Proc. Natl. Acad. Sci. USA Vol. 95, pp. 2498–2502, March 1998 Medical Science

Fatty acid-induced ␤ cell apoptosis: A link between obesity and diabetes

MICHIO SHIMABUKURO*†,YAN-TING ZHOU*†,MOSHE LEVI†, AND ROGER H. UNGER*†‡

*Gifford Laboratories for Diabetes Research, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, TX 75235; and †Veterans Administration Medical Center, Dallas, TX 75216

Contributed by Roger H. Unger, January 12, 1998

ABSTRACT Like obese humans, Zucker diabetic fatty vivo in the prediabetic phase of the disease causes the com- (ZDF) rats exhibit early ␤ cell compensation for insulin pensatory ␤ cell hyperplasia and hyperinsulinemia; a further resistance (4-fold ␤ cell hyperplasia) followed by decompen- increase in islet to ϳ50 times normal reverses the foregoing sation (>50% loss of ␤ cells). In prediabetic and diabetic ZDF compensatory changes and causes ␤ cell dysfunction, a reduc- islets, apoptosis measured by DNA laddering is increased 3- tion in the number of ␤ cells, and diabetes (7, 8). In other and >7-fold, respectively, compared with lean ZDF controls. words, the surfeit of fat in islets is associated with a dose- Ceramide, a -containing messenger in cytokine- related biphasic effect, initially enhancing insulin output by induced apoptosis, was significantly increased (P < 0.01) in stimulating hyperplasia (7, 8) but subsequently reversing these prediabetic and diabetic islets. Free fatty acids (FFAs) in compensatory changes when the fat content rises to extremely plasma are high (>1 mM) in prediabetic and diabetic ZDF high levels (7, 8). rats; therefore, we cultured prediabetic islets in 1 mM FFA. We have reported that the ␤ cell decompensation in this DNA laddering rose to 19.6% vs. 4.6% in lean control islets, form of diabetes may involve exaggerated induction by FFA of preceded by an 82% increase in ceramide. C2-Ceramide with- inducible nitric oxide synthase (iNOS) and excess nitric oxide out FFA induced DNA laddering, but fumonisin B1, a ceramide (NO) generation (9). Because intracellular NO is an important synthetase inhibitor, completely blocked FFA-induced DNA mediator of programmed cell death (10–12), it seemed pos- laddering in cultured ZDF islets. [3H]Palmitate incorporation sible that the loss of the ␤ cells observed late in the course of in [3H]ceramide in ZDF islets was twice that of controls, but adipogenic NIDDM (13) might be the result of NO-induced [3H]palmitate oxidation was 77% less. Triacsin C, an inhibitor apoptosis. Indeed, apoptosis has been reported in fat-laden of fatty acyl-CoA synthetase, and troglitazone, an enhancer of hepatocytes (14). Moreover, ceramide, a key component of the FFA oxidation in ZDF islets, both blocked DNA laddering. signal transduction pathway for apoptosis (15, 16), contains These agents also reduced inducible nitric oxide (NO) syn- long-chain fatty acids. Further, the susceptibility of ␤ cells to thase mRNA and NO production, which are involved in apoptotic stimuli such as interleukin 1␤ is tightly correlated to FFA-induced apoptosis. In ZDF obesity, ␤ cell apoptosis is islet fat content; measures that deplete islet fat, such as induced by increased FFA via de novo ceramide formation and hyperleptinemia, provide striking protection against interleu- increased NO production. kin 1␤ cytotoxicity (17, 18), perhaps by depleting the fatty acid source for ceramide synthesis. We therefore suspected that the ␤ ͞ The mechanism by which obesity, now the most common cell lipotoxicity in obesity might involve ceramide- and or American disease (1), leads to non-insulin-dependent diabetes NO-mediated apoptosis. This study was designed to test this mellitus (NIDDM), probably the second most common Amer- hypothesis. ican disease, is unknown. It is generally agreed that insulin resistance is an invariable accompaniment of obesity but that MATERIALS AND METHODS normoglycemia is maintained by compensatory hyperinsulin- ϩ͞ϩ emia until the pancreatic ␤ cells become unable to meet the Animals. Lean wild-type ( ) male ZDF rats and obese ͞ increased demand for insulin, at which point NIDDM begins. homozygous (fa fa) male ZDF rats were bred in our labora- ͞ The mechanism by which ␤ cells become unable to meet rising tory from [ZDF Drt-fa(F10)] rats purchased from R. Peterson insulin demand has never been elucidated, primarily because (University of Indiana School of Medicine, Indianapolis). of the unavailability of human pancreatic islets for appropriate Islet Isolation and Culture. Pancreatic islets were isolated by study. However, post-mortem studies in patients with NIDDM the method of Naber et al. (19) with modifications (7). Isolated indicate that the ␤ cell mass is reduced (2). islets were cultured as described (7, 9). In some experiments, Zucker Diabetic Fatty (ZDF) rats have provided a useful islets were cultured with or without 1 mM long-chain FFAs ͞ replica of the human phenotype of adipogenic NIDDM in (2:1 oleate palmitate; Sigma) in the absence and presence of ␮ ␮ which to study the islets in the obese prediabetic (7 weeks of 15 M fumonisin B1,15 MC2-ceramide, 0.5 mM aminogua- ␮ age) and obese diabetic (14 weeks of age) stages of the disease nidine (Sigma), 10 M triacsin C (Biomol, Plymouth Meeting, ␮ (3). Such studies implicate fat deposition in islets as the cause PA), and 10 M troglitazone (Sankyo). of the ␤ cell decompensation, so-called ‘‘lipotoxicity’’ (4, 5). DNA Fragmentation Assay. DNA fragmentation was as- Excess fat in ␤ cells and other nonadipocytes in this form of sayed by a modification of the method of Duke and Sellins (20). obesity is ascribed to the high plasma levels of free fatty acids Groups of freshly isolated or cultured islets were washed twice with ice-cold PBS and suspended in 100 ␮l of lysis buffer (10 (FFAs) (4, 5), coupled with a greatly enhanced capacity for ⅐ ͞ ͞ lipogenesis (6). There is compelling in vitro evidence that the mM Tris HCl 10 mM EDTA 0.5% Triton X-100, pH 8.0), modest 5- to 10-fold increase in islet fat content that occurs in vortex-mixed, sonicated, and incubated on ice for 20 min. After

The publication costs of this article were defrayed in part by page charge Abbreviations: ZDF, Zucker diabetic fatty; FFA, free fatty acid; NIDDM, non-insulin-dependent diabetes mellitus; iNOS, inducible payment. This article must therefore be hereby marked ‘‘advertisement’’ in nitric oxide synthase. accordance with 18 U.S.C. §1734 solely to indicate this fact. ‡To whom reprint requests should be addressed at: Center for © 1998 by The National Academy of Sciences 0027-8424͞98͞952498-5$2.00͞0 Diabetes Research, University of Texas Southwestern Medical Cen- PNAS is available online at http:͞͞www.pnas.org. ter, 5323 Harry Hines Boulevard, Dallas, TX 75235-8854.

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centrifugation for 20 min at 4°C (14,000 ϫ g), the supernatant using a first-strand cDNA synthesis kit (CLONTHECH). containing fragmented (soluble) DNA was transferred to iNOS and ␤-actin genes were amplified by PCR as described another tube. Lysis buffer (100 ␮l) was added to the pellet (9). The products were electrophoresed on a 1.2% agarose gel. containing insoluble DNA. Both samples were treated with After transferring to Hybond-N Nylon membrane (Amer- RNase A (0.5 mg͞ml) for 1 hr at 37°C and then with proteinase sham), DNA samples were hybridized with [32P]ATP-labeled K (Sigma, 0.4 mg͞ml) for 1 hr at 37°C. After adding 20 ␮lof specific probes and analyzed in the Molecular Imager (Bio- 5 M NaCl and 120 ␮l of isopropanol, the samples were Rad). incubated overnight at Ϫ20°C, and the DNA concentrations Nitrite Determination. NO formation was determined as were measured by the method of Hopcroft et al. (21). Frag- nitrite by a modification of the method of Green et al. (25) with mented DNA was calculated as 100% ϫ soluble DNA͞(soluble modifications (9). ϩ insoluble DNA). The soluble fraction of DNA was deter- mined by electrophoresis on 1.5% agarose gel and has a RESULTS ladder-like appearance. Ceramide Determination. Ceramide concentrations were Measurements of DNA Laddering and Ceramide in Predi- measured in freshly isolated or cultured islets by a modification abetic and Diabetic ZDF Islets. Our initial goal was to of the diacylglycerol kinase assay (22, 23). Islets were washed determine whether apoptosis was, in fact, the cause of the twice with ice-cold PBS and were extracted by the previously observed loss of ␤ cells that coincided with the onset method of Bligh and Dyer (24). The dried was solubilized of diabetes in ZDF rats. We therefore isolated islets from in 20 ␮l of detergent solution [7.5% octyl ␤-D-glucopyrano- obese ZDF rats in the early prediabetic stage (5 weeks of age), side͞5 mM cardiolipin in 1 mM diethylenetriamine pentaacetic in the late prediabetic stage (7 weeks), and about 4 weeks after acid (DETAPAC)]. After adding 50 ␮l of assay buffer (100 the onset of diabetes (14 weeks) and determined whether DNA mM imidazole hydrochloride, pH 6.6͞100 mM NaCl͞25 mM fragmentation, an index of apoptosis, was present. As shown in ͞ ␮ ␮ MgCl2 2 mM EGTA), 20 lof10mMDTT,and10 lof1:1 Fig. 1A, there was a more than 7-fold increase in DNA ladder diluted diacylglycerol kinase (Calbiochem), the reaction was formation in freshly isolated islets from 5-, 7-, and 14-week-old started by adding 10 ␮lof10mM[␥-32P]ATP (specific activity, ZDF rats, whereas none was detected in lean wild-type con- 30,000–40,000 cpm͞nmol) prepared in 100 mM imidazole͞1 trols (Fig. 1A). Moreover, in islets of wild-type rats apoptosis mM DETAPAC, pH 6.6. After mixing, the reaction was did not increase with age, whereas it increased almost 3-fold in continued for 45 min at 27°C. Lipids were extracted again and those of the obese rats. Thus, in ZDF islets, there was evidence the lower chloroform phase was washed twice with 2 ml of 1% of a high level of apoptosis in islet cells prior to the onset of perchloric acid (PCA), and dried under N2. The sample was diabetes and a further increase thereafter. resuspended in 80 ␮l of chloroform, spotted onto a Silica Gel To determine whether ceramide, a fatty acid-containing 60 (Merck) HPTLC plate, and developed with chloroform͞ messenger for certain cytokines in the apoptosis pathway, acetone͞methanol͞acetic acid͞water (10:4:3:2:1). The radio- might also be involved in the apoptosis of ZDF rats, it was active spot corresponding to ceramide-1-phosphate was quan- measured in freshly isolated islets from 7-week-old obese, tified by using the Molecular Imager (Bio-Rad). prediabetic, and 14-week-old obese diabetic fa͞fa ZDF rats De Novo Synthesis of [3H]Ceramide and Oxidation from and in age-matched lean wild-type controls. As shown in Fig. [3H]Palmitate. Freshly isolated islets were labeled for 0.5–4 hr 1B, the levels of ceramide were slightly but significantly higher with [3H]palmitate (Amersham) in the absence and presence (P Ͻ 0.01) in fa͞fa rats than in ϩ͞ϩ controls at 7 weeks of age of the ceramide synthase inhibitor fumonisin B1 (Sigma). and they increased further at 14 weeks. Lipids were extracted as described above. Lipid extracts dis- Effect of FFA on Ceramide and DNA Laddering. To deter- solved in 80 ␮l of chloroform were spotted onto high- mine whether an increase in ceramide and DNA laddering are performance thin-layer chromatography plate and developed induced by FFA, we measured, at various times, the effect of ͞ ͞ ͞ with CHCl3 CH2OH 7MNH4OH H2O (85:15:0.5:0.5). The FFA on ceramide levels on and DNA fragmentation in cul- radioactivity in the spot corresponding to [3H]palmitate was tured islets isolated from prediabetic fa͞fa ZDF rats and counted as described (5, 6). [3H]Palmitate oxidation were wild-type controls. In islets from the former rats cultured in 1 3 determined as [ H]H2O production in medium as described (6, mM FFA, DNA laddering rose from 6% at 0 time to 19% at 7). 24 hr, whereas in wild-type controls, it rose from 0.7% to only Semiquantitation of iNOS mRNA by Reverse Transcrip- 4.6% at 24 hr (Fig. 2A). The peak 82% increase in ceramide tion-Coupled PCR. iNOS mRNA expression was analyzed in in fa͞fa ZDF rats took place within 1 hr (Fig. 2B), before the cultured islets by using reverse transcription-coupled PCR as increase in DNA fragmentation had occurred (data not described in detail (9). Briefly, total RNA was extracted by shown). using a TRIzol isolation kit (Life Technologies) and treated Source of FFA-Induced Increase in Ceramide and Relation with RNase-free DNase. First-strand cDNA was obtained by to FFA Oxidation. Ceramide can be generated either by

FIG.1. (A) DNA fragmentation in freshly isolated islets from 5 (preobese)-, 7 (obese and prediabetic)-, and 14 (diabetic)-week-old fa͞fa ZDF rats and lean ϩ͞ϩ controls. Ladder (%), fragmented DNA percent; lane 1, 100-bp DNA size marker (Boeringer Mannheim). (B) Ceramide content in pancreatic islets of obese fa͞fa ZDF (solid bars) and lean ϩ͞ϩ controls (open bars). Values are the mean Ϯ SEM of three or four experiments. .P Ͻ 0.05 vs. ϩ͞ϩ;†,P Ͻ 0.05 vs. 7 week ,ء Downloaded by guest on October 3, 2021 2500 Medical Science: Shimabukuro et al. Proc. Natl. Acad. Sci. USA 95 (1998)

FIG.2. (A) Effect of FFA on DNA fragmentation in pancreatic islets from lean wild-type (ϩ͞ϩ) and obese prediabetic homozygous (fa͞fa) ZDF rats. Pancreatic islets were isolated from 7-week-old rats and cultured with 1 mM FFA as indicated. Ladder %, fragmented DNA percent. (B) Effect of FFA on ceramide content in pancreatic islets of ZDF rats. Islets from lean wild-type (F) and obese homozygous (ᮀ) ZDF rats were cultured with 0 or 1 mM FFA at the indicated times, and ceramide contents were determined. (C) De novo synthesis of [3H]ceramide from 3 [ H]palmitate in islets of wild type (ϩ͞ϩ) and fa͞fa ZDF rats in the absence and presence of the ceramide synthase inhibitor fumonisin B1 (FB1). 3 3 Rats were 7 weeks of age. Values are the mean Ϯ SEM of triplicate experiments. (D) Comparison of [ H]ceramide and [ H]H2O to assess relative rates of de novo ceramide synthesis vs. oxidation of [3H]palmitate by isolated islets of ϩ͞ϩ and fa͞fa rats. Values are the mean Ϯ SEM of triplicate .P Ͻ 0.05 vs. ϩ͞ϩ ,ء .experiments

degradation of sphingomyelin or by ceramide synthase- production in vitro and effectively prevents ␤ cell loss and catalyzed condensation of fatty acyl-CoA and sphinganine diabetes when administered to ZDF rats (9). In ZDF islets (26). To determine whether the increase in ceramide reflected cultured in 1 mM FFA plus aminoguanidine, iNOS expression de novo synthesis from FFA and to provide support for the and NO production were decreased and DNA fragmentation diacylglyceride kinase assay for ceramide (27), we incubated was lowered substantially (Fig. 4). islets in 1 mM [3H]palmitate and measured [3H]ceramide. In Effects of Fatty Acyl-CoA Blockade on DNA Fragmentation 3 addition we measured [ H]H2O as an index of FFA oxidation. and NO. For evidence that excess fatty acyl-CoA causes DNA 3 In wild-type islets, [ H]ceramide increased 3-fold within 2 hr. fragmentation via increased NO production, we blocked fatty ͞ 3 In islets from fa fa rats, [ H]ceramide was at least twice that acyl-CoA synthetase by adding 10 ␮M triacsin C to culture of wild-type rats at all time points (Fig. 2C), whereas medium containing 1 mM FFA (Fig. 4). Triacsin C has been 3 ͞ [ H]palmitate oxidation in islets of fa fa rats was only 23% of shown to inhibit palmitate esterification in INS-1 cells and that of wild-type rats (Fig. 2D). Because palmitoyl CoA is islets (ref. 30 and P. A. Antinozzi, M. Prentki, L. Segall, J. D. rate-limiting for ceramide synthesis in other systems (28), the McGarry, and C. B. Newgard, personal communication). Both reduction in FFA oxidation may have contributed increased NO production and iNOS mRNA were reduced and DNA substrate for ceramide synthesis. However, the increase in fragmentation was concomitantly lowered in the triacsin C- [3H]ceramide was greater than could be explained by even a treated cells. complete block in oxidation (Fig. 2D), suggesting an intrinsic increase in ceramide synthase activity. Effect of C2-Ceramide and Ceramide Synthase Inhibition on FFA-Induced Apoptosis. If ceramide is, in fact, the cause of FFA-induced apoptosis in ␤ cells, exogenous ceramide should induce it, and inhibition of ceramide synthesis should prevent it. In islets from prediabetic obese fa͞fa ZDF rats cultured in ␮ 15 MC2-ceramide without FFA, DNA fragmentation rose 2-fold (Fig. 3). By contrast, in culture medium containing 1 ␮ mM FFA, the presence of 50 M fumonisin B1 (the ceramide synthase inhibitor) almost completely prevented the FFA- induced increase in apoptosis (Fig. 3). These results point strongly to ceramide as the mediator of FFA-induced apopto- sis in these islets. Role of NO in FFA-Induced Apoptosis. We have observed an exaggeration of the normal FFA-induced increase of iNOS mRNA and NO production in fat-laden islets of fa͞fa rats (9). FIG. 3. Effect of exogenous ceramide and of blockade of ceramide Because inhibitors of iNOS prevent the diabetic phenotype in ͞ islets of fa͞fa rats, we have hypothesized that FFA-induced ␤ synthesis on DNA fragmentation in islets from obese fa fa ZDF rats. Islets isolated from 7-week-old obese prediabetic fa͞fa ZDF rats were cell dysfunction and damage is mediated by NO (9). If so, any cultured for 24 hr in medium containing 15 ␮MC2-ceramide (C2-Cer) maneuver that inhibits iNOS expression in islets should block without FFA or 1 mM FFA plus 50 ␮M fumonisin B1 (FB1), an DNA fragmentation and prevent the diabetes. Aminoguani- inhibitor of ceramide synthetase. Lane 1, 100-bp DNA size marker dine is an iNOS inhibitor (29) that profoundly reduces NO (Boeringer Mannheim); Ladder (%), fragmented DNA percent. Downloaded by guest on October 3, 2021 Medical Science: Shimabukuro et al. Proc. Natl. Acad. Sci. USA 95 (1998) 2501

FIG.4. (A) Inhibitory effect of triacsin C, troglitazone, and aminoguanidine on FFA-induced DNA fragmentation in islets from obese fa͞fa ZDF rats. Islets isolated from 7-week-old fa͞fa ZDF rats were cultured for 24 hr at 0 or 1 mM FFA with 10 ␮M triacsin C, 10 ␮M troglitazone, or 0.5 mM aminoguanidine. M, 100-bp DNA size marker. (B) Inhibitory effect of triacsin C, troglitazone, and aminoguanidine on FFA-induced iNOS mRNA induction. (C) FFA-induced NO production in islets of obese fa͞fa ZDF rats. Effect of triacsin C, troglitazone, and aminoguanidine on islets cultured as described in A except for 48 hr.

Effect of Reducing Islet Fat Content on DNA Fragmenta- of obese ZDF rats far more FFA than can be oxidized. Because tion. Finally, to determine whether a reduction of islet trig- fa͞fa islets exhibit a markedly increased lipogenic capacity and lycerides would rescue ␤ cells from apoptosis, we cultured islets a decreased oxidative capacity (6), unused FFA in islets are in 10 ␮M troglitazone, which has recently been shown to esterified and over time an excessive quantity of fat is depos- reduce the fat content of ZDF islets by increasing fatty acid ited (6). This is associated with an increase in ceramide, iNOS oxidation (17, 18). Troglitazone in the culture medium effec- expression, and NO production, which cause apoptosis. tively blocked the FFA-induced increase in iNOS mRNA, NO Because islets of obese humans are not available for assess- production, and apoptosis (Fig. 4). ment of their fat content, one cannot assume that this mech- anism necessarily explains obesity-associated diabetes in hu- DISCUSSION mans. Despite the similarities of the rodent and human syndromes, it is unlikely that fat can accumulate as rapidly in The foregoing results indicate that apoptosis is increased in islets of obese humans as it does in ZDF rats. Perhaps slower ͞ islets of fa fa ZDF rats that are progressing through the and more variable accumulation of ectopic fat stores in obese prediabetic and diabetic stages of their disease. They suggest humans accounts for the later onset and more variable inci- ␤ ͞ that the apoptotic effect of FFA on cells of fa fa ZDF rats dence of their diabetes. Even though the lipotoxic etiology of is somehow related to the high fat content of their islets and human adipogenic diabetes cannot be directly proven, the fact that this greatly exaggerates the apoptotic effect of fatty that troglitazone, an agent that reduces islet fat in ZDF rats acyl-CoA. In addition, they strongly implicate ceramide as a (17, 18) and prevents their diabetes (31–33), is equally effica- mediator of the FFA-induced apoptosis. FFA, a precursor of cious in the human form of the disease is consistent with a ceramide, raised ceramide levels in fat-laden ZDF islets via a common etiology. It is therefore possible that prophylactic greater rate of de novo biosynthesis, as indicated by increased interventions that reduce fat accumulation and NO production incorporation of [3H]palmitate into [3H]ceramide in the fa͞fa ͞ in islets will prevent the anticipated epidemic of obesity- islets. The reduction in fatty acid oxidation in islets of fa fa associated NIDDM in the U.S. ZDF rats may have contributed to their high ceramide levels by providing increased substrate for ceramide formation; We thank Drs. Daniel Foster and Christopher Newgard for critical indeed, it has been shown in another cell type that etomoxir, ␤ review of the paper, Xiaodong Wang and Michael Brown for impor- an inhibitor of mitochondrial oxidation, increases ceramide tant suggestions, Dr. Hua-Min Wang for technical help, and Ms. Tess synthesis (28). However, because even a complete blockade of Perico for secretarial support. We are particularly grateful to Drs. oxidation could not account for the striking increase in de novo Ruedi Aebersold and Julian Watts for their technical advice. This work ceramide formation, an underlying increase in ceramide syn- was supported by the National Institutes of Health (DK02700-37), thase activity seems likely. National Institutes of Health͞Juvenile Diabetes Foundation Interdis- The role of ceramide in the apoptosis was supported by the ciplinary Research Program, and Department of Veterans Affairs Institutional Support (SMI 821-109). demonstration that C2-ceramide caused DNA fragmentation in the absence of FFA while blockade of ceramide synthesis by 1. National Center for Health Statistics. (1994) Newsweek 127, 62. fumonisin B1 prevented FFA-induced DNA fragmentation in the presence of FFA. It is not clear, however, whether cer- 2. Rahier, J., Goebbles, R. M. & Henquin, J. C. (1983) Diabetologia 24, 366–371. amide mediates the FFA effect on NO or whether the FFA- 3. Peterson, R. G., Shaw, W. N., Neel, M., Little, L. A. & Eichberg, induced increase in iNOS represents a separate ceramide- J. (1990) ILAR News 32, 16–19. independent avenue for apoptosis. 4. Lee, Y., Hirose, H., Ohneda, M., Johnson, J. H., McGarry, J. D. In summary, we have demonstrated in islets of obese ZDF & Unger, R. H. (1994) Proc. Natl. Acad. Sci. USA 91, 10878– rats a pathway of lipotoxicity leading to diabetes. Elevated 10882. levels of circulating FFA (4) and lipoproteins transport to islets 5. Unger, R. H., (1995) Diabetes 44, 863–870. Downloaded by guest on October 3, 2021 2502 Medical Science: Shimabukuro et al. Proc. Natl. Acad. Sci. USA 95 (1998)

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