Oscillating clock gene expression gives rise to a molecular clock that is present not only in the body's master circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), but also in extra-SCN brain regions. These extra-SCN molecular clocks depend on the SCN for entrainment to a light:dark cycle. The SCN has limited neural efferents, so it may entrain extra-SCN molecular clocks through its well-established circadian control of glucocorticoid hormone secretion. Glucocorticoids can regulate the normal rhythmic expression of clock genes in some extra-SCN tissues. Untimely stress-induced glucocorticoid secretion may compromise extra-SCN molecular clock function. We examined whether acute restraint stress during the rat's inactive phase can rapidly (within 30 min) alter clock gene (Per1, Per2, Bmal1) and cFos mRNA (in situ hybridization) in the SCN, hypothalamic paraventricular nucleus (PVN), and prefrontal cortex (PFC) of male and female rats (6 rats per treatment group). Restraint stress increased Per1 and cFos mRNA in the PVN and PFC of both sexes. Stress also increased cFos mRNA in the SCN of male rats, but not when subsequently tested during their active phase. We also examined in male rats whether endogenous glucocorticoids are necessary for stress-induced Per1 mRNA (6–7 rats per treatment group). Adrenalectomy attenuated stress-induced Per1 mRNA in the PVN and ventral orbital cortex, but not in the medial PFC. These data indicate that increased Per1 mRNA may be a means by which extra-SCN molecular clocks adapt to environmental stimuli (e.g. stress), and in the PFC this effect is largely independent of glucocorticoids.
Insufficient sleep induces insulin resistance and is associated with an increased risk for developing obesity and cardiometabolic diseases, implicating sleep loss as a metabolic stressor. Fibroblast growth factor 21 (FGF21) is a hepatokine secreted in response to stress and is elevated in human obesity and diabetes. FGF21 expression can be induced by free fatty acid (FFA) activation of peroxisome proliferator‐activator receptor alpha. We previously reported that insufficient sleep results in elevated FFA; however, the impact of insufficient sleep on FGF21 has not been explored. In an ongoing study, we are comparing the effects of insufficient sleep on insulin sensitivity in physically active and inactive individuals to determine whether regular exercise attenuates the adverse effects of insufficient sleep. To explore the effects of physical activity and insufficient sleep on FGF21, we compared changes in FGF21 during an oral glucose tolerance test (OGTT). Eleven sedentary (SED: 6F, 24.9±4.2y, 22.3±1.7kg/m 2 ; mean±SD) and 11 physically active adults (PA: 6F, 23.5±3.3y, 22.0±2.3kg/m 2 ) participated in a 6‐day controlled inpatient protocol. Participants were provided isocaloric diets designed to meet energy requirements. Active participants continued to exercise during insufficient sleep by conducting 60 minutes of moderate physical activity (treadmill running) at 65–75% of maximum heart rate. An OGTT was conducted at baseline (9h sleep opportunity/night) and after 3 nights of insufficient sleep (5h sleep opportunity/night). Blood was sampled at T=0, +30, +60, +90, and +120 minutes following glucose ingestion and assayed for FGF21, glucose, insulin and FFA. FGF21 was elevated in response to insufficient sleep in PA participants (Fig 1a; baseline: 40.5±13.3 v. insufficient sleep: 95.3±18.2 pg/ml, p<0.05), but not SED (baseline: 68.9±19.4 v. insufficient sleep: 95.6±24.1 pg/ml, p=ns). Both SED and PA participants displayed elevated glucose in response to the OGTT during insufficient sleep (Fig 1b), whereas insulin was elevated only in SED participants (Fig 1c). PA participants had higher levels of FFA as compared to SED, though FFA were not altered by insufficient sleep in either group (Fig 1d). Changes in FGF21 were not related to glucose, insulin or FFA. FGF21 is elevated during insufficient sleep in physically active individuals. Furthermore, insulin response to an OGTT during insufficient sleep was attenuated only in the physically active participants. Though the physiological implications of elevated FGF21 in humans are unclear, we speculate that elevated FGF21 during insufficient sleep in active individuals may act as a compensatory response to mitigate metabolic impairments. Support or Funding Information This work was supported by the Sleep Research Society Early Career Development Award, the National Institutes of Health GCRC grant RR‐00036, R01HL109706 and K01DK110138, Society in Science, and The Branco Weiss Fellowship, administered by the ETH Zürich. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Findings from previous studies indicate daily variation in energy expenditure (EE) depends on circadian phase in humans, though findings have been inconsistent. Changes in energy expenditure throughout the day and night during non-fasting conditions are important to understand factors influencing energy metabolism. The aim of this study was to determine the circadian rhythm of resting EE, respiratory quotient (RQ), and substrate utilization using a constant routine protocol. Six healthy adults (3F, age 27.7±2.2 years, BMI 24.7±3.2; mean±SD) participated in a 26-hour constant routine study following 1 week of outpatient monitoring. Subjects arrived at the laboratory in the evening and were provided an 8-hour sleep opportunity. After waking at their habitual time, participants remained in constant conditions, which included dim light (<8 lux), constant posture (bed rest), ambient temperature, and consumed small identical hourly meals. Melatonin was measured from hourly saliva and EE was measured every 3 hours using hood indirect calorimetry. EE data were aligned to dim light melatonin onset (DLMO). Average DLMO occurred at 20:10h (±18min). EE began to decline before DLMO and was lowest early in the biological night. RQ and carbohydrate oxidation (CHO-ox) were highest during the biological day, began to decline before DLMO, and remained lower throughout the biological night. In contrast, fat oxidation (FAT-ox) was highest close to DLMO. Previous findings show nadirs of EE, RQ, and CHO-ox close to the core body temperature minimum, which occurs during the middle of the biological night. In contrast, our constant routine study found lowest EE, RQ, and CHO-ox earlier in the biological night. However, current and prior assessments cannot determine precise nadir timing due to the relatively low frequency of sampling. Non-fasting studies with more frequent sampling of EE and substrate utilization are needed to determine how circadian timing impacts energy metabolism. This work was supported by the Colorado Nutrition Obesity Research Center P30 DK048520-21, K01DK113063 to CR, K01DK110138 to JLB, and a University of Colorado Vice Chancellor of Research Innovative Seed Grant to JLB.
The molecular circadian clock is a self-regulating transcription/translation cycle of positive (Bmal1, Clock/Npas2) and negative (Per1,2,3, Cry1,2) regulatory components. While the molecular clock has been well characterized in the body's master circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN), only a few studies have examined both the positive and negative clock components in extra-SCN brain tissue. Furthermore, there has yet to be a direct comparison of male and female clock gene expression in the brain. This comparison is warranted, as there are sex differences in circadian functioning and disorders associated with disrupted clock gene expression. This study examined basal clock gene expression (Per1, Per2, Bmal1 mRNA) in the SCN, prefrontal cortex (PFC), rostral agranular insula, hypothalamic paraventricular nucleus (PVN), amygdala, and hippocampus of male and female rats at 4-h intervals throughout a 12:12 h light:dark cycle. There was a significant rhythm of Per1, Per2, and Bmal1 in the SCN, PFC, insula, PVN, subregions of the hippocampus, and amygdala with a 24-h period, suggesting the importance of an oscillating molecular clock in extra-SCN brain regions. There were 3 distinct clock gene expression profiles across the brain regions, indicative of diversity among brain clocks. Although, generally, the clock gene expression profiles were similar between male and female rats, there were some sex differences in the robustness of clock gene expression (e.g., females had fewer robust rhythms in the medial PFC, more robust rhythms in the hippocampus, and a greater mesor in the medial amygdala). Furthermore, females with a regular estrous cycle had attenuated aggregate rhythms in clock gene expression in the PFC compared with noncycling females. This suggests that gonadal hormones may modulate the expression of the molecular clock.
Abstract Post-traumatic stress disorder (PTSD) is associated with impaired conditioned fear extinction learning, a ventromedial prefrontal cortex (vmPFC)-dependent process. PTSD is also associated with dysregulation of vmPFC, circadian, and glucocorticoid hormone function. Rats have rhythmic clock gene expression in the vmPFC that requires appropriate diurnal circulatory patterns of corticosterone (CORT), suggesting the presence of CORT-entrained intrinsic circadian clock function within the PFC. We examined the role of vmPFC clock gene expression and its interaction with CORT profiles in regulation of auditory conditioned fear extinction learning. Extinction learning and recall were examined in male rats trained and tested either in the night (active phase) or in the day (inactive phase). Using a viral vector strategy, Per1 and Per2 clock gene expression were selectively knocked down within the vmPFC. Circulating CORT profiles were manipulated via adrenalectomy (ADX) ± diurnal and acute CORT replacement. Rats trained and tested during the night exhibited superior conditioned fear extinction recall that was absent in rats that had knock-down of vmPFC clock gene expression. Similarly, the superior nighttime extinction recall was absent in ADX rats, but restored in ADX rats given a combination of a diurnal pattern of CORT and acute elevation of CORT during the postextinction training consolidation period. Thus, conditioned fear extinction learning is regulated in a diurnal fashion that requires normal vmPFC clock gene expression and a combination of circadian and training-associated CORT. Strategic manipulation of these factors may enhance the therapeutic outcome of conditioned fear extinction related treatments in the clinical setting.
Objective The circadian system provides an organism with the ability to anticipate daily food availability and appropriately coordinate metabolic responses. Few studies have simultaneously assessed factors involved in both the anticipation of energy availability (i.e., hormones involved in appetite regulation) and subsequent metabolic responses (such as energy expenditure and substrate oxidation) under conditions designed to reveal circadian rhythmicity. Methods Eight healthy adults (four females; age: 28.0 ± 2.3 years; BMI: 24.3 ± 2.9 kg/m 2 ) participated in a 26‐hour constant routine protocol involving continuous wakefulness with constant posture, temperature, dim light, and hourly isocaloric snacks. Indirect calorimetry was performed every 3 hours for measurement of energy expenditure and substrate oxidation. Subjective hunger was obtained hourly using questionnaires. Saliva and plasma were obtained hourly to assess melatonin (circadian phase marker) and hormones (leptin, ghrelin, and peptide YY). Results Fat and carbohydrate oxidation was highest in the biological evening and morning, respectively. Subjective hunger ratings peaked during the middle of the biological day. Significant circadian rhythms were identified for ghrelin and peptide YY with peaks in the biological evening and morning, respectively. Conclusions These findings support a role for the circadian system in the modulation of nutrient oxidation, subjective measures of appetite, and appetitive hormones.
Circadian misalignment—sleeping during the biological day and eating at a time when the internal circadian clock promotes sleep—is a risk factor for obesity and diabetes, implicating circadian misalignment as a metabolic stressor. Fibroblast growth factor 21 (FGF-21) is a hepatokine secreted in response to stress and found to be elevated in human obesity and diabetes. Activation of peroxisome proliferator-activator receptor alpha by free fatty acids (FFA), can induce the expression of FGF-21 expression. It has been previously reported that circadian misalignment results in elevated fasting FFA. However, the impact of circadian misalignment on FGF-21 has not been explored. Fourteen healthy adults (6M; aged 26.4±1.2y, BMI 22.7±0.5 kg/m2; mean±SD) participated in a simulated shift-work protocol using a consecutive study design to examine the impact of circadian misalignment on diabetes- and obesity-related risk factors. Participants were provided with a 3d energy balance diet prior to admission and during the 5d inpatient study. Identical meals were consumed each day to minimize the effects of dietary intake on metabolic hormones. Blood was sampled every 1-2 hours for 24 hours during circadian alignment and circadian misalignment and assayed for melatonin and FGF-21. The melatonin rhythm was used as a marker of the master circadian clock and was unchanged during simulated shift-work, indicating circadian misalignment. Circadian misalignment led to elevated FGF-21 during scheduled wake (+123±41% mean±SEM; p<0.01) and across 24h (+36±16%; p<0.05) relative to circadian alignment. Circadian misalignment triggers a stress response that includes an increase in circulating FGF-21. The physiological implications of acute elevations in FGF-21 are unknown, and may contribute to metabolic dysregulation associated with circadian misalignment. Disclosure J.L. Broussard: Advisory Panel; Self; National Institutes of Health. Research Support; Self; National Institute of Diabetes and Digestive and Kidney Diseases, Sleep Research Society, Society in Science-The Branco Weiss Fellowship. S.J. Morton: None. K. Markan: None. A.W. McHill: None. J. Higgins: None. E. Melanson: None. K. Wright: Advisory Panel; Self; National Institutes of Health, Torvec, Inc. Research Support; Self; National Institutes of Health, Office of Naval Research, Pacific-12 Conference, SomaLogic, Inc. Other Relationship; Self; Circadian Therapeutics, Inc., Kellogg Company, Torvec, Inc. Funding National Institutes of Health (UL1RR025780, P30DK048520); Sleep Research Society Foundation; Branco Weiss Fellowship Society in Science (K01DK110138, R21DK092624)
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