Pancreatic Duodenum Homeobox Research Articles

PDX-1: A Promising Therapeutic Target to Reverse Diabetes.

The pancreatic duodenum homeobox-1 (PDX-1) is a transcription factor encoded by a Hox-like homeodomain gene that plays a crucial role in pancreatic development, β-cell differentiation, and the maintenance of mature β-cell functions. Research on the relationship between PDX-1 and diabetes has gained much attention because of the increasing prevalence of diabetes melitus (DM). Recent studies have shown that the overexpression of PDX-1 regulates pancreatic development and promotes β-cell differentiation and insulin secretion. It also plays a vital role in cell remodeling, gene editing, and drug development. Conversely, the absence of PDX-1 increases susceptibility to DM. Therefore, in this review, we summarized the role of PDX-1 in pancreatic development and the pathogenesis of DM. A better understanding of PDX-1 will deepen our knowledge of the pathophysiology of DM and provide a scientific basis for exploring PDX-1 as a potential target for treating diabetes.

Resistant starch produces antidiabetic effects by enhancing glucose metabolism and ameliorating pancreatic dysfunction in type 2 diabetic rats

The aim of the study was to evaluate the hypoglycemic effect of low, medium, and high doses of resistant starch type 2(RS2;100, 150, and 200g/kg) for 28days and explore its potential mechanism of this effect in type 2 diabetic rats treated with high-glucose-fat diet and low-dose streptozotocin(STZ). RS2 treatment induced better regulation of lipid in plasma and liver, fructosamine, oral glucose tolerance test, insulin, glucose metabolism and pancreatic damage in diabetic rats. The best hypoglycemic activity was observed after the medium-dose RS2 treatment. Western blot and real-time Polymerase Chain Reaction (RT-PCR)results revealed that phosphoenolpyruvate carboxykinase and glucose-6-phosphatase are involved in glyconeogenesis in the liver, and pancreatic duodenum homeobox 1 controls gluconeogenesis balance by regulating the expression levels of glucose kinase and glucose transport protein 2 in the liver and pancreas. Furthermore, the expression levels of insulin receptor substrate 1 and insulin receptor substrate 2 were enhanced in the pancreas. Results suggested that decreases in the blood glucose levels of diabetic rats fed with RS are regulated through the alteration of the expression levels of the genes related to glucose metabolism and amelioration of pancreatic dysfunction.

Human umbilical cord–derived mesenchymal stem cells can secrete insulin in vitro and in vivo

Diabetes mellitus is characterized by autoimmune destruction of pancreatic beta cells, leading to decreased insulin production. Differentiation of mesenchymal stem cells (MSCs) into insulin-producing cells offers novel ways of diabetes treatment. MSCs can be isolated from the human umbilical cord tissue and differentiate into insulin-secreting cells. Human umbilical cord-derived stem cells (hUDSCs) were obtained after birth, selected by plastic adhesion, and characterized by flow cytometric analysis. hUDSCs were transduced with nonintegrated lentivirus harboring PDX1 (nonintegrated LV-PDX1) and was cultured in differentiation medium in 21 days. Pancreatic duodenum homeobox protein-1 (PDX1) is a transcription factor in pancreatic development. Significant expressions of PDX1, neurogenin3 (Ngn3), glucagon, glucose transporter2 (Glut2), and somatostatin were detected by quantitative RT-PCR (P < 0.05). PDX1 and insulin proteins were shown by immunocytochemistry analysis. Insulin secretion of hUDSCs(PDX1+) in the high-glucose medium was 1.8 μU/mL. They were used for treatment of diabetic rats and could decrease the blood glucose level from 400 mg/dL to a normal level in 4 days. In conclusion, our results demonstrated that hUDSCs are able to differentiate into insulin-producing cells by transduction with nonintegrated LV-PDX1. These hUDSCs(PDX1+) have the potential to be used as a viable resource in cell-based gene therapy of type 1 diabetes.

Derivation of islet‐like cells from mesenchymal stem cells using PDX1‐transducing lentiviruses

Pancreatic duodenum homeobox protein-1 (PDX1) is a master regulatory gene in pancreatic development. Reprogramming of mesenchymal stem cells (MSCs) is a promising tool for producing insulin-producing cells. In this study, lentivirus harboring PDX1 (LV-PDX1) has been used for persistence gene expression in MSCs. The objective of this study was to evaluate the potential of lentivirus to introduce the PDX1 gene into MSCs to produce insulin-secreting cells and apply it for treatment of hyperglycemia in diabetic rats. MSCs were isolated from rat bone marrow, characterized, and transduced by LV-PDX1. Significant expressions of PDX1, neurogenin3, glucagon, glucose transporter2 (Glut2), and insulin were detected by quantitative reverse transcription-polymerase chain reaction (P < 0.05). PDX1 and insulin were detected at the protein level by immunofluorescence analysis. PDX1 could trigger a gene expression cascade that involved pancreatic endocrine differentiation and also revealed the glucose sensing ability by expressing Glut2 in high-glucose medium. The insulin secretion of MSCs(PDX1+) in the high-glucose medium was 1.75-fold higher than that secreted in the low-glucose medium (P < 0.05). MSCs(PDX1+) implanted into diabetic rats could decrease the blood glucose level from 485 mg/dL to the normal level in 3 days. This study showed MSCs(PDX1+) have the potential to be used as a viable resource in cell-based gene therapy of type 1 diabetes.

Nucleo-cytosolic Shuttling of FoxO1 Directly Regulates Mouse Ins2 but Not Ins1 Gene Expression in Pancreatic Beta Cells (MIN6)

The Forkhead box transcription factor FoxO1 regulates metabolic gene expression in mammals. FoxO1 activity is tightly controlled by phosphatidylinositol 3-kinase (PI3K) signaling, resulting in its phosphorylation and nuclear exclusion. We sought here to determine the mechanisms involved in glucose and insulin-stimulated nuclear shuttling of FoxO1 in pancreatic β cells and its consequences for preproinsulin (Ins1, Ins2) gene expression. Nuclear-localized endogenous FoxO1 translocated to the cytosol in response to elevated glucose (3 versus 16.7 mm) in human islet β cells. Real-time confocal imaging of nucleo-cytosolic shuttling of a FoxO1-EGFP chimera in primary mouse and clonal MIN6 β cells revealed a time-dependent glucose-responsive nuclear export, also mimicked by exogenous insulin, and blocked by suppressing insulin secretion. Constitutively active PI3K or protein kinase B/Akt exerted similar effects, while inhibitors of PI3K, but not of glycogen synthase kinase-3 or p70 S6 kinase, blocked nuclear export. FoxO1 overexpression reversed the activation by glucose of pancreatic duodenum homeobox-1 (Pdx1) transcription. Silencing of FoxO1 significantly elevated the expression of mouse Ins2, but not Ins1, mRNA at 3 mm glucose. Putative FoxO1 binding sites were identified in the distal promoter of rodent Ins2 genes and direct binding of FoxO1 to the Ins2 promoter was demonstrated by chromatin immunoprecipitation. A 915-bp glucose-responsive Ins2 promoter was inhibited by constitutively active FoxO1, an effect unaltered by simultaneous overexpression of PDX1. We conclude that nuclear import of FoxO1 contributes to the suppression of Pdx1 and Ins2 gene expression at low glucose, the latter via a previously unsuspected and direct physical interaction with the Ins2 promoter.

ParaHox genes in pancreatic cell cultures: effects on the insulin promoter regulation

The gene encoding PDX1 (pancreatic duodenum homeobox 1), the main transcription factor regulating the glucose-dependent transactivation of the insulin promoter in pancreatic beta-cells, clusters with two closely related homeobox genes (Gsh1 and Cdx2/3), all of them belonging to the ParaHox gene family. The ParaHox gene evolutionary history in the vertebrate lineage involved duplications of the cluster and subsequent loss of some members, so that eventually, the human and murine genomes contain only 6 ParaHox genes. The crucial role of PDX1 in pancreas development, beta-cell formation and insulin transcription regulation has long been established. There is some data on CDX2/3 function in alpha-cells, but remarkably, nothing is known on the role of the other ParaHox genes, which are also expressed in the endocrine pancreas. Homeobox transcription factors that belong to the same family show high conservation of the homeodomain and share similar target sites and oligomeric partners, and thus may act redundantly, synergistically or antagonistically on the same promoters. Therefore, we explored the effects of the Parahox proteins (GSH1, GSH2, CDX1, CDX2/3 and CDX4) on the regulation of the insulin promoter in transfected alpha- and beta- cultured cell lines at different glucose concentrations and compared them to those of PDX1. Noticeably, several ParaHox transcription factors are able to transactivate or inhibit the insulin promoter, depending on the cell type and glucose concentration, thus suggesting their possible participation in the regulation of similar target genes, such as insulin, either by silencing or activating them, in the absence of PDX1.

MicroRNA-124a Regulates Foxa2 Expression and Intracellular Signaling in Pancreatic β-Cell Lines

MicroRNAs (miRNAs) are short non-coding RNAs that have been implicated in fine-tuning gene regulation, although the precise roles of many are still unknown. Pancreatic development is characterized by the complex sequential expression of a gamut of transcription factors. We have performed miRNA expression profiling at two key stages of mouse embryonic pancreas development, e14.5 and e18.5. miR-124a2 expression was strikingly increased at e18.5 compared with e14.5, suggesting a possible role in differentiated beta-cells. Among the potential miR-124a gene targets identified by biocomputation, Foxa2 is known to play a role in beta-cell differentiation. To evaluate the impact of miR-124a2 on gene expression, we overexpressed or down-regulated miR-124a2 in MIN6 beta-cells. As predicted, miR-124a2 regulated Foxa2 gene expression, and that of its downstream target, pancreatic duodenum homeobox-1 (Pdx-1). Foxa2 has been described as a master regulator of pancreatic development and also of genes involved in glucose metabolism and insulin secretion, including the ATP-sensitive K(+) (K(ATP)) channel subunits, Kir6.2 and Sur-1. Correspondingly, miR-124a2 overexpression decreased, and anti-miR-124a2 increased Kir6.2 and Sur-1 mRNA levels. Moreover, miR-124a2 modified basal and glucose- or KCl-stimulated intracellular free Ca(2+) concentrations in single MIN6 and INS-1 (832/13) beta-cells, without affecting the secretion of insulin or co-transfected human growth hormone, consistent with an altered sensitivity of the beta-cell exocytotic machinery to Ca(2+). In conclusion, whereas the precise role of microRNA-124a2 in pancreatic development remains to be deciphered, we identify it as a regulator of a key transcriptional protein network in beta-cells responsible for modulating intracellular signaling.

Transcriptional profiling of type 1 diabetes genes on chromosome 21 in a rat beta-cell line and human pancreatic islets

We recently finemapped a type 1 diabetes (T1D)-linked region on chromosome 21, indicating that one or more T1D-linked genes exist in this region with 33 annotated genes. In the current study, we have taken a novel approach using transcriptional profiling in predicting and prioritizing the most likely candidate genes influencing beta-cell function in this region. Two array-based approaches were used, a rat insulinoma cell line (INS-1alphabeta) overexpressing pancreatic duodenum homeobox 1 (pdx-1) and treated with interleukin 1beta (IL-1beta) as well as human pancreatic islets stimulated with a mixture of cytokines. Several candidate genes with likely functional significance in T1D were identified. Genes showing differential expression in the two approaches were highly similar, supporting the role of these specific gene products in cytokine-induced beta-cell damage. These were genes involved in cytokine signaling, oxidative phosphorylation, defense responses and apoptosis. The analyses, furthermore, revealed several transcription factor binding sites shared by the differentially expressed genes and by genes demonstrating highly similar expression profiles with these genes. Comparable findings in the rat beta-cell line and human islets support the validity of the methods used and support this as a valuable approach for gene mapping and identification of genes with potential functional significance in T1D, within a region of linkage.

Localization of HB9 homeodomain protein and characterization of its nuclear localization signal during chick embryonic skin development.

We detected HB9 protein during tarsometatarsal scale skin and late feather development. Immunofluorescent analyses with N-terminal 14 amino acids antiserum revealed that HB9 was strongly expressed in epidermal basal cells of the outer scale face in tarsometatarsal scale skin. Specific expression was also detected in dermal cells at the root region of the feather and around the feather follicle. Furthermore, we observed precise distribution of HB9 protein by immunoelectron microscopy. We detected HB9 protein not only in the nucleus, but also in the cytoplasm in tarsometatarsal scale skin. However, in feather skin HB9 protein was found in the nucleus but not in the cytoplasm. Cytoplasmic localization of HB9 protein in tarsometatarsal scale skin was observed especially in the endoplasmic reticulum and the Golgi apparatus. To address the mechanism of nuclear-cytoplasmic translocation, we determined the nuclear localization signal (NLS) sequences by using eukaryotic green fluorescent protein fusion protein in primary keratinocyte culture. Chick HB9 homeoprotein has two types of the NLS sequences in its homeodomain. One of them is a bipartite type as representatively found in Xenopus nucleoplasmin. The other is very similar to hexapeptide NLS sequences identified in pancreatic duodenum homeobox 1 (PDX1). These sequences functioned not only in keratinocytes but also in dermal fibroblasts. They are conserved in Xenopus, mouse, and human HB9 ortholog. These results indicate that HB9 protein might be involved in chick tarsometatarsal scale and feather development and that nuclear import of HB9 protein might be regulated by these NLS sequences in the homeodomain.

Involvement of Per-Arnt-Sim (PAS) kinase in the stimulation of preproinsulin and pancreatic duodenum homeobox 1 gene expression by glucose.

Per-Arnt-Sim (PAS) domain-containing kinases are common in prokaryotes, but a mammalian counterpart has only recently been described. Although the PAS domain of the mammalian PAS kinase (PASK) is closely related to the bacterial oxygen sensor FixL, it is unclear whether PASK activity is changed in mammalian cells in response to nutrients and might therefore contribute to signal transduction by these or other stimuli. Here, we show that elevated glucose concentrations rapidly increase PASK activity in pancreatic islet beta cells, an event followed by the accumulation of both PASK mRNA and protein. Demonstrating a physiological role for PASK activation, comicroinjection into clonal beta cells of cDNA encoding wild-type PASK, or PASK protein itself, mimics the induction of preproinsulin promoter activity by high glucose concentrations. Conversely, anti-PASK antibodies block promoter activation by the sugar, and the silencing of PASK expression by RNA interference suppresses the up-regulation by glucose of preproinsulin and pancreatic duodenum homeobox 1 gene expression, without affecting glucose-induced changes in the levels of mRNAs encoding glucokinase or uncoupling protein 2. We conclude that PASK is an important metabolic sensor in nutrient-sensitive mammalian cells and plays an unexpected role in the regulation of key genes involved in maintaining the differentiated phenotype of pancreatic beta cells.

Pancreatic Duodenal Homeobox (PDX-1) in health and disease.

Insulin is expressed exclusively in the adult beta-cells of the islets of Langerhans. Pancreatic Duodenum Homeobox-1 (PDX-1) is a major regulator of transcription in these cells. It transactivates the insulin gene by binding to a specific DNA motif in its promoter region. Glucose, the main physiological regulator of insulin secretion, also regulates insulin gene transcription through PDX-1. While acute exposure to high glucose concentrations causes an increase in PDX-1 binding, and consequently in insulin mRNA levels, chronic hyperglycemia (toxic to the beta-cell) leads to a decrease in PDX-1 and insulin levels. PDX-1 is absolutely required for pancreas development. In view of the selective expression in adult beta-cells, pancreatic agenesis in both the pdx-1 null mouse and a human carrying a homozygous mutation of PDX-1 was an unexpected and remarkable finding. The homozygous clinical phenotype was neonatal diabetes mellitus (DM) and exocrine insufficiency. Heterozygosity for PDX-1 mutations was found in some individuals with a newly characterized subtype of maturity-onset diabetes of the young (MODY4) and in others with type 2 DM. This review underlines the unique role of PDX-1 in maintaining adult beta-cell-specific functions in normal and disease-related states.

The Role of Pancreatic Duodenum Homeobox Protein-1 in the Development of Diabetes Mellitus.

Pancreatic duodenum homeobox protein-1 (Pdx-1, also known as Idx-1, Ipf-1, Stf-1) is a homeodomain protein of 283 amino acids whose expression is required for pancreas development to occur. This protein is now recognized to be a critically important transcription factor involved in the regulation of the expression of multiple genes essential for the proper functioning of the beta cells in the endocrine pancreas (islets of Langerhans) that produce insulin, a lack of which results in the development of diabetes mellitus. Finding a way to increase Pdx-1 expression and the neogenesis of beta cells, perhaps through the stimulating effects of glucagon-like peptide-1, may be useful for the treatment of this crippling disease. (c) 2002 Prous Science. All rights reserved.

Glucagon-like peptide-1 induces cell proliferation and pancreatic-duodenum homeobox-1 expression and increases endocrine cell mass in the pancreas of old, glucose-intolerant rats.

Glucose homeostasis in mammals is maintained by insulin secretion from the beta-cells of the islets of Langerhans. Type 2 diabetes results either from primary beta-cell failure alone and/or a failure to secrete enough insulin to overcome insulin resistance. Here, we show that continuous infusion of glucagon-like peptide-1 (7-36) (GLP-1; an insulinotropic agent), to young and old animals, had effects on the beta-cell of the pancreas other than simply on the insulin secretory apparatus. Our previous studies on a rodent model of glucose intolerance, the aging Wistar rat, show that a plateau in islet size, insulin content, and beta-cell mass is reached at 13 months, despite a continuing increase in body weight. Continuous sc infusion of GLP-1 (1.5 pM/kg x min), over 5 days, resulted in normal glucose tolerance. Our current results in both young and old rats demonstrate that treatment caused an up-regulation of pancreatic-duodenum homeobox-1 (PDX-1) expression in islets and total pancreas, induced pancreatic cell proliferation, and beta-cell neogenesis. The effects on levels of PDX-1 messenger RNA were abrogated by simultaneous infusion of Exendin (9-39), a specific antagonist of GLP-1. PDX-1 protein levels increased 4-fold in whole pancreata and 6-fold in islets in response to treatment. Beta-cell mass increased to 7.2 +/- 0.58 from 4.88 +/- 0.38 mg, treated vs. control, respectively, P < 0.02. Total pancreatic insulin content also increased from 0.55 +/- 0.02 to 1.32 +/- 0.11 microg/mg total pancreatic protein. Therefore, GLP-1 would seem to be a unique therapy that can stimulate pancreatic cell proliferation and beta-cell differentiation in the pancreas of rodents.

Glucose-stimulated Preproinsulin Gene Expression and Nucleartrans-Location of Pancreatic Duodenum Homeobox-1 Require Activation of Phosphatidylinositol 3-Kinase but Not p38 MAPK/SAPK2

Exposure of islet beta-cells to elevated glucose concentrations (30 versus 3 mm) prompts enhanced preproinsulin (PPI) gene transcription and the trans-location to the nucleoplasm of pancreatic duodenum homeobox-1 (PDX-1; Rafiq, I., Kennedy, H., and Rutter, G. A. (1998) J. Biol. Chem. 273, 23241-23247). Here, we show that in MIN6 beta-cells, over-expression of p110.CAAX, a constitutively active form of phosphatidylinositol 3-kinase (PI3K) mimicked the activatory effects of glucose on PPI promoter activity, whereas Deltap85, a dominant negative form of the p85 subunit lacking the p110-binding domain, and the PI3K inhibitor LY 294002, blocked these effects. Similarly, glucose-stimulated nuclear trans-location of endogenous PDX-1 was blocked by Deltap85 expression, and wortmannin or LY 294002 blocked the trans-location from the nuclear membrane to the nucleoplasm of epitope-tagged PDX-1.c-myc. By contrast, SB 203580, an inhibitor of stress-activated protein kinase-2 (SAPK2)/p38 MAP kinase, had no effect on any of the above parameters, and PPI promoter activity and PDX-1.c-myc localization were unaffected by over-expression of the upstream kinase MKK6 (MAP kinase kinase-6) or wild-type p38/SAPK2, respectively. Furthermore, no change in the activity of extracted p38/SAPK2 could be detected after incubation of cells at either 3 or 30 mm glucose. These data suggest that stimulation of PI3K is necessary and sufficient for the effects of glucose on PPI gene transcription, acting via a downstream signaling pathway that does not involve p38/SAPK2.