Induction of beta-cell proliferation and retinoblastoma protein phosphorylation in rat and human islets using adenovirus-mediated transfer of cyclin-dependent kinase-4 and cyclin D1

The major regulator of the gap-1/synthesis phase (G(1)/S) cell cycle checkpoint is the retinoblastoma protein (pRb), and this is regulated in part by the activities of cyclin-dependent kinase (cdk)-4 and the D cyclins. Surprisingly, given the potential importance of beta-cell replication for islet replacement therapy, pRb presence, phosphorylation status, and function have not been explored in beta-cells. Here, adenoviruses expressing cdk-4 and cyclin D(1) were used to explore rat and human pRb phosphorylation and beta-cell cycle control. pRb is present in rat and human islets, and overexpression of cyclin D(1)/cdk-4 led to strikingly enhanced pRb phosphorylation in both species. Combined overexpression of both cdk-4 and cyclin D(1) caused a threefold increase in [(3)H]thymidine incorporation. This increase in proliferation was confirmed independently using insulin and bromodeoxyuridine immunohistochemistry, where human beta-cell replication rates were increased 10-fold. Cdk-4 or cyclin D(1) overexpression did not adversely effect beta-cell differentiation or function. The key cell cycle regulatory protein, pRb, can be harnessed to advantage using cyclin D(1)/cdk-4 for the induction of human and rodent beta-cell replication, enhancing replication without adversely affecting function or differentiation. This approach will allow detailed molecular study of the cellular mechanisms regulating the cell cycle in beta-cells, beta-cell lines, and stem cell-derived beta-cells.     

The cell cycle inhibitory protein p21cip is not essential for maintaining beta-cell cycle arrest or beta-cell function in vivo

p21(cip1), a regulatory molecule upstream of the G(1/0) checkpoint, is increased in beta-cells in response to mitogenic stimulation. Whereas p21(cip1) can variably stimulate or inhibit cell cycle progression, in vitro studies suggest that p21(cip1) acts as an inhibitor in the pancreatic beta-cell. To determine the functional role of p21(cip1) in vivo, we studied p21-null mice. Surprisingly, islet mass, beta-cell replication rates, and function were normal in p21-null mice. We next attempted to drive beta-cell replication in p21-null mice by crossing them with rat insulin II promoter-murine PL-1 (islet-targeted placental lactogen transgenic) mice. Even with this added replicative stimulus of PL, p21-null islets showed no additional stimulation. A G(1/S) proteome scan demonstrated that p21(cip1) loss was not associated with compensatory increases in other cell cycle inhibitors (pRb, p107, p130, p16, p19, and p27), although mild increases in p57 were apparent. Surprisingly, p18, which had been anticipated to increase, was markedly decreased. In summary, isolated p21(cip1) loss, as for pRb, p53, p18, and p27 and other inhibitors, results in normal beta-cell development and function, either because it is not essential or because its function is subserved or complimented by another protein. These studies underscore marked inhibitory pressure and the complexity and plasticity of inhibitory pathways that restrain beta-cell replication.     

Differential effects of p27 in regulation of beta-cell mass during development, neonatal period, and adult life

beta-Cell cycle progression and proliferation are critical to maintain beta-cell mass in adult mice. Of the cell cycle inhibitors, p27Kip1 is thought to be the primary modulator of the proliferative status in most cell types. p27 plays a role in beta-cell adaptation in genetic models of insulin resistance. To study the role of p27 in beta-cells during physiological conditions and at different stages of beta-cell differentiation, we studied mice deficient of or overexpressing p27. Experiments in p27-deficient mice showed improved glucose tolerance and hyperinsulinemia. These changes were associated with increased islet mass and proliferation. The experiments overexpressing p27 in beta-cells were performed using a doxycycline-inducible model. Interestingly, overexpression of p27 for 16 weeks in beta-cells from adult mice had no effect on glucose tolerance, beta-cell mass, or proliferation. In contrast, induction of p27 expression during beta-cell development or early neonatal period resulted in severe glucose intolerance and reduced beta-cell mass by decreased proliferation. These changes were reversible upon discontinuation of doxycycline. These experiments suggest that p27 is a critical molecule for beta-cell proliferation during beta-cell development and early postnatal life but not for maintenance of adult mass.     

Glucose infusion in mice: a new model to induce beta-cell replication

Developing new techniques to induce beta-cells to replicate is a major goal in diabetes research. Endogenous beta-cells replicate in response to metabolic changes, such as obesity and pregnancy, which increase insulin requirement. Mouse genetic models promise to reveal the pathways responsible for compensatory beta-cell replication. However, no simple, short-term, physiological replication stimulus exists to test mouse models for compensatory replication. Here, we present a new tool to induce beta-cell replication in living mice. Four-day glucose infusion is well tolerated by mice as measured by hemodynamics, body weight, organ weight, food intake, and corticosterone level. Mild sustained hyperglycemia and hyperinsulinemia induce a robust and significant fivefold increase in beta-cell replication. Glucose-induced beta-cell replication is dose and time dependent. Beta-cell mass, islet number, beta-cell size, and beta-cell death are not altered by glucose infusion over this time frame. Glucose infusion increases both the total protein abundance and nuclear localization of cyclin D2 in islets, which has not been previously reported. Thus, we have developed a new model to study the regulation of compensatory beta-cell replication, and we describe important novel characteristics of mouse beta-cell responses to glucose in the living pancreas.     

Effects of autoimmunity and immune therapy on beta-cell turnover in type 1 diabetes

beta-Cell mass can expand in response to demand: during pregnancy, in the setting of insulin resistance, or after pancreatectomy. It is not known whether similar beta-cell hyperplasia occurs following immune therapy of autoimmune diabetes, but the clinical remission soon after diagnosis and the results of recent immune therapy studies suggest that beta-cell recovery is possible. We studied changes in beta-cell replication, mass, and apoptosis in NOD mice during progression to overt diabetes and following immune therapy with anti-CD3 monoclonal antibodies (mAbs) or immune regulatory T-cells (Tregs). beta-Cell replication increases in pre-diabetic mice, after adoptive transfer of diabetes with increasing islet inflammation but before an increase in blood glucose concentration or a significant decrease in beta-cell mass. The pathogenic cells are responsible for increasing beta-cell replication because replication was reduced during diabetes remission induced by anti-CD3 mAb or Tregs. beta-Cell replication stimulated by the initial inflammatory infiltrate results in increased production of new beta-cells after immune therapy and increased beta-cell area, but the majority of this increased beta-cell area represents regranulated beta-cells rather than newly produced cells. We conclude that beta-cell replication is closely linked to the islet inflammatory process. A significant proportion of degranulated beta-cells remain, at the time of diagnosis of diabetes, that can recover after metabolic correction of hyperglycemia. Correction of the beta-cell loss in type 1 diabetes will, therefore, require strategies that target both the immunologic and cellular mechanisms that destroy and maintain beta-cell mass.     

Evaluation of beta-cell replication in mice transgenic for hepatocyte growth factor and placental lactogen: comprehensive characterization of the G1/S regulatory proteins reveals unique involvement of p21cip

We hypothesized that combined transgenic overexpression of hepatocyte growth factor (HGF) and placental lactogen in islets would lead to even greater increases in beta-cell mass and replication than either growth factor alone. This did not occur, suggesting that beta-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 beta-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.     

IRS proteins and beta-cell function

Insulin receptor substrate (IRS) proteins mediate a variety of the metabolic and growth-promoting actions of insulin and IGF-1. After phosphorylation by activated receptors, these intracellular signaling molecules recruit various downstream effector pathways including phosphatidylinositol 3-kinase and Grb2. Ablation of the IRS-2 gene produces a diabetic phenotype; mice lacking IRS-2 display peripheral insulin resistance and beta-cell dysfunction characterized by a 50% reduction in beta-cell mass. In contrast, deletion of IRS-1 retards somatic growth and enhances beta-cell mass. IRS1-/- mice are 50% smaller than controls but have a twofold increase in pancreatic beta-cell mass. Thus, observations from these recently developed animal models implicate the IRS signaling systems in the response of classical insulin target tissues, and they suggest a critical role for these proteins in the regulation of beta-cell function. In humans, type 2 diabetes generally occurs when insulin-secretory reserves fail to compensate for peripheral insulin resistance. Study and identification of the signals downstream of IRS proteins in beta-cells may provide unique insights into the compensatory mechanisms by which these cells respond to insulin resistance. Therefore, the intent of this review is to summarize recent observations regarding the regulation of beta-cell function by members of the IRS protein family.     

p27 Regulates the transition of beta-cells from quiescence to proliferation

Diabetes results from an inadequate mass of functional beta-cells. Such inadequacy could result from loss of beta-cells due to an immune assault or the inability to compensate for insulin resistance. Thus, mechanisms that regulate the number of beta-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 beta-cells formed before birth. We show that p27 accumulates in terminally differentiated beta-cells during embryogenesis. Disabling p27 allows newly differentiated beta-cells that are normally quiescent during embryogenesis to reenter the cell cycle and proliferate. As a consequence, excess beta-cells are generated in the p27(-/-) mice, doubling their beta-cell mass at birth. The early postnatal expansion of beta-cell mass was unaffected in p27(-/-) mice, indicating that the main function of p27 is to maintain the quiescent state of newly differentiated beta-cells generated during embryogenesis. The expanded beta-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 beta-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, beta-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 beta-cell mass and an important target for facilitating beta-cell regeneration in therapies for diabetes.     

beta-cell turnover: its assessment and implications

The pancreatic beta-cells are responsible for the maintenance of the body's glucose levels within a very narrow range; their population is dynamic and undergoes compensatory changes to maintain euglycemia. The structural parameters that allow mass changes (replication, neogenesis, cell volume changes, and cell death) can now be assessed and have proved to be powerful tools. Changes in one parameter can dramatically affect the beta-cell mass. Unfortunately, conclusions are often drawn on measurements that do not assess beta-cell mass but only relative volumes. Throughout the lifetime of a mammal, low levels of beta-cell replication and apoptosis are balanced and result in a slowly increasing mass. The balance allows gradual replacement of the beta-cell population; thus, beta-cells should be considered a slowly renewed tissue. Two major implications of beta-cell turnover are that 1) at any time, the beta-cells would be at different ages and 2) any limitation on replacement could have dire consequences for glucose homeostasis.     

Nuclear protein p8 is associated with glucose-induced pancreatic beta-cell growth

On its own, glucose is a major factor for proliferation of pancreatic beta-cells and is also an essential prerequisite for IGF-I and growth hormone-induced growth of these cells. p8 was originally identified as an emergency gene product upregulated in pancreatic acinar cells in response to acute pancreatitis. p8 was further shown to be involved in a broad range of biological functions, including cell growth, growth arrest, apoptosis, and tumor development. These in part opposite actions may be related to distinct stimuli and pathways in certain conditions and cell types. Here we demonstrate that p8 is widely expressed in human pancreatic islets in vivo and in several beta-cell lines in vitro. Based on this observation, we tested the hypothesis that p8 production in pancreatic beta-cells is regulated by glucose. Incubation of rat INS-1 beta-cells with 25 mmol/l glucose resulted in a continuous increase of proliferating cell numbers. This was accompanied by a strong upregulation of p8 mRNA and protein expression, indicating that p8 is a physiological mediator of glucose-induced pancreatic beta-cell growth. Binding of glucose-activated protein kinase C (PKC) to two PKC sites within a highly conserved region of the p8 protein may be a possible mechanism linking glucose and p8 pathways leading to proliferation.     

Heterogeneity in pancreatic beta-cell population.

All pancreatic beta-cells are identified by specific morphological characteristics. Similarity in microscopic features is not necessarily associated with identity in functional properties. In vitro studies on isolated rat beta-cells have indicated intercellular differences in the threshold for glucose-induced shifts in metabolic redox state. The cellular heterogeneity in glucose sensitivity results in a dose-dependent recruitment of glucose-exposed beta-cells into biosynthetic and secretory activities. The molecular basis of this diversity is not known. Indirect evidence supports the concept that the in situ pancreatic beta-cell population is also composed of functionally diverse subpopulations. The heterogeneity in glucose responsiveness is expected to create subpopulations of beta-cells with either constant, fluctuating, or occasional glucose-dependent functions; whether any subpopulation is preferentially responsive to other regulatory factors and/or committed to other activities is unknown. Morphological markers may help identify beta-cell subpopulations in situ and quantify their size in conditions known to affect total beta-cell mass or function. The concept of a functionally heterogeneous beta-cell population influences views on the role of pancreatic beta-cells in health and disease.     

Linear correlation between beta-cell mass and body weight throughout the lifespan in Lewis rats: role of beta-cell hyperplasia and hypertrophy

We determined the beta-cell replicative rate, beta-cell apoptosis, cross-sectional beta-cell area, and pancreatic beta-cell mass throughout the entire postweaning lifespan (months 1, 3, 7, 10, 15, and 20) of Lewis rats. Beta-cell replication was progressively reduced in the initial months of life but remained stable after month 7 (month 1, 0.99 +/- 0.10%; month 3, 0.24 +/- 0.04%; month 7, 0.12 +/- 0.02%; month 10, 0.14 +/- 0.02%; month 15, 0.10 +/- 0.03%; month 20, 0.13 +/- 0.03%; analysis of variance [ANOVA], P < 0.001). Beta-cell apoptosis was low and did not change significantly from month 1 to 20 of life. Cross-sectional area of individual beta-cells increased progressively in the initial months, remained stable from month 7 to 15, and increased again on month 20. The estimated number of beta-cells per pancreas, calculated as the ratio of total beta-cell mass to individual beta-cell mass, tripled from month 1 to 7 but did not change significantly thereafter. Beta-cell mass increased approximately 8 times from month 1 to 20 (month 1, 2.04 +/- 0.28 mg; month 20, 15.5 +/- 2.32 mg; ANOVA, P < 0.001) and showed a strong and significant linear correlation with body weight (r = 0.98, P < 0.001). In summary, we have shown that beta-cell replication was maintained throughout the lifespan in normal rats, clearly establishing that the beta-cell birth rate does not fall to 0, even in very old rats. Beta-cell mass increased throughout the lifespan, closely matching the increment in total body weight at any time point. This increment was selective for beta-cells, since the growth of the endocrine non-beta-cell mass was limited to the initial months of life. Both beta-cell hypertrophy and hyperplasia contributed to increased beta-cell mass in young animals, but only beta-cell hypertrophy was responsible for the increased beta-cell mass found in old animals. This study provides a global perspective for understanding the dynamics of beta-cell mass in young, adult, and aged animals.     

Replication increases beta-cell vulnerability to human islet amyloid polypeptide-induced apoptosis

Type 2 diabetes is characterized by a relative beta-cell deficit as a result of increased beta-cell apoptosis and islet amyloid derived from the beta-cell peptide islet amyloid polypeptide (IAPP). Human IAPP (h-IAPP) but not mouse IAPP (m-IAPP) induces apoptosis when applied to cells in culture, a property that depends on the propensity of h-IAPP to oligomerize. Since beta-cell mass is regulated, the question arises as to why it is not adaptively increased in response to insulin resistance and hyperglycemia in type 2 diabetes. This adaptation might fail if dividing beta-cells preferentially underwent apoptosis. We tested the hypothesis that beta-cells are preferentially vulnerable to h-IAPP-induced apoptosis. We established a microculture environment to perform time-lapse video microscopy (TLVM) and studied beta-cells (RIN) and HeLa cells undergoing replication or apoptosis. Sequential images (every 10 min for 36 h in RIN or 24 h in HeLa cells) of cells in vivo were analyzed, and each mitotic and apoptotic event was documented. Freshly dissolved h-IAPP caused a dose-dependent increased rate of apoptosis (P < 0.0001) in both cell types. At low and medium levels of toxicity, cells that had previously undergone mitosis were more vulnerable to h-IAPP-induced apoptosis than nondividing cells (P < 0.05). In the first 3 h after mitosis (full cell cycle length 26 +/- 0.6 h), beta-cells were particularly susceptible to h-IAPP-induced apoptosis (P < 0.05). Neither m-IAPP nor mature amyloid aggregates of h-IAPP were cytotoxic (P = 0.49). To corroborate these cell culture studies, we examined sections of human pancreatic tissue (five cases of type 2 diabetes) and human islets incubated for 48 h +/- h-IAPP. Both were stained for apoptosis with the transferase-mediated dUTP nick-end labeling method and analyzed for the presence of paired apoptotic cells anticipated in the event of postmitotic apoptosis. In human pancreatic tissue 26 +/- 5% (single plane of examination) and in human islets incubated with h-IAPP 44 +/- 4% of apoptotic islet cells were paired. In conclusion, replicating beta-cells are preferentially vulnerable to h-IAPP-induced apoptosis in cell culture. Postmitotic apoptosis was also documented in humans with type 2 diabetes and in human islet tissue. We postulate that beta-cell deficiency in type 2 diabetes may result in part from failure to adaptively increase beta-cell mass due to increased vulnerability of replicating beta-cells to undergo apoptosis. If this postulate is correct, then inhibition of apoptosis should allow recovery of beta-cell mass in type 2 diabetes.     

Exendin-4 stimulates both beta-cell replication and neogenesis, resulting in increased beta-cell mass and improved glucose tolerance in diabetic rats

Diabetes is a disease of increasing prevalence in the general population and of unknown cause. Diabetes is manifested as hyperglycemia due to a relative deficiency of the production of insulin by the pancreatic beta-cells. One determinant in the development of diabetes is an inadequate mass of beta-cells, either absolute (type 1, juvenile diabetes) or relative (type 2, maturity-onset diabetes). Earlier, we reported that the intestinal hormone glucagon-like peptide I (GLP-I) effectively augments glucose-stimulated insulin secretion. Here we report that exendin-4, a long-acting GLP-I agonist, stimulates both the differentiation of beta-cells from ductal progenitor cells (neogenesis) and proliferation of beta-cells when administered to rats. In a partial pancreatectomy rat model of type 2 diabetes, the daily administration of exendin-4 for 10 days post-pancreatectomy attenuates the development of diabetes. We show that exendin-4 stimulates the regeneration of the pancreas and expansion of beta-cell mass by processes of both neogenesis and proliferation of beta-cells. Thus, GLP-I and analogs thereof hold promise as a novel therapy to stimulate beta-cell growth and differentiation when administered to diabetic individuals with reduced beta-cell mass.     

Leptin effects on pancreatic beta-cell gene expression and function

The hormone leptin is secreted from white adipocytes, and serum levels of leptin correlate with adipose tissue mass. Leptin was first described to act on the satiety center in the hypothalamus through specific receptors (leptin receptor [ObR]) to restrict food intake and enhance energy expenditure. Important peripheral actions of leptin involve inhibition of insulin biosynthesis and secretion in pancreatic beta-cells. In turn, insulin stimulates leptin secretion from adipose tissue, establishing a hormonal regulatory feedback loop-the so-called "adipo-insular axis." Multiple signal transduction pathways are involved in leptin signaling in pancreatic beta-cells. We have identified the proinsulin gene and protein phosphatase 1 gene as leptin repressed genes and the gene for the suppressor of cytokine signaling 3 protein as a leptin-induced gene in pancreatic beta-cells. The molecular effects of leptin culminate to restrict insulin secretion and biosynthesis to adapt glucose homeostasis to the amount of body fat. In most overweight individuals, however, physiological regulation of body weight by leptin seems to be disturbed, representing "leptin resistance." This leptin resistance at the level of the pancreatic beta-cell may contribute to dysregulation of the adipo-insular axis and promote the development of hyperinsulinemia and manifest type 2 diabetes in overweight patients.     

Akt induces beta-cell proliferation by regulating cyclin D1, cyclin D2, and p21 levels and cyclin-dependent kinase-4 activity

Proliferation is the major component for maintenance of beta-cell mass in adult animals. Activation of phosphoinositide 3-kinase/Akt-kinase pathway is a critical regulator of beta-cell mass. Pancreatic beta-cell overexpression of constitutively active Akt in mice (caAkt(Tg)) resulted in marked expansion of beta-cell mass by increase in beta-cell proliferation and size. The current studies provide new insights into the molecular mechanisms involved in beta-cell proliferation by Akt. Proliferation of beta-cells in caAkt(Tg) was associated with increased cyclin D1, cyclin D2, and p21 levels and cyclin-dependent kinase-4 (cdk4) activity. To determine the role of cdk4 in beta-cell proliferation induced by Akt, we generated caAkt(Tg) mice that were homozygous, heterozygous, or nullizygous for cdk4. The results of these studies showed that deletion of one cdk4 allele significantly reduced beta-cell expansion in caAkt(Tg) mice by decreased proliferation. CaAkt(Tg) mice deficient in cdk4 developed beta-cell failure and diabetes. These experiments suggest that Akt induces beta-cell proliferation in a cdk4-dependent manner by regulation of cyclin D1, cyclin D2, and p21 levels. These data also indicate that alteration in levels of these cell cycle components could affect the maintenance of beta-cell mass in basal states and the adaptation of beta-cells to pathological states resulting in diabetes.     

Diabetes due to a progressive defect in beta-cell mass in rats transgenic for human islet amyloid polypeptide (HIP Rat): a new model for type 2 diabetes

The islet in type 2 diabetes is characterized by a deficit in beta-cell mass, increased beta-cell apoptosis, and impaired insulin secretion. Also, islets in type 2 diabetes often contain deposits of islet amyloid derived from islet amyloid polypeptide (IAPP), a 37-amino acid protein cosecreted with insulin by beta-cells. Several lines of evidence suggest that proteins with a capacity to develop amyloid fibrils may also form small toxic oligomers that can initiate apoptosis. The amino acid sequence of IAPP in rats and mice is identical and differs from that in humans by substitution of proline residues in the amyloidogenic sequence so that the protein no longer forms amyloid fibrils or is cytotoxic. In the present study, we report a novel rat model for type 2 diabetes: rats transgenic for human IAPP (the HIP rat). HIP rats develop diabetes between 5 and 10 months of age, characterized by an approximately 60% deficit in beta-cell mass that is due to an increased frequency of beta-cell apoptosis. HIP rats develop islet amyloid, but the extent of amyloid was not related to the frequency of beta-cell apoptosis (r = 0.10, P = 0.65), whereas the fasting blood glucose was (r = 0.77, P < 0.001). The frequency of beta-cell apoptosis was related to the frequency of beta-cell replication (r = 0.97, P < 0.001) in support of the hypothesis that replicating cells are more vulnerable to apoptosis than nondividing cells. The HIP rat provides additional evidence in support of the potential role of IAPP oligomer formation toward the increased frequency of apoptosis in type 2 diabetes, a process that appears to be compounded by glucose toxicity when hyperglycemia supervenes.     

Hepatocyte nuclear factor-1alpha modulates pancreatic beta-cell growth by regulating the expression of insulin-like growth factor-1 in INS-1 cells

Maturity-onset diabetes of the young type 3 (MODY3) is characterized by impaired insulin secretion. Heterozygous mutations in the gene encoding hepatocyte nuclear factor (HNF)-1alpha are the cause of MODY3. Transgenic mice overexpressing dominant-negative HNF-1alpha mutant in pancreatic beta-cells and HNF-1alpha knockout mice are animal models of MODY3. These mice exhibit defective glucose-stimulated insulin secretion and have reduced beta-cell mass and beta-cell proliferation rate. Here we examined the effect of HNF-1alpha on beta-cell proliferation by overexpressing a human naturally occurring dominant- negative mutation P291fsinsC in INS-1 cells under the control of doxycycline-induction system. INS-1 cells overexpressing P291fsinsC showed apparent growth impairment. The proliferation rate estimated by [(3)H]thymidine incorporation was significantly reduced in P291fsinsC-expressing INS-1 cells compared with noninduced or wild-type HNF-1alpha-overexpressing INS-1 cells. Growth inhibition occurred at the transition from G1 to S cell cycle phase, with reduced expression of cyclin E and upregulation of p27. cDNA array analysis revealed that the expression levels of IGF-1, a major growth factor for beta-cells, and macrophage migration inhibitory factor (MIF), a cytokine expressed in pancreatic beta-cells, were reduced in P291fsinsC-HNF-1alpha-expressing INS-1 cells. Although MIF seemed to have proliferative function, blockade of MIF action by anti-MIF antibody stimulated INS-1 cell proliferation, excluding its direct role in the growth impairment. However, addition of IGF-1 to P291fsinsC-expressing INS-1 cells rescued the growth inhibition. Our data suggest that HNF-1alpha is critical for modulating pancreatic beta-cell growth by regulating IGF-1 expression. IGF-1 might be a potential therapeutic target for the treatment of MODY3.