| Background and objective:In the past 30 years, rapidly increased incidences of obesity have unfortunately become a public health problem all over the world. Obesity is one of most important risk factor for diabetes, cardiovascular disease and tumor. Previous study revealed that two thirds hypertensive patients attribute to obesity. Recently study has revealed that adipokines play a pivotal link role in pathogenesis of obesity and its related metabolic disorder. Adipokines secrected by adipocyte play a key role in systemic inflammation and insulin resistance in obesity and hypertension. In our previous study, the relationship between retinol binding protein 4(RBP4) and insulin ressitance has been investigated, and RBP4 levels was elevated in obese patients, but has no significant influence on insulin resistance. Therefore, the further research should be performed in order to understand how different adipokines regulate insulin resistance of adipocyte.Recently, a new and highly conserved family of secreted proteins, C1q/tumour necrosis factor-related proteins(CTRPs), which include fifteen family members, were shown to possess structural homologies to adiponectin. C1q/TNF-related protein-3(CTRP3), one member of the CTRP superfamily, was identified as an anti-inflammatory adipokine that inhibits the inflammation induced by lipopolysaccharide, toll-like receptor 4 and fatty acid, as well as induces adiponectin and resistin release in murine adipocytes. Adiponectin is a most well-characterised member of the CTRP family, which tightly linked to insulin resistance and insulin sensitivity. Adiponectin also is an important biomarker and therapeutic target in obesity-associated metabolic diseases. CTRP3 has the similar function with adiponectin. So far, no data have been reported concerning the relationship between CTRP3 and drug-naive, newly diagnosed obesity and hypertension subjects. Therefore, we investigated the associations of CTRP3 with metabolic, inflammatory parameters and insulin resistance in obese and hypertensive patient in a clinical study(Clinical Trial NO. 02226471); secondly, the dynamic changes of CTRP3, adiponectin and IL-6 expression in the differentiation of 3T3-L1 cell were also observed; Thirdly, the influence of insulin resistance state and anti-insulin resistance drugs treatment to CTRP3, adiponectin and IL-6 in a 3T3-L1 adipocytes insulin resistance model were established in a basic study.Methods:(1) 826 Chinese subjects were enrolled from the general population who had undergone medical checkups at the Southwest Hospital Medical Center at the Third Military Medical University from March to November 2013. After excluding 580 of the 826 subjects, a total of 246 subjects were enrolled in our study. According to body mass index(BMI), the obesity group enrolled 113 obese subjects(Male 72, Female 41) and the norml weight group included 133 normal weight(NW) subjects(Male 91, Female 42). Based on blood pressure levels, the subjects were divided into four subgroups: obesity-hypertension subgroup(OB-HTN)(Male 35, Female 23), obesity-normal blood pressure subgroup(OB-NBP)(Male 37, Female 18), NW-hypertension subgroup(NW-HTN)(Male 43, Female 23), NW-normal blood pressure subgroup(NW-NBP)(Male 48, Female 19). Serum CTRP3 levels, as well as anthropometric, inflammatory and metabolic parameters were performed.(2) Cells of 3T3-L1 pre-adipocytes were cultivated via standard differentiation protocol induced by 3-isobutyl-1-methylxanthine, dexamethasone and insulin. The 3T3-L1 adipocytes and supernatants were collected at different differentiation times(pre-differentiation, 2nd, 6th and 9th day). The secretion and gene expression of CTRP3, adiponectin and IL-6 were detected by enzyme-linked immunosorbent assay(ELISA) and Quantitative real-time polymerase chain reaction(RT-PCR).(3) Insulin-resistant 3T3-L1 adipocytes construction: The differentiated adipocytes at 9th day were 48 hours incubated with 100 n M insulin and 10ng/ml TNFα. Glucose level in the cell culture media was determined by glucose oxidase method and glucose consumption was calculated as(25-glucose level) mmol/L.(4) 3T3-L1 insulin resistant adipocytes were treated for 48 hours with rosiglitazone, metformin and liraglutide, respectively. The gene expression and protein secretion of CTRP3, adiponectin and IL-6 in 3T3-L1 adipocytes and supernatants were detected by RT-PCR, ELISA and Western Blot under different intervention factors.(5) The experimental data were analysed using SPSS software(SPSS, Chicago, IL, USA), version 17.0. Continuous variables are presented as the means ± standard deviations, and categorical variables are presented as absolute and relative frequencies(%). Before the statistical analysis, the data were subjected to a normal distribution analysis using the Kolmogorov-Smirnov test. Non-normally distributed data(CTRP3, triglyceride, Fasting plasma insulin, HOMA-IR and HOMA-β) were converted by logarithmic transformation. Categorical variables were compared using the chi-squared test. The Student’s t-test and analysis of variance(ANOVA) were used to compare groups. In addition, a post-hoc least significant difference(LSD) test was used for equal variances of assumed variables, and Dunnett’s test was used for equal variances of variables that were not assumed. Interrelationships between variables were analyzed with Pearson’s correlation analysis. Partial correlation analysis was performed to control for gender and blood preesure. Multiple regression analysis was performed using stepwise linear regression to correct the effects of the covariates and to test the independent factors, and A P-value < 0.05 was considered to be statistically significant for all analyses.Result:(1) The serum CTRP3 levels in obese patients were lower than those in normal weight subjects; these levels were also lower in hypertensive subjects than in normotensive subjects.(2) After adjusting for gender and blood pressure, a modestly linear relationship was observed between CTRP3 and waist circumference(r =-0.168, P = 0.009), waist-to-hip ratio(r =-0.183, P = 0.004), homeostasis model assessment of IR(r =-0.264, P = 0.000), triglycerides(r =-0.136, P = 0.034), fasting blood glucose(r =-0.155, P = 0.016), fasting insulin(r =-0.248, P = 0.000) and homeostasis model assessment of β-cell insulin secretion(r =-0.128, P = 0.047).(3) Multiple stepwise regression analysis revealed that gender, diastolic blood pressure level and HOMA-IR were independently associated with serum CTRP3 levels.(4) CTRP3 and adiponectin had no expression in 3T3-L1 pre-adipocytes. With the differentiation and maturity of pre-adipocytes, the gene transcription level and protein secretion of CTRP3 and adiponectin gradually were up-regulated. The peak of m RNA expression was at the 9th day(98.65±2.68, P<0.01; 166.21±4.75, P<0.01, respectively). The m RNA expression of adiponectin was higher than CTRP3. TNFα was added to differentiation liquid A, and gene transcription level of CTRP3 and adiponectin were inhibited with time prolonged.(5) The m RNA expression of CTRP3 and adiponectin decreased by 38%(P<0.01) and 45%(P<0.01), but IL-6 increased by 86%(P<0.01) in the insulin-resistant 3T3-L1 adipocytes. The protein secretion levels of CTRP3 and adiponectin in the supernatant decreased by 30%(P<0.01) and 38%(P<0.01), but IL-6 increased by 75%(P<0.01) in the insulin-resistant 3T3-L1 adipocytes.(6) CTRP3 and adiponectin of m RNA expression in insulin-resistant 3T3-L1 adipocytes increased by 72%(P<0.01) and 82%(P<0.01), but IL-6 decreased by 46%(P<0.01) with intervention of rosiglitazone. CTRP3 and adiponectin of m RNA expression in insulin-resistant 3T3-L1 adipocytes increased by 60%(P<0.01) and 75%(P<0.01), but IL-6 decreased by 42%(P<0.01) with intervention of metformin. CTRP3 and adiponectin of m RNA expression in insulin-resistant 3T3-L1 adipocytes increased by 42%(P<0.01) and 62%(P<0.01), but IL-6 decreased by 28%(P<0.01) with intervention of liraglutide. The protein secretion levels in the supernatant showed a similar trend.Conclusion:(1) CTRP3 levels was close related to HOMA-IR, suggested the novel adipokine may paly an important role in the pathogenesis of obesity related insulin resistance.(2) CTRP3 plays a pivotal role in the differentiation of 3T3-L1 pre-adipocytes. CTRP3 may up-regulate expression of adiponectin.(3) The CTRP3 and adiponectin expression were down-regulated, but IL-6 was up-regulated in the insulin-resistant 3T3-L1 adipocytes.(4) Insulin resistance was improved by rosiglitazone, metformin and liraglutide through up-regulation of CTRP3 and adiponectin, as well as down-regulation of IL-6 in the insulin-resistant 3T3-L1 adipocytes. |
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