Estrogens and the circadian system.

Circadian rhythms are ~24 h cycles of behavior and physiology that are generated by a network of molecular clocks located in nearly every tissue in the body. In mammals, the circadian system is organized hierarchically such that the suprachiasmatic nucleus (SCN) is the main circadian clock that receives light information from the eye and entrains to the light-dark cycle. The SCN then coordinates the timing of tissue clocks so internal rhythms are aligned with environmental cycles. Estrogens interact with the circadian system to regulate biological processes. At the molecular level, estrogens and circadian genes interact to regulate gene expression and cell biology. Estrogens also regulate circadian behavior across the estrous cycle. The timing of ovulation during the estrous cycle requires coincident estrogen and SCN signals. Studies using circadian gene reporter mice have also elucidated estrogen regulation of peripheral tissue clocks and metabolic rhythms. This review synthesizes current understanding of the interplay between estrogens and the circadian system, with a focus on female rodents, in regulating molecular, physiological, and behavioral processes.

Estradiol regulates daily rhythms underlying diet-induced obesity in female mice.

The circadian system is a critical regulator of metabolism and obesity in males, but its role in regulating obesity in females is poorly understood. Because there are sex differences in the development of obesity and susceptibility to obesity-related disorders, we sought to determine the role of estrogens in regulating the circadian mechanisms underlying diet-induced obesity. When fed high-fat diet, C57BL/6J male mice gain weight, whereas females are resistant to diet-induced obesity. Here, we demonstrate that estradiol regulates circadian rhythms in females to confer resistance to diet-induced obesity. We found that ovariectomized females with undetectable circulating estrogens became obese and had disrupted daily rhythms of eating behavior and locomotor activity when fed a high-fat diet. The phase of the liver molecular circadian rhythm was also altered by high-fat diet feeding in ovariectomized mice. Estradiol replacement in ovariectomized females a fed high-fat diet rescued these behavioral and tissue rhythms. Additionally, restoring the daily rhythm of eating behavior in ovariectomized females with time-restricted feeding inhibited diet-induced obesity and insulin resistance. Together, these data suggest that the circadian system is a target for treating obesity and its comorbidities in women after menopause, when circulating levels of estrogens are too low to protect their circadian rhythms.

High-fat diet acutely affects circadian organisation and eating behavior.

The organisation of timing in mammalian circadian clocks optimally coordinates behavior and physiology with daily environmental cycles. Chronic consumption of a high-fat diet alters circadian rhythms, but the acute effects on circadian organisation are unknown. To investigate the proximate effects of a high-fat diet on circadian physiology, we examined the phase relationship between central and peripheral clocks in mice fed a high-fat diet for 1 week. By 7 days, the phase of the liver rhythm was markedly advanced (by 5 h), whereas rhythms in other tissues were not affected. In addition, immediately upon consumption of a high-fat diet, the daily rhythm of eating behavior was altered. As the tissue rhythm of the suprachiasmatic nucleus was not affected by 1 week of high-fat diet consumption, the brain nuclei mediating the effect of a high-fat diet on eating behavior are likely to be downstream of the suprachiasmatic nucleus.

Disconnected circadian and cell cycles in a tumor-driven cell line.

Cell division occurs at a specific time of day in numerous species, suggesting that the circadian and cell cycles are coupled in vivo. By measuring the cell cycle rhythm in real-time, we recently showed that the circadian and cell cycles are not coupled in immortalized fibroblasts, resulting in a rapid rate of cell division even though the circadian rhythm is normal in these cells. Here we report that tumor-driven Lewis lung carcinoma (LLC) cells have perfectly temperature compensated circadian clocks, but the periods of their cell cycle gene expression rhythms are temperature-dependent, suggesting that their circadian and cell cycles are not connected. These data support our hypothesis that decoupling of the circadian and cell cycles may underlie aberrant cell division in tumor cells.

Robust food anticipatory activity in BMAL1-deficient mice.

Food availability is a potent environmental cue that directs circadian locomotor activity in rodents. Even though nocturnal rodents prefer to forage at night, daytime food anticipatory activity (FAA) is observed prior to short meals presented at a scheduled time of day. Under this restricted feeding regimen, rodents exhibit two distinct bouts of activity, a nocturnal activity rhythm that is entrained to the light-dark cycle and controlled by the master clock in the suprachiasmatic nuclei (SCN) and a daytime bout of activity that is phase-locked to mealtime. FAA also occurs during food deprivation, suggesting that a food-entrainable oscillator (FEO) keeps time in the absence of scheduled feeding. Previous studies have demonstrated that the FEO is anatomically distinct from the SCN and that FAA is observed in mice lacking some circadian genes essential for timekeeping in the SCN. In the current study, we optimized the conditions for examining FAA during restricted feeding and food deprivation in mice lacking functional BMAL1, which is critical for circadian rhythm generation in the SCN. We found that BMAL1-deficient mice displayed FAA during restricted feeding in 12hr light:12hr dark (12L:12D) and 18L:6D lighting cycles, but distinct activity during food deprivation was observed only in 18L:6D. While BMAL1-deficient mice also exhibited robust FAA during restricted feeding in constant darkness, mice were hyperactive during food deprivation so it was not clear that FAA consistently occurred at the time of previously scheduled food availability. Taken together, our findings suggest that optimization of experimental conditions such as photoperiod may be necessary to visualize FAA in genetically modified mice. Furthermore, the expression of FAA may be possible without a circadian oscillator that depends on BMAL1.

NIBP, a novel NIK and IKK(beta)-binding protein that enhances NF-(kappa)B activation.

The transcription factor NF-kappaB plays an important role in both physiological and pathological events in the central nervous system. Nevertheless, the mechanisms of NF-kappaB-mediated regulation of gene expression, and the signaling molecules participating in the NF-kappaB pathway in the central nervous system are, to date, poorly understood. To identify such molecules, we conducted a yeast two-hybrid screen of a human brain cDNA library using NIK as bait. As a result, we identified a novel NIK and IKK(beta) binding protein designated NIBP that is mainly expressed in brain, muscle, heart, and kidney. Interestingly, low levels of expression were detected in immune tissues such as spleen, thymus, and peripheral blood leukocytes, where NF-kappaB is known to modulate immune function. We demonstrated by immunohistochemistry that NIBP expression in the brain is localized to neurons. NIBP physically interacts with NIK, IKK(beta), but not IKK(alpha) or IKK(gamma). NIBP overexpression potentiates tumor necrosis factor-alpha-induced NF-kappaB activation through increased phosphorylation of the IKK complex and its downstream I(kappa)B(alpha) and p65 substrates. Finally, knockdown of NIBP expression by small interfering RNA reduces tumor necrosis factor-alpha-induced NF-kappaB activation, prevents nerve growth factor-induced neuronal differentiation, and decreases Bcl-xL gene expression in PC12 cells. Our data demonstrate that NIBP, by interacting with NIK and IKK(beta), is a new enhancer of the cytokine-induced NF-(kappa)B signaling pathway. Because of its neuronal expression, we propose that NIBP may be a potential target for modulating the NF-(kappa)B signaling cascade in neuronal pathologies dependent upon abnormal activation of this pathway.