Endocrine Abstracts

Introduction: 3-M syndrome is associated with mutations in CUL7, OBSL1 and CCDC8 with the three proteins interacting within a novel growth pathway. The impact of this pathway on cellular growth has not been fully defined. We have shown that i) GH and IGF1 signalling are altered; ii) IGF2 expression is reduced and iii) expression of insulin receptor isoforms are altered in 3-M fibroblasts.

Aim: To characterise the activation of AKT by IGF2 and insulin in 3-M fibroblasts.

Methods: Fibroblast cells from 3-M patients with each of the three 3-M mutations and from normal controls were stimulated with 500 ng/ml IGF2 or 100 ng/ml insulin over 0, 5, 15 and 60 min. Relative phosphorylation of AKT was assessed by western blotting and image analysis. Statistical comparisons were made both within and between cell lines.

Results: Relative pAKT was reduced in 3-M fibroblasts compared to normal cells when stimulated with either IGF2 (P=0.033) or insulin (P=0.048) over the time course. In both cases, CUL7^−/− cells and OBSL1^−/− cells had a greater reduction in pAKT than CCDC8^−/− cells. CUL7^−/− cells had an earlier activation in response to insulin (P=0.016). Previous data after IGF1 stimulation showed CUL7^−/− cells had 10% of normal AKT activation while both OBSL1^−/− and CCDC8^−/− were ~50% of normal. However AKT activation was less impaired after IGF2 or insulin: for IGF2, pAKT at 60 min was reduced to 70–80% of normal in all 3-M cells: for insulin, pAKT at 60 min was reduced to 65–70% of normal for CUL7^−/− and OBSL1^−/− and to 90% for CCDC8^−/−.

Conclusions: Reduced AKT activation in response to IGF2 and insulin stimulation mirrors that previously seen although the reduction is less than IGF1. Impaired growth factor signalling is one mechanism that could account for the growth failure in 3-M and may contribute to insulin resistance in SGA children.