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Transcriptional Regulation of Adipocyte Hormone-Sensitive Lipase by Glucose

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Transcriptional Regulation of Adipocyte Hormone-Sensitive Lipase by Glucose Transcriptional Regulation of Adipocyte Hormone-Sensitive Lipase by Glucose Fatima Smih,1 Philippe Rouet,1 Ste´phanie Lucas,1 Aline Mairal,1 Coralie Sengenes,1 Max Lafontan,1 Sophie Vaulont,2 Marta Casado,2 and Dominique Langin1 Hormone-sensitive lipase (HSL) catalyzes the rate- maximum lipolytic capacity of human subcutaneous adi- limiting step in the mobilization of fatty acids from pocytes stimulated by a ␤-adrenergic agonist (2). More- adipose tissue, thus determining the supply of energy over, targeted disruption of the HSL gene in the mouse substrates in the body. HSL mRNA was positively regu- results in blunted ␤-adrenergic agonist-induced lipolysis lated by glucose in human adipocytes. Pools of stably (3,4). Clinical studies also support a role for HSL as a transfected 3T3-F442A adipocytes were generated with limiting factor in adipose tissue lipolysis and show that, ؊ human adipocyte HSL promoter fragments from 2,400/ besides the short-term modulation of activity by phosphor- ؉38 to ؊31/؉38 bp linked to the luciferase gene. A glucose-responsive region was mapped within the prox- ylation, variations in HSL expression are associated with imal promoter (؊137 bp). Electromobility shift assays changes in lipolytic capacity. Indeed, obese patients and showed that upstream stimulatory factor (USF)-1 and normal-weight subjects with a family trait for obesity show USF2 and Sp1 and Sp3 bound to a consensus E-box and decreased maximal lipolytic effect of catecholamines and -two GC-boxes in the ؊137-bp region. Cotransfection of blunted HSL expression (5,6). Furthermore, genetic stud -the ؊137/؉38 construct with USF1 and USF2 expres- ies suggest that HSL participates in the polygenic back sion vectors produced enhanced luciferase activity. ground of obesity and type 2 diabetes (7,8). Moreover, HSL mRNA levels were decreased in USF1- Increased mobilization of fatty acids from adipose tissue and USF2-deficient mice. Site-directed mutagenesis of the HSL promoter showed that the GC-boxes, although stores in diabetic patients leads to high free fatty acid contributing to basal promoter activity, were dispens- concentrations that contribute to the development of able for glucose responsiveness. Mutation of the E-box ketoacidosis, a major acute complication of type 1 diabe- led to decreased promoter activity and suppression of tes (9). Several mechanisms may account for the enhanced the glucose response. Analogs and metabolites were lipolysis. Hypoinsulinemia and an increase in the concen- used to determine the signal metabolite of the glucose trations of stimulatory hormones stimulate HSL activity response. The signal is generated downstream of glu- via posttranslational modifications. Moreover, isolated fat cose-6-phosphate in the glycolytic pathway before the cells from streptozotocin-induced diabetic rats show in- triose phosphate step. Diabetes 51:293–300, 2002 creased maximal lipolytic response to agents acting at the postreceptor level (10) and increased HSL mRNA and protein levels (11). Insulin deficiency and hyperglycemia could both account for the upregulation of HSL expression ormone-sensitive lipase (HSL) is a key enzyme in diabetes. Insulin per se had no effect on HSL mRNA for the hydrolysis of adipose tissue triacylglyc- concentrations in 3T3-F442A murine adipocytes (12). erol into fatty acids that are the major source of body energy in the absence of dietary lipids. However, in this cellular model, glucose deprivation re- H sulted in a twofold decrease in HSL mRNA and total Catecholamines and insulin are important regulators of lipolysis in adipocytes through a modulation of intracellu- activity levels (13). The effect of glucose was reversible lar cAMP levels and reversible phosphorylation of HSL (1). and was not due to an impairment of the differentiation The lipase is thought to catalyze the rate-limiting step in program, since the mRNA levels of CCAAT/enhancer bind- ␣ ␣ cAMP-dependent lipolysis. In agreement, a strong linear ing protein- (C/EBP ), a transcription factor important correlation was found between HSL protein levels and the for maintenance of the terminally differentiated state, and adipocyte triglyceride content were not affected by glu- cose deprivation. Moreover, it was shown that prolonged From 1INSERM Unite´ 317, Institut Louis Bugnard, Centre Hospitalier Univer- exposure of isolated rat adipocytes to glucose in the sitaire de Rangueil, Universite´ Paul Sabatier, Toulouse, France; and the 2Institut Cochin de Ge´ne´tique Mole´culaire, INSERM Unite´ 129, Paris, France. presence of insulin resulted in an increase of basal and Address correspondence and reprint requests to Philippe Rouet or Dominique stimulated lipolysis and a maintenance of HSL protein Langin, INSERM U317, Baˆtiment L3, CHU Rangueil, 31403 Toulouse Cedex 4, levels (14). France. E-mail: [email protected] or dominique.langin@toulouse .inserm.fr. The adipocyte form of HSL is encoded by nine exons F.S. and P.R. contributed equally to this work. M.C. is currently affiliated spanning 11 kb (15,16). The transcriptional start site has with the Instituto de Biomedicina de Valencia (CSIC), Jaume Roig 11, 46010 Ј Valencia, Spain. been mapped in a short 5 -noncoding exon located 1.5 kb Received for publication 4 June 2001 and accepted in revised form 5 upstream of the first coding exon. Transient transfections November 2001. of various portions of the 5Ј-flanking region linked to the DMEM, Dulbecco’s modified Eagle’s medium; EMSA, electromobility shift assay; FCS, fetal calf serum; HSL, hormone-sensitive lipase; USF, upstream luciferase gene into rat adipocytes showed the existence stimulatory factor. of an active promoter (17). However, the regulatory ele- DIABETES, VOL. 51, FEBRUARY 2002 293 HORMONE-SENSITIVE LIPASE REGULATION BY GLUCOSE ments within the human adipocyte HSL promoter have not Quantification of mRNA levels in human adipocytes. Adipose primary been characterized. Thus, we decided in this study to cell culture total RNA was extracted using the Qiagen RNeasy kit. Total RNA was stored at Ϫ80°C until analysis. Quantitation of HSL and cyclophilin mRNA characterize the cis-acting regions that are critical for levels were performed by reverse transcription–competitive PCR as described promoter activity and mediate the responsiveness to glu- previously (21). The reverse transcription step was performed on 100 ng of cose. total RNA with a specific antisense primer and Omniscript reverse transcrip- tase (Qiagen). cDNA samples were then amplified by PCR using sense and antisense primers in the presence of known amounts of a specific DNA RESEARCH DESIGN AND METHODS competitor. PCR products were separated by capillary electrophoresis and Plasmids. Constructs containing human adipocyte HSL 5Ј flanking regions quantified using the ABI PRISM 310 Genetic Analyzer system with the from Ϫ2,400 to Ϫ31 relative to the transcription start point and 38 bp of the Genescan program (PE Applied Biosystem). HSL mRNA levels were normal- 5Ј untranslated region in pGL3basic vector encoding a modified firefly ized with cyclophilin mRNA levels. luciferase (Promega, Madison, WI) have been described previously (17). Preparation of nuclear extracts and electromobility shift assay. 3T3- Mutant HSL promoter constructs were generated by synthetic nucleotide F442A preadipocyte and adipocyte nuclear extracts were prepared according assembly in pGL3basic using the KpnI site of the vector and the XbaI site of to standard procedures (18). Probe preparation and electromobility shift the HSL promoter. These constructs were made using standard procedures assay (EMSA) conditions were described previously (22). Briefly, 150,000 (18) and sequenced using a Perkin Elmer Dye terminator sequencing kit and cpm/lane (0.5 ng of end-labeled DNA) and 4 ␮g of nuclear extracts were an ABI 373 sequencer. Assembly of the double-stranded oligonucleotides led incubated for 15 min on ice. Upstream stimulatory factor (USF) and Sp1 to substitution of the characterized binding sites by a mutagenized box with competitor oligonucleotides were from Promega. The HNF1 oligonucleotide an XbaI site. pCR3-USF1 and -USF2a expression vectors, pCMV-Sp1, and used as nonspecific competitor has been described (22). Supershift assays pSVsport-ADD1 and pSVsport-ADD1403, were gifts from M. Raymondjean and using antibodies against USF1 and USF2 (gift of M. Raymondjean) and against M. Vasseur-Cognet (INSERM U 129, Paris), A.P. Butler (M.D. Anderson Cancer Sp1 (sc-59X; Santa Cruz) and Sp3 (sc-644X; Santa Cruz) were performed by Center, University of Texas, Houston, TX), and B. Spiegelman (Dana Farber incubating the nuclear extracts with the antibodies for 15 min on ice before Cancer Institute, Boston, MA), respectively. addition of the probe. After electrophoresis, the gel was fixed in 10% acetic Cell culture, transfection, and luciferase assay. In agreement with French acid/10% ethanol and dried. Gels were exposed and analyzed using a Molec- laws on biomedical research, subcutaneous abdominal adipose tissue was ular Dynamics SI445 Phosphorimager. obtained from female subjects undergoing plastic surgery. Human adipocytes Northern blot analysis of HSL mRNA in USF1- and USF2-deficient mice. in primary culture were differentiated as described by Hauner et al. (19), with Total RNA was isolated from perigonadal adipose tissue of fasted USF1- modifications. Briefly, all visible fibrous material and blood vessels were and USF2-deficient mice (23,24). Wild-type littermates were used as controls. discarded from adipose tissue samples.
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