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The growth and function of organs such as pancreatic islets adapt to meet physiological challenges and maintain metabolic balance, but the mechanisms controlling these facultative responses are unclear. Diabetes in patients treated with calcineurin inhibitors such as cyclosporin A indicates that calcineurin/nuclear factor of activated T-cells (NFAT) signalling might control adaptive islet responses, but the roles of this pathway in beta-cells in vivo are not understood. Here we show that mice with a beta-cell-specific deletion of the calcineurin phosphatase regulatory subunit, calcineurin b1 (Cnb1), develop age-dependent diabetes characterized by decreased beta-cell proliferation and mass, reduced pancreatic insulin content and hypoinsulinaemia. Moreover, beta-cells lacking Cnb1 have a reduced expression of established regulators of beta-cell proliferation. Conditional expression of active NFATc1 in Cnb1-deficient beta-cells rescues these defects and prevents diabetes. In normal adult beta-cells, conditional NFAT activation promotes the expression of cell-cycle regulators and increases beta-cell proliferation and mass, resulting in hyperinsulinaemia. Conditional NFAT activation also induces the expression of genes critical for beta-cell endocrine function, including all six genes mutated in hereditary forms of monogenic type 2 diabetes. Thus, calcineurin/NFAT signalling regulates multiple factors that control growth and hallmark beta-cell functions, revealing unique models for the pathogenesis and therapy of diabetes.

SUMMARY Intensive efforts are focused on identifying regulators of human pancreatic islet cell growth and maturation to accelerate development of therapies for diabetes. After birth, islet cell growth and function are dynamically regulated; however establishing these age-dependent changes in humans has been challenging. Here we describe a multimodal strategy for isolating pancreatic endocrine and exocrine cells from children and adults to identify age-dependent gene expression and chromatin changes on a genomic scale. These profiles revealed distinct proliferative and functional states of islet α-cells or β-cells, and histone modifications underlying age-dependent gene expression changes. Expression of SIX2 and SIX3, transcription factors without prior known functions in the pancreas and linked to fasting hyperglycemia risk, increased with age specifically in human islet β-cells. SIX2 and SIX3 were sufficient to enhance insulin content or secretion in immature β-cells. Our work provides a unique resource to study human-specific regulators of islet cell maturation and function.

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-...

Menin, the product of the Men1 gene mutated in familial multiple endocrine neoplasia type 1 (MEN1), regulates transcription in differentiated cells. Menin associates with and modulates the histone methyltransferase activity of a nuclear protein complex to activate gene expression. However, menin-dependent histone methyltransferase activity in endocrine cells has not been demonstrated, and the mechanism of endocrine tumor suppression by menin remains unclear. Here, we show that menin-dependent histone methylation maintains the in vivo expression of cyclindependent kinase (CDK) inhibitors to prevent pancreatic islet tumors. In vivo expression of CDK inhibitors, including p27 and p18, and other cell cycle regulators is disrupted in mouse islet tumors lacking menin. Chromatin immunoprecipitation studies reveal that menin directly associates with regions of the p27 and p18 promoters and increases methylation of lysine 4 (Lys-4) in histone H3 associated with these promoters. Moreover, H3 Lys-4 methylation associated with p27 and p18 is reduced in islet tumors from Men1 mutant mice. Thus, H3 Lys-4 methylation is a crucial function of menin in islet tumor suppression. These studies suggest an epigenetic mechanism of tumor suppression: by promoting histone modifications, menin maintains transcription at multiple loci encoding cell cycle regulators essential for endocrine growth control.islet of Langerhans ͉ Men1 ͉ multiple endocrine neoplasia ͉ tumor suppressor ͉ diabetes mellitus

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.