Autophagy Regulates Pancreatic Beta Cell Death in Response to Pdx1 Deficiency and Nutrient Deprivation
2009; Elsevier BV; Volume: 284; Issue: 40 Linguagem: Inglês
10.1074/jbc.m109.041616
ISSN1083-351X
AutoresKei Fujimoto, Piia T. Hanson, Hung D. Tran, Eric L. Ford, Zhiqiang Han, James D. Johnson, Robert E. Schmidt, Karen G. Green, Burton M. Wice, Kenneth S. Polonsky,
Tópico(s)Cannabis and Cannabinoid Research
ResumoThere are three types of cell death; apoptosis, necrosis, and autophagy. The possibility that activation of the macroautophagy (autophagy) pathway may increase beta cell death is addressed in this study. Increased autophagy was present in pancreatic islets from Pdx1+/− mice with reduced insulin secretion and beta cell mass. Pdx1 expression was reduced in mouse insulinoma 6 (MIN6) cells by delivering small hairpin RNAs using a lentiviral vector. The MIN6 cells died after 7 days of Pdx1 deficiency, and autophagy was evident prior to the onset of cell death. Inhibition of autophagy prolonged cell survival and delayed cell death. Nutrient deprivation increased autophagy in MIN6 cells and mouse and human islets after starvation. Autophagy inhibition partly prevented amino acid starvation-induced MIN6 cell death. The in vivo effects of reduced autophagy were studied by crossing Pdx1+/− mice to Becn1+/− mice. After 1 week on a high fat diet, 4-week-old Pdx1+/− Becn1+/− mice showed normal glucose tolerance, preserved beta cell function, and increased beta cell mass compared with Pdx1+/− mice. This protective effect of reduced autophagy had worn off after 7 weeks on a high fat diet. Increased autophagy contributes to pancreatic beta cell death in Pdx1 deficiency and following nutrient deprivation. The role of autophagy should be considered in studies of pancreatic beta cell death and diabetes and as a target for novel therapeutic intervention. There are three types of cell death; apoptosis, necrosis, and autophagy. The possibility that activation of the macroautophagy (autophagy) pathway may increase beta cell death is addressed in this study. Increased autophagy was present in pancreatic islets from Pdx1+/− mice with reduced insulin secretion and beta cell mass. Pdx1 expression was reduced in mouse insulinoma 6 (MIN6) cells by delivering small hairpin RNAs using a lentiviral vector. The MIN6 cells died after 7 days of Pdx1 deficiency, and autophagy was evident prior to the onset of cell death. Inhibition of autophagy prolonged cell survival and delayed cell death. Nutrient deprivation increased autophagy in MIN6 cells and mouse and human islets after starvation. Autophagy inhibition partly prevented amino acid starvation-induced MIN6 cell death. The in vivo effects of reduced autophagy were studied by crossing Pdx1+/− mice to Becn1+/− mice. After 1 week on a high fat diet, 4-week-old Pdx1+/− Becn1+/− mice showed normal glucose tolerance, preserved beta cell function, and increased beta cell mass compared with Pdx1+/− mice. This protective effect of reduced autophagy had worn off after 7 weeks on a high fat diet. Increased autophagy contributes to pancreatic beta cell death in Pdx1 deficiency and following nutrient deprivation. The role of autophagy should be considered in studies of pancreatic beta cell death and diabetes and as a target for novel therapeutic intervention. Normal pancreatic beta cell function is essential for normal glucose tolerance, and abnormal beta cell function leads to glucose intolerance and diabetes. A progressive reduction in beta cell mass has been shown to occur in the evolution of diabetes (1Butler P.C. Meier J.J. Butler A.E. Bhushan A. Nat. Clin. Pract. Endocrinol. Metab. 2007; 3: 758-768Crossref PubMed Scopus (224) Google Scholar). Thus understanding the mechanisms responsible for the reduction in beta cell mass is important for understanding the pathogenesis of diabetes and in developing novel approaches to prevention and treatment. There are three types of cell death; apoptosis, necrosis, and autophagy (2Scarlatti F. Granata R. Meijer A.J. Codogno P. Cell Death Differ. 2009; 16: 12-20Crossref PubMed Scopus (221) Google Scholar). Previous studies have focused on apoptosis as the mechanism underlying beta cell death (1Butler P.C. Meier J.J. Butler A.E. Bhushan A. Nat. Clin. Pract. Endocrinol. Metab. 2007; 3: 758-768Crossref PubMed Scopus (224) Google Scholar, 3Johnson J.D. Ahmed N.T. Luciani D.S. Han Z. Tran H. Fujita J. Misler S. Edlund H. Polonsky K.S. J. Clin. Invest. 2003; 111: 1147-1160Crossref PubMed Scopus (303) Google Scholar, 4Johnson J.D. Bernal-Mizrachi E. Alejandro E.U. Han Z. Kalynyak T.B. Li H. Beith J.L. Gross J. Warnock G.L. Townsend R.R. Permutt M.A. Polonsky K.S. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 19575-19580Crossref PubMed Scopus (172) Google Scholar, 5Li Y. Cao X. Li L.X. Brubaker P.L. Edlund H. Drucker D.J. Diabetes. 2005; 54: 482-491Crossref PubMed Scopus (187) Google Scholar). The possibility that activation of the macroautophagy (hereafter referred to as autophagy) pathway may increase beta cell death has not been systematically studied. Autophagy is a regulated lysosomal pathway leading to the degradation and recycling of long-lived proteins and organelles. During autophagy, cytoplasmic constituents are sequestered into autophagosomes with double membranes and fused to lysosomes (autolysosomes), where degradation occurs. Under certain circumstances such as in response to nutrient deprivation, autophagy may function as a pro-survival pathway by mediating cellular turnover of proteins and organelles (6Klionsky D.J. Nat. Rev. Mol. Cell Biol. 2007; 8: 931-937Crossref PubMed Scopus (1583) Google Scholar, 7Levine B. Kroemer G. Cell. 2008; 132: 27-42Abstract Full Text Full Text PDF PubMed Scopus (5647) Google Scholar, 8Mortimore G.E. Pösö A.R. Annu. Rev. Nutr. 1987; 7: 539-564Crossref PubMed Google Scholar). On the other hand, an increase in autophagy can cause autophagic cell death distinct from apoptosis (9Shimizu S. Kanaseki T. Mizushima N. Mizuta T. Arakawa-Kobayashi S. Thompson C.B. Tsujimoto Y. Nat. Cell Biol. 2004; 6: 1221-1228Crossref PubMed Scopus (1196) Google Scholar, 10Yu L. Alva A. Su H. Dutt P. Freundt E. Welsh S. Baehrecke E.H. Lenardo M.J. Science. 2004; 304: 1500-1502Crossref PubMed Scopus (1108) Google Scholar). It has been suggested that autophagy plays a key role in the turnover of insulin secretory granules and of mitochondria within the beta cell, thereby regulating insulin secretion (11Marsh B.J. Soden C. Alarcón C. Wicksteed B.L. Yaekura K. Costin A.J. Morgan G.P. Rhodes C.J. Mol. Endocrinol. 2007; 21: 2255-2269Crossref PubMed Scopus (152) Google Scholar, 12Twig G. Elorza A. Molina A.J. Mohamed H. Wikstrom J.D. Walzer G. Stiles L. Haigh S.E. Katz S. Las G. Alroy J. Wu M. Py B.F. Yuan J. Deeney J.T. Corkey B.E. Shirihai O.S. EMBO J. 2008; 27: 433-446Crossref PubMed Scopus (2230) Google Scholar). Complete genetic ablation of Atg7 in beta cells resulted in degradation of islets and impaired glucose tolerance, suggesting that “basal autophagy” is important for maintenance of normal islet architecture and function (13Ebato C. Uchida T. Arakawa M. Komatsu M. Ueno T. Komiya K. Azuma K. Hirose T. Tanaka K. Kominami E. Kawamori R. Fujitani Y. Watada H. Cell Metab. 2008; 8: 325-332Abstract Full Text Full Text PDF PubMed Scopus (630) Google Scholar, 14Jung H.S. Chung K.W. Won Kim J. Kim J. Komatsu M. Tanaka K. Nguyen Y.H. Kang T.M. Yoon K.H. Kim J.W. Jeong Y.T. Han M.S. Lee M.K. Kim K.W. Shin J. Lee M.S. Cell Metab. 2008; 8: 318-324Abstract Full Text Full Text PDF PubMed Scopus (533) Google Scholar). The present study was designed to determine whether activation of autophagy can contribute to pancreatic beta cell death that occurs with reduced expression of Pdx1 (pancreas duodenal homeobox 1). We chose to study Pdx1 deficiency because this homeodomain-containing transcription factor is essential for normal pancreatic beta cell function and survival. Complete deficiency of Pdx1 is associated with pancreatic agenesis, and partial deficiency leads to reduced insulin secretion and beta cell mass (15Ahlgren U. Jonsson J. Jonsson L. Simu K. Edlund H. Genes Dev. 1998; 12: 1763-1768Crossref PubMed Scopus (786) Google Scholar, 16Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Crossref PubMed Scopus (1574) Google Scholar, 17McKinnon C.M. Docherty K. Diabetologia. 2001; 44: 1203-1214Crossref PubMed Scopus (220) Google Scholar, 18Stoffers D.A. Ferrer J. Clarke W.L. Habener J.F. Nat. Genet. 1997; 17: 138-139Crossref PubMed Scopus (8) Google Scholar, 19Wang H. Hagenfeldt-Johansson K. Otten L.A. Gauthier B.R. Herrera P.L. Wollheim C.B. Diabetes. 2002; 51: S333-S342Crossref PubMed Google Scholar). Complementary studies were performed to determine whether nutrient deprivation leads to a similar increase in autophagy in the beta cell as it does in other cells and tissues (8Mortimore G.E. Pösö A.R. Annu. Rev. Nutr. 1987; 7: 539-564Crossref PubMed Google Scholar). Mouse insulinoma MIN6 3The abbreviations used are: MIN6mouse insulinoma 6KDknockdownLC3microtubule-associated protein 1 light chain 33-MA3-methyladenineshRNAsmall hairpin RNAFBSfetal bovine serumHBSSHanks' balanced salt solutionDAPI4′,6-diamidino-2-phenylindolePIpropidium iodideBafA1bafilomycin A1CHOChinese hamster ovaryGFPgreen fluorescent proteinWTwild type. cells were cultured in Dulbecco's modified Eagle's medium supplemented with 15% FBS, antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin), 1 mm sodium pyruvate, 10 mm HEPES, and 50 μm β-mercaptoethanol. For amino acid deprivation, the aforementioned media were changed to 5% FBS without amino acids. For experiments involving both amino acid and serum starvation, MIN6 cells were incubated in HBSS medium supplemented with 10 mm HEPES. Mouse fibroblastoid NIH 3T3 and HEK 293T cells were cultured in Dulbecco's modified Eagle's medium supplemented with antibiotics plus 10% horse serum or 10% FBS, respectively. All of the cells were maintained at 37 °C in an atmosphere of 5% CO2/balance air and 100% humidity. mouse insulinoma 6 knockdown microtubule-associated protein 1 light chain 3 3-methyladenine small hairpin RNA fetal bovine serum Hanks' balanced salt solution 4′,6-diamidino-2-phenylindole propidium iodide bafilomycin A1 Chinese hamster ovary green fluorescent protein wild type. Attached MIN6 and NIH 3T3 cells and islets were lysed in cell lysis buffer containing 1% Triton X-100, 1 mm EDTA, 1 mm EGTA, 10 mm dithiothreitol, 1 mm Na3VO4, and complete protease inhibitor mixture. Equal amounts of protein were fractionated by SDS-PAGE, and blots were probed with antibodies against Pdx1 (sc-14664; Santa Cruz Biotechnology Inc., Santa Cruz, CA), Becn1 (3738; Cell Signaling Technologies, Beverly, MA), LC3 (NB100-2331; Novus Biologicals Inc., Littleton, CO), cleaved caspase-3 (9661; Cell Signaling Technologies), actin (A-2066; Sigma), and Atg5 (NB110-53818; Novus Biologicals). For the last hour of incubation, 10 μg/ml propidium iodide (PI) and 20 μg/ml DAPI were added directly to the media. After this incubation, the MIN6 cells were washed three times with PBS and fixed with 3.7% formaldehyde for 15 min at 4 °C. Each condition reported represents >600 cells counted by randomized field selection. The percentage of cell death was calculated as the number of PI-stained nuclei over the total number of nuclei stained by DAPI. Mouse islets were isolated by using collagenase and filtration as previously described (3Johnson J.D. Ahmed N.T. Luciani D.S. Han Z. Tran H. Fujita J. Misler S. Edlund H. Polonsky K.S. J. Clin. Invest. 2003; 111: 1147-1160Crossref PubMed Scopus (303) Google Scholar), and human islets were provided by the Human Islet Isolation Core at Washington University in St. Louis. The islets were cultured in RPMI 1640 medium with antibiotics, 10% FBS, pH 7.4, with NaOH at 37 °C and 5% CO2. RNA isolation, first strand cDNA synthesis, and TaqMan gene expression assays were performed as previously described (20Althage M.C. Ford E.L. Wang S. Tso P. Polonsky K.S. Wice B.M. J. Biol. Chem. 2008; 283: 18365-18376Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). The RNA level was normalized to the amount of Hmbs mRNA in the same sample. Applied Biosystems (Foster City, CA) TaqMan assay numbers were: Hmbs, Mm00660262_g1; and Pdx1, Mm00435565_m1. The pLKO.1-puro lentivirus vector was generously provided by Dr. Sheila Stewart of Washington University Medical School (St. Louis, MO). Potential shRNA targets in the murine Pdx1 mRNA (GenBankTM accession number NM_008814) were identified using the Dharmacon on-line siDESIGN tool. Following a BLAST search of the NCBI data base, one potential target was identified that lacks significant homology to non-Pdx1 gene products. Pdx1 target sequence 5′-CAGTGAGGAGCAGTACTAC-3′ or control sequence 5′-ACTACCGTTGTTATAGGTG-3′ was subcloned into the AgeI/EcoRI restriction site of pLKO-1-puro. shRNA targeting mouse Becn1 and Atg5 were obtained from Sigma. Recombinant lentiviral particles were prepared by transfecting HEK 293T cells with the appropriate pLKO.1-puro plasmid plus pHR′CVM8.2 delta R and pCMV-VSV-G plasmids. Lentivirus was added to the medium on days 1 and 2 followed by 2 μg/ml puromycin selections on days 4, 5, and 7. The media including autophagy inhibitor were changed on days 3, 4, 5, and 7 (supplemental Fig. S1). The presence of autophagosomes in MIN6 cells or islets was evaluated using immunostaining. MIN6 cells were fixed with 3.7% formaldehyde at room temperature for 60 min followed by immunostaining with anti-LC3 antibody (NB100-2331; Novus Biologicals). For immunostaining of islets, the mice were anesthetized and perfused through the left ventricle with 3.7% formaldehyde. The pancreas was isolated and then fixed at room temperature overnight. To quantitate the extent of autophagy, the number of LC3 punctae in each cell was counted in five independent visual fields from at least two independent experiments. Bafilomycin A1 (BafA1) is known as a strong inhibitor of the vacuolar type H+-ATPase and thereby inhibits the final step of lysosomal digestion in autophagy (21Yamamoto A. Tagawa Y. Yoshimori T. Moriyama Y. Masaki R. Tashiro Y. Cell Struct. Funct. 1998; 23: 33-42Crossref PubMed Scopus (1088) Google Scholar). To determine whether reduced Pdx1 expression in MIN6 cells increases autophagic flux, 1 nm BafA1 (Sigma) was added to the medium 4 h prior to the end of the treatment. MIN6 cells were infected with the shPdx1 or control vector, and islets were isolated from 3-week-old male Pdx1+/− or Pdx1+/+ mice followed by fixation with modified Karnovsky's fixative containing 3% glutaraldehyde and 1% paraformaldehyde in sodium cacodylate buffer, pH 7.4, overnight. The cells or islets were then rinsed in sodium cacodylate buffer followed by post-fixation in cacodylate-buffered 1% OsO4, for 1 h, dehydrated in graded ethanol with a final dehydration in propylene oxide, and embedded in EMbed-812 (Electron Microscopy Sciences, Hatfield, PA). Tissue blocks were sectioned at ninety nanometers thick, post-stained with uranyl acetate and Venable's lead citrate, and viewed with a JEOL model 1200EX electron microscope (JEOL, Tokyo, Japan). Digital images were acquired using the AMT Advantage HR (Advanced Microscopy Techniques, Danvers, MA) high definition CCD, 1.3 megapixel transmission electron microscopy camera. The Pdx1+/− mice have been previously described (16Jonsson J. Carlsson L. Edlund T. Edlund H. Nature. 1994; 371: 606-609Crossref PubMed Scopus (1574) Google Scholar) and were kindly provided by Dr. Helena Edlund (University of Umea, Umea, Sweden). Becn1+/− mice (22Qu X. Yu J. Bhagat G. Furuya N. Hibshoosh H. Troxel A. Rosen J. Eskelinen E.L. Mizushima N. Ohsumi Y. Cattoretti G. Levine B. J. Clin. Invest. 2003; 112: 1809-1820Crossref PubMed Scopus (1889) Google Scholar) were kindly provided by Dr. Beth Levine of University of Texas Southwestern Medical Center (Dallas, TX). For high fat diet, male mice were fed food containing 42% fat (Harlan Laboratories, Inc., Indianapolis, IN) from 3 weeks of age and provided with water ad libitum. Wild type littermates were used as controls. Intraperitoneal glucose tolerance tests were performed after a 4 h fast (2 g of dextrose/kg of body weight). Autophagic flux was evaluated by intraperitoneal administration of BafA1 (Sigma) at 0.3 mg/kg for 24 h. For morphometric analysis, the sections were immunostained with antibody against cleaved caspase-3 (9661; Cell Signaling Technologies) or Ki-67 (Zymed Laboratories Inc./Invitrogen). Pancreatic area and beta cell area were each estimated using the intensity thresholding function of the integrated morphometry package in MetaMorph as previously described (3Johnson J.D. Ahmed N.T. Luciani D.S. Han Z. Tran H. Fujita J. Misler S. Edlund H. Polonsky K.S. J. Clin. Invest. 2003; 111: 1147-1160Crossref PubMed Scopus (303) Google Scholar). All of the experiments in this study using animal protocols were approved by the Washington University Animal Studies Committee. Statistical analyses were performed by Student's unpaired t test. The differences were considered significant when p < 0.05. The results are presented as the means ± S.E. To determine whether reduced Pdx1 expression affects autophagy, we developed a lentivirus-based system to deliver an shRNA construct to MIN6 cells a mouse insulinoma cell line to knock down-expression of Pdx1. Infection of MIN6 cells with lentivirus encoding this construct significantly reduced Pdx1 mRNA levels to 52 ± 3% of control on day 5 (Fig. 1A). A decrease in Pdx1 protein levels was also observed on day 6 (Fig. 1B), and an increase in cell death was observed on day 7 as assessed by PI staining, which stains dying and dead cells (Fig. 1C, panels a and b). Infection of MIN6 cells with lentivirus containing a control construct did not cause cell death, and the Pdx1 KD construct did not cause death in mouse fibroblastoid NIH 3T3 cells that do not express Pdx1 (data not shown). Thus the increase in cell death after lentiviral infection is due to a decrease in Pdx1 expression and not nonspecific cellular effect(s) of viral infection. To determine whether autophagy is present in MIN6 cells with reduced Pdx1 expression, Western blots of microtubule-associated protein 1 LC3 were performed on Pdx1 KD MIN6 cells at two time points: 48 h before cell death became microscopically evident (on day 5) and on day 7 when cell death was evident. LC3-II levels were increased at both time points (Fig. 1D, upper panel). In addition, we treated Pdx1 KD MIN6 cells with BafA1, which inhibits the final step of lysosomal digestion in autophagy to determine whether reduced Pdx1 expression in MIN6 cells increases autophagic flux. Reduced Pdx1 expression in the presence of 1 nm BafA1 resulted in accumulation of LC3-II in comparison with the absence of BafA1 in Pdx1 KD MIN6 cells on day 7 (Fig. 1D, lower panel), indicating that autophagic flux is increased in Pdx1-reduced MIN6 cells. Immunostaining revealed a diffuse pattern of LC3 fluorescence in control cells, whereas LC3 developed the punctate appearance characteristic of autophagy (23Kabeya Y. Mizushima N. Ueno T. Yamamoto A. Kirisako T. Noda T. Kominami E. Ohsumi Y. Yoshimori T. EMBO J. 2000; 19: 5720-5728Crossref PubMed Scopus (5510) Google Scholar) in the Pdx1 KD MIN6 cells on day 5. This effect was blocked by the autophagy inhibitor 1 mm 3-methyladenine (3-MA) (Fig. 2A). Quantitative morphometric analysis revealed a significant increase in LC3 punctae in the Pdx1 KD MIN6 cells compared with control MIN6 cells. Exposure of the cells to 1 mm 3-MA blocked this effect and reduced the number of LC3 punctae to levels seen in cells exposed to lentivirus containing a control vector (Fig. 2B). Thus 1 mm 3-MA prevents activation of autophagy but does not inhibit basal autophagy. Transmission electron microscopy provided additional morphological evidence for autophagy in the Pdx1 KD MIN6 cells. Autophagosomes and autolysosomes were evident 5 days after Pdx1 KD but not in MIN6 cells infected with lentivirus containing a control vector (Fig. 2C). All of these findings indicate that autophagy is increased in Pdx1-reduced MIN6 cells. MIN6 cells with reduced Pdx1 expression were exposed to agents that inhibit formation of autolysosomes by different mechanisms (24Klionsky D.J. Abeliovich H. Agostinis P. Agrawal D.K. Aliev G. Askew D.S. Baba M. Baehrecke E.H. Bahr B.A. Ballabio A. Bamber B.A. Bassham D.C. Bergamini E. Bi X. Biard-Piechaczyk M. Blum J.S. Bredesen D.E. Brodsky J.L. Brumell J.H. Brunk U.T. Bursch W. Camougrand N. Cebollero E. Cecconi F. Chen Y. Chin L.S. Choi A. Chu C.T. Chung J. Clarke P.G. Clark R.S. Clarke S.G. Clavé C. Cleveland J.L. Codogno P. Colombo M.I. Coto-Montes A. Cregg J.M. Cuervo A.M. Debnath J. Demarchi F. Dennis P.B. Dennis P.A. Deretic V. Devenish R.J. Di Sano F. Dice J.F. Difiglia M. Dinesh-Kumar S. Distelhorst C.W. Djavaheri-Mergny M. Dorsey F.C. Dröge W. Dron M. Dunn Jr., W.A. Duszenko M. Eissa N.T. Elazar Z. Esclatine A. Eskelinen E.L. Fésüs L. Finley K.D. Fuentes J.M. Fueyo J. Fujisaki K. Galliot B. Gao F.B. Gewirtz D.A. Gibson S.B. Gohla A. Goldberg A.L. Gonzalez R. González-Estévez C. Gorski S. Gottlieb R.A. Häussinger D. He Y.W. Heidenreich K. Hill J.A. Høyer-Hansen M. Hu X. Huang W.P. Iwasaki A. Jäättelä M. Jackson W.T. Jiang X. Jin S. Johansen T. Jung J.U. Kadowaki M. Kang C. Kelekar A. Kessel D.H. Kiel J.A. Kim H.P. Kimchi A. Kinsella T.J. Kiselyov K. Kitamoto K. Knecht E. Komatsu M. Kominami E. Kondo S. Kovács A.L. Kroemer G. Kuan C.Y. Kumar R. Kundu M. Landry J. Laporte M. Le W. Lei H.Y. Lenardo M.J. Levine B. Lieberman A. Lim K.L. Lin F.C. Liou W. Liu L.F. Lopez-Berestein G. López-Otín C. Lu B. Macleod K.F. Malorni W. Martinet W. Matsuoka K. Mautner J. Meijer A.J. Meléndez A. Michels P. Miotto G. Mistiaen W.P. Mizushima N. Mograbi B. Monastyrska I. Moore M.N. Moreira P.I. Moriyasu Y. Motyl T. Münz C. Murphy L.O. Naqvi N.I. Neufeld T.P. Nishino I. Nixon R.A. Noda T. Nürnberg B. Ogawa M. Oleinick N.L. Olsen L.J. Ozpolat B. Paglin S. Palmer G.E. Papassideri I. Parkes M. Perlmutter D.H. Perry G. Piacentini M. Pinkas-Kramarski R. Prescott M. Proikas-Cezanne T. Raben N. Rami A. Reggiori F. Rohrer B. Rubinsztein D.C. Ryan K.M. Sadoshima J. Sakagami H. Sakai Y. Sandri M. Sasakawa C. Sass M. Schneider C. Seglen P.O. Seleverstov O. Settleman J. Shacka J.J. Shapiro I.M. Sibirny A. Silva-Zacarin E.C. Simon H.U. Simone C. Simonsen A. Smith M.A. Spanel-Borowski K. Srinivas V. Steeves M. Stenmark H. Stromhaug P.E. Subauste C.S. Sugimoto S. Sulzer D. Suzuki T. Swanson M.S. Tabas I. Takeshita F. Talbot N.J. Tallóczy Z. Tanaka K. Tanaka K. Tanida I. Taylor G.S. Taylor J.P. Terman A. Tettamanti G. Thompson C.B. Thumm M. Tolkovsky A.M. Tooze S.A. Truant R. Tumanovska L.V. Uchiyama Y. Ueno T. Uzcátegui N.L. van der Klei I. Vaquero E.C. Vellai T. Vogel M.W. Wang H.G. Webster P. Wiley J.W. Xi Z. Xiao G. Yahalom J. Yang J.M. Yap G. Yin X.M. Yoshimori T. Yu L. Yue Z. Yuzaki M. Zabirnyk O. Zheng X. Zhu X. Deter R.L. Autophagy. 2008; 4: 151-175Crossref PubMed Scopus (1980) Google Scholar). One mm 3-MA, 0.3 μm chloroquine, and 30 nm wortmannin almost completely prevented Pdx1 KD-induced MIN6 cell death on day 7 (Fig. 1C). Optimal concentrations were 5–10 times lower than the concentrations used in previous studies (24Klionsky D.J. Abeliovich H. Agostinis P. Agrawal D.K. Aliev G. Askew D.S. Baba M. Baehrecke E.H. Bahr B.A. Ballabio A. Bamber B.A. Bassham D.C. Bergamini E. Bi X. Biard-Piechaczyk M. Blum J.S. Bredesen D.E. Brodsky J.L. Brumell J.H. Brunk U.T. Bursch W. Camougrand N. Cebollero E. Cecconi F. Chen Y. Chin L.S. Choi A. Chu C.T. Chung J. Clarke P.G. Clark R.S. Clarke S.G. Clavé C. Cleveland J.L. Codogno P. Colombo M.I. Coto-Montes A. Cregg J.M. Cuervo A.M. Debnath J. Demarchi F. Dennis P.B. Dennis P.A. Deretic V. Devenish R.J. Di Sano F. Dice J.F. Difiglia M. Dinesh-Kumar S. Distelhorst C.W. Djavaheri-Mergny M. Dorsey F.C. Dröge W. Dron M. Dunn Jr., W.A. Duszenko M. Eissa N.T. Elazar Z. Esclatine A. Eskelinen E.L. Fésüs L. Finley K.D. Fuentes J.M. Fueyo J. Fujisaki K. Galliot B. Gao F.B. Gewirtz D.A. Gibson S.B. Gohla A. Goldberg A.L. Gonzalez R. González-Estévez C. Gorski S. Gottlieb R.A. Häussinger D. He Y.W. Heidenreich K. Hill J.A. Høyer-Hansen M. Hu X. Huang W.P. Iwasaki A. Jäättelä M. Jackson W.T. Jiang X. Jin S. Johansen T. Jung J.U. Kadowaki M. Kang C. Kelekar A. Kessel D.H. Kiel J.A. Kim H.P. Kimchi A. Kinsella T.J. Kiselyov K. Kitamoto K. Knecht E. Komatsu M. Kominami E. Kondo S. Kovács A.L. Kroemer G. Kuan C.Y. Kumar R. Kundu M. Landry J. Laporte M. Le W. Lei H.Y. Lenardo M.J. Levine B. Lieberman A. Lim K.L. Lin F.C. Liou W. Liu L.F. Lopez-Berestein G. López-Otín C. Lu B. Macleod K.F. Malorni W. Martinet W. Matsuoka K. Mautner J. Meijer A.J. Meléndez A. Michels P. Miotto G. Mistiaen W.P. Mizushima N. Mograbi B. Monastyrska I. Moore M.N. Moreira P.I. Moriyasu Y. Motyl T. Münz C. Murphy L.O. Naqvi N.I. Neufeld T.P. Nishino I. Nixon R.A. Noda T. Nürnberg B. Ogawa M. Oleinick N.L. Olsen L.J. Ozpolat B. Paglin S. Palmer G.E. Papassideri I. Parkes M. Perlmutter D.H. Perry G. Piacentini M. Pinkas-Kramarski R. Prescott M. Proikas-Cezanne T. Raben N. Rami A. Reggiori F. Rohrer B. Rubinsztein D.C. Ryan K.M. Sadoshima J. Sakagami H. Sakai Y. Sandri M. Sasakawa C. Sass M. Schneider C. Seglen P.O. Seleverstov O. Settleman J. Shacka J.J. Shapiro I.M. Sibirny A. Silva-Zacarin E.C. Simon H.U. Simone C. Simonsen A. Smith M.A. Spanel-Borowski K. Srinivas V. Steeves M. Stenmark H. Stromhaug P.E. Subauste C.S. Sugimoto S. Sulzer D. Suzuki T. Swanson M.S. Tabas I. Takeshita F. Talbot N.J. Tallóczy Z. Tanaka K. Tanaka K. Tanida I. Taylor G.S. Taylor J.P. Terman A. Tettamanti G. Thompson C.B. Thumm M. Tolkovsky A.M. Tooze S.A. Truant R. Tumanovska L.V. Uchiyama Y. Ueno T. Uzcátegui N.L. van der Klei I. Vaquero E.C. Vellai T. Vogel M.W. Wang H.G. Webster P. Wiley J.W. Xi Z. Xiao G. Yahalom J. Yang J.M. Yap G. Yin X.M. Yoshimori T. Yu L. Yue Z. Yuzaki M. Zabirnyk O. Zheng X. Zhu X. Deter R.L. Autophagy. 2008; 4: 151-175Crossref PubMed Scopus (1980) Google Scholar) (data not shown). To determine the relative roles of apoptosis and autophagy on the observed cell death, MIN6 cells with reduced Pdx1 expression were treated with 3-MA (1 mm) or caspase-3 inhibitor DEVD-CHO (10 μm). Pdx1 KD MIN6 cells treated with DEVD-CHO showed nearly complete inhibition of Pdx1 KD-induced cell death compared with 1 mm 3-MA-treated Pdx1 KD MIN6 cells on day 9 (Fig. 2D). A progressive increase in cleaved caspase-3 levels was observed in 3-MA-treated Pdx1 KD MIN6 cells on days 5, 7, and 9, respectively (Fig. 2E). This increase in cleaved caspase-3 over time was completely prevented in DEVD-CHO-treated Pdx1 KD MIN6 cells. These results indicate that 3-MA delays Pdx1 KD-induced MIN6 cell death, but 3-MA-treated Pdx1 KD MIN6 cells finally die by caspase-3-dependent cell death. Thus in the temporal evolution of the cell death induced by reduced Pdx1 expression, an increase in autophagy appears to occur early, and this is followed by an increase in apoptosis. Because the autophagy inhibitors used in this study may have nonspecific effects (24Klionsky D.J. Abeliovich H. Agostinis P. Agrawal D.K. Aliev G. Askew D.S. Baba M. Baehrecke E.H. Bahr B.A. Ballabio A. Bamber B.A. Bassham D.C. Bergamini E. Bi X. Biard-Piechaczyk M. Blum J.S. Bredesen D.E. Brodsky J.L. Brumell J.H. Brunk U.T. Bursch W. Camougrand N. Cebollero E. Cecconi F. Chen Y. Chin L.S. Choi A. Chu C.T. Chung J. Clarke P.G. Clark R.S. Clarke S.G. Clavé C. Cleveland J.L. Codogno P. Colombo M.I. Coto-Montes A. Cregg J.M. Cuervo A.M. Debnath J. Demarchi F. Dennis P.B. Dennis P.A. Deretic V. Devenish R.J. Di Sano F. Dice J.F. Difiglia M. Dinesh-Kumar S. Distelhorst C.W. Djavaheri-Mergny M. Dorsey F.C. Dröge W. Dron M. Dunn Jr., W.A. Duszenko M. Eissa N.T. Elazar Z. Esclatine A. Eskelinen E.L. Fésüs L. Finley K.D. Fuentes J.M. Fueyo J. Fujisaki K. Galliot B. Gao F.B. Gewirtz D.A. Gibson S.B. Gohla A. Goldberg A.L. Gonzalez R. González-Estévez C. Gorski S. Gottlieb R.A. Häussinger D. He Y.W. Heidenreich K. Hill J.A. Høyer-Hansen M. Hu X. Huang W.P. Iwasaki A. Jäättelä M. Jackson W.T. Jiang X. Jin S. Johansen T. Jung J.U. Kadowaki M. Kang C. Kelekar A. Kessel D.H. Kiel J.A. Kim H.P. Kimchi A. Kinsella T.J. Kiselyov K. Kitamoto K. Knecht E. Komatsu M. Kominami E. Kondo S. Kovács A.L. Kroemer G. Kuan C.Y. Kumar R. Kundu M. Landry J. Laporte M. Le W. Lei H.Y. Lenardo M.J. Levine B. Lieberman A. Lim K.L. Lin F.C. Liou W. Liu L.F. Lopez-Berestein G. López-Otín C. Lu B. Macleod K.F. Malorni W. Martinet W. Matsuoka K. Mautner J. Meijer A.J. Meléndez A. Michels P. Miotto G. Mistiaen W.P. Mizushima N. Mograbi B. Monastyrska I. Moore M.N. Moreira P.I. Moriyasu Y. Motyl T. Münz C. Murphy L.O. Naqvi N.I. Neufeld T.P. Nishino I. Nixon R.A. Noda T. Nürnberg B. Ogawa M. Oleinick N.L. Olsen L.J. Ozpolat B. Paglin S. Palmer G.E. Papassideri I. Parkes M. Perlmutter D.H. Perry G. Piacentini M. Pinkas-Kramarski R. Prescott M. Proikas-Cezanne T. Raben N. Rami A. Reggiori F. Rohrer B. Rubinsztein D.C. Ryan K.M. Sadoshima J. Sakagami H. Sakai Y. Sandri M. Sasakawa C. Sass M. Schneider C. Seglen P.O. Seleverstov O. Settleman J. Shacka J.J. Shapiro I.M. Sibirny A. Silva-Zacarin E.C. Simon H.U. Simone C. Simonsen A. Smith M.A. Spanel-Borowski K. Srinivas V. Steeves M. Stenmark H. Stromhaug P.E. Subauste C.S. Sugimoto S. Sulzer D. Suzuki T. Swanson M.S. Tabas I. Takeshita F. Talbot N.J. Tallóczy Z. Tanaka K. Tanaka K. Tanida I. Taylor G.S. Taylor J.P. Terman A. Tettamanti G. Thompson C.B. Thumm M. Tolkovsky A.M. Tooze S.A. Truant R. Tumanovska L.V. Uchiyama Y. Ueno T. Uzcátegui N.L. van der Klei I. Vaquero E.C. Vellai T. Vogel M.W. Wang H.G. Webster P. Wiley J.W. Xi Z. Xiao G. Yahalom J. Yang J.M. Yap G. Yin X.M. Yoshimori T. Yu L. Yue Z. Yuzaki M. Zabirnyk O. Zheng X. Zhu X. Deter R.L. Autophagy. 2008; 4: 151-175Crossref PubMed Scopus (1980) Google Scholar), we also studied the effects of knocking down expression of Becn1 or Atg5 in MIN6 cells. Becn1 and Atg5 are key mediators of autophagosome formation (25Liang X.H. Jackson S. Seaman M. Brown K. Kempkes B. Hibshoosh H. Levine B. Nature. 1999; 402: 672-676Crossref PubMed Scopus (2771) Google
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