Surface-expressed insulin receptors as well as IGF-I receptors both contribute to the mitogenic effects of human insulin and its analogues.

There is a medical need for new insulin analogues. Yet, molecular alterations to the insulin molecule can theoretically result in analogues with carcinogenic effects. Preclinical carcinogenicity risk assessment for insulin analogues rests to a large extent on mitogenicity assays in cell lines.

Comparison of intracellular signalling by insulin and the hypermitogenic AspB10 analogue in MCF-7 breast adenocarcinoma cells.

We compared mitogenicity and intracellular signalling by human insulin and the AspB10 (X-10) human insulin analogue in MCF-7 human mammary adenocarcinoma cells. By flow analysis of phosphorylated histone H3 or cell cycle distributions, insulin and X-10 were mitogenic at physiologically relevant concentrations (2 nm to 74 pm range), with X-10 being approximately 3-fold more mitogenic than insulin. By western blotting with phospho-specific antibodies, insulin induced phosphorylation of IRS-1, Akt, p70S6K, S6 ribosomal protein, 4E-BP1, FoxO3a, FoxO1, p44/42 MAPK and the EGFR.

Synchronization in G0/G1 enhances the mitogenic response of cells overexpressing the human insulin receptor A isoform to insulin.

Evaluating mitogenic signaling specifically through the human insulin receptor (IR) is relevant for the preclinical safety assessment of developmental insulin analogs. It is known that overexpression of IR sensitizes cells to the mitogenic effects of insulin, but it is essentially unknown how mitogenic responses can be optimized to allow practical use of such recombinant cell lines for preclinical safety testing. We constitutively overexpressed the short isoform of the human insulin receptor (hIR-A, exon 11-negative) in L6 rat skeletal myoblasts.