Increased Number Of Beta Cells Research Articles

Pancreatic alpha cell glucagon–liver FGF21 axis regulates beta cell regeneration in a mouse model of type 2 diabetes

Aims/hypothesisGlucagon receptor (GCGR) antagonism ameliorates hyperglycaemia and promotes beta cell regeneration in mouse models of type 2 diabetes. However, the underlying mechanisms remain unclear. The present study aimed to investigate the mechanism of beta cell regeneration induced by GCGR antagonism in mice.MethodsThe db/db mice and high-fat diet (HFD)+streptozotocin (STZ)-induced mice with type 2 diabetes were treated with antagonistic GCGR monoclonal antibody (mAb), and the metabolic variables and islet cell quantification were evaluated. Plasma cytokine array and liver RNA sequencing data were used to screen possible mediators, including fibroblast growth factor 21 (FGF21). ELISA, quantitative RT-PCR and western blot were applied to verify FGF21 change. Blockage of FGF21 signalling by FGF21-neutralising antibody (nAb) was used to clarify whether FGF21 was involved in the effects of GCGR mAb on the expression of beta cell identity-related genes under plasma-conditional culture and hepatocyte co-culture conditions. FGF21 nAb-treated db/db mice, systemic Fgf21-knockout (Fgf21−/−) diabetic mice and hepatocyte-specific Fgf21-knockout (Fgf21Hep−/−) diabetic mice were used to reveal the involvement of FGF21 in beta cell regeneration. A BrdU tracing study was used to analyse beta cell proliferation in diabetic mice treated with GCGR mAb.ResultsGCGR mAb treatment improved blood glucose control, and increased islet number (db/db 1.6±0.1 vs 0.8±0.1 per mm2, p<0.001; HFD+STZ 1.2±0.1 vs 0.5±0.1 per mm2, p<0.01) and area (db/db 2.5±0.2 vs 1.2±0.2%, p<0.001; HFD+STZ 1.0±0.1 vs 0.3±0.1%, p<0.01) in diabetic mice. The plasma cytokine array and liver RNA sequencing data showed that FGF21 levels in plasma and liver were upregulated by GCGR antagonism. The GCGR mAb induced upregulation of plasma FGF21 levels (db/db 661.5±40.0 vs 466.2±55.7 pg/ml, p<0.05; HFD+STZ 877.0±106.8 vs 445.5±54.0 pg/ml, p<0.05) and the liver levels of Fgf21 mRNA (db/db 3.2±0.5 vs 1.8±0.1, p<0.05; HFD+STZ 2.0±0.3 vs 1.0±0.2, p<0.05) and protein (db/db 2.0±0.2 vs 1.4±0.1, p<0.05; HFD+STZ 1.6±0.1 vs 1.0±0.1, p<0.01). Exposure to plasma or hepatocytes from the GCGR mAb-treated mice upregulated the mRNA levels of characteristic genes associated with beta cell identity in cultured mouse islets and a beta cell line, and blockage of FGF21 activity by an FGF21 nAb diminished this upregulation. Notably, the effects of increased beta cell number induced by GCGR mAb were attenuated in FGF21 nAb-treated db/db mice, Fgf21−/− diabetic mice and Fgf21Hep−/− diabetic mice. Moreover, GCGR mAb treatment enhanced beta cell proliferation in the two groups of diabetic mice, and this effect was weakened in Fgf21−/− and Fgf21Hep−/− mice.Conclusions/interpretationOur findings demonstrate that liver-derived FGF21 is involved in the GCGR antagonism-induced beta cell regeneration in a mouse model of type 2 diabetes.Graphical abstract

1449-P: Reducing Ghrelin Levels as a Novel Method to Increase Pancreatic Beta-Cell Mass

Objective: Exogenous insulin remains the leading therapy for type 1 diabetes mellitus, T1D. However, current insulin formulations and delivery methods do not match the precision of functioning beta-cells, and even intensive monitoring of blood glucose and use of insulin pumps are not enough to halt frequent hypoglycemia, hyperglycemia and morbidity. Thus, regenerating beta-cells has become a major target of diabetes research to treat T1D. While the hormone ghrelin is well known to increase blood glucose, at least in part by inhibiting insulin secretion, ghrelin’s action on beta-cell mass and proliferation has heretofore not been explored in detail. Here, we sought to test the hypothesis that reducing ghrelin levels increases beta-cell mass. Methods: Using histologic methods, we compared islet anatomy in ghrelin-knockout (ghrelin-KO) mice vs. wildtype littermates at three different ages: newborns (P0-P2) , juveniles (4-week-old) , and adults (10-12-week-old) . We also compared islet anatomy in adult wildtype mice, 4 weeks following conditional ghrelin-cell ablation vs. wildtype mice with intact ghrelin-cells. n= 4-8 mice per group and 33-433 islets per mouse. Results: Ghrelin gene deletion or ghrelin-cell ablation increased mean islet area and mean islet insulin-immunoreactive (insulin-IR) area by 52-82 % and by 45-51 %, respectively, in juveniles and adults. Increased islet size was driven by an increased % of large islets. Increased insulin-IR area was associated with increased beta-cell number per islet. An increased % of large islets was also observed in newborn ghrelin-KO mice. Conclusions: In mice, ghrelin plays an important role in controlling beta-cell mass. Reducing ghrelin levels for even short periods can increase islet size by increasing beta-cell numbers. Disclosure D.Gupta: None. C.P.Richard: None. J.M.Zigman: Research Support; Novo Nordisk Research Center Indianapolis, Novo Nordisk Research Center Indianapolis, Stock/Shareholder; Medtronic, Medtronic. A.W.Burstein: None. K.Shankar: None. S.Varshney: None. O.Singh: None. S.Paul: None. S.B.Ogden: None. S.Osborne-lawrence: None. N.Metzger: None.

Butyrate and Class I Histone Deacetylase Inhibitors Promote Differentiation of Neonatal Porcine Islet Cells into Beta Cells.

Neonatal porcine islets-like clusters (NPICCs) are a promising source for cell therapy of type 1 diabetes. Freshly isolated NPICCs are composed of progenitor cells and endocrine cells, which undergo a maturation process lasting several weeks until the normal beta cell function has developed. Here, we investigated the effects of short-chain fatty acids on the maturation of islet cells isolated from two to three day-old piglets. NPICCs were cultivated with acetate, butyrate and propionate (0–2000 µM) for one to eight days. Incubation with butyrate resulted in a significant upregulation of insulin gene expression and an increased beta cell number, whereas acetate or propionate had only marginal effects. Treatment with specific inhibitors of G-protein-coupled receptor GPR41 (β-hydroxybutyrate) and/or GPR43 (GPLG0974) did not abolish butyrate induced insulin expression. However, incubation of NPICCs with class I histone deacetylase inhibitors (HDACi) mocetinostat and MS275, but not selective class II HDACi (TMP269, MC1568) mimicked the butyrate effect on beta cell differentiation. Our study revealed that butyrate treatment has the capacity to increase the number of beta cells, which may be predominantly mediated through its HDAC inhibitory activity. Butyrate and specific class I HDAC inhibitors may represent beneficial supplements to promote differentiation of neonatal porcine islet cells towards beta cells for cell replacement therapies.

FSTL3 Inhibition Restores Glucose Responsive Insulin Secretion in Nonfunctional Human Islets

Type 2 diabetes results from inadequate insulin production to control blood glucose. To address this need for replacing lost insulin-producing beta cells, most therapeutic development strategies are focused on enhancing functional beta cell number. Fairbanks Pharmaceuticals is pursuing therapies that enhance beta cell function and regeneration and is based on prior research in which inactivation of follistatin-like-3 (FSTL3) resulted in enlarged islets, increased beta cell number, and enhanced insulin secretion. At least some of these new beta cells arose from alpha to beta cell transdifferentiation in FSTL3 knockout mice. Fairbanks has produced a monoclonal antibody (FP-101) that can completely block FSTL3 from binding activin A and B, GDF11, and myostatin that could also disrupt pre-formed and otherwise irreversible complexes. FP-101 was also able to restore glucose-responsive insulin secretion in otherwise non-functional islets taken from HFD-fed mice. To determine if FSTL3 neutralization could be effective in humans, human islets from normal donors were first exposed to elevated glucose (25 nM) for 48-72 hours, a commonly utilized model for glucotoxicity. As expected, this caused loss of glucose response to subsequent GSIS. However, when treated for 24 hours with FP-101, GSIS was restored, indicating that neutralization of FSTL3 might be an effective strategy for restoring function to human non-functional islets. This observation agrees with previous research in which activin treatment directly restored GSIS in human islets from diabetic donors. Taken together with the potential for induction of alpha to beta cell transdifferentiation, these results suggest that neutralization of FSTL3 may have a dual benefit with acute restoration of GSIS in non-functional islets and chronic treatment leading to restoration of beta cell number through enhancement of alpha to beta cell transdifferentiation. In vivo studies will be initiated shortly. Disclosure A. Schneyer: Stock/Shareholder; Self; Fairbanks Pharmaceuticals, Inc.. Stock/Shareholder; Spouse/Partner; Fairbanks Pharmaceuticals, Inc.. Employee; Self; Fairbanks Pharmaceuticals, Inc.. M. Brown: None.

Metformin, beta-cell development, and novel processes following beta-cell ablation in zebrafish.

Type 1 and 2 diabetes are characterized by a loss of insulin-producing beta-cells. Current treatments help maintain blood glucose levels but cannot provide a cure. As such, a vital target for the cure of diabetes is a way to restore beta-cell mass. The drug metformin can protect cultured beta-cells/islets from hyperglycemia-induced dysfunction and death. Further, treatment of pregnant mice with metformin results in an enhanced beta-cell fraction in the embryos; however, whether this occurs via a direct effect is unknown. We utilized the external embryogenesis of the zebrafish to determine the direct effect of metformin treatment on the pancreas of the developing embryo and following beta-cell ablation. During development metformin did not alter beta-cell or alpha-cell mass but had a small effect to increase delta-cell mass as measured by in situ hybridization. Further metformin significantly increased beta-cell number. Following beta-cell ablation, both glucagon and somatostatin expression were upregulated (>2-fold). Additionally, while metformin showed no effect to alter beta-cell mass or number, somatostatin expression was further increased (>5-fold). We showed that direct exposure to metformin during embryogenesis does not increase insulin-expressing area but does increase beta-cell number. Further, we identified novel consequences of beta-cell ablation to alter the expression of other pancreatic hormones that were enhanced by metformin. Therefore, this study provides a greater understanding of the beta-cell development/regenerative processes and the effect of metformin, bringing us closer to identifying how to increase beta-cells in humans.

Topologically Heterogeneous Beta Cell Adaptation in Response to High-Fat Diet in Mice

AimsBeta cells adapt to an increased insulin demand by enhancing insulin secretion via increased beta cell function and/or increased beta cell number. While morphological and functional heterogeneity between individual islets exists, it is unknown whether regional differences in beta cell adaptation occur. Therefore we investigated beta cell adaptation throughout the pancreas in a model of high-fat diet (HFD)-induced insulin resistance in mice.MethodsC57BL/6J mice were fed a HFD to induce insulin resistance, or control diet for 6 weeks. The pancreas was divided in a duodenal (DR), gastric (GR) and splenic (SR) region and taken for either histology or islet isolation. The capacity of untreated islets from the three regions to adapt in an extrapancreatic location was assessed by transplantation under the kidney capsule of streptozotocin-treated mice.ResultsSR islets showed 70% increased beta cell proliferation after HFD, whereas no significant increase was found in DR and GR islets. Furthermore, isolated SR islets showed twofold enhanced glucose-induced insulin secretion after HFD, as compared with DR and GR islets. In contrast, transplantation of islets isolated from the three regions to an extrapancreatic location in diabetic mice led to a similar decrease in hyperglycemia and no difference in beta cell proliferation.ConclusionsHFD-induced insulin resistance leads to topologically heterogeneous beta cell adaptation and is most prominent in the splenic region of the pancreas. This topological heterogeneity in beta cell adaptation appears to result from extrinsic factors present in the islet microenvironment.

Maintenance of redox state and pancreatic beta‐cell function: Role of leptin and adiponectin

Whereas oxidative stress is linked to cellular damage, reactive oxygen species (ROS) are also believed to be involved in the propagation of signaling pathways. Studies on the role of ROS in pancreatic beta-cell physiology, in contrast to pathophysiology, have not yet been reported. In this study we investigate the importance of maintaining cellular redox state on pancreatic beta-cell function and viability, and the effects of leptin and adiponectin on this balance. Experiments were conducted on RINm and MIN6 pancreatic beta-cells. Leptin (1-100 ng/ml) and adiponectin (1-100 nM) increased ROS accumulation, as was determined by DCFDA fluorescence. Using specific inhibitors, we found that the increase in ROS levels was mediated by NADPH oxidase (Nox), but not by AMP kinase (AMPK) or phosphatidyl inositol 3 kinase (PI3K). Leptin and adiponectin increased beta-cell number as detected by the XTT method, but did not affect apoptosis, indicating that the increased cell number results from increased proliferation. The adipokines-induced increase in viability is ROS dependent as this effect was abolished by N-acetyl-L-cysteine (NAC) or PEG-catalase. In addition, insulin secretion was found to be regulated by alterations in redox state, but not by adipokines. Finally, the effects of the various treatments on activity and mRNA expression of several antioxidant enzymes were determined. Both leptin and adiponectin reduced mRNA levels of superoxide dismutase (SOD)1. Adiponectin also decreased SOD activity and increased catalase and glutathione peroxidase (GPx) activities in the presence of H2O2. The results of this study show that leptin and adiponectin, by inducing a physiological increase in ROS levels, may be positive regulators of beta-cell mass.