Combination Therapy With Glucagon-Like Peptide-1 and Gastrin Restores Normoglycemia in Diabetic NOD Mice

Combination therapy with epidermal growth factor (EGF) and gastrin induces beta-cell regeneration in rodents with chemically induced diabetes. We investigated whether EGF plus gastrin could correct hyperglycemia in NOD mice with autoimmune diabetes. Combined treatment with EGF (1 mug/kg) and gastrin (3 mug/kg) for 2 weeks restored normoglycemia after diabetes onset in NOD mice, whereas EGF or gastrin alone did not. Fasting blood glucose remained normal (3.5-6.5 mmol/l) or mildly elevated (<11 mmol/l) in five of six mice (83%) for 10 weeks after EGF plus gastrin treatment was stopped, whereas all mice treated with vehicle or EGF or gastrin alone became severely hyperglycemic (12-35 mmol/l). Pancreatic beta-cell mass was increased threefold and insulin content was increased eightfold in mice treated with EGF plus gastrin compared with pretreatment values. The correction of hyperglycemia correlated significantly with increases in pancreatic beta-cell mass and insulin content. In addition, splenic cells from mice treated with EGF plus gastrin delayed diabetes induction by adoptive transfer of diabetogenic cells into immunodeficient NOD-scid mice, suggesting the induction of immunoregulatory cells in NOD mice treated with EGF plus gastrin. We conclude that a short course of combined EGF and gastrin therapy increases pancreatic beta-cell mass and reverses hyperglycemia in acutely diabetic NOD mice; the impact of this combined therapy may result from the effects of EGF and gastrin on beta-cells, immune cells, or both.

To the Editor: Many beta cell growth factors have been identified, one of the most promising being glucagon-like peptide-1 (GLP-1), a peptide secreted from intestinal endocrine L-cells in response to nutrient ingestion. GLP-1 is rapidly cleaved and inactivated in vivo by dipeptidyl peptidase-4 (DPP-4), a ubiquitous serine protease. DPP-4 inhibitors (DPP-4i) have been shown to raise circulating levels of active GLP-1 and thus increase and maintain effective concentrations of this peptide reaching target tissues [1]. Studies in humans with type 2 diabetes have shown that DPP-4i therapy improves glucose tolerance [2]. In addition, DPP-4i treatments can preserve pancreatic beta cell mass in animal models of type 2 diabetes [3] and stimulate beta cell regeneration in streptozotocin-induced diabetic rats [4]. Regarding deficits in pancreatic beta cell mass resulting from autoimmune (type 1) diabetes, the GLP-1 analogue, exendin-4, reversed diabetes in NOD mice; however, this required additional immunosuppressive therapy [5]. Addition of the gastrointestinal peptide hormone, gastrin, to GLP-1 therapy increased pancreatic beta cell mass and restored normoglycaemia in diabetic NOD mice without the use of immunosuppressants [6]. The objective of this study was to determine whether combination therapy with a DPP4i to raise endogenous levels of GLP-1, together with a proton pump inhibitor (PPI) to raise endogenous levels of gastrin, could reverse diabetes in NOD mice. Here, we report that such a combination therapy increased circulating levels of GLP-1 and gastrin in acutely diabetic NOD mice, and that pancreatic insulin content, insulin secretion and normoglycaemia were restored. NOD female mice, age 6-8 weeks, were purchased from Taconic (Germantown, NY, USA). NOD-severe combined immunodeficient (NOD-scid) female mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA). The mice were housed and fed under specific pathogen-free conditions and cared for according to the guidelines of the Canadian Council on Animal Care. Diabetes onset was diagnosed by non-fasting blood glucose ≥10 mmol/l. Acutely diabetic NOD mice (blood glucose 10–16 mmol/l) were allocated to four groups within 4–7 days after diabetes onset and treated for 12 weeks with (1) the DPP-4i vehicle, 5 mg/ml methylcellulose in H2O given by oral gavage once daily and the PPI vehicle, PBS given by s. c. injection twice Diabetologia (2009) 52:1680–1682 DOI 10.1007/s00125-009-1390-z

Glial Cell Line-Derived Neurotrophic Factor Increases β-Cell Mass and Improves Glucose Tolerance

Pancreatic β-cell loss because of apoptosis is the major cause of type 1 diabetes (T1D) and late stage T2D. Puerarin possesses anti-diabetic properties; whether it acts directly on pancreatic β-cell is not clear. This study was designed to investigate the effects of puerarin on pancreatic β-cell survival and function. Diabetes was induced in male C57BL/6 mice by a single peritoneal injection of streptozotocin (STZ). Pancreatic β-cell survival and function were assessed in diabetic mice by measuring β-cell apoptosis, β-cell mass, pancreatic insulin content, and glucose tolerance, and in cultured islets and clonial MIN6 β-cells by measuring β-cell viability and apoptosis and glucose-stimulated insulin secretion. We found that pre-treatment with puerarin decreased the incidence of STZ-induced diabetes. Puerarin increased pancreatic β-cell mass via β-cell apoptosis inhibition in diabetic mice, and increased serum insulin, whereas it decreased blood glucose levels and improved glucose tolerance. In cultured islets and MIN6 cells, puerarin protected β-cell from cobalt chloride (CoCl2)-induced apoptosis and restored the impaired capacity of glucose-stimulated insulin secretion. Puerarin protection of β-cell survival involved the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. In conclusion, puerarin protects pancreatic β-cell function and survival via direct effects on β-cells, and its protection of β-cell survival is mediated by the PI3K/Akt pathway. As a safe natural plant extraction, puerarin might serve as a preventive and/or therapeutic approach for diabetes.

Diabetes mellitus, defined as a fasting plasma glucose of ≥126 mg/dL or a glycosylated hemoglobin A1c level (HbA1c) of ≥6.5%, afflicts ≈12.9% of adults in the United States and nearly 285 million adults worldwide.1,2 Diabetes mellitus is a major risk factor for the development of cardiovascular disease, independently conferring a 2-fold excess risk of coronary heart disease and stroke.3 Macrovascular events in diabetes mellitus remain the leading cause of mortality, and the burden of cardiovascular disease attributable to diabetes mellitus has increased over the past decade.4 An increase in the prevalence of obesity has contributed to the rise in diabetes mellitus. Additionally, obesity independently increases the risk of cardiovascular disease in patients with diabetes mellitus.5 Although strict glycemic control unequivocally reduces the microvascular complications of diabetes mellitus, the macrovascular benefits of intensive therapy have been difficult to establish, with conflicting results from large clinical trials.6–9 Multifactorial strategies are recommended to reduce cardiovascular risk in diabetes mellitus through enhanced glycemic control, blood pressure reduction, lipid management, weight loss, and physical activity.10 Unfortunately, despite aggressive interventions for hyperglycemia, <50% of patients achieve standard HbA1c targets with conventional therapy.11 Polypharmacy is required to achieve glycemic control in the majority of patients within 3 years of diagnosis.12 Although combinations of drug classes can synergistically target multiple pathophysiological defects, novel therapies are required to manage diabetes mellitus and mitigate cardiovascular risks. Dipeptidyl-peptidase IV (DPP-IV) inhibitor and glucagon-like peptide-1 (GLP-1) receptor agonist incretin therapies were developed to complement conventional treatment options for diabetes mellitus. Despite promising initial reports of cardioprotective effects, DPP-IV inhibitors have failed to demonstrate improved cardiovascular outcomes in large clinical trials.13–15 Randomized studies to evaluate cardiovascular outcomes associated with GLP-1 receptor agonists are currently underway. This review presents …

Bisphenol-A (BPA) is one of the highest volume chemicals produced worldwide. It is used as the base compound in the manufacture of polycarbonate plastics, epoxies and resins. Humans are consistently exposed to BPA and consistently it has been detected in the majority of individuals examined. Experimental research in animals, as well as human epidemiological studies, converge to conclude that BPA is a risk factor for the development of type 2 diabetes. In previous studies we have demonstrated that the exposure to BPA during embryonic development promote an increment of pancreatic β-cell mass. This was correlated with increased β-cell division and altered global gene expression in pancreatic β-cells. The aim of this work was to determinate whether ERβ was involved in the in the β-cell mass and proliferation increment observed in male mice offspring. ERβ+/- pregnant mice were treated with vehicle or BPA (10 μg/kg/day) from day 9 to 16 of gestation. Offspring pancreatic β-cell mass was measured at postnatal day 0 (P0) and 30 (P30). For ex vivo experiments Wild-type (WT) and ERβ-/- neonates as well as adult male and female mice were used. For in vitro, single islets cells were cultured for 48 h in the presence of 10 μmol/L BrdU, and vehicle, BPA (1, 10, 100 nM) or the specific ERβ agonist WAY200070 (1, 10, 100 nM). β-cell proliferation rate was quantified as the percentage of BrdU-positive pancreatic β-cells. In vivo exposure to BPA during pregnancy promoted an increment of pancreatic β-cell mass and proliferation in WT mice at P30 which was absent in ERβ -/- mice. In order to explore if these changes were related to a direct action of BPA on pancreatic β-cell division we performed a series of ex vivo experiments. Augmented β-cell proliferation rate was observed in BPA-exposed β-cells isolated from both adult male and female WT animals in comparison to controls. The increment was significant at all BPA doses tested. The effect was imitated by the selective ERβ agonist, WAY200070, and was abolished in cells from ERβ-/- mice. We also explored the effects of BPA in pancreatic β-cells from neonates and found an increment in BPA-exposed cells compared to controls, although the difference was only significant at the dose of 1 nM. A similar effect was observed in neonate cells treated with WAY200070 (10 nM). The effects on β-cell replication were abolished in cells from ERβ-/- neonate mice treated either with BPA or WAY200070. Our findings suggest that BPA modulate pancreatic β-cell growth and mass in an ERβ-dependent manner. This could have important implications for metabolic programming of T2DM. Ministerio de Economía y Competitividad, Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) grants BPU2017-86579-R (AN) and BFU2016-77125-R (IQ); Generalitat Valenciana PROMETEO II/2015/016 (AN). CIBERDEM is an initiative of the Instituto de Salud Carlos III.

Chromatin can exert a regulatory effect on gene transcription by modulating the access of transcription factors to target genes. In the present study, we examined whether nuclear actions of the incretin hormones, glucose-dependent insulinotropic polypeptide and glucagon-like peptide-1, involve modulation of beta-cell chromatin structure. Stimulation of INS-1(832/13) beta-cells or dispersed mouse islets with glucose-dependent insulinotropic polypeptide or glucagon-like peptide-1 resulted in the post-translational modification of core H3 histones, through acetylation and phosphorylation. Both increased histone H3 acetyltransferase and reduced histone deacetylase activities contributed. Subsequent studies demonstrated that incretin-mediated histone H3 modifications involved activation of protein kinase A, p42/44 mitogen-activated protein kinase (MAPK), and p38 MAPK signaling modules, resulting in the activation of mitogen- and stress-activated kinase-1. Additionally, modification of histone H3 increased its association with the transcription factor, phosphorylated cAMP-response element-binding protein (phospho-CREB) and with cAMP-responsive CREB coactivator 2. Incretin-activated CREB-related Bcl-2 transcription was greatly reduced by a histone acetyltransferase inhibitor, demonstrating the functional importance of histone H3 modification. This appears to be the first demonstration of beta-cell chromatin modification in response to the incretins and the studies indicate that their regulatory effects involve coordinated nuclear interactions between specific signaling modules, chromatin-modifying enzymes and transcription factors.

BackgroundThe control of the functional pancreatic β-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how β-cell mass is determined.Methodology/Principal FindingsConditional chicken ovalbumin upstream promoter transcription factor II (COUP-TFII)-deficient mice were generated and crossed with mice expressing Cre under the control of pancreatic duodenal homeobox 1 (pdx1) gene promoter. Ablation of COUP-TFII in pancreas resulted in glucose intolerance. Beta-cell number was reduced at 1 day and 3 weeks postnatal. Together with a reduced number of insulin-containing cells in the ductal epithelium and normal β-cell proliferation and apoptosis, this suggests decreased β-cell differentiation in the neonatal period. By testing islets isolated from these mice and cultured β-cells with loss and gain of COUP-TFII function, we found that COUP-TFII induces the expression of the β-catenin gene and its target genes such as cyclin D1 and axin 2. Moreover, induction of these genes by glucagon-like peptide 1 (GLP-1) via β-catenin was impaired in absence of COUP-TFII. The expression of two other target genes of GLP-1 signaling, GLP-1R and PDX-1 was significantly lower in mutant islets compared to control islets, possibly contributing to reduced β-cell mass. Finally, we demonstrated that COUP-TFII expression was activated by the Wnt signaling-associated transcription factor TCF7L2 (T-cell factor 7-like 2) in human islets and rat β-cells providing a feedback loop.Conclusions/SignificanceOur findings show that COUP-TFII is a novel component of the GLP-1 signaling cascade that increases β-cell number during the neonatal period. COUP-TFII is required for GLP-1 activation of the β-catenin-dependent pathway and its expression is under the control of TCF7L2.

Objective To investigate whether IL-10 gene combined with insulin-like growth factor-1(IGF-1) gene transfer could attenuate pancreatic insulitis, increase the percentage of CD4+ CD25+ Foxp3+ regulatory T cells, and protect β cells from autoimmune destruction. Methods An adenoviral vector containing IL-10 gene(Ad-IL-10) or IGF-1 gene(Ad-IGF-1) was constructed separately.Forty female non-obese diabetic(NOD) mice were injected intra-peritoneally with Ad-IL-10 and/or Ad-IGF-1, Ad-green fluorescent protein(GFP) and phosphate buffered saline(PBS)separately, repeated after 3 weeks. Blood glucose concentration was measured weekly.Serum insulin, cytokine production were tested by enzyme-linked immunosorbent assay.CD4+ CD25+ Foxp3+ Treg cells were determined by flow cyto-metry.Pancreatic histology was measured for determination of insulitis grades.Pancreatic insulin content and β-cell mass, proliferation were measured.Apoptosis was measured by using a terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Results A significantly lower diabetes incidence(P<0.01) was observed in NOD mice treated with Ad-IL-10 and/or Ad-IGF-1, compared with mice treated with Ad-GFP or PBS alone, especially combined group.Lower insulitis score compared to control mice was found in Ad-IL-10+ Ad-IGF-1 group(all P<0.01). The serum level of TNF-α and IFN-γ were decreased and the level of IL-10 increased in combination therapy.The CD4+ CD25+ Foxp3+ cells was (7.17±0.38)% in combined group, higher than that in the control groups.There was significantly less β-cell apoptosis(10.29±2.20)% in combined group than that in other groups(all P<0.05). Conclusions Combination therapy with IL-10 and IGF-1 gene is able to increase the percentage of CD4+ CD25+ Foxp3+ regulatory T cells, reduce autoimmunity and increase pancreatic β-cell mass, indicating promising potential of these therapies as a new treatment strategy for diabetes mellitus. Key words: Immunotherapy; Type 1 diabetes mellitus; T regulatory cells; Islet βcell

Better understanding how glucagon-like peptide 1 (GLP-1) promotes pancreatic β-cell function and/or mass may uncover new treatment for type 2 diabetes. In this study, we investigated the potential involvement of microRNAs (miRNAs) in the effect of GLP-1 on glucose-stimulated insulin secretion. miRNA levels in INS-1 cells and isolated rodent and human islets treated with GLP-1 in vitro and in vivo (with osmotic pumps) were measured by real-time quantitative PCR. The role of miRNAs on insulin secretion was studied by transfecting INS-1 cells with either precursors or antisense inhibitors of miRNAs. Among the 250 miRNAs surveyed, miR-132 and miR-212 were significantly up-regulated by GLP-1 by greater than 2-fold in INS-1 832/3 cells, which were subsequently reproduced in freshly isolated rat, mouse, and human islets, as well as the islets from GLP-1 infusion in vivo in mice. The inductions of miR-132 and miR-212 by GLP-1 were correlated with cAMP production and were blocked by the protein kinase A inhibitor H-89 but not affected by the exchange protein activated by cAMP activator 8-pCPT-2'-O-Me-cAMP-AM. GLP-1 failed to increase miR-132 or miR-212 expression levels in the 832/13 line of INS-1 cells, which lacks robust cAMP and insulin responses to GLP-1 treatment. Overexpression of miR-132 or miR-212 significantly enhanced glucose-stimulated insulin secretion in both 832/3 and 832/13 cells, and restored insulin responses to GLP-1 in INS-1 832/13 cells. GLP-1 increases the expression of miRNAs 132 and 212 via a cAMP/protein kinase A-dependent pathway in pancreatic β-cells. Overexpression of miR-132 or miR-212 enhances glucose and GLP-1-stimulated insulin secretion.

Type-I diabetes is a chronic disease mediated by autoimmune destruction of insulin-producing β-cells. Although progress has been made towards improving diabetes-associated pathologies and the quality of life for those living with diabetes, no therapy has been effective at eliminating disease manifestations or reversing disease progression. Here, we examined whether double-stranded adeno-associated virus serotype 8 (dsAAV8)-mediated gene delivery to endogenous β-cells of interleukin (IL)-4 in combination with β-cell growth factors can reverse early-onset diabetes in NOD mice. Our results demonstrate that a single treatment with dsAAV8 vectors expressing IL-4 in combination with glucagon-like peptide-1 or hepatocyte growth factor/NK1 under the regulation of the insulin promoter enhanced β-cell proliferation and survival in vivo, significantly delaying diabetes progression in NOD mice, and reversing disease in ∼10% of treated NOD mice. These results demonstrate the ability to reverse hyperglycemia in NOD mice with established diabetes by in vivo gene transfer to β-cells of immunomodulatory factors and β-cell growth factors.

Insulin and glucagon are well-known peptide hormones that keep glucose levels within a healthy range in the body. But they are only part of a complex network that controls concentrations of this ubiquitous sugar in blood and tissues. Other molecules regulate glucose by controlling insulin secretion from the pancreas or protecting pancreatic β cells against stresses that lead to cellular dysfunction or cell death (1). One of these protective regulators is glucagon-like peptide 1 (GLP-1), a 30-amino-acid-long peptide produced in specialized epithelial cells of the intestine, called L cells, and also in the brain and other organs and tissues (2). GLP-1 belongs to a group of peptides that mediate the “incretin effect,” an endocrine response to glucose arising from food digestion in the intestines (2, 3). This response helps regulate food intake and the fate of dietary glucose. Specifically, GLP-1 is released from the intestinal cells when food is ingested and then binds to and activates the GLP-1 receptor (GLP-1R), a G protein–coupled receptor on many cell types, including β cells in which GLP-1R signaling stimulates insulin synthesis and secretion (3). Notably, the incretin effect stimulates insulin secretion from pancreatic β cells more strongly than exposure to glucose alone. An article published in the Journal of Biological Chemistry (4), recognized as a Classic here, added to our understanding of the incretin effect by showing that GLP-1R signaling protects β cells from cell death (Fig. 1). This finding was significant for preventing or managing type 2 diabetes, in which β-cell apoptosis occurs (5) and may contribute to insufficient pancreatic insulin production (6). Open in a separate window Figure 1. Li et al. (4) have shown that binding of the receptor agonist exendin-4 to GLP-1R on pancreatic β cells protects the cells from cellular injury and cytokine-induced apoptosis and thereby preserves glucose homeostasis in mice. Binding of GLP-1 to its cognate receptor on pancreatic β cells up-regulates intracellular cAMP levels, in turn reducing streptozotocin-induced β-cell death. Images of exendin-4 and GLP-1R (with GLP-1 bound) are from Ref. 7; image of cAMP is from Wikimedia, used under Creative Commons.

P939 Aims: A substantial level of donor lymphocyte chimerism is required to protect donor-specific islet allografts in the mouse model with autoimmune diabetes. Donor islet allografts can be rejected in diabetic NOD mice with a low level of donor lymphocyte chimerism. In this study, we investigated whether posttransplant donor lymphocyte infusion increases donor chimerism and induces donor-specific tolerance to islet allografts in diabetic NOD mice. Methods: Donor BALB/c (H-2d) splenocytes were injected into prediabetic NOD/Lt mice (H-2g7) and diabetic NOD mice at day -3, followed by one dose of cyclophosphamide at day -1. BALB/c bone marrow cells (4x 107) were infused at day 0. Anti-CD40L mAb and rapamycin were given from day 0 to day 14. Donor islets were transplanted under left kidney capsule simultaneously with bone marrow transplantation in diabetic NOD mice. Donor lymphocyte infusion (1x107) was given in prediabetic NOD mice at 2 and 4 weeks post-bone marrow transplantation. In diabetic NOD mice, donor lymphocyte infusion (2x 107 or 3x 107) was given at 2, 4, and 6 weeks post-bone marrow transplantation. Mixed chimerism was measured by flow cytometric analysis. Islet graft function was monitored by measuring blood glucose. Left nephrectomy was performed in diabetic NOD mice with islet graft function at 100 days posttransplant. To test donor-specific tolerance, second donor-specific or third-party islets were transplanted under right kidney capsule of these NOD mice 1 week after left nephrectomy. Results: Donor lymphocyte chimerism was achieved in all prediabetic and diabetic NOD mice without or with posttransplant donor lymphocyte infusion. At 8 weeks post-bone marrow transplantation, the percentage of donor lymphocyte chimerism was 20.7±4.3% in prediabetic NOD mice without posttransplant donor lymphocyte infusion (N=11). Donor lymphocyte chimerism was significantly increased to 39.0±24.9% in prediabetic NOD mice with posttransplant donor lymphocyte infusion (N=9, P<0.05). The percentage of donor lymphocyte chimerism was 10.8±4.7% in diabetic NOD mice without posttransplant donor lymphocyte infusion (N=13). While posttransplant donor lymphocyte infusion was given, donor lymphocyte chimerism was significantly increased to 32.5±22.1% in diabetic NOD mice (N=9, P<0.05). Without posttransplant donor lymphocyte infusion, donor islet grafts survival was 30.8% in diabetic NOD mice at 100 days posttransplant (N=13). With posttransplant donor lymphocyte infusion, donor islet graft survival was 89% in diabetic NOD mice at 100 days posttransplant (N=9, P<0.01). The return of hyperglycemia in these NOD mice after nephrectomy confirmed islet graft function. Furthermore, long-term second donor-specific islet graft survival was achieved. Graft-versus-host disease was not observed in these NOD mice without or with posttransplant donor lymphocyte infusion. Conclusions: Our data demonstrate that adoptive immunotherapy with posttransplant donor lymphocyte infusion enhances donor chimerism and induces donor-specific tolerance to islet allografts in the setting of autoimmune diabetes.

betaTC-tet (H2(k)) is a conditional insulinoma cell line derived from transgenic mice expressing a tetracycline-regulated oncogene. Transgenic expression of several proteins implicated in the apoptotic pathways increase the resistance of betaTC-tet cells in vitro. We tested in vivo the sensitivity of the cells to rejection and the protective effect of genetic alterations in NOD mice. betaTC-tet cells and genetically engineered lines expressing Bcl-2 (CDM3D), a dominant negative mutant of MyD88 or SOCS-1 were transplanted in diabetic female NOD mice or in male NOD mice with diabetes induced by high-dose streptozotocin. Survival of functional cell grafts in NOD-scid mice was also analyzed after transfer of splenocytes from diabetic NOD mice. Autoreactive T-cell hybridomas and splenocytes from diabetic NOD mice were stimulated by betaTC-tet cells. betaTC-tet cells and genetically engineered cell lines were all similarly rejected in diabetic NOD mice and in NOD-scid mice after splenocyte transfer. In 3- to 6-week-old male NOD mice treated with high-dose streptozotocin, the cells temporarily survived, in contrast with C57BL/6 mice treated with high-dose streptozotocin (indefinite survival) and untreated 3- to 6-week-old male NOD mice (rejection). The protective effect of high-dose streptozotocin was lost in older male NOD mice. betaTC-tet cells did not stimulate autoreactive T-cell hybridomas, but induced IL-2 secretion by splenocytes from diabetic NOD mice. The autoimmune process seems to play an important role in the destruction of betaTC-tet cells in NOD mice. Genetic manipulations intended at increasing the resistance of beta cells were inefficient. Similar approaches should be tested in vivo as well as in vitro. High dose streptozotocin influences immune rejection and should be used with caution.

The precise fate of beta cells and the presence of islet infiltrates after onset of type 1 diabetes have not yet been fully characterized. Recently we showed that in newly diabetic NOD mice an appreciable number of beta cells remain. This was also observed during the first 2 weeks of diabetes in NOD mice without treatment with insulin. However, the mean number of beta cells per unit islet cross-sectional area decreased with increasing duration of disease. In contrast, glucagon and somatostatin cell numbers showed an increase. The persistence of insulitis in several islets until 4 weeks of diabetes suggests ongoing beta cell autoimmunity over a protracted phase. Combined daily treatment of newly diabetic NOD mice with epidermal growth factor (EGF) and gastrin for the first 14 days of diabetes resulted in temporary restoration of normoglycemia in 7 of 15 mice. We speculate that the residual beta cells present soon after onset of diabetes may respond to experimental regeneration. Treatment of newly diabetic NOD mice with the bioactive peptides EGF and gastrin resulted in partial and temporary reversal of diabetes. We propose that peptide therapies combined with other benign immunomodulatory approaches to rescue and preserve beta cells in the long term and to prevent recurring autoimmunity may be more effective than peptide therapy alone in reversing diabetes in NOD mice.