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-...

During pregnancy, maternal pancreatic islets grow to match dynamic physiological demands, but the mechanisms regulating adaptive islet growth in this setting are poorly understood. Here we show that menin, a protein previously characterized as an endocrine tumor suppressor and transcriptional regulator, controls islet growth in pregnant mice. Pregnancy stimulated proliferation of maternal pancreatic islet b-cells that was accompanied by reduced islet levels of menin and its targets. Transgenic expression of menin in maternal b-cells prevented islet expansion and led to hyperglycemia and impaired glucose tolerance, hallmark features of gestational diabetes. Prolactin, a hormonal regulator of pregnancy, repressed islet menin levels and stimulated b-cell proliferation. These results expand our understanding of mechanisms underlying diabetes pathogenesis and reveal potential targets for therapy in diabetes.

There is widespread interest in defining factors and mechanisms that stimulate proliferation of pancreatic islet cells. Wnt signaling is an important regulator of organ growth and cell fates, and genes encoding Wnt-signaling factors are expressed in the pancreas. However, it is unclear whether Wnt signaling regulates pancreatic islet proliferation and differentiation. Here we provide evidence that Wnt signaling stimulates islet ␤ cell proliferation. The addition of purified Wnt3a protein to cultured ␤ cells or islets promoted expression of Pitx2, a direct target of Wnt signaling, and Cyclin D2, an essential regulator of ␤ cell cycle progression, and led to increased ␤ cell proliferation in vitro. Conditional pancreatic ␤ cell expression of activated ␤-catenin, a crucial Wnt signal transduction protein, produced similar phenotypes in vivo, leading to ␤ cell expansion, increased insulin production and serum levels, and enhanced glucose handling. Conditional ␤ cell expression of Axin, a potent negative regulator of Wnt signaling, led to reduced Pitx2 and Cyclin D2 expression by ␤ cells, resulting in reduced neonatal ␤ cell expansion and mass and impaired glucose tolerance. Thus, Wnt signaling is both necessary and sufficient for islet ␤ cell proliferation, and our study provides previously unrecognized evidence of a mechanism governing endocrine pancreas growth and function.Cyclin D2 ͉ diabetes mellitus ͉ islets of Langerhans ͉ pancreas ͉ Pitx2

Determining the signalling pathways that direct tissue expansion is a principal goal of regenerative biology. Vigorous pancreatic β-cell replication in juvenile mice and humans declines with age, and elucidating the basis for this decay may reveal strategies for inducing β-cell expansion, a long-sought goal for diabetes therapy. Here we show that platelet-derived growth factor receptor (Pdgfr) signalling controls age-dependent β-cell proliferation in mouse and human pancreatic islets. With age, declining β-cell Pdgfr levels were accompanied by reductions in β-cell enhancer of zeste homologue 2 (Ezh2) levels and β-cell replication. Conditional inactivation of the Pdgfra gene in β-cells accelerated these changes, preventing mouse neonatal β-cell expansion and adult β-cell regeneration. Targeted human PDGFR-α activation in mouse β-cells stimulated Erk1/2 phosphorylation, leading to Ezh2-dependent expansion of adult β-cells. Adult human islets lack PDGF signalling competence, but exposure of juvenile human islets to PDGF-AA stimulated β-cell proliferation. The discovery of a conserved pathway controlling age-dependent β-cell proliferation indicates new strategies for β-cell expansion.

The release of reactive oxygen species (ROS) has been proposed as a cause of streptozotocin (STZ)-induced ␤-cell damage. This initiates a destructive cascade, consisting of DNA damage, excess activation of the DNA repair enzyme poly(ADP-ribose) polymerase, and depletion of cellular NAD ؉ . Metallothionein (MT) is an inducible antioxidant protein that has been shown to protect DNA from chemical damage in several cell types. Therefore, we examined whether overexpression of MT could protect ␤-cell DNA and thereby prevent STZinduced diabetes. Two lines of transgenic mice were produced with up to a 30-fold elevation in ␤-cell MT. Cultured islets from control mice and MT transgenic mice were exposed to STZ. MT was found to decrease STZ-induced islet disruption, DNA breakage, and depletion of NAD ؉ . To assess in vivo protection, transgenic and control mice were injected with STZ. Transgenic mice had significantly reduced hyperglycemia. Ultrastructural examination of islets from STZ-treated mice showed that MT prevented degranulation and cell death. These results demonstrate that MT can reduce diabetes and confirm the DNA damage mechanism of STZ-induced ␤-cell death. Diabetes 50:2040 -2046, 2001 P ancreatic ␤-cells (1) are extremely vulnerable to damage caused by reactive oxygen species (ROS). A striking example of ␤-cell vulnerability is the severe damage done by streptozotocin (STZ). This natural toxin has several destructive actions, including DNA methylation (2), protein modification (3), and ROS generation (4). STZ enters the cytoplasm via GLUT2, which is the ␤-cell's glucose transporter (5). The presence of this transporter may account for part of the specific vulnerability of the ␤-cell. However, GLUT2 is also present in the liver and kidney, tissues that are relatively resistant to STZ damage. The much greater sensitivity of the ␤-cell is probably due to its very low level of antioxidant enzyme expression and activity (6 -8), which leaves it unable to inactivate ROS. It has been demonstrated in vivo that overexpression of the antiapoptotic, antioxidant protein thioredoxin (9), as well as the antioxidant protein catalase (10), protects the ␤-cell from STZ. However, it is not possible to markedly induce these proteins in vivo. Therefore, we are exploring the potential of another antioxidant protein that is highly inducible, metallothionein (MT).MTs are a family of low-molecular weight cysteine-rich proteins that bind heavy metals with high affinity. MT appears to play important roles in zinc homeostasis and ROS protection. Both functions are due to the presence of abundant cysteine residues. MT protects against many agents known to act through ROS, including hydrogen peroxide, radiation, glutathione depletors, adriamycin, and xanthine oxidase (11,12). MT is distinct among ROS scavengers in the breadth of its protection, in that it scavenges nitric oxide radicals (13), superoxide radicals (14), and hydroxyl radicals (15), which are the most destructive ROS species (11). MT scavenging of hydroxyl radicals is especi...