Rapamycin in 3T3-L1 preadipocytes function and mechanism of
|School||Beijing Union Medical College|
|Course||Medical kidney disease learn|
|Keywords||Rapamycin 3T3-L1 cells Cholesterol Leptin PPARу TZD|
The background is a pleiotropic drug rapamycin, immunosuppressive, anti-tumor, anti-proliferative and anti-migration in recent years proved to be still in the treatment of pulmonary lymphatic muscle, except in transplantation immunology and eluting stents fibromatosis and tuberous sclerosis (LAM / TSC), kidney disease polycystic kidney disease, improve cognitive function, there is an apparent effect. However, the performance of both sides of the lipid metabolism has been a contradiction of rapamycin. Rapamycin proved to be good anti-atherosclerotic effect in experimental animal models, but at the same time, hyperlipidemia has been one of the major side effects of rapamycin. As we all know, hyperlipidemia is an independent risk factor for cardiovascular disease. The adipose tissue is the body's largest lipid storage organ, called \In addition, adipose tissue endocrine organ, the secretion of leptin, adiponectin hormone on body weight, blood lipids and lipid homeostasis play an important role. The study found that the use of rapamycin can cause a decrease in weight of the fat cells and storage lipid reduced fat cell dysfunction. PPARy is a key factor of fat cell differentiation and maturation of that expression before most of the fat cell gene transcriptional activation during adipocyte differentiation by positive feedback regulation of PPARy expression levels rising and reached the highest level, to the formation of mature fat cells. PPARγ the spoon activation can ultimately lead to the the adipose differentiation final gene activation. Rapamycin inhibits the generation of lipid and fat differentiation, causing fat cell dysfunction by inhibiting PPARy? This study concept Chalei Pa neomycin differentiation of 3T3-L1 preadipocytes and lipid homeostasis, and explore its Possible mechanisms aimed hyperlipidemia caused by rapamycin provide a possible solution. Purpose of rapamycin lipid homeostasis and secretory function in the differentiation of 3T3-L1 preadipocytes of the PPARy the impact. 3T3-L1 preadipocytes as experimental subjects, enzyme-linked immunosorbent oil red O staining and high performance liquid chromatography analysis (HPLC) qualitative and quantitative observation of lipid content in different concentrations of rapamycin cells; assay (ELISA) analysis of the effect of different concentrations of rapamycin cell secretion of adiponectin and leptin levels; Real-time PCR method to detect the effect of different concentrations of rapamycin 3T3-L1 cells PPARy expression impact: HPLC, ELISA, real-time PCR and Western blot method troglitazone rapamycin inhibited 3T3-L1 cell differentiation and secretion effect reversal. Results. Differentiation began four days plus with different concentrations of rapamycin, differentiation day 8, the cells were harvested oil red O staining can be seen at 50-200 nmol / L concentration range can reduce intracellular lipid droplets of accumulation. 2. HPLC quantitative determination the different differentiation period plus rapamycin obvious impact of differences in accumulation of intracellular cholesterol, different concentrations of class Pa neomycin dose-dependent manner to reduce accumulation of intracellular free cholesterol control, 50 , 100,200 nmol / L concentration of rapamycin FC content of intracellular ADR group were 12.89 ± 0.16,9.84 ± 0.45,9.39 ± 0.46,8.61 ± 0.34, rapamycin-treated cells within the FC concentration with the control group the difference was statistically significant (P lt; 0.05). Rapamycin, 50, 100, 200 nmol / L are so mature 3T3-L1 adipocytes cells secrete leptin levels decreased (P lt; 0.05), Determination of groups adiponectin in no significant difference (P gt ; 0.05). Role in 3T3-L1 cells 96 hours in the differentiation of the eight days cells were harvested, 50nmol / L, 100nmol / L and 200nmol / L group PPARy mRNA expression amount were in the control group 0.93 ± 0.14,0.62 ± 0.10,0.47 ± 0.19 times, the latter two groups were higher than those of the control group decreased (P lt; 0.05), in addition to the group and the comparison between groups was no significant difference between 50nmol / L and 100nmol / L, the other groups were statistically different. PPARγ protein expression levels in the corresponding group for the control group, 0.80 ± 0.12,0.74 ± 0.11,0.61 ± 0.10-fold, respectively, the latter two groups than in the control group decreased, with a significant difference (P lt; 0.05) between groups. 5. Rapamycin 100 nmol / L, the PPARy blockers GW966210μmol / L, PPARy sensitizing agent troglitazone (, HER-2/neu overexpressing gastric and colon, TZD) 10μmol / L, respectively, cells were treated 96 hours, cell differentiation day 8 PPARγmRNA expression for the control group, 0.67 ± 0.03,1.3 ± 0.14 compared with the control group, a statistically significant difference (P lt; 0.05). PPARγ protein corresponding cells for the control group, 0.57 ± 0.23,1.91 ± 0.12 respectively, compared with the control group, a statistically significant difference (P lt; 0.05). Control group, PPARγ blockers GW9662 10μmol / L of PPARγ sensitizer troglitazone (, HER-2/neu overexpressing gastric and colon, TZD) 10μmol / L, respectively, treated cells 96 hours total cholesterol HPLC measured in each group were 12.91 ± 0.5,11.88 ± 0.55,13.27 ± 0.71mg/mL the GW9662 group of intracellular free cholesterol less than the control group, TZD group total cholesterol is less than the control group (P lt; 0.05). By ELISA respective treatment groups, the amount of leptin changes were 19.02 ± 0.52,15.62 ± 0.47,17.45 ± 0.51ng/mLGW9662 group compared with the control group was statistically significant (P lt; 0.05). 7.TZD can the reversal rapamycin inhibition of 3T3-L1 adipocytes cell secretory function rapa100nmol / L TZD10μmol / L group of total and free cholesterol alcohol values ??were 11.28 ± 0.56,10.23 0.33 mg / mL, free cholesterol values ??rapa100nmol / L TZD10μmol / L group compared with rapa100nmol / L higher (P lt; 0.05). Control group rapa100nmol / L, rapa100nmol / L TZD10μmol / L treatment group intracellular leptin levels were 19.02 ± 0.52,15.62 ± 0.47,17.45 ± 0.51ng/mLrapa100nmol/L TZD10μmol / L leptin levels than rapa100nmol / L high, a significant difference (P lt; 0.05) between groups. Rapamycin effect with TZD intervention control group value standardized rapa100nmol / L rapa100nmol / L TZD10μmol / L group PPARy mRNA expression levels were 0.60 ± 0.141.12 ± 0.27, with the control compared between groups were statistically different (P lt; 0.05), a where, rapa100nmol / L TZD10μmol / L group PPARy expression levels significantly higher than rapa100nmol / L group (P lt; 0.05). PPARγ protein expression levels for the control group, 0.74 ± 0.11,1.37 ± 0.52, respectively, and the two groups and the control group, there were significant differences (P lt; 0.05), which rapa100nmol / L TZD10μmol / L group PPARy expression was significantly higher than rapa 100nmol / L group. Conclusion In the present experimental conditions: 1, rapamycin can reduce the 3T3-L1 preadipocytes differentiation and maturation of lipid accumulation and leptin secretion, and may inhibit PPARy expression. 2, troglitazone reversed the inhibitory effect of rapamycin in 3T3-L1 preadipocytes differentiation and maturation of lipid accumulation and secretion of leptin.