We hypothesized that combined transgenic overexpression of hepatocyte growth factor (HGF) and placental lactogen in islets would lead to even greater increases in ␤-cell mass and replication than either growth factor alone. This did not occur, suggesting that ␤-cell replication is saturable or subject to molecular restraint. We therefore performed the first comprehensive G 1 /S cell cycle survey in islets, cataloguing the broad range of kinases, cyclins, and kinase inhibitors that control the G 1 /S transition in islets from normal, HGF, placental lactogen, and doubly transgenic mice. Many of the G 1 /S checkpoint regulators (E2Fs; pRb; p107; p130; cyclins D 1 , 2 , 3 , A, and E; cdk-2; cdk-4; p15; p16; p18; p19; p21; p27; MDM2; p53; c-Myc; and Egr-1) are present in the murine islet. Most of these proteins were unaltered by overexpression of HGF or placental lactogen, either alone or in combination. In contrast, p21 cip was uniquely, dramatically, and reproducibly upregulated in placental lactogen and HGF islets. p21 cip was also present in, and upregulated in, proliferating human islets, localizing specifically in ␤-cells and translocating to the nucleus on mitogenic stimulation. Homozygous p21 cip loss releases islets from growth inhibition, markedly enhancing proliferation in response to HGF and placental lactogen. Diabetes 55:70 -77, 2006 P rogress in pancreatic islet transplantation (1) and in immunosuppression directed against the autoimmunity of type 1 diabetes (2) has focused attention on a need to understand the cellular mechanisms that control ␤-cell replication. Surprisingly, little is known regarding the molecular control of cell cycle events in the ␤-cell, in part, because of a longstanding belief that ␤-cells cannot replicate and that, if induced to replicate, they inevitably would de-differentiate. In contrast, we (3-5) and others (6 -10) have demonstrated that human and rodent ␤-cells can replicate and retain their differentiated phenotype in vivo. A recent study has suggested that replication of ␤-cells is the principal mechanism through which new ␤-cells are generated (6). Thus, a clear and comprehensive understanding of ␤-cell replication will be important for developing novel approaches to treating and preventing diabetes.Important lessons regarding ␤-cell replication have been revealed from mouse genetic models. For example, combined knockout of the p53 and pRb tumor suppressor genes leads to pancreatic islet tumors (11,12). Similarly, overexpression of simian virus 40 viral large T-antigen also leads to islet hyperplasia, and ultimately to islet tumorigenesis (13). Cyclin D 1 is the most abundant oncogene in human insulinomas (14), and cyclin D 1 overexpression leads to ␤-cell hyperplasia in transgenic mice (15). Cyclin D 2 has been shown to be essential for achieving and maintaining ␤-cell mass (16,17) and deletion of E2F1 and E2F2 transcription factors results in ␤-cell failure (18,19). Knockout of cdk-4 results in ␤-cell failure and diabetic ketoacidosis (20,21), and overexpression of co...

Transgenic overexpression of either parathyroid hormone-related peptide (PTHrP) or mouse placental lactogen type 1 (mPL1) in pancreatic ␤-cells, using the rat insulin II promoter (RIP), results in islet hyperplasia either through prolonged ␤-cell survival or through increased ␤-cell proliferation and hypertrophy, respectively. For determining whether the two proteins might exert complementary, additive, or synergistic effects on islet mass and function when simultaneously overexpressed in ␤-cells in vivo, RIP-PTHrP and RIP-mPL1 mice were crossed to generate mice doubly transgenic for PTHrP and mPL1. These double-transgenic mice displayed marked islet hyperplasia (threefold), hypoglycemia, increased ␤-cell proliferation (threefold), and resistance to the diabetogenic and cytotoxic effects of streptozotocin compared with their normal siblings. Although the phenotype of the double-transgenic mice was neither additive nor synergistic relative to their single-transgenic counterparts, it was indeed complementary, yielding the maximal salutary phenotypic features of both individual transgenes. Finally, mPL1, for the first time, was shown to exert a protective effect on the survival of ␤-cells, placing it among the few proteins that can improve function and proliferation and prolong the survival of ␤-cells. Placental lactogen 1 is an attractive target for future therapeutic strategies in diabetes. Diabetes 53:3120 -3130, 2004 W e have developed three types of transgenic mice in which islet mass and number are increased substantially (two-to threefold) by the targeted overexpression of growth factors to the ␤-cell and that result in insulin-mediated hypoglycemia (1-3). In these three mouse models, the rat insulin II promoter (RIP) drives expression in pancreatic ␤-cells of cDNAs encoding parathyroid hormone-related protein (PTHrP), mouse placental lactogen-1 (mPL1), or hepatocyte growth factor. These transgenic mouse models arrive at what seem to be superficially similar phenotypes through distinct cellular mechanisms, via distinct receptors, and via distinct intracellular signaling pathways (1-6). In this study, we selected two of these models, the RIP-mPL1 and RIP-PTHrP mice, for further study.RIP-mPL1 mice exhibit a twofold increase in ␤-cell replication, accompanied by mild (20%) ␤-cell hypertrophy. These events in combination seem to cause the augmented islet mass and hypoglycemia observed in these mice. Glucose-stimulated insulin secretion is normal in islets isolated from these mice (2). In contrast, RIP-PTHrP mice seem to derive their increases in ␤-cell number and overall islet mass, not as a result of an increase in ␤-cell proliferation (4) but from a prolongation of ␤-cell survival (6). Compared with their normal littermates, RIP-PTHrP mice are resistant to the diabetogenic effects of streptozotocin (STZ) and also display significantly diminished rates of ␤-cell death after STZ administration (4,6). Glucosestimulated insulin secretion is also normal in islets isolated from these mice (1).The signaling mechanis...

Pancreatic ␤-cell survival is critical in the setting of diabetes as well as in islet transplantation. Transgenic mice overexpressing parathyroid hormone-related protein (PTHrP) targeted to ␤-cells using the rat insulin II promoter (RIP) display hyperinsulinemia, hypoglycemia, and islet hyperplasia, without a concomitant increase in ␤-cell proliferation rate or enlargement of individual ␤-cell size. Thus, the mechanism for increased ␤-cell mass is unknown. In this study, we demonstrated that ␤-cells of transgenic mice are resistant to the cytotoxic effects of streptozotocin (STZ) in vivo, as documented by a sixfold reduction in the rate of STZ-induced ␤-cell death in RIP-PTHrP mice relative to their normal siblings. The reduced cell death in transgenic mice is due neither to their increased islet mass nor to a decrease in their sensing of STZ, but rather results from PTHrP-induced resistance to ␤-cell death. This is also demonstrated in vitro by markedly reduced cell death rates observed in ␤-cells of transgenic mice compared with normal mice when cultured in the absence of serum and glucose or in the presence of STZ. Finally, we demonstrated that NH 2 -terminal PTHrP inhibits ␤-cell death. These findings support the concept that PTHrP overexpression increases islet mass in transgenic mice through inhibition of ␤-cell death. Diabetes 51:3003-3013, 2002 F ailure of ␤-cell survival is critical to the etiology of type 1 (1-3) and type 2 (4,5) diabetes, as well as in the setting of islet transplantation (6,7). Despite an enormous increase in our understanding of islet differentiation and development, there is sparse information regarding the factors and pathways that regulate growth, survival, and death of islet cells. A number of approaches have been taken, including development of knockout and transgenic mouse models, to address these questions. Of the several ␤-cell-targeted transgenic mouse models generated to date, only a handful have displayed an increase in islet mass without displaying a concomitant negative effect on islet structure or function (8 -12). The mechanisms through which the targeted proteins have brought about islet mass expansion are quite distinct. These mechanisms include accelerated replication of preexisting ␤-cells (9 -12), an increase in ␤-cell size (hypertrophy) (9,11,12), a reduction in the rate of ␤-cell death (13,14), and an augmentation of islet neogenesis (11,12,15).Parathyroid hormone-related protein (PTHrP), widely expressed in most tissues of the body in the fetus and adult, is also expressed in the four endocrine cell types of the islet and in pancreatic ductal cells (16). PTHrP receptors have been shown to be present in islets (17) and the ␤-cell line RINm5F (18). Transgenic mice overexpressing PTHrP as well as PTHrP-knockout mice have demonstrated critical roles for this peptide in the development and differentiation of many organs, including the skeleton (19,20), mammary gland (21), skin (22), teeth (23), vascular system (24), and others. To examine the consequences of PTHrP ...

Animal studies show that G 1/S regulatory molecules (Dcyclins, cdk-4, p18, p21, p27) are critical for normal regulation of ␤-cell proliferation, mass, and function. The retinoblastoma protein, pRb, is positioned at the very end of a cascade of these regulatory proteins and is considered the final checkpoint molecule that maintains ␤-cell cycle arrest. Logically, removal of pRb from the ␤-cell should result in unrestrained ␤-cell replication, increased ␤-cell mass, and insulin-mediated hypoglycemia. Because global loss of both pRb alleles is embryonic lethal, this hypothesis has not been tested in ␤-cells. We developed two types of conditional knockout (CKO) mice in which both alleles of the pRb gene were inactivated specifically in ␤-cells. Surprisingly, although the pRb gene was efficiently recombined in ␤-cells of both CKO models, changes in ␤-cell mass, ␤-cell replication rates, insulin concentrations, and blood glucose levels were limited or absent. Other pRb family members, p107 and p130, were not substantially upregulated. In contrast to dogma, the pRb protein is not essential to maintain cell cycle arrest in the pancreatic ␤-cell. This may reflect fundamental inaccuracies in models of ␤-cell cycle control or complementation for pRb by undefined proteins. Diabetes 56:57-64, 2007