A Novel Hormone-sensitive Lipase Isoform Expressed in Pancreatic β-Cells
2004; Elsevier BV; Volume: 279; Issue: 5 Linguagem: Inglês
10.1074/jbc.m311365200
ISSN1083-351X
AutoresHåkan Lindvall, Pernilla Nevsten, Kristoffer Ström, Reine Wallenberg, F. Sundler, Dominique Langin, Maria Sörhede Winzell, Cecilia Holm,
Tópico(s)Adipose Tissue and Metabolism
ResumoHormone-sensitive lipase (HSL) is a key enzyme in fatty acid mobilization in many cell types. Two isoforms of HSL are known to date, namely HSLadi (84 kDa in rat) and HSLtes (130 kDa in rat). These are encoded by the same gene, with exons 1-9 encoding the parts that are common to both and an additional 5′-exon encoding the additional amino acids in HSLtes. HSL of various tissues, among these the islet of Langerhans, is larger than HSLadi, but not as large as HSLtes, indicating that there may be other 5′-coding exons. Here we describe the molecular basis for a novel 89-kDa HSL isoform that is expressed in β-cells, adipocytes, adrenal glands, and ovaries in the rat and that is encoded by exons 1-9 and exon A, which is spliced to exon 1 and thereby introducing an upstream start codon. The additional 5′-base pairs encode a 43-amino acid peptide, which is highly positively charged. Conglomerates of HSL molecules are in close association with the secretory granules of the β-cell, as determined by immunoelectron microscopy with antibodies targeting two separate regions of HSL. We have also determined that the human genomic sequence upstream of exon A has promoter activity in INS-1 cells as well as glucose sensing capability, mediating an increase in expression at high glucose concentration. The minimal promoter is present within 170 bp from the transcriptional start site and maximal glucose responsiveness is conferred by sequence within 850 bp from the transcriptional start site. Hormone-sensitive lipase (HSL) is a key enzyme in fatty acid mobilization in many cell types. Two isoforms of HSL are known to date, namely HSLadi (84 kDa in rat) and HSLtes (130 kDa in rat). These are encoded by the same gene, with exons 1-9 encoding the parts that are common to both and an additional 5′-exon encoding the additional amino acids in HSLtes. HSL of various tissues, among these the islet of Langerhans, is larger than HSLadi, but not as large as HSLtes, indicating that there may be other 5′-coding exons. Here we describe the molecular basis for a novel 89-kDa HSL isoform that is expressed in β-cells, adipocytes, adrenal glands, and ovaries in the rat and that is encoded by exons 1-9 and exon A, which is spliced to exon 1 and thereby introducing an upstream start codon. The additional 5′-base pairs encode a 43-amino acid peptide, which is highly positively charged. Conglomerates of HSL molecules are in close association with the secretory granules of the β-cell, as determined by immunoelectron microscopy with antibodies targeting two separate regions of HSL. We have also determined that the human genomic sequence upstream of exon A has promoter activity in INS-1 cells as well as glucose sensing capability, mediating an increase in expression at high glucose concentration. The minimal promoter is present within 170 bp from the transcriptional start site and maximal glucose responsiveness is conferred by sequence within 850 bp from the transcriptional start site. Because of the established association between type 2 diabetes and obesity, lipids have received much attention in research on the pathophysiology of type 2 diabetes. Lipids are believed to cause insulin resistance in tissues and to alter β-cell function. The effect of lipids on β-cells has been studied extensively, but the picture that emerges is complex. Different effects are seen depending on parameters such as duration of exposure, concentration, and fatty acid chain length and saturation. Briefly, the presence of free fatty acids is essential for normal glucose-stimulated insulin secretion (1Koyama K. Chen G. Wang M.-Y. Lee Y. Shimabukuro M. Newgard C.B. Unger R.H. Diabetes. 1997; 48: 1276-1280Google Scholar, 2Dobbins R.L. Chester M.W. Stevenson B.E. Daniels M.B. Stein D.T. McGarry J.D. J. Clin. Invest. 1998; 101: 2370-2376Google Scholar) and also has an amplifying action on glucose-stimulated insulin secretion. On the other hand, a prolonged exposure (24-48 h) to high concentrations (∼1 mm) of free fatty acids is detrimental to β-cell function and survival (3Elks M.L. Endocrinology. 1993; 133: 208-214Google Scholar, 4Sako Y. Grill V.E. Endocrinology. 1990; 127: 1580-1589Google Scholar).In view of this, knowledge of lipid mobilization, lipid storage, and the regulation of these in the β-cell are of great interest. Recently, we showed that hormone-sensitive lipase (HSL) 1The abbreviations used are: HSLhormone-sensitive lipaseGSTglutathione S-transferaseHSL-A-GSTfusion protein of GST and the peptide encoded by exon A of the HSL geneInrinitiatorNF-Ynuclear factor YSp1stimulating protein 1TSStranscriptional start siteRTreverse transcriptaseRACErapid amplification of cDNA ends.1The abbreviations used are: HSLhormone-sensitive lipaseGSTglutathione S-transferaseHSL-A-GSTfusion protein of GST and the peptide encoded by exon A of the HSL geneInrinitiatorNF-Ynuclear factor YSp1stimulating protein 1TSStranscriptional start siteRTreverse transcriptaseRACErapid amplification of cDNA ends. is expressed and active in islets of Langerhans and clonal β-cells (5Mulder H. Holst L.S. Svensson H. Degerman E. Sundler F. Ahren B. Rorsman P. Holm C. Diabetes. 1999; 48: 228-232Google Scholar). HSL is a key enzyme in fatty acid mobilization in the adipocytes and presumably also in several non-adipocyte cell types (see Ref. 6Holm C. Osterlund T. Laurell H. Contreras J.A. Annu. Rev. Nutr. 2000; 20: 365-393Google Scholar for review). It is unique among known lipases in that its activity is acutely controlled by hormones. Catabolic hormones, such as glucagon and catecholamines, activate the enzyme and anabolic hormones (e.g. insulin) decrease its activity. The activity of HSL is principally determined by the intracellular level of cAMP, which controls the activity of protein kinase A that phosphorylates HSL on critical serine residues (7Anthonsen M.W. Ronnstrand L. Wernstedt C. Degerman E. Holm C. J. Biol. Chem. 1998; 273: 215-221Google Scholar). In the adipocyte, this triggers translocation of HSL from the cytosol to the surface of the lipid droplet (8Brasaemle D.L. Levin D.M. Adler-Wailes D.C. Londos C. Biochim. Biophys. Acta. 2000; 1483: 251-262Google Scholar) and, possibly, also a change in specific activity. HSL has a broad substrate specificity, being able to hydrolyze, among others, mono-, di-, and triglycerides and cholesteryl esters. Besides β-cells and adipocytes, HSL is also present in testis, ovary, adrenal gland, heart, skeletal muscle (9Holm C. Belfrage P. Fredrikson G. Biochem. Biophys. Res. Commun. 1987; 148: 99-105Google Scholar), macrophages (10Khoo J.C. Reue K. Steinberg D. Schotz M.C. J. Lipid Res. 1993; 34: 1969-1974Google Scholar), and gastrointestinal tract mucosa (11Grober J. Lucas S. Sorhede-Winzell M. Zaghini I. Mairal A. Contreras J.A. Besnard P. Holm C. Langin D. J. Biol. Chem. 2003; 278: 6510-6515Google Scholar). The HSL gene is located on chromosome 19 (human) (12Holm C. Kirchgessner T.G. Svenson K.L. Fredrikson G. Nilsson S. Miller C.G. Shively J.E. Heinzmann C. Sparkes R.S. Mohandas T. Lusis A.J. Belfrage P. Schotz M.C. Science. 1988; 241: 1503-1506Google Scholar) and spans 27 kb (Fig. 1A) (11Grober J. Lucas S. Sorhede-Winzell M. Zaghini I. Mairal A. Contreras J.A. Besnard P. Holm C. Langin D. J. Biol. Chem. 2003; 278: 6510-6515Google Scholar, 13Langin D. Laurell H. Holst L.S. Belfrage P. Holm C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4897-4901Google Scholar, 14Holst L.S. Langin D. Mulder H. Laurell H. Grober J. Bergh A. Mohrenweiser H.W. Edgren G. Holm C. Genomics. 1996; 35: 441-447Google Scholar, 15Mairal A. Melaine N. Laurell H. Grober J. Holst L.S. Guillaudeux T. Holm C. Jégou B. Langin D. Biochem. Biophys. Res. Commun. 2002; 291: 286-290Google Scholar). All functional HSL proteins are encoded by, at least, exons 1-9. Upstream of exon 1 there are several exons that are spliced to exon 1 in a mutually exclusive manner. The 5′ exons can be coding or non-coding.The present investigation of HSL in the β-cell concerns structure, subcellular localization, and regulation of gene expression. HSL in the β-cell is larger than the predominant HSL isoform found in the adipocyte (89 versus 84 kDa) (5Mulder H. Holst L.S. Svensson H. Degerman E. Sundler F. Ahren B. Rorsman P. Holm C. Diabetes. 1999; 48: 228-232Google Scholar). Here we show that the difference in size between the two isoforms is explained by the addition of a 5′-exon to the HSL transcript, namely exon A, which contains coding sequence. We also present evidence that the larger HSL isoform is present in other cell types besides the β-cell. Using electron microscopy, we show the subcellular localization of HSL in rat islet β-cells. Formerly, we have shown that HSL expression is up-regulated in rat islets of Langerhans and clonal β-cells after long-term exposure to high concentrations of glucose (16Winzell M.S. Svensson H. Arner P. Ahren B. Holm C. Diabetes. 2001; 50: 2225-2230Google Scholar). Here we describe the genomic region responsible for this effect.EXPERIMENTAL PROCEDURESRNA Extraction—Total RNA was extracted from tissues or cells according to Chomczynski and Sacchi (17Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Google Scholar). Poly(A)+ RNA was prepared from total RNA with the Dynabeads mRNA purification kit (Dynal) according to the manufacturer's instructions.cDNA Cloning—The Marathon cDNA kit (Clontech) was used to create an adapter ligated cDNA library from 1 μg of poly(A)+ RNA from INS-1, passage 90. 5′-RACE was performed on a 1:50 dilution of the cDNA library. Adapter primer 1 of the Marathon kit was used in conjunction with a primer of the following sequence: 5′-TGAGGGCAGCCAGGTAGGCCTC-3′. A nested PCR was performed using the adapter primer 2 of the Marathon kit and a primer of the following sequence: 5′-AGGAATTCGCCCTGGCTTGAGAAGAAGGCCAT-3′ (EcoRI site underlined). The resulting product was cleaved with NotI and EcoRI and subcloned in pBluescript II SK (Stratagene). Plasmids were sequenced using BigDye reagents (Applied Biosystems) and comparative sequence analysis was performed using the MacVector software (Accelrys).Animals—Rats were obtained from B&K Universal, Sweden. Studies were approved by the local ethical committee.Northern Blotting—Tissues were dissected from a female rat weighing 300 g and frozen in liquid nitrogen until processing. Male genital organs were taken from a male rat of the same weight and luteal ovaries were taken from a 10-day pregnant female rat. Any adipose tissue was carefully removed from all tissues. Fifty μg of total RNA from the various tissues was electrophoresed in 0.9% agarose, 2.2 m formaldehyde gels. Nucleic acids were passively transferred to nylon membranes (Hybond N, Amersham Biosciences) and UV cross-linked.NotI and BsmI (cutting 5 bp downstream of the 5′-splice site of exon 1) were used to excise exon A sequence from the plasmid described under cDNA cloning. The resulting molecule was 32P-labeled using an Oligolabeling kit (discontinued; Amersham Biosciences). A 32P-end labeled 18 S probe (5′-TCCGAGGGCCTCACTAAACCATCCA-3′) was used for normalization between lanes. Hybridization was performed sequentially using ExpressHyb (Clontech). Hybridization, washing, and stripping of membranes were made according to the instructions of the manufacturer.Islet Isolation—Ten ml of cold Hanks' balanced salt solution with collagenase P (Roche Diagnostics), 1.35 units/ml, were injected into the bile duct, filling the pancreas retrogradely. The pancreas was removed and incubated at 37 °C for 20 min. After washing four times with Hanks' balanced salt solution, islets were manually separated from exocrine cells under a microscope.RT-PCR—The RT step was performed with 1.5 μg of islet total RNA, Moloney murine leukemia virus-RT (New England Biolabs), random hexamers, as well as a rat HSL-specific reverse primer from exon 8, at 37 °C for 60 min. A no RT reaction was run in parallel. The PCR was performed using a sense primer with the first 24 bp of exon A coding sequence and a reverse primer from exon 1 (5′-GGCGTAGTAGGCTAGAGCAC-3′), 1 μl of RT or no RT reaction, and AmpliTaq Gold polymerase (Applied Biosystems). Product size was determined on a 1% agarose gel. The expected size of the amplified HSL cDNA was 513 bp.Creation of Antibodies—Using pBluescript containing HSL-exon A cDNA as template, a PCR fragment containing the coding sequence of exon A was created. Primers used were 5′-GTGGATCCATGGAGCCGGCCGTGGAATC-3′ (BamHI site underlined) and 5′-TGAATTCTTTGACCTTGCCTTTTCGCCACC-3′ (EcoRI site underlined). The PCR fragment was gel purified (Qiaex II, Qiagen) and restriction cleaved with BamHI and EcoRI. The resulting molecule was ligated into the same sites of pGex-2T (Amersham Biosciences) containing the Schistosoma japonicum glutathione S-transferase (GST) cDNA. The resulting plasmid was sequenced across the insert and was found to be correct. The GST-HSL-A fusion protein was expressed in Escherichia coli and purified using glutathione-Sepharose 4B (Amersham Biosciences) according to the manufacturer's instructions.The GST-HSL-A fusion protein was used for immunization of rabbits. Antiserum from immunized rabbits was subjected to affinity purification on a CH-Sepharose 4B column (Amersham Biosciences) with GST-HSL-A fusion protein, immobilized according to the manufacturer's instructions.Western Blotting—Tissues were dissected from Sprague-Dawley rats. Tissues and INS-1 cells were homogenized with a glass Teflon homogenizer in homogenization buffer (0.25 mol/liter sucrose, 1 mmol/liter dithioerythritol, 1 mmol/liter EDTA, 10 μg/ml antipain, 10 liter μg/ml leupeptin, and 1 μg/ml pepstatin). A fat-depleted infranatant of adipose tissue was obtained after centrifugation at 50,000 × g, 45 min, 4 °C. Islets were isolated as described above and lysed in SDS-PAGE sample buffer. Determination of protein concentration was performed using a BCA protein kit (Pierce). Samples were subjected to SDS-PAGE on 8% acrylamide gels and then electroblotted onto nitrocellulose membranes (HyBond-C extra, Amersham Biosciences).Primary antibodies used were affinity purified anti-rat HSL antibodies, 1:5000 (raised against sequence emanating from exons 1 through 9 of the HSL gene, i.e. recombinant HSLadi (18Osterlund T. Danielsson B. Degerman E. Contreras J.A. Edgren G. Davis R.C. Schotz M.C. Holm C. Biochem. J. 1996; 319: 411-420Google Scholar)) and the affinity purified rabbit antibody directed toward the GST-HSL-A fusion protein (1:100), described above. A horseradish peroxidase-conjugated donkey anti-rabbit IgG (Amersham Biosciences) was used as secondary antibody (1:2000) and the blots were developed by enhanced chemiluminescence. Detection was performed using a CCD camera (LAS1000, Fuji).Tissue Processing and Immunoelectron Microscopy—Isolated islets were incubated in HEPES-balanced salt solution (114 mm NaCl, 4.7 mm KCl, 1.2 mm KH2PO4, 1.16 mm MgSO4, 20 mm HEPES, 2.5 mm CaCl2, 25.5 mm NaHCO3, 0.2% bovine serum albumin, pH 7.2) supplemented with 3 mm glucose during 1.5 h and then stimulated during 30 min at RT in HEPES-balanced salt solution containing 3 mm glucose, 15 mm glucose or 15 mm glucose with 5 μm forskolin. After washing with Sörensen buffer, the islets were fixed in 3% paraformaldehyde and 0.5% glutaraldehyde in Sörensen buffer during 1 h. The islets were again washed in pure Sörensen buffer and pre-embedded in 2% agar in distilled water. The pre-embedding was followed by dehydration in an ethanol series with increasing concentration of alcohol. The final embedding was performed in Lowicryl HM20 at -50 °C, which was UV-polymerized at -45 °C during 48 h followed by 32 h at RT. Ultrathin sections were cut with an ultramicrotome (Leica UCT) and deposited on Formvar-coated nickel grids.All washing steps were performed in a 400-ml vessel and the reactions were carried out in 100 μl-drop on Parafilm. Between all steps excess liquid was blotted off by touching the edge of the grids with a filter paper. The grids were first incubated, during 30 min at RT, in a blocking buffer consisting of phosphate-buffered saline supplemented with 0.5% acetylated bovine serum albumin (bovine serum albumin-c, Aurion) and 0.1% fish gelatin (Amersham Biosciences). All the grids, except the controls, were then incubated overnight at room temperature with the primary antibody rabbit anti-HSLadi or rabbit anti-GST-HSL-A diluted 1:25 in the blocking buffer. The controls were incubated in the blocking buffer overnight.After washing in a buffer (phosphate-buffered saline supplemented with 0.1% Tween 20) for 15 min the grids were incubated during 2 h at room temperature with a secondary antibody (goat anti-rabbit IgG conjugated with 6-nm gold particles (Aurion), diluted 1:10 in the blocking buffer). Finally the grids were washed during 15 min in washing buffer followed by washing in Milli-Q water for 6 min. After drying, counterstaining was performed with 3% uranyl acetate at 40 °C for 20 min followed by lead citrate (Reynolds' recipe) at room temperature for 4 min. The sections were then observed in a Philips CM 120 Biotwin instrument at 120 kV.Screening of Human Genomic Sequence—A cosmid containing human genomic sequence was cleaved with BamHI and BspEI. An exon A-containing fragment was identified using the exon A probe described above in Southern blotting. Southern blotting was performed using a nylon membrane (Hybond N, Amersham Biosciences) and ExpressHyb hybridization solution (Clontech) according to the manufacturer's instructions.The identified fragment of 2.5 kb was gel purified (Qiaex II, Qiagen) and subcloned into the BamHI and BspEI sites of the pTriAmp vector. The subcloned fragment was sequenced in both directions using the BigDye reagent (Applied Biosystems). The sequence was found to be essentially identical to a sequence with GenBank™ accession number AC011497 (human chromosome 19).Determination of Transcriptional Start Site (TSS)—Poly(A)+ RNA (250 ng) from INS-1 cells and rat tissues (adipose tissue or ovary), respectively, were treated with the FirstChoice RLM-RACE kit (Ambion). 5′-RACE was performed according to instructions of the manufacturer, using a gene-specific primer of the following sequence: 5′-GGCGTAGTAGGCTAGAGCAC-3′. Two μl of this PCR was used for nested PCR using a gene-specific primer of the following sequence: 5′-GATGAATTCACAGGTAGGCCTCCAGTTC-3′ (EcoRI site underlined). Both primers contain antisense sequence from exon 1 of the rat HSL gene. The resulting products were subcloned in pBluescript II SK and sequenced. Alternatively, the nested PCR was performed using an exon A-specific reverse primer (5′-TGAATTCTTTGACCTTGCCTTTTCGCCACC-3′; EcoRI site underlined). The size of the resulting products was determined on a 3% MetaPhor-agarose gel (FMC BioProducts).Creation of Luciferase Expressing Vectors—Sense and antisense primers that were complementary to sequence at the stated distances from the exon A TSS were created (DNA Technology, Aarhus, Denmark). Primers contained NheI or HindIII restriction sites in the 5′-end for cloning of PCR products into these sites of the pGL3basic vector (Promega). PCR was performed using proofreading DNA polymerase (Vent, New England Biolabs; or Platinum Pfx, Invitrogen).Dual Luciferase Reporter Assay—INS-1 cells (p85-98) were grown in 24-well plates and transfected at ∼80% confluence with empty pGL3basic vector (which contains a Firefly luciferase gene) or pGL3basic containing sequence upstream of exon A (1 μg/well) and pRL-CMV (which contains a Renilla luciferase gene) (10 ng/well). Transfection was performed using 2 μl of FuGENE 6 (Roche). Each pGL-construct was added to three wells for each experimental condition in each experiment. The cells were incubated for 24 h. After incubation, the cells were washed with phosphate-buffered saline and lysed. Twenty μl of cell lysate was used in the subsequent assay performed in a Wallac 1420 luminometer with dual luciferase reporter assay reagents (Promega). Lysate of uninfected cells was used as blank. The capacity of each DNA insert to yield firefly luciferase activity is expressed relative to that of empty pGL3basic at each experimental condition. Variation in transfection efficiency between wells was normalized for using Renilla luciferase activity.Statistical Analysis—Means and S.E. ± mean were calculated. Student's t test for unpaired data was used and differences were considered significant at p < 0.05.RESULTSHSL of the pancreatic β-cell is slightly larger (89 versus 84 kDa) than the predominant isoform found in adipocytes, referred to as HSLadi, as evident when lysates of clonal β-cell or islets from mouse and rat are subjected to immunoblot analysis (5Mulder H. Holst L.S. Svensson H. Degerman E. Sundler F. Ahren B. Rorsman P. Holm C. Diabetes. 1999; 48: 228-232Google Scholar). To resolve the sequence of the HSL transcript of the β-cell responsible for the difference in protein chain length, INS-1 cell mRNA was subjected to 5′-RACE using a gene-specific antisense primer with HSL exon 1 sequence. The RACE product obtained contained, in addition to the expected sequence from exon 1, sequence from exon A. Exon A has formerly been subcloned by use of the same technique from adipocytes of mouse (19Laurin N.N. Wang S.P. Mitchell G.A. Mamm. Genome. 2000; 11: 972-978Google Scholar) and human (20Grober J. Laurell H. Blaise R. Fabry B. Schaak S. Holm C. Langin D. Biochem. J. 1997; 328: 453-461Google Scholar) and noted to have a translation start codon, but its translation into protein has not been confirmed until now.Exon A sequence retrieved from the INS-1 cell cDNA was compared with corresponding sequences from mouse and human (Fig. 1B). The sequences were found to be similar (95% identity between rat and mouse and ∼70% identity between all three species). Mouse and human sequences both exhibit translation start codons in-frame with the exon 1 ATG. Fig. 1C depicts the predicted amino acid sequence encoded by exon A in rat, mouse, and human. The peptides display strong positive charge and have a molecular mass of ∼5 kDa, which well explains the observed difference in mobility between the two isoforms upon SDS-PAGE. Rat and mouse HSLadi share 94% identity at the protein level, whereas rat and human share 83% (12Holm C. Kirchgessner T.G. Svenson K.L. Fredrikson G. Nilsson S. Miller C.G. Shively J.E. Heinzmann C. Sparkes R.S. Mohandas T. Lusis A.J. Belfrage P. Schotz M.C. Science. 1988; 241: 1503-1506Google Scholar, 13Langin D. Laurell H. Holst L.S. Belfrage P. Holm C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4897-4901Google Scholar, 21Li Z. Sumida M. Birchbauer A. Schotz M.C. Reue K. Genomics. 1994; 24: 259-265Google Scholar). The novel HSL region described here consists of 43 amino acids and the level of homology between rat and mouse is similar to that of the rest of the protein. The homology, however, between rat and human is considerably lower (∼50%). A lower degree of sequence identity (∼30%) between rat and human has previously been described also for the testis-specific domain of HSL (14Holst L.S. Langin D. Mulder H. Laurell H. Grober J. Bergh A. Mohrenweiser H.W. Edgren G. Holm C. Genomics. 1996; 35: 441-447Google Scholar).To determine whether exon A-containing transcripts were present in cell types other than the β-cell, Northern blot analyses were performed using a radiolabeled exon A probe. Exon A sequences were detected in adipose tissue, adrenal glands, ovaries with and without corpora lutea, and INS-1 cells (Fig. 2A). Because of the difficulties to obtain large quantities of pure rat islets of Langerhans, a lesser amount of RNA from rat islets was used to perform RT-PCR. When using an exon A forward primer and an exon 1 reverse primer, an amplicon of the expected size was retrieved, whereas the negative control (no RT) was devoid of product (Fig. 2B).Fig. 2Detection of HSL exon A in transcripts from rat tissues.A, Northern blot analyses were performed using 50 μg of total RNA from each stated tissue and INS-1 cells and radiolabeled probes containing exon A sequence and 18 S sequence, respectively. Exon A-containing transcripts were found in RNA from adipose tissue, adrenal glands, ovaries, and INS-1 cells. B, RT-PCR analysis of islets of Langerhans using 1.5 μg of total RNA from rat islets and primers from exon A (sense) and exon 1 (antisense). The figure shows a band of the expected size (513 bp), resolved by agarose gel electrophoresis, and no product in the no RT control.View Large Image Figure ViewerDownload (PPT)Splicing of exon A to exons 1-9 yields a transcript that contains a translation start codon 129 bases upstream from the ATG in exon 1. Twenty of these nucleotides are derived from exon 1. It is not known whether the start codon in exon A is used in tissues. To find out whether the discovered transcript is translated into protein, affinity purified antibodies from a rabbit immunized with a fusion protein consisting of GST and a peptide encoded by rat exon A (GST-HSL-A) were used. Homogenates of adrenal glands, ovaries, islets, liver, and adipose tissue infranatant were subjected to Western blot analysis using an antibody directed against HSLadi (derived from exons 1-9) and anti-GST-HSL-A, respectively. Anti-HSLadi detected bands of 84 and 89 kDa in adipose tissue, adrenal glands, and ovaries (Fig. 3A). No HSL protein was detected in liver. In islets of Langerhans, a 89-kDa band was detected by anti-HSLadi (Fig. 3B). Anti-GST-HSL-A detected bands of 89 kDa in all tissues examined, except in liver (Fig. 3, A and B). These results demonstrate that exon A is, in fact, translated into protein sequence and that the exon A-containing transcript encodes a larger isoform than the predominant HSL isoform in adipocytes.Fig. 3Western blots with rat protein analyzed with HSL antibodies.A, a Western blot with two identical halves, each containing 20 μg of protein/lane from stated tissues, was cut down the middle and analyzed using one antibody for each half. Anti-HSLadi detects an 84-kDa band and an 89-kDa band in adipose tissue, ovaries, and adrenal glands. Recombinant rat-HSLadi was added as a positive control. Anti-HSL-A-GST, yields an 89-kDa band in all tissues except liver. Recombinant HSLadi is not recognized by this antibody. B, using the same strategy as above, a Western blot analysis with lysate of 200 rat islets, ∼100 μg of INS-1 protein, and recombinant HSLadi was performed. Both antibodies detect only the 89-kDa band in islets and INS-1 cells.View Large Image Figure ViewerDownload (PPT)Subcellular Localization of HSL in the β-Cell—Rat islets for immunoelectron microscopy were incubated in 3, 15, or 15 mm glucose with 5 μm forskolin, before fixation, to examine whether the subcellular localization of HSL varied under these conditions. We found no differences in localization between these experimental groups and therefore only results from one are shown (high glucose + forskolin). The two HSL antibodies, used for Western blot analysis, and gold-conjugated secondary antibodies were used to detect HSL in adjacent islet sections (Figs. 4 and 5). Immunolabeling was detected as conglomerates of gold particles in association with insulin granules in β-cells. The appearance of the HSL immunolabeling was virtually identical with the two antibodies.Fig. 4TEM micrographs of a section of β-cells from islets of Langerhans (adult rat), stained using anti-HSLadi. Labeling of HSL was performed with anti-rat HSLadi, as a primary antibody and a secondary antibody conjugated with a gold particle (6 nm). A, the arrows show areas that have been labeled by the gold-antibody conjugate. The precision of the HSL location was verified by labels attaching to the corresponding area in adjacent ultrathin sections. B-D, higher magnifications of the framed areas in A, showing the individual gold labels (arrows).View Large Image Figure ViewerDownload (PPT)Fig. 5TEM micrographs of a section of β-cells from islets of Langerhans (adult rat), stained using anti-HSL-A-GST. Labeling of HSL was performed as described in the legend to Fig. 4, but with anti-HSL-A-GST, as the primary antibody. A, the arrows show the location of the gold labels at low magnification. The labeling density is about the same as in Fig. 4. The gold particles can clearly be distinguished in images recorded with higher magnification (B-D).View Large Image Figure ViewerDownload (PPT)Determination of the Transcriptional Start Site of Exon A—Determination of the TSS was performed using RNA ligation-mediated RACE, a technique taking advantage of the presence of the cap structure to discriminate between full-length and partly degraded RNA molecules. mRNA from INS-1 cells, ovaries, and white adipose tissue were investigated. Using HSL-derived reverse primers and the RACE kit adapter primers, a single amplicon was obtained from each tissue at each experimental condition and these corresponded to 5′-untranslated regions between 41 and 47 bp, as detected either by subcloning with subsequent sequencing or size determination on MetaPhor-agarose gels (results not shown). This result was extrapolated to the human genomic sequence, when making constructs for the promoter studies described below.Promoter Activity of the Genomic Region Upstream of Exon A—A fragment of human genomic DNA containing exon A was identified using subcloned rat exon A as a probe in Southern blotting. The fragment was subcloned, sequenced, and used as template to make PCR products of lengths between -2150/+38 and -30/+38. These constructs were subcloned into pGL3basic and
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