Targeting D and the mitochondrial permeability transition enhances β-cell survival and prevents diabetes in Pdx1 deficiency

Kei Fujimotoa, Yun Chenb, Kenneth S. Polonskya,1, and Gerald W. Dorn IIb,1

aDivision of Endocrinology, and Lipid Research, and bCenter for Pharmacogenomics, Department of Medicine, Washington University School of Medicine, St. Louis, MO 63110

Edited* by Craig B. Thompson, University of Pennsylvania, Philadelphia, PA, and approved April 30, 2010 (received for review December 8, 2009) Mutations of the pancreatic duodenal homeobox -1, Pdx1, in the pathogenesis of Alzheimer’s disease and amyotrophic lat- cause heritable diabetes in humans and mice. A central abnormal- eral sclerosis (12, 13). ity with Pdx1 deficiency is increased death of β-cells, leading to Mitochondria are implicated in the pathogenesis of diabetes decreased β-cell mass. We show that lentiviral suppression of Pdx1 mellitus (14). Recently, a connection between Pdx1 insufficiency increases death of mouse insulinoma MIN6 β-cells associated with and mitochondrial dysfunction was described, implicating dys- dissipation of the mitochondrial inner membrane electrochemical regulation of the mitochondrial transcription factor TFAM in Δψ gradient, m. Preventing mitochondrial permeability transition abnormal insulin secretion (15). However, a cause–effect re- pore opening with the cyclophilin D inhibitor cyclosporin A re- lationship between mitochondria and programmed β-cell death Δψ β stored m and rescued cell viability. Reduced -cell mass, markers in diabetes has not been identified. Here, we combined studies in of β-cell , necrosis, and decreased proliferation are pres- insulinoma cells with in vivo genetic complementation to exam- ent in Pdx1 haploinsufficient mice. Genetic ablation of the Ppif ine the role of cell death by MPTP-dependent programmed gene, encoding cyclophilin D, restored β-cell mass and decreased necrosis in Pdx1 insufficiency. Our results reveal an essential role TUNEL and complement complex labeling without affecting β-cell for MPTP opening in β-cell death and show that pharmacolog- proliferation. In adult mice maintained on a high-fat diet, Ppif ical or genetic inhibition of the mitochondrial permeability ablation normalized fasting glucose and glucose and insulin transition can protect against programmed β-cell necrosis and responses to acute glucose challenge. Thus, cyclophilin D and the prevent diabetes in Pdx1 insufficiency. mitochondrial permeability transition are critical regulators of β-cell death caused by Pdx1 insufficiency. Results Cyclosporin A Protects MIN6 Cells Against Death Induced by Pdx1 cell necrosis | apoptosis | insulin Insufficiency. Suppression or genetic ablation of Pdx1 in vitro and in vivo has previously revealed that Pdx1 insufficiency impairs oss of insulin-producing β-cells contributes to type 1 and type 2 islet growth both by inhibiting β-cell proliferation and stimulating Ldiabetes (1, 2). Normal postnatal β-cell homeostasis is main- β-cell programmed death (4, 9, 16). In our studies of Pdx1 de- tained largely by self-replication, not differentiation from progenitor ficient β-cell death, we observed markers for both apoptosis cells (3). β-Cell proliferation is also essential for compensatory islet (cleaved -3) and autophagy (LC3 punctae, autophagic hyperplasia under various conditions of metabolic stress, and vesicles). However, inhibition of autophagy only temporarily therefore for prevention of diabetes (4). Proliferation is opposed by diminished β-cell death (16). Because caspase activation and programmed β-cell elimination; β-cell mass in the adult pancreas is autophagy after loss of ATP production might be secondary dynamically regulated by constant adjustment of the balance be- consequences of primary mitochondrial disruption caused by tween these two processes (5). permeability transition (17), we postulated that the primary The transcription factor, pancreatic duodenal homeobox gene-1 cause of Pdx1-deficient β-cell death could be programmed ne- (Pdx1) regulates β-cell proliferation and other events in insulin crosis, rather than apoptosis or autophagy. Accordingly, we used secretion during normal pancreatic development and in response a lentivirus–shRNA system to suppress Pdx1 in mouse MIN6 to insulin-resistant states (4, 6). In humans, loss-of-function mu- insulinoma cells (18) and measured cell viability, TUNEL posi- tations of this factor are a cause of familial early-onset diabetes tivity, and mitochondrial inner membrane potential as a function fi β – fi (7). In mice, germ-line Pdx1 haploinsuf ciency and -cell speci c of Pdx1 level. As previously described (16), expression of Pdx1 Pdx1 ablation produce glucose intolerance associated with de- protein was decreased by ≈70% by shRNA in MIN6 cells (Fig. β α creased -cell mass, a proportional increase in islet -cells, and 1A). Compared with scrambled control shRNA, Pdx1 shRNA markers of β-cell apoptosis (4, 8–10). The observation that in- fi β markedly increased cell death measured as propidium iodide suf ciency of this -cell proliferative factor not only impairs nor- (PI) staining (Fig. 1B) and TUNEL labeling (Fig. 1C). Strikingly, mal islet development and reactive islet hyperplasia, but also Pdx1 suppression also dissipated the inner mitochondrial mem- induces apoptosis in its target cells, suggests a mechanistic con- Δψ fl β brane potential, m, measured as loss of uorescence for the nection between Pdx1 regulation of -cell cycling and pro- voltage-sensitive mitochondrial dye rhodamine 123 (Fig. 1D). grammed elimination. There are three distinct mechanisms of programmed cell death: apoptosis, autophagy, and necrosis. Although the stimuli Author contributions: K.F. and Y.C. performed research; K.F., K.S.P., and G.W.D. analyzed for, and signaling of, these cell death pathways are distinct, they data; Y.C. contributed new reagents/analytic tools; K.S.P. and G.W.D. designed research; share a critical involvement of mitochondria (11). Mitochondrial and K.F., K.S.P., and G.W.D. wrote the paper. dysfunction resulting in decreased levels of ATP is a potent The authors declare no conflict of interest. stimulus for autophagy. Permeabilization of the mitochondrial *This Direct Submission article had a prearranged editor. outer membranes releases that activates the apo- 1To whom correspondence may be addressed. E-mail: [email protected] or Polonsky@ ptosis caspase cascade. Finally, opening of the mitochondrial dom.wustl.edu. permeability transition pore (MPTP) is the irreversible step in This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. programmed necrosis. The latter process plays an important role 1073/pnas.0914209107/-/DCSupplemental.

10214–10219 | PNAS | June 1, 2010 | vol. 107 | no. 22 www.pnas.org/cgi/doi/10.1073/pnas.0914209107 Downloaded by guest on September 24, 2021 Fig. 2. Ultrastructural abnormalities in β-cell mitochondria of Pdx1+/− mice. Transmission electron microscopy showing mitochondrial paucity and struc- +/− β Fig. 1. Cyclosporin A decreases MIN6 cell death caused by Pdx1 deficiency. tural abnormalities (black arrows) in Pdx1 -cells. Electron-dense core (A) Immunoblot of Pdx1 expression in MIN6 cells 7 d after infection with bodies (white arrowheads) are insulin-containing granules. lentiviral shRNA against Pdx1 or scrambled control shRNA. (B) PI staining in control (white) and Pdx1 knockdown (KD, black) MIN6 cells as a function of 1 nM CsA treatment. (C) TUNEL labeling in control (white) and Pdx1 KD (black) of β-cells ringed by a mantle of α-cells (Fig. 3A, Left) is charac- MIN6 cells, as a function of 1 nM CsA treatment. (D) Fluorescence cell sorting teristically disturbed, with α-cells distributed throughout the islet − analysis of rhodamine 123 (Rh123) in control (white) and Pdx1 KD (black) “core”. Islets of adult Pdx1+/ mice exhibited these features, MIN6 cells, as a function of 1 nM CsA treatment. Mean ± SEM; n = 3 per appearing abnormally small with a paucity of insulin-staining group. β-cells (Fig. 3A, Center, and Fig. 3 B and C) and proportionately increased glucagon-staining α-cells (Fig. 3 A, Right, and D). Islet number per pancreas did not change. Δψ − − Although loss of m is not essential in apoptotic or auto- Pancreatic islets in Ppif / mice appeared normal (Fig. 3A). − − − phagic cell death, it indicates opening of the MPTP that initiates Unexpectedly, islets in Pdx1+/ Ppif / mice exhibited a pheno- programmed cell necrosis. We therefore determined whether type that was neither totally normal nor completely typical of − − − MPTP opening was responsible for MIN6 cell death during Pdx1 Pdx1+/ mice. Ppif / ablation improved all features of the − suppression by treating cells with cyclosporin A (CsA), which Pdx1+/ phenotype relating to β-cell loss (Fig. 3A, Right, and Fig. inhibits an essential component of the MPTP, cyclophylin D 3 B–D). However, the abnormal distribution of α-cells in the islet Δψ fi (CypD) (19). CsA normalized m in Pdx1-de cient MIN6 core was not normalized (Fig. 3A, Right), suggesting that the fi cells (Fig. 1D) and signi cantly (but not completely) protected benefits of Ppif ablation in Pdx1 haploinsufficiency are limited to against cell death (Fig. 1B) and apoptosis (Fig. 1C). These results protecting β-cells from death, without correcting abnormalities MEDICAL SCIENCES demonstrate that suppression of Pdx1 predisposes MIN6 cells to related to developmental orchestration of islet architecture. − opening of MPTPs, which contributes to their programmed Pdx1+/ β-cells exhibited increased TUNEL labeling (Fig. 3E). elimination. Although TUNEL staining is widely used as a marker for apo- ptosis, it is not specific, and can also be present in necrotic and Pdx1+/− β − − − Primary Mitochondrial Abnormalities in Pancreatic -Cells. MPTP-dependent cell death (21, 22). Islets of Pdx1+/ Ppif / mice Δψ Because it is not possible to directly measure m in vivo, it is had reduced TUNEL positivity to levels that were ≈50% lower fi − dif cult to demonstrate that activation of the MPTP is re- than seen in Pdx1+/ mice (Fig. 3E). Because it has been de- β +/− sponsible for -cell loss and the diabetic phenotype of Pdx1 termined that Ppif ablation does not affect intrinsic pathway ap- mice. However, MPTP opening results in characteristic de- optosis (19), these results suggested that the β-cell–sparing effects generative changes in mitochondria, including matrix swelling seen with Ppif ablation are the consequence of decreased MPTP- and outer membrane rupture (20). Ultrastructural examination β +/− dependent necrotic -cell death. Indeed, staining of complement 9 of pancreatic islets from adult Pdx1 mice revealed severe (C5b9), a component of the membrane attack complex that is − mitochondrial degeneration having these characteristics (Fig. 2). more specific for cellular necrosis, was increased in Pdx1+/ islets Because these mitochondrial changes are not necessarily seen in and reduced by concomitant Ppif ablation (Fig. 3F). purely apoptotic or autophagic cell death, they support an in vivo To determine whether the islet-sparing effects of Ppif ablation +/− role for MPTP-mediated β-cell necrosis in the Pdx1 diabetic were solely the result of inhibition of β-cell death, or whether − − − phenotype. there was also an effect on β-cell proliferation, Pdx1+/ Ppif /

+/− islets were stained for the proliferative marker Ki-67. Pro- β-Cell Protection in Adult Pdx1 Mice Lacking Functional MPTP. We β − liferation of -cells was diminished by an order of magnitude in +/ β +/− next assessed the possible role of MPTP in in vivo Pdx1 -cell Pdx1 islets compared with WT, and was not significantly im- death using a functional read-out, phenotypic rescue. CypD is an proved by Ppif ablation (Fig. 3G). essential component of the MPTP, and genetic ablation of the peptidyl- (Ppif) gene encoding CypD prevents Preventing Diabetes in Pdx1+/− Mice by Genetic Ablation of the MPTP. − MPTP-dependent cell death (12, 13, 19). Accordingly, we crossed Pdx1+/ mice maintained on a high-fat diet develop increased − Pdx1+/ mice, which have a well-described phenotype of glucose fasting blood glucose and impaired glucose clearance in glucose intolerance associated with decreased β-cell mass (4, 8–10, 16), tolerance tests (16). This phenotype, which is due to β-cell in- − − onto the homozygous Ppif / background. All mice were fed sufficiency and defective insulin secretion, was confirmed in our − − − a high-fat diet to promote insulin resistance and accelerate the studies (Fig. 4 A–C). Pdx1+/ Ppif / mice exhibited significant diabetic phenotype. lower fasting blood glucose, (Fig. 4A), a normal response to The pathological hallmark of diabetes in mice with β-cell– glucose challenge (Fig. 4 A and B), and markedly increased se- specific Pdx1 ablation is loss of β-cells and increased α-cells (8, rum insulin levels at baseline and in response to glucose (Fig. 10). Furthermore, the normal islet architecture of a central core 4C). These results were similar after 2 wk, 4 wk (Fig. 4), or 8 wk

Fujimoto et al. PNAS | June 1, 2010 | vol. 107 | no. 22 | 10215 Downloaded by guest on September 24, 2021 − − − − − Fig. 3. Restoration of β-cells in pancreatic islets of adult Pdx1+/ mice lacking functional MPTP. (A) Islet morphology in WT, Pdx1+/ , and Pdx1+/ Ppif / mice: α-cells are red (anti-glucagon), and β-cells are green (anti-insulin). (B–D) Group data for β-cell studies. (E) TUNEL labeling of pancreatic islets. (Right) Quantitative TUNEL data. (F) C5b9 complement staining of pancreatic islets. (Right) Quantitative data. (G) Ki-67 staining of β-cells; group Ki-67 data are on the right. All group data are mean ± SEM of n = 5 12-wk-old mice.

− − of high-fat diet. As previously described (23), Ppif / mice are tion rescues the characteristic diabetes phenotype of Pdx1 hap- prone to obesity, which was manifested as enhanced weight gain loinsufficiency by reducing necrotic β-cell death mediated through on high-fat diet (Fig. S1). However, this did not alter their glu- the mitochondrial permeability transition. cose or insulin tolerance (Fig. S1), and there was no difference in − − − − − +/− +/− body mass between Ppif / and Pdx1+/ Ppif / mice on high-fat Islet Structure and β-Cell Number in Neonatal Pdx1 and Pdx1 −/− diet (Fig. S1). Thus, phenotypic rescue was achieved despite Ppif Mice. The above studies demonstrate that preventing the − continued obesity. mitochondrial permeability transition protects Pdx1+/ β-cells − − The Ppif / mouse is a germ-line deletion. Therefore, we de- from their characteristic degeneration and prevents diabetes. All termined whether enhanced insulin sensitivity played any role in of these studies were performed in adult mice maintained on − reversal of the Pdx1+/ diabetic phenotype by performing insulin a high-fat diet, and it is therefore possible that the salutary effects tolerance tests. The blood glucose response to insulin was nor- of Ppif ablation applied only to reactive islet hyperplasia in the − − − − mal in both Pdx1+/ and Pdx1+/ Ppif / mice (Fig. 4D). Together adult (4). To test this possibility, we examined islets of 1-d-old with the above results, these data indicate that Ppif gene abla- neonatal mice that had not been subjected to any extrinsic meta-

10216 | www.pnas.org/cgi/doi/10.1073/pnas.0914209107 Fujimoto et al. Downloaded by guest on September 24, 2021 TUNEL labeling (Fig. 5B), but without normalization of islet architecture (Fig. 5A) or improvement in β-cell proliferation (Fig. − 5C). These results indicate that protection of Pdx1+/ mice con- ferred by Ppif ablation is inclusive of both islet development and reactive growth. Discussion These studies establish the mitochondrial permeability transition as a primary cause of β-cell loss induced by Pdx1 insufficiency. Reduced β-cell mass in Pdx1 deficiency is widely attributed to combinatorial effects of decreased proliferation and increased apoptosis (4, 24, 25). Indeed, here and previously we observe caspase activation and increased TUNEL staining in Pdx1-de- ficient β-cells (9, 16, 26). Others have described transcriptional down-regulation of antiapoptotic factors and up-regulation of by Pdx1 deficiency (27). However, activation of intrinsic pathway apoptosis by outer membrane permeabilization does not directly induce, nor require, dissipation of Δψ as observed Fig. 4. MPTP inhibition by genetic ablation of Ppif prevents diabetes in m − fi Pdx1+/ mice. (A) Glucose levels after i.p. injection of dextrose (2 g/kg). (B) in Pdx1-de cient MIN6 cells. This is the functional signature of − − − − Areas under the GTT curves (AUC) from Fig. 3A. Pdx1+/ , n =7;Pdx1+/ Ppif / , MPTP-dependent necrosis, which results in collateral caspase n = 8; WT, n =4;*P < 0.05. (C) Fasting and 15 min after i.p. dextrose insulin activation and positive TUNEL staining (17). Moreover, we levels in Pdx1+/− (n = 7) and Pdx1+/−Ppif−/− mice (n = 8). (D) Glucose levels in show that pharmacological inhibition of MPTP opening with response to 0.75 U insulin/kg body weight; Pdx1+/−, n =4;Pdx1+/−Ppif−/−, n = CsA attenuates MIN6 cell death and TUNEL positivity induced 4; WT, n = 4. All data are presented as mean ± SEM. by Pdx1 suppression, and restores Δψm. In agreement with the in vitro results, genetic ablation of the essential MPTP component, − CypD, increased β-cell mass, decreased TUNEL labeling in islets +/ − bolic stress. Pancreata of neonatal Pdx1 mice exhibited struc- of Pdx1+/ mice and normalized glucose and insulin levels. Thus, tural abnormalities similar to those observed in the adults, in- programmed β-cell necrosis is important in the Pdx1-deficient β cluding decreased islet size and -cell content, with abnormal diabetic phenotype, and the MPTP plays a causative role in β-cell α distribution of -cells in the central core of the pancreatic islet death. These results are relevant to the human condition of (Fig. 5A). TUNEL positivity in islets was increased (Fig. 5B) and PDX1 insufficiency, hereditary maturity-onset diabetes of the +/− β-cell proliferation was decreased (Fig. 5C) in neonatal Pdx1 young (7, 28). Given recent evidence linking the PDX1 locus to mice to similar extents as in adults. Thus, the characteristic his- sporadic type 2 diabetes (29), these findings may be even more tological abnormalities are similar in nonstressed neonatal and broadly applicable to the human diabetic condition. − high-fat-fed adult Pdx1+/ mice. Likewise, the benefits of Ppif Our findingsimplicate themitochondrion as a central regulator of

ablation were the same in neonates as adults, with partial nor- β-cell fate. The evidence supporting a meaningful role for apoptotic MEDICAL SCIENCES malization of β-cell number and islet size (Fig. 5A) and decreased β-cell death via the mitochondrial pathway, at least in Pdx1 de-

− − − − Fig. 5. Pancreatic islet morphology in neonatal Pdx1+/ and Pdx1+/ Ppif / mice. (A) Pancreatic islets from 1-day-old mice comparing α-cells (red; anti-glucagon) and β-cells (green; anti-insulin). Right: Quantitative group datat. (B) TUNEL labeling of neonatal islets. (Right) Quantitative data. (C) Ki-67 staining. (Right) Group data. All data are mean ± SEM of n = 5 mice per group.

Fujimoto et al. PNAS | June 1, 2010 | vol. 107 | no. 22 | 10217 Downloaded by guest on September 24, 2021 ficiency, seems incontrovertible (4, 9, 16, 24, 26, 30). Because Ppif for Pdx1 were recently described (16). Blots were probed with antibodies ablation specifically does not prevent Bax/Bak-mediated mito- against Pdx1 (#sc-14664; Santa Cruz Biotechnology) and actin (#A-2066; chondrial outer membrane permeabilization or apoptosis (19), the Sigma Chemical). incomplete cellular rescue we observed with either pharmacological Death Pathway Studies in MIN6 Cells. PI (10 μg/mL) and DAPI (20 μg/mL) were or genetic inhibition of CypD is consistent with roles for apoptosis fi via the mitochondrial pathway, and for programmed necrosis via added to the culture medium 1 h before formalin xation. Excitation fre- β quencies were 536 nm for PI and 355 nm for DAPI, with emission monitored opening of the MPTP, in -cell death. at 617 nm and 460 nm, respectively. TUNEL labeling was assessed using the Recent studies have suggested specific events that would ac- β DeadEnd Fluorometric TUNEL System kit and according to the manu- tivate the -cell mitochondrial permeability transition in Pdx1- facturer’s instructions (Promega). The integrity of the MPTP was assessed fi de cient cells. Gauthier et al. (15) implicated the mitochondrial using the voltage-sensitive dye rhodamine 123 to measure Δψm and quan- transcription factor TFAM in loss of mitochondrial gene in- tified by fluorescence cell sorting. Excitation was at 490 nm and emission tegrity and disruption of respiratory chain function in the Pdx1- monitored at 530 nm. To inhibit MPTP opening, CsA (Sigma Chemical) was deficient model. The resulting β-cell ATP depletion will starve dissolved in DMSO and used at a final concentration of 1 nM, which pre- energy-dependent calcium pumps, causing cytosolic calcium liminary studies showed inhibited peroxide-stimulated MIN6 MPTP opening overload that is a potent stimulus for MPTP opening (19). In- without causing cell toxicity. Each condition represents >600 cells counted terestingly, MPTP opening itself further impairs ATP production by randomized field selection. by dissipating Δψm, reversing oxidative phosphorylation, and contributing to a vicious cycle leading to cell necrosis. We ob- Generation of Genetic Mouse Models and Characterization of Diabetic Phenotype. All animal studies used protocols approved by the Washington served decreased TFAM expression in pancreatic islets isolated +/− – +/− University Animal Studies Committee. The previously described Pdx1 (4, 8 10) − − − − − − from Pdx1 mice that did not normalize with phenotypic rescue and Ppif / mice (19) were bred to obtain Pdx1+/ Ppif / mice. Pdx1+/ mice were by concomitant Ppif ablation (Fig. S2), consistent with a mecha- kindly provided by Dr. Helena Edlund (University of Umea). For neonatal nism whereby TFAM is proximal and MPTP opening is studies, pancreata were removed from 1-d-old pups whose dame had been a downstream effector of β-cell death mediated by Pdx1 hal- maintained on standard laboratory chow. For adult studies, mice were placed poinsufficiency. on 42% fat chow (Harlan Laboratories) at weaning. Glucose tolerance tests Endoplasmic reticulum (ER) stress has been implicated in β-cell (GTTs) were performed after a 16-h fast by administering 2 g dextrose/kg body lipotoxicity and programmed death in response to hyperglycemia weight, administered i.p., and serially monitoring blood glucose concen- (31). Recent findings have established that opening of the MPTP is trations. Insulin levels were measured at fasting and 15 min after glucose a component of the ER stress response, including in β-cells (32, 33). challenge. Insulin tolerance tests were performed after a 4-h fast by adminis- Sachdeva et al. recently reported that Pdx1 deficiency enhances tering 0.75 U human recombinant insulin/kg body weight. β -cell susceptibility to ER stress-associated apoptosis (34). Thus, fi accumulated data indicate that the mitochondrial permeability Imaging Studies of Pancreatic Islets. Formalin- xed pancreas sections un- fi derwent antigen retrieval in boiling citrate buffer (pH 6.0) for 10 min before transition plays a signi cant role in programmed cell death via labeling with antibodies against insulin (#4010-01L; Millipore), glucagon metabolic shutdown and the ER stress response. Because opening (#G2654; Sigma Chemical), Ki-67 (#180191Z; Zymed Laboratories/Invitrogen), of the MPTP is a common death effector for multiple pathways, or C5b-9 (Abcam). Fluorescent images were obtained on a Nikon C1si D- preventing the permeability transition has the potential to increase eclipse confocal microscope system and camera with a Nikon plan Apro VC β-cell survival under many conditions. ×60/1.40 oil objective. β-Cell area was quantified from anti-insulin stained The present studies establish a causal link between the mito- pancreas sections counterstained with hemotoxylin using the intensity chondrial permeability transition, β-cell death, and the insulin thresholding function of the integrated morphometry package in Meta- secretory response to glucose challenge. The ability to respond Morph. TUNEL labeling used the DeadEnd Fluorometric TUNEL System normally to metabolic stresses requires a physiological reserve of (Promega). insulin secretion and β-cell mass. Absence of this reserve leads to diabetes. Although β-cell protection from death was incomplete Statistical Analysis. Multiple experimental groups were compared using one- ’ ’ after either pharmacological or genetic inhibition of MPTP way ANOVA and Tukey s post hoc test. Pairwise comparisons used Student s unpaired t test. Differences were considered significant at P < 0.05. opening, the degree of rescue was sufficient to prevent the in vivo +/− diabetic phenotype that is characteristic of Pdx1 mice. In view ACKNOWLEDGMENTS. We thank Mr. H. Tran, Mr. E. L. Ford, and Mr. J. of our results, it will be interesting to determine whether com- Engelhard for excellent technical assistance and Dr. Helena Edlund for bined inhibition of β-cell necrosis and apoptosis signaling can providing Pdx1+/− mice. This work was supported by National Institutes of further enhance β-cell survival in diabetes. Health Grants R01 HL059888 (to G.W.D.) and R01 DK031842 (to K.S.P.), the Radioimmunoassay and Morphology Cores of the Diabetes Research and Materials and Methods Training Center of Washington University (GP60 DK-20579), the Washington University Digestive Diseases Research Center Morphology Core (P30 MIN6 Cell Culture, Pdx1 Suppression, and Immunoblot Analysis. MIN6 cell (18) DK052574), the Clinical and Translational Science Award to Washington Uni- culture and recombinant lentivirus expressing scrambled shRNA and shRNA versity (UL1 RR024992), and the Blum Kovler Foundation.

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