UBC Theses and Dissertations
Modulation of muscle cell insulin receptor signalling, transcription and trafficking by insulin Cen, Haoning
Hyperinsulinemia is commonly viewed as a compensatory response to insulin resistance, yet studies have suggested that chronically elevated insulin may also drive insulin resistance. The molecular mechanisms underpinning this potentially cyclic process remain poorly defined. Particularly, the regulation of insulin receptor (INSR) mRNA levels, protein abundance, cell-surface dynamics, and internalization in the presence and absence of insulin are incompletely understood in muscle cells. To study the direct effects of insulin on INSR in muscle cells, we conducted in vitro studies on C2C12 myotubes and myoblasts, and analyzed publicly available human muscle transcriptomic data. Our in vitro studies established that acute AKT and ERK signalling were attenuated by 16 hours of hyperinsulinemia. RNA-sequencing of cells both before and after nutrient withdrawal highlighted genes in the insulin receptor (INSR) signalling, FOXO signalling, and glucose metabolism pathways indicative of ‘hyperinsulinemia’ and ‘starvation’ programs. We observed that hyperinsulinemia led to a substantial reduction in Insr gene expression, and subsequently a reduced surface INSR and total INSR protein, both in vitro and in vivo. Transcriptomic meta-analysis in >450 human samples demonstrated a reliable negative correlation between fasting insulin and INSR mRNA in skeletal muscle. Bioinformatic modelling combined with RNAi identified SIN3A as a negative regulator of Insr mRNA and JUND, MAX, and MXI as positive regulators of Irs2 mRNA. To study INSR internalization, we used surface labelling and live-cell imaging and observed robust basal internalization of INSR and relatively modest effects of insulin in C2C12 myoblasts. We performed a stringent mass spectrophotometry analysis of INSR interactors to provide clues as to the molecular mechanisms associated with internalization, which identified previously unappreciated interactors such as ANXA2. Mapping these interactors into a protein-protein interaction network pointed to a role for caveolin-mediated endocytosis. Interestingly, INSR interacted with both caveolin and clathrin in mouse skeletal muscle and C2C12 myoblasts, with the interactions modulated by insulin. Together, this work identifies novel mechanisms which may explain the cyclic processes underlying hyperinsulinemia-induced insulin resistance in muscle, a process directly relevant to the etiology of type 2 diabetes.
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