Regulation of ATP-sensitive potassium channel subunit Kir6.2 expression in rat intestinal insulin-producing progenitor cells

We have reported that the combined expression of Pdx-1 (pancreatic duodenal homeobox 1) and Isl-1 (islet 1) enables immature rat enterocytes (IEC-6) to produce and release insulin. A key component regulating the release of insulin is the ATP-sensitive potassium channel subunit Kir6.2. To investigate the regulation of Kir6.2 gene expression, we assessed Kir6.2 expression in IEC-6 cells expressing Pdx-1 and/or Isl-1. We observed that Kir6.2 protein was expressed de novo in IEC-6 cells expressing both Pdx-1 and Isl-1 but not in cells expressing Pdx-1 alone. Next, we analyzed the regions of the Kir6.2 promoter (-1677/-45) by performing a luciferase assay and electrophoretic mobility shift assay. The results have demonstrated that Kir6.2 promoter possesses two regions regulating the promoter activity: a Foxa2-binding site (-1364 to -1210) and an Sp1/Sp3-binding site (-1035 to -939). The additional expression of Isl-1 in IEC-6 cells expressing Pdx-1 attenuated overexpression of Foxa2 protein and enhanced Kir6.2 expression. Finally, knockdown of Isl-1 using the iRNA technique resulted in decreased expression of Kir6.2 protein in a rat pancreatic beta-cell line (RIN-5F cells). These results indicate that expression of Kir6.2 in the rat intestine is moderated by Isl-1.     

Cellular interaction between mouse pancreatic alpha-cell and beta-cell lines: possible contact-dependent inhibition of insulin secretion

The endocrine cells in the pancreatic islet have cellular communication between the heterotypic cells as well as the homotypic cells. The present study was conducted to elucidate the cellular interaction between pancreatic alpha cells and beta cells utilizing differentiated mouse cell lines (i.e., alphaTC clone 6 and betaTC cells). Co-culture of these two cell lines on a gyratory shaker generated numerous cellular aggregates of homogenous size within 48 h. Immunohistochemical staining for insulin and glucagon demonstrated that betaTC cells were located in the central core of each aggregate, while alphaTC cells formed a mantle layer surrounding the betaTC cells. This segregation was observed regardless of the ratios of the two cell types employed. Although glucagon at concentrations of 10(-8) M or higher stimulated insulin secretion from betaTC cells in both monolayer and aggregates, an increase in the ratio of alphaTC/betaTC cells in aggregate cultures was accompanied by a decrease in secreted insulin and a rise in intracellular insulin content of the betaTC component. The inhibitory effect of alphaTC cells on betaTC insulin secretion was not limited to aggregate culture, since insulin secretion from betaTC cells was also suppressed, and intracellular insulin content increased, by co-culture of alphaTC with betaTC cells in monolayer. On the other hand, the secreted and intracellular insulin of betaTC cells was not affected by alphaTC cells in a Transwell co-culture system in which direct cell-to-cell contacts were prevented by a semipermeable membrane that permitted chemical communication via medium metabolites. These data suggest that the insulin secretion from betaTC cells may be inhibited possibly as a result of the contact with alphaTC cells.     

Expression of Pdx-1 in bone marrow mesenchymal stem cells promotes differentiation of islet-like cells in vitro

Bone marrow mesenchymal stem cells (BMSCs) have the ability of self-renewal and multi-directional differentiation. Recent reports showed that BMSCs could differentiate into endocrine cells of pancreas. However, the differentiation is not efficient enough to produce insulin-producing cells for the future therapeutic use. Pdx-1 is a crucial regulator for pancreatic development. Therefore we constructed a eukaryotic expression vector containing Pdx-1 to determine the effect of Pdx-1 expression on differentiation of BMSCs in vitro. The results showed that BMSCs could self-assemble to form functional pancreatic islet-like structures after differentiation in vitro. The proportion of insulin-producing cells differentiated from Pdx-1⁺BMSCs was 28.23%±2.56%, higher than that from BMSCs transfected with vacant vector and Pdx-1 ⁻ BMSCs (7.23%±1.56% and 4.08%±2.69% respectively) by flow cytometry. Immunocytochemical examination also testified the expression of multiple β-cells-specific genes such as insulin, glucagons, somatostatin in differentiated BMSCs. The results also revealed that the expressions of genes mentioned above in Pdx-1⁺BMSCs were higher than that in Pdx-1⁻BMSCs, which was confirmed by Western blotting analysis and RT-PCR. Glucose-induced insulin secretion from Pdx-1⁺BMSCs in 5mmol/L and 25mmol/L glocuse was (56.61±4.82) μU/mL and (115.29±2.56) μU/mL respectively, which were much higher than those from Pdx-1⁻BMSCs((25.53±6.49) μU/mL and (53.26±7.56) μU/mL respectively). Grafted animals were able to maintain their body weight and survive for relatively longer periods of time than hyperglycemic sham-grafted controls, which demonstrated an overall beneficial effect of the grafted cells on the health of the animals. These findings thus suggested that exogenous expression of Pdx-1 should provide a promising approach for efficiently producing islet-like cells from BMSCs for the future therapeutic use in diabetic patients.     

Increased DNA methylation and decreased expression of PDX-1 in pancreatic islets from patients with type 2 diabetes

Mutations in pancreatic duodenal homeobox 1 (PDX-1) can cause a monogenic form of diabetes (maturity onset diabetes of the young 4) in humans, and silencing Pdx-1 in pancreatic β-cells of mice causes diabetes. However, it is not established whether epigenetic alterations of PDX-1 influence type 2 diabetes (T2D) in humans. Here we analyzed mRNA expression and DNA methylation of PDX-1 in human pancreatic islets from 55 nondiabetic donors and nine patients with T2D. We further studied epigenetic regulation of PDX-1 in clonal β-cells. PDX-1 expression was decreased in pancreatic islets from patients with T2D compared with nondiabetic donors (P = 0.0002) and correlated positively with insulin expression (rho = 0.59, P = 0.000001) and glucose-stimulated insulin secretion (rho = 0.41, P = 0.005) in the human islets. Ten CpG sites in the distal PDX-1 promoter and enhancer regions exhibited significantly increased DNA methylation in islets from patients with T2D compared with nondiabetic donors. DNA methylation of PDX-1 correlated negatively with its gene expression in the human islets (rho = -0.64, P = 0.0000029). Moreover, methylation of the human PDX-1 promoter and enhancer regions suppressed reporter gene expression in clonal β-cells (P = 0.04). Our data further indicate that hyperglycemia decreases gene expression and increases DNA methylation of PDX-1 because glycosylated hemoglobin (HbA1c) correlates negatively with mRNA expression (rho = -0.50, P = 0.0004) and positively with DNA methylation (rho = 0.54, P = 0.00024) of PDX-1 in the human islets. Furthermore, while Pdx-1 expression decreased, Pdx-1 methylation and Dnmt1 expression increased in clonal β-cells exposed to high glucose. Overall, epigenetic modifications of PDX-1 may play a role in the development of T2D, given that pancreatic islets from patients with T2D and β-cells exposed to hyperglycemia exhibited increased DNA methylation and decreased expression of PDX-1. The expression levels of PDX-1 were further associated with insulin secretion in the human islets.