Circadian clock and muscle wasting associated with impaired insulin signalling: in vitro effect of hydrocortisone circadian administrations.

M. Negri^a (Dr), C. Pivonello*^a (Dr), F. Amatrudo^a (Dr), R. Patalano^a (Dr), T. Montò^a (Dr), C. Simeoli^a (Dr), C. De Angelis^a (Dr), V. Hasenmajer^b (Dr), M. Minnetti^b (Dr), RS. Auriemma^a (Dr), A. Colao^c (Prof), AM. Isidori^b (Prof), R. Pivonello^c (Prof)

^a Dipartimento di Medicina Clinica e Chirurgia, Università Federico II, Naples, ITALY ; ^b Department of Experimental Medicine, Sapienza University of Rome, Rome, ITALY ; ^c Dipartimento di Medicina Clinica e Chirurgia, Università Federico II; UNESCO Chair for Health Education and Sustainable Development, Federico II University, Naples, ITALY

* cpivonello@gmail.com

Introduction: Transcriptional activator Bmal1, reaching the acrophase in the morning and the bathyphase in the evening, is the main component of the circadian clock. A mutual interaction between circadian clock and glucocorticoids (GCs) exists and recent evidence demonstrated the circadian control of both muscle metabolism and insulin signalling. The current in vitro study aims at evaluating GCs physiological and non-physiological circadian administrations on muscle wasting focusing on anabolic insulin signalling.

Materials and methods: mouse myocytes C2C12 were serum-shocked to synchronize clock genes oscillation. Using acrophase and bathyphase of Bmal1, in vitro hydrocortisone (HC) administrations were aligned to daytime of human cortisol peaks and nadir. During Bmal1 acrophase, cells were exposed for 1h to physiological concentration of 450 nM HC and to non-physiological concentrations of 650 and 750 nM HC. During Bmal1 bathyphase, cells were exposed for 1h to the physiological concentration of 150 nM HC and to non-physiological concentrations of 177 and 300 nM HC. HC-induced muscle wasting was evaluated by analyzing atrogenes by RT-qPCR; mTOR pathway and KLF15 proteins by western blot (WB). HC effects on insulin signalling were evaluated by analyzing insulin pathway by WB.

Results: At Bmal1 acrophase and bathyphase, no significant change in gene expression levels MuRF-1and Atrogin-1, was revealed. Conversely, 300 nM HC at Bmal1 bathyphase significantly inhibited mTORC1 protein expression (65%; p<0.01 vs 150 nM), significantly decreased pP70S6K (46%; p<0.01 vs 150 nM) and significantly stimulated KLF15 expression (50%; p<0.05 vs 150 nM). At Bmal1 bathyphase, preliminary results demonstrated that 300 nM HC combined with insulin strongly decreased pIRS-1 on Tyr608 and pAKT on Ser473 compared to 150 nM HC. Moreover, only at Bmal1 bathyphase, 300 nM HC combined with insulin strongly stimulated p110αPI3K and glucocorticoid receptor proteins expression compared to 150 nM HC.

Discussion: These data demonstrated that in Bmal1 bathyphase, non-physiological HC concentration induces muscle wasting by reducing the anabolic action of insulin signalling.

The author has declared no conflict of interest.