1. 4 / EFFECT OF INSULIN IS VIA PI3K BUT IS GLUCOSE INDEPENDENT Introduction The mammalian circadian clock is an endogenous daily rhythm in behavioural and cellular activity that is reset by appropriate physiological and environmental cues. Prominent among these is feeding time, which can reset the clock in peripheral tissue. When this occurs out of phase with other timing cues, such as light, the result is a disruption of internal timing — associated with an increased risk of pathologies such as type II diabetes and obesity 1. Previous work has suggested that the metabolic hormone insulin plays a role in food-resetting circadian rhythms in liver and adipose tissue 2, but has not been able to explain the basis of this resetting in other “non- metabolic” tissue types. Here, we show that administration of insulin at physiologically-relevant concentrations stimulates acute expression of the circadian clock gene PER2 in a range of non-metabolic cell types. Introduction Priya Crosby 1 and John S. O’Neill 1 1. MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, UK Insulin resets the circadian clock in “non-metabolic” cells via induction of clock gene per2 REFERENCES 1.Reddy, A. B. & O’Neill, J. S. Healthy clocks, healthy body, healthy mind. Trends Cell Biol (2010) 2.Sato et al., The Role of the Endocrine System in Feeding-Induced Tissue-Specific Circadian Entrainment, Cell Reports (2014) ACKNOWLEDGMENTS Many thanks to the O’Neill lab and to Michael Hastings for discussions, and to Mario de Bono and Rob Kay for equipment loans Introduction a) In the absence of serum and B27, insulin acts as a major zeitgeber for cellular time keeping and its application affects the b) phase (n=4, ±SEM) c) period (n=7, ±SD) and d) amplitude (n=4, ±SD) of the cellular circadian rhythm, as observed using PER2:: LUC bioluminescence e) The magnitude of this effect is dose dependent, but is evident even with insulin concentrations as low as 1nM (n=6, ±SEM) e Introduction mTORC 1 LY29400 2 UO126 glucose starvatio n rapamyci n GLUT4 vesicle PIP 3 IRS-1 MEK 1/2 PI3K PKB PKC p70S6 K eIF4E mPer2 PER2 IRS mTORC 1 ? insulin receptor d c Introduction mPer 2 Lu c LuciferasePER 2 + Luciferin LIGHT bmal1/cloc k BMAL1/CL OCK Luciferase ba cd b a c INTRODUCTION 3 / ACUTE AND LONG TERM INSULIN APPLICATION AFFECTS THE CIRCADIAN CLOCK IN ‘NON- METABOLIC’ CELL TYPES a) Schematic of the mammalian cycle of core clock gene expression and the use of the PERIOD2:: LUCIFERASE reporter with this system to provide a real-time report of cellular circadian state b) An acute (3hr) bolus of insulin (600nM) induces a phase-shift in PER2 expression in perfused immortalised fibroblasts (representative, n=3). This acute induction in PER2 and subsequent phase shift is also observed in c) neuronal cultures (n=8, ±SEM) but d) is not observed in fibroblast that express luciferase constitutively (NIH 3T3 SV40::LUC, n=3, ±SEM) 1 / INSULIN IMPACTS ON ALL ASPECTS OF CELLULAR CIRCADIAN TIMEKEEPING 2 / INSULIN INDUCES PRODUCTION OF NASCENT PER2 Schematic highlighting components of the insulin signalling pathway implicated in the induction of PERIOD2 protein as a result of extracellular insulin application Introduction a)PER2 induction and subsequent phase-shift occurs in the absence of extracellular glucose (representative, n=3) b) PER2 induction is attenuated in the presence PI3K inhibitor LY294002 (n=4, ±SEM) and c) in the presence of mTOR inhibitor rapamycin but not in the presence of MEK1/2 inhibitor UO126 (n=4, ±SEM) ab Introduction c Induction of PER2 by insulin is sufficient to modulate key parameters of the cellular clock This effect is dose-dependent and is independent of prior circadian phase. These effects are independent of glucose availability, but are reliant on PI3K and its downstream components. Our data suggest that insulin alone may be sufficient to account for entrainment of mammalian circadian rhythms by feeding time CONCLUSIO N