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ABSTRACT: Ghrelin has wide effects on cardiovascular and endocrine system. The aims of this study are to investigate the direct damage effect of high glucose and high palmitate on cardiomyocyte, and to study the effect of ghrelin on insulin resistance induced by glucotoxicity/lipotoxicity in cardiomyocyte and the possible mechanism underlying the cardioprotective activities of ghrelin. The changes of [(3)H]-2-deoxy-d-glucose ((3)H-G) intake rates were detected by isotope tracer method and the gene expressions in insulin signal transduction pathway were detected by real-time PCR and Western blot assay. The (3)H-G intake rate significantly reduced in high glucose (25mmol/l) or high palmitate (0.5mmol/l) treated primary rat ventricular myocytes. After the treatment of ghrelin (10(-7)mol/l), the (3)H-G intake rate recovered to the normal level. In addition, the phosphorylation of AKT occurred in 10min and was the highest in 30min after the stimulation with ghrelin, which can be blocked by phosphoinositide 3-kinase (PI3K) inhibitor, LY2940002. Ghrelin also increased the mRNA levels of glucose transporter 4 (GLUT4), peroxisome proliferators (PPARr) and AMP activated protein kinase (AMPK) genes in insulin signal transduction pathway. These results indicate that the direct damage of high glucose and high palmitate on cardiomyocyte might be through insulin resistance (IR). Ghrelin can inhibit gluco/lipotoxicity induced insulin resistance by PI3K/AKT pathway. This may provide a clue for therapy for myocardial disease in diabetes mellitus.
Peptides 02/2011; 32(2):209-15. · 2.43 Impact Factor
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ABSTRACT: Ghrelin is the endogenous ligand of the growth hormone secretagogue receptor. It has been reported to have cardiovascular protective activities. Elevated plasma free fatty acids trigger apoptosis in vascular endothelial cells. We investigated the effect of ghrelin on palmitate-induced apoptosis in rat aortic endothelial cells and the involved transduction pathway.
Rat aortic endothelial cells were treated with palmitate in the presence or absence of ghrelin. Apoptosis was detected using an annexin V-FITC/PI binding assay and a caspase-3 activity assay. The intracellular reactive oxygen species formation in endothelial cells was detected by fluorescence measurement with dichlorofluorescein diacetate (DCFH-DA). Western blot analysis was used to examine the changes in the expression levels of Akt, Bcl-2, and Bax.
Exposure of rat aortic endothelial cells to palmitate (0.3 mM) for 24 hours caused a significant increase in cell apoptosis. Ghrelin attenuated palmitate-induced apoptosis in endothelial cells. Exposure of cells to ghrelin caused a rapid activation of Akt. These effects were abolished by [D-Lys3]-GHRH-6, the special antagonist of growth hormone secretagogue receptor. Phosphatidyl inositol 3-kinase (PI3K) inhibitor, LY294002, extenuated the antiapoptotic effect of ghrelin. Reactive oxygen species levels were increased by palmitate, but this effect was inhibited by ghrelin. In addition, ghrelin increased the expression of Bcl-2 and decreased the expression of Bax; thus, the Bcl-2/Bax ratio was increased.
Ghrelin inhibits palmitate-induced apoptosis involved in activating the PI3K/Akt pathway and suppressing palmitate-induced oxidative stress in endothelial cells. Ghrelin may be protective against palmitate-induced endothelial injury.
Medical science monitor: international medical journal of experimental and clinical research 11/2010; 16(12):BR396-403. · 1.70 Impact Factor
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ABSTRACT: Lipotoxicity plays an important role in underlying mechanism of type 2 diabetes. Prolonged exposure of pancreatic beta-cells to elevated levels of fatty acid is associated with beta-cell apoptosis. Ghrelin is a 28-amino acid peptide, mainly secreted from X/A like cells of gastric fungus. The effects of ghrelin are considered to be broadly including cell protection. However, the mechanism of ghrelin protecting pancreatic beta-cells against lipotoxicity is unknown. Our study showed that ghrelin promoted cell survival and attenuated palmitate-induced apoptosis in pancreatic beta-cells (MIN6). Exposure of MIN6 cells to palmitate (0.4mM) for 24h caused a significant increase in cell apoptosis, which could be protected by ghrelin. Exposure of MIN6 cells to ghrelin caused a rapid activation of protein kinase B (PKB) and inhibition of c-Jun N-terminal kinase (JNK) under lipotoxic state. Furthermore, LY294002, a PI3K inhibitor, abolished the anti-lipotoxic effect of ghrelin, as well as ghrelin-induced inhibition of JNK, while JNK inhibitor, SP600125 enhanced protective effect of ghrelin on MIN6 cells. Ghrelin also inhibited the mitochondrial pathway of apoptosis and it down-regulated Bax in MIN6 cells. For secretion experiment, ghrelin suppressed insulin release under palmitate-incubated state. Our findings suggest that ghrelin may prevent lipotoxicity-induced apoptosis in MIN6 cells through activation of PKB, inhibition of JNK and mitochondrial pathway.
Regulatory Peptides 04/2010; 161(1-3):43-50. · 2.11 Impact Factor
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ABSTRACT: DNA is a valid drug target for development of target-based therapeutics against cancer. Screening DNA-targeted anticancer drugs is a key process for the research and development of new anticancer drugs. The traditional anticancer drug screening methods, including animal experiments and cell-based screening assays, have unavoidable drawbacks. In this contribution, the new instrument-based screening assay for DNA-targeted anticancer drugs in vitro using resonance light scattering (RLS) technique was proposed. The experiments suggested that the increment of RLS intensity was directly proportional to the antitumor effect of anticancer drugs. Therefore, it was intuitive to obtain the sequence of the antitumor activity of four anticancer drugs without data processing as follows: mitoxantrone (MIT) > pirarubicin (PIR) > daunorubicin (DAU) > doxorubicin (DOX) by RLS screening spectra. Moreover, the apparent equilibrium constant (K) was 1.23 x 10(4), 2.22 x 10(4), 4.66 x 10(4) L/mol for DOX, DAU, and PIR, respectively. The inhibitory concentration 50 (IC50) was 0.148, 0.102, 0.025, 0.013 micromol/L for DOX, DAU, PIR, MIT, respectively. Therefore, the antitumor effect of four drugs was as follows: MIT > PIR > DAU > DOX, which was in good agreement with the result obtained from RLS screening assays. The mechanism between DNA and anthracycline drugs was investigated using UV-vis spectroscopy, fluorescence spectroscopy, and electrophoresis experiments. The proposed assay is a rapid, intuitive, and easy-to-conduct bioassay with good accuracy and reproducibility.
Combinatorial chemistry & high throughput screening 01/2010; 13(5):383-92. · 2.46 Impact Factor
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ABSTRACT: Ghrelin is a 28-amino-acid peptide secreted predominantly by X/A-like cells of the gastric fundus. Ghrelin increases pancreatic beta-cell proliferation and survival via sequential activation of phosphatidylinositol-3 kinase (PI3K) and Akt. The transcription regulator Foxo1 is a prominent effector of PI3K/Akt; when it is inhibited, pancreatic beta-cells are protected against fatty-acid-induced apoptosis. We investigated the role of Foxo1 in the protective effect of ghrelin under lipotoxic conditions in the MIN6 pancreatic beta-cell line. Results showed that ghrelin promoted cell proliferation and attenuated palmitate-induced apoptosis in cultured MIN6 cells. Nuclear exclusion of Foxo1 was necessary for the function of ghrelin. Treatment of MIN6 cells with palmitate and ghrelin-induced rapid nuclear exclusion and phosphorylation of Foxo1. Unlike the JNK inhibitor SP600125, Akt inhibitor IV blocked the anti-lipotoxic effect of ghrelin and stimulated Foxo1 nuclear translocation. In addition, treatment with ghrelin combined with SP600125 showed a synergistic antiapoptotic effect in palmitate-treated MIN6 cells. Ghrelin also inhibited the endoplasmic reticulum stress pathway of apoptosis in MIN6 cells, decreased expression of cytoplasmic triglyceride, and downregulated gene expression of Bcl-2-associated X (BAX), sterol-response element-binding protein 1c (SREBP1c), and C/EBP homologous protein (CHOP-10). These findings suggest that ghrelin protects pancreatic beta-cells from lipotoxicity by inhibiting the nuclear translocation of Foxo1.
Peptides 11/2009; 31(2):307-14. · 2.43 Impact Factor
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ABSTRACT: To explore safety and efficacy of BIAsp 30 as initiation or replacement therapy in routine clinical practice in patients with poorly controlled diabetes in the Chinese cohort of the IMPROVE study.
In the 26-week, non-interventional, observational study, Chinese subjects with diabetes started BIAsp 30 treatment in routine care. Data from patients' diaries and medical records were transferred to CRFs by participating physicians.
NCT00659282.
Of the 21,729 subjects enrolled (mean age 54.0 years, BMI 24.6 kg/m 2 , diabetes duration 4.86 years), 32.3% were treatment-naïve, 59.3% were from oral anti-diabetic drugs (OADs) only and 8.1% were from insulin +/- OADs. Overall, mean HbA(1c) and FBG decreased by 2.82% and 5 mmol/L, respectively. In the subgroups, changes were: -3.27% and -6.06 mmol/L (treatment-naïve), -2.57% and -4.54 mmol/L (OADs only), -2.96% and 3.51 mmol/L (insulin +/- OADs) all p < 0.05. HbA(1c) < 7% was achieved by 71.4% of patients. Only 0.1% of subjects reported major hypoglycaemia and 73 SADRs were observed without significant difference compared to those at baseline. Body weight did not change significantly.
Regardless of previous treatments, insulin initiation or replacement with BIAsp 30 improved glycaemic control without increasing major hypoglycaemia or weight gain in Chinese patients with diabetes.
Current Medical Research and Opinion 11/2009; 26(1):101-7. · 2.38 Impact Factor
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ABSTRACT: A method for the determination of ofloxacin (OFL) has been developed, based on the enhancement of resonance light scattering (RLS) of OFL in the presence of alizarin violet 3B (AV3B). Under the experimental conditions, the RLS intensity of AV3B was greatly enhanced by adding OFL. At pH 5.09, the enhancement of the RLS intensity at 439.5 nm was proportional to the concentration of OFL in the range 0.10-2.50 microg/ml. The detection limit (3sigma) was 0.013 microg/ml. At pH 6.90, the enhancement of the RLS intensity at 405.0 nm was proportional to the concentration of OFL in the range 0.05-3.00 microg/ml. The detection limit (3sigma) was 0.021 microg/ml. The proposed method with high sensitivity, selectivity and reproducibility was satisfactorily applied to the determination of OFL in human serum.
Analytical Sciences 08/2009; 25(7):891-6. · 1.25 Impact Factor
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ABSTRACT: Phosphodiesters quaternary ammonium salt (PQAS) displayed quite intense light scattering in aqueous solution under the optimum condition. In addition, the resonance light scattering (RLS) signal of PQAS was remarkably decreased after adding trace amount polysaccharide with the maximum peak located at 391 nm. It was found that the decreased RLS intensity of the PQAS-PPGL system (DeltaI(RLS)) was in proportion to PPGL concentration in the range of 0.1-30 ng mL(-1), with a lower detection limit of 0.05 ng mL(-1). Based on this rare decreased RLS phenomenon, the novel method of the determination of purified polysaccharide of Gracilaria Lemaneiformis (PPGL) at nanogram level was proposed in this contribution. The proposed approach was used to determine purified polysaccharide extracted from Gracilaria Lemaneiformis with satisfactory results. Compared with the reported polysaccharide assays, this proposed method has good selectivity, high sensitivity and is especially simple and convenient. Moreover, the mechanism of the reaction between PQAS and polysaccharide was investigated by RLS, fluorescence, and fluorescence lifetime spectra.
Talanta 08/2009; 79(2):171-6. · 3.79 Impact Factor
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ABSTRACT: An in vitro screening model using resonance light scattering (RLS) technique with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reagent as the reactive probe to target cancer cell was firstly developed. In this model, MTT was reduced by viable cancer cells to produce a purple formazan. Cell viability was proportional to the number of formazan induced strong light scattering signal. The inhibition rate of anticancer drug was found to vary inversely with the H(22)-MTT system RLS intensity. So it was intuitive to see the sequence of the tumor suppressive activity of six anticancer drugs without data processing by RLS/MTT screening spectra. Compared with the traditional MTT method, this method has high sensitivity, low detection limit and quite intuitive screening results which were identical to those obtained from the MTT colorimetric assay.
Talanta 03/2009; 77(4):1365-9. · 3.79 Impact Factor
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ABSTRACT: A new high-sensitivity detection of protein assay at the nanogram level is proposed based on the decreased resonance light scattering (RLS) signals of zwitterionic gemini surfactant (phosphodiesters quaternary ammonium salt [PQAS]). It was found that PQAS self-assembled into nanometer-scale PQAS aggregates, which induced intense RLS signal in Britton-Robinson (BR) buffer solution (pH 10.5). Under the optimum conditions, the RLS intensity quenching extent of PQAS aggregation was in proportion to the concentration of proteins in the range of 0.0012-1.08 microg/ml for bovine serum albumin, 0.0015-0.95 microg/ml for human serum albumin, and 0.0025-1.3 microg/ml for gamma-globulin. The detection limits were 0.8, 1.2, and 2.0 ng/ml, respectively. The proposed method was successfully applied to determine total protein in human serum samples, and the results were identical to those obtained by the Bradford assay. The mechanism of interaction between PQAS and protein was studied using RLS, fluorescence, and time-resolved fluorescence, which indicated that the new complex formed between them, disaggregating self-aggregation of PQAS, resulted in the dominated quenching of RLS signal of the assay system.
Analytical Biochemistry 11/2008; 384(2):337-42. · 3.00 Impact Factor
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ABSTRACT: Plasminogen activator inhibitor type 1 (PAI-1), produced partly from liver is a risk factor for macrovascular and microvascular complications of diabetes. Ghrelin, a recently described orexigenic peptide hormone, attenuates PAI-1 induced by TNF-alpha in the human hepatoma cell line (HepG2). Exposure to TNF-alpha (1 ng/ml) for 24h caused a significant increase in PAI-1 mRNA expression and protein secretion, as evaluated by RT-PCR and ELISA, but pretreatment with ghrelin (1-100 ng/ml) inhibited both basal and TNF-alpha-induced PAI-1 release in a dose and time-dependent manner in HepG2. PDTC, selective NF-kappaB inhibitor, had no additive inhibitory effects with ghrelin. The results indicate that ghrelin inhibits both basal and TNF-alpha-induced PAI-1 production via NF-kappaB pathway in HepG2 cells, and suggest that the peptide plays a therapeutic role in atherosclerosis, especially in obese patients with insulin resistance, in whom ghrelin levels were reduced.
Cell Biology International 08/2008; 32(10):1310-7. · 1.48 Impact Factor
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ABSTRACT: Endothelial dysfunction is thought to be a major cause of vascular complications in diabetes. Our research shows that ghrelin attenuates high glucose-induced apoptosis in cultured human umbilical vein endothelial cells (ECV-304). Exposure to glucose (33.3mM) for 72 h caused a significant increase in apoptosis, as evaluated by TUNEL and flow cytometry, but pretreatment of ghrelin (10(-7)M) eliminated high glucose-induced apoptosis in ECV-304. Ghrelin also prevented the induction of caspase-3 activation, in cells incubated with glucose (33.3 mM). Exposure of cells to ghrelin (10(-7)M) caused rapid activation of Akt. PI3K inhibitor, LY294002 attenuated ghrelin's inhibitory effect on caspase-3 activity. Ghrelin protected endothelial cells from high glucose by inhibiting reactive oxygen species (ROS) generation. Results of our study indicate that ghrelin inhibits both high glucose-induced apoptosis via PI3K/Akt pathway and ROS production in ECV-304. This peptide may have potential in preventing diabetic complications, especially in obese patients.
Biochemical and Biophysical Research Communications 11/2007; 362(3):677-81. · 2.48 Impact Factor
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ABSTRACT: Lipotoxicity plays an important role in underlying mechanism of type 2 diabetes. Prolonged exposure of pancreatic β-cells to elevated levels of fatty acid is associated with β-cell apoptosis. Ghrelin is a 28-amino acid peptide, mainly secreted from X/A like cells of gastric fungus. The effects of ghrelin are considered to be broadly including cell protection. However, the mechanism of ghrelin protecting pancreatic β-cells against lipotoxicity is unknown. Our study showed that ghrelin promoted cell survival and attenuated palmitate-induced apoptosis in pancreatic β-cells (MIN6). Exposure of MIN6 cells to palmitate (0.4 mM) for 24 h caused a significant increase in cell apoptosis, which could be protected by ghrelin. Exposure of MIN6 cells to ghrelin caused a rapid activation of protein kinase B (PKB) and inhibition of c-Jun N-terminal kinase (JNK) under lipotoxic state. Furthermore, LY294002, a PI3K inhibitor, abolished the anti-lipotoxic effect of ghrelin, as well as ghrelin-induced inhibition of JNK, while JNK inhibitor, SP600125 enhanced protective effect of ghrelin on MIN6 cells. Ghrelin also inhibited the mitochondrial pathway of apoptosis and it down-regulated Bax in MIN6 cells. For secretion experiment, ghrelin suppressed insulin release under palmitate-incubated state. Our findings suggest that ghrelin may prevent lipotoxicity-induced apoptosis in MIN6 cells through activation of PKB, inhibition of JNK and mitochondrial pathway.
Regulatory Peptides.
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ABSTRACT: Ghrelin is a 28-amino-acid peptide secreted predominantly by X/A-like cells of the gastric fundus. Ghrelin increases pancreatic β-cell proliferation and survival via sequential activation of phosphatidylinositol-3 kinase (PI3K) and Akt. The transcription regulator Foxo1 is a prominent effector of PI3K/Akt; when it is inhibited, pancreatic β-cells are protected against fatty-acid-induced apoptosis. We investigated the role of Foxo1 in the protective effect of ghrelin under lipotoxic conditions in the MIN6 pancreatic β-cell line. Results showed that ghrelin promoted cell proliferation and attenuated palmitate-induced apoptosis in cultured MIN6 cells. Nuclear exclusion of Foxo1 was necessary for the function of ghrelin. Treatment of MIN6 cells with palmitate and ghrelin-induced rapid nuclear exclusion and phosphorylation of Foxo1. Unlike the JNK inhibitor SP600125, Akt inhibitor IV blocked the anti-lipotoxic effect of ghrelin and stimulated Foxo1 nuclear translocation. In addition, treatment with ghrelin combined with SP600125 showed a synergistic antiapoptotic effect in palmitate-treated MIN6 cells. Ghrelin also inhibited the endoplasmic reticulum stress pathway of apoptosis in MIN6 cells, decreased expression of cytoplasmic triglyceride, and downregulated gene expression of Bcl-2-associated X (BAX), sterol-response element-binding protein 1c (SREBP1c), and C/EBP homologous protein (CHOP-10). These findings suggest that ghrelin protects pancreatic β-cells from lipotoxicity by inhibiting the nuclear translocation of Foxo1.
Peptides.
