Effects Of C-Reactive Protein On Total And Hmw Adiponectin In 3T3-L1 Cells And The Mechanisms Involved In

ObjectiveTo investigate the effects of CRP on the expressions of total adiponectin and different adiponectin oligomers. To elucidate the mechanisms involved in CRP to adiponectin.Methods1. The fully differentiated 3T3-L1 adipocytes were collected with CRP treatment. Real-time PCR was used to detect the expression of total adiponectin mRNA expression of 3T3-L1 adipocytes. Western blot was employed for the detection of total and HMW adiponectin protein expression.2. Immunofluorescence measured the inhibition of CRP on adiponectin.3. Western bolt was used to find the changes of total adiponectin and different oligomers after CRP treatment. The ratios of HMW/total adiponectin were calculated. The Erp44, Ero1-α and DsbA-L mRNA expression were examined by real-time PCR.4. In fully differentiated 3T3-L1 adipocytes, CRP, the PI3 K inhibitor LY294002, and phosphorylation of Akt inhibitor T3830 were added respectively. The mRNA and protein expressions of total adiponectin and HMW adiponectin were detected by real-time PCR and Western blot.Results1. In mature adipocytes, after 24 h CRP treatment, 25μg/mL CRP and 50μg/m L CRP resulted in a substantial reduction of adiponectin while 5μg/mL CRP showed no change. CRP reduced adiponectin expression in a dose-dependent manner. With 50μg/mL CRP added, 12 h and 24 h CRP treatment inhibited the expression of adiponectin, but remained nochange with 5μg/mL CRP treatment. CRP inhibited adiponectin expression in a time-dependent manner.2. Immunofluorescence illustrated that 50μg/m L CRP inhibited adiponectin expression in 3T3-L1 adipocytes.3. Western bolt showed that 5μg/m L CRP did not downregulate HMW adiponectin protein expression, 25μg/m L CRP inhibited HMW adiponectin expression while 50μg/m L CRP showed a more dramatic reduction after 24 h treatment. CRP inhibited HMW adiponectin expression in a dose-dependent manner. 50μg/mL CRP decreased HMW adiponectin protein expression for 12 h treatment, and HMW adiponectin dropped more after 24 h treatment. CRP decreased HMW adiponectin expression in a time-dependent manner.4. Western blot analyses showed that after 50μg/m L CRP for 24 h, total, MMW and HMW adiponectin decreased while HMW dropped the most. LMW showed no change. Ratios of HMW/total adiponectin also decreased. Real-time PCR illustrated that the mRNA expressions of adiponectin relative HMW assembly regulators Erp44 increased, meanwhile, Ero1-α and DsbA-L decreased.5. After 3T3-L1 adipocytes were fully differentiated, PI3 K inhibitor LY294002 and the inhibitor T3830 which suppresses the phosphorylation of Akt partially abolished the CRP impact on adiponectin.Conclusion:1. CRP inhibited total and HMW adiponectin expression in a time- and dose-dependent manner.2. CRP inhibited HMW more than other olimgers, and changed adiponectin assembly regulators.3. PI3K-Akt might be involved in CRP mediated inhibition of adiponectin expression.