Proliferation of pancreatic islet b cells is an important mechanism for self-renewal and for adaptive islet expansion. Increased expression of the Ink4a/Arf locus, which encodes the cyclin-dependent kinase inhibitor p16INK4a and tumor suppressor p19 Arf , limits b-cell regeneration in aging mice, but the basis of b-cell Ink4a/Arf regulation is poorly understood. Here we show that Enhancer of zeste homolog 2 (Ezh2), a histone methyltransferase and component of a Polycomb group (PcG) protein complex, represses Ink4a/Arf in islet b cells. Ezh2 levels decline in aging islet b cells, and this attrition coincides with reduced histone H3 trimethylation at Ink4a/ Arf, and increased levels of p16INK4a and p19Arf . Conditional deletion of b-cell Ezh2 in juvenile mice also reduced H3 trimethylation at the Ink4a/Arf locus, leading to precocious increases of p16INK4a and p19 Arf . These mutant mice had reduced b-cell proliferation and mass, hypoinsulinemia, and mild diabetes, phenotypes rescued by germline deletion of Ink4a/Arf. b-Cell destruction with streptozotocin in controls led to increased Ezh2 expression that accompanied adaptive b-cell proliferation and re-establishment of b-cell mass; in contrast, mutant mice treated similarly failed to regenerate b cells, resulting in lethal diabetes. Our discovery of Ezh2-dependent b-cell proliferation revealed unique epigenetic mechanisms underlying normal b-cell expansion and b-cell regenerative failure in diabetes pathogenesis.[Keywords: Pancreas; islet of Langerhans; histone; epigenetics; diabetes; cell cycle] Supplemental material is available at http://www.genesdev.org. Received September 18, 2008; revised version accepted March 13, 2009. Proliferation of insulin-secreting b cells in pancreatic islets is an important mechanism for establishing, maintaining, and adapting islet organ function to meet host physiological demands (for review, see Cozar-Castellano et al. 2006;Heit et al. 2006a). Harnessing our understanding of these mechanisms could accelerate development of islet replacement strategies in diseases like autoimmune (type 1) diabetes, but the molecular basis of self-renewal in organs like islets is poorly understood. Islet b cells expand in neonatal humans, mice, and other species, but this proliferation decays thereafter Meier et al. 2008), which may promote pandemic (type 2) forms of diabetes mellitus. Thus, investigation of regenerative failure in b cells may elucidate important mechanisms underlying diabetes pathogenesis. p16 INK4a and p19 Arf (hereafter Ink4a and Arf) encoded by the Cdkn2a locus are negative regulators of the cell cycle and are thought to limit proliferation in islet b cells (Krishnamurthy et al. 2006) and other tissues (Zindy et al. 1997). Ink4a inhibits specific cyclin-dependent kinases (CDK), including CDK4, a key regulator of b-cell proliferation (Rane et al. 1999), while Arf inhibits the ubiquitin ligase activity of MDM2, thereby stabilizing p53 (for review, see Matheu et al. 2008). Germline Ink4a deficiency in mice permits increased b-...

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Although ϳ1 million islets exist in the adult human pancreas, current pancreas preservation and islet isolation techniques recover <50%. Presently, cadaveric donors remain the sole source of pancreatic tissue for transplantation. Brain death is characterized by activation of proinflammatory cytokines and organ injury during preservation and reperfusion. In this study, we assessed the effects of brain death on islet isolation yields and functionality. Brain death was induced in male 250-to 350-g Lewis rats by inflation of a Fogarty catheter placed intracranially. The rats were mechanically ventilated for 2, 4, and 6 h before removal of the pancreas (n ‫؍‬ 6). In controls, the catheter was not inflated (n ‫؍‬ 6). Shortly after brain death induction, a significant increase in serum tumor necrosis factor-␣ (TNF-␣), interleukin (IL)-1␤, and IL-6 was demonstrated in a time-dependent manner. Upregulation of TNF-␣, IL-1␤, and IL-6 mRNA was noted in the pancreas. Brain death donors presented lower insulin release after glucose stimulation assessed by in situ perfusion of the pancreas. Islet recovery was reduced in brain death donors compared with controls (at 6 h 602.3 ؎ 233.4 vs. 1,792.5 ؎ 325.4 islet equivalents, respectively; P < 0.05). Islet viability assessed in dissociated islet cells and in intact cultured islets was reduced in islets recovered from brain death donors, an effect associated with higher nuclear activities of NF-B p50, c-Jun, and ATF-2. Islet functionality evaluated in vitro by static incubation and in vivo after intraportal transplantation in syngeneic streptozotocin-induced diabetic rats was significantly reduced in preparations obtained from brain death donors. In conclusion, brain death significantly reduced islet yields and functionality. These observations may lead to strategies to reduce the effects of brain death on pancreatic islets and improve the results in clinical transplantation.

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