Objective: To explore effects and potential mechanisms of high insulin environment on high density lipoprotein (HDL) generation-related functional protein ABCA1.
Methods: [(3)H] labeled cholesterol efflux from mature 3T3-L1 adipocytes was detected by liquid scintillation counting. ABCA1 mRNA and protein expression in mature 3T3-L1 adipocytes post stimulation with various concentrations of insulin was detected by real-time fluorescence-based quantitative techniques and Western blot, respectively, in the absence and presence of CHX (cycloheximide, CHX), calpeptin (calpain pathway inhibitor) or MG-132 (proteasome pathway inhibitor).
Results: Cholesterol efflux rates were reduced post insulin stimulation in a dose-dependent manner ((7.06 ± 0.27)%, (6.59 ± 0.30)%, (6.34 ± 0.24)%, (5.07 ± 0.40)%, and (4.71 ± 0.40)% at 0, 1, 10, 10², and 10³ nmol/L of insulin, P < 0.05). Cholesterol efflux rates decreased in a time-dependent manner post 10³ nmol/L insulin stimulation (6.52 ± 0.30)%, (5.59 ± 0.71)%, (5.44 ± 0.37)%, (4.52 ± 0.32)%, and (4.38 ± 0.33)% at 0, 2, 4, 6, 12 h, respectively). ABCA1mRNA levels were not affected by insulin (P > 0.05). ABCA1 protein level was significantly downregulated in 10³ nmol/L insulin group compared to 0 nmol/L insulin group (P < 0.01). Compared with the 0 h group, ABCA1 protein level was significantly reduced in 6 h group (P < 0.05) and further reduced in 12 h group (P < 0.01). Both calpeptin and MG-132 could partly reduce insulin-induced degradation of ABCA1. Compared with the negative control group, ABCA1 protein levels were significantly upregulated by cotreatment with calpeptin and MG-132, respectively (both P < 0.01).
Conclusion: Our data suggest that high insulin level could promote the ABCA1 protein degradation and reduce cholesterol efflux from mature 3T3-L1 adipocytes through calpain and proteasome pathway, thus, produce a circumference not suitable for nascent HDL formation in 3T3-L1 adipocytes.