1.Plasma membrane-related Ca(2+)-ATPase-1 gene silencing promotes insulin secretion in islet beta cells NIT.
Yan-ping FANG ; Ai-min JI ; Yue-lian YANG ; Qiao-fei FENG ; Liang SUN ; Hong LIU
Journal of Southern Medical University 2009;29(8):1565-1567
OBJECTIVETo assess the effect of RNA interference-mediated gene silencing of plasma membrane-related Ca(2+)-ATPase-1 (PMR1) gene on the insulin secretion in islet beta cells NIT-1 in vitro.
METHODSA small interfering RNA duplex (siPMR1) corresponding to the nucleotides 337-357 of mouse PMR1 cDNA was introduced into NIT-1 cells via liposomes. The gene silencing effect was assessed by RT-PCR, and the total insulin level in the transfected cells was measured by radioimmunoassay.
RESULTSTransfection with siPMR1 resulted in obviously reduced PMR1 expression and increased insulin secretion in NIT-1 cells.
CONCLUSIONThe synthesized siPMR1 can significantly silence the expression of PMR1 and promote the secretion of insulin in the islet cells in vitro, which shed light on further studies of RNAi-based therapy of diabetes.
Animals ; Calcium-Transporting ATPases ; deficiency ; genetics ; Cell Line ; Gene Expression Regulation ; Insulin ; secretion ; Insulin-Secreting Cells ; metabolism ; secretion ; Mice ; RNA Interference ; RNA, Messenger ; genetics ; metabolism
2.Effects of NYGGF4 gene over-expression on the insulin sensitivity and secretory function of adipocytes.
Chun-Mei ZHANG ; Jie QIU ; Xiao-Hui CHEN ; Bin WANG ; Min ZHANG ; Xi-Rong GUO
Chinese Journal of Contemporary Pediatrics 2009;11(10):846-849
OBJECTIVETo study the effect of a new obesity-related gene NYGGF4 on the insulin sensitivity and secretory function of adipocytes.
METHODS3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1; control group) or an NYGGF4 expression vector (NYGGF4-pcDNA3.1) were cultured in vitro and differentiated into the matured adipocytes with the standard insulin plus dexamethasone plus 3-isobutyl-methylxanthine (MDI) induction cocktail. 2-deoxy-D-[3H] glucose uptake was determined by liquid scintillation counting. Western blot was performed to detect the protein content and translocation of glucose transporter 4 (GLUT4). The supernatant concentrations of TNF-alpha, IL-6, adiponectin and resistin were measured using ELISA.
RESULTSNYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake. NYGGF4 over-expression impaired insulin-stimulated GLUT4 translocation without affecting the total protein content of GLUT4. The concentrations of TNF-alpha, IL-6, adiponectin and resistin in the culture medium of 3T3-L1 transfected with NYGGF4 were not significantly different from those in the control group.
CONCLUSIONSNYGGF4 over-expression impairs the insulin sensitivity of 3T3-L1 adipocytes through decreasing GLUT4 translocation and had no effects on the secretory function of adipocytes.
3T3-L1 Cells ; Adipocytes ; drug effects ; secretion ; Adiponectin ; secretion ; Animals ; Carrier Proteins ; genetics ; physiology ; Glucose ; metabolism ; Glucose Transporter Type 4 ; analysis ; metabolism ; Insulin ; pharmacology ; Interleukin-6 ; secretion ; Mice ; Resistin ; analysis ; Transfection ; Tumor Necrosis Factor-alpha ; secretion
4.Observation of insulin exocytosis by a pancreatic β cell line with total internal reflection fluorescence microscopy.
Zhao-ying FU ; Ya-ping WANG ; Yu CHEN
Chinese Medical Sciences Journal 2011;26(1):60-63
Animals
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Exocytosis
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drug effects
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physiology
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Glucose
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pharmacology
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Insulin
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secretion
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Insulin-Secreting Cells
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cytology
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drug effects
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metabolism
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Mice
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Microscopy, Fluorescence
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methods
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Potassium
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pharmacology
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Recombinant Fusion Proteins
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genetics
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metabolism
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Vesicle-Associated Membrane Protein 2
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genetics
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metabolism
5.Effective glycemic control achieved by transplanting non-viral cationic liposome-mediated VEGF-transfected islets in streptozotocin-induced diabetic mice.
Hee Young CHAE ; Byung Wan LEE ; Seung Hoon OH ; You Ran AHN ; Jae Hoon CHUNG ; Yong Ki MIN ; Myung Shik LEE ; Moon Kyu LEE ; Kwang Won KIM
Experimental & Molecular Medicine 2005;37(6):513-523
Hypoxic damage is one of the major causes of islet graft failure and VEGF is known to play a crucial role in revascularization. To address the effectiveness of a cationic lipid reagent as a VEGF gene carrier, and the beneficial effect of VEGF-transfected islets on glycemic control, we used effectene lipid reagent in a transfection experiment using mouse islets. Transfection efficiencies were highest for 4 microgram/microliter cDNA and 25 microliter effectene and cell viabilities were also satisfactory under this condition, and the overproduction of VEGF mRNA and protein were confirmed from conditioned cells. A minimal number of VEGF-transfected islets (100 IEQ/animal) were transplanted into streptozotocin (STZ)-induced diabetic mice. Hyperglycemia was not controlled in the islet transplantation (IT)-alone group (0/8) (non- diabetic glucose mice number/total recipient mice number) or in the IT-pJDK control vector group (0/8). However, hyperglycemia was completely abrogated in the IT-pJDK-VEGF transduced group (8/8), and viable islets and increased VEGF-transfected grafts vascularization were observed in renal capsules.
Animals
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Body Weight
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Cell Survival
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Diabetes Mellitus, Experimental/*complications/metabolism
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Disease Models, Animal
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Glucose/pharmacology
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Glucose Tolerance Test
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Hyperglycemia/complications/*metabolism/*therapy
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Insulin/secretion
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Islets of Langerhans/blood supply/cytology/secretion
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*Islets of Langerhans Transplantation
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Liposomes/*administration & dosage/chemistry
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Male
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Mice
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Mice, Inbred BALB C
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Neovascularization, Physiologic
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RNA, Messenger/genetics/metabolism
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Research Support, Non-U.S. Gov't
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Streptozocin
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Transfection
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Vascular Endothelial Growth Factors/biosynthesis/*genetics/*metabolism/secretion
6.B-cell translocation gene 2 positively regulates GLP-1-stimulated insulin secretion via induction of PDX-1 in pancreatic beta-cells.
Seung Lark HWANG ; Okyun KWON ; Sun Gyun KIM ; In Kyu LEE ; Yong Deuk KIM
Experimental & Molecular Medicine 2013;45(5):e25-
Glucagon-like peptide-1 (GLP-1) is a potent glucoincretin hormone and an important agent for the treatment of type 2 diabetes. Here we demonstrate that B-cell translocation gene 2 (BTG2) is a crucial regulator in GLP-1-induced insulin gene expression and insulin secretion via upregulation of pancreatic duodenal homeobox-1 (PDX-1) in pancreatic beta-cells. GLP-1 treatment significantly increased BTG2, PDX-1 and insulin gene expression in pancreatic beta-cells. Notably, adenovirus-mediated overexpression of BTG2 significantly elevated insulin secretion, as well as insulin and PDX-1 gene expression. Physical interaction studies showed that BTG2 is associated with increased PDX-1 occupancy on the insulin gene promoter via a direct interaction with PDX-1. Exendin-4 (Ex-4), a GLP-1 agonist, and GLP-1 in pancreatic beta-cells increased insulin secretion through the BTG2-PDX-1-insulin pathway, which was blocked by endogenous BTG2 knockdown using a BTG2 small interfering RNA knockdown system. Finally, we revealed that Ex-4 and GLP-1 significantly elevated insulin secretion via upregulation of the BTG2-PDX-1 axis in pancreatic islets, and this phenomenon was abolished by endogenous BTG2 knockdown. Collectively, our current study provides a novel molecular mechanism by which GLP-1 positively regulates insulin gene expression via BTG2, suggesting that BTG2 has a key function in insulin secretion in pancreatic beta-cells.
Animals
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Gene Expression Regulation/drug effects
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Glucagon-Like Peptide 1/*pharmacology
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Homeodomain Proteins/*genetics/metabolism
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Humans
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Immediate-Early Proteins/genetics/*metabolism
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Insulin/genetics/*secretion
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Insulin-Secreting Cells/drug effects/*metabolism
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Male
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Mice
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Mice, Inbred C57BL
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Peptides/pharmacology
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Promoter Regions, Genetic/genetics
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Protein Binding/drug effects/genetics
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Rats
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Trans-Activators/*genetics/metabolism
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Tumor Suppressor Proteins/genetics/*metabolism
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Venoms/pharmacology
7.Effect of ginsenoside Rb1 on insulin signal transduction pathway in hippocampal neurons of high-glucose-fed rats.
Wen-Juan GU ; Di LIU ; Meng-Ren ZHANG ; Hong ZHANG
China Journal of Chinese Materia Medica 2014;39(6):1064-1068
OBJECTIVETo study the effect of ginsenoside Rb1 on GSKbeta/IDE signal transduction pathway and Abeta protein secretion in hippocampal neurons of high glucose-treated rats.
METHODHippocampal neurons of 24 h-old newly born SD rats were primarily cultured, inoculated in culture medium under different conditions, and then divided into the normal group, the high glucose group, the LiCl group and the Rb1 group. After being cultured for 72 h, the expressions of their phosphorylated GSK3beta, total GSK3beta and IDE protein were detected by Western blotting analysis. The mRNA expressions of GSK3beta and IDE were determined by RT-PCR. The ELISA assay was used to detect the secretion of Abeta protein in cell supernatant.
RESULTCompared with the normal group, the high glucose group showed increase in the p/tGSK3beta protein ratio and the secretion of Abeta protein and decrease in IDE protein and mRNA (P < 0.05). Compared with the high glucose group, both Rb1 and LiCl groups showed decrease in the p/tGSK3beta protein ratio and the expression of Abeta protein and increase in IDE protein and mRNA expression (P < 0.05). Compared with the LiCl group, the Rb1 group showed no significant difference in the expressions of p/tGSK3beta protein, IDE protein, mRNA and Abeta protein expression. In addition, the GSK3beta mRNA expression of the four groups had no significant difference.
CONCLUSIONGinsenoside Rb1 may reduce the secretion of Abeta protein in hippocampal neurons by reducing the phosphorylation of GSK3beta, down-regulating the ratio of pGSK3beta/GSK3beta and upregulating the expression of IDE.
Amyloid beta-Peptides ; genetics ; metabolism ; secretion ; Animals ; Dietary Carbohydrates ; adverse effects ; Gene Expression Regulation ; drug effects ; Ginsenosides ; pharmacology ; Glucose ; adverse effects ; Glycogen Synthase Kinase 3 ; genetics ; metabolism ; Glycogen Synthase Kinase 3 beta ; Hippocampus ; cytology ; Insulin ; metabolism ; Insulysin ; genetics ; metabolism ; Neurons ; cytology ; drug effects ; metabolism ; secretion ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; drug effects
8.Secretory expression and biological function of IGFBP-3 in eukaryotic cells.
Chen WU ; Yaojun SHAN ; Minji ZOU ; Jiaxi WANG ; Donggang XU
Chinese Journal of Biotechnology 2008;24(4):558-562
The cDNA of Insulin-like growth factor binding protein 3 was subcloned into a eukaryotic secretory expression vector pSectagA to construct pSectag-IGFBP3. Human renal cell carcinoma (RCC) 786-0 cells were transfected with pSectag-IGFBP3 using lipofectamine 2000. After 48 h, the secretory IGFBP-3 was tested and identified by western blotting. Meanwhile, Annexin V-EGFP stain was used to analyze the apoptosis of 786-0 cells induced by IGFBP-3. Secretory IGFBP-3 protein could express successfully in the 786-0 cells and the expressed IGFBP-3 directly displayed an apoptotic effect on the host cells. This work provides a basis for further study on the apoptosis-inducing mechanism of IGFBP-3 and the development of a new anti-tumor drug.
Apoptosis
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drug effects
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Base Sequence
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Carcinoma, Renal Cell
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metabolism
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pathology
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Humans
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Insulin-Like Growth Factor Binding Protein 3
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genetics
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secretion
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Kidney Neoplasms
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metabolism
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pathology
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Molecular Sequence Data
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Recombinant Fusion Proteins
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biosynthesis
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genetics
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physiology
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Transfection
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Tumor Cells, Cultured
9.Advances of the mechanism study on berberine in the control of blood glucose and lipid as well as metabolism disorders.
Ning SHEN ; Cai-Na LI ; Yi HUAN ; Zhu-Fang SHEN
Acta Pharmaceutica Sinica 2010;45(6):699-704
Berberine, an isoquinoline alkaloid isolated from some Chinese medicinal herbs such as Coptidis rhizoma, has been used for the treatment of diarrhea and other gastrointestinal infections as an antibacterial drug in Chinese medicine. In recent years, it was reported to have beneficial effects on the metabolism disorders states of diabetes. The mechanisms involve many aspects of the diabetes, including regulating the blood cholesterol and triglyceride, lowering blood glucose, ameliorating the insulin resistant state and influencing the function of the pancreatic beta cell.
Animals
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Berberine
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isolation & purification
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pharmacology
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Blood Glucose
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metabolism
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Coptis
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chemistry
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Diabetes Mellitus
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metabolism
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Drugs, Chinese Herbal
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isolation & purification
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pharmacology
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Humans
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Insulin
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metabolism
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secretion
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Insulin-Secreting Cells
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drug effects
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Metabolic Diseases
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metabolism
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Nicotinamide Phosphoribosyltransferase
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biosynthesis
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genetics
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Plants, Medicinal
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chemistry
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Protein Kinases
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metabolism
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RNA, Messenger
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metabolism
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Receptors, LDL
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genetics
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metabolism
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Signal Transduction
10.Role of phospholipase D1 in glucose-induced insulin secretion in pancreatic beta cells.
Wei na MA ; Shin Young PARK ; Joong Soo HAN
Experimental & Molecular Medicine 2010;42(6):456-464
As glucose is known to induce insulin secretion in pancreatic beta cells, this study investigated the role of a phospholipase D (PLD)-related signaling pathway in insulin secretion caused by high glucose in the pancreatic beta-cell line MIN6N8. It was found that the PLD activity and PLD1 expression were both increased by high glucose (33.3 mM) treatment. The dominant negative PLD1 inhibited glucose-induced Beta2 expression, and glucose-induced insulin secretion was blocked by treatment with 1-butanol or PLD1-siRNA. These results suggest that high glucose increased insulin secretion through a PLD1-related pathway. High glucose induced the binding of Arf6 to PLD1. Pretreatment with brefeldin A (BFA), an Arf inhibitor, decreased the PLD activity as well as the insulin secretion. Furthermore, BFA blocked the glucose-induced mTOR and p70S6K activation, while mTOR inhibition with rapamycin attenuated the glucose induced Beta2 expression and insulin secretion. Thus, when taken together, PLD1 would appear to be an important regulator of glucose-induced insulin secretion through an Arf6/PLD1/mTOR/p70S6K/Beta2 pathway in MIN6N8 cells.
ADP-Ribosylation Factors/metabolism/physiology
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Animals
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Basic Helix-Loop-Helix Transcription Factors/metabolism/physiology
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Cells, Cultured
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Gene Expression Regulation, Enzymologic/drug effects
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Glucose/*pharmacology
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Insulin/*secretion
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Insulin-Secreting Cells/*drug effects/enzymology/metabolism/secretion
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Intracellular Signaling Peptides and Proteins/metabolism/physiology
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Mice
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Models, Biological
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Oligodeoxyribonucleotides, Antisense/pharmacology
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Phospholipase D/antagonists & inhibitors/genetics/metabolism/*physiology
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Protein-Serine-Threonine Kinases/metabolism/physiology
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Ribosomal Protein S6 Kinases, 70-kDa/metabolism/physiology
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Signal Transduction/drug effects/genetics