Synchronizing rhythms of behavior and metabolic processes is important for cardiovascular health and preventing metabolic diseases. The nuclear receptors REV-ERBα and REV-ERBβ play an integral role in regulating the expression of core clock proteins driving rhythms in activity and metabolism. Here we describe the identification of potent synthetic REV-ERB agonists with in vivo activity. Administration of synthetic REV-ERB ligands alters circadian behavior and the circadian pattern of core clock gene expression in the hypothalami of mice. The circadian pattern of expression of an array of metabolic genes in the liver, skeletal muscle, and adipose tissue was also altered resulting in increased energy expenditure. Treatment of diet-induced obese mice with a REV-ERB agonist decreased obesity by reducing fat mass and markedly improving dyslipidemia and hyperglycemia. These results suggest that synthetic REV-ERB ligands that pharmacologically target the circadian rhythm may hold utility in the treatment of sleep disorders as well as metabolic diseases.

Circadian rhythms in mammals are generated by a negative transcriptional feedback loop in which PERIOD (PER) is rate-limiting for feedback inhibition. Casein kinases I␦ and I (CKI␦/) can regulate temporal abundance/activity of PER by phosphorylation-mediated degradation and cellular localization. Despite their potentially crucial effects on PER, it has not been demonstrated in a mammalian system that these kinases play essential roles in circadian rhythm generation as does their homolog in Drosophila. To disrupt both CKI␦/ while avoiding the embryonic lethality of CKI␦ disruption in mice, we used CKI␦-deficient Per2 Luc mouse embryonic fibroblasts (MEFs) and overexpressed a dominant-negative mutant CKI (DN-CKI) in the mutant MEFs. CKI␦-deficient MEFs exhibited a robust circadian rhythm, albeit with a longer period, suggesting that the cells possess a way to compensate for CKI␦ loss. When CKI activity was disrupted by the DN-CKI in the mutant MEFs, circadian bioluminescence rhythms were eliminated and rhythms in endogenous PER abundance and phosphorylation were severely compromised, demonstrating that CKI␦/ are indeed essential kinases for the clockwork. This is further supported by abolition of circadian rhythms when physical interaction between PER and CKI␦/ was disrupted by overexpressing the CKI␦/ binding domain of PER2 (CKBD-P2). Interestingly, CKBD-P2 overexpression led to dramatically low levels of endogenous PER, while PER-binding, kinase-inactive DN-CKI did not, suggesting that CKI␦/ may have a non-catalytic role in stabilizing PER. Our results show that an essential role of CKI␦/ is conserved between Drosophila and mammals, but CKI␦/ and DBT may have divergent noncatalytic functions in the clockwork as well.casein kinase I delta ͉ casein kinase I epsilon ͉ dominant-negative mutant ͉ PERIOD

Hepatocyte nuclear factor 4 alpha (HNF4α) is a master regulator of liver-specific gene expression with potent tumor suppressor activity, yet many liver tumors express HNF4α. This study reveals that P1-HNF4α, the predominant isoform expressed in the adult liver, inhibits expression of tumor promoting genes in a circadian manner. In contrast, an additional isoform of HNF4α, driven by an alternative promoter (P2-HNF4α), is induced in HNF4α-positive human hepatocellular carcinoma (HCC). P2-HNF4α represses the circadian clock gene ARNTL (BMAL1), which is robustly expressed in healthy hepatocytes, and causes nuclear to cytoplasmic re-localization of P1-HNF4α. We reveal mechanisms underlying the incompatibility of BMAL1 and P2-HNF4α in HCC, and demonstrate that forced expression of BMAL1 in HNF4α-positive HCC prevents the growth of tumors in vivo. These data suggest that manipulation of the circadian clock in HNF4α-positive HCC could be a tractable strategy to inhibit tumor growth and progression in the liver.

No abstract

Modern society characterized by a 24/7 lifestyle leads to misalignment between environmental cycles and endogenous circadian rhythms. Persisting circadian misalignment leads to deleterious effects on health and healthspan. However, the underlying mechanism remains not fully understood. Here, we subjected adult, wild-type mice to distinct chronic jet-lag paradigms, which showed that long-term circadian misalignment induced significant early mortality. Non-biased RNA sequencing analysis using liver and kidney showed marked activation of gene regulatory pathways associated with the immune system and immune disease in both organs. In accordance, we observed enhanced steatohepatitis with infiltration of inflammatory cells. The investigation of senescence-associated immune cell subsets from the spleens and mesenteric lymph nodes revealed an increase in PD-1 + CD44 high CD4 T cells as well as CD95 + GL7 + germinal center B cells, indicating that the long-term circadian misalignment exacerbates immune senescence and consequent chronic inflammation. Our results underscore immune homeostasis as a pivotal interventional target against clock-related disorders. From the cellular to the organismal levels, circadian clocks regulate various essential biological processes to enable anticipation of and adaptation to the daily environmental changes from Earth rotation 1. Modernization of our society is accompanied by a dramatic change in human lifestyle, with unprecedented increases in, for example, night shift work and nocturnal feeding/recreational activities 2. Recent epidemiological studies have revealed shift workers as being at a higher risk of various diseases, such as mood disorders, metabolic syndrome, cardiovascular disease, and some types of cancers, suggesting that the misalignment between environmental cycles and endogenous circadian clocks exacerbates systemic pathological consequences 3-8. However, the pathophysiological mechanisms underlying the deleterious effects of long-term circadian misalignment in health and healthspan remain unclear. Recent studies have investigated the perturbation of circadian systems by environmental and/or genetic manipulation in animal models 9,10. For example, Davidson et al. reported that an experimental model of environmental perturbation induced by the scheduled shifts of light-dark cycles-called chronic jet-lag (CJL)-for 8 weeks using aged mice (27-31 months old) showed the mortality rate to be higher in the phase advance condition (6-hour phase advance every 7 days) than in the phase delay (6-hour phase delay every 7 days) condition or control LD condition 9. These studies principally investigated the acute or subacute (for up to a few months) effects of circadian misalignment; it thus remains uncertain how long-term perturbation of environmental light-dark cycle induces physiological transformation and pathological consequences.