A JournAl of the AmericAn DiAbetes AssociAtion

Islet Biology/Insulin Secretion137-LBEngaging Canonical and Non-Canonical Wnt Signaling with Wnt3aand R-Spondin3 and Inhibition of RhoA/ROCK Signaling IncreasesProliferation of Adult Human β-CellsHAYTHAM ALY, NIDHI ROHATGI, CONNIE MARSHALL, HIROYUKI MIYOSHI,THADDEUS STAPPENBECK, MICHAEL MCDANIEL, St. Louis, MOOur previous studies demonstrated that Wnt/GSK-3/β-catenin and mTORsignaling are necessary for increasing regenerative processes in adult humanβ-cells. Direct inhibition of GSK-3 increased β-catenin nuclear translocationand β-cell proliferation but resulted in a loss of insulin content. Our currentgoal was to engage canonical and non-canonical Wnt signaling at thereceptor level, identify additional pathways involved in β-cell proliferation,and maintain β-cell phenotype.Treatment of human islets with a GSK-3 inhibitor or conditioned media (CM)containing Wnt3a and R-spondin3, enhanced DNA synthesis ~2 fold. However,treatment with CM+ [containing CM, ROCK inhibitor (Y-27632) and SB-431542(that results in RhoA inhibition)] significantly enhanced DNA synthesis ~6fold in a rapamycin-sensitive manner. Moreover, CM+ increased humanβ-cell proliferation ~20 fold above glucose alone and enhanced β-cateninmediatedgene expression as well as IRS1/2 gene expression. PKA signalingis also important for enhancement of Wnt signaling. Inhibition of PKA byH89 significantly blocked CM+ mediated human islet DNA synthesis. DNAsynthesis was not enhanced by exendin-4 alone, but significantly increasedin the presence of RhoA/ROCK inhibitors. However, these effects weresignificantly lower than the CM+ treatments. Activation of PKA with forskolinincreased Wnt signaling through phosphorylation of GSK-3 and CREB, and ledto increased expression of CREB target genes, NR4A2 and IRS2. Treatment ofhuman islets with CM or CM+ also increased expression of NR4A2 and IRS2and resulted in activation of Akt.Our data demonstrate that engaging Wnt signaling at the receptor levelleads to crosstalk between Wnt/β-catenin, PKA/CREB and mTOR signaling.Regulation of these pathways with CM+ or exendin-4 plus RhoA/ROCKinhibitors, substantially enhances human β-cell proliferation and maymaintain β-cell differentiation.Supported by: NIH138-LBAlpha-Cells are Dispensable for Post-Natal Islet Morphogenesisand Maturation of Beta-CellsCHIYO SHIOTA, KRISHNA PRASADAN, PING GUO, YOUSEF EL-GOHARY, XIANG-WEI XIAO, GEORGE K. GITTES, Pittsburgh, PAGlucagon-producing α-cells are the second most abundant cell type inislets. While α-cells make up less than 20% of the cells in a mature islet, theyoccupy a much larger proportion of the pancreatic endocrine cell populationduring the early post-natal period, the time when extensive β-cell proliferationand dynamic morphogenesis occur to form mature islets. To determine ifα-cells have a role in this postnatal process of islet development, we haveestablished a diphtheria toxin-mediated α-cell ablation mouse model in whichα-cells can be ablated by toxin injection. Rapid and persistant depletion ofα-cells was achieved by daily injections of the toxin for 2 weeks starting atpost-natal day1 (P1). Total pancreatic glucagon content was undetectable atP14, and still only 2.8±0.7ng at 4 months of age in the α-cell ablated mice,compared with 393±39ng and 1,087±18ng, respectively, at those ages incontrol mice. Histological analyses revealed that formation of spherical isletsfrom primitive aggregations of endocrine cells occurred normally despitethe near total lack of α-cells. Furthermore, islet size distribution, whichwas evaluated histologically was not changed by the lack of α-cells in the4-month old mice. Insulin content of the α-cell ablated pancreata was notsignificantly different from control pancreata during the post-natal period, butit was somewhat higher (a 26% increase) at 4 months of age. Blood glucoseand plasma insulin levels in the α-cell ablated mice were slightly lowerthan that of the control mice, but the differences were not significant. Bothglucose and insulin tolerance tests indicated that the α-cell ablated mice hadnormal glucose homeostasis physiology. In perifusion experiments, glucosestimulatedinsulin secretion in islets isolated from the α-cell ablated micewas not different from control islets. Taken together, these results indicatethat α-cells do not play a critical role in post-natal islet morphogenesis norfunctional maturation of β-cells.139-LBMicroRNA-7a Regulates the mTOR Pathway and Proliferation inAdult Pancreatic β-CellsYOU WANG, JIANGYING LIU, CHENGYANG LIU, ALI NAJI, DORIS A. STOFFERS,Philadelphia, PAElucidating the mechanism underlying the poor proliferative capacity ofadult pancreatic beta cells is critical to regenerative therapeutic approachesfor diabetes. We find that the miR7/7ab family member microRNA-7a (miR-7a)is enriched in adult pancreatic islets and, remarkably, targets five componentsof the mTOR signaling pathway, including the two main downstream effectorsof TORC1, p70S6K and eukaryotic translation initiation factor 4E (eIF4E), twoMAPK-interacting kinases Mknk1 and Mknk2 that phosphorylate eIF4E, andan essential TORC2 component, Mapkap1. miR-7a acts on the 3’-UTR of thesegenes. Inhibition of miR-7a increases protein levels of these componentsas well as the activity of their downstream signaling targets (p-Akt S473,pS6, and pEIF4E) in Min6 cells and primary mouse islets. Expression of themitosis marker, phospho-histone H3, was increased, and immunofluorescenceanalysis further showed a 2.6 and 2.9 fold increase in the number of insulin+cells expressing p-HH3 and Ki67, respectively, in dispersed adult murineislets transfected with anti-miR-7a, demonstrating that miR-7a negativelyregulates adult primary beta cell proliferation. There was no change in cellsurvival as measured by TUNEL or in glucose stimulated insulin secretion inmiR-7a deficient and control islet cells. Importantly, this miR-7a-mTOR-betacell proliferation axis is conserved in primary human beta cells. Dispersedhuman islets transfected with control oligonucleotide showed no detectablereplication, whereas miR-7a inhibitor transfection resulted in 0.5% insulin+Ki67+ human islet cells. To establish the causal role of mTOR signaling inthis response, we used the mTOR inhibitor rapamycin and found completeabrogation of the enhanced beta cell replication induced by miR-7a deficiencyin both mouse and human islets. These data suggest that miR-7a acts as abrake on adult beta cell proliferation by inhibiting mTOR signaling and theyimplicate miR-7a as a therapeutic target for diabetes.Supported by: NIH (P01DK49210), State of PA (4100043362)140-LBSignaling Pathway of β Cell Proliferation in Pancreatectomy isDistinct from that in High-Fat Diet-Loading in MiceYU TOGASHI, JUN SHIRAKAWA, KAZUKI ORIME, KOICHIRO SATO, KAZUKITAJIMA, AKINOBU NAKAMURA, YASUO TERAUCHI, Yokohama, JapanHigh-fat (HF) diet-loading and partial pancreatectomy are good modelsfor the evaluation of adaptive proliferation of β cells in rodents. Here wecompared the role of glucokinase (Gck) and insulin receptor substrate 2 (IRS-2) in these mouse models. As we previously reported, both β cell-specificGck haploinsufficient (Gck +/- ) mice and IRS-2-deficient (IRS-2 -/- ) mice failed toincrease β cell mass on a HF diet in association with insufficient upregulationof IRS-2, Pdx1 and cyclin D2 in islet. Next, Gck +/- mice, IRS-2 -/- mice andwild type (WT) mice were subjected to 60% partial pancreatectomy (Px) ora sham operation (Sham). Both Gck +/- mice and IRS-2 -/- mice, as well as WTmice, exhibited sufficient expansion of β cell mass and significant increasein β cell proliferation after Px, compared to Sham. There were no significantdifferences in the expression levels of Gck, IRS-2, Pdx1, cyclin D1 and cyclinD2 in islets between Sham and Px in all genotypes. Gene expressions ofFoxM1, cyclin B1, Aurora kinase B and Birc5 in islets were increased by Pxin WT mice and Gck +/- mice, while those were not increased by Px in IRS-2 -/-mice. Thus, neither Gck nor IRS-2 was required for β cell proliferation after Px.Furthermore, the gene expression profiles of transcriptional factors and cyclinfamilies demonstrated quite different patterns between Px and HF. SinceIRS-2 -/- mice demonstrated β cell expansion without upregulation of FoxM1and its downstream molecules, β cell proliferation after pancreatectomy inIRS-2 -/- mice might be mediated by independent pathway of both FoxM1and insulin signaling. In conclusion, varying signaling pathways of β cellproliferation exist in respective experimental models. Partial pancreatectomyin mice may be an attractive model for therapy development by exploring theunique nature of β cell proliferation.ADA-Funded ResearchFor author disclosure information, see page LB45.LB35