Silencing the circadian clock gene Clock using RNAi reveals dissociation of the circatidal clock from the circadian clock in the mangrove cricket

Whether a clock that generates a circatidal rhythm shares the same elements as the circadian clock is not fully understood. The mangrove cricket, Apteronemobius asahinai, shows simultaneously two endogenous rhythms in its locomotor activity; the circatidal rhythm generates active and inactive phases, and the circadian rhythm modifies activity levels by suppressing the activity during subjective day. In the present study, we silenced Clock (Clk), a master gene of the circadian clock, in A. asahinai using RNAi to investigate the link between the circatidal and circadian clocks. The abundance of Clk mRNA in the crickets injected with double-stranded RNA of Clk (dsClk) was reduced to a half of that in control crickets. dsClk injection also reduced mRNA abundance of another circadian clock gene period (per) and weakened diel oscillation in per mRNA expression. Examination of the locomotor rhythms under constant conditions revealed that the circadian modification was disrupted after silencing Clk expression, but the circatidal rhythm remained unaffected. There were no significant changes in the free-running period of the circatidal rhythm between the controls and the crickets injected with dsClk. Our results reveal that Clk is essential for the circadian clock, but is not required for the circatidal clock. From these results we propose that the circatidal rhythm of A. asahinai is driven by a clock, the molecular components of which are distinct from that of the circadian clock.     

A circadian oscillator in Aspergillus spp. regulates daily development and gene expression

We have established the presence of a circadian clock in Aspergillus flavus and Aspergillus nidulans by morphological and molecular assays, respectively. In A. flavus, the clock regulates an easily assayable rhythm in the development of sclerotia, which are large survival structures produced by many fungi. This developmental rhythm exhibits all of the principal clock properties. The rhythm is maintained in constant environmental conditions with a period of 33 h at 30°C, it can be entrained by environmental signals, and it is temperature compensated. This endogenous 33-h period is one of the longest natural circadian rhythms reported for any organism, and this likely contributes to some unique responses of the clock to environmental signals. In A. nidulans, no obvious rhythms in development are apparent. However, a free running and entrainable rhythm in the accumulation of gpdA mRNA (encoding glyceraldehyde-3-phosphate dehydrogenase) is observed, suggesting the presence of a circadian clock in this species. We are unable to identify an Aspergillus ortholog of frequency, a gene required for normal circadian rhythmicity in Neurospora crassa. Together, our data indicate the existence of an Aspergillus circadian clock, which has properties that differ from that of the well-described clock of N. crassa.     

AMPK regulates circadian rhythms in a tissue- and isoform-specific manner

Background

AMP protein kinase (AMPK) plays an important role in food intake and energy metabolism, which are synchronized to the light-dark cycle. In vitro, AMPK affects the circadian rhythm by regulating at least two clock components, CKIα and CRY1, via direct phosphorylation. However, it is not known whether the catalytic activity of AMPK actually regulates circadian rhythm in vivo.

Methodology/Principal Finding

The catalytic subunit of AMPK has two isoforms: α1 and α2. We investigate the circadian rhythm of behavior, physiology and gene expression in AMPKα1−/− and AMPKα2−/− mice. We found that both α1−/− and α2−/− mice are able to maintain a circadian rhythm of activity in dark-dark (DD) cycle, but α1−/− mice have a shorter circadian period whereas α2−/− mice showed a tendency toward a slightly longer circadian period. Furthermore, the circadian rhythm of body temperature was dampened in α1−/− mice, but not in α2−/− mice. The circadian pattern of core clock gene expression was severely disrupted in fat in α1−/− mice, but it was severely disrupted in the heart and skeletal muscle of α2−/− mice. Interestingly, other genes that showed circadian pattern of expression were dysreguated in both α1−/− and α2−/− mice. The circadian rhythm of nicotinamide phosphoryl-transferase (NAMPT) activity, which converts nicotinamide (NAM) to NAD⁺, is an important regulator of the circadian clock. We found that the NAMPT rhythm was absent in AMPK-deficient tissues and cells.

Conclusion/Significance

This study demonstrates that the catalytic activity of AMPK regulates circadian rhythm of behavior, energy metabolism and gene expression in isoform- and tissue-specific manners.     

Circadian clock rhythms in different adipose tissue model systems

ABSTRACT

Circadian clock-controlled 24-h oscillations in adipose tissues play an important role in the regulation of energy homeostasis, thus representing a potential drug target for prevention and therapy of metabolic diseases. For pharmacological screens, scalable adipose model systems are needed that largely recapitulate clock properties observed in vivo. In this study, we compared molecular circadian clock regulation in different ex vivo and in vitro models derived from murine adipose tissues. Explant cultures from three different adipose depots of PER2::LUC circadian reporter mice revealed stable and comparable rhythms of luminescence ex vivo. Likewise, primary pre- and mature adipocytes from these mice displayed stable luminescence rhythms, but with strong damping in mature adipocytes. Stable circadian periods were also observed using Bmal1-luc and Per2-luc reporters after lentiviral transduction of wild-type pre-adipocytes. SV40 immortalized adipocytes of murine brown, subcutaneous and epididymal adipose tissue origin showed rhythmic mRNA expression of the core clock genes Bmal1, Per2, Dbp and REV-erbα in pre- and mature adipocytes, with a maturation-associated increase in overall mRNA levels and amplitudes. A comparison of clock gene mRNA rhythm phases revealed specific changes between in vivo and ex vivo conditions. In summary, our data indicate that adipose culture systems to a large extent mimic in vivo tissue clock regulation. Thus, both explant and cell systems may be useful tools for large-scale screens for adipose clock regulating factors.