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  • IntroductionType 2 diabetes mellitus results from an inadequateadaptation of the functional pancreatic cell mass inthe face of insulin resistance. In turn, hyperglycemiaby itself has secondary adverse effects on cells.Indeed, several studies have shown that chronic eleva-tion of blood glucose concentration impairs cellfunction, leading to the concept of glucotoxicity (17).Moreover, elevated glucose concentrations induce cell apoptosis in cultured islets from diabetes-pronePsammomys obesus (8) and from humans (9, 10); some-what higher concentrations of glucose are required toinduce cell apoptosis in rodent islets (8, 11, 12). Var-ious molecular mechanisms have been proposed to

    underlie glucose-induced cell dysfunction, includingformation of advanced glycation end products (13),direct impairment of insulin gene transcription andproinsulin biosynthesis (14, 15), and reduced bindingactivity of pancreatic duodenal homeobox 1 (PDX-1)(7). Recently, we proposed a mechanism underlyingglucose-induced cell apoptosis in human islets thatinvolves upregulation of Fas receptors by elevated glu-cose levels (9). However, the mediator of glucose-induced Fas expression and its role in glucotoxicityremains unknown.

    IL-1 has been proposed to mediate both impairedfunction and destruction of pancreatic cells duringthe development of autoimmune type 1 diabetes (16).In keeping with this, treatment of rodent islets with IL-1 results in a potent inhibition of insulin secretionfollowed by islet destruction (1723). In human islets,IL-1 has been shown to impair insulin release and toinduce Fas expression, enabling Fas-triggered apopto-sis (9, 2428). Finally, activation of the nuclear tran-scription factor NF-B is required for IL-1inducedFas expression (2931). Part of these IL-1 effects arereminiscent of the toxic effects of elevated glucose con-centrations. Together, the above findings led us to pos-tulate that glucose may induce IL-1 secretion from cells in the absence of an autoimmune process. We now

    The Journal of Clinical Investigation | September 2002 | Volume 110 | Number 6 851

    Glucose-induced cell production of IL-1 contributes to glucotoxicity in human pancreatic islets

    Kathrin Maedler,1 Pavel Sergeev,1 Frdric Ris,2, 3 Jos Oberholzer,3

    Helen I. Joller-Jemelka,4 Giatgen A. Spinas,1 Nurit Kaiser,5 Philippe A. Halban,2

    and Marc Y. Donath1

    1Division of Endocrinology and Diabetes, University Hospital, Zurich, Switzerland2Louis-Jeantet Research Laboratories, University of Geneva Medical Center, Geneva, Switzerland3Division of Surgical Research, Department of Surgery, University of Geneva Medical Center, Geneva, Switzerland4Division of Clinical Immunology, University Hospital, Zurich, Switzerland5Department of Endocrinology and Metabolism, Hebrew University Hadassah Medical Center, Jerusalem, Israel

    In type 2 diabetes, chronic hyperglycemia is suggested to be detrimental to pancreatic cells, caus-ing impaired insulin secretion. IL-1 is a proinflammatory cytokine acting during the autoimmuneprocess of type 1 diabetes. IL-1 inhibits cell function and promotes Fas-triggered apoptosis in partby activating the transcription factor NF-B. Recently, we have shown that increased glucose con-centrations also induce Fas expression and cell apoptosis in human islets. The aim of the presentstudy was to test the hypothesis that IL-1 may mediate the deleterious effects of high glucose onhuman cells. In vitro exposure of islets from nondiabetic organ donors to high glucose levels result-ed in increased production and release of IL-1, followed by NF-B activation, Fas upregulation, DNAfragmentation, and impaired cell function. The IL-1 receptor antagonist protected cultured humanislets from these deleterious effects. cells themselves were identified as the islet cellular source ofglucose-induced IL-1. In vivo, IL-1producing cells were observed in pancreatic sections of type2 diabetic patients but not in nondiabetic control subjects. Similarly, IL-1 was induced in cells ofthe gerbil Psammomys obesus during development of diabetes. Treatment of the animals with phlo-rizin normalized plasma glucose and prevented cell expression of IL-1. These findings implicatean inflammatory process in the pathogenesis of glucotoxicity in type 2 diabetes and identify the IL-1/NF-B pathway as a target to preserve cell mass and function in this condition.

    J. Clin. Invest. 110:851860 (2002). doi:10.1172/JCI200215318.

    Received for publication February 20, 2002, and accepted in revised formJuly 16, 2002.

    Address correspondence to: Marc Y. Donath, Division ofEndocrinology and Diabetes, Department of Medicine,University Hospital, CH-8091 Zurich, Switzerland. Phone: 41-1-255-3625; Fax: 41-1-255-4447; E-mail: marc.donath@dim.usz.ch.Pavel Sergeev and Frdric Ris contributed equally to this work.Conflict of interest: No conflict of interest has been declared.Nonstandard abbreviations used: IL-1 receptor antagonist (IL-1Ra); Fas ligand (FasL); pyrrolidinedithiocarbamate (PDTC);inducible nitric oxide synthase (iNOS); inhibitory B (I-B).

  • identify cells as the cellular source of glucose-inducedIL-1 in cultured human islets and confirm this usingtissue sections from the pancreas of type 2 diabeticpatients and of Psammomys obesus. The role of suchendogenously produced IL-1 in cell glucotoxicitywas also explored.

    MethodsIslet isolation and culture. Islets were isolated from pan-creata of 11 organ donors at the Department ofSurgery, University of Geneva Medical Center, asdescribed (3234). Islet purity was greater than 95% asjudged by dithizone staining (if this degree of puritywas not achieved by routine isolation, islets were handpicked). The donors, aged 4070 years, were heart-beat-ing cadaver organ donors, and none had a previous his-tory of diabetes or metabolic disorders. For long-termin vitro studies, the islets were cultured on extracellu-lar matrixcoated plates derived from bovine cornealendothelial cells (Novamed Ltd., Jerusalem, Israel),allowing the cells to attach to the dishes and spread,preserving their functional integrity (7, 35). Islets werecultured in CMRL 1066 medium (hereafter referred toas culture medium) containing 100 U/ml penicillin,100 g/ml streptomycin, and 10% FCS (Invitrogen Ltd.,Carlsbad, California, USA). Two days after plating,when most islets were attached and began to flatten,the medium was changed to culture medium contain-ing 5.5, 11.1, or 33.3 mM glucose. In some experiments,islets were additionally cultured with 2 ng/ml recom-binant human IL-1, 1,000 U/ml recombinant humanIFN- (ReproTech EC Ltd., London, United Kingdom),500 ng/ml IL-1 receptor antagonist (IL-1Ra; R&D Sys-tems Inc., Minneapolis, Minnesota, USA), 1 ng/mlmembrane-bound Fas ligand (FasL; Upstate Biotech-nology Inc., Lake Placid, New York, USA) (36), or with100 M pyrrolidinedithiocarbamate (PDTC) for2 hours for every 2 days in culture (Sigma-Aldrich, St.Louis, Missouri, USA).

    Animals. Psammomys obesus of both sexes (age 2.03.5months) from the diabetes-prone and diabetes-resist-ant lines of the Hebrew University colonies were origi-nally obtained from Harlan Laboratories Ltd.(Jerusalem, Israel). After weaning, diabetes-pronePsammomys obesus were maintained on a low-energy dietcontaining 2.38 kcal/g (Koffolk Ltd., Petach Tikva,Israel) until the start of the experiments, whereas dia-betes-resistant Psammomys obesus were maintained on ahigh-energy diet containing 2.93 kcal/g (WeizmannInstitute of Science, Rehovot, Israel) to identify animalsthat develop diabetes and exclude them from the study(3040% of the animals in the diabetes-resistantcolony). All nonfasted animals with random blood glu-cose concentrations below 7.8 mmol/l (tested with theGlucometer Elite from Bayer Corp., Elkhart, Indiana,USA) were considered nondiabetic. Diabetes-pronePsammomys obesus switched to a high-energy dietreceived an injection of 0.4 g/kg phlorizin (Sigma-Aldrich) or solvent (40% propylene glycol) every 12

    hours and were killed after 8 days. Psammomys obesuswere anesthetized with ketamine (Ketalar; Parke-Davis& Co., Gwent, United Kingdom) and exsanguinated bycardiac puncture. The pancreas was rapidly removedand immersion-fixed in 10% phosphate-buffered for-malin. The animal studies were approved by the Insti-tutional Animal Care and Use Committee of HebrewUniversity and the Hadassah Medical Organization.

    Detection of IL-1expressing cells. Pancreata fromroutine necropsies and from Psammomys obesus wereimmersion-fixed in formalin, followed by paraffinembedding. Sections were deparaffinized and rehy-drated, and endogenous peroxidase was blocked bysubmersion in 0.3% H2O2 for 15 minutes. Sectionswere then incubated in methanol for 4 minutes. Afterwashing with PBS, cultured islets and isolated cellswere fixed in 4% paraformaldehyde (30 minutes atroom temperature) followed by permeabilization with0.5% Triton X-100 (4 minutes at room temperature).Both tissue sections and cultured cells were double-labeled for IL-1 and insulin by 1 hour of exposure to10% BSA followed by incubation for 1 hour at 37Cwith mouse antiIL-1 antibody (1:30 dilution; R&DSystems Inc.). Detection was performed using donkeyanti-mouse Cy3-conjugated antibody (1:100 dilution;Jackson ImmunoResearch Laboratories, West Grove,Pennsylvania, USA). Subsequently, specimens wereincubated for 30 minutes at 37C with guinea piganti-insulin antibody diluted 1:50 (DAKO Corp.,Carpinteria, California, USA), followed by a 30-minuteincubation with a 1:20 dilution of fluorescein-conju-gated rabbit antiguinea pig antibody (DAKO Corp.).For positive control of IL-1 staining, humanmononuclear cells were isolated as described previ-ously (37) and exposed for 2 hours at 37C to 1 g/mlLPS (Becton, Dickinson and Co., Franklin Lakes, NewJersey, USA). Coverslips were air-dried and mountedonto slides, then fixed and permeabilized for