The circadian clock coordinates physiology and metabolism. mTOR (mammalian/mechanistic target of rapamycin) is a major intracellular sensor that integrates nutrient and energy status to regulate protein synthesis, metabolism, and cell growth. Previous studies have identified a key role for mTOR in regulating photic entrainment and synchrony of the central circadian clock in the suprachiasmatic nucleus (SCN). Given that mTOR activities exhibit robust circadian oscillations in a variety of tissues and cells including the SCN, here we continued to investigate the role of mTOR in orchestrating autonomous clock functions in central and peripheral circadian oscillators. Using a combination of genetic and pharmacological approaches we show that mTOR regulates intrinsic clock properties including period and amplitude. In peripheral clock models of hepatocytes and adipocytes, mTOR inhibition lengthens period and dampens amplitude, whereas mTOR activation shortens period and augments amplitude. Constitutive activation of mTOR in Tsc2–/–fibroblasts elevates levels of core clock proteins, including CRY1, BMAL1 and CLOCK. Serum stimulation induces CRY1 upregulation in fibroblasts in an mTOR-dependent but Bmal1- and Period-independent manner. Consistent with results from cellular clock models, mTOR perturbation also regulates period and amplitude in the ex vivo SCN and liver clocks. Further, mTOR heterozygous mice show lengthened circadian period of locomotor activity in both constant darkness and constant light. Together, these results support a significant role for mTOR in circadian timekeeping and in linking metabolic states to circadian clock functions.

In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed 1,2 ). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere 1,2 . Individual cells are the functional units for generation and maintenance of circadian rhythms 3,4 , and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous [5][6][7] . Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects 5,8 . Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms 5,[8][9][10][11][12][13] . Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals 14,15 , as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection 13,16,17 or stable transduction 5,10,18,19 . Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells 20 . Once a reporter cell line is established, the dynamics of clock function can be examined through bioluminescence recording. We first describe the generation of P(Per2)-dLuc reporter lines, and then present data from this and other circadian reporters. In these assays, 3T3 mouse fibroblasts and U2OS human osteosarcoma cells are used as cellular models. We also discuss various ways of using these clock models in circadian studies. Methods described here can be applied to a great variety of cell types to study the cellular and molecular basis of circadian clocks, and may prove useful in tackling problems in other biological systems. Video LinkThe video component of this article can be found at http://www.jove.com/video/4234/ Protocol Construction of Lentiviral Luciferase ReportersA mammalian circadian reporter construct usually contains an expression cassette in which a circadian promoter is fused with the lucifer...

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In mammals, many aspects of behavior and physiology such as sleep-wake cycles and liver metabolism are regulated by endogenous circadian clocks (reviewed 1,2). The circadian time-keeping system is a hierarchical multi-oscillator network, with the central clock located in the suprachiasmatic nucleus (SCN) synchronizing and coordinating extra-SCN and peripheral clocks elsewhere 1,2 . Individual cells are the functional units for generation and maintenance of circadian rhythms 3,4, and these oscillators of different tissue types in the organism share a remarkably similar biochemical negative feedback mechanism. However, due to interactions at the neuronal network level in the SCN and through rhythmic, systemic cues at the organismal level, circadian rhythms at the organismal level are not necessarily cell-autonomous 5-7 . Compared to traditional studies of locomotor activity in vivo and SCN explants ex vivo, cell-based in vitro assays allow for discovery of cell-autonomous circadian defects 5,8 . Strategically, cell-based models are more experimentally tractable for phenotypic characterization and rapid discovery of basic clock mechanisms 5,8-13 . Because circadian rhythms are dynamic, longitudinal measurements with high temporal resolution are needed to assess clock function. In recent years, real-time bioluminescence recording using firefly luciferase as a reporter has become a common technique for studying circadian rhythms in mammals 14,15, as it allows for examination of the persistence and dynamics of molecular rhythms. To monitor cell-autonomous circadian rhythms of gene expression, luciferase reporters can be introduced into cells via transient transfection 13,16,17 or stable transduction 5,10,18,19 . Here we describe a stable transduction protocol using lentivirus-mediated gene delivery. The lentiviral vector system is superior to traditional methods such as transient transfection and germline transmission because of its efficiency and versatility: it permits efficient delivery and stable integration into the host genome of both dividing and non-dividing cells 20