Preserving expression of Pdx1 improves ß-cell failure in diabetic mice.

Pdx1, a ß-cell-specific transcription factor, has been shown to play a crucial role in maintaining ß-cell function through transactivation of ß-cell-related genes. In addition, it has been reported that the expression levels of Pdx1 are compromised under diabetic conditions in human and rodent models. We therefore aimed to clarify the possible beneficial role of Pdx1 against ß-cell failure and generated the transgenic mouse that expressed Pdx1 conditionally and specifically in ß cells (ßPdx1) and crossed these mice with Ins2Akita diabetic mice. Whereas Pdx1 mRNA levels were reduced in Ins2Akita mice compared with their non-diabetic littermates, the mRNA levels of Pdx1 were significantly recovered in the islets of ßPdx1; Ins2Akita mice. The ßPdx1; Ins2Akita mice exhibited significantly improved glucose tolerance, compared with control Ins2Akita littermates, accompanied by increased insulin secretion after glucose loading. Furthermore, histological examination demonstrated that ßPdx1; Ins2Akita mice had improved localization of SLC2A2 (GLUT2), and quantitative RT-PCR showed the recovered expression of Mafa and Gck mRNAs in the islets of ßPdx1; Ins2Akita mice. These findings suggest that the sustained expression of Pdx1 improves ß-cell failure in Ins2Akita mice, at least partially through the preserving expression of ß-cell-specific genes as well as improved localization of GLUT2.

Short-term selective alleviation of glucotoxicity and lipotoxicity ameliorates the suppressed expression of key ß-cell factors under diabetic conditions.

Alleviation of hyperglycaemia and hyperlipidemia improves pancreatic ß-cell function in type 2 diabetes. However, the underlying molecular mechanisms are still not well clarified. In this study, we aimed to elucidate how the expression alterations of key ß-cell factors are altered by the short-term selective alleviation of glucotoxicity or lipotoxicity. We treated db/db mice for one week with empagliflozin and/or bezafibrate to alleviate glucotoxicity and/or liptotoxicity, respectively. The gene expression levels of Pdx1 and Mafa, and their potential targets, insulin 1, Slc2a2, and Glp1r, were higher in the islets of empagliflozin-treated mice, and levels of insulin 2 were higher in mice treated with both reagents, than in untreated mice. Moreover, compared to the pretreatment levels, Mafa and insulin 1 expression increased in empagliflozin-treated mice, and Slc2a2 increased in combination-treated mice. In addition, empagliflozin treatment enhanced ß-cell proliferation assessed by Ki-67 immunostaining. Our date clearly demonstrated that the one-week selective alleviation of glucotoxicity led to the better expression levels of the key ß-cell factors critical for ß-cell function over pretreatment levels, and that the alleviation of lipotoxicity along with glucotoxicity augmented the favorable effects under diabetic conditions.

A novel function of Onecut1 protein as a negative regulator of MafA gene expression.

The transcription factor MafA is a key regulator of insulin gene expression and maturation of islet ß cells. Despite its importance, the regulatory mechanism of MafA gene expression is still unclear. To identify the transcriptional regulators of MafA, we examined various transcription factors, which are potentially involved in ß cell differentiation. An adenovirus-mediated overexpression study clearly demonstrated that Onecut1 suppresses the promoter activity of MafA through the Foxa2-binding cis-element on the MafA enhancer region (named area A). However, ChIP analysis showed that Foxa2 but not Onecut1 could directly bind to area A. Furthermore, overexpression of Onecut1 inhibited the binding of Foxa2 onto area A upon ChIP analysis. Importantly, insertion of a mutation in the Foxa2-binding site of area A significantly decreased the promoter activity of MafA. These findings suggest that Onecut1 suppresses MafA gene expression through the Foxa2-binding site. In the mouse pancreas, MafA expression was first detected at the latest stage of ß cell differentiation and was scarcely observed in Onecut1-positive cells during pancreas development. In addition, Onecut1 expression was significantly increased in the islets of diabetic db/db mice, whereas MafA expression was markedly decreased. The improved glucose levels of db/db mice with insulin injections significantly reduced Onecut1 expression and rescued the reduction of MafA expression. These in vivo experiments also suggest that Onecut1 is a negative regulator of MafA gene expression. This study implicates the novel role of Onecut1 in the control of normal ß cell differentiation and its involvement in ß cell dysfunction under diabetic conditions by suppressing MafA gene expression.

Sequential introduction and dosage balance of defined transcription factors affect reprogramming efficiency from pancreatic duct cells into insulin-producing cells.

While the exogenous expression of a combination of transcription factors have been shown to induce the conversion of non-ß cells into insulin-producing cells, the reprogramming efficiency remains still low. In order to develop an in vitro screening system for an optimized reprogramming protocol, we generated the reporter cell line mPac-MIP-RFP in which the reprogramming efficiency can be quantified with red fluorescent protein expressed under the control of the insulin promoter. Analysis with mPac-MIP-RFP cells sequentially infected with adenoviruses expressing Pdx1, Neurog3, and Mafa revealed that expression of Pdx1 prior to Neurog3 or Mafa augments the reprogramming efficiency. Next, infection with a polycistronic adenoviral vector expressing Pdx1, Neurog3 and Mafa significantly increased the expression level of insulin compared with the simultaneous infection of three adenoviruses carrying each transcription factor, although excessive expression of Mafa together with the polycistronic vector dramatically inhibited the reprogramming into insulin-producing cells. Thus, in vitro screening with the mPac-MIP-RFP reporter cell line demonstrated that the timing and dosage of gene delivery with defined transcription factors influence the reprogramming efficiency. Further investigation should optimize the reprogramming conditions for the future cell therapy of diabetes.

Consistency of plasma glucagon levels in patients with type 1 diabetes after a 1-year period.

Recent progress in research on glucagon and α-cells highlights their pathophysiological roles in diabetes. We previously showed that plasma glucagon levels measured by newly developed enzyme-linked immunosorbent assay were dysregulated in patients with type 1 diabetes with respect to plasma glucose levels, suggesting dysregulated secretion. In the current study, the annual change in plasma glucagon levels was assessed in these same patients. We found that the plasma glucagon levels in the 66 Japanese patients involved in the study were significantly correlated between both years. In addition, they were significantly associated with serum blood urea nitrogen levels in a multivariate linear regression analysis, as reported in our previous study. The statistical correlation in glucagon levels between annual checkups and the sustained significant correlation between glucagon and blood urea nitrogen suggest a constant dysregulation of glucagon in association with altered amino acid metabolism in patients with type 1 diabetes.

Mafa Enables Pdx1 to Effectively Convert Pancreatic Islet Progenitors and Committed Islet α-Cells Into ß-Cells In Vivo.

Among the therapeutic avenues being explored for replacement of the functional islet ß-cell mass lost in type 1 diabetes (T1D), reprogramming of adult cell types into new ß-cells has been actively pursued. Notably, mouse islet α-cells will transdifferentiate into ß-cells under conditions of near ß-cell loss, a condition similar to T1D. Moreover, human islet α-cells also appear to poised for reprogramming into insulin-positive cells. Here we have generated transgenic mice conditionally expressing the islet ß-cell-enriched Mafa and/or Pdx1 transcription factors to examine their potential to transdifferentiate embryonic pan-islet cell Ngn3-positive progenitors and the later glucagon-positive α-cell population into ß-cells. Mafa was found to both potentiate the ability of Pdx1 to induce ß-cell formation from Ngn3-positive endocrine precursors and enable Pdx1 to produce ß-cells from α-cells. These results provide valuable insight into the fundamental mechanisms influencing islet cell plasticity in vivo.