These data are in accordance with a functional partnership between BMAL2 and PER2. Indeed, daily temporal regulation of BMAL2 protein levels seemed also to differ between SHR and Wistar rats under RF; in SHR under RF, the highest BMAL2 levels were detected during subjective day when Per2 transcription was Y-27632 ROCK inhibitor increased compared with Wistar rats. Hence, in SHR maintained under RF, BMAL2CLOCK-mediated transactivation may support the conventional BMAL1-CLOCK mediated pathways, leading to an increased clock oscillation amplitude, as demonstrated in our study. Consequently, the higher amplitude of the Per2 expression rhythm might be related to higher levels of the corresponding protein that facilitates PER2-mediated inhibition of the BMAL2CLOCK. This mechanism is Crizotinib likely not present in Wistar rats, in which the Bmal2 gene does not seem to be sensitive to RF, and the clock amplitude is thus suppressed under RF conditions. Bmal2 is hypothesized to have been generated by gene duplication at the beginning of vertebrate evolution. In early studies, the role of Bmal2 in the circadian clock mechanism was not recognized because it was downregulated in Bmal1-deficient mice together with its paralog. However, recently accumulated evidence and our current data suggest its plausible physiological role in mediating the changes in feeding regimes by the hepatic clock. Our hypothesis that Bmal2 plays a role in mediating the interaction between the clock and metabolism is further supported by a recent finding that the constitutive expression of Bmal2 rescues the rhythmicity of the insulin action, locomotor activity and oxygen consumption in Bmal1-knockout mice. The Bmal2 gene is considered to be associated with type 1 diabetes in non-obese mice. Moreover, it has been suggested that alternative splicing of Bmal2 may provide tissues with a pathway regulating CLOCK-BMAL2 heterodimer function to respond to varied metabolic demands. In addition, BMAL2 was found to selectively bind to the E-box on the promoter of Pai-1 and regulate its expression. Pai-1 is elevated and associated with metabolic syndrome. Hence, a role of BMAL2 in mediating the interaction between the clock and metabolic state is likely, and our current data suggest that the interaction is likely bi-directional. The strain-selective sensitivity of Bmal2 to RF in SHR might be related to i) polymorphisms of the gene, and/or ii) to a higher metabolic challenge impinging on the clock in the rat strain. The former possibility is unlikely because the Bmal2 coding sequence in SHR and Wistar rats does not differ, although there might be undocumented polymorphisms in the regulatory regions. Several polymorphisms associated with metabolic syndrome were identified in the SHR Bmal1 promoter. Two of the SNPs affected the transcriptional binding sites for the GATA and Pax6 nuclear factors and significantly lowered the promoter activity. Therefore, it is possible that this deficiency is compensated by the higher Bmal2 sensitivity in SHR. The latter possibility of a higher metabolic pressure on the clock in SHR is supported by the previously demonstrated relationship between hypertension and hepatic physiology, as manifested by the metabolic aberrances of SHRs and the differences in the expression of metabolism-relevant proteins in SHR liver, as well as by the different sensitivity of the metabolic markers to RF, as discussed below. The higher amplitude and the advance of the hepatic clock rhythmicity under RF in the SHR compared with the Wistar rats corresponded to an enhanced and phase-advanced rhythmicity of Wee1 expression. WEE1 is a 
kinase which inactivates the complex Cdc2/cyclin B, thus controlling the G2/M transition of the cell cycle in a circadian manner. We used this gene as a marker of conventional E-box driven rhythmicity to confirm the effect of RF on the circadian clock in SHR.