Liraglutide Improves Pancreatic Beta Cell Mass and Function in Alloxan-Induced Diabetic Mice

Glucagon-like peptide-1 (GLP-1) receptor agonists potentiate glucose-induced insulin secretion. In addition, they have been reported to increase pancreatic beta cell mass in diabetic rodents. However, the precise mode of action of GLP-1 receptor agonists still needs to be elucidated. Here we clarify the effects of the human GLP-1 analog liraglutide on beta cell fate and function by using an inducible Cre/loxP-based pancreatic beta cell tracing system and alloxan-induced diabetic mice. Liraglutide was subcutaneously administered once daily for 30 days. The changes in beta cell mass were examined as well as glucose tolerance and insulin secretion. We found that chronic liraglutide treatment improved glucose tolerance and insulin response to oral glucose load. Thirty-day treatment with liraglutide resulted in a 2-fold higher mass of pancreatic beta cells than that in vehicle group. Liraglutide increased proliferation rate of pancreatic beta cells and prevented beta cells from apoptotic cells death. However, the relative abundance of YFP-labeled beta cells to total beta cells was no different before and after liraglutide treatment, suggesting no or little contribution of neogenesis to the increase in beta cell mass. Liraglutide reduced oxidative stress in pancreatic islet cells of alloxan-induced diabetic mice. Furthermore, the beneficial effects of liraglutide in these mice were maintained two weeks after drug withdrawal. In conclusion, chronic liraglutide treatment improves hyperglycemia by ameliorating beta cell mass and function in alloxan-induced diabetic mice.     

TNF-Like Weak Inducer of Apoptosis (TWEAK) Promotes Beta Cell Neogenesis from Pancreatic Ductal Epithelium in Adult Mice

Aim/Hypothesis

The adult mammalian pancreas has limited ability to regenerate in order to restore adequate insulin production from multipotent progenitors, the identity and function of which remain poorly understood. Here we test whether the TNF family member TWEAK (TNF-like weak inducer of apoptosis) promotes β-cell neogenesis from proliferating pancreatic ductal epithelium in adult mice.

Methods

C57Bl/6J mice were treated with Fc-TWEAK and pancreas harvested at different time points for analysis by histology and immunohistochemistry. For lineage tracing, 4 week old double transgenic mice CAII-CreER^TM: R26R-eYFP were implanted with tamoxifen pellet, injected with Fc-TWEAK or control Ig twice weekly and analyzed at day 18 for TWEAK-induced duct cell progeny by costaining for insulin and YFP. The effect of TWEAK on pancreatic regeneration was determined by pancytokeratin immunostaining of paraffin embedded sections from wildtype and TWEAK receptor (Fn14) deficient mice after Px.

Results

TWEAK stimulates proliferation of ductal epithelial cells through its receptor Fn14, while it has no mitogenic effect on pancreatic α- or β-cells or acinar cells. Importantly, TWEAK induces transient expression of endogenous Ngn3, a master regulator of endocrine cell development, and induces focal ductal structures with characteristics of regeneration foci. In addition, we identify by lineage tracing TWEAK-induced pancreatic β-cells derived from pancreatic duct epithelial cells. Conversely, we show that Fn14 deficiency delays formation of regenerating foci after Px and limits their expansion.

Conclusions/Interpretation

We conclude that TWEAK is a novel factor mediating pancreatic β-cell neogenesis from ductal epithelium in normal adult mice.     

Conversion of pancreatic α cells into insulin-producing cells modulated by β-cell insufficiency and supplemental insulin administration

The emergence of bihormonal (BH) cells expressing insulin and glucagon has been reported under diabetic conditions in humans and mice. Whereas lineage tracing studies demonstrated that glucagon-producing α cells can be reprogrammed into BH cells, the underlying dynamics of the conversion process remain poorly understood. In the present study, we investigated the identities of pancreatic endocrine cells by genetic lineage tracing under diabetic conditions. When β-cell ablation was induced by alloxan (ALX), a time-dependent increase in BH cells was subsequently observed. Lineage tracing experiments demonstrated that BH cells originate from α cells, but not from β cells, in ALX-induced diabetic mice. Notably, supplemental insulin administration into diabetic mice resulted in a significant increase in α-cell-derived insulin-producing cells that did not express glucagon. Furthermore, lineage tracing in Ins2^Akita diabetic mice demonstrated a significant induction of α-to-β conversion. Thus, adult α cells have plasticity, which enables them to be reprogrammed into insulin-producing cells under diabetic conditions, and this can be modulated by supplemental insulin administration.     

Neurogenin3 Activation Is Not Sufficient to Direct Duct-to-Beta Cell Transdifferentiation in the Adult Pancreas

It remains controversial whether adult pancreatic ducts harbor facultative beta cell progenitors. Because neurogenin3 (Ngn3) is a key determinant of pancreatic endocrine cell neogenesis during embryogenesis, many studies have also relied upon Ngn3 expression as evidence of beta cell neogenesis in adults. Recently, however, Ngn3 as a marker of adult beta cell neogenesis has been called into question by reports of Ngn3 expression in fully-developed beta cells. Nevertheless, direct evidence as to whether Ngn3 activation in adult pancreatic duct cells may lead to duct-to-beta cell transdifferentiation is lacking. Here we studied two models of Ngn3 activation in adult pancreatic duct cells (low-dose alloxan treatment and pancreatic duct ligation) and lineage-traced Ngn3-activated duct cells by labeling them through intraductal infusion with a cell-tagging dye, CFDA-SE No dye-labeled beta cells were found during the follow-up in either model, suggesting that activation of Ngn3 in duct cells is not sufficient to direct their transdifferentiation into beta cells. Therefore, Ngn3 activation in duct cells is not a signature for adult beta cell neogenesis.     

Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis

Glucagon-like peptide-1 (GLP-1) stimulates insulin secretion and augments beta cell mass via activation of beta cell proliferation and islet neogenesis. We examined whether GLP-1 receptor signaling modifies the cellular susceptibility to apoptosis. Mice administered streptozotocin (STZ), an agent known to induce beta cell apoptosis, exhibit sustained improvement in glycemic control and increased levels of plasma insulin with concomitant administration of the GLP-1 agonist exendin-4 (Ex-4). Blood glucose remained significantly lower for weeks after cessation of exendin-4. STZ induced beta cell apoptosis, which was significantly reduced by co-administration of Ex-4. Conversely, mice with a targeted disruption of the GLP-1 receptor gene exhibited increased beta cell apoptosis after STZ administration. Exendin-4 directly reduced cytokine-induced apoptosis in purified rat beta cells exposed to interleukin 1beta, tumor necrosis fator alpha, and interferon gamma in vitro. Furthermore, Ex-4-treated BHK-GLP-1R cells exhibited significantly increased cell viability, reduced caspase activity, and decreased cleavage of beta-catenin after treatment with cycloheximide in vitro. These findings demonstrate that GLP-1 receptor signaling directly modifies the susceptibility to apoptotic injury, and provides a new potential mechanism linking GLP-1 receptor activation to preservation or enhancement of beta cell mass in vivo.     

Effects of growth factors on an intestinal epithelial cell line: transforming growth factor beta inhibits proliferation and stimulates differentiation

The effects of epidermal growth factor transforming growth factor beta (TGF beta) and other growth factors on the proliferation and differentiation of a cell line derived from rat intestinal crypt epithelium (IEC-6) were defined. Incorporation of [3H]-thymidine was stimulated 1.4-2.4 fold by insulin, insulin like growth factor (IGF), platelet derived growth factor (PDGF), epidermal growth factor (EGF) and 2% fetal calf serum (FCS) respectively. Additive stimulation was observed when FCS was supplemented by insulin,IGF-I or PDGF but not EGF. Incorporation of [3H]-thymidine by IEC-6 was strongly inhibited by TGF beta with greater than 80% inhibition of incorporation at concentration approximately equal to 2.0 pM. IEC-6 cells bound 4.1 +/- 0.15 X 10(4) molecules TGF beta/cell and appeared to have only a single class of high affinity receptors (Kd approximately equal to 0.5 pM). TGF beta inhibition was unaffected by the presence of insulin or IGF-I suggesting it inhibits proliferation at a step subsequent to that at which these growth factors stimulate [3H]-thymidine incorporation. TGF beta also reduced the stimulation induced by FCS by 65%. In contrast EGF reduced TGF beta inhibition by 60%. IEC-6 cells demonstrated the appearance of sucrase activity after greater than 18 hours treatment with TGF beta. These findings suggest that TGF beta may inhibit proliferative activity and promote the development of differentiated function in intestinal epithelial cells.     

Mitogenic stimulation of human breast cancer cells in a growth factor-defined medium: synergistic action of insulin and estrogen

The cooperative action of 17 beta-estradiol (E2) and polypeptide growth factors in stimulating proliferation of human breast cancer cells in vitro was investigated. To prevent background estrogenic stimulation, only phenol red-free media were used. When cultured in media supplemented with steroid-stripped serum in which all polypeptide growth factor activity had been chemically inactivated, MCF7 cells were unable to proliferate and became virtually quiescent. In the additional presence of insulin, epidermal growth factor (EGF), and E2, however, cells proliferated as rapidly as did cells cultured in media supplemented with fetal calf serum. Analysis by DNA flow cytometry showed that in the absence of external growth factors, MCF7 cells became arrested predominantly in the G1/G0 phase of the cell cycle. Upon addition of insulin in combination with EGF and E2, however, cells reentered the cell cycle with a high degree of synchrony. When added alone, E2 induced only slight mitogenic effects under these growth factor-defined conditions. In contrast, this steroid induced optimal proliferation in conventional steroid-stripped serum, which in itself contained considerable mitogenic activity. Insulin (at 10 micrograms/ml) was the most potent stimulator of MCF7 cell proliferation under growth factor-defined conditions, resulting in a more than sixfold increase in cell number after 96 hours. Other growth factors such as platelet-derived growth factor (PDGF), transforming growth factor beta (TGF beta), and EGF had little effect by themselves and only slightly influenced insulin-induced proliferation. At suboptimal concentrations of insulin (10-100 ng/ml), however, strong synergism was observed between E2 and insulin in inducing MCF7 proliferation. Using the CG5 cell line, a highly E2-sensitive MCF7 variant, synergism with E2 was already observed at 1 ng/ml insulin. It is concluded that MCF7 cells require insulin (or insulin-like growth factors) for proliferation. At suboptimal insulin concentrations, E2 acts synergistically with insulin, possibly by inducing autocrine production of polypeptide growth factors by these cells.     

Enhancement of mouse pancreatic regeneration and HIT-T15 cell proliferation with rat pancreatic extract

In this study, the effects of rat pancreatic extract (RPE) on regeneration of impaired mouse pancreas and proliferation of beta-cell line (HIT-T15) were investigated. RPE from the regenerating pancreas (2 days after 60% pancreatectomy) was treated to cure streptozotocin (STZ) induced diabetes in BALB/c mice. RPE-treated BALB/c mice for 21 consecutive days became euglycemic by day 30 and remained normoglycemic during a 150 day follow-up. Saline treated mice remained hyperglycemic sustained uncontrolled hyperglycemia. Islet neogenesis was observed in RPE-treated mice and confirmed by use of immunocytochemistry. Morphometric analysis of pancreas in reverted RPE-treated mice showed a new population of small islets compared with saline controls and an increased islet number. When HIT-T15 cells were treated with RPE, HIT-T15 cell proliferation and insulin secretion increased with increases in the RPE concentration. These results imply that RPE have the regeneration factors and help in the cure of diabetes.     

Beta Cell Count Instead of Beta Cell Mass to Assess and Localize Growth in Beta Cell Population following Pancreatic Duct Ligation in Mice

Background

Pancreatic-tail duct ligation (PDL) in adult rodents has been reported to induce beta cell generation and increase beta cell mass but increases in beta cell number have not been demonstrated. This study examines whether PDL increases beta cell number and whether this is caused by neogenesis of small clusters and/or their growth to larger aggregates.

Methodology

Total beta cell number and its distribution over small (<50 µm), medium, large (>100 µm) clusters was determined in pancreatic tails of 10-week-old mice, 2 weeks after PDL or sham.

Principal findings

PDL increased total beta cell mass but not total beta cell number. It induced neogenesis of small beta cell clusters (2.2-fold higher number) which contained a higher percent proliferating beta cells (1.9% Ki67+cells) than sham tails (<0.2%); their higher beta cell number represented <5% of total beta cell number and was associated with a similar increase in alpha cell number. It is unknown whether the regenerative process is causally related to the inflammatory infiltration in PDL-tails. Human pancreases with inflammatory infiltration also exhibited activation of proliferation in small beta cell clusters.

Conclusions/significance

The PDL model illustrates the advantage of direct beta cell counts over beta cell mass measurements when assessing and localizing beta cell regeneration in the pancreas. It demonstrates the ability of the adult mouse pancreas for neogenesis of small beta cell clusters with activated beta cell proliferation. Further studies should investigate conditions under which neoformed small beta cell clusters grow to larger aggregates and hence to higher total beta cell numbers.     

TWEAK, via its receptor Fn14, is a novel regulator of mesenchymal progenitor cells and skeletal muscle regeneration

Inflammation participates in tissue repair through multiple mechanisms including directly regulating the cell fate of resident progenitor cells critical for successful regeneration. Upon surveying target cell types of the TNF ligand TWEAK, we observed that TWEAK binds to all progenitor cells of the mesenchymal lineage and induces NF-kappaB activation and the expression of pro-survival, pro-proliferative and homing receptor genes in the mesenchymal stem cells, suggesting that this pro-inflammatory cytokine may play an important role in controlling progenitor cell biology. We explored this potential using both the established C2C12 cell line and primary mouse muscle myoblasts, and demonstrated that TWEAK promoted their proliferation and inhibited their terminal differentiation. By generating mice deficient in the TWEAK receptor Fn14, we further showed that Fn14-deficient primary myoblasts displayed significantly reduced proliferative capacity and altered myotube formation. Following cardiotoxin injection, a known trigger for satellite cell-driven skeletal muscle regeneration, Fn14-deficient mice exhibited reduced inflammatory response and delayed muscle fiber regeneration compared with wild-type mice. These results indicate that the TWEAK/Fn14 pathway is a novel regulator of skeletal muscle precursor cells and illustrate an important mechanism by which inflammatory cytokines influence tissue regeneration and repair. Coupled with our recent demonstration that TWEAK potentiates liver progenitor cell proliferation, the expression of Fn14 on all mesenchymal lineage progenitor cells supports a broad involvement of this pathway in other tissue injury and disease settings.     

IL-2 Immunotherapy Reveals Potential for Innate Beta Cell Regeneration in the Non-Obese Diabetic Mouse Model of Autoimmune Diabetes

Type-1 diabetes (T1D) is an autoimmune disease targeting insulin-producing beta cells, resulting in dependence on exogenous insulin. To date, significant efforts have been invested to develop immune-modulatory therapies for T1D treatment. Previously, IL-2 immunotherapy was demonstrated to prevent and reverse T1D at onset in the non-obese diabetic (NOD) mouse model, revealing potential as a therapy in early disease stage in humans. In the NOD model, IL-2 deficiency contributes to a loss of regulatory T cell function. This deficiency can be augmented with IL-2 or antibody bound to IL-2 (Ab/IL-2) therapy, resulting in regulatory T cell expansion and potentiation. However, an understanding of the mechanism by which reconstituted regulatory T cell function allows for reversal of diabetes after onset is not clearly understood. Here, we describe that Ab/IL-2 immunotherapy treatment, given at the time of diabetes onset in NOD mice, not only correlated with reversal of diabetes and expansion of Treg cells, but also demonstrated the ability to significantly increase beta cell proliferation. Proliferation appeared specific to Ab/IL-2 immunotherapy, as anti-CD3 therapy did not have a similar effect. Furthermore, to assess the effect of Ab/IL-2 immunotherapy well after the development of diabetes, we tested the effect of delaying treatment for 4 weeks after diabetes onset, when beta cells were virtually absent. At this late stage after diabetes onset, Ab/IL-2 treatment was not sufficient to reverse hyperglycemia. However, it did promote survival in the absence of exogenous insulin. Proliferation of beta cells could not account for this improvement as few beta cells remained. Rather, abnormal insulin and glucagon dual-expressing cells were the only insulin-expressing cells observed in islets from mice with established disease. Thus, these data suggest that in diabetic NOD mice, beta cells have an innate capacity for regeneration both early and late in disease, which is revealed through IL-2 immunotherapy.     

Small-molecule discovery in the pancreatic beta cell

The pancreatic beta cell is the only cell type in the body responsible for insulin secretion, and thus plays a unique role in the control of glucose homeostasis. The loss of beta-cell mass and function plays an important role in both type 1 and type 2 diabetes. Thus, using chemical biology to identify small molecules targeting the beta cell could be an important component to developing future therapeutics for diabetes. This strategy provides an attractive path toward increasing beta-cell numbers in vivo. A regenerative strategy involves enhancing proliferation, differentiation, or neogenesis. On the other hand, protecting beta cells from cell death, or improving maturity and function, could preserve beta-cell mass. Here, we discuss the current state of chemical matter available to study beta-cell regeneration, and how they were discovered.     

In vitro toxicity of alloxan for guinea pig B cells: comparison with rat B cells

Previous studies have shown that guinea pigs are resistant to the in vivo diabetogenic action of alloxan and that this resistance may be accompanied by a regeneration of B cells in the initial days following administration of the drug. In the studies reported here, we used the measurement of insulin and glucagon released over a 7-day culture period as indices of islet cell viability and examined effects of in vitro exposure to alloxan upon subsequent release of insulin and glucagon from guinea pig (alloxan-resistant) and rat (alloxan-sensitive) islet cell cultures. An alloxan dose-dependent decrease in subsequent insulin release was found. However, whereas the lowest concentration of the drug (1 mM) produced a significant depression in insulin release in rat islet cultures, with maximal depression occurring after exposure to 5 mM alloxan, insulin release from guinea pig cultures was not significantly depressed by 1 or 2 mM alloxan, and 5 mM alloxan treatment produced a submaximal depression. Furthermore, insulin release from guinea pig but not rat cultures increased transiently at between 6 and 18 hr during the first day following exposure to all doses of alloxan. Treatment with high doses of the drug (40 mM or greater) caused the same maximal chronic depression of insulin release for both species. In contrast, glucagon release from cultures of both species was not affected significantly following alloxan treatment. Thus, guinea pig B cells are more resistant than those of the rat to the action of alloxan, but this resistance can be overcome by employing high doses of the drug. Other factions unidentified by the present studies may also be involved in the failure of guinea pigs to develop diabetes following in vivo treatment with alloxan.     

Neogenesis vs. apoptosis As main components of pancreatic beta cell ass changes in glucose-infused normal and mildly diabetic adult rats.

We have investigated in adult rats made mildly diabetic by a low dose of streptozotocin (35 mg/kg; STZ rats) and in nondiabetic rats (ND rats) the mechanisms leading to adaptive changes in the beta cell mass, during glucose infusion and several days after stopping infusion. As early as 24 h of glucose infusion, the beta cell mass was maximally increased in ND and STZ rats. In both groups, this increase was due mainly to a rapid activation of neogenesis of new endocrine cells rather than to an increase in beta cell proliferation. Seven days after stopping glucose infusion, the beta cell mass returned to basal values in both groups as a result of stimulation of beta cell apoptosis and a decrease in beta cell replication rate. In glucose-infused ND rats, changes in the beta cell mass were correlated to insulin secretion, whereas in STZ rats, insulin secretion in response to glucose was still impaired whatever the beta cell mass. In conclusion, the data stress the impressive plasticity of the endocrine pancreas of adult rats. They also show that changes in beta cell mass in ND and STZ rats resulted from a disruption in the balance between neogenesis and apoptosis.     

Formation and regeneration of the endocrine pancreas

The elaboration of the pancreas from epithelial buds to the intricate organ requires complex patterning information that controls fundamental cellular processes such as differentiation and proliferation of pancreatic progenitor cells. During pancreatic organogenesis, endocrine cells are generated from a population of pancreatic progenitor cells. The progenitor cells during the early development simultaneously receive multiple signals, some mitogenic and some inducing differentiation. These extrinsic signals are interpreted through an intrinsic mechanism that either commits the progenitor cell to the mitotic cell cycle or leads to exit from the cell cycle in order to differentiate. The endocrine cells that differentiate from progenitor cells are postmitotic, and direct lineage tracing analyses indicate that a population of progenitor cells persists throughout embryogenesis to allow the differentiation of new endocrine cells. At the end of embryogenesis an early postnatal period is characterized by high rates of beta cell proliferation leading to massive increases in beta cell mass. The beta cell mass expansion considerably slows down in adult animals, though variations in insulin demand due to physiological and pathological states such as pregnancy and obesity can lead to adaptive changes in the beta cells that include hyperplasia, hypertrophy, and increased insulin synthesis and secretion. Deciphering the mechanisms that regulate the plasticity of beta cell mass can be an important step in developing effective strategies to treat diabetes.     

Betacellulin-induced beta cell proliferation and regeneration is mediated by activation of ErbB-1 and ErbB-2 receptors

Background

Betacellulin (BTC), a member of the epidermal growth factor family, is known to play an important role in regulating growth and differentiation of pancreatic beta cells. Growth-promoting actions of BTC are mediated by epidermal growth factor receptors (ErbBs), namely ErbB-1, ErbB-2, ErbB-3 and ErbB-4; however, the exact mechanism for beta cell proliferation has not been elucidated. Therefore, we investigated which ErbBs are involved and some molecular mechanisms by which BTC regulates beta cell proliferation.

Methodology/Principal Findings

The expression of ErbB-1, ErbB-2, ErbB-3, and ErbB-4 mRNA was detected by RT-PCR in both a beta cell line (MIN-6 cells) and C57BL/6 mouse islets. Immunoprecipitation and western blotting analysis showed that BTC treatment of MIN-6 cells induced phosphorylation of only ErbB-1 and ErbB-2 among the four EGF receptors. BTC treatment resulted in DNA synthetic activity, cell cycle progression, and bromodeoxyuridine (BrdU)-positive staining. The proliferative effect was blocked by treatment with AG1478 or AG825, specific tyrosine kinase inhibitors of ErbB-1 and ErbB-2, respectively. BTC treatment increased mRNA and protein levels of insulin receptor substrate-2 (IRS-2), and this was blocked by the ErbB-1 and ErbB-2 inhibitors. Inhibition of IRS-2 by siRNA blocked cell cycle progression induced by BTC treatment. Streptozotocin-induced diabetic mice injected with a recombinant adenovirus expressing BTC and treated with AG1478 or AG825 showed reduced islet size, reduced numbers of BrdU-positive cells in the islets, and did not attain BTC-mediated remission of diabetes.

Conclusions/Significance

These results suggest that BTC exerts proliferative activity on beta cells through the activation of ErbB-1 and ErbB-2 receptors, which may increase IRS-2 expression, contributing to the regeneration of beta cells.     

Intramuscular adipose is derived from a non-Pax3 lineage and required for efficient regeneration of skeletal muscles

Ectopic accumulation of adipose in the skeletal muscle is associated with muscle wasting, insulin resistance and diabetes. However, the developmental origin of postnatal intramuscular adipose and its interaction with muscle tissue are unclear. We report here that compared to the fast EDL muscles, slow SOL muscles are more enriched with adipogenic progenitors and have higher propensity to form adipose. Using Cre/LoxP mediated lineage tracing in mice, we show that intramuscular adipose in both EDL and SOL muscles is exclusively derived from a Pax3(-) non-myogenic lineage. In contrast, inter-scapular brown adipose is derived from the Pax3(+) lineage. To dissect the interaction between adipose and skeletal muscle tissues, we used Myf5-Cre and aP2-Cre mice in combination with ROSA26-iDTR mice to genetically ablate myogenic and adipogenic cell lineages, respectively. Whereas ablation of the myogenic cell lineage facilitated adipogenic differentiation, ablation of the adipogenic cell lineage surprisingly impaired the regeneration of acutely injured skeletal muscles. These results reveal striking heterogeneity of tissue-specific adipose and a previously unappreciated role of intramuscular adipose in skeletal muscle regeneration.     

pdx-1 function is specifically required in embryonic beta cells to generate appropriate numbers of endocrine cell types and maintain glucose homeostasis

The pdx1 gene is essential for pancreatic organogenesis in humans and mice; pdx1 mutations have been identified in human diabetic patients. Specific inactivation of pdx1 in adult beta cells revealed that this gene is required for maintenance of mature beta cell function. In the following study, a Cre-lox strategy was used to remove pdx1 function specifically from embryonic beta cells beginning at late-gestation, prior to islet formation. Animals in which pdx1 is lost in insulin-producing cells during embryogenesis had elevated blood glucose levels at birth and were overtly diabetic by weaning. Neonatal and adult mutant islets showed a dramatic reduction in the number of insulin(+) cells and an increase in both glucagon(+) and somatostatin(+) cells. Lineage tracing revealed that excess glucagon(+) and somatostatin(+) cells did not arise by interconversion of endocrine cell types. Examination of mutant islets revealed a decrease in proliferation of insulin-producing cells just before birth and a concomitant increase in proliferation of glucagon-producing cells. We propose that pdx1 is required for proliferation and function of the beta cells generated at late gestation, and that one function of normal beta cells is to inhibit the proliferation of other islet cell types, resulting in the appropriate numbers of the different endocrine cell types.