In vivo differential effects of fasting, re-feeding, insulin and insulin stimulation time course on insulin signaling pathway components in peripheral tissues

Background: Components of the insulin receptor signaling pathway are probably some of the best studied ones. Even though methods for studying these components are well established, the in vivo effects of different fasting regimens, and the time course of insulin receptor phosphorylation and that of its downstream components in insulin-sensitive peripheral tissues have not been analyzed in detail. Rationale: When assessing insulin signaling, it may be beneficial to drive insulin levels as low as possible by performing an overnight fast before injecting a supra-physiological dose of insulin. Recent studies have shown however that 5 or 6 h fast in mice is sufficient to assess physiological responses to insulin and/or glucose in glucose tolerance tests [1], insulin tolerance tests [2], [3] and [4] and euglycemic hyperinsulinemic clamp studies [5] and [6]. Moreover, mice are nocturnal feeders, with ∼70% of their daily caloric intake occurring during the dark cycle [5], and their metabolic rate is much higher than humans. Therefore, an overnight fast in mice is closer to starvation than just food withdrawal. Thus our aim was to assess insulin signaling components from the insulin receptor to downstream targets IRS1, Akt/PKB, GSK3, Erk1/2 and ribosomal protein S6 in muscle, liver and adipose tissue in 5 h versus 16 h (overnight) fasted mice, and the time course (0–30 min) of these phosphorylation events. We also assessed whether re-feeding under 5 h and 16 h fasting conditions was a more robust stimulus than insulin alone. Conclusions: Our study determines that a short food withdrawal from mice, for a period of 5 h, results in a similar insulin-stimulated response in phosphorylation events as the long overnight fast, presenting a more physiological experimental set up. We also demonstrate that in vivo, insulin-stimulated phosphorylation of its signaling components is different between different peripheral tissues, and depending on the tissue(s) and protein(s) of interest, an appropriate time course should be chosen.

Research highlights
► We determine that short, 5 h fasting of mice results in similar insulin-stimulated response in phosphorylation events as long overnight fast.
► We demonstrate that in adipose tissue only, Akt/PKB phosphorylation is regulated by re-feeding, and not in muscle or liver.
► We demonstrate that in vivo, the time course of insulin-stimulated phosphorylation events is divergent in different insulin-sensitive tissues.
► Insulin receptor phosphorylation in muscle, liver and adipose peaks by 15 min post-insulin stimulation.
► Insulin signaling post-IR (i.e. IRS1, Akt/PKB, GSK3, Erk1/2 and S6) exhibits completely different time courses in different tissues.