Inhibition of CD36, a fatty acid transporter, has been reported to prevent glucotoxicity and ameliorate high glucose induced beta cell dysfunction. Ezetimibe is a selective cholesterol absorption inhibitor that blocks Niemann Pick C1-like 1 protein, but may exert its effect through suppression of CD36. We attempted to clarify the beneficial effect of ezetimibe on insulin secreting cells and to determine whether this effect is related to change of CD36 expression. mRNA expression of insulin and CD36, intracellular peroxide level and glucose stimulated insulin secretion (GSIS) under normal (5.6 mM) or high glucose (30 mM) condition in INS-1 cells and primary rat islet cells were compared. Changes of the aforementioned factors with treatment with ezetimibe (20 μM) under normal or high glucose condition were also assessed. mRNA expression of insulin was decreased with high glucose, which was reversed by ezetimibe in both INS-1 cells and primary rat islets. CD36 mRNA expression was increased with high glucose, but decreased by ezetimibe in INS-1 cells and primary rat islets. Three-day treatment with high glucose resulted in an increase in intracellular peroxide level; however, it was decreased by treatment with ezetimibe. Decrease in GSIS by three-day treatment with high glucose was reversed by ezetimibe. Palmitate uptake following exposure to high glucose conditions for three days was significantly elevated, which was reversed by ezetimibe in INS-1 cells. Ezetimibe may prevent glucotoxicity in pancreatic β-cells through a decrease in fatty acid influx via inhibition of CD36.
Animals ; Anticholesteremic Agents/*pharmacology ; Antigens, CD36/antagonists & inhibitors/genetics/*metabolism ; Cells, Cultured ; Ezetimibe/*pharmacology ; Flow Cytometry ; Glucose/toxicity ; Insulin/genetics/metabolism/secretion ; Insulin-Secreting Cells/cytology/*drug effects/metabolism ; Male ; Palmitic Acid/metabolism ; RNA, Messenger/metabolism ; Rats ; Rats, Sprague-Dawley ; Reactive Oxygen Species/metabolism ; Real-Time Polymerase Chain Reaction

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

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 ; Gene Expression Regulation/drug effects ; Glucagon-Like Peptide 1/*pharmacology ; Homeodomain Proteins/*genetics/metabolism ; Humans ; Immediate-Early Proteins/genetics/*metabolism ; Insulin/genetics/*secretion ; Insulin-Secreting Cells/drug effects/*metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Peptides/pharmacology ; Promoter Regions, Genetic/genetics ; Protein Binding/drug effects/genetics ; Rats ; Trans-Activators/*genetics/metabolism ; Tumor Suppressor Proteins/genetics/*metabolism ; Venoms/pharmacology

The protective effects of Da Chai Hu Granules (DCHKL) on islet cells which were incubated with 4 mmol x L(-1) alloxan (AXN) were studied. The viability of islet cells were measured with MTT. Insulin released into medium and in islets was detected by radioimmunoassay. Cell apoptosis rate was determined by flow cytometry. The expression of anti-apoptotic gene Bcl-2 and pro-apoptotic gene Bax in islet cells were measured with RT-PCR (reverse transcription polymerase chain reaction). Serum containing DCHKL can promote the activity of islet cells significantly (P < 0.01). Basal insulin secretion and high glucose-stimulated insulin secretion increased significantly (P < 0.01). Serum containing DCHKL can inhibit apoptosis of islet cells, the ratio of apoptosis was decreased. Serum containing DCHKL increased expression of Bcl-2 mRNA and decreased expression of Bax mRNA. DCHKL can significantly promote proliferation of islet cells and increase the amount of basal secretion of pancreatic islet cells and high glucose-stimulated insulin secretion. The expression of Bcl-2 increased significantly. The expression of Bax decreased significantly. DCHKL have a protective effect on the islet cells.
Alloxan ; toxicity ; Animals ; Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Insulin ; metabolism ; secretion ; Islets of Langerhans ; cytology ; drug effects ; metabolism ; Plants, Medicinal ; chemistry ; Protective Agents ; pharmacology ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Rats ; Rats, Sprague-Dawley ; bcl-2-Associated X Protein ; genetics ; metabolism

Animals ; Exocytosis ; drug effects ; physiology ; Glucose ; pharmacology ; Insulin ; secretion ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Mice ; Microscopy, Fluorescence ; methods ; Potassium ; pharmacology ; Recombinant Fusion Proteins ; genetics ; metabolism ; Vesicle-Associated Membrane Protein 2 ; genetics ; metabolism

BACKGROUNDType 2 diabetes mellitus (T2DM) results from the complex association of insulin resistance and pancreatic β-cell failure. Recent studies have shown that patients diagnosed with T2DM present with a significant decrease in β-cell function, which can be further compromised during the progression of the disease. Several mechanisms have been shown to play a role in this process such as glucotoxicity and lipotoxicity, which contribute to accelerating insulin secretion. In this regard, Chinese medicine has a certain advantage. This experiment was performed to observe the effect of a Chinese medicine named Kaiyuqingre formula (KYQRF) on β-cell function and its mechanisms of action therein.

METHODSHigh glucose was used to set up a model of β-cell function failure. At the same time, medicated serum of KYQRF with different doses were administered to the cells. Rosiglitazone was taken as a control to observe the changes in insulin secretion, ATP-sensitive K(+) channels (K(ATP) channel) and uncoupling protein-2 (UCP-2) in each group.

RESULTSKYQRF had some effects on the insulin secretion. In a low glucose environment, no effective change in insulin secretion was observed (P > 0.05). However, insulin levels increased significantly when INS-1 cells were exposed to a high glucose environment (P < 0.05). KYQRF could also enhance cell viability (P < 0.05) in an effect similar to rosiglitazone. Although KYQRF had no effect on inwardly rectifying potassium channels (Kir6.2) (P > 0.05), it could decrease the overexpression of both UCP-2 and sulfonylurea receptor 1 (P < 0.05).

CONCLUSIONKYQRF can protect islet function by decreasing UCP-2 and sulfonylurea receptor 1.

ATP-Binding Cassette Transporters ; genetics ; Animals ; Cell Survival ; drug effects ; Drugs, Chinese Herbal ; pharmacology ; Glucose ; pharmacology ; Insulin ; secretion ; Insulin-Secreting Cells ; cytology ; drug effects ; metabolism ; Ion Channels ; genetics ; Male ; Mitochondrial Proteins ; genetics ; Potassium Channels, Inwardly Rectifying ; genetics ; Rats ; Rats, Sprague-Dawley ; Receptors, Drug ; genetics ; Sulfonylurea Receptors ; Thiazolidinediones ; pharmacology ; Uncoupling Protein 2

BACKGROUNDTissue-engineered bioartificial muscle-based gene therapy represents a promising approach for the treatment of heart diseases. Experimental and clinical studies suggest that systemic administration of insulin-like growth factor-1 (IGF-1) protein or overexpression of IGF-1 in the heart exerts a favorable effect on cardiovascular function. This study aimed to investigate a chronic stage after myocardial infarction (MI) and the potential therapeutic effects of delivering a human IGF-1 gene by tissue-engineered bioartificial muscles (BAMs) following coronary artery ligation in Sprague-Dawley rats.

METHODSLigation of the left coronary artery or sham operation was performed. Primary skeletal myoblasts were retrovirally transduced to synthesize and secrete recombinant human insulin-like growth factor-1 (rhIGF-1), and green fluorescent protein (GFP), and tissue-engineered into implantable BAMs. The rats that underwent ligation were randomly assigned to 2 groups: MI-IGF group (n = 6) and MI-GFP group (n = 6). The MI-IGF group received rhIGF-secreting BAM (IGF-BAMs) transplantation, and the MI-GFP group received GFP-secreting BAM (GFP-BAMs) transplantation. Another group of rats served as the sham operation group, which was also randomly assigned to 2 subgroups: S-IGF group (n = 6) and S-GFP group (n = 6). The S-IGF group underwent IGF-1-BAM transplantation, and S-GFP group underwent GFP-BAM transplantation. IGF-1-BAMs and GFP-BAMs were implanted subcutaneously into syngeneic rats after two weeks of operation was performed. Four weeks after the treatment, hemodynamics was performed. IGF-1 was measured by radioimmunoassay, and then the rats were sacrificed and ventricular samples were subjected to immunohistochemistry. Reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the mRNA expression of bax and Bcl-2. TNF-α and caspase 3 expression in myocardium was examined by Western blotting.

RESULTSPrimary rat myoblasts were retrovirally transduced to secrete rhIGF-1 and tissue-engineered into implantable BAMs containing parallel arrays of postmitotic myofibers. In vitro, they secreted consistent levels of hIGF (0.4 - 1.2 µg×BAM(-1)×d(-1)). When implanted into syngeneic rat, IGF-BAMs secreted and delivered rhIGF. Four weeks after therapy, the hemodynamics was improved significantly in MI rats treated with IGF-BAMs compared with those treated with GFP-BAMs. The levels of serum IGF-1 were increased significantly in both MI and sham rats treated with IGF-BAM. The mRNA expression of bax was lower and Bcl-2 expression was higher in MI-IGF group than MI-GFP group (P < 0.05). Western blotting assay showed TNF-α and caspase 3 expression was lower in MI-IGF group than MI-GFP group after therapy.

CONCLUSIONSrhIGF-1 significantly improves left ventricular function and suppresses cardiomyocyte apoptosis in rats with chronic heart failure. Genetically modified tissue-engineered BAMs provide a method delivering recombinant protein for the treatment of heart failure.

Animals ; Apoptosis ; Caspase 3 ; analysis ; Desmin ; analysis ; Genetic Therapy ; Heart Failure ; pathology ; physiopathology ; therapy ; Insulin-Like Growth Factor I ; genetics ; secretion ; Myoblasts, Skeletal ; metabolism ; Myocytes, Cardiac ; pathology ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; secretion ; Retroviridae ; genetics ; Tissue Engineering ; Tumor Necrosis Factor-alpha ; analysis ; Ventricular Function, Left

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 ; Berberine ; isolation & purification ; pharmacology ; Blood Glucose ; metabolism ; Coptis ; chemistry ; Diabetes Mellitus ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Humans ; Insulin ; metabolism ; secretion ; Insulin-Secreting Cells ; drug effects ; Metabolic Diseases ; metabolism ; Nicotinamide Phosphoribosyltransferase ; biosynthesis ; genetics ; Plants, Medicinal ; chemistry ; Protein Kinases ; metabolism ; RNA, Messenger ; metabolism ; Receptors, LDL ; genetics ; metabolism ; Signal Transduction

BACKGROUNDUncoupling protein (UCP) 2 is related to the dysfunction of beta cells induced by fatty acids. However, whether UCP2 has similar effects on alpha cell is still not clear. This study aimed to investigate the effects of UCP2 and its possible mechanisms in lipotoxicity-induced dysfunction of pancreatic alpha cells.

METHODSThe alpha TC1-6 cells were used in this study to evaluate the effects of palmitate and/or UCP2 inhibit factors on the glucagon secretory function, glucagon content, the glucagon mRNA level and the nitrotyrosine level in the supernatant. Meantime, the expression levels of UCP2 and peroxisome proliferator-activated receptor-γ coactivator-1 alpha (PGC-1 alpha) were measured by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blotting. Furthermore, the possible relationship between UCP2 and insulin signal transduction pathway was analyzed.

RESULTSPalmitate stimulated alpha cell glucagon secretion and the expression of UCP2 and PGC-1 alpha, which could be partially decreased by the inhibition of UCP2. Palmitate increased nitrotyrosine level and suppressed insulin signal transduction pathway in alpha cells. Inhibition of UCP2 influenced the effects of free fatty acid on alpha cells and may relate to glucagon secretion.

CONCLUSIONUCP2 played an important role on alpha cell dysfunction induced by free fatty acid in vitro, which may be related to its effects on oxidative stress and insulin signal transduction pathway.

Animals ; Cells, Cultured ; Glucagon ; secretion ; Glucagon-Secreting Cells ; drug effects ; physiology ; Insulin ; pharmacology ; Insulin Receptor Substrate Proteins ; metabolism ; Ion Channels ; genetics ; physiology ; Iridoid Glycosides ; pharmacology ; Iridoids ; Mice ; Mitochondrial Proteins ; genetics ; physiology ; Oxidative Stress ; Palmitic Acid ; toxicity ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; Phosphorylation ; RNA, Messenger ; analysis ; Signal Transduction ; Trans-Activators ; genetics ; physiology ; Transcription Factors ; Tyrosine ; analogs & derivatives ; metabolism ; Uncoupling Protein 2

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 ; Animals ; Basic Helix-Loop-Helix Transcription Factors/metabolism/physiology ; Cells, Cultured ; Gene Expression Regulation, Enzymologic/drug effects ; Glucose/*pharmacology ; Insulin/*secretion ; Insulin-Secreting Cells/*drug effects/enzymology/metabolism/secretion ; Intracellular Signaling Peptides and Proteins/metabolism/physiology ; Mice ; Models, Biological ; Oligodeoxyribonucleotides, Antisense/pharmacology ; Phospholipase D/antagonists & inhibitors/genetics/metabolism/*physiology ; Protein-Serine-Threonine Kinases/metabolism/physiology ; Ribosomal Protein S6 Kinases, 70-kDa/metabolism/physiology ; Signal Transduction/drug effects/genetics