Pdx1 maintains ?-cell identity and function by repressing an ?-cell program
ABSTRACT: To get a more complete picture of the transcriptional changes associated with Pdx1 loss in ?-cells, we conducted an mRNA microarray comparing normal islet ?-cells and a-cells to the reprogrammed cells from PKO mice. Islet beta cells are from mice which has a single copy of Pdx1 flox (Pdx1L/+) allele, but is considered normal based on normal islet morphology, gene profiling, and euglycemic status. We chose to use heterozygous mice as control to avoid the litter effect. Islet alpha cells are from normal mice. To enrich for genes directly affected by Pdx1 loss, we chose the early time-point for analysis of PKO mice (5d after TAM administration). Control mRNA profiling was performed on FACS sorted islet YFP+ ?-cells and a-cells obtained from 2 month-old glucagon-Cre; RosaYFP and RIP-CreER; Pdx1fl/+, RosaYFP mice, respectively.
Project description:To get a more complete picture of the transcriptional changes associated with Pdx1 loss in β-cells, we conducted an mRNA microarray comparing normal islet β-cells and a-cells to the reprogrammed cells from PKO mice. Islet beta cells are from mice which has a single copy of Pdx1 flox (Pdx1L/+) allele, but is considered normal based on normal islet morphology, gene profiling, and euglycemic status. We chose to use heterozygous mice as control to avoid the litter effect. Islet alpha cells are from normal mice. To enrich for genes directly affected by Pdx1 loss, we chose the early time-point for analysis of PKO mice (5d after TAM administration). Control mRNA profiling was performed on FACS sorted islet YFP+ β-cells and a-cells obtained from 2 month-old glucagon-Cre; RosaYFP and RIP-CreER; Pdx1fl/+, RosaYFP mice, respectively.
Project description:Pancreas specific deletion of the Haster promoter region results in a variegated phenotype in pancreatic islets with overexpression or silencing of the Hnf1a gene. To determine the transcriptional consequence of the overexpression or silencing of Hnf1a is islet cells from the Haster pKO mice (Haster loxP/loxP;Pdx1-Cre), we performed scRNA-seq of pancreatic islets from control and adult female Haster pKO mice.
Project description:Pancreas/duodenum homeobox protein 1 (PDX1) is an important transcription factor that regulates islet β-cell proliferation, differentiation, and function. Reduced expression of PDX1 is thought to contribute to β-cell loss and dysfunction in diabetes. Thus, promoting PDX1 expression can be an effective strategy to preserve β-cell mass and function. Previously, we established a <i>PDX1</i> promoter-dependent luciferase system to screen agents that can promote PDX1 expression. Natural compound tectorigenin (TG) was identified as a promising candidate that could enhance the activity of the promoter for the <i>PDX1</i> gene. In this study, we first demonstrated that TG could promote the expression of PDX1 in β-cells via activating extracellular signal-related kinase (ERK), as indicated by increased phosphorylation of ERK; this effect was observed under either normal or glucotoxic/lipotoxic conditions. We then found that TG could suppress induced apoptosis and improved the viability of β-cells under glucotoxicity and lipotoxicity by activation of ERK and reduction of reactive oxygen species and endoplasmic reticulum (ER) stress. These effects held true <i>in vivo</i> as well: prophylactic or therapeutic use of TG could obviously inhibit ER stress and decrease islet β-cell apoptosis in the pancreas of mice given a high-fat/high-sucrose diet (HFHSD), thus dramatically maintaining or restoring β-cell mass and islet size, respectively. Accordingly, both prophylactic and therapeutic use of TG improved HFHSD-impaired glucose metabolism in mice, as evidenced by ameliorating hyperglycemia and glucose intolerance. Taken together, TG, as an agent promoting PDX1 expression exhibits strong protective effects on islet β-cells both <i>in vitro</i> and <i>in vivo</i>.
Project description:Transcription factor pancreatic and duodenal homeobox 1 (Pdx1) plays an essential role in the pancreas to regulate its development and maintain proper islet function. However, less is known about the function of Pdx1 in the small intestine. We aim to investigate the role of Pdx1 in mature proximal small intestine by profiling the expression of genes differentially regulated in response to Pdx1 inactivation restricted to the intestinal epithelium in mice. Pdx1 was conditionally inactivated in the intestinal epithelium of Pdx1flox/flox;VilCre mice, by crossing mutant mice homozygous for loxP site-flanked Pdx1 alleles with transgenic mice expressing Cre recombinase under the control of the mouse villin 1 gene promoter. Total RNA was isolated from the first five centimeters of the small intestine from adult Pdx1flox/flox;VilCre and littermate control mice. Microarray analysis was performed to investigate genome-wide transcriptional profiles in the proximal small intestine.
Project description:Mutations in pancreatic duodenal homeobox (PDX1) are linked to human type 2 diabetes and maturity-onset diabetes of the young type 4. Consistent with this, Pdx1-haploinsufficient mice develop diabetes. Both apoptosis and necrosis of β cells are mechanistically implicated in diabetes in these mice, but a molecular link between Pdx1 and these 2 forms of cell death has not been defined. In this study, we introduced an shRNA into mouse insulinoma MIN6 cells to deplete Pdx1 and found that expression of proapoptotic genes, including NIP3-like protein X (Nix), was increased. Forced Nix expression in MIN6 and pancreatic islet β cells induced programmed cell death by simultaneously activating apoptotic and mitochondrial permeability transition-dependent necrotic pathways. Preventing Nix upregulation during Pdx1 suppression abrogated apoptotic and necrotic β cell death in vitro. In Pdx1-haploinsufficient mice, Nix ablation normalized pancreatic islet architecture, β cell mass, and insulin secretion and eliminated reactive hyperglycemia after glucose challenge. These results establish Nix as a critical mediator of β cell apoptosis and programmed necrosis in Pdx1-deficient diabetes.
Project description:Among the therapeutic avenues being explored for replacement of the functional islet ?-cell mass lost in type 1 diabetes (T1D), reprogramming of adult cell types into new ?-cells has been actively pursued. Notably, mouse islet ?-cells will transdifferentiate into ?-cells under conditions of near ?-cell loss, a condition similar to T1D. Moreover, human islet ?-cells also appear to poised for reprogramming into insulin-positive cells. Here we have generated transgenic mice conditionally expressing the islet ?-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 ?-cell population into ?-cells. Mafa was found to both potentiate the ability of Pdx1 to induce ?-cell formation from Ngn3-positive endocrine precursors and enable Pdx1 to produce ?-cells from ?-cells. These results provide valuable insight into the fundamental mechanisms influencing islet cell plasticity in vivo.
Project description:Transcription factor pancreatic and duodenal homeobox 1 (Pdx1) plays an essential role in the pancreas to regulate its development and maintain proper islet function. However, the functions of Pdx1 in mature small intestine are less known. We aimed to investigate the intestinal role of Pdx1 by profiling the expression of genes differentially regulated in response to inactivation of Pdx1 specifically in the intestinal epithelium. Pdx1 was conditionally inactivated in the intestinal epithelium of Pdx1(flox/flox);VilCre mice. Total RNA was isolated from the first 5 cm of the small intestine from mature Pdx1(flox/flox);VilCre and littermate control mice. Microarray analysis identified 86 probe sets representing 68 genes significantly upregulated or downregulated 1.5-fold or greater in Pdx(flox/flox);VilCre mice maintained under standard conditions. Ingenuity Pathway Analysis revealed that functions of the differentially expressed genes are significantly associated with metabolism of nutrients including lipids and iron. Network analysis examining the interactions among the differentially expressed genes further supports the notion that Pdx1 may modulate metabolism of lipids and iron from mature intestinal epithelium. Following forced oil feeding, Pdx1(flox/flox);VilCre mice showed diminished lipid staining in the duodenal epithelium and decreased serum triglyceride levels, indicating reduced lipid absorption compared with control duodenal epithelium. Blood samples from Pdx1(flox/flox);VilCre mice have significantly lower mean values for mean corpuscular volume and mean corpuscular hemoglobin, consistent with iron deficiency. The absence of nonheme iron in the villous epithelium and lamina propria of Pdx1(flox/flox);VilCre duodenum indicates that the duodenal epithelium lacking Pdx1 may have defects in importing iron through enterocytes, resulting in iron deficiency in Pdx1(flox/flox);VilCre mice.
Project description:<h4>Objective</h4>The transcription factors (TF) Foxa2 and Pdx1 are key regulators of beta-cell (?-cell) development and function. Mutations of these TFs or their respective cis-regulatory consensus binding sites have been linked to maturity diabetes of the young (MODY), pancreas agenesis, or diabetes susceptibility in human. Although Foxa2 has been shown to directly regulate Pdx1 expression during mouse embryonic development, the impact of this gene regulatory interaction on postnatal ?-cell maturation remains obscure.<h4>Methods</h4>In order to easily monitor the expression domains of Foxa2 and Pdx1 and analyze their functional interconnection, we generated a novel double knock-in homozygous (FVFPBF<sup>DHom</sup>) fluorescent reporter mouse model by crossing the previously described Foxa2-Venus fusion (FVF) with the newly generated Pdx1-BFP (blue fluorescent protein) fusion (PBF) mice.<h4>Results</h4>Although adult PBF homozygous animals exhibited a reduction in expression levels of Pdx1, they are normoglycemic. On the contrary, despite normal pancreas and endocrine development, the FVFPBF<sup>DHom</sup> reporter male animals developed hyperglycemia at weaning age and displayed a reduction in Pdx1 levels in islets, which coincided with alterations in ?-cell number and islet architecture. The failure to establish mature ?-cells resulted in loss of ?-cell identity and trans-differentiation towards other endocrine cell fates. Further analysis suggested that Foxa2 and Pdx1 genetically and functionally cooperate to regulate maturation of adult ?-cells.<h4>Conclusions</h4>Our data show that the maturation of pancreatic ?-cells requires the cooperative function of Foxa2 and Pdx1. Understanding the postnatal gene regulatory network of ?-cell maturation will help to decipher pathomechanisms of diabetes and identify triggers to regenerate dedifferentiated ?-cell mass.
Project description:Pdx1 and Oc1 are co-expressed in multipotent pancreatic progenitors and regulate the pro-endocrine gene Neurog3. Their expression diverges in later organogenesis, with Oc1 absent from hormone+ cells and Pdx1 maintained in mature ? cells. In a classical genetic test for cooperative functional interactions, we derived mice with combined Pdx1 and Oc1 heterozygosity. Endocrine development in double-heterozygous pancreata was normal at embryonic day (E)13.5, but defects in specification and differentiation were apparent at E15.5, the height of the second wave of differentiation. Pancreata from double heterozygotes showed alterations in the expression of genes crucial for ?-cell development and function, decreased numbers and altered allocation of Neurog3-expressing endocrine progenitors, and defective endocrine differentiation. Defects in islet gene expression and ?-cell function persisted in double heterozygous neonates. These results suggest that Oc1 and Pdx1 cooperate prior to their divergence, in pancreatic progenitors, to allow for proper differentiation and functional maturation of ? cells.
Project description:The transcription factor encoded by the Pdx1 gene is a critical transcriptional regulator, as it has fundamental actions in the formation of all pancreatic cell types, islet ?-cell development, and adult islet ?-cell function. Transgenic- and cell line-based experiments have identified 5'-flanking conserved sequences that control pancreatic and ?-cell type-specific transcription, which are found within areas I (bp -2694 to -2561), II (bp -2139 to -1958), III (bp -1879 to -1799), and IV (bp -6200 to -5670). Because of the presence in area IV of binding sites for transcription factors associated with pancreas development and islet cell function, we analyzed how an endogenous deletion mutant affected Pdx1 expression embryonically and postnatally. The most striking result was observed in male Pdx1?IV mutant mice after 3 weeks of birth (i.e., the onset of weaning), with only a small effect on pancreas organogenesis and no deficiencies in their female counterparts. Compromised Pdx1 mRNA and protein levels in weaned male mutant ?-cells were tightly linked with hyperglycemia, decreased ?-cell proliferation, reduced ?-cell area, and altered expression of Pdx1-bound genes that are important in ?-cell replication, endoplasmic reticulum function, and mitochondrial activity. We discuss the impact of these novel findings to Pdx1 gene regulation and islet ?-cell maturation postnatally.