Pdx1 Research Articles

Enhanced expression of PDX1 accumulates vitamin B6 in transgenic wheat seeds

Humans fulfill their daily requirement of vitamin B6, a vital micronutrient for strong metabolism and immunity, from various food components. Bio-fortified wheat, a staple crop, holds the potential to overcome vitamin B6 deficiency. Thus, in the present work, endogenous PDX1 gene was expressed in wheat under endosperm promoter. Calli from immature embryos of two wheat varieties, FSD-08 and Galaxy were transformed via Agrobacterium with transformation efficiency of 1.82% and 1.09%, respectively. After BASTA and PCR confirmation, T1 generation of twenty transgenic lines was analyzed for mRNA expression via quantitative real-time PCR and vitamin B6 levels via HPLC along with various biochemical and morphological traits. The transgenic lines exhibited 1.04–19.46 fold increase in PDX1 gene expression and upto 34.96 fold increase in vitamin B6 content of seeds as compared to control. Enhanced carbohydrate content was observed in half of the transgenics while majority of the lines had insignificant variation in their chlorophyll, protein and starch content. Among the morphological traits studied, only seed yield increased in majority of transgenic lines. In conclusion, over-expression of PDX1 in wheat endosperm successfully augmented vitamin B6 content of seeds without posing adverse effects on biochemical or morphological traits in either varieties.

Utilization of zebrafish larvae to monitor gastric motility in diabetes mellitus: targeting GLP‐1 and GIP receptors

BackgroundDiabetes mellitus (DM) is a prominent chronic disease associated with mortality and morbidity. The impact of DM is extensive, impacting with several major organs systems, including the gastrointestinal (GI) tract. For instance, the prevalence of DM in gastroparetic patients is approximately 30%. Glucagon‐like peptide‐1 (GLP‐1) and gastric inhibitory peptide (GIP) are hormones involved in glucose homeostasis but may also affect gastric motility. GLP‐1 and GIP receptors belong to class B G‐protein coupled receptors (GPCRs) and zebrafish GPCRs (zGPCRs) share structural similarities with the human GLP‐1 (hGLP‐1) and human glucagon receptors (hGCGR). Studies found that zebrafish GLP‐1 (zGLP‐1) receptors exhibit all the structural features of class B GPCRs and have ~50% homology with human GLP‐1 receptors. The aim of the studies is to establish experimental models of DM and investigate if GLP‐1 and GIP receptor agonists alter gastric function.MethodologyDiet‐induced DM zebrafish larvae models (from 5 days post fertilization (dpf) to 7 dpf) were established by (i) overfeeding, (ii) feeding with a high‐fat diet (egg yolk), (iii) exposure to high levels of glucose (30 mmol/L) or (iv) a knockdown of the pdx1 (insulin promoter factor 1) gene mutant. A body mass index (BMI) analyses and enzyme‐linked immunosorbent assays (ELISA) for insulin levels and glucose assay were used to grade DM. As obesity is associated with development of diabetes, adipose deposits in the abdominal area was also determined by an inverted microscope. Exendin‐4 and tirzepatide (100 µM – 1 nM) were administered via microgavage. Peristalsis was captured in anaesthetised animals using light microscopy and quantified in MATLAB using imaging velocimetry and pixel differences.ResultsExcept for the overfed and pdx1 mutated models, significant differences in BMI, insulin and glucose levels were observed relative to controls. The excess diet model showed high level of adipose deposits (2.4 ± 0.17 mm) and at 7 dpf an 11% increase in adipose area is seen but without DM properties (i.e. increase glucose levels and altered insulin levels). There were significant differences between DM models and control‐fed larvae in terms of mixing power, anterograde and retrograde peristalsis of several parameters including contractile intervals and velocity. Preliminary data indicated that larvae at7 dpf fed a standard diet, exendin‐4 (≥ 100 nM/larvae) and tirzepatide (≥100 µM/larvae) reduced motility in the anterior and mid‐intestine.ConclusionThree DM models and an obesity model using zebrafish larvae were successfully established. Advanced imaging and processing techniques revealed intricate subtypes of dysmotility in DM and obesity. Exendin‐4 and tirzepatide at high doses reduced motility in normal fish which may be consistent with their clinical side effect profile.

Precision Diagnosis of Maturity-Onset Diabetes of the Young with Next-Generation Sequencing: Findings from the MODY-IST Study in Adult Patients.

Maturity-onset diabetes of the young (MODY) is a highly heterogeneous group of monogenic and nonautoimmune diseases. Misdiagnosis of MODY is a widespread problem and about 5% of patients with type 2 diabetes mellitus and nearly 10% with type 1 diabetes mellitus may actually have MODY. Using next-generation DNA sequencing (NGS) to facilitate accurate diagnosis of MODY, this study investigated mutations in 13 MODY genes (HNF4A, GCK, HNF1A, PDX1, HNF1B, NEUROD1, KLF11, CEL, PAX4, INS, BLK, ABCC8, and KCNJ11). In addition, we comprehensively investigated the clinical phenotypic effects of the genetic variations identified. Fifty-one adult patients with suspected MODY and 64 healthy controls participated in the study. We identified 7 novel and 10 known missense mutations localized in PDX1, HNF1B, KLF11, CEL, BLK, and ABCC8 genes in 29.4% of the patient sample. Importantly, we report several mutations that were classified as "deleterious" as well as those predicted as "benign." Notably, the ABCC8 p.R1103Q, ABCC8 p.V421I, CEL I336T, CEL p.N493H, BLK p.L503P, HNF1B p.S362P, and PDX1 p.E69A mutations were identified for the first time as causative variants for MODY. More aggressive clinical features were observed in three patients with double- and triple-heterozygosity of PDX1-KLF11 (p.E69A/p.S182R), CEL-ABCC8-KCNJ11 (p.I336, p.G157R/p.R1103Q/p.A157A), and HNF1B-KLF11 (p.S362P/p.P261L). Interestingly, the clinical effects of the BLK mutations appear to be exacerbated in the presence of obesity. In conclusion, NGS analyses of the adult patients with suspected MODY appear to be informative in a clinical context. These findings warrant further clinical diagnostic research and development in different world populations suffering from diabetes with genetic underpinnings.

Hypermethylation of PDX1, EN2, and MSX1 predicts the prognosis of colorectal cancer

Despite numerous observations regarding the relationship between DNA methylation changes and cancer progression, only a few genes have been verified as diagnostic biomarkers of colorectal cancer (CRC). To more practically detect methylation changes, we performed targeted bisulfite sequencing. Through co-analysis of RNA-seq, we identified cohort-specific DNA methylation markers: CpG islands of the intragenic regions of PDX1, EN2, and MSX1. We validated that these genes have oncogenic features in CRC and that their expression levels are increased in correlation with the hypermethylation of intragenic regions. The reliable depth of the targeted bisulfite sequencing data enabled us to design highly optimized quantitative methylation-specific PCR primer sets that can successfully detect subtle changes in the methylation levels of candidate regions. Furthermore, these methylation levels can divide CRC patients into two groups denoting good and poor prognoses. In this study, we present a streamlined workflow for screening clinically significant differentially methylated regions. Our discovery of methylation markers in the PDX1, EN2, and MSX1 genes suggests their promising performance as prognostic markers and their clinical application in CRC patients.

Mitigation of streptozotocin-induced alterations by natural agents via upregulation of PDX1 and Ins1 genes in male rats.

Diabetes is the most common endocrine disorder contributing to high morbidity and mortality globally. Due to the side effects reported for anti-diabetic drugs, there is a rising interest in herbal medicine for diabetes mitigation. However, rare studies were performed to analyze the molecular effects of natural agents on diabetes. Therefore, this study is the first to assess the possible ameliorating effects of avocado and cinnamon extracts on streptozotocin (STZ)-induced disturbances in the gene expression of PDX1 and Ins1 in type-2 diabetic rats, in comparison to metformin. A total number of 50 male Albino rats were randomly divided into five groups; normal control, STZ-induced diabetic group (65mg/kg b.w divided into three doses 5days apart), three other STZ-diabetic groups were treated orally for 6weeks with metformin (500mg/kg b.w/day), an avocado fruit ethanolic extract (300mg/kg b.w/day), or a cinnamon aqueous extract (200mg/kg b.w/day). All rats were fasted overnight then euthanized, blood was collected, and serum was separated for biochemical analyses. The pancreas was processed for the evaluation of PDX1 and Ins1 gene expression by qRT-PCR. Oral administration of the avocado/cinnamon extract significantly improved the altered levels of blood glucose, insulin, lipid profile, and total antioxidant capacity (TAC) and upregulated the pancreatic expression of PDX1 and Ins1 genes. In conclusion, this study added another mechanism of anti-diabetic action for the plant extracts via upregulating the gene expression of PDX1 and Ins1 in STZ-diabetic rats. Metformin has a more profound effect than the plant extracts; however, cinnamon has a comparable effect. PRACTICAL APPLICATIONS: Diabetes is a worldwide chronic serious problem; therefore, different efficacious drug treatments are currently available. However, several side effects of these drugs also negatively affect patient welfare. Therefore, the present study provides an additive/adjunct biochemical rationale for further natural intervention strategy for type-2 diabetes through the introduction of plant-derived products that revealed a wide range of therapeutic effects without causing untoward actions. This study revealed that cinnamon and avocado extracts exhibited marked anti-diabetic and antioxidant effects in rats, as compared to the standard drug, metformin. These results augmented the previous ones and added a new molecular mechanism of action through upregulation of Ins1 and PDX1 in type-2 diabetic rats.

OVEREXPRESSION OF PDX-1 GENE INCREASES INS1 GENE mRNA EXPRESSION, NOT INS2 GENE mRNA EXPRESSION, IN INSULINOMA CELL LINE RIN-5F.

Insulinoma is one of the functional neuroendocrine tumors, which induce hypoglycemia caused by inadequate high secretion of insulin. In rats, two genes coding for insulin, insulin 1 (Ins1) and insulin 2 (Ins2) are found on chromosome 1. Some structural feature studies have shown that Ins1 was generated from a transcript of Ins2 and was inserted into the genome by an RNA-mediated duplication-transposition event. In this study, the author has investigated how the expression of insulin genes, Ins 1 and Ins2, are altered by Pdx-1 gene overexpression in the insulinoma cell line, Rin-5F. Overexpression of the Pdx-1 gene increases Ins1 gene mRNA expression, not Ins2 gene mRNA expression, in the insulinoma cell line. Thus, levels of the rat insulin 1 and insulin 2 peptides may be changed under specific conditions. This is the first report that Ins1, but not Ins2, is significantly increased by Pdx-1 gene overexpression in the insulinoma cell line. This could indicate more research and analysis of insulinoma tumorigenesis and Pdx-1 gene expression.

Hepatocyte Nuclear Factor 4-α (HNF4α) controls the insulin resistance-induced pancreatic β-cell mass expansion

BackgroundRegardless of the etiology, any type of DM presents a reduction of insulin-secreting cell mass, so it is important to investigate pathways that induce the increase of this cell mass. AimBased on the fact that (1) HNF4α is crucial for β-cell proliferation, (2) DEX-induced IR promotes β-cell mass expansion, and (3) the stimulation of β-cell mass expansion may be an important target for DM therapies, we aimed to investigate whether DEX-induced proliferation of β pancreatic cells is dependent on HNF4α. MethodsWe used WildType (WT) and Knockout (KO) mice for HNF4-α, treated or not with 100 mg/Kg/day of DEX, for 5 consecutive days. One day after the last injection of DEX the IR was confirmed by ipITT and the mice were euthanized for pancreas removal. ResultsIn comparison to WT, KO mice presented increased glucose tolerance, lower fasting glucose and increased glucose-stimulates insulin secretion (GSIS). DEX induced IR in both KO and WT mice. In addition, DEX-induced β-cell mass expansion and an increase in the Ki67 immunostaining were observed only in WT mice, evidencing that IR-induced β-cell mass expansion is dependent on HNF4α. Also, we observed that DEX-treatment, in an HNF4α-dependent way, promoted an increase in PDX1, PAX4 and NGN3 gene expression. ConclusionsOur results strongly suggest that DEX-induced IR promotes β-cell mass expansion through processes of proliferation and neogenesis that depend on the HNF4α activity, pointing to HNF4α as a possible therapeutic target in DM treatment.

Oxidative Stress Leads to β-Cell Dysfunction Through Loss of β-Cell Identity.

Pancreatic β-cell failure is a critical event in the onset of both main types of diabetes mellitus but underlying mechanisms are not fully understood. β-cells have low anti-oxidant capacity, making them more susceptible to oxidative stress. In type 1 diabetes (T1D), reactive oxygen species (ROS) are associated with pro-inflammatory conditions at the onset of the disease. Here, we investigated the effects of hydrogen peroxide-induced oxidative stress on human β-cells. We show that primary human β-cell function is decreased. This reduced function is associated with an ER stress response and the shuttling of FOXO1 to the nucleus. Furthermore, oxidative stress leads to loss of β-cell maturity genes MAFA and PDX1, and to a concomitant increase in progenitor marker expression of SOX9 and HES1. Overall, we propose that oxidative stress-induced β-cell failure may result from partial dedifferentiation. Targeting antioxidant mechanisms may preserve functional β-cell mass in early stages of development of T1D.

Overexpression of PDX1 and NKX6.1 as a Treatment for Type 1 Diabetes: A Research Protocol

Introduction: Type 1 diabetes is an autoimmune disorder characterized by the destruction of pancreatic islet beta cells responsible for insulin production, leading to insulin deficiency. The PDX1 and NKX6-1 genes are essential to beta cell development and maturation in the pancreas. Based on previous studies, Pdx1 is downregulated in diabetics, and higher Nkx6.1 expression has been shown to induce high cell turnover. This proposal aims to investigate the potential therapeutic effects of gene therapy on streptozotocin-induced non-obese diabetic/severe-combined immunodeficient mice in treating Type 1 Diabetes. It is hypothesized that the use of gene therapy to overexpress PDX1 and NKX6-1 into the pancreas of experimental diabetic mice will result in increased beta cell production and improved glucose metabolism. Methods: Both male and female streptozotocin-induced non-obese diabetic/severe-combined immunodeficient experimental mice will receive human islets from individuals 32-55 years old. The PDX1 and NKX6-1 genes will be overexpressed in a shuttle vector and incorporated into the gutless adenovirus vector backbone through co-transfection using the Cre293/lox P cell line. Verification of successful vectors containing the gutless adenovirus coupled with a helper plasmid will be done using gel electrophoresis. Incubation with beta cells will stimulate the transduction process after which beta cells will be delivered into mice pancreas’ through endoscopic retrograde cholangiopancreatography. An intraperitoneal glucose tolerance test, pancreatic biopsy with immunohistochemical staining, and islet calculations will be done. Results: An intraperitoneal glucose tolerance test is expected to show improved blood glucose levels in experimental mice post-treatment, while a pancreatic biopsy with immunohistochemical staining and subsequent islet diameter and volume calculations are expected to depict an increase in the number of beta cells. Discussion: The experimental mice are anticipated to show an improvement in blood glucose levels and increased production of beta cells following treatment due to the overexpression of PDX1 and NKX6-1. The normal control mice and diabetic untreated mice are expected to show normal and high glucose levels, as well as normal and deficient beta cells respectively. Conclusion: This proposal may provide a better understanding of the pathophysiology in Type 1 Diabetes and pave a new path for its treatment.

Preconditioned human dental pulp stem cells with cerium and yttrium oxide nanoparticles effectively ameliorate diabetic hyperglycemia while combatting hypoxia

The development of efficient insulin producing cells (IPC) induction system is fundamental for the regenerative clinical applications targeting Diabetes Mellitus. This study was set to generate IPC from human dental pulp stem cells (hDPSCs) capable of surviving under hypoxic conditions in vitro and in vivo. MethodshDPSCs were cultured in IPCs induction media augmented with Cerium or Yttrium oxide nanoparticles along with selected growth factors & cytokines. The generated IPC were subjected to hypoxic stress in vitro to evaluate the ability of the nanoparticles to combat hypoxia. Next, they were labelled and implanted into diabetic rats. Twenty eight days later, blood glucose and serum insulin levels, hepatic hexokinase and glucose-6-phosphate dehydrogenase activities were measured. Pancreatic vascular endothelial growth factor (VEGF), pancreatic duodenal homeobox1 (Pdx-1), hypoxia inducible factor 1 alpha (HIF-1α) and Caspase-3 genes expression level were evaluated. ResultshDPSCs were successfully differentiated into IPCs after incubation with the inductive media enriched with nanoparticles. The generated IPCs released significant amounts of insulin in response to increasing glucose concentration both in vitro &in vivo. The generated IPCs showed up-regulation in the expression levels of anti-apoptotic genes in concomitant with down-regulation in the expression levels of hypoxic, and apoptotic genes. The in vivo study confirmed the homing of PKH-26-labeled cells in pancreas of treated groups. A significant up-regulation in the expression of pancreatic VEGF and PDX-1 genes associated with significant down-regulation in the expression of pancreatic HIF-1α and caspase-3 was evident. ConclusionThe achieved results highlight the promising role of the Cerium & Yttrium oxide nanoparticles in promoting the generation of IPCs that have the ability to combat hypoxia and govern diabetes mellitus.

The Application of Next Generation Sequencing Maturity Onset Diabetes of the Young Gene Panel in Turkish Patients from Trakya Region

Objective:The aim of this study was to investigate the molecular basis of maturity-onset diabetes of the young (MODY) by targeted-gene sequencing of 20 genes related to monogenic diabetes, estimate the frequency and describe the clinical characteristics of monogenic diabetes and MODY in the Trakya Region of Turkey.Methods:A panel of 20 monogenic diabetes related genes were screened in 61 cases. Illumina NextSeq550 system was used for sequencing. Pathogenicity of the variants were assessed by bioinformatics prediction software programs and segregation analyses.Results:In 29 (47.5%) cases, 31 pathogenic/likely pathogenic variants in the GCK, ABCC8, KCNJ11, HNF1A, HNF4A genes and in 11 (18%) cases, 14 variants of uncertain significance (VUS) in the GCK, RFX6, CEL, PDX1, KCNJ11, HNF1A, G6PC2, GLIS3 and KLF11 genes were identified. There were six different pathogenic/likely pathogenic variants and six different VUS which were novel.Conclusion:This is the first study including molecular studies of twenty monogenic diabetes genes in Turkish cases in the Trakya Region. The results showed that pathogenic variants in the GCK gene are the leading cause of MODY in our population. A high frequency of novel variants (32.4%-12/37) in the current study, suggests that multiple gene analysis provides accurate genetic diagnosis in MODY.

Exendin-4 may improve type 2 diabetes by modulating the epigenetic modifications of pancreatic histone H3 in STZ-induced diabetic C57BL/6J mice.

Type 2 diabetes (T2D) is a complicated systemic disease that might be improved by exendin-4, although the epigenetic role remains unclear. In the current study, C57BL/6J mice were used to generate a T2D model, followed by treatment with exendin-4 (10μg/kg). Histone H3K9 and H3K23 acetylation, H3K4 mono-methylation, and H3K9 di-methylation were explored by western blot analysis of pancreatic histone extracts. Real-time polymerase chain reaction (PCR) was used to examine the expression levels of pancreatic beta cell development-related genes, and chromatin immunoprecipitation (ChIP) was applied to analyze H3 and H3K9 acetylation, H3K4 mono-methylation, and H3K9 di-methylation in the promoter region of the pancreatic and duodenal homeobox 1 (Pdx1) gene. The results showed that total H3K9 di-methylation and H3K9 and H3K23 acetylation increased in pancreatic tissues of diabetic mice, whereas H3K4 mono-methylation was reduced. All of these changes could be abrogated by treatment with exendin-4. Our data indicated that T2D progression might be improved by exendin-4 treatment through the reversal of global pancreatic histone H3K9 and H3K23 acetylation, H3K4 mono-methylation, and H3K9 di-methylation. A better understanding of these epigenetic alterations may, therefore, lead to novel therapeutic strategies for T2D.

Effect of the Internal Septum Extract of the Walnut Kernel on the Mesenchymal Stem Cells Cycle and MSCs-derived Insulin-Producing Β-cells Differentiation and Glucose Uptake

Background: For years, scientists have been striving to identify compounds to improve the characteristics and functions of cells for therapeutic applications. Objectives: This study aimed to evaluate the effect of the aqueous decoction of the internal septum of the walnut kernel (DISWK) on the survival, total antioxidant capacity (TAC), and cell cycle of rat bone marrow-derived mesenchymal stem cells (rBM-MSCs). We also evaluated the expression of genes involved in pancreatic insulin-producing β-cell (IPCs) commitment and glucose uptake in MSCs-derived IPCs. Methods: The rBM-MSCs were isolated, and then the cytotoxic and antioxidant effects of DISWK on the cells were examined by the MTT and TAC assays, respectively. Moreover, the influence of DISWK on the rBM-MSCs cell cycle was evaluated by PI-staining and flow-cytometry analysis. Next, the rBM-MSCs were transdifferentiated into IPCs treated with DISWK. Finally, Ins1/2, Insr, Glut1, Glut4, and Pdx1 genes expression was investigated in the DISWK-treated IPCs by qRT-PCR. Results: The results indicated that DISWK significantly reduced the viability of rBM-MSCs in a dose- and time-dependent manner and decreased their TAC in long-term cultures. Moreover, it suppressed the rBM-MSCs cell cycle at S and G2 phases. The gene expression analysis showed that DISWK significantly upregulated Ins1/2, Insr, and Glut1 genes and downregulated Pdx1 gene in the rBM-MSCs-derived IPCs. Conclusions: Decoction of the internal septum of walnut kernel suppresses the MSCs cell cycle and may prevent IPCs development via inhibiting Pdx1. It may also help glucose uptake by upregulating Ins1/2, Insr, and Glut1 genes expression in MSCs-derived IPCs.

WITHDRAWN: Combined therapy of adenovirus vector mediated IGF-1 gene with anti-CD20 mAbs exerts potential beneficial role on type 1 diabetes in nonobese diabetic mice

To assess the protective effects of combined treatment with anti-CD20 monoclonal antibody (mAb) and adenovirus mediated mouse insulin-like growth factor 1 (Adv-mIGF-1) gene on type 1 diabetes (T1D) in nonobese diabetic (NOD) mice at early stage. To simultaneously restore the proportion of Th cells and block the interaction of B cells, NOD model mice were assigned to four groups which received PBS, Adv-mIGF-1 gene and anti-CD20 mAbs alone or combination, respectively. After 16 weeks of therapeutic intervention, blood samples and pancreatic tissues of mice were measured via the methods of ELISA, RT-PCR, western blotting, H&E staining, TUNEL and immunohistochemistry assays. Chronic combination intervention with Adv-mIGF-1 gene and anti-CD20 mAbs reduced the T1D-related morbidity, promoted the secretion of insulin, controlled the blood glucose levels (BGLs) and alleviated insulitis of experimental mice. In addition, current combination intervention also protected the pancreatic β cells via suppressing the expression of Fas and TNF-α, inhibiting Caspase-3/8 related apoptotic pathway, and activating the Bcl-2-related antiapoptotic pathway. Furthermore, current combination therapy also increased the expression levels of PDX-1 and CK-19 genes, and finally accelerated the proliferation and differentiation of pancreatic β-cells. In addition, combination therapy could also ameliorate the pathological characteristics of diabetic nephropathy in NOD mice. Combination treatment with Adv-mIGF-1 gene and anti-CD20 mAbs may exert a potential beneficial role on T1D in NOD mice.

Dysregulation of the Pdx1/Ovol2/Zeb2 axis in dedifferentiated β-cells triggers the induction of genes associated with epithelial–mesenchymal transition in diabetes

Objectiveβ-cell dedifferentiation has been revealed as a pathological mechanism underlying pancreatic dysfunction in diabetes. We previously showed that increased miR-7 levels trigger β-cell dedifferentiation and diabetes. We used β-cell-specific miR-7 overexpressing mice (Tg7) to test the hypothesis that loss of β-cell identity triggered by miR-7 overexpression alters islet gene expression and islet microenvironment in diabetes. MethodsWe performed bulk and single-cell RNA sequencing (RNA-seq) in islets obtained from β-cell-specific miR-7 overexpressing mice (Tg7). We carried out loss- and gain-of-function experiments in MIN6 and EndoC-bH1 cell lines. We analysed previously published mouse and human T2D data sets. ResultsBulk RNA-seq revealed that β-cell dedifferentiation is associated with the induction of genes associated with epithelial-to-mesenchymal transition (EMT) in prediabetic (2-week-old) and diabetic (12-week-old) Tg7 mice. Single-cell RNA-seq (scRNA-seq) indicated that this EMT signature is enriched specifically in β-cells. These molecular changes are associated with a weakening of β-cell: β-cell contacts, increased extracellular matrix (ECM) deposition, and TGFβ-dependent islet fibrosis. We found that the mesenchymal reprogramming of β-cells is explained in part by the downregulation of Pdx1 and its inability to regulate a myriad of epithelial-specific genes expressed in β-cells. Notable among genes transactivated by Pdx1 is Ovol2, which encodes a transcriptional repressor of the EMT transcription factor Zeb2. Following compromised β-cell identity, the reduction in Pdx1 gene expression causes a decrease in Ovol2 protein, triggering mesenchymal reprogramming of β-cells through the induction of Zeb2. We provided evidence that EMT signalling associated with the upregulation of Zeb2 expression is a molecular feature of islets in T2D subjects. ConclusionsOur study indicates that miR-7-mediated β-cell dedifferentiation induces EMT signalling and a chronic response to tissue injury, which alters the islet microenvironment and predisposes to fibrosis. This research suggests that regulators of EMT signalling may represent novel therapeutic targets for treating β-cell dysfunction and fibrosis in T2D.

Ovariectomy Increases Pancreatic Endoplasmic Reticulum Stress Genes in Simian Immunodeficiency Virus Infection

Background Antiretroviral therapy (ART) has significantly reduced mortality of people living with HIV (PLWH). Older PLWH, especially menopausal women, experience complex interactions of HIV, ART, hormonal status, and lifestyle behaviors such as at-risk alcohol use, increasing the risk of metabolic comorbidities. Our studies have demonstrated that chronic binge alcohol (CBA) significantly decreases acute insulin response to a glucose challenge in Simian Immunodeficiency Virus (SIV)-infected macaques, suggesting pathophysiological changes in pancreatic endocrine function. Endoplasmic Reticulum (ER) stress is one of the major mechanisms associated with alcoholic pancreatitis and endocrine pancreatic dysfunction. The objective of this study was to examine the effects of CBA administration and gonadal hormone loss, resulting from ovariectomy (OVX), on pancreatic mechanisms that contribute to impaired glucose-insulin dynamics in SIV-infected female macaques. Hypothesis Chronic binge alcohol and ovariectomy increase pancreatic ER stress and decrease expression of regulators of insulin synthesis. Methods CBA or isovolumetric water (VEH) was administered through an intragastric catheter for 30 minutes, 5 days a week. Three months after initiation of CBA/VEH administration, macaques were infected with SIVMac251 and 2.5 months later initiated on ART. After 1 month of ART, macaques underwent sham surgery or ovariectomy and euthanized 9 months post-SIV-infection. Total RNA was isolated from pancreas collected from macaques (VEH/SHAM= 8, CBA/SHAM= 7, VEH/OVX= 7, CBA/OVX n=7). cDNA was synthesized and qPCR was used to analyze gene expression of markers for ER stress (X-Box Binding Protein 1, XBP1; Activating Transcription Factor 4, ATF4; and C/EBP Homologous Protein, CHOP) and regulators of insulin synthesis (Homeobox Protein Nkx-2.2, NKX2.2; insulin promoter factor 1, PDX1; Transcription factor MafA, MAFA; Glucose transporter 2, GLUT2; and Glucokinase). Results There was a main effect of OVX to increase mRNA expression of ATF4 and XBP1 (p<0.05) in SIV-infected macaques. There was not a statistically significant effect of CBA or OVX to alter gene expression of CHOP, or insulin synthesis markers, NKX2.2, PDX1, MAFA, GLUT2 and Glucokinase. Conclusions OVX increased pancreatic expression of ER stress markers without significantly altering expression of regulators of insulin synthesis. We speculate that the increased expression of ER stress markers may result in increased beta cell apoptosis. These findings provide evidence for pancreatic mechanisms that can contribute to the observed impairment of glucose insulin dynamics in SIV infection.

Glycerol‐3‐phosphate phosphatase/ Pgp in Pancreatic ß‐cells Functions as a Glucose Excess Security Valve Preventing Oversecretion of Insulin Secretion and Glucotoxicity

Objective The recently identified enzyme in mammalian cells, glycerol-3-phosphate (Gro3P) phosphatase (G3PP), gene name Pgp, was proposed to regulate intermediary metabolism. G3PP was shown in in vitro studies to regulate metabolism and glucose stimulated insulin secretion (GSIS) in ß-cells. We now examined the in vivo role of G3PP in the control of insulin secretion and ß-cell glucotoxicity. Methods Glucose and insulin tolerance were studied in ß-cell specific G3PP-KO (BKO) mice. Body weight gain, fed glycemia and insulinemia were measured. Pancreatic islets were isolated for ex vivo GSIS and biochemical measurements. Results BKO mice show increased body weight gain, unaltered fed glycemia and insulinemia, and enhanced insulin secretion in response to glucose load in an intraperitoneal but not oral glucose tolerance test, reaching 21 and 14 mM glucose, respectively. Insulin sensitivity in vivo remains unchanged in the BKO mice. GSIS response ex vivo at 16 mM but not 8 mM glucose is higher in BKO mouse islets. BKO islets show reduced glucose-induced glycerol release and elevated O2 consumption and ATP production at high (16 mM) but not low (4 mM) glucose levels. Glucotoxicity at 30 mM glucose for 7 days led to increased apoptosis, reduced insulin content and expression of Pdx-1 and Ins-2 genes in BKO islets. Conclusion G3PP impacts insulin secretion in vivo and ex vivo only under conditions of high but not intermediate and low glucose levels. G3PP plays a role in preventing ß-cell glucotoxicity. These effect likely result from the capacity of G3PP to redirect the excess glucose carbons from intermediate metabolism to glycerol that exits ß-cells. We propose that G3PP acts as a glucose excess security valve to prevent excessive insulin secretion and ß-cell dysfunction when blood glucose reaches very high levels.

Effect of Rosolic acid on endothelial dysfunction under ER stress in pancreatic microenvironment

Endothelial cell (EC) dysfunction is the underlying cause for the development of several pathologies, and the interdependency between the pancreatic β-cells and ECs has been established in the pathophysiology of diabetes. ECs release several factors that govern the expression of genes involved in the proliferation, physiology, and survival of the β-cells. Of the known factors that collapse this intricately balanced system, endothelial dysfunction is the crucial condition that manifests as the causative factor for micro and macrovascular diseases. Our earlier studies demonstrated that activation of nuclear factor erythroid-related factor (Nrf2) renders protection to the ECs experiencing ER stress. In this study, using a co-culture system, the crosstalk between pancreatic cells under ER stress and ECs and the effect of a novel Nrf2 activator Rosolic Acid (RA), on the crosstalk was investigated. ECs pre-treated with different concentrations RA and co-cultured with thapsigargin-induced ER stressed pancreatic β-cells showed increased levels of Nrf2 and its downstream targets such as heme oxygenase-1 (HO-1) and NADPH-quinone oxidoreductase-1 (NQO-1), and reduction of ER stress evinced by the decreased levels of glucose-regulated protein (GRP) 78 and C/ERB homologous protein (CHOP). The sensitization of ECs using RA, offered protection to pancreatic cells against ER stress as displayed by increased intracellular insulin and upregulated expression of cell survival and proliferative genes BCl2 and PDX-1. In addition, RA treatment resulted in elevated levels of various angiogenic factors, while inflammatory (TNF-α and IL-1β) and apoptotic markers (CXCL10 and CCL2) decreased. RA treatment normalized the levels of 115 proteins of the 277, which were differentially regulated as revealed by proteomic studies of ER stressed pancreatic β-cells in co-culture conditions. These findings clearly indicate the role of small molecule activators of Nrf2 not only in restoring the functioning of pancreatic cells but also in increasing the cell mass. Further, the study impinges on the strategies that can be developed to balance the pancreatic microenvironment, leading to the restoration of β-cell mass and their normophysiology in diabetic patients.

PDX1-MODY: A rare missense mutation as a cause of monogenic diabetes

Maturity-Onset Diabetes of the Young type 4 is a rare form of diabetes mellitus, caused by mutations in the PDX1 gene. However, only a few mutations in this gene have been associated as a cause of monogenic diabetes up to date. It makes difficult to create a clinical manifestation profile of this disease and, consequently, to improve the therapeutic management for these patients. Here we report a normal weight woman, diagnosed with diabetes mellitus at 27 years old, during her first pregnancy. At the time of the recruitment, she was 40 years old and had a body mass index of 23.9 kg/m2, glycated hemoglobin level of 9.6%, and fasting plasma glucose (FPG) of 254 mg/dL. She presented no diabetic complications and she was being treated with insulin. She reported a family history of diabetes mellitus characteristic of an autosomal dominant mode of inheritance. Molecular analysis of the PDX1 gene revealed the missense variant c.532G > A (p.(Glu178Lys)) segregating from the patient to her son, reported as diabetic. It was absent in her healthy daughter. The c.532G > A seems to be a rare variant, absent in human variants databases, and among 86 normoglycemic controls. Eight in silico algorithms classified this variant as probably pathogenic. Additionally, analysis of the evolutionary conservation showed the glutamic acid in the position 178 of PDX-1 protein as conserved among several species. Our findings reinforce the importance of screening rare MODY genes among families with suspicion of monogenic diabetes to help better understand the clinical manifestations of this disease.

Embryonic Exposure to Low Concentrations of Bisphenol A and S Altered Genes Related to Pancreatic β-Cell Development and DNA Methyltransferase in Zebrafish.

Bisphenol A (BPA) and bisphenol S (BPS) are implicated in the development of metabolic disorders, such diabetes mellitus. However, the epigenetic mechanism underlying the pancreatic β-cell dysregulation for both BPA/BPS needs clarification. This exploratory study was designed to investigate whether embryonic exposure to low BPA/BPS concentrations impair early pancreatic β-cell differentiation as well as DNA methylation in its gene expression profile using an in vivo model, zebrafish. Zebrafish embryos were exposed to 0, 0.01, 0.03, 0.1, 0.3, and 1.0µM BPA/BPS at 4-h post fertilization (hpf) until 120 hpf. BPA/BPS-induced effects on pancreatic-related genes, insulin gene, and DNA methylation-associated genes were assessed at developmental stages (24-120 hpf), while glucose level was measure at the 120 hpf. The insulin expression levels decreased at 72-120 hpf for 1.0µM BPA, while 0.32 and 0.24-fold of insulin expression were elicited by 0.3 and 1µM BPS respectively at 72 hpf. Significant elevation of glucose levels; 16.3% (for 1.0µM BPA), 7.20% (for 0.3µM BPS), and 74.09% (for 1.0µM BPS) higher than the control groups were observed. In addition, pancreatic-related genes pdx-1, foxa2, ptfla, and isl1 were significantly interfered compared with the untreated group. Moreover, the maintenance methylation gene, dnmt1, was monotonically and significantly decreased at early stage of development following BPA exposure but remained constant for BPS treatment relative to the control group. DNMT3a and DNMT3b orthologs were distinctively altered following BPA/BPS embryonic exposure. Our data indicated that embryonic exposure to low concentration of BPA/BPS can impair the normal expressions of pancreatic-associated genes and DNA methylation pattern of selected genes in zebrafish early development.