Stimulating Beta-cell Replication Research Articles

Exogenous Lactogenic Signaling Stimulates Beta Cell Replication In Vivo and In Vitro.

As patients recently diagnosed with T1D and patients with T2D have residual beta cell mass, there is considerable effort in beta cell biology to understand the mechanisms that drive beta cell regeneration as a potential cellular therapy for expanding patients’ residual beta cell population. Both mouse and human studies have established that beta cell mass expansion occurs rapidly during pregnancy. To investigate the mechanisms of beta cell mass expansion during pregnancy, we developed a novel in vivo and in vitro models of pseudopregnancy. Our models demonstrate that pseudopregnancy promotes beta cell mass expansion in parous mice, and this expansion is driven by beta cell proliferation rather than hypertrophy. Importantly, estrogen, progesterone, and placental lactogen induce STAT5A signaling in the pseudopregnancy model, demonstrating that this model successfully recapitulates pregnancy-induced beta cell replication. We then created an in vitro model of pseudopregnancy and found that the combination of estrogen and placental lactogen induced beta cell replication in human islets and rat insulinoma cells. Therefore, beta cells both in vitro and in vivo increase proliferation when subjected to the pseudopregnancy cocktail compared to groups treated with estradiol or placental lactogen alone. The pseudopregnancy models described here may help inform novel methods of inducing beta cell replication in patients with diabetes.

Role of growth factors in control of pancreatic beta cell mass: focus on betatrophin.

Betatrophin is a newly described hormone, which potently stimulates beta cell replication in mice. This discovery has engendered great hope that it could prove clinically important in the treatment of type 1 and type 2 diabetes. Betatrophin, a 198-amino acid protein secreted by liver and adipose tissue, stimulates growth of pancreatic beta cell mass in insulin-resistant mice. Betatrophin has previously been named RIFL, lipasin, and ANGPLT8, and its salutory effects on lipid metabolism have been described in mouse and human studies. Serum betatrophin levels in humans correlate with improved adipose tissue lipid storage and lower serum triglyceride levels in the fed state, but do not correlate with insulin resistance or carbohydrate tolerance in humans. Betatrophin has not yet been shown to have an effect on beta cell replication in human pancreatic islets. Many endocrine and paracrine factors, of which betatrophin is the newest described, increase beta cell mass in murine models. None of these factors, including betatrophin, have displayed the same activity in clinical studies. This may reflect a profound species difference in beta cell regeneration pathways in mice and humans.

In vivo activation of the PI3K–Akt pathway in mouse beta cells by the EGFR mutation L858R protects against diabetes

EGF receptor (EGFR) signalling is required for normal beta cell development and postnatal beta cell proliferation. We tested whether beta cell proliferation can be triggered by EGFR activation at any age and whether this can protect beta cells against apoptosis induced by diabetogenic insults in a mouse model. We generated transgenic mice with doxycycline-inducible expression of constitutively active EGFR (L858R) (CA-EGFR) under the insulin promoter. Mice were given doxycycline at various ages for different time periods, and beta cell proliferation and mass were analysed. Mice were also challenged with streptozotocin and isolated islets exposed to cytokines. Expression of EGFR (L858R) led to increased phosphorylation of EGFR and Akt in pancreatic islets. CA-EGFR expression during pancreatic development (embryonic day [E]12.5 to postnatal day [P]1) increased beta cell proliferation and mass in newborn mice. However, CA-EGFR expression in adult mice did not affect beta cell mass. Expression of the transgene improved glycaemia and markedly inhibited beta cell apoptosis after a single high dose, as well as after multiple low doses of streptozotocin. In vitro mechanistic studies showed that CA-EGFR protected isolated islets from cytokine-mediated beta cell death, possibly by repressing the proapoptotic protein BCL2-like 11 (BIM). Our findings show that the expression of CA-EGFR in the developing, but not in the adult pancreas stimulates beta cell replication and leads to increased beta cell mass. Importantly, CA-EGFR protects beta cells against streptozotocin- and cytokine-induced death.

Stimulating Beta Cell Replication and Improving Islet Graft Function by a GPR40 and GPR120 Dual Agonist

Stimulating Beta Cell Replication and Improving Islet Graft Function by a GPR40 and GPR120 Dual Agonist

Stimulating beta cell replication and improving islet graft function by GPR119 agonists

G protein-coupled receptor 119 (GPR119) is predominantly expressed in β cells and intestinal L cells. In this study, we investigated whether oleoylethanolamide (OEA), a GPR119 endogenous ligand, and PSN632408, a GPR119 synthetic agonist, can stimulate β-cell replication in vitro and in vivo and improve islet graft function in diabetic mice. We found that OEA and PSN632408 significantly increased numbers of insulin(+)/5-bromo-2'-deoxyuridine (BrdU)(+) β cells in cultured mouse islets in a dose-dependent manner. All diabetic recipient mice, given marginal syngeneic islet transplants with OEA or PSN632408 or vehicle, achieved normoglycemia at 4 weeks after transplantation. However, normoglycemia was achieved significantly faster in OEA- or PSN632408-treated diabetic mice than in vehicle-treated diabetic mice (P < 0.05). The percentage of insulin(+)/BrdU(+) β cells in islet grafts in OEA- and PSN632408-treated mice was significantly higher than in vehicle-treated mice (P < 0.01). Our data demonstrated that OEA and PSN632408 can stimulate β-cell replication in vitro and in vivo and improve islet graft function. Targeting GPR119 is a novel therapeutic approach to increase β-cell mass and to improve islet graft function by stimulating β-cell replication.

Upregulating CD4+CD25+FOXP3+ Regulatory T Cells in Pancreatic Lymph Nodes in Diabetic NOD Mice by Adjuvant Immunotherapy

Immunotherapy with Complete Freund's adjuvant (CFA) is effective in ameliorating autoimmunity in diabetic nonobese diabetic (NOD) mice. We investigated whether CFA treatment up-regulates CD4+CD25+Foxp3+ regulatory T cells and increases transforming growth factor (TGF)-beta1 production in diabetic NOD mice. New-onset diabetic NOD mice were treated with CFA and exendin-4, a potent analog of glucagon-like peptide-1. Reversal of diabetes was determined by monitoring blood glucose level. Ameliorating autoimmunity through immunoregulation was assessed by adoptive transfer. Regulatory T cells in the peripheral blood, spleen, thymus, and pancreatic nodes were measured. TGF-beta1 in plasma and the insulin content in the pancreas were also measured. Immunostainings for insulin and BrdU were performed. New-onset diabetes could be reversed in 38% of NOD mice treated with CFA alone and in 86% of NOD mice treated with both CFA and exendin-4. Diabetes adoptive transfer by splenocytes from CFA-treated NOD mice was delayed. The percentage of CD4+CD25+Foxp3+ regulatory T cells in the pancreatic lymph nodes of CFA-treated NOD mice was significantly increased at 1, 5, and 15 to 17 weeks after treatment. TGF-beta1 in the plasma of CFA-treated NOD mice was also significantly increased. Combining CFA with exendin-4 treatment significantly increased the insulin content and the numbers of insulin and BrdU double-labeled beta cells in the islets. Our results demonstrated that CFA treatment ameliorates autoimmunity in diabetic NOD mice by up-regulating CD4=CD25+Foxp3+ regulatory T cells and increasing TGF-beta1 production. Exendin-4 enhanced the effect of CFA on reversing diabetes in NOD mice by stimulating beta-cell replication.

Rapid Ca2+ Influx and Diacylglycerol Synthesis in Growth Hormone-mediated Islet β-Cell Mitogenesis

Growth hormone (GH) is an important mitogenic stimulus for the insulin-producing beta-cell. We investigated the effects of GH on Ca(2+) handling and diacylglycerol (DAG) and cAMP formation in the beta-cell. GH elicited a rapid increase in the cytoplasmic free [Ca(2+)], which required extracellular Ca(2+) and was also blocked by pertussis toxin or protein kinase C (PKC) inhibition. GH also elevated islet DAG content, which should lead to PKC activation. Pertussis toxin and PKC inhibitors obliterated the mitogenicity of GH, suggesting involvement of GTP-binding proteins. PKC activation stimulated beta-cell proliferation, and it also activated phospholipase D. Islet cAMP content was not elevated by GH. Addition of a specific protein kinase A antagonist failed to influence the mitogenicity of GH, whereas a stimulatory cAMP agonist stimulated beta-cell replication. We conclude that GH rapidly increases the beta-cell cytoplasmic free [Ca(2+)] and also evokes a similar increase in DAG content via a phosphatidylcholine-specific phospholipase C, but does not affect mitogen-activated protein kinases, phospholipase D, or the cAMP signaling pathway. This rise in DAG may be of importance in translation of the stimulatory signal of GH into a proliferative response by the beta-cell, which seems to occur through GTP-binding proteins and PKC-dependent mechanisms.

Prolactin induction of insulin gene expression: the roles of glucose and glucose transporter-2.

Previous studies have shown that lactogenic hormones stimulate beta-cell proliferation and insulin production in pancreatic islets. However, all such studies have been conducted in cells incubated in medium containing glucose. Since glucose independently stimulates beta-cell replication and insulin production, it is unclear whether the effects of prolactin (PRL) on insulin gene expression are exerted directly or through the uptake and/or metabolism of glucose. We examined the interactions between glucose and PRL in the regulation of insulin gene transcription and the expression of glucose transporter-2 (glut-2) and glucokinase mRNAs in rat insulinoma (INS-1) cells. In the presence of 5.5 mM glucose, the levels of preproinsulin and glut-2 mRNAs in PRL-treated cells exceeded the levels in control cells (1.7-fold, P<0.05 and 2-fold, P<0.05 respectively). The maximal effects of PRL were noted at 24-48 h of incubation. PRL had no effect on the levels of glucokinase mRNA. The higher levels of glut-2 mRNA were accompanied by an increase in the number of cellular glucose transporters, as demonstrated by a 1. 4- to 2.4-fold increase in the uptake of 2-deoxy-d-[(3)H]glucose in PRL-treated INS-1 cells (P<0.001). These findings suggested that the insulinotropic effect of PRL is mediated, in part, by induction of glucose transport and/or glucose metabolism. Nevertheless, even in the absence of glucose, PRL stimulated increases in the levels of preproinsulin mRNA (3.4-fold higher than controls, P<0.0001) and glut-2 mRNA (2-fold higher than controls, P<0.01). These observations suggested that PRL exerts glucose-independent as well as glucose-dependent effects on insulin gene expression. Support for this hypothesis was provided by studies of insulin gene transcription using INS-1 cells transfected with a plasmid containing the rat insulin 1 promoter linked to a luciferase reporter gene. Glucose and PRL, alone and in combination, stimulated increases in cellular luciferase activity. The relative potencies of glucose (5.5 mM) alone, PRL alone, and glucose plus PRL in combination were 2.2 (P<0.001), 3.4 (P<0.01), and 7.9 (P<0.0001) respectively. Our findings suggest that glucose and PRL act synergistically to induce insulin gene transcription.

The Reg gene family and Reg proteins: with special attention to the regeneration of pancreatic beta-cells.

Pancreatic beta-cells of the islets of Langerhans are the only cells that produce insulin in humans, as well as in most animals, but they have been thought to have a limited capacity for regeneration, which is a predisposing factor for the development of diabetes mellitus. Strategies for influencing the replication and growth of the beta-cell mass are therefore important for the prevention and/or treatment of diabetes. We have established a model for islet regeneration in 90% depancreatized rats by the administration of poly(ADP-ribose) synthetase inhibitors such as nicotinamide. The regenerating islets in the remaining pancreas of poly(ADP-ribose) synthetase inhibitor-treated rats were markedly enlarged and consisted largely of insulin-producing beta-cells, preventing the development of diabetes mellitus that would otherwise be caused by the 90% pancreatectomy. In screening the regenerating islet-derived cDNA library, we found a novel gene and named it Reg (i.e., regenerating gene). The rate Reg cDNA had a single open reading frame that encoded a 165-amino acid protein with a 21-amino acid signal peptide. We also isolated the human REG cDNA which encoded a 166-amino acid protein with a 22-amino acid signal peptide. The amino acid sequence of human REG protein has 68% homology to that of rat Reg protein. Recombinant rat Reg protein without the signal peptide, produced in yeast, stimulated beta-cell replication and increased the beta-cell mass in the residual pancreas of 90% depancreatized rats, ameliorating the surgical diabetes. Recombinant human REG protein also induced an expansion of the beta-cell mass in non-obese diabetic (NOD) mice, resulting in the amelioration of diabetes. These results, as well as several other lines of evidence, indicate that Reg protein is a growth factor for pancreatic beta-cells and also suggest that the administration of Reg protein and/or activation of the Reg gene can be used as a potential therapeutic approach for diabetes mellitus. We have further isolated several Reg and Reg-related genes from human, rat and mouse, and revealed that they constitute a multigene family, the Reg gene family. Based on the primary structures of proteins encoded by the Reg gene family, we have grouped the members of the family into three subclasses, type I, II, and III. Type I Reg (Reg I) encodes a beta-cell growth factor, Reg I protein, as mentioned above. Some of the type III Reg (Reg III) have recently been suggested to play roles in the regeneration of cells other than pancreatic beta-cells, such as neuronal cells and epithelial cells in the alimentary tract.

Lithium increases DNA replication, polyamine content, and insulin secretion by rat pancreatic beta-cells.

The impact of long-term lithium exposure on the replication, polyamine content, and insulin production of rat pancreatic beta-cells was examined. Fetal rat pancreatic islets enriched in beta-cells were isolated and cultured for 3 days in the presence of different concentrations of LiCl. It was found that lithium dose dependently stimulated beta-cell replication, evoking a 40% increase in beta-cell replication at 1 mM, which was further increased to 70% at 10 mM of the ion. In contrast, the islet contents of insulin mRNA and insulin and insulin biosynthesis rates remained unaltered. The long-term insulin accumulation in the culture medium was nevertheless increased in LiCl-treated groups. In addition, neither the mitogenic effect nor the increased insulin accumulation in the medium by lithium was further augmented by growth hormone, which itself stimulated these functions. Lithium was also found to elevate the islet content of polyamines. Treatment with enzymatic inhibitors of polyamine synthesis failed to preclude the mitogenic and secretagogic impact of lithium, suggesting that the increased contents of polyamines did not convey the lithium effect. We conclude that lithium treatment stimulates rat beta-cell replication and long-term insulin secretion in vitro. These direct effects of the ion on the beta-cell may contribute to the antidiabetic effect of lithium encountered in animal models and patients.

Pancreatic beta cell replication: effects of hexose sugars.

Neonatal rat pancreatic monolayer cultures were utilized to investigate the effects of various hexose sugars on insulin release and beta cell replication. Sugars tested were D-glucose, L-glucose, 3-O-methyl-D-glucose, D-mannose and D-fructose. These agents were added at varying concentrations to a control medium containing a baseline level of 5.5 mM D-glucose. Replication was estimated by incubating cultures with [3H]thymidine and determining the frequency of beta cell labelling in aldehyde-thionin stained radioautographs. Although the addition of D-glucose to the control medium resulted in a concentration-dependent increase in both insulin release and beta cell replication, the sensitivity of these two processes to glucose differed. Insulin release was stimulated by a 2.75 mM elevation in the D-glucose concentration of the control medium, while an elevation of 11 mM D-glucose was required to increase the frequency of beta cell replication. Moreover, while insulin release was maximally stimulated by an 11 mM elevation in the concentration of D-glucose, the frequency of replication continued to rise as the concentration of D-glucose was further increased. The specificity of these responses to D-glucose was demonstrated by the inability of 11 mM L-glucose or 11 mM 3-O-methyl-D-glucose to stimulate beta cell replication. D-mannose and D-fructose were less potent insulin secretagogues than D-glucose on an equimolar basis and neither compound enhanced beta cell replication at a concentration of 11 mM. However, higher concentrations of D-mannose (16.5 and 22 mM) enhanced beta cell replication, while comparable concentrations of D-fructose did not. These results indicate that the sensitivity of insulin release and beta cell replication to D-glucose differ, and that in addition to D-glucose, D-mannose is also capable of stimulating beta cell replication.