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ObjectiveTo investigate the effects of lipopolysaccharide ( LPS ) on cell apoptosis,proliferation, and insulin secretion in a β-cell line, NIT-1. MethodsNIT-1 cells were stimulated with 1 μg/ml LPS for 0-120 h. Cell apoptosis was evaluated by Hochest33342 staining and Annexin V/PI flow cytometry. Cell proliferation was evaluated by CCK-8 and BrdU assay. Intracellular insulin content, basal insulin secretion, and glucose-stimulated insulin secretion(GSIS) were detected by RIA. The IRS-2 tyrosine phosphorylation was determined by Western blot. ResultsCell apoptosis was not significantly changed by treatment with LPS for 120 h. Cell proliferation was stimulated by LPS before 48 h, and inhibited after 96 h. Intracellular insulin content or GSIS was not altered, but basal insulin secretion was decreased significantly by LPS after 48 h ( all P＜0.01 ). LPS decreased the tyrosine phosphorylation level of IRS-2 ( 0. 45 ± 0. 08 vs 0. 22 ± 0. 06, P ＜ 0. 05 ) and stimulated IκBα phosphorylation. Pretreatment with a specific IκBα phosphorylation inhibitor, Bay1 1-7082 for 1 h, remarkably blunted the LPS-induced phosphorylation of IκBα and cell proliferation( both P＜0.01 ). ConclusionsLow-dosages of LPS regulate proliferation and basal insulin secretion of NIT-1 β-cells, in which activation of NF-κB and inhibition of IRS2 tyrosine-phosphorylation may be involved.
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BACKGROUND: Recent study showed that osteocalcin may elevate Insulin secretion and sensitivity, prevent the fat accumulation, play a role in the metablism of glucose and lipid. Undercarboxylated osteocalcin works as the main role. OBJECTIVE: To investigate the effect of different concentrations of glucose on osteoblast undercarboxylated osteocalcin. METHODS: The rib trabeculae were resected and broken, trypsinizated and washed completely by PBS. Bone surface and non-adhesive floating cells in cleaning fluid were observed with inverted microscope. Rib trabeculae was washed by DMEM culture medium once, and cultured in culture bottle. The culture liquid was replaced by new one once a week. The osteoblast was moved from the scledte a week later. The cells were fused monolayer and could be subcultured 4 to 6 weeks later. The active second or third generation cells were inoculated to 6-pore plate forming 5 groups. Osteoblast were stimulated by 5.6 mmol/L., 7.6 mmol/L, 9.6 mmol/L, 12.6 mmol/L, 20.6 mmol/L glucose medium respectively after the 80% cells were fused, the vitamin K_2 was added into the culture liquid until the concentration of it to be 10~(-5) mol/L. Supernatant was collected after half hour culturing, the undercarboxylated osteocalcin level were detected with RIA test kit, and corrected it as the total the undercarboxylated osteccalcin, calculated the carboxylated incomplete osteocalcin rate. RESULTS AND CONCLUSION: The rate of ostecblast carboxylated incomplete osteocalcin was different under different concentration glucose. The rate of 7.6 mmol/L, 9.6 mmol/L, 20.6 mmol/L concentration glucose groups were higher than that of 5.6 mmol/L glucose group [(0.27±0.02)%, (0.29±0.04)%, (0.12±0.02)%, P < 0.05]. It is indicated that osteoblast could sense the change of glucose concentration by regulating the secretion of the undercarboxylated osteocalcin between the concentration of 5.6mmol/L to 9.6mmol/L, while the carboxylated incomplete osteocalcin decreased as the concentration of glucose increased.
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Objective To investigate the effect of miR-375 inhibited by 2'-O-me-375 on lipoapoptosis of NIT-1 pancreatic β cells. Methods NIT-1 cells were divided and treated according to the optimal condition: mock (without lipofectamine) ,lipofectamine( transfected only with lipofectamine) ,NC-miRNA (transfected with negative control miRNA) ,and 2'-O-me-375( transfected with 2'-O-me-375) groups. 72 hours later, all cells in each group were cultured with 500 μmol/L palmitate for 48 h. The percentage of apoptotic cells was detected by Hochest33342 staining and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL). The protein expression of myotrophin ( V1 ) , a target gene of miR-375, was detected by Western blotting. Results Compared to the other three groups,the cell apoptosis rate of 2'-O-me-375 group was the lowest (P<0.01) .along with the highest VI expression level(P<0. 01). Conclusion Inhibition of miR-375 decreases pancreatic (3-cell lipoapoptosis.
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The angiotensinogen(AGT) expression and angiotensin Ⅱ (AngⅡ ) secretion levels in cultured SD rat mesangial cells were determined. High glucose up-regulated AGT mRNA(0. 29±0.07 vs 0. 20±0. 05,P< 0.05)and protein(0.66±0.23 vs 0.37±0. 15,P<0.05) expression and Ang Ⅱ secretion [(9.85±2.08 vs 7.50± 1. 51) pg/ml,P<0. 05]levels, which were down-regulated by pyrrolidine dithiocarbamate( PDTC) treatment via inhibiting NF-κB activity.
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The effect of globular adiponectiin (gAd)on the function of NIT-1 cells under high glucose medium was investigated. The results showed that gad could completely block the increase of NADPH oxidase components p47phox expression and recover mRNA expression of pancreatic duodenal homeobox-I ,paired box gene 6,glucose transpoter 2,and glucokinase except neurogenic differentiation factor 1 (P<0.05 or P<0.01). Whereas,impaired insulin secretion and mRNA expression at high glucose concentration were not significantly improved by gAd.
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The relationship between C936T polymorphism at 3'-untranslated region of vascular endothelial growth factor (VEGF) gene and diabetic nephropathy (DN) was analysed in 194 type 2 diabetic patients. The frequencies of genotype CC and allele C were significantly higher in DN group than those in non-DN group and control group. Allele C and genotype CC of VEGF may be a genetic marker susceptible to DN.
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Objective To compare the efficacy and safety of three regiments of transient intensive insulin therapy for type 2 diabetes mellitns: thrice preprandial injection of premixed insulin aspart 30, thrice preprandial injection of insulin aspart and injection of glargine at bedtime, thrice preprandial injection of regular insulin and injection of NPH at bedtime. Methods Patients were randomly divided into 3 groups, treated with 3 kinds of intensive insulin therapy. After achieving the target goal, continuous glucose monitoring system was used to compare the blood glucose level, therapeutic time, dosage of insulin, occurrence of hypoglycemia. Results Detected by continuous glucose monitoring system, there was no statistical difference in average blood glucose [(8.3±2.1,7.5±1.9, 6.8±0.8) mmol/L, P > 0.05], blood glucose area under curve 3 hours (AUC1-3) after breakfast, therapeutic time [ (8.3±2.5, 9.1±3.8, 8.4±1.7)d, P > 0.05], dosage of insulin [(0.63± 80%, P > 0.05) among three kinds of transient intensive insulin therapy. There were no patients complaining of hypoglycemic symptom. Conclusion The short-term efficacy and safety among three intensive insulin therapeutic methods are similar. More attention should be paid to monitor the blood glucose during sleep.
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BACKGROUND:Adiponectin possess functions of lowering blood glucose and blood lipids, and improve insulin sensitivity. But, controversy results about the effect of adiponectin on skeletal muscle have been reported.OBJECTIVE:To study the effects of eukaryon expressed adiponectin on the glycogen synthesis and glucose oxidation in skeletal muscle cell strain C2C12 myotubes by transfecting plasmids carrying mouse adiponectin.DESIGN: A controlled experiment.SETTING: The Second Affiliated Hospital of Sun Yat-sen University.MATERIALS: PcDNA3.0 plasmid with mouse adiponectin cDNA, pcDNA3.0-mad (generously presented from Dr. Gong,University of Maryland), C2C12 cell strain (purchased from ATCC, GRL-1722), DMEM high glucose (Gibco), MEM (Hyclone), fetal bovine serum (Hanagzhou Sijiqing), equine serum (Hyclone), lipofectamine 2000 (Invitrogene), G418 (Gibco), rabbit anti-mouse adiponectin IgG (ACRP303-A, Alpha Diagnostic International), chemiluminescence kit (ECL+PLUS,Amersham), SABC instant immunohistochemistry kit (Boster), D-[U-14C] glucose (specific activity 9.25-13.32 GBq/mmol,NEC), scintillation fluid POP, POPOP (SIGMA), liquid scintillation counter (LS3801, Beckman, USA).METHODS:This study was carried out in the Central Laboratory of the Second Affiliated Hospital of Sun Yat-sen University from March to August, 2003. ① After extraction of plasmid, double digest with Xho Ⅰ and Xba Ⅰ and identification with HindⅢ digest were carried out. ② Plasmid pcDNA3.0-mad and pcDNA3.0 blank vector were transfected using liposome to C2C12 cells, and the stably transfected cells were screened by 500 mg/L G418 for 3 weeks, G418 resistant C2C12 cells were thereafter harvested, therefore stable transfected pcDNA3.0-mad and pcDNA 3.0 C2C12 cell strains were established.③ Adiponectin protein expression was determined by Western blot analysis and immunohistochemistry. ④ Glucose oxidation and glycogen synthesis detections were divided into control, vector and pcDNA3.0-mad (mad)group. Each group was further divided into 4 subgroups with 0, 0.5, 5 and 100 nmol/L insulin (n =6), respectively. Detection of glucose oxidation and glycogen synthesis was carried out with 14C-labeled glucose by counting radioactivity of 14CO2 or 14C labeled glycogen with scintillation, respectively.MAIN OUTCOME MEASURES:Changes of glycogen synthesis and glucose oxidation in skeletal muscle cell strain C2C12myotubes.RESULTS: ① Results of plasmid transfection and restrict digest: After plasmid extraction, double digest with Xba Ⅰ and Xho Ⅰ was carried out along with HindⅢ digest identification.Digest fragments were in accordance with expectation.Length of adiponectin cDNA fragment was 781 bp, plasmid fragment was 5 446 bp, adiponectin cDNA was inserted between digest sites (Xho Ⅰ and Xba Ⅰ ) of eukaryotic expression vector pcDNA3.0. ② Plasmid transfection of C2C12 cell and positive clone screening: On the 10th day of G418 media culture screening after transfection, most C2C12 cells died.Positive clone appeared at the 2nd week. G418 resistant C2C12 colonies were harvested at the 3rd week. ③ Western blot and immunohistochemical identifications: Both confirmed that adipoenctin gene was stably transfected into cells in the Mad group, with successful adipoenctin expression. ④ Effect of stably transfected adiponectin gene to myocyte glucose metabolism:The myocyte glycogen synthesis and glucose oxidation increased along with the increasing of insulin concentration. The linear regression analyses of measured myocyte glucose oxidation amount showed that the regression coefficients of the control group, blank vector group and mad group were 23.34, 2;3.23 and 26.06 respectively. This result indicated that in C2C12 cell stably transfected with adiponectin gene, when insulin concentration increased, the acceleration rate of glucose oxidation increasing was higher than other 2 groups. However, no significant difference could be observed in glycogen synthesis and glucose oxidation of C2C12 cells under basic status without insulin stimulus and treatment status with different insulin concentrations between control group, blank vector group and mad group (P＞ 0.05).CONCLUSION: ① We have successfully established stably adiponectin gene transfected C2C12 cell strain with adiponectin protein expression ability. ② Transfection with adiponectin cDNA had no significant effect on the glucose oxidation and glycogen synthesis of C2C12 myotubes.③ The glucose oxidation and glycogen synthesis of C2C12 myotubes increased with the increasing of insulin concentration. ④ Adipoenctin may coordinate with insulin in improving myocyte glucose oxidation and increasing myocyte glucose uptake.
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BACKGROUND: Nowadays, angiotensin Ⅱ plays an important role in onset of diabetic nephropathy. Therefore, the nuclear factor-κB may have adjustive effects on angiotonin system of kidney tissue of diabetic rats. OBJECTIVE: To observe the relationship of activity of inhibitive nuclear factor-κB with angiotensin Ⅱ and its type 1 receptor mRNA expression of renal tissue of diabetic rats. DESIGN: Completely randomized group design, control experiment. MATERIALS: The experiment was conducted at the Experimental Animal Center, Sun Yat-sen University of Medical Sciences between March and April 2000. Fifty-one pure breed clean grade male Wistar rats were select ed. METHODS: ①Models were established in 39 rats. Streptozotocin dissolv ing in citric acid buffer (0.1 mmol/L,pH=4.5) were given to establish dia betic models with 60 mg/kg intraperitoneal injection. If the fasting blood glucose maintained above 13.9 mmol/L, the establishment of models was successful. The thirty-nine rats were randomly assigned into 3 groups: model group (n=17, without other interventional measure, feeding normally) and pyrrolidine dithiocar2. Bamate (PDTC) (active inhibitor of nuclear fac tor-κB) interventional group [n=22, PDTC at the dose of 20 mg/kg were given with intraperitoneal injection, twice a day]. Other 12 rats were as normal control group, did not make into diabetic models with normal breeding. ②After feeding for 18 weeks kidneys were got in every group. The activity of nuclear factor-κB was detected with electrophoretic mobility shift assay. The expression of type 1 receptor mRNA of angiotensin Ⅱ was measured with reverse transcription polymerase chain reaction (RT-PCR). Contents of angiotonin Ⅰ and angiotensin Ⅱ were tested with Radio Im munoassay (RIA). Activity of rennin was referred to that the result of the level of angiotonin Ⅰ at 37 ℃ water bath subduced to that at 4 ℃. ③Dif ference of measurement data was compared with single factor analysis of variance. After normal transformation, the non-normal distribution data were conducted with statistical disposal. MAIN OUTCOME MEASURES: Comparison of contents of angiotensin Ⅰ and Ⅱ, activities of rennin and nuclear factor-κB and expression of type 1 receptor mRNA of angiotensin Ⅱ in renal tissues of rats of each group. RESULTS: In the normal control group, model group and PDTC interven tional group 1, 6 and 13 rats were dropped out, respectively, so 11, 11 and 9 rats in each group were involved in the result analysis. ①Activity of nu clear factor-κB: It was higher significantly in the model group than that in the normal control group and PDTC interventional group (P ＜ 0.01 ). It was similar between the normal control group and the PDTC interventional group. ②Activity of rennin of renal tissue: It was similar among the 3 groups. ③Content of angiotonin Ⅰ of renal tissue: It was higher obviously in the model group that that in the normal control group and the PDTC interventional group (P ＜ 0.01 ). ④Content of angiotensin Ⅱ in renal tissue: It was similar between the model group and the normal control group. It was lower markedly in the PDTC interventional group than that in the model group and the normal control group (P ＜ 0.01 ). Expression of type 1 receptor mRNA of angiotensin Ⅱ: It was lower remarkably in the model group than that in the normal control group (P ＜ 0.01 ). It was lower dis tinctly in the PDTC interventional group than that in the model group and the normal control group (P ＜ 0.01 ). CONCLUSION: The increase of activity of nuclear factor-κB in renal tissue of diabetic rats can inhibit the activity of nuclear factor-κB, which will induce the reduction of the level of angiotensin Ⅱ and expression of type 1 receptor mRNA of angiotensin Ⅱ in renal tissue of diabetic rats.
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The study of glomerular mesangial cells of normal rats showed that angiotensin Ⅱ (ATⅡ) down-regulated the expression of MMP-2 mRNA, did not have significant effect on TIMP-2 mRNA. And in consequence, ATⅡ down-regulated the ratio of MMP-2 mRNA to TIMP-2 mRNA.
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<p><b>OBJECTIVE</b>To investigate the association of Pro12Ala variant in peroxisome proliferator-activated receptor-gamma2 gene with type 2 diabetes mellitus and its clinical characteristics.</p><p><b>METHODS</b>The genotypes of Pro12Ala variant in peroxisome proliferator-activated receptor-gamma2 gene were determined by polymerase chain reaction-restriction fragment length polymorphisms assay in 401 unrelated subjects of the Han population in the southern part of China (including 180 subjects with normal glucose tolerance and 221 type 2 diabetic patients). The clinical data were also analyzed.</p><p><b>RESULTS</b>The allele frequencies in the case and control groups were 96.15%,96.11% for P and 3.85%, 3.89% for A; the genotype frequencies were 92.77%, 92.22% for PP, 6.78%, 7.78% for PA and 0.45%, 0 for AA. The Pro12Ala variant of peroxisome proliferator-activated receptor-gamma2 was not significantly associated with type 2 diabetes. The Pro12Ala polymorphism of peroxisome proliferator-activated receptor-gamma2 in diabetes patients was associated with increased waist circumference and waist to hip ratio. The Pro12Ala polymorphism in Chinese population was similar to that in Japanese population and was different from that in European and American population.</p><p><b>CONCLUSION</b>The above data showed that the Pro12Ala variant of peroxisome proliferator-activated receptor-gamma2 was not significantly associated with type 2 diabetes, but it could be associated with abdominal obesity in type 2 diabetes. The significant difference of Pro12Ala of peroxisome proliferator-activated receptor-gamma2 among various races was observed.</p>
Subject(s)
Adult , Female , Humans , Male , Middle Aged , Alanine , Genetics , Alleles , Amino Acid Substitution , Blood Glucose , Metabolism , Body Constitution , Body Mass Index , Cholesterol , Blood , Cholesterol, HDL , Blood , Cholesterol, LDL , Blood , Diabetes Mellitus, Type 2 , Blood , Genetics , Pathology , Gene Frequency , Genotype , Insulin , Blood , Proline , Genetics , Receptors, Cytoplasmic and Nuclear , Genetics , Transcription Factors , Genetics , Triglycerides , BloodABSTRACT
【Objective】 To study the expression of leptin mRNA in Chinese obese subjects using dot blot hybridization with a digoxigenin labele d probe. 【Methods】 11 Chinese nonobese subjects (bodymass index, BMI 21.0 kg/ m2±1.5 kg/m2) and 12 Chinese obese subjects(BMI 28.5 kg/m2±2.3 kg/m 2) parti cipated in the study. The human leptin cDNA probe was labeled wih digoxigenin(DI G) b y the random priming method. Expression of leptin mRNA in abdominal adipose tiss ue has been examined using dot blot hybridization with this probe. 【Results】 T he expression of leptin mRNA was significantly higher in obese subjects than in non-obese ones (312.8±108.9 vs 175.9±81.5, relative units, P<0.0 5), an d correlated with BMI (r=0.56, P<0.05). 【Conclusions】 Leptin mRNA le vel is high in obese subjects, and correlated with BMI.
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AIM: To elucidate if the cytoprotective effects of IGF-1 and insulin on free fatty acid-treated pancreatic ? cells involve alteration in NF-?B activity.METHODS: Apoptosis was characterized by morphological analysis with invert microscope as well as Hoechst 33342 staining under a fluorescence microscope.Influence of co-incubation with free fatty acid(FFA) and IGF-1 or regular insulin(RI) on NF-?B activity were determined by Western blotting.Impacts of Bay-117082,which is NF-?B inhibitor,on cytoprotective effects of IGF-1 and RI were measured by flow cytometry.RESULTS: Apoptosis measured by flow cytometry was inhibited by IGF-1 and RI and semi-quantitative determination by Western blotting showed co-incubation with FFA and IGF-1 or RI caused more potent activation of NF-?B compared with incubation with FFA solely.Furthermore,flow cytometry showed suppression of NF-?B activity abolished the cytoprotective effects of IGF-1 and RI.CONCLUSION: Our data suggest that anti-apoptotic effects of IGF-1 and regular insulin on FFA-treated RIN-m cells are mediated via NF-?B pathway.
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A Review Resistin, a new hormone found in the year 2001 and secreted by adipocytes, is related to type 2 diabetes and obesity. It brings some hope to solve the medical hamper of insulin resistance. The resistin discovery, molecule structure, function and expression, secretion regulation as well as gene polymorphism are reviewed in the article. [
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AIM: To study the culture and characteristics of mouse adult bone marrow-derived pluripotential mesenchymal stem cells and its potential to differentiate into insulin secretion cells. METHODS: Cells were plated on 60% DMEM-LG and 40% MCDB-201 medium supplemented with 2% fetal calf serum and 10 ?g/L PDGF-BB, 10 ?g/L EGF and 1?10~6 U/L LIF. The proliferation rate, phenotype and oct-4 mRNA were tested. After it was plated on serum-free medium DMEM/F12 with GLP-1 and nicotinamide, the nkx2.2 ngn3, pdx-1 and insulin 2 mRNA were tested. RESULTS: The cells were round with large nucleus and scant cytoplasma. They were CD13~+, CD44~-, CD45~- and MHCⅡ~-. Oct-4 mRNA were present. The nkx2.2 pdx-1 and insulin 2 mRNA were presented in cells plated on the inducing medium at 14 days. CONCLUSION: The adult bone marrow-derived pluripotent stem cells were cultured and they has the possibilities to be induced into insulin-secreting cells.
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Objective To study the relationship between large multifunctional proteasome (LMP) 7 gene polymorphism and susceptibility of type 1 diabetes mellitus (DM). Methods The genotyping of LMP7 gene was determined by polymerase chain reaction restriction fragment length polymorphism (PCR RFLP) in 71 type 1 DM patients and 86 healthy persons (as controls). Furthermore, the type 1 DM patients were divided into 3 groups according to the age of diabetic onset. Group A was ≤14 years, group B 15～30 years, group C≥31 years.Results The frequency of LMP7 B/B was decreased significantly (39% vs 58%, P
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Objective To observe the effect of leptin on glucose oxidation with increased mRNA expression of complete length long intracellular domain of leptin receptor ( OBRb) in primary cultured skeletal muscle cells. Methods Skeletal muscle cells were isolated from SD sucking rat and cultured in four groups (recombinant transfection, empty plasmid transfection, non-transfection and control).The recombinant with complete length OBRb cDNA or the empty plasmid were introduced into skeletal muscle cells by clonfectin when the cultured cells were 70% conlluency, leptin and regular insulin were applied into wells and incubated for 1 h, then D-[ U-I4C]-glucose was added into wells and incubated for 2 h. The radioactivity of collected 14CO2 was assayed by scintillation. Expression level of OBRb mRNA in transfected cells was assayed by RT-PCR. Results The ratios of OBRb intraceUular domain mRNA and p-actin mRNA in cells transfected with recombinant, transiected with empty plasimd, and non-transfected were 1.22?0.10,0.41?0.08 and 0.49?0.09, respectively.The expression of OBRb intraceUular domain mRNA in cells transfected with recombinants was significantly increased as compared to the other groups of cells (P
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The HLA DQA1 genes of 40 IDDM patients (including 8 cases with onset of diabetes before 14 years of age, 19 between 15-30 years and 13 after 31 years) and 51 healthy controls were studied by using for allele specific oligonucleotide probes. All of our research subjects are of Southern Chinese origin. The results showed that the HLA DQA1 52Arg(+) associated IDDM susceptibility is significantly higher in the group younger than 14 years of age (P