Identification of insulin-sensitizing molecules acting by disrupting the interaction between the Insulin Receptor and Grb14.

Metabolic diseases are characterized by a decreased action of insulin. During the course of the disease, usual treatments frequently fail and patients are finally submitted to insulinotherapy. There is thus a need for innovative therapeutic strategies to improve insulin action. Growth factor receptor-bound protein 14 (Grb14) is a molecular adapter that specifically binds to the activated insulin receptor (IR) and inhibits its tyrosine kinase activity. Molecules disrupting Grb14-IR binding are therefore potential insulin-sensitizing agents. We used Structure-Based Virtual Ligand Screening to generate a list of 1000 molecules predicted to hinder Grb14-IR binding. Using an acellular bioluminescence resonance energy transfer (BRET) assay, we identified, out of these 1000 molecules, 3 compounds that inhibited Grb14-IR interaction. Their inhibitory effect on insulin-induced Grb14-IR interaction was confirmed in co-immunoprecipitation experiments. The more efficient molecule (C8) was further characterized. C8 increased downstream Ras-Raf and PI3-kinase insulin signaling, as shown by BRET experiments in living cells. Moreover, C8 regulated the expression of insulin target genes in mouse primary hepatocytes. These results indicate that C8, by reducing Grb14-IR interaction, increases insulin signalling. The use of C8 as a lead compound should allow for the development of new molecules of potential therapeutic interest for the treatment of diabetes.

[Control of insulin signalisation and action by the Grb14 protein]. / Contrôle de la signalisation et de l'action de l'insuline par la protéine Grb14.

The action of insulin on metabolism and cell growth is mediated by a specific receptor tyrosine kinase, which, through phosphorylation of several substrates, triggers the activation of two major signaling pathways, the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway and the Ras/extracellular signal-regulated kinase (ERK) pathway. Insulin-induced activation of the receptor and downstream signaling is also subjected to a negative feedback control involving several mechanisms, among which the interaction of the insulin receptor and its substrates with inhibitory proteins. After summarizing the major mechanisms underlying the activation and attenuation of insulin signaling, this review focuses on its control by the Grb14 adaptor protein. Grb14 has been identif-ied as an inhibitor of insulin signaling and action, and is involved in insulin resistance associated with type 2 diabetes and obesity. Studies on the molecular mechanism of action of Grb14 have shown that, through interaction with the activated insulin receptor, Grb14 inhibits its catalytic activity and the activation of downstream signaling. However, the consequences of Grb14 gene invalidation are complex and tissue-specific, and some effects of Grb14 on insulin signaling appear to be linked to its interaction with effector proteins downstream the insulin receptor. Pharmacological inhibition of Grb14 should allow to enhance insulin sensitivity and improve energy homeostasis in insulin-resistant states.