| Objectives:This study aims to observe the intervention effect of temisartan on insulin sensitivity and the incidence rate of type2diabetes mellitus(T2DM) of pre-diabetic OLETF (Otsuka Long-Evans Tokushima Fatty) rats fed with long-term high-fat diet (HFD). To explore the regulation effect of telmisartan on expressions of adiponectin and its receptors, leptin,, MCP-1、IL-6、TNF-α, PPARγ1, PPARγ2and AT1&AT2in subcutaneous (SAT) and visceral (VAT) adipose tissue in OLETF rats and its depot-specific difference. Investigate the correlation of RAS in adipose tissue regions and insulin resistance and the amelioration of temisartan on insulin resistance and the incidence of diabetes and its mechanism in OLETF rats.Content:Fifty four-week-old male OLETF rats were selected; twelve gender-and age-matched LETO (Long-evans Tokushima otsuka) rats were used as normal controls. At22weeks of age, there was no diabetes or IGT in all the groups. OLETF rats were randomly divided:O-T group (telmisartan,5mg·kg-1·d-1, n=10); O-TL group (low telmisartan,0.5mg·kg-1·d-1,n=9); O-P group (pioglitazone10mg·kg-1·d-1, n=8), O-M grou (metformin,100mg·kg-1·d-1,n=10). The OLETF control (O-C,n=10) and LETO control (LETO, n=12) rats were treated with a vehicle. The weight, fasting plasma glucose and blood pressure were measured every two weeks. At the age of48weeks, euglycaemic-hyperinsulinaemic clamp experiments were performed in rats anesthetized with a single intraperitoneal injection of20%urethane (0.3ml/100g body weight). All rats were then sacrificed and serum samples were collected. The SAT and VAT fat pads were dissected and stored at-80℃. The mRNA and protein expressions of adipocykines (e.g.adiponectin, leptin, TNF-α, MCP-1, IL-6, etc.) in SAT and VAT adipose tissue were determined. The morphological changes of adipose tissues were observed.Methods:1. Experimental methods included:ⅰ) T2DM animal model was established by high-fat diet containing high unsaturated fatty acid in four-week-old spontaneously diabetic male OLETF rats; ⅱ) The levels of adiponectin, leptin, free fatty acid (FFA), TNF-α, MCP-1, IL-6and PPARγ etc.in the serum were determined by ELISA. The total cholesterol (TC), triglyceride (TG), fasting plasma glucose (FPG), and fasting insulin (FINS) levels were measured, iii) The gene expressions of adiponectin and its receptors, leptin, TNF-α, IL-6, MCP-1and PPARys etc. were determined by real-time PCR.iv) The protein expressions levels of adiponectin, pro-inflammatory cytokines, etc. were measured by Western blotting. v) SAT and VAT from rats were obtained for HE dyeing, and morphology of adipocytes was observed.2. Statistics:Continuous data were expressed as the mean±S.E.M. Comparisons between groups were made by covariance analyses. The general linear model procedure of SPSS was used in the analyses to identify independent effects of the different treatments after adjusting for the SBP and DBP. Fisher’s exact test was performed for categorical variables. The correlations of HOMA-IR with adipocytokines were analyzed using the Spearman’s rank correlation coefficient (ρ). Bivariate logistic regression modeling was performed to detect associations between cytokine levels and glycometabolism dysfunction.Results:1. The body weights of the OLETF rats were significantly higher than those of the LETO rats, no significant difference was found between O-C and O-T group(P>0.05), but the visceral fat index in O-T group is significant lower than O-C group. The weight of O-P group was increased continually from30to48weeks, and was higher than other group including O-C group (P＜0.05).2.Serum FPG, FINS, FFA, TC, LDL-C, HOMA-IR,24hour urine volume and urine glucose were markedly increased in O-C group, the incidence rates of T2DM was90%. The biochemical markers above, the glucose level in OGTT and HOMA-IR were significant decreased in O-T group, the incidence rates of T2DM was also significant decreased (x2=5.495, P=0.019).3. TNF-α, MCP-1and leptin expression levels in both serum and in SAT and VAT were markedly increased, whereas the adiponectin and PPARy expressions were decreased. Both telmisartan and pioglitazone down-regulated the leptin and TNF-a in SAT and VAT, and decreased the serum leptin level, but serum TNF-a and IL-6were only decreased by telmisartan. The mRNA and protein expressions of MCP-1in SAT were down-regulated by telmisartan, pioglitazone and metformin, but MCP-1in serum and in VAT was deceased only by telmisartan.4. AT1mRNA expression in SAT and VAT was significantly increased in O-C group, but down-regulation of was AT1observed in O-P, O-T and O-TL, no significant difference was noted between O-P and O-T group. AT2mRNA expression was significant higher than other groups except O-TL group. Only telmisartan has up-regulated AdipoRl&R2mRNA in both SAT and VAT. The mRNA expressions of PPARyl and PPARy2in SAT were significantly up-regulated in O-P group, whereas PPARγ1and PPARy2expressions in VAT were markedly up-regulated in O-T group, In addition, PPARy2mRNA in VAT was up-regulated by telmisartan, but no significant difference was observed between O-P and O-T group.Adipocyte size increased and adipocyte morphous appeared irregular, and in disorder. Compare to O-C group, adipocyte size was downsizing58%,38%,56%and19%respectively in O-P, O-M, O-T and O-TL groups, and the arrangement of adipocyte was tend to regular.5. There was no significant correlation between the BP levels and HOMA-IR, suggested that telmisartan lowered the BP levels and HOMA-IR without a correlation between their changes. Telmisartan improved insulin resistance beyond its BP-lowering effect. The correlation analysis revealed that there was a significant negative correlation between HOMA-IR and PPARy2mRNA expression in VAT, but not in SAT. By contrast, PPARγ1mRNA expression was not dependent on HOMA-IR in SAT or in VAT adipose tissue.Conclusion:1. OLETF rats fed with long-time HFD were diagnosed with hyperglycemia, mild obesity, glucose and lipid metabolic disorder and developed insulin resistance, it suggested that the animal model was established successfully.2. ARB telmisartan can ameliorate visceral fat and glucolipid metabolism, reduced the incidence of T2DM. Telmisartan improved insulin resistance, which may be correlated with its ameliorating the expression of adipocytokines in serum and adipose tissues, especially in VAT, but beyond its BP-lowering effect.3. Telmisartan can regulate the expressions of adipocytokines profile, including adiponectin, leptin, IL-6, TNF-a and MCP-1, and improve the inflammatory microenvironment in adipose tissue.4. Telmisartan has stronger effects on VAT in regulating the expression of adipocytokines, while pioglitazone shows the stronger effects in SAT. This might due to the depot-specific in distribution of angiotensin type1receptors and PPARys.5. Telmisartan can ameliorate inflammatory state in adipose tissue, which is related to the double effects of ARB/PPARy ligand, there have two pathways involved: telmisartan can inhibit the biological effect of Angll by blocking the ATIR and improve the insulin resistance activated by RAS. In addition, telmisartan can also regulate the inflammatory markers involved in glucolipid metabolism, downsize the adipose cell size, improve insulin sensitivity and reduce the incidence of T2DM through activating PPAR-y partially. |
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