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Glucose Induces b-Cell Apoptosis Via Upregulation ofthe Fas Receptor in Human IsletsKathrin Maedler,

1Giatgen A. Spinas,

1Roger Lehmann,

1Pavel Sergeev,

1Markus Weber,

2

Adriano Fontana,3

Nurit Kaiser,4

and Marc Y. Donath1

In autoimmune type 1 diabetes, Fas–to–Fas-ligand (FasL)interaction may represent one of the essential pro-apoptotic pathways leading to a loss of pancreatic b-cells.In the advanced stages of type 2 diabetes, a decline inb-cell mass is also observed, but its mechanism is notknown. Human islets normally express FasL but not theFas receptor. We observed upregulation of Fas in b-cellsof type 2 diabetic patients relative to nondiabetic controlsubjects. In vitro exposure of islets from nondiabeticorgan donors to high glucose levels induced Fas expres-sion, caspase-8 and -3 activation, and b-cell apoptosis. Theeffect of glucose was blocked by an antagonistic anti-Fasantibody, indicating that glucose-induced apoptosis is dueto interaction between the constitutively expressed FasLand the upregulated Fas. These results support a new rolefor glucose in regulating Fas expression in human b-cells.Upregulation of the Fas receptor by elevated glucoselevels may contribute to b-cell destruction by the consti-tutively expressed FasL independent of an autoimmunereaction, thus providing a link between type 1 and type 2diabetes. Diabetes 50:1683–1690, 2001

Type 2 diabetes manifests itself in individuals wholose the ability to produce sufficient quantities ofinsulin to maintain normoglycemia in the face ofinsulin resistance (1). Indeed, the contribution of

a relative insulin deficiency to the establishment of overtdiabetes is now widely accepted (2–5). The ability tosecrete adequate amounts of insulin depends on b-cellfunction and mass. The endocrine pancreas has a remark-able capacity to adapt to conditions of increased insulindemand (e.g., in pregnancy, obesity, and cortisol or growthhormone excess) by increasing its functional b-cell mass;only 20% of the individuals under these conditions fail toadapt and become diabetic with time (5). Long-termadaptation of the b-cell mass to conditions of increased

demand occurs by increasing the b-cell number throughhyperplasia and neogenesis (5,6). However, b-cell expan-sion can be offset by concomitant apoptosis (7,8).

In a previous study, we analyzed b-cell turnover inpancreases of Psammomys obesus, a rodent with a naturaltendency toward diet-induced type 2–like diabetes, ini-tially characterized by hyperinsulinemia, but progressingto hypoinsulinemia and reduced b-cell mass (8). Analysisof b-cell turnover in P. obesus during nutrition-inducedtransition from normoglycemia to hyperglycemia revealedan initial and transient increase in b-cell replication thatwas followed by a prolonged increase in the number ofapoptotic b-cells, leading to a marked reduction in thefunctional b-cell mass. Elevated glucose concentrationsdirectly induced b-cell apoptosis in cultured islets fromdiabetes-prone P. obesus, but not in islets from normal rats(8,9). Glucose-induced b-cell proliferation was observed inboth rat (10) and P. obesus islets; however, the lattershowed only a limited capacity. It is not known whetherelevated glucose concentrations can also adversely affectb-cell turnover in human islets and, if it can, by whichmechanism.

In type 1 diabetes, the failure of the islet is alreadydetectable at the onset of hyperglycemia because of b-cellapoptosis (11–13). However, the precise mechanisms lead-ing to b-cell destruction remain unclear. The cell deathreceptor Fas (CD95) is able to signal apoptosis via anintracellular death domain (14). Cytokines can induceupregulation of Fas expression on b-cells, making themsusceptible to apoptosis in the presence of agonisticanti-Fas antibodies, or interaction with Fas-ligand (FasL)–expressing T-cells (15,16) as well as neighboring b-cells(17). Nonobese diabetic (NOD) mice develop spontaneousautoimmune diabetes, but Fas-deficient NOD mice(NODlpr) are protected against the disease (18,19). There-fore, Fas has been postulated to play an important role inthe b-cell demise of type 1 diabetes. However, the role ofFas in diabetes has been challenged by several studies(20,21). Islet grafts from Fas-deficient NOD mice areprotected only marginally when grafted into diabetic mice(20). Furthermore, Thomas et al. (21) detected only few, ifany, Fas-expressing b-cells in islets of NOD mice close tothe onset of hyperglycemia.

We hypothesized that Fas expression and activationmay constitute an immune-independent event induced bytransient hyperglycemic excursions. If true, this processmay not be limited to type 1 diabetes but may also be

From the 1Division of Endocrinology and Diabetes, the 2Department ofSurgery, and the 3Division of Clinical Immunology, University Hospital,Zurich, Switzerland; and the 4Department of Endocrinology and Metabolism,Hebrew University-Hadassah Medical Center, Jerusalem, Israel.

Address correspondence and reprint requests to Marc Y. Donath MD,Division of Endocrinology and Diabetes, Department of Medicine, UniversityHospital, CH-8091 Zurich, Switzerland. E-mail: marc.donath@dim.usz.ch.

Received for publication 28 March 2001 and accepted in revised form15 May 2001. Posted on the World Wide Web at www.diabetes.org/diabetes on21 June 2001.

DTT, dithiothreitol; FasL, Fas-ligand; PBS, phosphate-buffered saline; TUNEL,terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.

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present in type 2 diabetes. Indeed, using cultured isletsderived from normal individuals, we show here that patho-logically elevated glucose concentrations induce apoptosisin human b-cells and that this is initiated by upregulationof the Fas receptor.

RESEARCH DESIGN AND METHODS

Islet isolation and culture. Islets were isolated from the pancreases of eightorgan donors at the Division of Endocrinology and Diabetes, UniversityHospital of Zurich, as previously described (22). The donors, aged 17, 26, 38,50, 56, 60, 61, and 67 years, were heart-beating cadaver organ donors, andnone had a previous history of diabetes or metabolic disorders. For long-termin vitro studies, the islets were cultured on extracellular matrix-coated platesderived from bovine corneal endothelial cells (Novamed, Jerusalem), allowingthe cell to attach to the dishes and spread, preserving their functional integrity(23–24). Islets were cultured in CMRL 1066 medium containing 100 units/mlpenicillin, 100 mg/ml streptomycin, and 10% fetal calf serum (Gibco, Gaithers-burg, MD). Two days after plating, when most islets were attached and hadbegun to flatten, the culture medium was changed to CMRL containing 5.5,11.1, or 33.3 mmol/l glucose. In some experiments, islets were cultured with 2pg/ml interleukin-1b (R&D Systems, Minneapolis, MN) or 500 ng/ml antago-nistic Fas antibody (ZB4; MBL, Nogoya, Japan).b-cell replication. For b-cell proliferation studies, a monoclonal antibodyagainst the human Ki-67 antigen was used (Zymed, San Francisco, CA). Ki-67is a nuclear antigen expressed by proliferating cells and is used as a markerfor late G1, S, G2, and M phases of the cell cycle (8,25). After washing withphosphate-buffered saline (PBS), cultured islets were fixed in 4% paraformal-dehyde (30 min, room temperature) followed by permeabilization with 0.5%triton X-100 (4 min, room temperature). Afterward, islets were incubated for1 h at room temperature with monoclonal mouse anti–Ki-67 antibody diluted1:10, followed by detection using a streptavidin-biotin-peroxidase complex(Histostain-Plus Kit; Zymed). Subsequently, islets were incubated for 30 min at37°C with guinea pig anti-insulin antibody diluted 1:50 (Dako, Carpinteria,CA), followed by a 10-min incubation with a 1:10 dilution of fluorescein-conjugated rabbit anti–guinea pig antibody (Dako).b-cell apoptosis. The free 3-OH strand breaks resulting from DNA degrada-tion were detected by the terminal deoxynucleotidyl transferase–mediateddUTP nick-end labeling (TUNEL) technique (26). Islet cultures were fixed andpermeabilized as described above, then they underwent the TUNEL assay,which was performed according to the manufacturer’s instructions (in situcell death detection kit AP; Boehringer Mannheim, Mannheim, Germany). Thepreparations were then rinsed with Tris-buffered saline and incubated (10 min,room temperature) with 5-bromo-4-chloro-indolyl phosphate/nitro blue tetra-zolium liquid substrate system (Sigma). Thereafter, islets were incubated witha guinea pig anti-insulin antibody as above, followed by detection using thestreptavidin-biotin-peroxidase complex (Zymed). Afterward, islets were incu-bated for 2 h at 37°C with a rabbit anti–cleaved-caspase-3 antibody (1:50dilution, D 175; Cell Signaling, Beverly, MA), followed by incubation (30 min,37°C) with a fluorescein-conjugated donkey anti-rabbit antibody (1:100 dilu-tion; Jackson ImmunoResearch Laboratories, West Grove, PA). In parallel tothe TUNEL reaction, we used the DNA-binding dye propidium iodide (Sigma)to assess the effects of glucose on necrosis. Unfixed cultured islets wereincubated for 10 min on ice with 10 mg/ml propidium iodide in PBS, washedwith PBS, and embedded in fluorescent mounting medium (Dako). Thesamples were immediately evaluated by fluorescent microscopy for positivelystained necrotic nuclei.Detection of Fas- and FasL-expressing b-cells. Pancreases from routinenecropsies were immersion-fixed in formalin, followed by paraffin embedding.Sections were deparaffinized and rehydrated, and then they were endogenousperoxidase–blocked by submersion in 0.3% H2O2 for 15 min. For FasLdetection, the sections were incubated in methanol for 2 min. Islet cultureswere fixed and permeabilized as described above. Tissue sections andcultured islets were double-labeled for Fas receptor or for FasL and insulin by1-h exposure to 10% bovine serum albumin, followed by incubation (30 min,37°C) with mouse anti-Fas antibody (1:50 dilution; Transduction Laboratories,Lexington, KY) or by incubation (2 h, 37°C) with anti-FasL antibody (1:25dilution, NOK-1; Transduction Laboratories). Detection was then performedusing the streptavidin-biotin-peroxidase complex (Zymed) or donkey anti-mouse Cy3-conjugated antibody (1:100 dilution; Jackson ImmunoResearch).Subsequently, the specimens were stained for insulin, as described above, andinsulin was detected with a fluorescein-conjugated rabbit anti–guinea pigantibody. The specificity of the Fas and FasL antibodies was assessed by anabsorption test using soluble Fas (27) and soluble FasL (28). Human malignantglioma cells transfected with a human Fas cDNA expression vector were usedas a positive control for Fas, and untransfected glioma cells were used as a

negative control (27). For mRNA in situ hybridization, we used FasL and Fasfull-length cDNA that were cloned into pCR-Script SK(1) plasmid (Stratagene,La Jolla, CA) and linearized. Using RNA polymerase and RNA digoxigeninlabeling mix (Roche, Switzerland), we prepared sense and antisense digoxi-genin-labeled RNA probes. Tissue sections were treated with 20 mg/mlproteinase K (Roche) and prehybridized for 2 h at 55°C in hybridization buffercontaining 50% formamide, 5 3 sodium chloride–sodium citrate, 50 mg/mlsalmon sperm (Sigma), 1 3 Denhart’s solution, and 250 mg/ml RNA type IVfrom calf liver (Sigma). The hybridization was performed overnight at 52°C in100 ml hybridization buffer containing 30 ng of digoxigenin-labeled RNA probe.Sections were then blocked with 5% milk powder at room temperature andincubated 1 h at 37°C with anti–digoxigenin-rhodamine Fab fragment (20mg/ml; Roche) followed by insulin immunostaining as described above.

After staining, samples were embedded in Kaiser’s glycerol gelatin (Merck,Darmstadt, Germany) and analyzed by light and fluorescence microscopy(microscope Axiolab; Zeiss, Jena, Germany).Western blot analysis. Islets were cultured in suspension in CMRL 1066medium containing 5.5 mmol/l glucose, as described above. One day afterisolation, medium was changed and groups of 200 islets were incubated for36 h in medium containing 5.5, 11.1, or 33.3 mmol/l glucose. At the end of theincubations, islets were washed in PBS. For analysis of the human islets’subcellular fractions, mitochondrial and cytosolic fractions were preparedfrom islets suspended in 70 ml of ice-cold buffer containing 20 mmol/lHEPES-KOH (pH 7.5), 10 mmol/l KCl, 15 mmol/l MgCl2, 1 mmol/l Na-EDTA, 1mmol/l dithiothreitol (DTT), 0.1 mmol/l phenylmethylsulfonylfluorid, and 250mmol/l sucrose. Mechanical homogenization was achieved by repeated aspi-ration through a pipette. Unlysed cells and nuclei were pelleted by 10 mincentrifugation (750g, 4°C). The supernatant was centrifuged at 10 000g for 15min at 4°C. The resulting pellet, representing the mitochondrial fraction, wasthen resuspended in 10 ml of the buffer described above. Finally, thesupernatant was centrifuged at 100 000g for 1 h at 4°C. The supernatant fromthis final centrifugation represented the cytosolic fraction. Both fractionswere frozen at 280°C until used. Mitochondrial and cytosolic fractions werediluted in sample buffer (187.5 mmol/l Tris-HCl, pH 6.8, 6% SDS, 30% glycerol,150 mmol/l DTT, and 0.3% bromphenol blue) and then boiled for 5 min.Equivalent amounts of each treatment group at a 5:3 cytosolic–to–mitochon-drial fraction ratio were run on 15% SDS polyacrylamide gels. Proteins wereelectrically transferred to nitrocellulose filters and incubated with a mouseanti-cytochrome c monoclonal antibody (PharMingen, San Diego, CA) fol-lowed by incubation with horseradish-peroxidase–linked anti-mouse IgG(Santa Cruz Biotechnology, Santa Cruz, CA). The emitted light was capturedon X-ray film after adding Lumiglo reagent (Phototope-HRP Western blotdetection kit; Biolabs, Beverly, MA). Human islets exposed for 36 h to 0.5mmol/l palmitic acid were used as positive control for cytochrome c release(29). As a marker, biotinylated protein molecular weight standard (Biolabs)was run in parallel.

For analysis of FasL, Fas, caspase-3, and caspase-8, islets were suspendedin 50 ml sample buffer (125 mmol/l Tris-HCl, pH 6.8, 4% SDS, 10% glycerol, 0.3%bromphenol blue, and 1.8% b-mercaptoethanol) and boiled for 5 min. Humanfibroblast lysate derived from foreskin was used as a positive control for Fas(Transduction Laboratories). Equivalent amounts of each treatment groupwere run on 15% SDS polyacrylamide gels. Proteins were electrically trans-ferred to nitrocellulose filters and incubated with mouse anti-Fas (Transduc-tion Laboratories), anti-FasL (NOK-1; Transduction Laboratories), anti–cas-pase-3 (PharMingen), or anti–caspase-8 (Biosource, Camarillo, CA) antibod-ies, followed by incubation with horseradish-peroxidase–linked anti-mouse oranti-rabbit IgG. After adding Lumiglo reagent, the emitted light was capturedon X-ray film. Between the incubations, nitrocellulose membranes werestripped for 30 min at 50°C in 40 ml of a watery solution containing 280 mlb-mercaptoethanol, 5 ml 0.5 mol/l Tris-HCl, pH 6.8, and 10% SDS, and thenthey were washed for 1 h in Tris-buffered saline containing 0.1% Tween-20.Statistical analysis. Data are presented as means 6 SE and were analyzedby Student’s t test or by analysis of variance with a Bonferroni correction formultiple group comparisons. Cultures were evaluated in a randomized mannerby a single investigator (K.M.) who was blinded to the treatment conditions.Care was taken to score islets of similar size.

RESULTS

Modulation of human b-cell proliferation and apopto-

sis by elevated glucose concentrations. Human islets,isolated from pancreases of heart-beating donors andcultured on extracellular matrix-coated plates, were ex-posed to elevated glucose concentrations for 5 days.Analysis of b-cell nuclei for DNA fragmentation (TUNEL-

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positive) (Fig. 1A-1 and A-2) revealed a 2.4- and 3.5-foldincrease in islets cultured at medium glucose concentra-tions of 11.1 and 33.3 mmol/l, respectively, relative to isletsat 5.5 mmol/l glucose (Fig. 1B-1). A typical feature of theDNA fragmentation in b-cells was the occasional appear-ance of TUNEL-positive nuclei in doublets, suggestive ofpostmitotic apoptosis. The TUNEL reaction may also stainnecrotic cells; therefore, in parallel to the TUNEL reaction,we used the DNA-binding dye propidium iodide to assessthe effect of glucose on necrosis. Exposure of islet cul-tures to increasing glucose concentrations (from 5.5 to33.3 mmol/l) did not lead to propidium iodide uptake intothe cultured cells (not shown). Moreover, triple-immunos-taining for DNA fragmentation, insulin, and cleavedcaspase-3 demonstrated cleaved caspase-3 in the TUNEL-positive b-cells (Fig. 1A-2 insert), confirming an apoptoticprocess. To exclude a nonspecific effect of the highconcentration of D-glucose (33.3 mmol/l), osmolarity wascorrected with the metabolically inactive L-glucose. A5-day exposure to 27.8 mmol/l L-glucose together with 5.5mmol/l D-glucose resulted in a b-cell DNA fragmentationrate similar to that induced by 5.5 mmol/l D-glucose alone.The time-course of the effect of 33.3 mmol/l glucose onDNA fragmentation reveals a significant increase in b-celldeath after 1 day of exposure to the high glucose level andan increased number of TUNEL-positive b-cells persistingthroughout the 10 days of the study (2.0-, 7.4-, 3.0-, 4.0-, and5.0-fold increase after 1, 3, 5, 7, and 10 days of treatment,respectively, compared with 5.5 mmol/l glucose) (Fig.1B-2).

Exposure of cultured human islets to elevated glucoseconcentrations for 5 days decreased the number of prolif-erating (Ki-67–positive) b-cells (Fig. 1A-3 to A-6). Prolifer-ation was reduced by 42 and 61% in medium containing11.1 and 33.3 mmol/l glucose, respectively, relative to isletsat 5.5 mmol/l glucose (Fig. 1B-3). Exposure of the islets to27.8 mmol/l L-glucose together with 5.5 mmol/l D-glucosedid not change the baseline proliferative activity observedwith 5.5 mmol/l D-glucose alone. An initial increase of 1.5-and 2.5-fold in Ki-67–positive b-cells was observed after a1- and 3-day exposure, respectively, to 33.3 mmol/l glucose(relative to islets at 5.5 mmol/l glucose); longer exposuretimes resulted in a marked inhibition of the b-cells’ prolif-erative capacity (three- and twofold decrease after 5 and10 days, respectively, of exposure to 33.3 mmol/l glucose)(Fig. 1B-4).

These studies indicate that, unlike the long-term increasein b-cell death generated in human islets by a continuousexposure to elevated glucose levels, b-cell proliferativecapacity exhibited only a transient increase followed by aprolonged decrease.Glucose induces Fas expression and activation in

human b-cells. We next studied the cellular mechanismof glucose-induced apoptosis in human b-cells; specifi-cally, we investigated the possible involvement of the Fasreceptor pathway. Exposure of cultured human islets toincreasing glucose concentrations (from 5.5 to 33.3 mmol/lglucose) for 5 days resulted in a dose-dependent increasein Fas receptor expression in the b-cells, as determined bydouble-immunostaining (with anti-Fas and anti-insulin an-tibodies) islets plated on extracellular matrix–coateddishes (Fig. 2A). The large majority of the Fas-positive

cells observed were insulin-positive. However, a few cellswere Fas-positive and insulin-negative. These cells can beinsulin-depleted b-cells or non–b-cells. The effect of ele-vated glucose on Fas expression was verified by Westernblot analysis (Fig. 2B). Glucose-dependent cleavage ofprocaspase-3 to activated caspase-3 was also identified byWestern blotting (Fig. 2B), supporting the idea that glu-cose-induced DNA fragmentation, as determined by theTUNEL assay, represents apoptotic b-cell death. Treat-ment of islets with 33.3 mmol/l glucose induced activationof caspase-8 (Fig. 2C). Because caspase-8 is the mostupstream caspase in the Fas apoptotic pathway, its acti-vation by glucose further supports a role for Fas inglucose-induced apoptosis. Changes in glucose concentra-tion in the culture medium did not induce release ofcytochrome c from the mitochondria to the cytosol (Fig.2D), suggesting that glucose-dependent apoptosis is inde-pendent of the mitochondrial–cytochrome c pathway inhuman islets.Constitutive expression of FasL in human islets and

upregulation of the Fas receptor in islets of hyper-

glycemic type 2 diabetic patients. Loweth et al. (17)showed constitutive expression of FasL in human islets.However, this finding was not confirmed by two subse-quent studies (30,31). The negative results were imputedto the specificity of the antibody used, which in subse-quent studies has been reported to produce a false-positivereaction (32). We tested an antibody shown to be specificfor FasL (31,32) and found substantial amounts of FasL inthe human b-cells of control and diabetic patients (Fig. 3,panels 1–6). The expression of FasL was verified byimmunostaining of isolated islets (not shown) as well asby Western blot analysis (Fig. 2B). Moreover, the presenceof FasL mRNA transcripts was verified by in situ hybrid-ization in the b-cells of diabetic (Fig. 3, panels 7 and 8) andnormal individuals (Fig. 3, panels 9 and 10), whereas theexocrine pancreas was negative. For controls, we used adigoxigenin-labeled sense probe and found no signal (Fig.3, panel 11). We concluded that the previously reportedfailure to detect FasL was probably caused by differencesin the preparation of the pancreas sections because it waspossible to detect FasL in the b-cell only after performingan antigen retrieval procedure with methanol, as de-scribed by Loweth et al. (17).

The Fas receptor is not expressed in normal humanpancreatic islets (17,31). However, based on our in vitrostudies, we anticipated its expression in the islets of type2 diabetic patients as a result of repeated hyperglycemicepisodes. Expression of Fas was therefore studied insections of pancreases from five poorly controlled type 2diabetic patients, all with documented fasting blood glu-cose .8 mmol/l. Double-immunostaining and mRNA insitu hybridization of the pancreatic sections for Fas andinsulin revealed localization of the Fas receptor to theb-cells (Fig. 3, panels 13, 14, 19, and 20). Fas expressioncould not be detected in b-cells of nondiabetic controlsubjects (Fig. 3, panels 15, 16, 21, and 22).Glucose-induced b-cell apoptosis and impaired pro-

liferation are mediated by Fas-FasL interaction in

human islets. To examine whether the induction ofapoptosis and impaired proliferation by glucose is causedby interaction between constitutively expressed FasL and

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FIG. 1. Characterization of the effect of elevated glucose concentrations on human b-cell apoptosis and proliferation. A: Islets were exposed for5 days to media containing 5.5 mmol/l glucose (panels 1, 3, and 5) or 33.3 mmol/l glucose (panels 2, 4, and 6). Triple-immunostaining (panels 1and 2) for insulin (orange), DNA fragmentation by the TUNEL assay (black), and cleaved caspase-3 (green, bottom insert). Detection of b-cellproliferation with anti–Ki-67 (red; panels 3 and 4) and with anti-insulin antibody (green; panels 5 and 6). The white arrows mark nuclei stainedpositive for the TUNEL reaction, and the black arrows mark nuclei stained positive for Ki-67. Original magnification 3400. B: Relative numberof TUNEL-positive (panel 1) and Ki-67–positive b-cells (panel 3) per islet after 5-day culture in 5.5, 11.1, and 33.3 mmol/l D-glucose or in 5.5mmol/l D-glucose plus 27.8 mmol/l L-glucose, normalized to control incubations at 5.5 mmol/l glucose alone (100%; in absolute value: 0.53TUNEL-positive b-cells per islet and 1.16 Ki-67–positive b-cells per islet). TUNEL-positive (panel 2) and Ki-67–positive (panel 4) b-cells per isletduring 10-day culture at 5.5 mmol/l or 33.3 mmol/l glucose. The mean number of islets scored was 141 for each treatment condition. Islets wereisolated from eight heart-beating cadaver organ donors. Results are shown as means 6 SE. *P < 0.01 relative to islets at 5.5 mmol/l glucose; **P <0.01 relative to islets at 33.3 mmol/l glucose.

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FIG. 2. Glucose induces Fas expression and caspase activation in human b-cells. A: Double-immunostaining for the Fas receptor (panels 1, 3, and5) and insulin (panels 2, 4, and 6) in human islets cultured on extracellular matrix–coated dishes and exposed for 5 days to media containing 5.5mmol/l glucose (panels 1 and 2), 11.1 mmol/l glucose (panels 3 and 4), or 33.3 mmol/l glucose (panels 5 and 6). Original magnification 3400, withhigher magnification of b-cells stained for the Fas receptor (panel 5a) and insulin (panel 6a). Immunostaining for the Fas receptor (panels 7 and8) in untransfected human malignant glioma cells (negative control) (panel 7) and in cell transfected with human Fas cDNA (positive control)(panel 8). B: Immunoblotting of Fas receptor, FasL, procaspase-3, and activated caspase-3. Human islets cultured in suspension at 5.5, 11.1, or33.3 mmol/l glucose were analyzed after a 36-h incubation. Human fibroblast derived from foreskin was used as a positive control for Fas. Theantibodies were blotted on the same membrane after stripping. C: Caspase-8 activation. D: Subcellular localization of cytochrome c in humanislets cultured at 5.5 or 33.3 mmol/l glucose for 36 h or in the presence of 0.5 mmol/l Palmitic acid (positive control). Immunoblotting ofcytochrome c was performed on mitochondrial and cytosolic fractions. Each experiment was repeated three times.

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upregulated Fas, we used the antagonistic anti-Fas anti-body ZB4. Because interleukin-1b–induced b-cell apopto-sis has recently been shown to involve an association

between Fas and its ligand (17,31), ZB4 was also tested forits effect on interleukin-1b–mediated injury. In humanislets, interleukin-1b increased b-cell apoptosis and de-

FIG. 3. Constitutive expression of FasL in human isletsand upregulation of the Fas receptor in islets of hypergly-cemic type 2 diabetic patients. Double-immunostaining forFasL (orange; panels 1, 3, and 5) and insulin (green;panels 2, 4, and 6) in tissue sections of pancreas from apatient with type 2 diabetes—without (panels 1 and 2) andwith a preabsorbtion by FasL peptide (negative control;panels 5 and 6)—and from a nondiabetic patient (panels 3and 4). mRNA in situ hybridization for FasL (red; panels 7,9, and 11) double-immunostained for insulin (green; pan-els 8, 10, and 12) in a tissue section of a pancreas from apatient with type 2 diabetes—using anti-sense probe (pan-el 7) and sense probe (negative control; panel 11)—andfrom a nondiabetic patient using anti-sense probe (panel9). Double-immunostaining for the Fas receptor (orange;panels 13, 15, and 17) and insulin (green; panels 14, 16,and 18) in tissue sections of a pancreas from a patient withtype 2 diabetes—without (panels 13 and 14) and with apreabsorbtion by Fas peptide (negative control; panels 17and 18)—and from a nondiabetic patient (panels 15 and16). mRNA in situ hybridization for Fas (red; panels 19, 21,and 23) double-immunostaining for insulin (green; panels20, 22, and 24) in a tissue section of a pancreas of a patientwith type 2 diabetes—using anti-sense probe (panel 19)and sense probe (negative control; panel 23)—and of anondiabetic patient using anti-sense probe (panel 21).Original magnification 3400.

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creased b-cell proliferation to a similar extent as 33.3mmol/l glucose (Fig. 4), but no additive effect was ob-served. ZB4 inhibited the deleterious effect of both inter-leukin-1b and glucose. Using the same antagonistic anti-Fas antibody, Loweth et al. (17) did not observe inhibitionof interleukin-1b–induced apoptosis. As the authors pro-posed, this was probably caused by limited access of theZB4 antibody to cells in the center of the islets. In thepresent study, the islets were cultured on extracellularmatrix–coated dishes (23), allowing the cells to attach tothe dishes and to spread, thus permitting access of theantibody to all islet cells.

DISCUSSION

In the present study, we show that increased glucoseconcentration by itself induces apoptosis in human pan-creatic b-cells. The mechanism underlying glucose-in-duced b-cell death involves the upregulation of Fasreceptors, which can interact with the constitutively ex-pressed FasL on neighboring b-cells. Fas-FasL interactionleads to cleavage of procaspase-8 to caspase-8. Activatedcaspase-8, the most upstream caspase in the Fas apoptoticpathway, promotes caspase-3 activation and DNA frag-mentation (33). We demonstrate here that caspase-8 and -3

are indeed activated by high glucose in human islets; themitochondrial apoptotic pathway does not appear to beinvolved.

In contrast to that seen in human islets, an increase inglucose concentration to 11 mmol/l in rat islets promotesb-cell survival (8,9,29,34). When glucose concentrationswere further increased, glucose proved to be pro- oranti-apoptotic, depending on culture conditions. The dif-ference in glucose sensitivity between human and rat isletscan be explained by the mechanism of glucose-inducedb-cell apoptosis. Human islets constitutively express FasL(17) (Fig. 3), whereas islets from 2- to 3-month-old rats—the age at which rats are usually investigated—do notexpress FasL (35). Thus, whereas glucose-induced Fas inhuman b-cells interact with FasL expressed in adjacentb-cells, Fas ligation does not occur in young rats. Theseinterspecies differences raise the possibility that differ-ences in sensitivity to glucose may also exist amongindividuals of the same species. Indeed, although glucosewas capable of inducing b-cell apoptosis in each samplefrom the eight organ donors studied, large variations wereobserved in the response. It may be postulated that bothgenetic background and age may determine the suscepti-bility to glucose-induced b-cell apoptosis: type 2 diabetesoccurs more often in older people with an inheritedpredisposition.

Elevated glucose concentrations induced Fas expres-sion in almost all b-cells; however, apoptosis was ob-served in only a few cells. An interesting feature of theglucose-induced apoptosis was the appearance of frag-mented nuclei doublets, suggestive of postmitotic apopto-sis. Therefore, susceptibility to apoptosis via Fasactivation may be increased in proliferating cells. Becauseglucose also induces b-cell proliferation, a relationshipbetween induction of proliferation and apoptosis seemsplausible. In line with this suggestion, exposure of b-cellsto elevated glucose concentrations induced a short-lastingincrease in proliferation accompanied by long-lastingb-cell apoptosis. The same sequence is also observed invivo during the evolution of diabetes (1,8,36,37). Thus,reduced compensatory proliferative response coupledwith increased b-cell death in response to hyperglycemiamay well contribute to the progressive decline of b-cellmass in diabetic patients.

So far, induction of Fas receptors on b-cells was con-sidered to be limited to type 1 diabetes in response tocytokines during the process of autoimmune destruction.Here, we demonstrate that glucose may directly induce theFas receptor on human b-cells, leading to apoptosis due tointeraction with the constitutively expressed FasL of sur-rounding b-cells. Moreover, we observed the expression ofFas in islets of type 2 diabetic patients. Therefore, a similarmechanism for b-cell destruction probably exists in bothtype 1 and 2 diabetes. However, in type 1 diabetes, isletcell destruction may not result solely from activation ofthe Fas pathway but also from the action of cytokines andcytolytic perforin/granzyme released from cytotoxic T-cells. Nevertheless, our results underscore the importanceof tight glucose control in limiting b-cell destruction in alldiabetic patients as well as in patients undergoing islettransplantation.

FIG. 4. Effect of blockade of the Fas receptor on glucose- and inter-leukin-1b–induced b-cell DNA fragmentation and proliferative activity.Human islets were cultured on extracellular matrix-coated dishes for 5days in 5.5 or 33.3 mmol/l glucose alone (control) or in the presence ofinterleukin-1b (IL-1), antagonistic Fas antibody (ZB4), or both. Re-sults are means 6 SE of the relative number of TUNEL-positive (panel1) and Ki-67–positive b-cells (panel 2) per islet normalized to controlincubations at 5.5 mmol/l glucose alone (100%; in absolute values: 0.18TUNEL-positive b-cells per islet and 1.16 Ki-67–positive b-cells perislet). The mean number of islets scored was 33 for each treatmentcondition. *P < 0.001 relative to islets at 5.5 mmol/l glucose; **P < 0.05relative to control at the same glucose concentration; 1P < 0.05relative to the interleukin-1b–treated cells.

K. MAEDLER AND ASSOCIATES

DIABETES, VOL. 50, AUGUST 2001 1689

ACKNOWLEDGMENTS

This work was supported by grants from the Swiss Na-tional Science Foundation (3200-057595.99 to M.Y.D.) andfrom the Juvenile Diabetes Foundation International (I-1998-9 to N.K.). M.Y.D is supported by the Max CloettaFoundation.

We thank Erol Cerasi for comments and critical readingof the manuscript, Kristian Lobner for helpful suggestions,Phillipp U. Heitz, Paul Komminoth and Parvin Saremaslanifor providing the sections of human pancreas, and GarethaSiegfried-Kellenberger and Heidi Seiler for technical assis-tance.

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