Hepatocyte growth factor (HGF) is produced in pancreatic mesenchyme-derived cells and in islet cells. In vitro, HGF increases the insulin content and proliferation of islets. To study the role of HGF in the islet in vivo, we have developed three lines of transgenic mice overexpressing mHGF using the rat insulin II promoter (RIP). Each RIP-HGF transgenic line displays clear expression of HGF mRNA and protein in the islet. RIPmHGF mice are relatively hypoglycemic in post-prandial and fasting states compared with their normal littermates. They display inappropriate insulin production, striking overexpression of insulin mRNA in the islet, and a 2-fold increase in the insulin content in islet extracts. Importantly, beta cell replication rates in vivo are two to three times higher in RIP-HGF mice. This increase in proliferation results in a 2-3-fold increase in islet mass. Moreover, the islet number per pancreatic area was also increased by approximately 50%. Finally, RIP-mHGF mice show a dramatically attenuated response to the diabetogenic effects of streptozotocin. We conclude that the overexpression of HGF in the islet increases beta cell proliferation, islet number, beta cell mass, and total insulin production in vivo. These combined effects result in mild hypoglycemia and resistance to the diabetogenic effects of streptozotocin. Hepatocyte growth factor (HGF)1 is a mesenchyme-derived protein originally identified as a circulating factor implicated in liver regeneration after hepatic injury or hepatectomy (1-3). It is now recognized that HGF also exhibits its mitogenic, motogenic, and morphogenic activities in a wide variety of cells (4, 5). The active form of HGF is a disulfide-linked heterodimeric protein, which is composed of a 69-kDa ␣-chain and a 34-kDa ␤-chain, containing four kringle domains and a serine protease-like domain, respectively. Active HGF derives from an inactive single chain precursor that is processed and activated by proteolysis. Four proteases have been reported to date to activate HGF in vitro, including blood coagulation factor XIIa, urokinase, tissue-type plasminogen activator, and a serumderived serine protease named HGF activator (6 -9). HGF is primarily a paracrine factor produced by mesenchymal cells that acts on epithelial cells through a membrane-spanning tyrosine kinase receptor, the protein product of the proto-oncogene, c-met (5, 10, 11). The receptor, like the ligand, has a widespread distribution.Messenger RNAs encoding HGF and the HGF receptor, cmet, are highly expressed during the early development of the pancreas, and then maintained at a low level during puberty and adult life (12)(13)(14). HGF has been detected immunohistochemically in the exocrine portion of rabbit pancreas, and in rat and human pancreatic islet cells (15-17). Tissue-type plasminogen activator has been detected in the rat endocrine pancreas, preferentially in somatostatin cells (18). In addition, confocal immunofluorescent studies have preferentially colocalized the c-Met receptor protein to insulin-conta...

Highlights d Adult human pancreatic beta cells can be induced to proliferate at high rates d Driven by synergy between DYRK1A inhibitors and TGFb superfamily inhibitors d Reflects activation of cyclins and CDKs accompanied by CDK inhibitor suppression d Proliferation occurs in type 2 diabetic beta cells, with enhanced differentiation SUMMARYSmall-molecule inhibitors of dual-specificity tyrosine-regulated kinase 1A (DYRK1A) induce human beta cells to proliferate, generating a labeling index of 1.5%-3%. Here, we demonstrate that combined pharmacologic inhibition of DYRK1A and transforming growth factor beta superfamily (TGFbSF)/SMAD signaling generates remarkable further synergistic increases in human beta cell proliferation (average labeling index, 5%-8%, and as high as 15%-18%), and increases in both mouse and human beta cell numbers. This synergy reflects activation of cyclins and cdks by DYRK1A inhibition, accompanied by simultaneous reductions in key cell-cycle inhibitors (CDKN1C and CDKN1A). The latter results from interference with the basal Trithorax-and SMAD-mediated transactivation of CDKN1C and CDKN1A.Notably, combined DYRK1A and TGFb inhibition allows preservation of beta cell differentiated function. These beneficial effects extend from normal human beta cells and stem cell-derived human beta cells to those from people with type 2 diabetes, and occur both in vitro and in vivo.

Type 1 and type 2 diabetes both result from inadequate production of insulin by the beta-cells of the pancreatic islet. Accordingly, strategies that lead to increased pancreatic beta-cell mass, as well as retained or enhanced function of islets, would be desirable for the treatment of diabetes. Although pancreatic beta-cells have long been viewed as terminally differentiated and irreversibly arrested, evidence now indicates that beta-cells can and do replicate, that this replication can be enhanced by a variety of maneuvers, and that beta-cell replication plays a quantitatively significant role in maintaining pancreatic beta-cell mass and function. Because beta-cells have been viewed as being unable to proliferate, the science of beta-cell replication is undeveloped. In the past several years, however, this has begun to change at a rapid pace, and many laboratories are now focused on elucidating the molecular details of the control of cell cycle in the beta-cell. In this review, we review the molecular details of cell cycle control as they relate to the pancreatic beta-cell. Our hope is that this review can serve as a common basis and also a roadmap for those interested in developing novel strategies for enhancing beta-cell replication and improving insulin production in animal models as well as in human pancreatic beta-cells.

Recent advances in human islet transplantation have highlighted the need for expanding the pool of beta-cells available for transplantation. We have developed three transgenic models in which growth factors (hepatocyte growth factor [HGF], placental lactogen, or parathyroid hormone-related protein) have been targeted to the beta-cell using rat insulin promoter (RIP). Each displays an increase in islet size and islet number, and each displays insulin-mediated hypoglycemia. Of these three models, the RIP-HGF mouse displays the least impressive phenotype under basal conditions. In this study, we show that this mild basal phenotype is misleading and that RIP-HGF mice have a unique and salutary phenotype. Compared with normal islets, RIP-HGF islets contain more insulin per beta-cell (50 +/- 5 vs. 78 +/- 9 ng/islet equivalent [IE] in normal vs. RIP-HGF islets, P < 0.025), secrete more insulin in response to glucose in vivo (0.66 +/- 0.06 vs. 0.91 +/- 0.10 ng/ml in normal vs. RIP-HGF mice, P < 0.05) and in vitro (at 22.2 mmol/l glucose: 640 +/- 120.1 vs. 1,615 +/- 196.9 pg. microg protein(-1). 30 min(-1) in normal vs. RIP-HGF islets, P < 0.01), have two- to threefold higher GLUT2 and glucokinase steady-state mRNA levels, take up and metabolize glucose more effectively, and most importantly, function at least twice as effectively after transplantation. These findings indicate that HGF has surprisingly positive effects on beta-cell mitogenesis, glucose sensing, beta-cell markers of differentiation, and transplant survival. It appears to have a unique and unanticipated effective profile as an islet mass- and function-enhancing agent in vivo.

A lthough it is currently very clear that ␤-cells can replicate, albeit slowly, in vitro and in vivo, both basally and in response to a variety of maneuvers and stimuli (rev. in 1-3), the key components of the cell cycle machinery in the ␤-cell and the factors that regulate them are poorly understood at a molecular level. The retinoblastoma protein (pRb) (rev. in 4 -6) is a key gap-1/synthesis phase (G 1 /S) checkpoint gatekeeper. In its dephosphorylated (or active) state, pRb binds to the E2F family of cell cycle regulatory genes and leads to the transcriptional repression of downstream genes, with resultant cell cycle arrest. Conversely, when pRb is phosphorylated to form ppRb, it becomes inactive and releases E2Fs, removing transcriptional repression of critical cell cycle genes, and the cell cycle progresses. pRb can be phosphorylated by a number of kinases. These include cyclin-dependent kinase (cdk)-4 and -6, which form complexes with the D cyclins, as well as cyclin E and cdk-2, which also form a complex. Because of their importance, these cyclins and cdks are under tight regulatory control themselves. This regulation is principally inhibitory and is accomplished by inhibitory kinases or cyclin inhibitor proteins such as p16, p18, p21, p27, p53, p57, and others.The majority of the information described above on pRb and ppRb has been obtained in human and animal cancers and fibroblasts (4 -6). Surprisingly, given the current attention on ␤-cell replication, little is known regarding molecular control of the cell cycle in the ␤-cell. For example, we are unaware of any study examining either the presence of, or the phosphorylation status of, pRb in human or animal ␤-cells. On the other hand, there are some data that point to this pathway as being critical to the control of the cell cycle in ␤-cells. For example, SV-40 large T-antigen (TAg) is a transforming viral protein that interacts with p53 and pRb. TAg has been overexpressed in the ␤-cell of transgenic "RIP-TAg" mice and in cultured ␤-cell lines by Hanahan (7) and , and increased ␤-cell replication resulted. Ultimately, autonomous ␤-cell tumors develop in RIP-TAg mice.Homozygous disruption of the pRb gene in mice results in embryonic lethality (10). Heterozygous deletion results in adult animals characterized by the development of