Cell Lipotoxicity Induced by Overexpression of Hormone-Sensitive Lipase
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Pancreatic -Cell Lipotoxicity Induced by Overexpression of Hormone-Sensitive Lipase Maria So¨rhede Winzell,1 Håkan Svensson,1 Sven Enerba¨ck,2 Kim Ravnskjaer,3 Susanne Mandrup,3 Victoria Esser,4 Peter Arner,5 Marie-Clotilde Alves-Guerra,6 Bruno Miroux,6 Frank Sundler,7 Bo Ahre´n,8 and Cecilia Holm1 Lipid perturbations associated with triglyceride over- storage in -cells impair insulin secretion, a process termed lipotoxicity. To assess the role of hormone- he established relation between obesity and type sensitive lipase, which is expressed and enzymatically 2 diabetes has brought much attention to the active in -cells, in the development of lipotoxicity, we pathophysiological role of lipids for the develop- generated transgenic mice overexpressing hormone- Tment of type 2 diabetes (1–3). Besides the link sensitive lipase specifically in -cells. Transgenic mice between lipids and the major complication of diabetes, i.e., developed glucose intolerance and severely blunted macroangiopathic disease (4), the lipid abnormalities may glucose-stimulated insulin secretion when challenged also be a key factor for the development of both insulin with a high-fat diet. As expected, both lipase activity resistance and impaired glucose-stimulated insulin secre- and forskolin-stimulated lipolysis was increased in tion (GSIS), the two hallmarks of type 2 diabetes (5). Thus, transgenic compared with wild-type islets. This was inappropriate accumulation of triglycerides in non- reflected in significantly lower triglycerides levels in adipose cells may be an initial event, which in turn may transgenic compared with wild-type islets in mice re-  ceiving the high-fat diet, whereas no difference in islet abrogate GSIS in -cells and decrease insulin sensitivity triglycerides was found between the two genotypes (6), processes referred to as lipotoxicity. In support of the under low-fat diet conditions. Our results highlight the lipotoxicity theory, a strong correlation between accumu- importance of mobilization of the islet triglyceride pool lation of triglycerides in myocytes and insulin resistance in the development of -cell lipotoxicity. We propose has been observed (5,7), and infusion of lipids interferes that hormone-sensitive lipase is involved in mediating with glucose disposal in skeletal muscle (8). With regard -cell lipotoxicity by providing ligands for peroxisome to -cells, their failure in the Zucker diabetic fatty (ZDF) proliferator–activated receptors and other lipid-acti- rat is preceded by a rise in plasma nonesterified fatty acid vated transcription factors, which in turn alter the and accumulation of triglycerides in islets (9). Reduction expression of critical genes. One such gene might be of -cell lipid accumulation before the development of uncoupling protein-2, which was found to be upregu- frank diabetes improves -cell function (10), suggesting a lated in transgenic islets, a change that was accompa- causal relation between triglyceride overstorage and -cell nied by decreased ATP levels. Diabetes 52:2057–2065, failure. The mechanisms underlying lipotoxicity are yet 2003 poorly understood. Despite the accumulating data suggesting the impor- tance of lipids for the development of the -cell secretory defects that characterize type 2 diabetes, little is known about how fatty acids are mobilized from the intracellular pool(s) of triglycerides in -cells and how this process is regulated. We recently showed that hormone-sensitive From the 1Department of Cell and Molecular Biology, Lund University, Lund, Sweden; the 2Department of Molecular Biology, Go¨ teborg University, Go¨te- lipase (HSL), which is mostly known as the hormonally borg, Sweden; the 3Department of Biochemistry and Molecular Biology, regulated acylglycerol lipase in adipocytes (11), is ex- University of Southern Denmark, Odense, Denmark; the 4Department of pressed and enzymatically active in -cells (12). To gain Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas; the 5Department of Medicine, Huddinge University Hospital Karolinska further insight into the pathophysiology of lipotoxicity, Institute, Stockholm, Sweden; 6CNRS UPR 9078, Meudon, France; the 7De- including the role of HSL in this process, we have gener- partment of Physiological Sciences, Lund University, Lund, Sweden; and the  8Department of Medicine, Lund University, Lund Sweden. ated transgenic mice with -cell–specific overexpression Address correspondence and reprint requests to Dr. Maria So¨ rhede Winzell, of HSL. These mice exhibit impaired GSIS when chal- Department of Cell and Molecular Biology, Section for Molecular Signaling, lenged with a high-fat diet, suggesting that HSL may be Lund University, Biomedical Center (BMC), C11, SE-221 84 Lund, Sweden. E-mail: [email protected]. involved in -cell lipotoxicity by providing lipid ligands for Received for publication 13 December 2002 and accepted in revised form transcription factors. The gene expression changes that 13 May 2003.  CPT I, carnitine palmitoyltransferase I; FFA, free fatty acid; GSIS, glucose- follow may be responsible for the development of -cell stimulated insulin secretion; HSL, hormone-sensitive lipase; IVGTT, intrave- dysfunction. We have identified one such change, which nous glucose tolerance test; MCAD, medium-chain acyl dehydrogenase; may fully or partly explain the observed phenotype, i.e., PDX-1, pancreatic/duodenal homeobox-1; PPAR, peroxisome proliferator– activated receptor; UCP2, uncoupling protein 2. the upregulation of the uncoupling protein 2 (UCP2) gene, © 2003 by the American Diabetes Association. which recently was found to negatively regulate GSIS (13). DIABETES, VOL. 52, AUGUST 2003 2057 OVEREXPRESSION OF HSL LEADS TO -CELL FAILURE RNA isolation and Northern blot analysis. Total RNA was isolated from islets (18), electrophoresed, and transferred to nylon membranes (Hybond- Nϩ). The blots were probed with a 32P-labeled mouse UCP2 cDNA using ExpressHyb (Clontech). Membranes were analyzed and quantified by digital imaging (Fujix Bas 2000; Fuji). Quantitative PCR. Pooled total RNA isolated from islets was DNase-treated, and three independent reverse transcription reactions were performed using Superscript II reverse transcriptase (Invitrogen) following the manufacturer’s instructions. The mRNA expression levels were determined using real-time fluorescent detection and the following primer combinations: 5Ј-GTACCAC- TACGGAGTTCACGCAT-f-3Ј and 5Ј-CGCCGAAAGAAGCCCTTAC-r-3Ј for per- oxisome proliferator–activated receptor (PPAR)-␣,5Ј-CAGATAATTGGCACC TACGCC-f-3Ј and 5Ј-AAGATGAGTTCCGTGGCCC-r-3Ј for acylCoA oxidase, 5Ј-TCTGGATGGCTATGGTCAAG-f-3Ј and 5Ј-TACCACTGCAATTTGGGTCC- f-3Ј for CPT-I, 5Ј-AGCTGCTAGTGGAGCACCAAG-f-3Ј and 5Ј-TCGCCATTTCT GCGAGC-r-3Ј for medium-chain acyl dehydrogenase (MCAD), and 5Ј-CCTAC AGCGCCAGATGAGCT-f-3Ј and 5Ј-GAGTCGTAGAGGCCAATGCG-r-3Ј for UCP2. The mRNA expression levels were determined twice in each first-strand synthesis reaction and normalized to the expression levels of 18S rRNA as described by the vendor (Applied Biosystems). Glucose tolerance tests and insulin release. Glucose tolerance tests were FIG. 1. Specific overexpression of HSL in pancreatic -cells. A: Sche- matic representation of a vector for transgenic overexpression of HSL performed in anesthetized mice after intravenous administration of D-glucose in -cells. Details are given in the text. B: Analysis of HSL expression (1 g/kg) or arginine (0.25 g/kg). Blood samples were taken at 0, 1, 5, 20, and 50 in islets and selected tissues from transgenic (tg) and wild-type (wt) min. female and male mice using Western blot analysis. Equal amounts of Insulin secretion, insulin content, and ATP in isolated islets. Insulin total protein of homogenate from each tissue were loaded on the gel. secretion from isolated islets was measured as described (15). Total insulin C: Diglyceride (DG) and triglyceride (TG) lipase activity measured in content in islets was measured after extraction of insulin with acidic ethanol homogenates of islets from wild-type (wt) and transgenic (tg) mice. (0.2 mol/l HCl in 75% ethanol) (Linco). Increased triglyceride lipase activity correlated with increased release For determination of ATP levels, batches of 10 islets were incubated in of glycerol, an end product of lipolysis. Values are means ؎ SE of three different experiments; **P < 0.01 vs. wild-type islets. BAT, brown RPMI-1640 medium containing 16.7 mmol/l glucose at 37°C for 5 min. The ATP adipose tissue; WAT, white adipose tissue. concentration was measured using a luciferase-based assay (ATP monitoring kit; Thermo Labsystems). Triglyceride measurements in islets. Acylglycerols were extracted from isolated islets (19). After hydrolyzation, the amount of released glycerol was RESEARCH DESIGN AND METHODS measured using a bioluminescence assay (20). DNA construct and generation of transgenic mice. A pBluescript vector Enzyme activity assays and measurement of lipolysis. Homogenates, containing the rat adipocyte HSL cDNA (nucleotides 610–3,225) downstream prepared as for Western blot analysis, were assayed for diglyceride and of the scavenger receptor gene promoter was used as starting plasmid (kind triglyceride lipase activity, as described (21) using mono-oleoyl-2-O- gift from Drs. Karen Reue and Jean-Louis Escary, VA Lipid Research Labora- monooleylglycerol, a diacylglycerol analog, and triolein, respectively, as tory, Los Angeles, CA). After excision of the scavenger receptor gene substrates. One unit of enzyme