CRY Links the Circadian Clock and CREB-Mediated Gluconeogenesis

CRY links the circadian clock and CREB-mediated gluconeogenesis

Megumi Hatori1, Satchidananda Panda1

1The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA 92037, USA Cell Research (2010) 20:1285-1288. doi:10.1038/cr.2010.152; published online 9 November 2010

Circadian oscillators based on a to the cis-acting E-box elements in transcription. Subsequently, PER and transcriptional feedback loop exist in Period (Per1-Per3) and Cryptochrome CRY proteins enter the nucleus and almost all cells of animals. The cellular (Cry1 and Cry2) genes and drive their inhibit the transactivation by CLOCK- oscillators synchronize each other via paracrine or systemic communications, resulting in rhythmic changes of tis- sue- and whole body-level physiologies and behaviors. Circadian regulation of metabolism is well documented and disruption of such temporal regulation is known to predispose organisms to metabolic diseases. However, the underlying mechanism of circadian regulation of metabolism is complex and incom- pletely understood. The research has mainly focused on the transcriptional mechanisms by which circadian clock components regulate metabolic gene expression. A recent paper by Zhang et al. in Nature Medicine [1] for the first time elucidated a non-nuclear role of a circadian clock protein CRYPTO- CHROME (CRY) in mice; CRY regu- lates the circadian changes of hepatic gluconeogenesis, possibly through the interaction with a G protein, Gs α sub- Figure 1 Circadian clock protein CRYPTOCHROME controls gluconeogenesis unit, which leads to temporal regulation in the liver. The circadian core loop (blue box) is constituted of transcriptional of glucagon signaling triggering CREB activators CLOCK and BMAL1 and repressors PER and CRY. In the mouse phosphorylation (Figure 1). liver, CLOCK-BMAL1 heterodimer activates expression of Per and Cry genes The circadian oscillator is based on at early night (right panel). Then translated PER and CRY proteins inhibit CLOCK-BMAL1 activity in the morning (left panel) to form a negative feedback transcriptional mechanisms in which loop which cycles once a day. Fasting signal induces gluconeogenesis program the BMAL1 and CLOCK (or NPAS2) through activation of cAMP/CREB signaling (red box). Zhang et al. [1] found transcription factor heterodimer binds that CRY inhibits glucagon-stimulated cAMP production possibly through interaction with a G-protein Gs α subunit. The circadian rhythm of CRY protein levels form a “gate” for the response of the gluconeogenesis program to fasting: in Correspondence: Satchidananda Panda the morning when CRY levels are high, the response is small (left panel), while Tel: +1-858-453-4100 ext 1912 at early night when CRY levels are low and mice normally start to eat, the re- E-mail: [email protected] sponse is large (right panel). npg 1286 BMAL1, thus producing alternating ated by the natural cycles of feeding and luciferase (luc) reporter into mice. The cycles of transcriptional activation and fasting even in the complete absence of activity of reporter in the liver can be repression. Several additional gene a clock [10]. However, the oscillations monitored by an in vivo imaging sys- products including REV-ERB and ROR are robust in the presence of a functional tem. Fasted mice showed high hepatic participate in maintaining the accuracy oscillator, which suggests both the cir- CRE-luc activity during early nighttime. of this rhythm (see a review [2] for de- cadian clock and feeding and fasting In contrast, in the morning, fasted mice tails). The circadian oscillator exists in regulated mechanisms synergize to gen- showed less CRE-luc activity compared many tissues and organs [3]. The master erate robust transcriptional rhythms. to nighttime. Even exogenous injection circadian pacemaker resident in the The groups of Drs Steve Kay and of excess glucagon at both times led to hypothalamic suprachiasmatic nucleus Marc Montminy with complementary the day-night differences in CRE-luc generates daily rhythms of activity-rest expertise in the circadian clock and the activity, thus ruling out rhythmic glu- (and hence feeding and fasting), and the metabolism focused on the dynamic cagon production as a potential cause. peripheral oscillator in the liver plays a regulation of hepatic gluconeogenesis. This morning dip in CRE-luc activity major role in energy homeostasis in the Gluconeogenesis is the process of glu- reflected reduced CREB transcriptional face of periodic fasting and feeding. cose synthesis to maintain the blood activity and coincided with similar dip Given the transcriptional nature of glucose levels under fasting (starving) in CLOCK-BMAL1 activity. Since the the core oscillator, it is presumed that conditions. Several components of morning dip in CLOCK-BMAL1 activ- circadian regulation of output physiol- gluconeogenic pathway show circadian ity is due to repression by peak levels ogy and metabolism is at least partly rhythms in transcript levels and are also of the PER-CRY complex, the authors mediated by transcriptional oscillation known to be regulated by feeding and speculated that either one or more of of the relevant genes. Accordingly, fasting. Disrupted gluconeogenesis is a PER1-3 and CRY1-2 may downregulate genome-wide temporal expression hallmark feature of several metabolic CRE-mediated gene expression. The profiling studies have shown that up diseases. circadian oscillator can also regulate to 15% of the protein coding genes in Gluconeogenesis is tightly controlled gene expression via non-cell autono- the liver of mice fed ad libitum show by multiple signaling mechanisms mous systemic cues [11]. Therefore, circadian rhythmic expression. Several among which the peptide hormone they performed cell-based assays to of these rhythmic transcripts encode glucagon is a prominent player. Dur- test whether overexpression of clock rate-limiting enzymes of metabolic ing fasting, glucagon secreted by the proteins can modulate CRE-luc activ- pathways including those involved in pancreas binds to a G-protein coupled ity triggered by a Gs coupled-GPCR energy homeostasis [4]. The relevance receptor (GPCR) called the glucagon and its ligand. Among various clock of circadian regulation to metabolism receptor (Figure 1) in hepatocytes. components tested, overexpression of has been established by the observation This binding activates the downstream CRY1 and CRY2 inhibited CRE-luc that mice lacking normal CLOCK or G-protein Gs, which triggers increased activity, suggesting the specific roles of BMAL1 function in the entire body or intracellular cyclic AMP (cAMP) and CRY on inhibition of gluconeogenesis in specific metabolic organs show ab- phosphorylation of CREB (cAMP in the morning. normal glucose homeostasis leading to response element-binding). Tran- This inhibitory effect by CRY was metabolic diseases [5-8]. Therefore, un- scriptionally active p-CREB drives also observed in vivo with CRE-luc derstanding the mechanism of circadian transcription of gluconeogenic genes, mice. Adenoviral introduction of CRY1 regulation of metabolism would open such as Pck1 [encoding PEPCK (phos- reduced the hepatic CRE activity of novel approaches to treat the growing phoenolpyruvate carboxykinase)] and fasted mice during early nighttime, epidemic of metabolic diseases. G6pc (encoding G6Pase [glucose-6- when endogenous CRY protein level is The mechanisms underlying circa- phosphatase]), from cis-acting CRE low. In accordance to the reduced CRE dian and metabolic regulation of liver (cAMP response element) sites in their activity, the expression of gluconeo- function are complex and reciprocal in promoter regions. genic genes was also inhibited, resulting nature. Expression profiling of theClock The levels of p-CREB and a large in low blood glucose concentration. In mutant liver has shown that most of the number of CREB-regulated transcripts contrast to the CRY overexpression, rhythmic transcripts are not directly show rhythmic expression in the liver Cry1 and Cry2 double KO mice or regulated by the clock components, [10]. Zhang et al. first tested whether the acute RNAi inhibition of Crys in the rather indirect mechanisms generate glucagon-mediated activation of CREB liver increased the CRE activity, the such rhythms [9]. Furthermore, daily target gene transcription shows rhythmic expression of gluconeogenic genes, and transcriptional oscillation in a large pattern. They intravenously delivered the blood glucose level of fasted mice number of hepatic transcripts is gener- an adenovirus encoding CRE-driven in the morning. The inhibitory effect

Cell Research | Vol 20 No 12 | December 2010 npg 1287 of CRY on the glucose production was Such a physical interaction suggests a between the cell-autonomous circadian also observed in db/db mice, a mouse potential role of CRY in inhibiting Gs oscillator and feeding-fasting driven model of obesity and diabetes with function. rhythms in sustaining robust genome- excess gluconeogenesis. When CRY1 Novel findings about CRY regulation wide transcriptional rhythms. was adenovirally overexpressed in the of Gs signaling leading to CREB activa- liver of db/db mice, the mice showed tion is a paradigm shifting observation References reduced blood glucose levels in fasted that has widespread repercussions. Al- condition, thus establishing that acute though CRY proteins have been studied 1 Zhang EE, Liu Y, Dentin R, et al. Cryp- manipulation of the function of a clock in multiple organisms, their functions tochrome mediates circadian regulation component can be a strategy to perturb have always been described as tran- of cAMP signaling and hepatic gluconeogenesis. Nat Med 2010; 16:1152- gluconeogenesis and regulate blood scriptional regulators, and their interact- 1156. sugar levels. ing partners have no protein structure 2 Takahashi JS, Hong HK, Ko CH, et al. Which step(s) of the gluconeogen- similarity with the Gs proteins. Hence, The genetics of mammalian circadian esis cascade are inhibited by CRY? the observation raises a new function order and disorder: implications for From the observation that fasting- or of CRY proteins outside the nucleus. physiology and disease. Nat Rev Genet glucagon-induced cAMP concentration It also raises a question whether CRY 2008; 9:764-775. fluctuates in ~24 h rhythms with trough inhibition of G-protein is specific to Gs 3 Dibner C, Schibler U, Albrecht U. The mammalian circadian timing system: level coinciding with the peak level of or it can modulate other G-proteins. organization and coordination of cen- CRY expression, the authors speculated On the other hand, the mechanism of tral and peripheral clocks. Annu Rev that CRY might inhibit a step upstream downregulation of G proteins is of Physiol 2010; 72:517-549. of cAMP production. When mice were intense scrutiny. Typically, activated 4 Panda S, Antoch MP, Miller BH, et al. starving, adenoviral transduction of and GTP-bound G-proteins catalyze Coordinated transcription of key path- CRY reduced the hepatic concentration the breakdown of GTP to GDP, which ways in the mouse by the circadian of cAMP, suggesting that the point of leads to inactive GDP-bound G-protein. clock. Cell 2002; 109:307-320. CRY function might be at the level of Several proteins including the Regu- 5 Rudic RD, McNamara P, Curtis AM, et al. BMAL1 and CLOCK, two essential Gs, or at the level of adenylate cyclase lators of G-protein Signaling (RGS) components of the circadian clock, are (AC) that produces cAMP from ATP. proteins accelerate the GTPase activity involved in glucose homeostasis. PLoS The observation however, did not dis- of G-proteins and are thus said to be Biol 2004; 2:e377. tinguish between a direct role of CRY GTPase-activating proteins (GAPs) 6 Turek FW, Joshu C, Kohsaka A, et al. or an unknown CRY-regulated protein [12]. A potential mechanism by which Obesity and metabolic syndrome in in cAMP regulation. To differentiate CRY can inhibit Gs activity is being a circadian Clock mutant mice. Science this, the authors used a membrane GAP, facilitating recruitment of a GAP 2005; 308:1043-1045. 7 Lamia KA, Storch KF, Weitz CJ. Physi- preparation to measure isoproterenol to Gs or hindering the activation of AC ological significance of a peripheral tis- activation of Gs-coupled β adrenergic by activated Gs. Irrespective of these sue circadian clock. Proc Natl Acad Sci receptor leading to increased cAMP mechanisms, the observation that CRY USA 2008; 105:15172-15177. production. In this assay, membrane can modulate p-CREB levels also brings 8 Marcheva B, Ramsey KM, Buhr ED, et preps from wild-type cells mixed with up potential roles for a CRY-CREB al. Disruption of the clock components CRY1-expressing cytoplasm produced functional interaction in other func- CLOCK and BMAL1 leads to hypoin- less cAMP while antibody-mediated tions of CREB. For example, the clock sulinaemia and diabetes. Nature 2010; 466:627-631. depletion of CRY increased cAMP component Per genes also harbor CRE 9 Miller BH, McDearmon EL, Panda S, production, thus suggesting a direct role sites, and p-CREB has been shown to et al. Circadian and CLOCK-controlled of CRY (not of its downstream tran- activate Per transcription. This may un- regulation of the mouse transcriptome scriptional target) in inhibiting GPCR- derlie the simple feeding-fasting driven and cell proliferation. Proc Natl Acad stimulated cAMP production. Per transcript oscillation in the absence Sci USA 2007; 104:3342-3347. Finally, the authors tested which step of CRY proteins [10]. In the presence 10 Vollmers C, Gill S, DiTacchio L, et al. in the cAMP production is inhibited by of a fully functional circadian clock Time of feeding and the intrinsic cir- CRY. CRY failed to inhibit forskolin and the CRY proteins, CRY fine-tunes cadian clock drive rhythms in hepatic gene expression. Proc Natl Acad Sci activation of AC, thus ruling out AC p-CREB activity rhythms and enhances USA 2009; 106:21453-21458. as a point of CRY regulation. That left the robustness of the circadian clock 11 Kornmann B, Schaad O, Bujard H, et Gs as a potential point of regulation. In and its downstream targets. Hence, al. System-driven and oscillator-de- fact, the authors showed that CRY can the mechanism described here likely pendent circadian transcription in mice bind to Gs in reciprocal binding assays. explains the synergistic interaction with a conditionally active liver clock. www.cell-research.com | Cell Research npg 1288

PLoS Biol 2007; 5:e34. ing proteins for heterotrimeric G pro- (RGS) and RGS-like proteins. Annu 12 Ross EM, Wilkie TM. GTPase-activat- teins: regulators of G protein signaling Rev Biochem 2000; 69:795-827.

Cell Research | Vol 20 No 12 | December 2010