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Fatty Acid-Induced Я Cell Apoptosis

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Fatty Acid-Induced Я Cell Apoptosis Proc. Natl. Acad. Sci. USA Vol. 95, pp. 2498–2502, March 1998 Medical Science Fatty acid-induced b 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 b cell compensation for insulin pensatory b cell hyperplasia and hyperinsulinemia; a further resistance (4-fold b cell hyperplasia) followed by decompen- increase in islet fat to ;50 times normal reverses the foregoing sation (>50% loss of b cells). In prediabetic and diabetic ZDF compensatory changes and causes b cell dysfunction, a reduc- islets, apoptosis measured by DNA laddering is increased 3- tion in the number of b 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 fatty acid-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 b 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 b 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 b cells to thase mRNA and NO production, which are involved in apoptotic stimuli such as interleukin 1b is tightly correlated to FFA-induced apoptosis. In ZDF obesity, b 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 1b cytotoxicity (17, 18), perhaps by depleting the fatty acid source for ceramide synthesis. We therefore suspected that the b y 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- 1y1 emia until the pancreatic b cells become unable to meet the Animals. Lean wild-type ( ) male ZDF rats and obese y increased demand for insulin, at which point NIDDM begins. homozygous (fa fa) male ZDF rats were bred in our labora- y The mechanism by which b 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 b 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 y replica of the human phenotype of adipogenic NIDDM in (2:1 oleate palmitate; Sigma) in the absence and presence of m m which to study the islets in the obese prediabetic (7 weeks of 15 M fumonisin B1,15 MC2-ceramide, 0.5 mM aminogua- m age) and obese diabetic (14 weeks of age) stages of the disease nidine (Sigma), 10 M triacsin C (Biomol, Plymouth Meeting, m (3). Such studies implicate fat deposition in islets as the cause PA), and 10 M troglitazone (Sankyo). of the b cell decompensation, so-called ‘‘lipotoxicity’’ (4, 5). DNA Fragmentation Assay. DNA fragmentation was as- Excess fat in b 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 ml of lysis buffer (10 (FFAs) (4, 5), coupled with a greatly enhanced capacity for z y y 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 0027-8424y98y952498-5$2.00y0 Diabetes Research, University of Texas Southwestern Medical Cen- PNAS is available online at http:yywww.pnas.org. ter, 5323 Harry Hines Boulevard, Dallas, TX 75235-8854. 2498 Downloaded by guest on October 3, 2021 Medical Science: Shimabukuro et al. Proc. Natl. Acad. Sci. USA 95 (1998) 2499 centrifugation for 20 min at 4°C (14,000 3 g), the supernatant using a first-strand cDNA synthesis kit (CLONTHECH). containing fragmented (soluble) DNA was transferred to iNOS and b-actin genes were amplified by PCR as described another tube. Lysis buffer (100 ml) 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 mgyml) for 1 hr at 37°C and then with proteinase sham), DNA samples were hybridized with [32P]ATP-labeled K (Sigma, 0.4 mgyml) for 1 hr at 37°C. After adding 20 mlof specific probes and analyzed in the Molecular Imager (Bio- 5 M NaCl and 120 ml of isopropanol, the samples were Rad). incubated overnight at 220°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% 3 soluble DNAy(soluble modifications (9). 1 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 lipids were extracted by the previously observed loss of b cells that coincided with the onset method of Bligh and Dyer (24). The dried lipid was solubilized of diabetes in ZDF rats. We therefore isolated islets from in 20 ml of detergent solution [7.5% octyl b-D-glucopyrano- obese ZDF rats in the early prediabetic stage (5 weeks of age), sidey5 mM cardiolipin in 1 mM diethylenetriamine pentaacetic in the late prediabetic stage (7 weeks), and about 4 weeks after acid (DETAPAC)]. After adding 50 ml of assay buffer (100 the onset of diabetes (14 weeks) and determined whether DNA mM imidazole hydrochloride, pH 6.6y100 mM NaCly25 mM fragmentation, an index of apoptosis, was present. As shown in y m m 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 mlof10mM[g-32P]ATP (specific activity, ZDF rats, whereas none was detected in lean wild-type con- 30,000–40,000 cpmynmol) prepared in 100 mM imidazoley1 trols (Fig. 1A).
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