Decreased fatty acid esterification compensates for the reduced lipolytic activity in hormone-sensitive lipase-deficient white adipose tissue
2003; Elsevier BV; Volume: 44; Issue: 11 Linguagem: Inglês
10.1194/jlr.m300190-jlr200
ISSN1539-7262
AutoresRobert Zimmermann, Guenter Haemmerle, Elke Wagner, Juliane Gertrude Bogner‐Strauß, Dagmar Kratky, Rudolf Zechner,
Tópico(s)Fatty Acid Research and Health
ResumoIt has been observed previously that hormone-sensitive lipase-deficient (HSL-ko) mice have reduced white adipose tissue (WAT) stores compared to control mice. These findings contradict the expectation that the decreased lipolytic activity in WAT of HSL-ko mice would cause accumulation of triglycerides (TGs) in that tissue. Here we demonstrate that the cellular TG synthesis in HSL-deficient WAT is markedly reduced due to downregulation of the enzymatic activities of glycerophosphate acyltransferase, dihydroxyacetonphosphate acyltransferase, lysophosphatidate acyltransferase, and diacylglycerol acyltransferase. Fatty acid de novo synthesis is also decreased due to reduced cellular glucose uptake, reduced glucose incorporation into adipose tissue lipids, and reduced activities of acetyl:CoA carboxylase and fatty acid synthase. Finally, the activities of phosphoenolpyruvate carboxykinase (PEPCK), acyl:CoA synthetase (ACS), and glucose 6-phosphate dehydrogenase, the enzymes that provide glycerol-3-phosphate, acyl-CoA, and NADPH for TG synthesis, respectively, are decreased in HSL-ko mice. The reduced expression of the peroxisome proliferator-activated receptor γ (PPARγ) target genes PEPCK, ACS, and aP2, as well as reduced mRNA levels of PPARγ itself, suggest the involvement of this transcription factor in the downregulation of lipogenesis.Taken together, these results establish that in the absence of HSL, the reduced NEFA production is counteracted by a drastic reduction of NEFA reesterification that provides sufficient quantities of NEFA for release into the circulation. These metabolic adaptations result in decreased fat mass in HSL-ko mice. It has been observed previously that hormone-sensitive lipase-deficient (HSL-ko) mice have reduced white adipose tissue (WAT) stores compared to control mice. These findings contradict the expectation that the decreased lipolytic activity in WAT of HSL-ko mice would cause accumulation of triglycerides (TGs) in that tissue. Here we demonstrate that the cellular TG synthesis in HSL-deficient WAT is markedly reduced due to downregulation of the enzymatic activities of glycerophosphate acyltransferase, dihydroxyacetonphosphate acyltransferase, lysophosphatidate acyltransferase, and diacylglycerol acyltransferase. Fatty acid de novo synthesis is also decreased due to reduced cellular glucose uptake, reduced glucose incorporation into adipose tissue lipids, and reduced activities of acetyl:CoA carboxylase and fatty acid synthase. Finally, the activities of phosphoenolpyruvate carboxykinase (PEPCK), acyl:CoA synthetase (ACS), and glucose 6-phosphate dehydrogenase, the enzymes that provide glycerol-3-phosphate, acyl-CoA, and NADPH for TG synthesis, respectively, are decreased in HSL-ko mice. The reduced expression of the peroxisome proliferator-activated receptor γ (PPARγ) target genes PEPCK, ACS, and aP2, as well as reduced mRNA levels of PPARγ itself, suggest the involvement of this transcription factor in the downregulation of lipogenesis. Taken together, these results establish that in the absence of HSL, the reduced NEFA production is counteracted by a drastic reduction of NEFA reesterification that provides sufficient quantities of NEFA for release into the circulation. These metabolic adaptations result in decreased fat mass in HSL-ko mice. Storage and mobilization of metabolic energy in mammals are coordinated by tight hormonal control of the synthesis and the catabolism of triglycerides (TGs) in white adipose tissue (WAT). Imbalances in these anabolic and catabolic processes might be involved in dysregulation of body weight control and the pathogenesis of obesity and related disorders. Hormone-sensitive lipase (HSL) is considered to be the central enzyme in the mobilization of WAT-TG stores. This multifunctional enzyme has been shown to hydrolyse TGs, diglycerides, and monoglycerides, as well as cholesteryl ester and other small water-soluble substrates (1Yeaman S.J. Smith G.M. Jepson C.A. Wood S.L. Emmison N. The multifunctional role of hormone-sensitive lipase in lipid metabolism.Adv. Enzyme Regul. 1994; 34: 355-370Crossref PubMed Scopus (65) Google Scholar). During periods of increased energy demand, HSL is activated by hormones such as catecholamines, which leads to an increase in the intracellular cAMP levels, resulting in the activation of protein kinase A (PKA) and phosphorylation of HSL (2McKnight G.S. Cummings D.E. Amieux P.S. Sikorski M.A. Brandon E.P. Planas J.V. Motamed K. Idzerda R.L. Cyclic AMP, PKA, and the physiological regulation of adiposity.Recent Prog. Horm. Res. 1998; 53: 139-159PubMed Google Scholar). In parallel, activation of PKA leads to the phosphorylation of perilipin A, which elicits the translocation of phosphorylated HSL from the cytoplasm to the lipid droplet (3Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation.J. Cell Biol. 2003; 161: 1093-1103Crossref PubMed Scopus (420) Google Scholar), a process that might also involve lipotransin (4Syu L.J. Saltiel A.R. Lipotransin: a novel docking protein for hormone-sensitive lipase.Mol. Cell. 1999; 4: 109-115Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Once activated and translocated, HSL can hydrolyze lipid droplet-associated TG, and the mobilized nonesterified fatty acids (NEFAs) are released from WAT into the circulation. Hormone-sensitive lipase-deficient (HSL-ko) mice exhibited a marked decrease of acylglyceride hydrolase activity in WAT, mainly because of decreased diglyceride hydrolase activity (5Haemmerle G. Zimmermann R. Hayn M. Theussl C. Waeg G. Wagner E. Sattler W. Magin T.M. Wagner E.F. 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 (487) Google Scholar). Accordingly, fasted HSL-ko mice had reduced plasma NEFA and TG levels, and liver TG stores were depleted, indicating that under starving conditions, the HSL-ko WAT is not able to supply the body with sufficient NEFA (6Haemmerle G. Zimmermann R. Strauss J.G. Kratky D. Riederer M. Knipping G. Zechner R. Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle.J. Biol. Chem. 2002; 277: 12946-12952Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). In the fed state, HSL-ko mice have normal NEFA and TG levels in plasma, and experiments with ex vivo fat pads and isolated adipocytes showed that the basic NEFA release in HSL-ko cells is similar to that of controls and can be stimulated by β-adrenergic agonists (5Haemmerle G. Zimmermann R. Hayn M. Theussl C. Waeg G. Wagner E. Sattler W. Magin T.M. Wagner E.F. 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 (487) Google Scholar, 7Okazaki H. Osuga J. Tamura Y. Yahagi N. Tomita S. Shionoiri F. Iizuka Y. Ohashi K. Harada K. Kimura S. Gotoda T. Shimano H. Yamada N. Ishibashi S. Lipolysis in the absence of hormone-sensitive lipase: evidence for a common mechanism regulating distinct lipases.Diabetes. 2002; 51: 3368-3375Crossref PubMed Scopus (101) Google Scholar). Accordingly, at least one more as yet unidentified lipase(s), in addition to HSL, is thought to be present in adipose tissue (AT) (5Haemmerle G. Zimmermann R. Hayn M. Theussl C. Waeg G. Wagner E. Sattler W. Magin T.M. Wagner E.F. 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 (487) Google Scholar, 7Okazaki H. Osuga J. Tamura Y. Yahagi N. Tomita S. Shionoiri F. Iizuka Y. Ohashi K. Harada K. Kimura S. Gotoda T. Shimano H. Yamada N. Ishibashi S. Lipolysis in the absence of hormone-sensitive lipase: evidence for a common mechanism regulating distinct lipases.Diabetes. 2002; 51: 3368-3375Crossref PubMed Scopus (101) Google Scholar). However, this hypothetical lipolytic enzyme(s) cannot fully compensate for the absence of HSL, as evident from the reduced total acylhydrolase activity observed in HSL-ko mice (5Haemmerle G. Zimmermann R. Hayn M. Theussl C. Waeg G. Wagner E. Sattler W. Magin T.M. Wagner E.F. 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 (487) Google Scholar, 8Osuga J. Ishibashi S. Oka T. Yagyu H. Tozawa R. Fujimoto A. Shionoiri F. Yahagi N. Kraemer F.B. Tsutsumi O. Yamada N. Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.Proc. Natl. Acad. Sci. USA. 2000; 97: 787-792Crossref PubMed Scopus (504) Google Scholar). Additionally, HSL-ko mice exhibited reduced WAT mass (9Wang S.P. Laurin N. Himms-Hagen J. Rudnicki M.A. Levy E. Robert M.F. Pan L. Oligny L. Mitchell G.A. The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice.Obes. Res. 2001; 9: 119-128Crossref PubMed Scopus (195) Google Scholar). This unexpected finding indicated that in addition to HSL-independent lipolytic activities, other metabolic adaptations must exist that affect TG synthesis in HSL-deficient WAT. Therefore it is conceivable that a reduction of NEFA re-esterification could cause reduced fat synthesis, thereby increasing the NEFA supply for the vascular system. In this study, we demonstrate that in the absence of HSL, the esterification of NEFA is drastically reduced, leading to TG resynthesis and providing a compensatory mechanism for the reduced lipolytic activity. Together with the observed decrease in de novo synthesis of fatty acids, the downregulation of TG synthesis also provides a plausible explanation for the observed loss in WAT mass in HSL-ko mice. HSL-ko mice were generated by targeted homologous recombination as previously described (5Haemmerle G. Zimmermann R. Hayn M. Theussl C. Waeg G. Wagner E. Sattler W. Magin T.M. Wagner E.F. 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 (487) Google Scholar). For breeding experiments, mice heterozygous for the deleted HSL allele were used to generate homozygous HSL-ko animals. Mice were maintained on a regular light-dark cycle (14 h light, 10 h dark) and kept on a standard laboratory chow diet (4.5% wt/wt fat). Only male animals at the age of 22 to 26 weeks were used for metabolic experiments. Blood samples and epididymal fat pads were collected from fed (ad libitum access to food overnight) or fasted (food was removed for 20 h) animals between 9 AM and 10 AM. cDNA probes for Northern blot analysis of mouse diacylglycerol acyltransferase-1 and -2 (DGAT-1, DGAT-2), mitochondrial glycerol-3-phosphate acyltransferase (GPAM), lysophosphatidate acyltransferase (LPAAT), glycerophosphate acyltransferase (GNPAT), acyl-CoA synthetase (ACS), and sterol regulatory element binding protein (SREBP) were prepared by RT-PCR by using first-strand cDNA from mouse fat mRNA. The PCR primers used to generate these probes were as follows. DGAT-1: forward, 5′-TCT GAG GTG CCA TCG TCT GC-3′, and reverse, 5′-CGG CAC CAC AGG TTG ACA TC-3′; DGAT-2: forward, 5′-TGG CTC CAG CAT CCT CTC AG-3′, and reverse, 5′-AGA TCA GCT CCA TGG CGC AG-3′; GPAM: forward, 5′-ATT CCT GCA CGC CAC AGA GC-3′, and reverse, 5′-TGA TAA CGC CTC TCG CCA CA-3′; LPAAT: forward, 5′-AGA GAT ACA GCC AGC CGC CA-3′, and reverse, 5′-CCG GAA GAT GGT GAG CAT GG-3′; GNPAT: forward, 5′-CGC ATA GGA GCC ATT CGG TT-3′, and reverse, 5′-AGT GGT GGA CTC CTT CGG CT-3′; ACS: forward, 5′-GAA GAT CAA GCG AGG CTC CA-3′, and reverse, 5′-CCT TCC TGC ATT CCA TCG TC-3′; SREBP-1: forward, 5′-GCAAATCACTGAAGGACCTGG-3′, and reverse, 5′-GCTGGTGCAGCTTATGGTAGAC-3′. A 977 bp XhoI fragment derived from the cytosolic isoform of mouse phosphoenolpyruvate carboxykinase (PEPCK) cDNA was used to detect PEPCK mRNA. The cDNA-specific PCR products containing single 3′-dA overhangs generated by Taq polymerase (AdvanTaq DNA polymerase, Clontech) were cloned into the linearized acceptor vector pST-Blue-1 (Novagen, Madison, WI). The following primer sequences specific for β-actin, glucose-6-phosphate dehydrogenase (G6PDH), and fatty acid synthase (FAS) were used for real-time PCR. β-actin: forward, 5′-GAC AGG ATG CAG AAG GAG ATT ACT G-3′, and reverse, 5′-GCC ACC GAT CCA CAC AGA GT-3′, probe 5′-CAA GAT CAT TGC TCC TCC TGA GCG CA-3′; G6PDH: forward, 5′-GGG CAA AGA GAT GGT CCA GA-3′, and reverse, 5′-CAA TGT TGT CTC GAT TCC AGA TG-3′, probe 5′-ATC CTG TTG GCA AAC CTC AGC ACC A-3′; FAS: forward, 5′-ATG TGA ACA GCG CAG GCA C-3′, and reverse, 5′-ACA ATG CCC ACG TCA CCA AT-3′, probe 5′-TGT CCT CCC AGG CCT TGC CGT-3′. For measurement of AT and ACS activity, intraperitoneal fat pads were surgically removed and washed in PBS containing 1 mM EDTA. Homogenization was performed on ice in lysis buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithioerythritol) using a motor-driven teflon glass homogenizer (8 strokes at medium speed; Schütt Labortechnik, Germany). The homogenized tissue was centrifuged at 1,000 g for 15 min. The supernatant was centrifuged at 100,000 g to obtain membrane and cytosolic fractions. For photometric assays of acetyl-CoA carboxylase (ACC), FAS, and G6PDH, fat pad homogenates were prepared as follows. Abdominal fat pads were shock frozen in liquid nitrogen and homogenized in 3 vol of cold homogenization buffer [9 mM KH2PO4, 85 mM K2HPO4, 1 mM DTT, and 70 mM KHCO3, (pH 7)]. After centrifugation for 12 min at 20,000 g, the fat cake was discarded, and the cytosolic fraction was obtained after centrifugation of the supernatant at 100,000 g for 1 h at 4°C. ACS activity was measured as synthesis of acyl-CoA in the presence of 2 mM [3H]9,10-oleic acid (3,000 cpm/nmol) and 0.6 mM CoA as described (10Iritani N. Ikeda Y. Kajitani H. Selectivities of 1-acylglycerophosphorylcholine acyltransferase and acyl-CoA synthetase for n-3 polyunsaturated fatty acids in platelets and liver microsomes.Biochim. Biophys. Acta. 1984; 793: 416-422Crossref PubMed Scopus (36) Google Scholar). The reaction was performed in a volume of 0.2 ml and contained 0.1 M Tris-HCl (pH 8), 5 mM DTT, 0.15 M KCl, 15 mM MgCl2, 15 mM ATP, and 5 μg protein of the membrane fraction. The mixture was incubated for 10 min at 37°C, and the reaction was stopped by the addition of 2.25 ml isopropanol-hexane-1 M H2SO4 (40:10:1; v/v/v). After centrifugation (5 min, 1,000 g), the upper phase was removed, and the lower phase containing the water-soluble acyl-CoA was washed twice with hexane containing 4 mg/ml oleic acid. The radioactivity in the aqueous phase was determined by liquid scintillation. Glycerol-3-phosphate acyltransferase (GPAT) and GNPAT activities were measured as described by Jones and Hajra (11Jones K.M. Hajra A.K. Assay of dihydroxyacetone phosphate acyltransferase with 32P-labeled substrate.Clin. Chem. 1994; 40: 946-947Crossref PubMed Scopus (7) Google Scholar) using 32P-labeled glycerol-3-phosphate or dihydroxyacetone phosphate (500 cpm/nmol) and palmitoyl-CoA as substrate. The reaction was performed in a volume of 0.6 ml and contained 5 μg cell membrane protein, 75 mM Tris-HCl, pH 7.5 (for GPAT activity), or 2-[N-morpolino]ethansulfonic acid, pH 5.5, (for GNPAT activity), 1.7 mg/ml BSA, 8.3 mM NaF, 8.3 mM MgCl2, 83 μM palmitoyl-CoA, 420 μM 32P-labeled glycerol or dihydroxyacetone, and 100 μl of an egg yolk-lecithin suspension (Sigma P7318, 20 mg/ml) prepared by sonification in 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA and centrifuged at 40,000 g for 30 min. To distinguish between mitochondrial and microsomal GPAT activity (GPAM and GPATer, respectively), the membrane proteins were incubated in the absence or in the presence of 2 mM N-ethylmaleimide for 15 min on ice (12Igal R.A. Wang S. Gonzalez-Baro M. Coleman R.A. Mitochondrial glycerol phosphate acyltransferase directs the incorporation of exogenous fatty acids into triacylglycerol.J. Biol. Chem. 2001; 276: 42205-42212Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). After incubation for 10 min at 37°C, the reaction was stopped by the addition of 2.25 ml CHCl3-methanol (1:2; v/v). Phase separation was achieved by the addition of 0.75 ml CHCl3 and 0.75 ml 2 M KCl in 0.2 M H3PO4. After centrifugation (5 min, 1,000 g), the upper phase was removed, and the organic phase was washed twice with chloroform-methanol-0.5 N H3PO4 (1:12:12; v/v/v). The radioactivity in the organic phase was determined by liquid scintillation. LPAAT activity was measured with 1-oleoyl-sn-glycero-3-phosphate as acceptor and [1-14C]palmitoyl-CoA as acyl donor (25,000 cpm/nmol) as described (13Eberhardt C. Gray P.W. Tjoelker L.W. Human lysophosphatidic acid acyltransferase. cDNA cloning, expression, and localization to chromosome 9q34.3.J. Biol. Chem. 1997; 272: 20299-20305Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar). The reaction mixture contained 0.1 M Hepes (pH 7.5), 0.2 M NaCl, 5% glycerol, 10 mM EDTA, 5 mM β-mercaptoethanol, 20 μM lysophosphatidate (LPA), 40 μM acyl-CoA, and 5 μg cell membrane protein in a volume of 0.2 ml. After incubation for 10 min at 37°C, the lipids were extracted as described (14Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). The organic phase was dried in a SpeedVac concentrator, redissolved in CHCl3 containing phosphatidic acid as standard, and applied onto TLC plates (Silica gel 60, plastic, VWR). The TLC plates were developed in CHCl3-methanol-water (65:25:4; v/v/v). The lipids were visualized with iodine vapor, the radioactive spots corresponding to phosphatidic acid were cut out, and the radioactivity was quantitated by liquid scintillation. DGAT activities were determined as described (15Coleman R.A. Diacylglycerol acyltransferase and monoacylglycerol acyltransferase from liver and intestine.Methods Enzymol. 1992; 209: 98-104Crossref PubMed Scopus (81) Google Scholar), with sn-1,2-dioleoylglycerol as acceptor and 1-[14C]palmitoyl-CoA as acyl donor (25,000 cpm/nmol). The reaction mixture contained 175 mM Tris-HCl (pH 8), 1 mg/ml BSA (fatty acid-free), 16 mM MgCl2, 0.2 mM diacylglycerol, 40 μM palmitoyl-CoA, and 5 μg cell membrane protein in a volume of 0.2 ml. The reaction mixture was incubated for 10 min at 25°C. The lipids were extracted as described (14Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), dried, redissolved in CHCl3 containing trioleylglycerol as standard, and applied to TLC plates. The TLC plates were developed in hexan-diethylether-acetic acid (70:29:1; v/v/v). Visualization and quantitation of the radioactive TG spots were performed as described above. ACC activity was measured using an NADH-linked assay, with slight modifications (16Tanabe T. Nakanishi S. Hashimoto T. Ogiwara H. Nikawa J. Numa S. Acetyl-CoA carboxylase from rat liver.Methods Enzymol. 1981; 71: 5-16Crossref PubMed Scopus (76) Google Scholar). The medium [56 mM Tris-HCl (pH 8), 10 mM MgCl2, 11 mM EDTA, 4 mM ATP, 52 mM KHCO3, 0.75 mg/ml BSA, 0.5 mM NADH, 1.4 mM phosphoenolpyruvate] was mixed with 5.6 U/ml pyruvate kinase and 5.6 U/ml lactate dehydrogenase. The baseline was followed at 30°C until a constant slope was reached. Per 170 μl medium, 75 μl of activated homogenate was added, and the reaction was started with acetyl-CoA (0.125 mM final concentration). For enzyme activation, 1 vol homogenate was incubated with 1 vol activation buffer [20 mM citrate, 100 mM Tris-HCl (pH 8), 1.5 mg/ml BSA, 20 mM MgCl2, and 20 mM GSH (pH 7.5)] for 15 min at 37°C. FAS activity was determined as the decrease of the NADPH absorption at 340 nm as described earlier (17Nepokroeff C.M. Lakshmanan M.R. Porter J.W. Fatty-acid synthase from rat liver.Methods Enzymol. 1975; 35: 37-44Crossref PubMed Scopus (231) Google Scholar). G6PDH was investigated following the increase of NADPH absorption at 340 nm. For detecting G6PDH, a medium containing 50 mM Tris buffer (pH 8), 1 mM MgCl2, and 5 mM NADP was mixed with 60 μg cytosolic protein/ml, and the reaction was started with glucose-6-phosphate (8 mM final concentration) at 37°C. WAT was surgically removed, extensively washed with ice-cold PBS, and extracted as described (14Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) under acidic conditions (0.1% acidic acid). An aliquot of the extract was dried in a SpeedVac concentrator, and the NEFA content was determined using a commercial kit (Wako Chemicals, Germany). Pieces of intraperitoneal WAT (30–40 mg) were incubated in DMEM medium containing 1% BSA (NEFA-free) for 6 h at 37°C in the presence of 200 μM oleic acid (5 μCi/well [3H]9,10-oleic acid). The lipids were extracted as described (14Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). An aliquot of the lipid extract was analyzed by TLC using hexan-diethylether-acetic acid (70:29:1; v/v/v) as mobile phase. The lipids were visualized with iodine vapor, and the radioactive spots corresponding to TG and phospolipids (PLs) were cut out and quantitated by liquid scintillation. For measurement of [3H]deoxyglucose uptake, fat pads (30–40 mg) were incubated for 30 min at 37°C in Krebs-Ringer buffer (pH 7.4) containing 1% BSA and 1 μCi/ml [3H]deoxyglucose. Thereafter, the fat pads were extensively washed and digested in lysis buffer A containing 1% SDS, 400 μg/ml proteinase K, 100 mM NaCl, 10 mM Tris (pH 7.5), and 1 mM EDTA. Aliquots of the lysate were used for determination of radioactivity and quantitation of DNA. To measure d-glucose incorporation into the lipid moiety, pieces of intraperitoneal WAT (30–40 mg) were incubated in DMEM medium containing 1 g/l d-glucose, 1% BSA (NEFA-free), and 0.5 μCi/ml d-[14C]glucose for 6 h at 37°C. Lipids were extracted from fat pads as described (14Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar), and the organic phase was washed two times and dried under nitrogen. TLC analysis was performed as described above. The spots corresponding to PL, NEFA, and TG were cut out and quantitated by liquid scintillation. For determination of incorporation of d-glucose into TG-associated fatty acids (TG-NEFAs), the lipid extracts were digested with a TG lipase from Candida rugosa (4 U/ml, Sigma Chemicals Co.) for 3 h at 37°C, and the released fatty acids were separated by TLC. Fed mice were injected intraperitoneally with a solution containing 0.15 M NaCl, 100 mg/ml d-glucose, 4 μCi/ml [3H]deoxyglucose, and 4 μCi/ml d-[14C]glucose (18 μl/g mouse). After 2 h, mice were sacrificed, and pieces of the gonadal fat pads were extensively washed in PBS containing 0.01% EDTA. For determination of [3H]deoxyglucose uptake and quantitation of DNA, the fat pads were lysed in lysis buffer A as described above. For measurement of d-[14C]glucose incorporation into WAT lipids, fat pads were extracted as described (14Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar) and subsequently lysed in lysis buffer A for DNA determination. Aliquots of the WAT lysate and of the lipid extract were quantitated by liquid scintillation. WAT DNA was isolated by overnight digestion in lysis buffer A and precipitated in ethanol. The tissue DNA content was quantitated fluorimetrically by the method of Labarca and Paigen (18Labarca C. Paigen K. A simple, rapid, and sensitive DNA assay procedure.Anal. Biochem. 1980; 102: 344-352Crossref PubMed Scopus (4552) Google Scholar). Protein concentrations were measured with the BCA reagent (Pierce) using bovine albumin as standard. Total RNA was isolated from WAT using the TRI Reagent procedure according to manufacturer's protocol (Molecular Research Center, Karlsruhe, Germany). Specific mRNAs were detected using standard Northern blotting techniques with 10 μg total RNA (19Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar). Probes for specific hybridization were generated using random priming. Northern blots were visualized by exposure to a PhosphorImager Screen (Apbiotech, Freiburg, Germany) and analyzed using ImageQuant software. Quantitative real-time PCR analysis was used to determine the relative levels of ACC, G6PDH, PEPCK, and PDH. Reverse transcription and PCR were performed according to the manufacturer's instructions (TaqMan™ One-step RT-PCR Kit, Applied Biosystems, Vienna, Austria) on the 5700 AbiPrism Sequence Detection System (Applied Biosystems). Primers and TaqMan™ probes were designed using Primer Express software (Applied Biosystems) and excluded detection of genomic DNA. Sequence-specific amplification was detected with an increasing fluorescence signal, with FAM as the reporter dye, during the amplification cycle. Amplification of murine β-actin was performed on all samples tested as an internal control for variations in RNA amounts. Levels of the different mRNAs were subsequently normalized to β-actin mRNA levels. For the preparation of protein extracts, epididymal WAT was homogenized in a buffer containing 1% SDS, 1% Triton X-100, 50 mM Tris-HCL (pH 7.4), 5 mM EDTA, 15 U/ml DNase I (Boehringer Mannheim, Inc.), 1× complete protease inhibitor cocktail (Roche Diagnostics Inc., Germany). Proteins (20 μg) were separated by SDS-PAGE and transferred to a nitrocellulose membrane (Schleicher and Schuell, Inc.). Blots were incubated with 1:300-diluted affinity-purified rabbit polyclonal antibody raised against SREBP-1 p125 and p68 (SREBP-1, K-10; Santa Cruz Biotechnology, Inc.). Bound immunoglobulins were detected with a horseradish peroxidase anti-rabbit IgG conjugate (Vector Laboratories) and visualized by enhanced chemiluminescence detection (Amersham Biosciences) according to the manufacturers' instructions. The effects of HSL deficiency on body mass composition was analyzed by the determination of total body weight and AT mass from several fat depots. No significant differences were found in body weight of male and female HSL-ko mice compared with control mice (Table 1). In contrast, gonadal WAT mass was significantly reduced in 15–18-week-old female mice (−30%). Gonadal fat mass was even more decreased in older mice. At the age of 40–45 weeks, female and male mice exhibited a 70% and 71% reduction, respectively. In histological examinations, epididymal WAT of HSL-ko and control mice appeared as unilocular AT with a large single lipid droplet in mature adipocytes. By visual inspection, the cell size of adipocytes appeared much more heterogeneous in HSL-ko mice than in control animals (not shown). Although an extensive morphometric analysis to determine the mean cell size was not performed, the tissue mass per μg DNA was decreased by 46% (n = 5; P < 0.05) in HSL-ko mice, suggesting a reduced mean size of adipocytes.TABLE 1Body weight and gonadal WAT mass of control (wt) and HSL-ko animalsAge, Genderwt (n)HSL-ko (n)t-testBody weight (g) 15–18 Weeks, female29.2 ± 1.9 (8)30.7 ± 2.1 (8)ns 40–45 Weeks, female29.8 ± 5.8 (8)30.0 ± 3.9 (8)ns 40–45 Weeks, male35.7 ± 2.8 (9)35.8 ± 3.2 (11)nsGonadal WAT mass (% of body weight) 15–18 Weeks, female1.21 ± 0.23 (8)0.85 ± 0.20 (8)P < 0.05 40–45 Weeks, female4.26 ± 1.21 (8)1.25 ± 0.39 (8)P < 0.001 40–45 Weeks, male1.98 ± 0.71 (9)0.57 ± 0.28 (11)P < 0.001HSL, hormone-sensitive lipase; HSL-ko, hormone-sensitive lipase-deficient; ns, nonsignificant; WAT, white adipose tissue; wt, wild-type. Animals used for this study were littermates and were kept on a standard laboratory chow diet (4.5% wt/wt fat). Data are expressed as means ± SD. Open table in a new tab HSL, hormone-sensitive lipase; HSL-ko, hormone-sensitive lipase-deficient; ns, nonsignificant; WAT, white adipose tissue; wt, wild-type. Animals used for this study were littermates and were kept on a standard laboratory chow diet (4.5% wt/wt fat). Data are expressed as means ± SD. In a previous study, we demonstrated that despite decreased lipolytic activity, HSL-ko fat pads exhibited a basic NEFA release, which can be stimulated by isoproterenol, similar to that of controls (5Haemmerle G. Zimmermann R. Hayn M. Theussl C. Waeg G. Wagner E. Sattler W. Magin T.M. Wagner E.F. 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 (487) Google Scholar). The determination of intracellular steady-state NEFA levels revealed identical results in nonstimulated fat pads of HSL-ko mice and control animals (Fig. 1). After stimulation of lipolysis for 1 h with isoprot
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