W. Dunn M.J. Current two-dimensional electrophoresis technology for proteomics.Proteomics. 2004; 4: 3665-3685Crossref PubMed Scopus (1534) Google Scholar). In the first dimension, 45 μg or 510 μg protein were separated by isoelectric focusing in 7 cm or 18 cm immobilized nonlinear pH 3–10 gradients (IPG-strips, GE Healthcare, Germany) using Amersham Biosciences Multiphor II (GE Healthcare). Isoelectric focusing was performed using a discontinuos voltage gradient starting at 0 V reaching 200 V within the first minute. The voltage was then increased to 3500 V during the following 1.5 h, and held at this level for another 1.5 h. In the second dimension, proteins were separated by 10% SDS-PAGE on 7 cm gels at 20 mA constant current for 1.5 h. Gels were fixed in 7.5% acetic acid and 10% ethanol and scanned at a resolution of 100 μm (BIORAD Molecular ImagerTM FX Pro Plus). Cy2b fluorescence was detected at 530 nm and an excitation wavelength of 488 nm. Cy3 fluorescence was determined at 605 nm and an excitation wavelength of 532 nm. Cy5 fluorescence was measured at 695 nm and an excitation wavelength of 633 nm. For visualization of the whole protein pattern, gels were stained with RuBPS following the manufacturer's instructions (Molecular Probes, Eugene OR) and scanned at an emission wavelength of 605 nm and an excitation wavelength of 488 nm. The signals obtained with RuBPS depended on prestaining with the Cy-tagged inhibitors. Proteins giving fluorescent lanes/spots with the ARPs showed less intense lanes/spots with RuBPS. The PMT voltage of the Molecular Imager was individually set for each Cy-tagged inhibitor using the same sample to reach comparable fluorescence signal intensities. Quantification of the fluorescence signals was performed using Quantity One 1D analysis software (BIORAD, Vienna, Austria) and Progenesis PG 220 versus 2006 2D analysis software (Nonlinear Dynamics, Newcastle upon Tyne, UK). Fluorescent protein spots were excised and tryptically digested according to the method by Shevchenko et al. (28Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels.Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7807) Google Scholar). Peptide extracts were dissolved in 0.1% formic acid and separated on a nano-HPLC-system (FAMOS™ -autosampler, SWITCHOS™ -loading system, ULTIMATE™ - dual gradient system, LC-Packings, Amsterdam, Netherlands). Twenty-microliter samples were injected and concentrated on the loading column (LC Packings PepMap™ C-18 5 μm 100 Å, 300 μm ID × 1 mm) for 5 min using 0.1% formic acid as isocratic solvent at a flow rate of 20 μl/min. The column was then switched into the nano-flow-circuit and the sample was loaded on the nano-column (LC-Packings C-18 PepMap™ 75 μm ID × 150 mm) at a flow rate of 300 nl/min and separated using a gradient from 0.3% formic acid and 5% acetonitrile to 0.3% formic acid and 50% acetonitrile over 60 min. The sample was ionized in a Finnigan nano-ESI source equipped with NanoSpray tips (PicoTip™ Emitter, New Objective) and analyzed in a Thermo-Finnigan LCQ Deca XPplus iontrap mass-spectrometer. The MS/MS data were analyzed by searching the NCBI public database with SpectrumMill 03.03.084 SR4 (Agilent). Details and acceptance parameters can be found in the supplementary data (see supplemental file S1). Identified protein sequences were subjected to BLAST and motif search for identification of potential serine hydrolases. The coding sequences of HSL (NCBI Accession # 677884), Es1 (NCBI Accession # 247269928), Es10 (esterase D/formylglutathione hydrolase (NCBI Accession # 146134463)) and Es31-like (NCBI Accession # 226874913) were amplified by PCR from cDNA prepared from mouse BAT using Reverse transcription system (Promega Corporation, Madison, WI) and cloned into the eukaryotic expression vector pcDNA3.1 (Invitrogen Inc.) as described previously for HSL and ATGL (14Zimmermann R. Strauss J.G. Haemmerle G. Schoiswohl G. Birner-Gruenberger R. Riederer M. Lass A. Neuberger G. Eisenhaber F. Hermetter A. Zechner R. Fat mobilization in adipose tissue is promoted by adipose triglyceride lipase.Science. 2004; 306: 1383-1386Crossref PubMed Scopus (1505) Google Scholar). Transfection of COS-7 cells was performed with Metafectene™ (Biontex) according to the manufacturer's instruction. Apparent molecular weights of the proteins were about 88 kDa for HSL, 61 kDa for Es1, 35 kDa for Es10, and 63 kDa for Es31-like. Apolipoprotein A-IV was isolated from human plasma by lipoprotein depletion according to literature (29Haiman M. Salvenmoser W. Scheiber K. Lingenhel A. Rudolph C. Schmitz G. Kronenberg F. Dieplinger H. Immunohistochemical localization of apolipoprotein A-IV in human kidney tissue.Kidney Int. 2005; 68: 1130-1136Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). p-Nitrophenyl acetate, butyrate, and laurate were used as substrates. Fifty millimolar substrate solutions in ethanol were prepared and stored at −25 °C. 30 μl of the substrates were added to 10 ml assay buffer (20 mm Tris/HCl, pH 8.0, 150 mm NaCl, 0.01% Triton X-100). In a 96-well microtiterplate, 1 μg protein was mixed with 200 μl of substrate solution. Absorbance at 405 nm was measured every 3 min for 30 min using a spectrophotometer (Anthos 2010, Labtec Instruments, Wals, Austria). Solutions containing 1 μmol of pyrene-labeled acylglycerol and 1 μmol of PC/PI (3:1 mol:mol) were mixed. The solvent of the resultant solution was removed under a stream of nitrogen and the substrate was solubilized in 10 ml of 50 mm Tris/HCl buffer (pH 7.4) followed by vigorous vortexing for 5 min and pulsed sonication for 5 min. For the assay, 50 μl of the substrate solution, was diluted with 150 μl of 50 mm Tris/HCl buffer. The reaction was started by adding 50 μg of protein. The assay mixture was incubated for 1 h at 37 °C and 1000 rpm. The reaction was stopped by adding CHCl3/MeOH (2:1) and 20 μl HCl followed by vigorous vortexing for 5 min. For better phase separation, the mixture was centrifuged for 2 min at 9000 × g followed by removal of the aqueous layer. The organic solvent was removed under a stream of nitrogen and the residue was dissolved in 20 μl CHCl3. The entire organic lipid solution was applied onto a TLC plate and separated using CHCl3/EtAc (90:10, V:V) as mobile phase. The fluorescent spots were quantified with a CCD camera using an excitation wavelength of 365 nm. The amounts of the fluorescent components were determined from their respective fluorescence intensities. It was the aim of this study to determine the effects of ATGL- and HSL-deficiency on the expression of other lipolytic activities in BAT and WAT. For this purpose, we analyzed the lipolytic proteomes of both tissues of ATGL- and HSL-deficient mice. The substrate preferences of the enzymes responding to lipase deficiencies were determined using synthetic mono-, di-, and triacylglycerols. The lipolytic proteomes of adipose tissue homogenates isolated from ATGL−/− and HSL−/− mice were analyzed and compared with the enzyme patterns of the wt tissues using the DABGE method (20Morak M. Schmidinger H. Krempl P. Rechberger G. Kollroser M. Birner-Gruenberger R. Hermetter A. Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities.J. Lipid Res. 2009; 50: 1281-1292Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). This technique was developed for the comparison of the enzyme components of two different samples in one electrophoresis gel. It is based on protein labeling with activity recognition probes (ARPs, Fig. 1) possessing the same substrate analogous structure but carrying different cyanine-dyes (Cy2b, Cy3, and Cy5) as reporter tags. In a typical experiment, the wild-type (wt) tissue was labeled with Cy3-Ethyl-P (green emission) whereas the knock-out (ko) tissue was labeled with Cy5-Ethyl-P (red emission). Both samples were labeled with Cy2b-Ethyl-P as internal standard. To avoid artifacts because of fluorescence labeling, we performed a dye-swap experiment. In this case, the wild-type and knock-out samples were labeled with the Cy5- and Cy3-Ethyl-P, respectively. After labeling, the samples were mixed as described in the method section, followed by protein precipitation and 1D or 2D gel electrophoresis. In the 2D gel some of the labeled enzymes appeared as horizontal polypeptide ladders, which are typical for post-translational modifications. For identification enzyme spots and lanes were excised from the gel, tryptically digested and analyzed by nanoHPLC-MS/MS. The fluorescence patterns of the tagged proteins on the gels were detected by laser scanning. Fig. 2 shows the fluorescence images of 1D and 2D electrophoresis gels containing the labeled BAT and WAT proteins from ATGL- and HSL-ko mice as compared with the wild-type. Active enzymes more abundant in the wt tissue appear green (red/dye-swap), those more abundant in the ko sample appear red (green/dye-swap), and enzymes which are equally abundant in both samples are yellow. The green and red fluorescence intensities of the spots were determined and the enzymes ratios of the ko versus wt mice were calculated as described before (20Morak M. Schmidinger H. Krempl P. Rechberger G. Kollroser M. Birner-Gruenberger R. Hermetter A. Differential activity-based gel electrophoresis for comparative analysis of lipolytic and esterolytic activities.J. Lipid Res. 2009; 50: 1281-1292Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). Results for 2D and 1D gels are shown in Table I, Table II, respectively. Detailed results are shown in supplemental files S2-S4. The raw data can be found under the following links: ftp://ftp.tugraz.at/pub/genau and http://ftp.tugraz.at/pub/genau.Table IDifferential lipolytic proteomes of adipose tissue from ATGL- and HSL-deficient mice (2D PAGE) Open table in a new tab Table IIDifferential lipolytic proteomes of adipose tissue from ATGL- and HSL-deficient mice (1D SDS-PAGE) Open table in a new tab The lipolytic p
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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.
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The perilipins are a family of 5 proteins that are among the most abundant lipid droplet‐associated proteins in animal cells; most cells in the body express 2 – 4 perilipins. Perilipins form organizing scaffolds at the surfaces of lipid droplets to coordinate the activity of lipases and other factors. Different cells express different combinations of lipases; hence, lipolysis is controlled by a unique combination of perilipins and lipases in a cell‐specific manner. In adipocytes, perilipin 1 serves a critical function in the control of lipolysis catalyzed by hormone‐sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). Under basal (fed) conditions, perilipin 1 restricts the access of lipases to lipid droplets, thus promoting the storage of triacylglycerols. Following binding of catecholamines to cell surface receptors of adipocytes, phosphorylation of perilipin 1 by protein kinase A promotes lipolysis through multiple mechanisms including facilitating recruitment and binding of HSL to lipid droplets and release of CGI‐58, a co‐activator of ATGL, from the perilipin scaffold. Perilipin 5 is highly expressed in oxidative tissues including heart and skeletal muscle, where ATGL and HSL catalyze lipolysis. Perilipin 5 binds both CGI‐58 and ATGL at lipid droplet surfaces, and serves as a negative regulator of lipolysis under basal conditions. Supported by NIH R01 DK54797.
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Obesity is associated with increased triacylglycerol (TAG) storage in adipose tissue and insulin resistance. The mobilization of stored TAG is mediated by hormone-sensitive lipase (HSL) and the recently discovered adipose triglyceride lipase (ATGL). The aim of the present study was to examine whether ATGL and HSL mRNA and protein expression are altered in insulin-resistant conditions. In addition, we investigated whether a possible impaired expression could be reversed by a period of weight reduction.Adipose tissue biopsies were taken from obese subjects (n = 44) with a wide range of insulin resistance, before and just after a 10-wk hypocaloric diet. ATGL and HSL protein and mRNA expression was determined by Western blot and quantitative RT-PCR, respectively.Fasting insulin levels and the degree of insulin resistance (using the homeostasis model assessment index for insulin resistance) were negatively correlated with ATGL and HSL protein expression, independent of age, gender, fat cell size, and body composition. Both mRNA and protein levels of ATGL and HSL were reduced in insulin-resistant compared with insulin-sensitive subjects (P < 0.05). Weight reduction significantly decreased ATGL and HSL mRNA and protein expression. A positive correlation between the decrease in leptin and the decrease in ATGL protein level after weight reduction was observed. Finally, ATGL and HSL mRNA and protein levels seem to be highly correlated, indicating a tight coregulation and transcriptional control.In obese subjects, insulin resistance and hyperinsulinemia are strongly associated with ATGL and HSL mRNA and protein expression, independent of fat mass. Data on weight reduction indicated that also other factors (e.g. leptin) relate to ATGL and HSL protein expression.
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Obesity, insulin resistance, and type 2 diabetes are associated with elevated concentration of circulating free fatty acids (FFAs), which are critically governed by the process of triglyceride lipolysis in adipocytes. Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) are two major enzymes in the control of triacylglycerol hydrolysis in adipose tissue. ATGL expressed predominantly in white adipose tissue specifically initiates triacylglycerol hydrolysis to generate diacylglycerols and FFA, a role distinguished from HSL that mainly hydrolyzes diacylglycerols. The transcription of ATGL is regulated by several factors. ATGL activity is regulated by CGI-58. Under basal conditions, interaction of CGI-58 with a lipid droplet associating protein, perilipin, results in an inactivation of ATGL activity. During PKA-stimulated lipolysis, CGI-58 is released from phosphorylated perilipin and in turn, binds to ATGL. This action facilitates triglyceride lipolysis. This review focuses on the regulation and function of ATGL in adipose lipolysis and metabolism.
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Cardiotrophin-1 (CT-1) is a cytokine with antiobesity properties and with a role in lipid metabolism regulation and adipose tissue function. The aim of this study was to analyze the molecular mechanisms involved in the lipolytic actions of CT-1 in adipocytes. Recombinant CT-1 (rCT-1) effects on the main proteins and signaling pathways involved in the regulation of lipolysis were evaluated in 3T3-L1 adipocytes and in mice. rCT-1 treatment stimulated basal glycerol release in a concentration- and time-dependent manner in 3T3-L1 adipocytes. rCT-1 (20 ng/ml for 24 h) raised cAMP levels, and in parallel increased protein kinase (PK)A-mediated phosphorylation of perilipin and hormone sensitive lipase (HSL) at Ser660. siRNA knock-down of HSL or PKA, as well as pretreatment with the PKA inhibitor H89, blunted the CT-1-induced lipolysis, suggesting that the lipolytic action of CT-1 in adipocytes is mainly mediated by activation of HSL through the PKA pathway. In ob/ob mice, acute rCT-1 treatment also promoted PKA-mediated phosphorylation of perilipin and HSL at Ser660 and Ser563, and increased adipose triglyceride lipase (desnutrin) content in adipose tissue. These results showed that the ability of CT-1 to regulate the activity of the main lipases underlies the lipolytic action of this cytokine in vitro and in vivo, and could contribute to CT-1 antiobesity effects.
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Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)
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