Identification of in vivo interactions of insulin receptor substrate 1 in murine liver

The development of obesity, insulin resistance and type 2 diabetes is highly interlinked. In the past, differential phosphorylation and protein interaction of the insulin receptor substrates (IRS) have been identified in the regulation of insulin signaling. However, the exact molecular changes on the IRS protein network that occur upon insulin resistance have yet to be determined completely. In this study, a transgenic mouse model was generated featuring a streptavidin binding peptide tag allowing for IRS1 affinity purification in the liver and the assessment of in vivo interactions of IRS1 in diet-induced obesity and insulin signaling. To this end, affinity purification of hepatic streptavidin binding peptide-tagged IRS1 was performed and subsequent mass spectrometry and label-free quantification of the results led to the identification of 809 putative IRS1 interactions. Of the interacting proteins, association of 53 was reduced on HFD in the non-insulin stimulated state. Comparing the different diets upon insulin signaling, IRS1 association of 31 proteins was increased in NCD-fed mice, while in HFD-fed mice only 18 proteins were increasingly associated with IRS1. Notably, the association of p110α and β with the IRS1-p85 complex was increased upon insulin treatment in NCD-fed mice. However, this effect was blunted on HFD, indicating a reduced insulin signaling capacity in HFD-fed mice and a contribution to the development of insulin resistance. A tendency towards increased Lyn association with IRS1 upon HFD may likely lead to the development of insulin resistance, as for Fyn, a kinase related to Lyn, a regulation of energy expenditure and fatty acid oxidation has been described previously. Moreover, 14-3-3 proteins ε, α/β, γ, η and ζ/δ increasingly interacted with IRS1 upon insulin signaling in NCD conditions, however on HFD interaction was reduced to levels seen in the non-insulin stimulated state. In this context, 14-3-3 proteins seem to serve as adaptor proteins regulating the association of kinases with IRS1, a mechanism that may affect the signaling functionality of IRS1 and thereby contribute to insulin resistance upon diet-induced obesity. IRS1 interaction with two serine/threonine kinases, protein kinase C (PKC) and salt inducible kinase 3 (QSK) was identified in this study. IRS1-PKC interaction has been described previously in connection with 14-3-3 interaction. QSK, however, is a novel interacting protein of IRS1, which has been shown to also interact with 14-3-3 proteins. These findings strongly suggest that not only PKC-, but also QSK-mediated regulation of insulin signaling at the level of IRS1 may contribute to insulin resistance.