AMP-activated protein kinase (AMPK) senses energetic stress and, in turn, promotes catabolic and suppresses anabolic metabolism coordinately to restore energy balance. We found that a diverse array of AMPK activators increased mTOR complex 2 (mTORC2) signaling in an AMPK-dependent manner in cultured cells. Activation of AMPK with the type 2 diabetes drug metformin (GlucoPhage) also increased mTORC2 signaling in liver in vivo and in primary hepatocytes in an AMPK-dependent manner. AMPK-mediated activation of mTORC2 did not result from AMPK-mediated suppression of mTORC1 and thus reduced negative feedback on PI3K flux. Rather, AMPK associated with and directly phosphorylated mTORC2 (mTOR in complex with rictor). As determined by two-stage in vitro kinase assay, phosphorylation of mTORC2 by recombinant AMPK was sufficient to increase mTORC2 catalytic activity toward Akt. Hence, AMPK phosphorylated mTORC2 components directly to increase mTORC2 activity and downstream signaling. Functionally, inactivation of AMPK, mTORC2, and Akt increased apoptosis during acute energetic stress. By showing that AMPK activates mTORC2 to increase cell survival, these data provide a potential mechanism for how AMPK paradoxically promotes tumorigenesis in certain contexts despite its tumor-suppressive function through inhibition of growth-promoting mTORC1. Collectively, these data unveil mTORC2 as a target of AMPK and the AMPK-mTORC2 axis as a promoter of cell survival during energetic stress.

Fibroblast growth factor 21 (FGF21) is a potent antidiabetic and triglyceride-lowering hormone whose hepatic expression is highly responsive to food intake. FGF21 induction in the adaptive response to fasting has been well studied, but the molecular mechanism responsible for feeding-induced repression remains unknown. In this study, we demonstrate a novel link between FGF21 and a key circadian output protein, E4BP4. Expression of Fgf21 displays a circadian rhythm, which peaks during the fasting phase and is anti-phase to E4bp4, which is elevated during feeding periods. E4BP4 strongly suppresses Fgf21 transcription by binding to a D-box element in the distal promoter region. Depletion of E4BP4 in synchronized Hepa1c1c-7 liver cells augments the amplitude of Fgf21 expression, and overexpression of E4BP4 represses FGF21 secretion from primary mouse hepatocytes. Mimicking feeding effects, insulin significantly increases E4BP4 expression and binding to the Fgf21 promoter through AKT activation. Thus, E4BP4 is a novel insulin-responsive repressor of FGF21 expression during circadian cycles and feeding.The mammalian circadian rhythm system plays a fundamental role in coordinating various physiological processes, which are manifested by a precise 24-h cycle and responsiveness to light or food cues (1-4). Recent genetic and biochemical studies of mammals, Drosophila, and bacteria have provided a general model of the circadian clock that is based on a transcriptional-translational feedback loop consisting of both positive and negative circadian clock proteins (1, 4). Besides controlling the core circadian oscillation loop, the clock proteins also actively participate in rhythmic expression of various output genes, which may account for the rhythmic activities in peripheral tissues (1, 3). As demonstrated in various microarray studies, genes important for gluconeogenesis, lipogenesis, and cholesterol synthesis are potential targets of clock proteins (5-8). Therefore, for drug administration and drug design, it becomes critical to understand how the cycling of individual metabolic genes is regulated in a 24-h rhythm (9, 10). E4BP4 (E4-binding protein 4), also called NFIL3, is a b-ZIP (basic leucine zipper) transcription factor initially identified as an IL-3-inducible factor in pro-B lymphocytes (11-13). The biological function of E4BP4 has been largely explored in the immune system, in which E4BP4 knock-out mice are defective in natural killer cell development and IgE class switch (14 -16). E4BP4 was first identified as a clock-controlled gene in mouse liver (17,18). Its mRNA and protein levels oscillate in a circadian fashion, which is anti-phase to DBP (D-site of albumin promoter-binding protein), another clock-controlled output gene (19). The mRNA of E4BP4 peaks at circadian time (CT) 2 0 and troughs at CT 12 (17,20). Although the role of E4BP4 in the mammalian circadian system is unclear, its homologue in Drosophila, vrille, serves as a key component of the core circadian network via a negative feedback loop (21-23). E...

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The metazoan circadian clock mechanism involves cyclic transcriptional activation and repression by proteins whose degradation is highly regulated via the ubiquitin-proteasome pathway. The heme receptor Rev-erbα, a core negative component of the circadian network, controls circadian oscillation of several clock genes, including Bmal1 Rev-erbα protein degradation can be triggered by inhibitors of glycogen synthase kinase 3β, such as lithium, and also by serum shock, which synchronizes circadian rhythms in cultured cells. Here we report that two E3 ligases, Arf-bp1 and Pam (Myc-bp2), are copurified with Rev-erbα and required for its ubiquitination. RNAinterference-mediated depletion of Arf-bp1 and Pam stabilizes the Rev-erbα protein and protects Rev-erbα from degradation triggered by either lithium or serum shock treatment. This degradation pathway modulates the expression of Rev-erbα-regulated Clock gene and circadian function in mouse hepatoma cells. Thus, Arf-bp1 and Pam are novel regulators of circadian gene expression that target Rev-erbα for degradation.ubiquitin | circadian rhythm T he physiology and behavior of mammals are subject to daily oscillation driven by endogenous circadian rhythm systems (1-3). In mammals, the core circadian network is generated and maintained via a tightly regulated transcriptional-translational feedback loop (4-6). BMAL1 and CLOCK, the positive components of the circadian clock, activate many circadian output genes, as well as the negative limbs of the circadian loops (2, 7-9). The core negative limb is constituted by the PERIOD (PER) and CRYPTOCHROME (CRY) proteins, which bind to and inhibit BMAL1/CLOCK (4). The other negative limb is mediated by the nuclear receptor Rev-erbα (10), which directly represses Bmal1 gene expression and plays a crucial role in circadian oscillation of Bmal1 mRNA (11).Emerging evidence has demonstrated that the negative components of the circadian clock are subjected to ubiquitination and subsequent proteasome-mediated degradation (12, 13). For example, the F-box protein FBXL3 regulates the stability of CRY proteins (14-16), whereas another F-box protein containing E3 ligase SCF-βTrcp1 targets phosphorylated PER2 protein for degradation in both Drosophila and mammals (17)(18)(19)(20). During a normal circadian cycle, hepatic Rev-erbα protein peaks during the circadian day and becomes undetectable during the circadian night (10). Similar to CRY and PER proteins, Rev-erbα proteins also undergo ubiquitination and proteasome degradation (21), but the mechanism controlling this temporal rhythm of Rev-erbα protein remains unknown.Lithium, a common treatment for bipolar disorder, can lengthen the period of circadian rhythm in a wide range of experimental systems including insects, mice, and humans (22,23). In vitro studies suggest that lithium might target multiple clock proteins via its inhibition of GSK3β (21, 24), although whether GSK3β is the sole circadian target of lithium remains unclear. We previously reported that Rev-erbα degradation is accelerate...

Self-renewing embryonic stem cells (ESCs) respond to environmental cues by exiting pluripotency or entering a quiescent state. The molecular basis underlying this fate choice remains unclear. Here, we show that histone acetyltransferase MOF plays a critical role in this process through directly activating fatty acid oxidation (FAO) in the ground-state ESCs. We further show that the ground-state ESCs particularly rely on elevated FAO for oxidative phosphorylation (OXPHOS) and energy production. Mof deletion or FAO inhibition induces bona fide quiescent ground-state ESCs with an intact core pluripotency network and transcriptome signatures akin to the diapaused epiblasts in vivo. Mechanistically, MOF/FAO inhibition acts through reducing mitochondrial respiration (i.e., OXPHOS), which in turn triggers reversible pluripotent quiescence specifically in the ground-state ESCs. The inhibition of FAO/OXPHOS also induces quiescence in naive human ESCs. Our study suggests a general function of the MOF/FAO/OXPHOS axis in regulating cell fate determination in stem cells.