Anil Bhushan

β-Cell Replication Is the Primary Mechanism Subserving the Postnatal Expansion of β-Cell Mass in Humans

OBJECTIVE— Little is known about the capacity, mechanisms, or timing of growth in β-cell mass in humans. We sought to establish if the predominant expansion of β-cell mass in humans occurs in early childhood and if, as in rodents, this coincides with relatively abundant β-cell replication. We also sought to establish if there is a secondary growth in β-cell mass coincident with the accelerated somatic growth in adolescence. RESEARCH DESIGN AND METHODS— To address these questions, pancreas volume was determined from abdominal computer tomographies in 135 children aged 4 weeks to 20 years, and morphometric analyses were performed in human pancreatic tissue obtained at autopsy from 46 children aged 2 weeks to 21 years. RESULTS— We report that 1) β-cell mass expands by severalfold from birth to adulthood, 2) islets grow in size rather than in number during this transition, 3) the relative rate of β-cell growth is highest in infancy and gradually declines thereafter to adulthood with no secondary accelerated growth phase during adolescence, 4) β-cell mass (and presumably growth) is highly variable between individuals, and 5) a high rate of β-cell replication is coincident with the major postnatal expansion of β-cell mass. CONCLUSIONS— These data imply that regulation of β-cell replication during infancy plays a major role in β-cell mass in adult humans.

β cell replication is the primary mechanism for maintaining postnatal β cell mass

The endocrine pancreas undergoes major remodeling during neonatal development when replication of differentiated β cells is the major mechanism by which β cell mass is regulated. The molecular mechanisms that govern the replication of terminally differentiated β cells are unclear. We show that during neonatal development, cyclin D2 expression in the endocrine pancreas coincides with the replication of endocrine cells and a massive increase in islet mass. Using cyclin D2–/– mice, we demonstrate that cyclin D2 is required for the replication of endocrine cells but is expendable for exocrine and ductal cell replication. As a result, 14-day-old cyclin D2–/– mice display dramatically smaller islets and a 4-fold reduction in β cell mass in comparison to their WT littermates. Consistent with these morphological findings, the cyclin D2–/– mice are glucose intolerant. These results suggest that cyclin D2 plays a key role in regulating the transition of β cells from quiescence to replication and may provide a target for the development of therapeutic strategies to induce expansion and/or regeneration of β cells.

Sustained beta cell apoptosis in patients with long-standing type 1 diabetes: indirect evidence for islet regeneration?

Aims/hypothesisType 1 diabetes is widely held to result from an irreversible loss of insulin-secreting beta cells. However, insulin secretion is detectable in some people with long-standing type 1 diabetes, indicating either a small population of surviving beta cells or continued renewal of beta cells subject to ongoing autoimmune destruction. The aim of the present study was to evaluate these possibilities.Materials and methodsPancreatic sections from 42 individuals with type 1 diabetes and 14 non-diabetic individuals were evaluated for the presence of beta cells, beta cell apoptosis and replication, T lymphocytes and macrophages. The presence and extent of periductal fibrosis was also quantified.ResultsBeta cells were identified in 88% of individuals with type 1 diabetes. The number of beta cells was unrelated to duration of disease (range 4–67 years) or age at death (range 14–77 years), but was higher (p<0.05) in individuals with lower mean blood glucose. Beta cell apoptosis was twice as frequent in type 1 diabetes as in control subjects (p<0.001), but beta cell replication was rare in both groups. The increased beta cell apoptosis in type 1 diabetes was accompanied by both increased macrophages and T lymphocytes and a marked increase in periductal fibrosis (p<0.001), implying chronic inflammation over many years, consistent with an ongoing supply of beta cells.Conclusions/interpretationMost people with long-standing type 1 diabetes have beta cells that continue to be destroyed. The mechanisms underlying increased beta cell death may involve both ongoing autoimmunity and glucose toxicity. The presence of beta cells despite ongoing apoptosis implies, by definition, that concomitant new beta cell formation must be occurring, even after long-standing type 1 diabetes. We conclude that type 1 diabetes may be reversed by targeted inhibition of beta cell destruction.

Age-Dependent Decline in β-Cell Proliferation Restricts the Capacity of β-Cell Regeneration in Mice

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 p16Ink4aexpression in the β-cells. Young Bmi1−/− mice that prematurely upregulate p16Ink4afailed to expand β-cell mass in response to exendin-4, indicating that p16Ink4alevels 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/p16Ink4apathway.

The replication of beta cells in normal physiology, in disease and for therapy.

Replication of β cells is an important source of β-cell expansion in early childhood. The recent linkage of type 2 diabetes with several transcription factors involved in cell cycle regulation implies that growth of the β-cell mass in early childhood might be an important determinant of risk for type 2 diabetes. Under some circumstances, including obesity and pregnancy, the β-cell mass is adaptively increased in adult humans. The mechanisms by which this adaptive growth occurs and the relative contributions of β-cell replication or of mechanisms independent of β-cell replication are unknown. Also, although there is interest in the potential for β-cell regeneration as a therapeutic approach in both type 1 and 2 diabetes, little is yet known about the potential sources of new β cells in adult humans. In common with other cell types, replicating β cells have an increased vulnerability to apoptosis, which is likely to limit the therapeutic value of inducing β-cell replication in the proapoptotic environment of type 1 and 2 diabetes unless applied in conjunction with a strategy to suppress increased apoptosis.

Bmi-1 regulates the Ink4a/Arf locus to control pancreatic β-cell proliferation

The molecular mechanisms that regulate the age-induced increase of p16(INK4a) expression associated with decreased beta-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 beta-cell regeneration these histone modifications are reversed resulting in reduced p16(INK4a) 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 beta-cell proliferation during aging and regeneration.

Pancreatic β Cell Identity Is Maintained by DNA Methylation-Mediated Repression of Arx

Adult pancreatic β cells can replicate during growth and after injury to maintain glucose homeostasis. Here, we report that β cells deficient in Dnmt1, an enzyme that propagates DNA methylation patterns during cell division, were converted to α cells. We identified the lineage determination gene aristaless-related homeobox (Arx), as methylated and repressed in β cells, and hypomethylated and expressed in α cells and Dnmt1-deficient β cells. We show that the methylated region of the Arx locus in β 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 α cell lineage determination genes to maintain pancreatic β cell identity.

RLIM inhibits functional activity of LIM homeodomain transcription factors via recruitment of the histone deacetylase complex

LIM domains are required for both inhibitory effects on LIM homeodomain transcription factors and synergistic transcriptional activation events. The inhibitory actions of the LIM domain can often be overcome by the LIM co-regulator known as CLIM2, LDB1 and NLI (referred to hereafter as CLIM2; refs 2, 3, 4). The association of the CLIM cofactors with LIM domains does not, however, improve the DNA-binding ability of LIM homeodomain proteins, suggesting the action of a LIM-associated inhibitor factor. Here we present evidence that LIM domains are capable of binding a novel RING-H2 zinc-finger protein, Rlim (for RING finger LIM domain-binding protein), which acts as a negative co-regulator via the recruitment of the Sin3A/histone deacetylase corepressor complex. A corepressor function of RLIM is also suggested by in vivo studies of chick wing development. Overexpression of the gene Rnf12, encoding Rlim, results in phenotypes similar to those observed after inhibition of the LIM homeodomain factor LHX2, which is required for the formation of distal structures along the proximodistal axis, or by overexpression of dominant-negative CLIM1. We conclude that Rlim is a novel corepressor that recruits histone deacetylase-containing complexes to the LIM domain.

Nkx2.2 repressor complex regulates islet β-cell specification and prevents β-to-α-cell reprogramming

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 β 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 β-cell specification. Furthermore, we demonstrate that Nkx2.2 preferentially recruits Grg3 and HDAC1 to the methylated Aristaless homeobox gene (Arx) promoter in β cells. The Nkx2.2 TN mutation results in ectopic expression of Arx in β cells, causing β-to-α-cell transdifferentiation. A corresponding β-cell-specific deletion of DNMT3a is also sufficient to cause Arx-dependent β-to-α-cell reprogramming. Notably, subsequent removal of Arx in the β cells of Nkx2.2(TNmut/TNmut) mutant mice reverts the β-to-α-cell conversion, indicating that the repressor activities of Nkx2.2 on the methylated Arx promoter in β cells are the primary regulatory events required for maintaining β-cell identity.

p27 Regulates the Transition of β-Cells From Quiescence to Proliferation

Diabetes results from an inadequate mass of functional β-cells. Such inadequacy could result from loss of β-cells due to an immune assault or the inability to compensate for insulin resistance. Thus, mechanisms that regulate the number of β-cells will be key to understanding both the pathogenesis of diabetes and for developing therapies. In this study, we show that cell cycle regulator p27 plays a crucial role in establishing the number of β-cells formed before birth. We show that p27 accumulates in terminally differentiated β-cells during embryogenesis. Disabling p27 allows newly differentiated β-cells that are normally quiescent during embryogenesis to reenter the cell cycle and proliferate. As a consequence, excess β-cells are generated in the p27−/− mice, doubling their β-cell mass at birth. The early postnatal expansion of β-cell mass was unaffected in p27−/− mice, indicating that the main function of p27 is to maintain the quiescent state of newly differentiated β-cells generated during embryogenesis. The expanded β-cell mass was accompanied by increased insulin secretion; however, the p27−/− mice were glucose intolerant, as these mice were insulin insensitive. To assess the role of p27 to affect regeneration of β-cells in models of diabetes, p27−/− mice were injected with streptozotocin (STZ). In contrast to control mice that displayed elevated blood glucose levels, p27−/− mice showed decreased susceptibility to develop STZ-induced diabetes. Furthermore, β-cells retained the ability to reenter the cell cycle at a far greater frequency in p27−/− mice after developing STZ-induced diabetes compared with wild-type littermates. These data indicate that p27 is a key regulator in establishing β-cell mass and an important target for facilitating β-cell regeneration in therapies for diabetes.