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...

Islet transplantation is a promising therapy for Type 1 diabetes, but many attempts have failed due to early graft hypoxia or immune rejection, which generate reactive oxygen species (ROS). In the current study, we determined that transgenic overexpression of the antioxidant metallothionein (MT) in pancreatic beta cells provided broad resistance to oxidative stress by scavenging most kinds of ROS including H 2 O 2 , peroxynitrite radical released from streptozotocin, 3-morpholinosydnonimine (SIN-1), and superoxide radical produced by xanthine/ xanthine oxidase. MT also reduced nitric oxide-induced beta cell death. A direct test of hypoxia/reperfusion sensitivity was made by exposing FVB and MT islets to hypoxia (1% O 2 ). MT markedly reduced ROS production and improved islet cell survival. Because MT protected beta cells from a broad spectrum of ROS and from hypoxia, we considered it to be an ideal candidate for improving islet transplantation. We first tested syngeneic transplantation by implanting islets under the kidney capsule of the same strain, FVB mice, thereby eliminating the immune rejection component. Under these conditions, MT islets maintained much greater insulin content than control islets. Allotransplantation was then tested. MT transgenic and normal FVB islets were implanted under the kidney capsule of BALB/c mice that were previously treated with streptozotocin to induce diabetes. We found that MT islets extended the duration of euglycemia 2-fold longer than nontransgenic islets. The benefit of MT was due to protection from ROS since nitrotyrosine staining, an indicator of free radical damage, was much lower in MT grafts than in FVB grafts. The time course of protection suggested that the major mode of MT action may have been protection from hypoxia or hypoxia/reperfusion. These data demonstrate that treatment with a broad spectrum antioxidant protects islets from ROS damage such as that produced during the early phase of islet transplantation.Transplantation of pancreatic islets is considered to be one of the most effective treatments for Type 1 diabetes (1). Recently, islet transplantation using the Edmonton protocol (2) achieved insulin independence in 12 out of 15 diabetic patients for 1 year. However, widespread application of transplantation therapy is still limited by the need for more than one donor pancreas per recipient and difficulties in maintaining long term euglycemia (1). One obstacle has been that many islets are lost during the initial stages of transplantation (3, 4). Shortly after implantation, islet grafts function poorly, and many transplanted beta cells undergo apoptosis prior to stable engraftment. This increases the mass of islets needed to achieve euglycemia (5). Unfortunately, there is an extreme shortage of human pancreatic islet donors. Therefore, instead of increasing the number of islets implanted, a more desirable strategy is to improve islet graft survival during the early stages of transplantation. However, to date, no impressive regimen has been devised to preve...

Reactive oxygen species (ROS) and nitric oxide (NO) are proposed mediators of cytokine-induced ␤-cell destruction in type 1 diabetes. We produced transgenic mice with increased ␤-cell expression of manganese superoxide dismutase (MnSOD) and catalase. Expression of these antioxidants increased ␤-cell ROS scavenging and improved ␤-cell survival after treatment with different sources of ROS. MnSOD or catalase conferred protection against streptozotocin (STZ)-induced ␤-cell injury. Coexpression of MnSOD and catalase provided synergistic protection against peroxynitrite and STZ. To determine the potential effect of these antioxidants on cytokine-induced toxicity, we exposed isolated islets to a cytokine mixture, including interleukin-1␤ and interferon-␥. Cytokine toxicity was measured as reduced metabolic activity after 6 days and reduced insulin secretion after 1 day. Cytokines increased ROS production, and both antioxidants were effective in reducing cytokine-induced ROS. However, MnSOD and/or catalase provided no protection against cytokine-induced injury. To understand this, the nuclear factor-B (NF-B) signaling cascade was investigated. Antioxidants reduced NF-B activation by ROS, but none of the antioxidants altered activation by cytokines, as measured by inhibitor of B phosphorylation, NF-B translocation, inducible NO synthase activation, and NO production. Our data agree with previous reports that antioxidants benefit ␤-cell survival against ROS damage, but they are not consistent with reports that antioxidants reduce cytokine toxicity. ROS appear to have no role in cytokine toxicity in primary ␤-cells. Diabetes 54:1437-1446, 2005 R eactive oxygen species (ROS) have been proposed to play an important role in ␤-cell destruction. Several studies have shown that exposure of pancreatic islets to cytokines (1-3) greatly increases ROS production and leads to oxidative damage to ␤-cells. Since pancreatic ␤-cells contain very low levels of antioxidant enzymes (4), they may be more susceptible to the toxic actions of cytokines (5). In agreement with the proposed role of ROS, overexpression of antioxidant enzymes in insulin-producing tumor cell lines has achieved great success in improving resistance to both free radicals and cytokines. For instance, overexpression of antioxidant enzymes conferred protection to several insulinoma cell lines against the toxic effects of ROS (6), nitric oxide (NO) (7), interleukin-1␤ (IL-1␤) (8), and a mixture of cytokines (9). However, other studies on primary islet cells, using various antioxidant compounds or enzymes, reported no protection against cytokine toxicity (10 -13).Inhibition of nuclear factor-B (NF-B) activity decreases cytokine-induced ␤-cell death (14 -16). NF-B activation is modulated by oxidative stress (17); compounds that generate ROS induce 19), and some antioxidants can block . Recently, it was reported that increased expression of mitochrondrial manganese superoxide dismutase (MnSOD), but not other antioxidant enzymes, prevented cytokine-induced NF-B activation and...

It is widely proposed that reactive oxygen species (ROS) contribute to ␤-cell death in type 1 diabetes. We tested this in nonobese diabetic (NOD) mice using ␤-cell-specific overexpression of three antioxidant proteins: metallothionein (MT), catalase (Cat), or manganese superoxide dismutase (MnSOD). Unexpectedly, the cytoplasmic antioxidants, MT and catalase, greatly accelerated diabetes after cyclophosphamide and accelerated spontaneous diabetes in male NOD mice. This occurred despite the fact that they reduced cytokine-induced ROS production and MT reduced streptozotocin diabetes in NOD mice. Accelerated diabetes onset coincided with increased ␤-cell death but not with increased immune attack. Islets from MTNOD mice were more sensitive to cytokine injury. In vivo and in vitro studies indicated reduced activation of the Akt/pancreatic duodenal homeobox-1 survival pathway in MTNOD and CatNOD islets. Our study indicates that cytoplasmic ROS may have an important role for protecting the ␤-cell from autoimmune destruction. Diabetes 55:1592-1604, 2006