Overexpression of Cyclin D1 in Pancreatic -Cells In Vivo Results in Islet Hyperplasia Without Hypoglycemia Xiaoboo Zhang, 1 John P. Gaspard, 1 Yusuke Mizukami, 1 Jingnan Li, 1 Fiona Graeme-Cook, 2 and Daniel C. Chung 1 Cyclin D1 can stimulate proliferation by driving cells from the G1 into the S-phase of the mammalian cell cycle. Previous animal studies have implicated the G1-S transition as a key regulatory checkpoint governing the proliferation of pancreatic islet cells. We expressed cyclin D1 in the -cells of mice and islet hyperplasia developed in a time-dependent manner. The hyperplas- tic -cells exhibited higher rates of proliferation. How- ever, blood glucose levels in fasting as well as nonfasting conditions remained normal. Furthermore, glucose tolerance tests demonstrated nearly normal responses, and diabetes did not develop in any of the animals. No islet cell tumors were observed, even among animals >2 years of age. Under our experimental con- ditions, the proliferative stimulus provided by cyclin D1 is not tumorigenic, does not result in diabetes, and does not result in hypoglycemia. Cyclin D1 may thus be considered a potential candidate to augment the -cell population ex vivo as a prelude to islet transplantation for diabetes. Diabetes 54:712–719, 2005 N ew -cells can arise from fully differentiated -cells through the process of replication, and growth factors including parathyroid hormone– related peptide, hepatocyte growth factor, glucagon-like peptide-1/exendin-4, prolactin, growth hor- mone, IGF-I, and IGF-II have demonstrated important roles (1– 6). Understanding the complex molecular mech- anisms that regulate growth and proliferation of the -cell has tremendous implications for the design of strategies aimed at -cell replacement therapy in diabetes. The replication rate of -cells in the mature pancreatic islet is low and estimated to be 3% in rodents (7). Replication of islet cells is balanced by apoptosis. Inter- estingly, total -cell mass in the rodent has been demon- strated to gradually increase as animals age (8), and this is partially attributable to ongoing -cell replication. Several genetic mouse models have provided unique insights into some of the molecular signals that may control islet cell replication. Specifically, it has been demonstrated that genetic manipulations of the cell cycle machinery uniquely alter the proliferation of pancreatic islet cells. No pancre- atic islet phenotype was observed in mice deficient in either the Rb or p53 gene, but compound Rb +/- , p53 -/- knockout animals developed striking islet cell hyperplasia as well as islet cell tumors (9,10). One critical point at which the Rb and p53 pathways overlap is the G1 check- point of the cell cycle; Rb inhibits the E2F transcription factor that is necessary for progression of the cell cycle into S-phase (rev. in 11), and p53 also functions to arrest cells at the G1 checkpoint, in part through the induction of p21 (rev. in 12). These findings provided suggestive evi- dence that pancreatic islet cell growth may be tightly regulated at the G1 checkpoint. The hypothesis that the G1 checkpoint may be a key regulator of pancreatic -cell proliferation was strength- ened by in vivo studies of cyclin-dependent kinase (cdk)-4, an enyzme that directly regulates the G1 checkpoint through its phosphorylation of Rb. When cdk-4 was de- leted in the germline of mice, there were no widespread developmental defects (13,14). Rather, the primary pheno- types were infertility due to reduced cell numbers in the pituitary gland and diabetes from a marked reduction in -cells (15). When the constitutively active cdk-4 R24C allele was “knocked in” to the germline, dramatic -cell hyper- plasia was observed. These studies illustrated the pivotal role of cdk-4, a specific regulator of the G1 checkpoint, in -cell proliferation. Another critical regulator of the G1 checkpoint is cyclin D1, a specific catalyst for cdk-4 (rev. in 16). Overexpres- sion of cyclin D1 has been demonstrated in benign prolif- erations of several human endocrine tissues including parathyroid adenomas (17) and pancreatic islet cell ade- nomas (18), suggesting a role in the proliferation of islet cells in humans. In some highly differentiated cell types, such as neurons, overexpression of cyclin D1 induces apoptosis (19), but in other endocrine tissues, such as the parathyroid gland, there is enhanced proliferation (20). Recently, in vitro studies of cultured human and rat islets demonstrated that adenoviral transfer of cyclin D1 in- duced the replication of -cells (21). We therefore sought to determine whether expression of cyclin D1 would stimulate islet cell proliferation in vivo. Our studies re- vealed that targeted expression of cyclin D1 in pancreatic -cells of mice resulted in marked islet hyperplasia. Nota- From the 1 Gastrointestinal Unit, Department of Medicine, Massachusetts General Hospital Harvard Medical, Boston, Massachusetts; and the 2 Depart- ment of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts. Address correspondence and reprint requests to Daniel C. Chung, GRJ 825, Gastrointestinal Unit, Massachusetts General Hospital, 70 Blossom St., Bos- ton, MA 02114. E-mail: chung.daniel@mgh.harvard.edu. Received for publication 16 February 2004 and accepted in revised form 29 November 2004. cdk, cyclin-dependent kinase; H+E, hematoxylin and eosin; PCNA, prolif- erating cell nuclear antigen; TUNEL, transferase-mediated UTP nick-end labeling. © 2005 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 712 DIABETES, VOL. 54, MARCH 2005