Time-of-Day Effects on Metabolic and Clock-Related Adjustments to Cold.

BACKGROUND: Daily cyclic changes in environmental conditions are key signals for anticipatory and adaptive adjustments of most living species, including mammals. Lower ambient temperature stimulates the thermogenic activity of brown adipose tissue (BAT) and skeletal muscle. Given that the molecular components of the endogenous biological clock interact with thermal and metabolic mechanisms directly involved in the defense of body temperature, the present study evaluated the differential homeostatic responses to a cold stimulus at distinct time-windows of the light/dark-cycle. METHODS: Male Wistar rats were subjected to a single episode of 3 h cold ambient temperature (4°C) at one of 6 time-points starting at Zeitgeber Times 3, 7, 11, 15, 19, and 23. Metabolic rate, core body temperature, locomotor activity (LA), feeding, and drinking behaviors were recorded during control and cold conditions at each time-point. Immediately after the stimulus, rats were euthanized and both the soleus and BAT were collected for real-time PCR. RESULTS: During the light phase (i.e., inactive phase), cold exposure resulted in a slight hyperthermia (p < 0.001). Light phase cold exposure also increased metabolic rate and LA (p < 0.001). In addition, the prevalence of fat oxidative metabolism was attenuated during the inactive phase (p < 0.001). These metabolic changes were accompanied by time-of-day and tissue-specific changes in core clock gene expression, such as DBP (p < 0.0001) and REV-ERBα (p < 0.01) in the BAT and CLOCK (p < 0.05), PER2 (p < 0.05), CRY1 (p < 0.05), CRY2 (p < 0.01), and REV-ERBα (p < 0.05) in the soleus skeletal muscle. Moreover, genes involved in substrate oxidation and thermogenesis were affected in a time-of-day and tissue-specific manner by cold exposure. CONCLUSION: The time-of-day modulation of substrate mobilization and oxidation during cold exposure provides a clear example of the circadian modulation of physiological and metabolic responses. Interestingly, after cold exposure, time-of-day mostly affected circadian clock gene expression in the soleus muscle, despite comparable changes in LA over the light-dark-cycle. The current findings add further evidence for tissue-specific actions of the internal clock in different peripheral organs such as skeletal muscle and BAT.

Differential effects of diet composition and timing of feeding behavior on rat brown adipose tissue and skeletal muscle peripheral clocks.

The effects of feeding behavior and diet composition, as well as their possible interactions, on daily (clock) gene expression rhythms have mainly been studied in the liver, and to a lesser degree in white adipose tissue (WAT), but hardly in other metabolic tissues such as skeletal muscle (SM) and brown adipose tissues (BAT). We therefore subjected male Wistar rats to a regular chow or free choice high-fat-high sugar (fcHFHS) diet in combination with time restricted feeding (TRF) to either the light or dark phase. In SM, all tested clock genes lost their rhythmic expression in the chow light fed group. In the fcHFHS light fed group rhythmic expression for some, but not all, clock genes was maintained, but shifted by several hours. In BAT the daily rhythmicity of clock genes was maintained for the light fed groups, but expression patterns were shifted as compared with ad libitum and dark fed groups, whilst the fcHFHS diet made the rhythmicity of clock genes become more pronounced. Most of the metabolic genes in BAT tissue tested did not show any rhythmic expression in either the chow or fcHFHS groups. In SM Pdk4 and Ucp3 were phase-shifted, but remained rhythmically expressed in the chow light fed groups. Rhythmic expression was lost for Ucp3 whilst on the fcHFHS diet during the light phase. In summary, both feeding at the wrong time of day and diet composition disturb the peripheral clocks in SM and BAT, but to different degrees and thereby result in a further desynchronization between metabolically active tissues such as SM, BAT, WAT and liver.

Light at night acutely impairs glucose tolerance in a time-, intensity- and wavelength-dependent manner in rats.

AIMS/HYPOTHESIS: Exposure to light at night (LAN) has increased dramatically in recent decades. Animal studies have shown that chronic dim LAN induced obesity and glucose intolerance. Furthermore, several studies in humans have demonstrated that chronic exposure to artificial LAN may have adverse health effects with an increased risk of metabolic disorders, including type 2 diabetes. It is well-known that acute exposure to LAN affects biological clock function, hormone secretion and the activity of the autonomic nervous system, but data on the effects of LAN on glucose homeostasis are lacking. This study aimed to investigate the acute effects of LAN on glucose metabolism. METHODS: Male Wistar rats were subjected to i.v. glucose or insulin tolerance tests while exposed to 2 h of LAN in the early or late dark phase. In subsequent experiments, different light intensities and wavelengths were used. RESULTS: LAN exposure early in the dark phase at ZT15 caused increased glucose responses during the first 20 min after glucose infusion (p < 0.001), whereas LAN exposure at the end of the dark phase, at ZT21, caused increased insulin responses during the first 10 min (p < 0.01), indicating that LAN immediately induces glucose intolerance in rats. Subsequent experiments demonstrated that the effect of LAN was both intensity- and wavelength-dependent. White light of 50 and 150 lx induced greater glucose responses than 5 and 20 lx, whereas all intensities other than 5 lx reduced locomotor activity. Green light induced glucose intolerance, but red and blue light did not, suggesting the involvement of a specific retina-brain pathway. CONCLUSIONS/INTERPRETATION: Together, these data show that exposure to LAN has acute adverse effects on glucose metabolism in a time-, intensity- and wavelength-dependent manner.

Feeding during the resting phase causes profound changes in physiology and desynchronization between liver and muscle rhythms of rats.

Shiftworkers run an increased risk of developing metabolic disorders, presumably as a result of disturbed circadian physiology. Eating at a time-of-day that is normally dedicated to resting and fasting, may contribute to this association. The hypothalamus is the key brain area that integrates different inputs, including environmental time information from the central biological clock in the suprachiasmatic nuclei, with peripheral information on energy status to maintain energy homeostasis. The orexin system within the lateral hypothalamus is an important output of the suprachiasmatic nuclei involved in the control of sleep/wake behavior and glucose homeostasis, among other functions. In this study, we tested the hypothesis that feeding during the rest period disturbs the orexin system as a possible underlying contributor to metabolic health problems. Male Wistar rats were exposed to an 8-week protocol in which food was available ad libitum for 24-h, for 12-h during the light phase (i.e., unnatural feeding time) or for 12-h during the dark phase (i.e., restricted feeding, but at the natural time-of-day). Animals forced to eat at an unnatural time, i.e., during the light period, showed no changes in orexin and orexin-receptor gene expression in the hypothalamus, but the rhythmic expression of clock genes in the lateral hypothalamus was absent in these animals. Light fed animals did show adverse changes in whole-body physiology and internal desynchronization of muscle and liver clock and metabolic gene expression. Eating at the 'wrong' time-of-day thus causes internal desynchronization at different levels, which in the long run may disrupt body physiology.

The role of feeding rhythm, adrenal hormones and neuronal inputs in synchronizing daily clock gene rhythms in the liver.

The master clock in the hypothalamic suprachiasmatic nucleus (SCN) is assumed to distribute rhythmic information to the periphery via neural, humoral and/or behavioral connections. Until now, feeding, corticosterone and neural inputs are considered important signals for synchronizing daily rhythms in the liver. In this study, we investigated the necessity of neural inputs as well as of the feeding and adrenal hormone rhythms for maintaining daily hepatic clock gene rhythms. Clock genes kept their daily rhythm when only one of these three signals was disrupted, or when we disrupted hepatic neuronal inputs together with the adrenal hormone rhythm or with the daily feeding rhythm. However, all clock genes studied lost their daily expression rhythm after simultaneous disruption of the feeding and adrenal hormone rhythm. These data indicate that either a daily rhythm of feeding or adrenal hormones should be present to synchronize clock gene rhythms in the liver with the SCN.