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.

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

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. [

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

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.

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.

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

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.

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.

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.