Regeneration Of Beta-cell Mass Research Articles

Glycemic control releases regenerative potential of pancreatic beta cells blocked by severe hyperglycemia.

Diabetogenic ablation of beta cells in mice triggers a regenerative response whereby surviving beta cells proliferate and euglycemia is regained. Here, we identify and characterize heterogeneity in response to beta cell ablation. Efficient beta cell elimination leading to severe hyperglycemia (>28mmol/L), causes permanent diabetes with failed regeneration despite cell cycle engagement of surviving beta cells. Strikingly,correction of glycemia via insulin, SGLT2 inhibition, or a ketogenic diet for about 3weeks allows partial regeneration of beta cell mass and recovery from diabetes, demonstrating regenerative potential masked by extreme glucotoxicity. We identify gene expression changes in beta cells exposed to extremely high glucose levels, pointing to metabolic stress and downregulation of key cell cycle genes, suggesting failure of cell cycle completion. These findings reconcile conflicting data on the impact of glucose on beta cell regeneration and identify a glucose threshold converting glycemic load from pro-regenerative to anti-regenerative.

1452-P: PDX1 and CKas Markers of Cell Maturation in the Human Pancreas

Regeneration of beta-cell mass is one of the fundamental problems in diabetology. Embryonic source of all pancreatic cell types is the epithelial cells of the primitive ducts. During pancreas development, epithelial progenitors differentiate in three directions, which is accompanied by changes in cell phenotype, including the expression of various types of cytokeratins, transcription factors, as well as hormones and some other antigens. The early markers of pancreatic epithelial progenitors are the homeodomain transcription factor Pdx 1 (pancreatic and duodenal homeobox 1) and an intermediate filament protein cytokeratin (CK19) . The aim of our work was to analyze the distribution of Pdx1 and CKduring endocrine pancreas development in humans. The study was performed on the pancreatic samples from 27 fetuses (gestational age 14-40 weeks) using double and triple immunohistochemistry with antibodies to Pdx1, CK19, and insulin or glucagon. During human prenatal development, the co-expression of CKand Pdx1 was detected in several cell types. Firstly, it was observed in the epithelial cells of the pancreatic ducts. During the branching of pancreatic ducts, CKexpression decreased in outgrows, while the Pdx1 expression was lowered in large ducts. Secondly, co-expression of CKand Pdx1 was detected in endocrine cells (mainly in alpha) . However, the intensity of the reaction to both markers in these cells was low. Phenotype of beta cells was Pdx1+/ CK19−. At the same time, during development, some cells with an intense reaction to CKand Pdx1 were detected in the forming islets. Apparently, an intense reaction to CK19/Pdx1 may characterized low-differentiated (progenitor) cells that are present in the epithelium of ducts and in the forming islets during the development of the human pancreas. Disclosure A.Proshchina: n/a. Y.Krivova: None. D.Otlyga: None. S.Saveliev: None.

Translation of curative therapy concepts with T cell and cytokine antibody combinations for type 1 diabetes reversal in the IDDM rat

Proinflammatory cytokines released from the pancreatic islet immune cell infiltrate in type 1 diabetes (T1D) cause insulinopenia as a result of severe beta cell loss due to apoptosis. Diabetes prevention strategies targeting different cytokines with antibodies in combination with a T cell antibody, anti-TCR, have been assessed for therapy success in the LEW.1AR1-iddm (IDDM) rat, an animal model of human T1D. Immediately after diabetes manifestation, antibody combination therapies were initiated over 5 days with anti-TNF-α (tumour necrosis factor), anti-IL-1β (interleukin), or anti-IFN-γ (interferon) together with anti-TCR for the reversal of the diabetic metabolic state in the IDDM rat. Anti-TCR alone showed only a very limited therapy success with respect to a reduction of immune cell infiltration and beta cell mass regeneration. Anti-TCR combinations with anti-IL-1β or anti-IFN-γ were also not able to abolish the increased beta cell apoptosis rate and the activated immune cell infiltrate leading to a permanent beta cell loss. In contrast, all anti-TCR combinations with anti-TNF-α provided sustained therapy success over 60 to 360 days. The triple combination of anti-TCR with anti-TNF-α plus anti-IL-1β was most effective in regaining sustained normoglycaemia with an intact islet structure in a completely infiltration-free pancreas and with a normal beta cell mass. Besides the triple combination, the double antibody combination of anti-TCR with anti-TNF-α proved to be the most suited therapy for reversal of the T1D metabolic state due to effective beta cell regeneration in an infiltration free pancreas.Key messagesAnti-TCR is a cornerstone in combination therapy for autoimmune diabetes reversal.The combination of anti-TCR with anti-TNF-α was most effective in reversing islet immune cell infiltration.Anti-TCR combined with anti-IL-1β was not effective in this respect.The combination of anti-TCR with anti-TNF-α showed a sustained effect over 1 year.

Imaging Beta Cell Regeneration and Interactions with Islet Vasculature in Transparent Adult Zebrafish

Blood vessel networks provide nutrients and gaseous exchange that are essential for functions. Pancreatic islet capillaries deliver oxygen to endocrine cells while transporting hormones to organs and peripheral locations throughout the body. We have developed a zebrafish diabetes model in which adult islets can be followed in vivo during beta cell regeneration while calibrating changes in beta cell mass and fasting blood glucose levels. After genetic ablation, beta cells are initially dysfunctional or dying, and blood glucose levels increase fourfold. During a 2-week period, hyperglycemia eventually normalizes as beta cell mass regenerates. We show that mCherry-fluorescent, insulin-positive beta cells re-emerge in close contact with the vascular endothelium. Alterations in the dense vascular network of zebrafish islets were visualized by the expression of green fluorescent protein (GFP) in endothelial cells derived from the Fli transcription factor promoter. The rapid destruction and regeneration of beta cell mass was evaluated in the same animal over time, providing a functional model for investigating the interactions of islet cell types with vascular cells as well as the consequences of hyperglycemia on other tissues. Regenerating adult zebrafish can be utilized as vertebrate, metabolically active models for generating new insights into treatments for type 2 diabetes.

Lineage tracing and resulting phenotype of haemopoietic-derived cells in the pancreas during beta cell regeneration

Transplantation of bone marrow-derived haemopoietic stem cells following streptozotocin (STZ) treatment to induce pancreatic beta cell loss in mice causes the partial regeneration of beta cell mass, with many haemopoietic cells demonstrating endothelial cell markers. This study used genetically tagged haemopoietic lineage-derived cells to determine how endogenous cells are mobilised following beta cell loss and subsequent replacement. A double transgenic mouse model, Vav-iCre; R26R-enhanced yellow fluorescent protein (YFP), was used where only haemopoietic lineage cells expressed the Vav1 gene promoter allowing expression of the YFP reporter gene. Between postnatal days 2 and 4 mice were injected with STZ or vehicle (control) and body weight and glycaemia were monitored. Mice were killed between days 10 and 130, and the pancreases were examined by immunofluorescence microscopy. YFP-expressing cells infiltrated the pancreas at all ages, being present around newly forming islets at the pancreatic ducts, and within larger islets. Small numbers of YFP-positive cells (<5%) co-stained for the macrophage markers F4/80 or Mac1, for cytokeratin 19, or for the transcription factor pancreatic and duodenal homeobox 1 (PDX-1), but no co-localisation was seen with insulin or other endocrine hormones. Within islets approximately 30% of YFP-positive cells co-stained for the endothelial cell marker CD31, and following STZ the number of haemopoietic-derived cells, and the proportion that were CD31-positive, both significantly increased after 21 and 40 days, coincident with a partial replacement of beta cells. Our results suggest that following beta cell loss endogenous haemopoietic-lineage cells contribute to intra-islet angiogenesis, which supports a partial recovery of beta cell mass.

Therapeutic approaches based on beta-cell mass preservation and/or regeneration

Beta-cell deficiency is a pathophysiologic component of diabetes mellitus and a primary cause of islet dysfunction. Islet dysfunction is a prerequisite for the development of diabetes mellitus since individuals with insulin resistance (e.g. obesity, pregnancy) do not develop hyperglycemia unless beta-cell compensation fails. Therefore, understanding of the biology and mechanisms involved in normal beta-cell adaptation may provide novel therapeutic targets for preservation and/or regeneration of beta-cell mass in diabetes mellitus. Normal adaptation of beta-cell mass occurs by beta-cell replication and/or neogenesis from precursor cells inside the pancreas. However, the relative importance of both processes for successful adaptation is unknown. In type-2 diabetes, the primary defect is increased beta-cell apoptosis. Since replicating beta-cells are more vulnerable to apoptosis, the proapoptotic diabetic milieu limits the regenerative capacity of the islet and directly causes accelerated islet loss. Therapeutic approaches need to address both processes of islet turnover (regeneration and cell loss) in order to be successful. It may be anticipated that such an intervention is also effective early in the course of diabetes or in prediabetic conditions.

Pancreatic beta cell mass regeneration and expansion — a role for FoxM1?

Pancreatic beta cell mass regeneration and expansion — a role for FoxM1?

Inhibition of GSK3 Promotes Replication and Survival of Pancreatic Beta Cells

Recent developments indicate that the regeneration of beta cell function and mass in patients with diabetes is possible. A regenerative approach may represent an alternative treatment option relative to current diabetes therapies that fail to provide optimal glycemic control. Here we report that the inactivation of GSK3 by small molecule inhibitors or RNA interference stimulates replication of INS-1E rat insulinoma cells. Specific and potent GSK3 inhibitors also alleviate the toxic effects of high concentrations of glucose and the saturated fatty acid palmitate on INS-1E cells. Furthermore, treatment of isolated rat islets with structurally diverse small molecule GSK3 inhibitors increases the rate beta cell replication by 2-3-fold relative to controls. We propose that GSK3 is a regulator of beta cell replication and survival. Moreover, our results suggest that specific inhibitors of GSK3 may have practical applications in beta cell regenerative therapies.

WITHDRAWN: Pancreatic beta cell mass regeneration and expansion — a role for FoxM1?

WITHDRAWN: Pancreatic beta cell mass regeneration and expansion — a role for FoxM1?

Pancreatic islets from cyclin-dependent kinase 4/R24C (Cdk4) knockin mice have significantly increased beta cell mass and are physiologically functional, indicating that Cdk4 is a potential target for pancreatic beta cell mass regeneration in Type 1 diabetes.

Cyclin-dependent kinase 4 (Cdk4) is crucial for beta cell development. A mutation in the gene encoding for Cdk4, Cdk4R24C, causes this kinase to be insensitive to INK4 cell cycle inhibitors and induces beta cell hyperplasia in Cdk4R24C knockin mice. We aimed to determine whether this Cdk4R24C mutation also affects proper islet function, and whether it promotes proliferation in human islets lentivirally transduced with Cdk4R24C cDNA. Our study was conducted on wild-type and Cdk4R24C knockin mice. Pancreases were morphometrically analysed. Intraperitoneal glucose tolerance tests and intravenous insulin tolerance tests were performed on wild-type and Cdk4R24C mice. We also did in vitro islet perifusion studies and islet metabolic labelling analysis. Human islets were transduced with Cdk4R24C cDNA. Pancreatic islets from Cdk4R24C knockin mice exhibit a larger insulin-producing beta cell area and a higher insulin content than islets from wild-type littermates. Insulin secretion in response to glucose is faster and reaches a higher peak in Cdk4R24C mice without leading to hypoglycaemia. Conversion of pro-insulin into insulin and its intermediates is similar in Cdk4R24C and wild-type mice. Glucose utilisation and oxidation measured per islet were similar in both experimental groups. Insulin secretion was faster and enhanced in Cdk4R24C islets perifused with 16.7 mmol/l glucose, with slower decay kinetics when glucose returned to 2.8 mmol/l. Moreover, human islets expressing Cdk4R24C cDNA exhibited higher beta cell proliferation. Despite their hyperplastic growth, Cdk4R24C insulin-producing islet cells behave like differentiated beta cells with regard to insulin production, insulin secretion in response to glucose, and islet glucose metabolism. Therefore Cdk4 could possibly be used to engineer a source of beta cell mass for islet transplantation.

Mechanisms of glucose hypersensitivity in beta-cells from normoglycemic, partially pancreatectomized mice.

Increased beta-cell sensitivity to glucose precedes the loss of glucose-induced insulin secretion in diabetic animals. Changes at the level of beta-cell glucose sensor have been described in these situations, but it is not clear whether they fully account for the increased insulin secretion. Using a euglycemic-normolipidemic 60% pancreatectomized (60%-Px) mouse model, we have studied the ionic mechanisms responsible for increased beta-cell glucose sensitivity. Two weeks after Px (Px14 group), Px mice maintained normoglycemia with a reduced beta-cell mass (0.88 +/- 0.18 mg) compared with control mice (1.41 +/- 0.21 mg). At this stage, the dose-response curve for glucose-induced insulin release showed a significant displacement to the left (P < 0.001). Islets from the Px14 group showed oscillatory electrical activity and cytosolic Ca2+ ([Ca2+]i) oscillations in response to glucose concentrations of 5.6 mmol/l compared with islets from the control group at 11.1 mmol/l. All the above changes were fully reversible both in vitro (after 48-h culture of islets from the Px14 group) and in vivo (after regeneration of beta-cell mass in islets studied 60 days after Px). No significant differences in the input resistance and ATP inhibition of ATP-sensitive K+ (K(ATP)) channels were found between beta-cells from the Px14 and control groups. The dose-response curve for glucose-induced MTT (C,N-diphenyl-N''-4,5-dimethyl thiazol 2 yl tetrazolium bromide) reduction showed a significant displacement to the left in islets from the Px14 group (P < 0.001). These results indicate that increased glucose sensitivity in terms of insulin secretion and Ca2+ signaling was not due to intrinsic modifications of K(ATP) channel properties, and suggest that the changes are most likely to be found in the glucose metabolism.