Endocrine Abstracts

JOINT1382

Objectives: The mechanisms behind the increased height velocity and accelerated epiphyseal growth plate maturation since early childhood aren’t well-defined.Hyperinsulinemia has been implicated as one of the causative factors.The aim of this study is to explore the mechanisms by which insulin regulates the expression of local aromatase in the growth plate,thereby leading to accelerated epiphyseal closure.

Methods: Culturing and identification of primary chondrocytes from the growth plate of 20 - day - old Sprague - Dawley rats were performed.The CCK8 assay procedure was utilized to identify the optimal concentrations of leptin antagonist and PI3K inhibitor. After culturing primary chondrocytes for 4 days and subjecting them to serum starvation for 24 hours,the leptin antagonist and insulin were sequentially added. qPCR and Western blot were employed to analyze the effects of different action durations and concentrations of insulin on the mRNA and protein expression of CYP19A1. This step was to determine the optimal action time and concentration of insulin for subsequent signal pathway studies. The phosphorylation of AKT in chondrocytes was analyzed by Western blot, and the changes in AKT phosphorylation, CYP19A1 mRNA and protein levels following the addition of a PI3K inhibitor were examined. After the construction, the four recombinant plasmids (pGL3.0-CYP19A1-I.3, pGL3.0-CYP19A1-II, pGL3.0-CYP19A1-I.4, and pGL3.0-CYP19A1-I.6) containing different lengths ofthe aromatase promoter fragments were co-transfected with the the dual-luciferase reporter system (firefly luciferase and Renilla luciferase) into the target cells. The activity of each promoter was quantitatively evaluated by the Dual-Luciferase Reporter Assay Kit.

Results: qPCR and Western blot analysis showed that 12-hour treatment with 20 nmol/l insulin maximally upregulated CYP19A1 mRNA and protein expression in a dose and time dependent manner (P<0.01).Western Blot was employed to detect the phosphorylation of AKT at specific time points,namely 1, 5, 15, 30, and 60 minutes. The results indicated that AKT phosphorylation reached its peak at 30 minutes.In the PI3K inhibitor group, the degree of AKT phosphorylation was significantly decreased at 30 minutes (P<0.01). After insulin (20 nmol/l) acted for 12 hours, the expression of CYP19A1 mRNA and protein in the PI3K inhibitor group was significantly reduced (P<0.01). Luciferase reporter gene analysis showed that after 12 hours of incubation, insulin significantly increased the activity of aromatase promoters I.4/I.6.

Conclusions: Insulin not only promotes the expression of aromatase within growth plate chondrocytes through the PI3K/AKT signal transduction pathway, but also significantly upregulates the activity of aromatase promoters I.4/I.6.