OBJECTIVE The aim of this study was to elucidate whether age plays a role in the expansion or regeneration of β-cell mass. RESEARCH DESIGN AND METHODS We analyzed the capacity of β-cell expansion in 1.5- and 8-month-old mice in response to a high-fat diet, after short-term treatment with the glucagon-like peptide 1 (GLP-1) analog exendin-4, or after streptozotocin (STZ) administration. RESULTS Young mice responded to high-fat diet by increasing β-cell mass and β-cell proliferation and maintaining normoglycemia. Old mice, by contrast, did not display any increases in β-cell mass or β-cell proliferation in response to high-fat diet and became diabetic. To further assess the plasticity of β-cell mass with respect to age, young and old mice were injected with a single dose of STZ, and β-cell proliferation was analyzed to assess the regeneration of β-cells. We observed a fourfold increase in β-cell proliferation in young mice after STZ administration, whereas no changes in β-cell proliferation were observed in older mice. The capacity to expand β-cell mass in response to short-term treatment with the GLP-1 analog exendin-4 also declined with age. The ability of β-cell mass to expand was correlated with higher levels of Bmi1, a polycomb group protein that is known to regulate the Ink4a locus, and decreased levels of p16 Ink4a expression in the β-cells. Young Bmi1 −/− mice that prematurely upregulate p16 Ink4a failed to expand β-cell mass in response to exendin-4, indicating that p16 Ink4a levels are a critical determinant of β-cell mass expansion. CONCLUSIONS β-Cell proliferation and the capacity of β-cells to regenerate declines with age and is regulated by the Bmi1/p16 Ink4a pathway.

Summary Adult pancreatic beta cells can replicate during growth and after injury to maintain glucose homeostasis. Here we report that beta cells deficient in Dnmt1, an enzyme that propagates DNA methylation patterns during cell division, were converted to alpha cells. We identified the lineage determination gene aristaless related homeobox (Arx), as methylated and repressed in beta cells, and hypo-methylated and expressed in alpha cells and Dnmt1-deficient beta cells. We show that the methylated region of the Arx locus in beta cells was bound by methyl binding protein MeCP2 which recruited PRMT6, an enzyme that methylates histone H3R2 resulting in repression of Arx. This suggests that propagation of DNA methylation during cell division also ensures recruitment of enzymatic machinery capable of modifying and transmitting histone marks. Our results reveal that propagation of DNA methylation during cell division is essential for repression of alpha cell lineage determination genes to maintain pancreatic beta cell identity.

The molecular mechanisms that regulate the ageinduced increase of p16INK4a expression associated with decreased b-cell proliferation and regeneration are not well understood. We report that in aged islets, derepression of the Ink4a/Arf locus is associated with decreased Bmi-1 binding, loss of H2A ubiquitylation, increased MLL1 recruitment, and a concomitant increase in H3K4 trimethylation. During b-cell regeneration these histone modifications are reversed resulting in reduced p16INK4a expression and increased proliferation. We suggest that PcG and TrxG proteins impart a combinatorial code of histone modifications on the Ink4a/Arf locus to control b-cell proliferation during aging and regeneration.Supplemental material is available at http://www.genesdev.org.Received September 18, 2008; revised version accepted March 6, 2009. Emerging evidence indicates that proliferation of pancreatic b cells is an important mechanism not only to maintain homeostasis in the endocrine pancreas but also for adapting islet function to changes in metabolic demands (Dor et al. 2004;Kulkarni et al. 2004;Okamoto et al. 2006;Zhong et al. 2007). The inability of the b cells to expand and compensate for the changing insulin demand can contribute to the pathogenesis of diabetes. Several studies suggest that b-cell proliferation declines with age (Teta et al. 2005;Meier et al. 2008) and this age-dependent decline in the b-cell proliferation could curtail the ability of the endocrine pancreas to respond to metabolic changes. However, the cell-intrinsic genetic and epigenetic mechanisms regulating the age-dependent decline of b-cell proliferation are not very clear.The products of the Ink4a/Arf (Cdkn2a) locus, p16 Ink4a(a negative regulator of CDK4-D-type cyclins) (Kim and Sharpless 2006) and p19 Arf show increased expression with age (Nielsen et al. 1999;Krishnamurthy et al. 2004) and have been linked to reduction in the proliferative capacity of aged b cells . Genome-wide studies have linked the Ink4/Arf locus with type 2 diabetes and impaired insulin release, suggesting that failure of islets to expand may play an important role in type 2 diabetes (for review, see Doria et al. 2008). Thus, understanding the regulation of Ink4a/ Arf locus could reveal the molecular basis of reduced b-cell proliferation with aging and also be extremely useful in devising strategies to foster b-cell regeneration.Several genetic studies show that a polycomb group (PcG) protein, Bmi-1, is essential for maintenance of adult stem cell self-renewal primarily due to its ability to repress the Ink4a/Arf locus (Jacobs et al. 1999;Lessard and Sauvageau 2003;Molofsky et al. 2003;Bruggeman et al. 2005). Bmi-1, along with Ring1 and Ring2, is a part of the polycomb-repressive complex 1 (PRC1), which possesses histone H2A-K119 ubiquitin E3 ligase activity, required for the repression of Hox genes (Wang et al. 2004). While Ring2 is the catalytic subunit, Bmi-1 maintains the integrity of the complex and is essential for enzymatic activity (Cao et al. 2005). Analysis in ...

Regulation of cell differentiation programs requires complex interactions between transcriptional and epigenetic networks. Elucidating the principal molecular events responsible for the establishment and maintenance of cell fate identities will provide important insights into how cell lineages are specified and maintained and will improve our ability to recapitulate cell differentiation events in vitro. In this study, we demonstrate that Nkx2.2 is part of a large repression complex in pancreatic b cells that includes DNMT3a, Grg3, and HDAC1. Mutation of the endogenous Nkx2.2 tinman (TN) domain in mice abolishes the interaction between Nkx2.2 and Grg3 and disrupts b-cell specification. Furthermore, we demonstrate that Nkx2.2 preferentially recruits Grg3 and HDAC1 to the

The glucoincretin hormone glucagon-like peptide-1 (GLP-1) and its analog exendin-4 (Ex-4) promote β-cell growth and expansion. Here we report an essential role for Skp2, a substrate recognition component of SCF (Skp, Cullin, F-box) ubiquitin ligase, in promoting glucoincretin-induced β-cell proliferation by regulating the cellular abundance of p27. In vitro, GLP-1 treatment increases Skp2 levels, which accelerates p27 degradation, whereas in vivo, loss of Skp2 prevents glucoincretin-induced β-cell proliferation. Using inhibitors of phosphatidylinositol 3-kinase and Irs2 silencing RNA, we also show that the effects of GLP-1 in facilitating Skp2-dependent p27 degradation are mediated via the Irs2-phosphatidylinositol-3 kinase pathway. Finally, we show that down-regulation of p27 occurs in islets from aged mice and humans, although in these islets, age-dependent accumulation of p16(Ink4a) prevent glucoincretin-induced β-cell proliferation; however, ductal cell proliferation is maintained. Taken together, these data highlight a critical role for Skp2 in glucoincretin-induced β-cell proliferation.