Hyperactivation of the Insulin Signaling Pathway Improves Intracellular Proteostasis by Coordinately Up-regulating the Proteostatic Machinery in Adipocytes

Annabel Y Minard,Martin K.L Wong,Rima Chaudhuri,Shi-Xiong Tan,Sean J Humphrey,Benjamin L Parker,Jean Y Yang,D Ross Laybutt,Gregory J Cooney,Adelle C.F Coster,Jacqueline Stöckli,David E James

doi:10.1074/jbc.m116.741140

Abstract

Hyperinsulinemia, which is associated with aging and metabolic disease, may lead to defective protein homeostasis (proteostasis) due to hyperactivation of insulin-sensitive pathways such as protein synthesis. We investigated the effect of chronic hyperinsulinemia on proteostasis by generating a time-resolved map of insulin-regulated protein turnover in adipocytes using metabolic pulse-chase labeling and high resolution mass spectrometry. Hyperinsulinemia increased the synthesis of nearly half of all detected proteins and did not affect protein degradation despite suppressing autophagy. Unexpectedly, this marked elevation in protein synthesis was accompanied by enhanced protein stability and folding and not by markers of proteostasis stress such as protein carbonylation and aggregation. The improvement in proteostasis was attributed to a coordinate up-regulation of proteins in the global proteostasis network, including ribosomal, proteasomal, chaperone, and endoplasmic reticulum/mitochondrial unfolded protein response proteins. We conclude that defects associated with hyperactivation of the insulin signaling pathway are unlikely attributed to defective proteostasis because up-regulation of protein synthesis by insulin is accompanied by up-regulation of proteostatic machinery.

Highlights

The insulin signaling pathway (ISP)4 is a master regulator of protein metabolism
Our studies focus on postmitotic 3T3-L1 adipocytes because exposure of these cells to chronic hyperinsulinemia results in insulin resistance [29], and defects in insulin action in adipocytes contribute to whole body insulin resistance [30]
Mapping Large Scale Protein Synthesis and Degradation—To assess the effect of chronic insulin signaling on global proteostasis, we exposed 3T3-L1 adipocytes to insulin over 4 days and analyzed protein turnover by pulse-chase metabolic labeling and mass spectrometry (Fig. 1A)

Summary

Results

Mapping Large Scale Protein Synthesis and Degradation—To assess the effect of chronic insulin signaling on global proteostasis, we exposed 3T3-L1 adipocytes to insulin over 4 days and analyzed protein turnover by pulse-chase metabolic labeling and mass spectrometry (Fig. 1A). Insulin-treated adipocytes demonstrated a similar decrease in heavy labeled proteins as compared with untreated cells but a greater increase in medium labeled proteins (Fig. 1E) These changes correspond to increased protein synthesis with no change in protein degradation rates in response to insulin treatment. Some studies of mTORC1-mediated protein synthesis have suggested that chronic activation of mTORC1 reduces protein synthesis as either inhibitory proteostatic pathways are activated or there is increased degradation of newly made proteins due to increased misfolding (38 – 40) In contrast to this view, in insulin-stimulated 3T3-L1 adipocytes we observed that all but one of the 663 insulin-regulated proteins increased in abundance (median increase of 49% after 48 h) (Fig. 2C). This may be because cell division is associated with activation of DECEMBER 2, 2016 VOLUME 291 NUMBER 49

B Long Lived Proteins

Discussion

Experimental Procedures