Pancreatic Duodenal Homeobox-1 Expression Research Articles

RANKL/RANK contributes to the pathological process of type 2 diabetes mellitus through TRAF3 activation of NIK

Diabetic osteoporosis is a complication of diabetes mellitus (DM). Denosumab (DMB) is an effective anti-osteoporotic drug functions by inhibiting NF-κB ligand receptor-activating factor (RANKL). Previous study found that osteoprotegerin (OPG) regulated βcell homeostasis through the RNAK/RANKL pathway. The present study aimed to investigate the effect of RANKL/RANK on the pathological process of DM and the underlying mechanism. We used D-glucose-induced RINm5F cells to construct in vitro type 2 diabetes models (T2DM). A high-fat diet combined with intraperitoneal injection of streptozotocin (STZ) was used to establish a T2DM model in SD rats. The apoptosis of β-cells was determined by TdT-mediated dUTP nick-end labeling (TUNEL) analysis. qRT-PCR and western blotting assays were used to explore the mRNA and protein expression of the TRAF3 (Tumor necrosis factor receptor-associated factor)/NIK (NF-κB-inducible kinase) pathway. Furthermore, insulin expression was detected by ELISA and immunohistochemistry assay. The islet morphology was analyzed by H&E. In vivo experiments demonstrated that sRANKL-IN-3 down-regulated insulin secretion levels by significantly ameliorating pancreatic tissue damage and mitigating apoptosis of high glucose induced β-cells. Subsequently, sRANKL-IN-3, acting as an inhibitor of RANKL, mitigated functional decline in β-cells induced by high glucose, mainly manifested by the low expression of PDX-1 (pancreatic duodenal homeobox 1), BETA2 (beta-2 adrenoceptors), INS-1 (insulin 1), and INS-2 (insulin 2). Mechanistic studies revealed that deletion of TRAF3 combined with sRANKL-IN-3 administration reduced the activity of NIK, NF-κB2, and RelB in RINm5F cells. In addition, our study demonstrated that inhibition of either RANKL or TRAF3 had a protective effect on high glucose induced apoptosis. Moreover, the combined action of sRANKL-IN-3 and shTRAF3 had a more pronounced inhibitory effect on high glucose-induced apoptosis. In summary, RANKL/RANK deficiency may attenuate apoptosis of β-cells, a phenomenon associated with the TRAF3/NIK pathway. Therefore, RANKL/RANK could be regarded as a potential therapeutic strategy for DM.

Astragalus polysaccharide restores insulin secretion impaired by lipopolysaccharides through the protein kinase B /mammalian target of rapamycin/glucose transporter 2 pathway

BackgroundLipopolysaccharide (LPS)-induced dysfunction of pancreatic β-cells leads to impaired insulin (INS) secretion. Astragalus polysaccharide (APS) is a bioactive heteropolysaccharide extracted from Astragalus membranaceus and is a popular Chinese herbal medicine. This study aimed to elucidate the mechanisms by which APS affects INS secretion from β-cells under LPS stress.MethodsRat insulinoma (INS-1) cells were treated with LPS at a low, medium, or high concentration of APS. Glucose-stimulated insulin secretion (GSIS) was evaluated using an enzyme-linked immunosorbent assay (ELISA). Transcriptome sequencing was used to assess genome-wide gene expression. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was used to determine the signaling pathways affected by APS. Quantitative reverse transcription–polymerase chain reaction (qRT-PCR) was performed to evaluate the gene expression of glucose transporter 2 (GLUT2), glucokinase (GCK), pancreatic duodenal homeobox-1 (PDX-1), and INS. Western blot analysis was used to detect the protein expression of phosphorylated protein kinase B (p-Akt), total Akt (t-Akt), phosphorylated mammalian target of rapamycin (p-mTOR), total mTOR (t-mTOR), and GLUT2.ResultsLPS decreased GLUT2, GCK, PDX-1, and INS expression and reduced GSIS. These LPS-induced decreases in gene expression and GSIS were restored by APS treatment. In addition, transcriptome sequencing in combination with KEGG enrichment analysis revealed changes in the INS signaling pathway following APS treatment. LPS decreased p-Akt and p-mTOR expression, which was restored by APS treatment. The restorative effects of APS on GSIS as well as on the expression of GLUT2, GCK, PDX-1, and INS were abolished by treatment with the Akt inhibitor MK2206 or the mTOR inhibitor rapamycin (RPM).ConclusionsAPS restored GSIS in LPS-stimulated pancreatic β-cells by activating the Akt/mTOR/GLUT2 signaling pathway.

Human amniotic fluid stem cell therapy can help regain bladder function in type 2 diabetic rats.

BACKGROUNDDiabetes mellitus (DM) is a serious and growing global health burden. It is estimated that 80% of diabetic patients have micturition problems such as poor emptying, urinary incontinence, urgency, and urgency incontinence. Patients with diabetic bladder dysfunction are often resistant to currently available therapies. It is necessary to develop new and effective treatment methods.AIMTo examine the therapeutic effect of human amniotic fluid stem cells (hAFSCs) therapy on bladder dysfunction in a type 2 diabetic rat model.METHODSSixty female Sprague-Dawley rats were divided into five groups: Group 1, normal-diet control (control); group 2, high-fat diet (HFD); group 3, HFD plus streptozotocin-induced DM (DM); group 4, DM plus insulin treatment (DM + insulin); group 5, DM plus hAFSCs injection via tail vein (DM + hAFSCs). Conscious cystometric studies were done at 4 and 12 wk after insulin or hAFSCs treatment to measure peak voiding pressure, voided volume, intercontraction interval, bladder capacity, and residual volume. Immunoreactivities and/or mRNA expression of muscarinic receptors, nerve growth factor (NGF), and sensory nerve markers in the bladder and insulin, MafA, and pancreatic-duodenal homeobox-1 (PDX-1) in pancreatic beta cells were studied.RESULTSCompared with DM rats, insulin but not hAFSCs treatment could reduce the bladder weight and improve the voided volume, intercontraction interval, bladder capacity, and residual volume (P < 0.05). However, both insulin and hAFSCs treatment could help to regain the blood glucose and bladder functions to the levels near controls (P > 0.05). The immunoreactivities and mRNA expression of M2- and M3-muscarinic receptors (M2 and M3) were increased mainly at 4 wk (P < 0.05), while the number of beta cells in islets and the immunoreactivities and/or mRNA of NGF, calcitonin gene-related peptide (CGRP), substance P, insulin, MafA, and PDX-1 were decreased in DM rats (P < 0.05). However, insulin and hAFSCs treatment could help to regain the expression of M2, M3, NGF, CGRP, substance P, MafA, and PDX-1 to near the levels of controls at 4 and/or 12 wk (P > 0.05).CONCLUSIONInsulin but not hAFSCs therapy can recover the bladder dysfunction caused by DM; however, hAFSCs and insulin therapy can help to regain bladder function to near the levels of control.

Beneficial effects of proximal intestinal bypass reconstruction on glucose metabolism in a type 2 diabetes animal model: a possible reconstruction strategy for diabetic gastric cancer patients.

PurposeProximal intestinal bypass (PIB), such as Billroth II or Roux-en-Y gastrojejunostomy after curative distal gastrectomy for gastric cancer induces beneficial effects on glycemic control in patients with type 2 diabetes. We aimed to characterize the long-term evolution of pancreatic beta cells and insulin signaling in target tissue after a PIB procedure.MethodsZucker diabetic fatty rats were randomly assigned to the PIB, sham-operated PIB pair-fed, and ad libitum fed groups. Oral glucose tolerance (GT) and plasma insulin levels were measured periodically at 16 weeks postoperatively. Histomorphometric analyses were performed to evaluate changes in islet architectures and intranuclear pancreatic duodenal homeobox 1 (PDX1) expression in beta cells. Insulin signaling changes in visceral adipocytes were measured by the phosphorylated Akt/Akt ratio.ResultsContrary to the progressively deteriorating GT and plasma insulin levels in sham-operated animals, these were preserved in PIB animals (P < 0.01) at 16 weeks postoperatively. The proportion of the islets having asteroid-like expanding projection was higher in PIB animals than in sham-operated animals (P < 0.01). PIB animals had 3-fold wider fractional area of beta cells (P < 0.01) and 3-fold higher proportion of beta-cell nuclear PDX1 expression (P < 0.01) than sham-operated animals. PIB animals had significantly higher levels of Akt phosphorylation in the visceral adipocytes (P < 0.05). The PIB did not substantially affect weight and food intake postoperatively.ConclusionThe PIB preserved the plasma insulin levels and the wider beta-cell area over time and facilitated insulin signaling in the visceral fats. It could be considered as a possible reconstruction strategy for diabetic gastric cancer patients.

GSTO1 regulates insulin biosynthesis in pancreatic β cells

Insulin biosynthesis and secretion by pancreatic β cells are critical for the maintenance of blood glucose homeostasis. Here, we show that the expression of glutathione S-transferase omega-1 (GSTO1) is upregulated in the primary islet cells of diabetic Goto-Kakizaki (GK) rats. Knocking out GSTO1 upregulated insulin transcripts and increased the insulin content in both INS-1 cells and primary islet cells. In contrast, overexpression of GSTO1 reduced the insulin content. Furthermore, knocking out GSTO1 increased the expression of pancreatic duodenal homeobox-1 (PDX1) at both the transcription and protein levels. These results indicate that GSTO1 may be involved in the regulation of insulin biosynthesis by modulating the transcriptional expression of PDX1.

Effects of Sclerocarya birrea Stem-Bark Extracts on Glucose Uptake, Insulin Synthesis and Expression of Selected Genes Involved in the Synthesis and Secretion of Insulin in Rat Insulinoma Pancreatic Beta Cells

The present study reported the effects of Sclerocarya birrea stem-bark (SBSB) extracts on glucose uptake, insulin synthesis and the expression of glucose transporter 2 (GLUT2), glucokinase, pancreatic duodenal homeobox-1 (PDX-1), musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) and pre-proinsulin genes in rat insulinoma (RIN)-m5F pancreatic beta cells. The amount of glucose takenup by RIN-m5F cells was measured using a glucose oxidase-based assay kit. Intracellular and secreted insulin were measured using an enzyme linked immunoassay kit. Pre-proinsulin gene expression was determined using the conventional polymerase chain reaction (PCR) technique, while the expressions of GLUT2, glucokinase, PDX-1 and MafA genes were evaluated using quantitative real-time PCR technique. Of the four SBSB extracts investigated in the study, only the SBSB hexane extract positively affected all the study variables in RIN-m5F cells compared with the DMSO control. Thus, the SBSB hexane extract contains phytochemicals capable of enhancing insulin synthesis partly through up-regulation of the expression of GLUT2, glucokinase, PDX-1, MafA and pre-proinsulin genes.

Danzhi Jiangtang Capsule Mediates NIT-1 Insulinoma Cell Proliferation and Apoptosis by GLP-1/Akt Signaling Pathway.

Objective This study aimed to investigate the effects of Danzhi Jiangtang Capsule (DJC) on the proliferation and apoptosis functions of NIT-1 pancreatic β-cells exposed to high-glucose load through GLP-1 activated Akt/ FoxO1 signaling pathway. Methods Cellular apoptosis of NIT-1 pancreatic β-cells was induced by culturing in medium with 33.3mmol/L high glucose (HG). Then low-dose DJC (HG +LD), high-dose DJC (HG +HD), high-dose DJC+ GLP-1 inhibition (HG +HD +GI), and high-dose DJC+AKT inhibition (HG +HD+AI) were added, respectively. Cellular proliferation was accessed by cell counting kit (CCK-8) and cellular apoptosis was measured by Annexin V-FITC/PI staining. The protein levels of phosphorylated phosphatidylinositol-3-kinase (p-PI3K), phosphorylated AKT (p-AKT), phosphorylated Forkhead box protein O1 (p-FoxO1), and cleaved caspase-3 were detected by Western blotting. The mRNA expression of pancreatic duodenal homeobox-1 (PDX-1), CyclinD1, Bcl-2, and insulin was tested by Q-PCR. Results Comparing to HG group, (HG+HD) group showed a significantly increased cellular proliferation. The apoptosis of NIT-1 cells also was obviously reduced, with downregulated cleaved caspase-3 protein level and upregulated PDX-1, CyclinD1, and Bcl-2 mRNA levels (P<0.05). Additionally, (HG+HD) group manifested increased insulin mRNA expression; the protein levels of p-PI3K and p-AKT were markedly increased and p-FoxO1 was decreased. All of the above therapeutic effects by DJC intervention had been reversed by GLP-1 inhibition in (HG+HD+GI) group or AKT inhibition in (HG+HD+AI) group. Conclusion DJC was able to attenuate the toxicity of high-glucose load in NIT-1 pancreatic β-cells, ascribed to the improvement of cellular proliferation and apoptosis by GLP-1/Akt signaling pathway. This study could supply a new mechanism of DJC effects on type 2 diabetes mellitus (T2DM) treatment.

Naringin (4′,5,7-Trihydroxyflavanone 7-Rhamnoglucoside) Attenuates β-Cell Dysfunction in Diabetic Rats through Upregulation of PDX-1

Background: Pancreatic duodenal homeobox-1 (PDX-1) is a key transcription factor which regulates Insulin gene expression and insulin secretion in adult β-cells and helps to maintain β-cells mass. Naringin, a flavanone, owing to its anti­oxidant property, is reported to have antidiabetic effects. Objectives: The present study tries to evaluate the role of naringin on the β-cell-specific transcription factor PDX-1 in diabetic rats. Methods: Diabetes was induced in male rats using streptozotocin and treated with naringin (100 mg/kg) orally for 4 and 8 weeks. Serum insulin level, Pdx-1 and Insulin gene expression, and PDX-1 protein expression were assessed in the rat pancreas. Histopathological and ultrastructural changes in the islet and β-cells were observed. Results: Naringin prevented leukocytic infiltration in the pancreas of diabetic rats and recouped the β-cells with adequate secretory granules. Naringin-treated diabetic rats showed significantly increased mRNA expression of Pdx-1 and Insulin genes, increased expression of transcription factor PDX-1, and higher serum insulin levels than the diabetic control animals. These changes were more pronounced in the 8-week naringin-treated diabetic animals. Conclusions: Naringin was found to be an effective antidiabetic agent which increased Insulin gene expression and insulin secretion by upregulating the PDX-1 gene and protein expression.

Increased apoptosis, but not pancreatic duodenal homeobox-1 expression in pancreatic islets is associated with intermittent glucose loads in mice

Background: Several caloric restriction studies revealed good for diabetes prevention. However, prevalence of it seems rising yearly. It needs alternative technique thus people can choose suitable way for them.&#x0D; Aim: To determine the effect of glucose diet intermittently on pancreatic duodenal homeobox-1 (PDX-1), apoptosis in pancreatic islets, and pancreatic islets area.&#x0D; Materials and methods: Balb/c mice were divided into five groups. Control group was given standard diet. The Continuous group was given standard diet and added with 7.4% calories continuously. The 1x, 2x, and 3x intermittent groups were given standard diet and added 7.4% calories for 1x, 2x, and 3x/week respectively. The 7.4% calorie addition was a glucose solution by oral galvage and ad libitum for 8 weeks.&#x0D; Results: There was a significantly difference on apoptosis density (p=0.043), but not in PDX-1. The islets Int2x and Int3x groups showed a significant decrease than control group (p=0.048). Insulin serum levels increased significantly in Continuous group compared to control group (p=0.04). In addition, the insulin serum level of 1x and 3x intermittent groups were significantly lower than Continuous group (p0.05). Pre-post blood glucose levels on treatment groups decreased significantly compared to control group (p=0.012).&#x0D; Conclusions: Continuous and 1-3x/week intermittent addition of 7.4% calories of glucose for 8 weeks indicate a compensation mechanism for maintaining homeostasis, such as increase insulin serum level and seem to initiate the changes of morphologic-biomolecular (mainly apoptosis density in islets). The better mode is 1x/week of additional calories. However it needs further exploration to find out other influenced factors for these mechanism discovery.

GPR120 protects lipotoxicity-induced pancreatic β-cell dysfunction through regulation of PDX1 expression and inhibition of islet inflammation.

G-protein coupled receptor 120 (GPR120) has been shown to act as an omega-3 unsaturated fatty acid sensor and is involved in insulin secretion. However, the underlying mechanism in pancreatic β cells remains unclear. To explore the potential link between GPR120 and β-cell function, its agonists docosahexaenoic acid (DHA) and GSK137647A were used in palmitic acid (PA)-induced pancreatic β-cell dysfunction, coupled with GPR120 knockdown (KD) in MIN6 cells and GPR120 knockout (KO) mice to identify the underlying signaling pathways. In vitro and ex vivo treatments of MIN6 cells and islets isolated from wild-type (WT) mice with DHA and GSK137647A restored pancreatic duodenal homeobox-1 (PDX1) expression levels and β-cell function via inhibiting PA-induced elevation of proinflammatory chemokines and activation of nuclear factor κB, c-Jun amino (N)-terminal kinases1/2 and p38MAPK signaling pathways. On the contrary, these GPR120 agonism-mediated protective effects were abolished in GPR120 KD cells and islets isolated from GPR120 KO mice. Furthermore, GPR120 KO mice displayed glucose intolerance and insulin resistance relative to WT littermates, and β-cell functional related genes were decreased while inflammation was exacerbated in islets with increased macrophages in pancreas from GPR120 KO mice. DHA and GSK137647A supplementation ameliorated glucose tolerance and insulin sensitivity, as well as improved Pdx1 expression and islet inflammation in diet-induced obese WT mice, but not in GPR120 KO mice. These findings indicate that GPR120 activation is protective against lipotoxicity-induced pancreatic β-cell dysfunction, via the mediation of PDX1 expression and inhibition of islet inflammation, and that GPR120 activation may serve as a preventative and therapeutic target for obesity and diabetes.

Hypericin maintians PDX1 expression via the Erk pathway and protects islet β-cells against glucotoxicity and lipotoxicity.

A decrease in islet β-cell mass is closely associated with the development and progression of diabetes. Therefore, protection against β-cell loss is an essential measure to prevent and treat diabetes. In this study, we investigated the protective effects of non-photoactivated hypericin, a natural compound, on β-cells both in vitro and in vivo. In vitro, hypericin greatly improved INS-1 cell viability under high-glucose and high-fatty-acid conditions by inhibiting glucotoxicity- and lipotoxicity-induced apoptosis and nitric oxide (NO) production. Then, we further demonstrated that hypericin elicited its protective effects against glucotoxicity and lipotoxicity in INS-1 cells by attenuating the reduction in pancreatic duodenal homeobox-1 (PDX1) expression and Erk activity. In vivo, prophylactic or therapeutic use of hypericin inhibited islet β-cell apoptosis and enhanced the anti-oxidative ability of pancreatic tissue in high-fat/high-sucrose (HFHS)-fed mice, thus alleviating β-cell loss and maintaining or improving β-cell mass and islet size. More importantly, hypericin treatment decreased fasting blood glucose, improved glucose intolerance and insulin intolerance, and alleviated hyperinsulinaemia in HFHS-fed mice. Therefore, hypericin showed preventive and therapeutic effects against HFHS-induced onset of type II diabetes in mice. Hypericin possesses great potential for development as an anti-diabetes drug in the future.

Improvement of isolated caprine islet survival and functionality in vitro by enhancing of PDX1 gene expression

Dead islets replaced with viable islets are a promising offer to restore normal insulin production to a person with diabetes. The main reason for establishing a new islet source for transplantation is the insufficiency of human donor pancreas while using xenogeneic islets perhaps assists this problem. The expression of PDX1 is essential for the pancreas expansion. In mature β-cells, PDX1 has several critical roles such as glucose sensing, insulin synthesis, and insulin secretion. In this study, we aimed to evaluate the expression of pancreatic duodenal homeobox-1 (PDX1) in treated caprine islets in culture and to assess the protective effects of antioxidant factors on the PDX1 gene in cultured caprine islets. Purified islets were treated with serum-free, serum, IBMX, tocopherol, or IBMX and tocopherol media. Quantitative polymerase chain reaction and Western blotting were carried out to compare the expression levels of PDX1 in treated purified islets cultured with different media. Islets treated with IBMX/tocopherol exhibited the highest fold change in the relative expression of PDX1 on day 5 post-treatment (relative expression: 6.80±2.08), whereas serum-treated islets showed the lowest fold changes in PDX1 expression on day 5 post-treatment (0.67±0.36), as compared with the expression on day 1 post-treatment. Insulin production and viability tests of purified islets showed superiority of islet at supplemented serum-free media with IBMX/tocopherol compared to other cultures (53.875%±1.59%). Our results indicated that supplemented serum-free medium with tocopherol and IBMX enhances viability and PDX1 gene expression compared to serum-added and serum-free media.

The impact of a novel peach gum-derived polysaccharide on postprandial blood glucose control in streptozotocin-induced diabetic mice

Peach [Prunus persica (L.)] gum exudates are produced by the trunks and fruits in peach gummosis. Clinically, these exudates have been used to treat diabetes in China, though the molecular mechanism underlying remains unclear. In the current study, a novel peach gum-derived polysaccharide was isolated, designated as PGPSD, and its anti-diabetic effect was assessed in mice. This polysaccharide was composed of arabinose, xylose and galactose in the molar ratio of 5.98:1:3.55, with the average molecular weight at 1.00×106Da. The animal study demonstrated that the PGPSD polysaccharide significantly lowered the postprandial blood glucose in streptozotocin-induced diabetic mice. Histology and immunohistochemistry results further confirmed that the application of PGPSD polysaccharide partially restored the pancreatic islets in diabetic mice, and enhanced the expression of pancreatic duodenal homeobox-1, insulin and hexokinase1. Collectively, the data suggested that the peach gum-derived polysaccharide had a meaningful potential as a non-insulin therapeutic compound in the treatment of diabetes.

Long-Term Exposure of Pancreatic β-Cells to Palmitate Results in SREBP-1C-Dependent Decreases in GLP-1 Receptor Signaling via CREB and AKT and Insulin Secretory Response.

The effects of prolonged exposure of pancreatic β-cells to high saturated fatty acids on glucagon-like peptide-1 (GLP-1) action were investigated. Murine islets, human pancreatic 1.1B4 cells, and rat INS-1E cells were exposed to palmitate for 24 hours. mRNA and protein expression/phosphorylation were measured by real-time RT-PCR and immunoblotting, respectively. Specific short interfering RNAs were used to knockdown expression of the GLP-1 receptor (Glp1r) and Srebf1. Insulin release was assessed with a specific ELISA. Exposure of murine islets, as well as of human and INS-1E β-cells, to palmitate reduced the ability of exendin-4 to augment insulin mRNA levels, protein content, and release. In addition, palmitate blocked exendin-4-stimulated cAMP-response element-binding protein and v-akt murine thymoma viral oncogene homolog phosphorylation, whereas phosphorylation of MAPK-ERK kinase-1/2 and ERK-1/2 was not altered. Similarly, RNA interference-mediated suppression of Glp1r expression prevented exendin-4-induced cAMP-response element-binding protein and v-akt murine thymoma viral oncogene homolog phosphorylation, but did not impair exendin-4 stimulation of MAPK-ERK kinase-1/2 and ERK-1/2. Both islets from mice fed a high fat diet and human and INS-1E β-cells exposed to palmitate showed reduced GLP-1 receptor and pancreatic duodenal homeobox-1 (PDX-1) and increased sterol regulatory element-binding protein (SREBP-1C) mRNA and protein levels. Furthermore, suppression of SREBP-1C protein expression prevented the reduction of PDX-1 and GLP-1 receptor levels and restored exendin-4 signaling and action. Finally, treatment of INS-1E cells with metformin for 24 h resulted in inhibition of SREBP-1C expression, increased PDX-1 and GLP-1 receptor levels, consequently, enhancement of exendin-4-induced insulin release. Palmitate impairs exendin-4 effects on β-cells by reducing PDX-1 and GLP-1 receptor expression and signaling in a SREBP-1C-dependent manner. Metformin counteracts the impairment of GLP-1 receptor signaling induced by palmitate.

Metformin Restrains Pancreatic Duodenal Homeobox-1 (PDX-1) Function by Inhibiting ERK Signaling in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most potent and perilous diseases known, with a median survival rate of 3-5 months due to the combination of only advanced stage diagnosis and ineffective therapeutic options. Metformin (1,1-Dimethylbiguanide hydrochloride), the leading drug used for type 2 diabetes mellitus, emerges as a potential therapy for PDAC and other human cancers. Metformin exerts its anticancer action via a variety of adenosine monophosphate (AMP)-activated protein kinase (AMPK)- dependent and/or AMPK-independent mechanisms. We present data here showing that metformin downregulated pancreatic transcription factor pancreatic duodenal homeobox-1 (PDX-1), suggesting a potential novel mechanism by which metformin exerts its anticancer action. Metformin inhibited PDX-1 expression at both protein and mRNA levels and PDX-1 transactivity as well in PDAC cells. Extracellular signal-regulated kinase (ERK) was identified as a PDX-1-interacting protein by antibody array screening in GFP-PDX-1 stable HEK293 cells. Co-transfection of ERK1 with PDX-1 resulted in an enhanced PDX-1 expression in HEK293 cells in a dose-dependent manner. Immunoprecipitation/Western blotting analysis confirmed the ERK-PDX-1 interaction in PANC-1 cells stimulated by epidermal growth factor (EGF). EGF induced an enhanced PDX-1 expression in PANC-1 cells and this stimulation was inhibited by MEK inhibitor PD0325901. Metformin inhibited EGF-stimulated PDX-1 expression with an accompanied inhibition of ERK kinase activation in PANC- 1 cells. Taken together, our studies show that PDX-1 is a potential novel target for metformin in PDAC cells and that metformin may exert its anticancer action in PDAC by down-regulating PDX-1 via a mechanism involving inhibition of ERK signaling.

Fetuin A promotes lipotoxicity in β cells through the TLR4 signaling pathway and the role of pioglitazone in anti-lipotoxicity

ObjectiveFetuin A (FetA), a secreted glycoprotein, is known to affect inflammation and insulin resistance (IR) in obese humans and animals. Lipotoxicity from chronic hyperlipidemia damages pancreatic β cells, hastening the onset of diabetes. We sought to determine whether FetA promotes lipotoxicity through modulation of the toll-like receptor 4 (TLR4) inflammatory signaling pathway as well as the protective effect of pioglitazone(PIO) on lipotoxicity. MethodsβTC6, a glucose-sensitive mouse pancreatic β cell line, and Sprague–Dawley rats with diet-induced obesity, were used to investigate FetA-mediated lipotoxicity. Protein expression/activation were measured by Western blotting. Small interfering (si)RNAs for TLR4 were used. Cell apoptosis was quantified by TUNEL analysis or flow cytometry, respectively. Insulin release was assessed with an insulin ELISA. ResultsFetA dose-dependently aggravated palmitic acid (PA)-induced βTC6 cell apoptosis, insulin secretion impairment, and inhibition of the expression of G-protein-coupled receptor 40 (GPR40) and pancreatic duodenal homeobox-1(PDX-1). Combined FetA + PA induced TLR4 expression, and subsequent inhibition of TLR4 signaling or expression was shown to prevent the strengthening effect of FetA on PA-induced lipotoxicity in βTC6 cells. FetA + PA induced p-JNK and nuclear factor-κB (NF-κB) subunit P65 expression, and inhibition of this activity reduced PA+ FetA lipotoxicity in βTC6 cells. PIO could ameliorate PA+ FetA-induced damage to βTC6 cells. Similarly, PIO improved insulin secretion disorder, reduced apoptosis, decreased FetA, TLR4, p-JNK, NF-κB subunit P65 and cleaved caspase 3 expression, and increased GPR40 and PDX-1 expression in islet β cells of diet-induced obese rats. The correlative bivariate analysis showed that increases in Fetuin A were directly proportional to the development of β cell injury. ConclusionsFetA can promote lipotoxicity in β cells through the TLR4-JNK-NF-κB signaling pathway. The protective effects of PIO on lipotoxicity in β cells may involve the inhibition of the activation of the FetA and TLR4 signaling pathway.

Isolation and Characterization of Human Umbilical Cord Mesenchymal Stem Cells and Their Differentiation into Pdx-1&amp;lt;sup&amp;gt;+&amp;lt;/sup&amp;gt; Cells

Background: Recent studies have focused on generating of insulin-producing cells (IPCs) from pluripotent stem cells. Producing of precursor's population with pancreatic endoderm properties is a challenging issue in front of regenerative medicine investigators. Previous studies have shown that during pancrease development in lower portion of foregut, signals from notochord suppress sonic hedgehog (Shh) expression and lead to increase expression of pancreatic duodenal homeobox-1 (Pdx-1) as a marker for pancreatic precursor's cells. Therefore, Shh repression is considered as a critical step in IPCs generation protocols. Objective: Isolation and characterization of human umbilical cord mesenchymal stem cells (HUC-MSCs) is the aim of current study. As well as the role of basic fibroblast growth factor (bFGF) alone and in combination with cyclopamine are investigated in creating cells with Pdx-1 expression ability. Methods: Cells differentiate into definitive endoderm by adding activin A and wnt-3α into RPMI medium supplemented with for 3 days. At the second stage, the cells are washed with phosphate-buffered saline (PBS). One group (group A) is treated with bFGF for 5 days. Second group (group B) is treated with cyclopamine-KADD for 5 days. Third group (group C) is treated with bFGF and cyclopamine-KAAD for 5 days. Forth group (group D) is untreated as control. Result: Our results show that bFGF and cyclopamine in combination induce more expression of Pdx-1 in HUC-MSCs.

PDX-1 mRNA expression in endoscopic ultrasound-guided fine needle cytoaspirate: Perspectives in the diagnosis of pancreatic cancer

Background and aimsEndoscopic ultrasound-guided fine needle aspiration is routinely used in the diagnostic work up of pancreatic cancer but has a low sensitivity. Studies showed that Pancreatic Duodenal Homeobox-1 (PDX-1) is expressed in pancreatic cancer, which is associated with a worse prognosis. We aimed to verify whether the assessment of PDX-1 in endoscopic ultrasound-guided fine needle aspiration samples may be helpful for the diagnosis of pancreatic cancer. MethodsmRNA of 54 pancreatic cancer and 25 cystic lesions was extracted. PDX-1 expression was assessed by Real-Time PCR. ResultsIn all but two patients with pancreatic cancer, PDX-1 was expressed and was found positive in 7 patients with pancreatic cancer in which cytology was negative. The positivity was associated with a probability of 0.98 (95% CI 0.90–1.00) of having cancer and the negativity with one of 0.08 (95% CI 0.01–0.27). The probability of cancer rose to 1.00 (95% CI 0.97–1.00) for patients positive to both PDX-1 and cytology and fell to 0.0 (95% CI 0.00–0.15) in patients negative for both. ConclusionsPDX-1mRNA is detectable in samples of pancreatic cancer. Its quantification may be helpful to improve the diagnosis of pancreatic cancer.

Branched-chain amino acids supplementation protects streptozotocin-induced insulin secretion and the correlated mechanism.

Significant evidence demonstrates that oxidative stress can impair insulin secretion and contribute to the development of type 2 diabetes. Branched-chain amino acids (BCAAs) are reported to be positively related to insulin secretion. This study aimed to determine how oxidative stress affects the function of islets and whether BCAAs can ameliorate the oxidative stress, and accompanying c-jun N-terminal kinase (JNK), protein kinase D1 (PKD1), and pancreatic/duodenal homeobox-1 (PDX-1) changes induced by streptozotocin (STZ). Plasma glucose, plasma insulin, and JNK, PKD1 and PDX-1 mRNA and protein expression were measured in rats treated with STZ and BCAAs. The glucose level in STZ-induced diabetic rats was much higher than that in control animals, and the elevated plasma glucose level in diabetic rats could be significantly inhibited by BCAAs treatment. Consistent with the change in glucose levels, the levels of insulin were also affected by BCAAs treatment. The mRNA and protein expression of JNK, PDX-1, and PKD1 were significantly altered in diabetic rats compared with the control group (P<0.01) and treatment with a low dose of BCAA reversed these changes in those above markers significantly (P<0.01). The present study demonstrated that STZ-induced oxidative stress could reduce serum insulin levels and alter the JNK, PDX-1, and PKD1 expression. BCAAs restored the levels of serum insulin reversed changes in JNK, PDX-1, and PKD1 expression.

The effect of glucagon-like peptide-1 in the management of diabetes mellitus: cellular and molecular mechanisms.

Incretins, such as glucagon-like peptide-1 (GLP)-1, have been shown to elevate plasma insulin concentration. The purpose of this study is to investigate the cellular and molecular basis of the beneficial effects of GLP-1. Normal and diabetic male Wistar rats were treated with GLP-1 (50 ng/kg body weight) for 10 weeks. At the end of the experiment, pancreatic tissues were taken for immunohistochemistry, immunoelectron microscopy and real-time polymerase chain reaction studies. Samples of blood were retrieved from the animals for the measurement of enzymes and insulin. The results show that treatment of diabetic rats with GLP-1 caused significant (P < 0.05) reduction in body weight gain and blood glucose level. GLP-1 (10(-12)-10(-6) M) induced significant (P < 0.01) dose-dependent increases in insulin release from the pancreas of normal and diabetic rats compared to basal. Diabetes-induced abnormal liver (aspartate aminotransferase and alanine aminotransferase) and kidney (blood urea nitrogen and uric acid) parameters were corrected in GLP-1-treated rats compared to controls. GLP-1 treatment induced significant (P < 0.05) elevation in the expression of pancreatic duodenal homeobox-1, heat shock protein-70, glutathione peroxidase, insulin receptor and GLP-1-receptor genes in diabetic animals compared to controls. GLP-1 is present in pancreatic beta cells and significantly (P < 0.05) increased the number of insulin-, glutathione reductase- and catalase-immunoreactive islet cells. The results of this study show that GLP-1 is co-localized with insulin and seems to exert its beneficial effects by increasing cellular concentrations of endogenous antioxidant genes and other genes involved in the maintenance of pancreatic beta cell structure and function.