Central Clock Genes Research Articles

Hypertension disrupts the vascular clock in both sexes.

Blood pressure (BP) displays a circadian rhythm and disruptions in this pattern elevate cardiovascular risk. Although both central and peripheral clock genes are implicated in these processes, the importance of vascular clock genes is not fully understood. BP, vascular reactivity, and the renin-angiotensin-aldosterone system display overt sex differences, but whether changes in circadian patterns underlie these differences is unknown. Therefore, we hypothesized that circadian rhythms and vascular clock genes would differ across sex and would be blunted by angiotensin II (ANG II)-induced hypertension. ANG II infusion elevated BP and disrupted circadian patterns similarly in both males and females. In females, an impact on heart rate (HR) and locomotor activity was revealed, whereas in males hypertension suppressed baroreflex sensitivity (BRS). A marked disruption in the vascular expression patterns of period circadian regulator 1 (Per1) and brain and muscle aryl hydrocarbon receptor nuclear translocator like protein 1 (Bmal1) was noted in both sexes. Vascular expression of the G protein-coupled estrogen receptor (Gper1) also showed diurnal synchronization in both sexes that was similar to that of Per1 and Per2 and disrupted by hypertension. In contrast, vascular expression of estrogen receptor 1 (Esr1) showed a diurnal rhythm and hypertension-induced disruption only in females. This study shows a strikingly similar impact of hypertension on BP rhythmicity, vascular clock genes, and vascular estrogen receptor expression in both sexes. We identified a greater impact of hypertension on locomotor activity and heart rate in females and on baroreflex sensitivity in males and also revealed a diurnal regulation of vascular estrogen receptors. These insights highlight the intricate ties between circadian biology, sex differences, and cardiovascular regulation.NEW & NOTEWORTHY This study reveals that ANG II-induced hypertension disrupts the circadian rhythm of blood pressure in both male and female mice, with parallel effects on vascular clock gene and estrogen receptor diurnal patterns. Notably, sex-specific responses to hypertension in terms of locomotor activity, heart rate, and baroreflex sensitivity are revealed. These findings pave the way for chronotherapeutic strategies tailored to mitigate cardiovascular risks associated with disrupted circadian rhythms in hypertension.

Sex-dependent circadian alterations of both central and peripheral clock genes expression and gut–microbiota composition during activity-based anorexia in mice

RationalePatients with anorexia nervosa (AN) often present sleep disorders and circadian hormonal dysregulation. The role of the microbiota–gut–brain axis in the regulation of feeding behavior has emerged during the last decades but its relationships with the circadian rhythm remains poorly documented. Thus, we aimed to characterize the circadian clock genes expression in peripheral and central tissues in the activity-based anorexia mouse model (ABA), as well as the dynamics of the gut–microbiota composition.MethodsFrom day 1 to day 17, male and female C57Bl/6 mice were submitted or not to the ABA protocol (ABA and control (CT) groups), which combines a progressive limited access to food and a free access to a running wheel. At day 17, fasted CT and ABA mice were euthanized after either resting (EoR) or activity (EoA) phase (n = 10–12 per group). Circadian clock genes expression was assessed by RT-qPCR on peripheral (liver, colon and ileum) and central (hypothalamic suprachiasmatic nucleus or SCN) tissues. Cecal bacterial taxa abundances were evaluated by qPCR. Data were compared by two-way ANOVA followed by post-tests.ResultsABA mice exhibited a lower food intake, a body weight loss and an increase of diurnal physical activity that differ according with the sex. Interestingly, in the SCN, only ABA female mice exhibited altered circadian clock genes expression (Bmal1, Per1, Per2, Cry1, Cry2). In the intestinal tract, modification of clock genes expression was also more marked in females compared to males. For instance, in the ileum, female mice showed alteration of Bmal1, Clock, Per1, Per2, Cry1, Cry2 and Rev-erbα mRNA levels, while only Per2 and Cry1 mRNAs were affected by ABA model in males. By contrast, in the liver, clock genes expression was more markedly affected in males compared to females in response to ABA. Finally, circadian variations of gut–bacteria abundances were observed in both male and female mice and sex-dependent alteration were observed in response to the ABA model.ConclusionsThis study shows that alteration of circadian clock genes expression at both peripheral and central levels occurs in response to the ABA model. In addition, our data underline that circadian variations of the gut–microbiota composition are sex-dependent.

Exercise alters molecular rhythms in the central nervous system that negatively correlate with depression-like behavior.

Major depressive disorder (MDD) ranks among the top 10 leading causes of death. However, exercise is known to improve depressive symptoms but the mechanism responsible is still unknown. To date, numerous studies have shown that molecular rhythms and exercise are associated with MDD. Thus, we hypothesized that exercise could affect the expression of central nervous system clock genes to improve depressive symptoms. Ninety adult male Sprague-Dawley rats (250 g) were divided into a control Normal Group, an unpredictable chronic mild stress (CMS) treated CMS Group and an Exercise Group, which was intervened by a moderate-intensity exercise training on a treadmill at 2 p.m. every day for 4 weeks after CMS treatment. The open field test, elevated plus maze and forced swim test were employed to test mood-related behaviors. The telemetry recording method recorded voluntary locomotor activity and core body temperature. Expression of core clock genes in the suprachiasmatic nucleus (SCN) was tested by qRT-PCR. Compared with the CMS Group, depressive symptoms were improved in the Exercise Group ( P < 0.05). Moreover, the periodic changes of molecular rhythms in the Exercise Group were close to those of rats in Normal Group. Next, exercise increased oscillations of expression of core clock genes in SCN after CMS treatment, and the amplitudes of core clock gene expression oscillations were negatively correlated with depressive-like behavior. Our findings suggested that exercise could change the expressions of central clock genes in MDD animals, and this effect was positively correlated with the improvement of depressive symptoms by exercise.

Sweet treats before sleep disrupt the clock system and increase metabolic risk markers in healthy rats.

Biological rhythms are endogenously generated natural cycles that act as pacemakers of different physiological mechanisms and homeostasis in the organism, and whose disruption increases metabolic risk. The circadian rhythm is not only reset by light but it is also regulated by behavioral cues such as timing of food intake. This study investigates whether the chronic consumption of a sweet treat before sleeping can disrupt diurnal rhythmicity and metabolism in healthy rats. For this, 32 Fischer rats were administered daily a low dose of sugar (160 mg/kg, equivalent to 2.5 g in humans) as a sweet treat at 8:00 a.m. or 8:00 p.m. (ZT0 and ZT12, respectively) for 4 weeks. To elucidate diurnal rhythmicity of clock gene expression and metabolic parameters, animals were sacrificed at different times, including 1, 7, 13, and 19 h after the last sugar dose (ZT1, ZT7, ZT13, and ZT19). Increased body weight gain and higher cardiometabolic risk were observed when sweet treat was administered at the beginning of the resting period. Moreover, central clock and food intake signaling genes varied depending on snack time. Specifically, the hypothalamic expression of Nampt, Bmal1, Rev-erbα, and Cart showed prominent changes in their diurnal expression pattern, highlighting that sweet treat before bedtime disrupts hypothalamic control of energy homeostasis. These results show that central clock genes and metabolic effects following a low dose of sugar are strongly time-dependent, causing higher circadian metabolic disruption when it is consumed at the beginning of the resting period, that is, with the late-night snack.

CD36 regulates diurnal glucose metabolism and hepatic clock to maintain glucose homeostasis in mice

The mammalian circadian clock and glucose metabolism are highly interconnected, and disruption of this coupling is associated with multiple negative health outcomes. Liver is the major source of endogenous glucose production and liver clock is one of the most vital peripheral clock systems. We demonstrate that fatty acid translocase (CD36) is expressed rhythmically in mouse liver and autonomously modulates the diurnal oscillations of liver clock and glucose homeostasis. CD36 knockout in hepatocytes inhibits the relay of insulin signaling and provokes FoxO1 nuclear shuttling, consequently increasing Per1 nuclear expression. Moreover, FoxO1 can activate the central clock gene Per1 at the transcriptional level. These changes lead to a disrupted clock oscillation and behavioral rhythm. Our study first reveal that CD36 is a key regulator of the circadian oscillator and its deficiency may cause liver clock disruption, which aggravates the imbalance of glucose homeostasis and contribute to augmentation and progression of metabolic disease.

Repetitive mild traumatic brain injury alters central and peripheral clock gene expression in the adolescent rat

Mild traumatic brain injury (mTBI) or concussion is a common injury worldwide leading to substantial medical costs and a high burden on society. In adolescents, falls and sports related trauma are often the causes of mTBI. Importantly, critical brain growth and development occurs during this sensitive period making the prospect of a brain injury a worrying phenomenon. Upwards of 70% of patients report circadian disruption following these injuries and this has been shown to impede recovery. Therefore, we sought to determine if core circadian clock gene expression was disrupted in rat model of repetitive mTBI (RmTBI). Male and female adolescent rats (n = 129) received sham or RmTBI. The animals were then euthanized at different times throughout the day and night. Tissue from the hypothalamus, cerebellum, hippocampus, liver, and small intestine were evaluated for the expression of per1, per2, cry1, clock, bmal1 and rev-erb-α. We found most clock genes varied across the day/night indicating circadian expression patterns. In the hypothalamus we found RmTBI altered the expression of cry1 and bmal1 in addition to sex differences in per2, cry1, clock, bmal1 and rev-erb- α. In the cerebellum, per1, per2, cry1, clock, bmal1 and rev-erb-α rhythms were all knocked out by RmTBI in addition to sex differences in cry1, clock and bmal1 expression. We also detected a significant decrease in overall expression of all clock genes in males in the middle of the night. In the hippocampus we found that RmTBI changed the rhythm of rev-erb-α expression in addition to sex differences in bmal1 expression. In the liver we detected strong rhythms in all genes examined, however only per2 expression was knocked out by RmTBI, in addition we also detected sex differences in per2 and cry1. We also detected an overall decrease in female clock gene expression in the early night. In the small intestine, RmTBI altered cry1 expression and there were sex differences in rev-erb-α. These results indicate that RmTBI alters core circadian clock gene expression in the central and peripheral nervous system in a time, tissue and sex dependent manner. This may be disrupting important phase relationships between the brain and peripheral nervous system and contributing to post-injury symptomology and also highlights the importance for time and sex dependent assessment of injury outcomes.

Administration time effect of dietary proanthocyanidins on the metabolome of Fischer 344 rats is sex- and diet-dependent.

Proanthocyanidins (PAs) are one of the most commonly ingested polyphenols in the human diet, with a wide range of beneficial health effects. Remarkably, PAs have been reported to influence core and peripheral clock genes expression, and their effects may change in a time-of-day dependent manner. Therefore, the aim of this study was to investigate whether the capacity of PAs to modulate the metabolome is conditioned by the time-of-day in which these compounds are consumed in a diet- and sex-dependent manner. To do this, a grape seed proanthocyanidin extract (GSPE) was administered to female and male Fischer 344 rats at ZT0 (in the morning) and ZT12 (at night) and the GSPE administration time effect was evaluated on clock genes expression, melatonin hormone and serum metabolite levels in a healthy and obesogenic context. The results showed an administration time effect of GSPE on the metabolome in a sex and diet-dependent manner. Specifically, there was an effect on amino acid, lipid and cholate metabolite levels that correlated with the central clock genes expression. Therefore, this study shows a strong influence of sex and diet on the PAs effects on the metabolome, modulated in turn by the time-of-day.

Circadian desynchronization in pregnancy of Golden hamster following long time light exposure: Involvement of Akt/FoxO1 pathway.

Coordination between central and peripheral reproductive clocks in females is poorly understood. Long light is having a hazardous effect on reproductive health. Hence, explored the effect of long-time light exposure (LLD; 16L:8D) on the central and peripheral reproductive (ovary and uterus) clock genes (Bmal1, Clock, Per1, Per2, Cry1 and Cry2) and its downstream regulators (Aanat, Egf, Cx26, Cx43, ERα, pAktS-473, pAktT-308, pFoxO1T-24, 14-3-3, HoxA10, HoxA11 and Pibf) expression in non-pregnant and pregnant Golden hamster. Young adult Golden hamsters were exposed to LLD for 30days and then were mated. We observed that LLD exposure increased the thickness of the endometrium and reduced myometrium thickness, resembling uterine adenomyosis. In non-pregnant females LLD altered the expressions of clock genes in suprachiasmatic nuclei (SCN), ovary and the uterus along with serum estradiol rhythm. LLD upregulated Egf and downregulated Aanat, Cx26, and Cx43 mRNA levels in uterus. LLD upregulated Akt/FoxO1 phosphorylation and 14-3-3 expressions in the uterus of nonpregnant females. LLD exposure to pregnant females lowered serum progesterone, Aanat, Pibf, Hoxa10, and Hoxa11 mRNA expressions on D4 (peri-implantation) and D8 (post-implantation) resulting in a low implantation rate on D8 (post-implantation). Hence it is evident that the frequent pregnancy anomalies noted under a long light schedule might be due to desynchronization in Aanat, Pibf, Hoxa10, and Hoxa11 as well as the central and peripheral clock genes (Bmal1, Clock, Per1, Per2, Cry1 and Cry2). LLD exposure desynchronized the central and peripheral reproductive clock affecting uterine physiology via Akt/FoxO1 pathway in Golden hamsters. Thus, LLD is a risk factor for female reproductive health and fertility.

Universal Stress Protein regulates the circadian rhythm of central oscillator genes in Arabidopsis.

Environmental stresses restrict plant growth and development and decrease crop yield. The circadian clock is associated with the ability of a plant to adapt to daily environmental fluctuations and the production and consumption of energy. Here, we investigated the role of Arabidopsis Universal Stress Protein (USP; At3g53990) in the circadian regulation of nuclear clock genes. The Arabidopsis usp knockout mutant line exhibited critically diminished circadian amplitude of the central oscillator CIRCADIAN CLOCK ASSOCIATED 1 (CCA1) but enhanced the amplitude of TIMING OF CAB EXPRESSION 1 (TOC1). However, the expression of USP under the control of its own promoter restored the circadian timing of both genes, suggesting that USP regulates the circadian rhythm of Arabidopsis central clock genes, CCA1 and TOC1.

Dual Role for FHY3 in Light Input to the Clock.

The red-light regulated transcription factors FHY3 and FAR1 form a key point of light input to the plant circadian clock in positively regulating expression of genes within the central clock. However, the fhy3 mutant shows an additional red light-specific disruption of rhythmicity which is inconsistent with this role. Here we demonstrate that only fhy3 and not far1 mutants show this red specific disruption of rhythmicity. We examined the differences in rhythmic transcriptome in red versus white light and reveal differences in patterns of rhythmicity among the central clock proteins suggestive of a change in emphasis within the central mechanism of the clock, changes which underlie the red specificity of the fhy3 mutant. In particular, changes in enrichment of promoter elements were consistent with a key role for the HY5 transcription factor, a known integrator of the ratio of red to blue light in regulation of the clock. Examination of differences in the rhythmic transcriptome in the fhy3 mutant in red light identified specific disruption of the CCA1-regulated ELF3 and LUX central clock genes, while the CCA1 target TBS element, TGGGCC, was enriched among genes that became arrhythmic. Coupled with the known interaction of FHY3 but not FAR1 with CCA1 we propose that the red-specific circadian phenotype of fhy3 may involve disruption of the previously demonstrated moderation of CCA1 activity by FHY3 rather than a disruption of its own transcriptional regulatory activity. Together, this evidence suggests a conditional redundancy between FHY3 and HY5 in the integration of red and blue light input to the clock in order to enable a plasticity in response to light and optimise plant adaptation. Furthermore, our evidence also suggests changes in CCA1 activity between red and white light transcriptomes. This, together with the documented interaction of HY5 with CCA1, leads us to propose a model whereby this integration of red and blue signals may at least partly occur via direct FHY3 and HY5 interaction with CCA1 leading to moderation of CCA1 activity.

Continuous artificial light potentially disrupts central and peripheral reproductive clocks leading to altered uterine physiology and reduced pregnancy success in albino mice.

The mechanism behind clock coordination in female reproductive disorders is poorly understood despite the known importance of coordinated and synchronized timing of central and clocks in reproductive organs. We investigated the effect of continuous artificial light (LL) on the central and peripheral reproductive clock gene (Bmal1, Clock, Per1, Per2 and Cry1) and its downstream regulators (Hgf, PR-A and HOXA10) during non-pregnancy and pregnancy phases of female mice. Mice (n = 60) in two sets, were maintained under continuous light (LL) and natural day cycle (LD;12L: 12D) for both non-pregnant and pregnant study. Tissues from hypothalamus-containing SCN, ovary, uterus and serum were collected at different zeitgeber time points (ZT; at 4-h intervals across 24-h periods). LL exposure desynchronized the expressions of the clock mRNAs (Bmal1, Clock, Per1, Per2 and Cry1) in SCN, ovary, and uterus along with Hgf mRNA rhythm. LL significantly increased the thickness of endometrial tissues. Furthermore, the pregnant study revealed lower serum progesterone level during peri- and post-implantation under LL along with downregulated expression of progesterone receptor (PR) as well as progesterone dependent uterine Homeobox A-10 (Hoxa10) proteins with lowered pregnancy outcomes. Our result suggests that LL disrupted the circadian coordination between central and clock genes in reproductive tissue leading to interrupted uterine physiology and altered pregnancy in mice. This led us to propose that duration of light exposure at work-places or home for females is very important in prevention of pregnancy anomalies.

BMAL1 regulates Propionibacterium acnes-induced skin inflammation via REV-ERBα in mice.

Acne vulgaris is a common skin disease, affecting over 80% of adolescents. Inflammation is known to play a central role in acne development. Here, we aimed to investigate the role of the central clock gene Bmal1 in acne-associated inflammation in mice. To this end, mice were injected intradermally with Propionibacterium acnes (P. acnes) to induce acne-associated skin inflammation. We found that Bmal1 and its target genes Rev-erbα, Dbp, Per1 and Cry2 were down-regulated in the skin of P. acnes-treated mice, suggesting a role of Bmal1 in the condition of acne. Supporting this, Bmal1-deleted or jet-lagged mice showed exacerbated P. acnes-induced inflammation in the skin. Regulation of P. acnes-induced inflammation by Bmal1 was further confirmed in RAW264.7 cells and primary mouse keratinocytes. Transcriptomic and protein expression analyses suggested that Bmal1 regulated P. acnes-induced inflammation via the NF-κB/NLRP3 axis, which is known to be repressed by REV-ERBα (a direct target of BMAL1). Moreover, loss of Rev-erbα in mice exacerbated P. acnes-induced inflammation. In addition, Rev-erbα silencing attenuated the inhibitory effects of Bmal1 on P. acnes-induced inflammation. Bmal1 knockdown failed to modulate P. acnes-induced inflammation in Rev-erbα-silenced cells. It was thus proposed that Bmal1 restrained P. acnes-induced skin inflammation via its target REV-ERBα, which acts on the NF-κB/NLRP3 axis to repress inflammation. In conclusion, Bmal1 disruption is identified as a potential pathological factor of acne-associated inflammation. The findings increase our understanding of the crosstalk between skin clock and acne and suggest targeting circadian rhythms as a promising approach for management of acne.

Disturbance of the Circadian System in Shift Work and Its Health Impact

The various non-standard schedules required of shift workers force abrupt changes in the timing of sleep and light-dark exposure. These changes result in disturbances of the endogenous circadian system and its misalignment with the environment. Simulated night-shift experiments and field-based studies with shift workers both indicate that the circadian system is resistant to adaptation from a day- to a night-oriented schedule, as determined by a lack of substantial phase shifts over multiple days in centrally controlled rhythms, such as those of melatonin and cortisol. There is evidence that disruption of the circadian system caused by night-shift work results not only in a misalignment between the circadian system and the external light-dark cycle, but also in a state of internal desynchronization between various levels of the circadian system. This is the case between rhythms controlled by the central circadian pacemaker and clock genes expression in tissues such as peripheral blood mononuclear cells, hair follicle cells, and oral mucosa cells. The disruptive effects of atypical work schedules extend beyond the expression profile of canonical circadian clock genes and affects other transcripts of the human genome. In general, after several days of living at night, most rhythmic transcripts in the human genome remain adjusted to a day-oriented schedule, with dampened group amplitudes. In contrast to circadian clock genes and rhythmic transcripts, metabolomics studies revealed that most metabolites shift by several hours when working nights, thus leading to their misalignment with the circadian system. Altogether, these circadian and sleep-wake disturbances emphasize the all-encompassing impact of night-shift work, and can contribute to the increased risk of various medical conditions. Here, we review the latest scientific evidence regarding the effects of atypical work schedules on the circadian system, sleep and alertness of shift-working populations, and discuss their potential clinical impacts.

An in-depth neurobehavioral characterization shows anxiety-like traits, impaired habituation behavior, and restlessness in male Cryptochrome-deficient mice.

Many psychiatric disorders, for example, anxiety, are accompanied by disturbances of circadian rhythms, including disturbed sleep/wake cycles, changes in locomotor activity, and abnormal endocrine function. Conversely, alternations of circadian rhythms are a risk factor for the development of psychiatric disorders. This assumption is supported by animals with clock gene mutations which often display behaviors that resemble human psychiatric disorders. In this study, we performed an in-depth behavioral analysis with male mice lacking the central clock genes Cryptochrome 1 and 2 (Cry1/2-/- ), which are thus unable to express endogenous circadian rhythms. With wild-type and Cry1/2-/- mice, we performed an extensive behavioral analysis to study their cognitive abilities, social behavior, and their expression of depression-like and anxiety-like behavior. While Cry1/2-/- mice showed only mild abnormalities at cognitive and social behavioral levels, they were consistently more anxious than wildtype mice. Anxiety-like behavior was particularly evident in reduced mobility in new environments, altered ability to habituate, compensatory behavior, and consistent restless behavior across many behavioral tests. In line with their anxiety-like behavioral phenotype, Cry1/2-/- mice have higher c-Fos activity in the amygdala after exposure to an anxiogenic stressor than wild-type mice. In our study, we identified Cry1/2-/- mice as animals that qualify as a translational mouse model for anxiety disorder in humans because of its consistent behavior of restlessness, increased immobility, and dysfunctional habituation in new environments.

Impaired Renal Endothelin‐1 Excretion in Diet‐induced Obesity Contributes to Salt Sensitivity in Male Sprague‐Dawley Rats

The renal endothelin system is an important regulator of sodium excretion in response to high salt intake. Urinary Na+ excretion (UNaV) as well as endothelin‐1 (ET‐1) excretion follow a diurnal rhythm, which is controlled by central and peripheral clock genes. Decreased renal medullary ET‐1 secretion has been attributed to salt sensitive (SS) hypertension. Diet‐induced obesity leads to clock gene dysfunction and Na+ imbalance. However, it is not clear how a high fat (HF) diet could influence the diurnal regulation of these systems. Our overarching hypothesis is that HF diet contributes to the development of salt‐sensitive hypertension through renal ET‐1 dysfunction. Male Sprague‐Dawley rats were put on either high fat (45 Kcal% fat, HF) or normal fat (10 Kcal% fat, NF) diet for 8 weeks duration starting at 6 weeks of age. Half‐way through the feeding protocol, rats were implanted with telemetry transmitters for continuous blood pressure measurement. For the last 2 weeks of feeding, HF and NF groups were divided into 2 subgroups to receive an additional high salt (4% NaCl, HS) diet or continue on the normal salt (0.3% NaCl, NS) diet with continuous blood pressure monitoring. For the last 4 days of the feeding protocol, rats were put in metabolic cages for 2 days of acclimation, and then urine was collected and food and water intakes were monitored in 12‐hr increments (inactive‐lights on/active‐lights off) for the subsequent 2 days. Systolic blood pressure (BP) was significantly increased in HF compared to NF rats after 6 weeks of feeding (145 ± 1 vs 138 ± 2 mmHg, n =14 and 11 respectively) during their active period. Chronic HS diet led to a further increase in active‐time systolic BP in HF rats compared to their NS control (153 ± 2 vs 146 ± 2 mmHg, n =6/group), while addition of HS to the NF group showed no significant increase in systolic BP (145 ± 2 vs 140 ± 3 mmHg, n =6/group) during the active period. The NF group on HS diet had a significant increase in active period ET‐1 excretion compared to NF/NS (4.34 ± 0.35 vs 1.65 ± 0.38 pg/12hrs, n= 6/group). However, HS diet failed to increase active period ET‐1 excretion in the HF (2.56 ±0.29 vs 1.32 ± 0.12 pg/12 hrs, n=6/group) compared to their NS controls. Rats on HF/NS and NF/NS diets were given an acute salt load of 900 μEq NaCl in 1 ml water, i.p. to assess their acute natriuretic response. The natriuretic response to an acute salt load given at the beginning of the active period was significantly reduced in rats on HF vs. NF with ⊗UNaV of 629 ± 91 vs 985 ± 75 μEq/12 hrs in the first 12 hours post‐load, respectively, n= 6/group. This was associated with attenuated ET‐1 excretion in HF versus NF diet (0.93 ± 0.15 vs 1.69 ± 0.16 pg/12hrs, respectively, n=11/group). There were no significant differences in the natriuretic response when the salt load was given at the beginning of the inactive period. These data show that HF diet reduces renal ET‐1 and salt handling in a time of day dependent manner. These findings are also consistent with our hypothesis that timing of Na+ handling may contribute to salt‐sensitive hypertension in diet‐induced obesity.Support or Funding InformationFunded by P01 HL136267 to DMP

Effects of desflurane on central and peripheral clock genes in mice

Effects of desflurane on central and peripheral clock genes in mice

Abstract P1127: Reverse Feeding Abolishes Circadian Blood Pressure Rhythms in Bmal1-Knockout Rats

Blood pressure follows a strong circadian rhythm regulated by the molecular clock, which can be entrained by light, feeding, and other cues. Using a whole body knockout of a central clock gene, Bmal1 (Bmal1KO), we tested the hypothesis that food availability would entrain circadian blood pressure rhythms with a functional molecular clock. Male and female rats were implanted with a telemetry transmitter to measure 24-hour mean arterial pressure (MAP). Following recovery, rats were placed in metabolic cages to measure food and water intake. Rats were allowed ad lib access to food for three days and then food was restricted to the inactive period (lights on, 7 am to 7 pm) for five days. Under ad lib feeding conditions, male littermate control rats (n=8) had a MAP of 113.3±2.6 mmHg during the inactive period and a significantly higher MAP during the active period (120.6±3.1 mmHg, repeated measures ANOVA with Sidak’s post hoc, p&lt;0.0001). Similarly, Bmal1KO rats (n=12) had a significantly higher MAP during the active period (114.5±1.8 mmHg) versus the inactive period (108.9±2.0 mmHg, p&lt;0.0001). Under reverse feeding conditions, control rats had an average MAP of 118.3±2.5 mmHg during the inactive period and a significantly higher MAP to 121.7±2.8 mmHg during the active period (p&lt;0.0001). Bmal1KO rats had similar MAPs during the inactive and active periods (113.1±1.7 vs. 113.3±1.8 mmHg, respectively, p=0.99). During ad lib feeding, female control rats (n=7) had a significant diurnal pattern with an average MAP of 110.6±1.4 mmHg in the inactive period and 118.9±1.9 mmHg during the active period (p&lt;0.0001). Female Bmal1KO rats (n=3) had an average MAP of 104.7±0.4 mmHg in the inactive period and a significantly higher MAP of 110.5±0.3 mmHg during the active period (p&lt;0.001). Under reverse feeding conditions, female control animals maintained a significant diurnal MAP from inactive to active periods (110.6±1.5 vs. 115.3±1.9 mmHg, respectively, p&lt;0.0001), but female Bmal1KO animals had no significant difference in inactive and active period MAP (105.0±0.3 vs. 106.8±0.4 mmHg, respectively, p&lt;0.0001). Together, these data suggest that food availability contributes to the circadian blood pressure rhythms in male and female Bmal1 knockout rats.

Angptl8 mediates food-driven resetting of hepatic circadian clock in mice

Diurnal light-dark cycle resets the master clock, while timed food intake is another potent synchronizer of peripheral clocks in mammals. As the largest metabolic organ, the liver sensitively responds to the food signals and secretes hepatokines, leading to the robust regulation of metabolic and clock processes. However, it remains unknown which hepatokine mediates the food-driven resetting of the liver clock independent of the master clock. Here, we identify Angptl8 as a hepatokine that resets diurnal rhythms of hepatic clock and metabolic genes in mice. Mechanistically, the resetting function of Angptl8 is dependent on the signal relay of the membrane receptor PirB, phosphorylation of kinases and transcriptional factors, and consequently transient activation of the central clock gene Per1. Importantly, inhibition of Angptl8 signaling partially blocks food-entrained resetting of liver clock in mice. We have thus identified Angptl8 as a key regulator of the liver clock in response to food.

Histone H3 lysine 4 methyltransferase is required for facultative heterochromatin at specific loci

BackgroundHistone H3 lysine 4 tri-methylation (H3K4me3) and histone H3 lysine 9 tri-methylation (H3K9me3) are widely perceived to be opposing and often mutually exclusive chromatin modifications. However, both are needed for certain light-activated genes in Neurospora crassa (Neurospora), including frequency (frq) and vivid (vvd). Except for these 2 loci, little is known about how H3K4me3 and H3K9me3 impact and contribute to light-regulated gene expression.ResultsIn this report, we performed a multi-dimensional genomic analysis to understand the role of H3K4me3 and H3K9me3 using the Neurospora light response as the system. RNA-seq on strains lacking H3 lysine 4 methyltransferase (KMT2/SET-1) and histone H3 lysine 9 methyltransferase (KMT1/DIM-5) revealed some light-activated genes had altered expression, but the light response was largely intact. Comparing these 2 mutants to wild-type (WT), we found that roughly equal numbers of genes showed elevated and reduced expression in the dark and the light making the environmental stimulus somewhat ancillary to the genome-wide effects. ChIP-seq experiments revealed H3K4me3 and H3K9me3 had only minor changes in response to light in WT, but there were notable alterations in H3K4me3 in Δkmt1/Δdim-5 and H3K9me3 in Δkmt2/Δset-1 indicating crosstalk and redistribution between the modifications. Integrated analysis of the RNA-seq and ChIP-seq highlighted context-dependent roles for KMT2/SET1 and KMT1/DIM-5 as either co-activators or co-repressors with some overlap as co-regulators. At a small subset of loci, H3K4 methylation is required for H3K9me3-mediated facultative heterochromatin including, the central clock gene frequency (frq). Finally, we used sequential ChIP (re-ChIP) experiment to confirm Neurospora contains K4/K9 bivalent domains.ConclusionsCollectively, these data indicate there are obfuscated regulatory roles for H3K4 methylation and H3K9 methylation depending on genome location with some minor overlap and co-dependency.

Sex‐specific Impairment of Diurnal Renal Na+ Excretion in Obese Sprague‐Dawley Rats

The role of the renal endothelin system in obesity is poorly understood despite playing a key role in excreting a high salt diet. Sodium excretion follows a diurnal rhythm that is controlled by central and peripheral clock genes. The aim of this study was to determine whether the renal endothelin system is disrupted in obesity and interferes with the diurnal rhythm of Na+ excretion. Our overarching hypothesis is that diet‐induced obesity impairs salt handling in a time of day‐dependent manner through renal endothelin dysfunction. Male and female Sprague‐Dawley rats were put on either 45 Kcal% fat (high fat, HF) or control 10 Kcal% fat (normal fat, NF) diet starting at 6 weeks of age. After 8 weeks, rats were put in metabolic cages for 2 days of acclimation, followed by 12‐hour collections of baseline urine output, food and water intake for an additional 2 days. On day 5, rats were given an acute salt load of (900 mEq NaCl in 1ml H2O, i.p.) either at the beginning of their inactive period (Zeitgeber time 0, ZT0) or active period (Zeitgeber time 12, ZT12). Urine was collected over the following 24 hours in 12‐hour intervals. Both male and female rats on HF diet showed significantly greater body weights (414 ± 6 vs 386 ± 6 gm in males, n= 12 and 303 ± 9 vs 266 ± 7 gm in females, n= 8) and total body fat measured by QMR (10.8 ± 0.5 vs 6.7 ± 0.4% in males, n= 12 and 15.3 ± 0.9 vs 9.8 ± 0.5% in females, n= 8) compared to NF controls. Male rats on HF given salt load at ZT0 showed similar urine and Na+ excretion (UNaV) rates compared to lean controls (DUNaV was 395 ± 46 vs 496 ± 71 μEq/12 hrs in the first 12 hours post‐load, n= 12). However, HF rats showed significantly delayed natriuresis in response to ZT12 salt load (DUNaV was 605 ± 98 vs 960 ± 59 μEq/12 hrs in the first 12 hours post‐load, n= 8). On the other hand, obese female rats showed an intact natriuretic response at both ZT0 (772 ± 32 vs 673 ± 84 μEq/12 hrs in the first 12 hours post‐load, n= 8) and ZT12 (671 ± 153 vs 801. ± 106 μEq/12 hrs in the first 12 hours post‐load, n=8) compared to NF. Assessment of urinary endothelin‐1 (ET1) showed significantly decreased active period ET1 excretion rates in obese males (0.3 ± 0.04 vs 0.3 ± 0.04 pg/12 hrs inactive and 0.8 ± 0.1 vs 1.5 ± 0.1 pg/12 hrs active period, n=11) and following salt challenge (0.9 ± 0.1 vs 1.7 ± 0.2 pg/12 hrs in the second 12 hrs of ZT0 salt load and 1.5 ± 0.1 vs 2.7 ± 0.1 pg/12 hrs in the first 12 hrs of ZT12 salt load, n=11). The reduced ET1 in the HF group was evident during their active period, while inactive period ET1 was similar among groups. Obese female rats, on the other hand, showed comparable urine ET1 values to their lean control, at both baseline (0.5 ± 0.2 vs 0.3 ± 0.1 pg/12 hrs at inactive and 1.1 ± 0.2 vs 1.4 ± 0.2 pg/12 hrs at active period, n=5) and following the acute salt load regardless of time of day (0.8 ± 0.1 vs 0.7 ± 0.1 pg/12 hrs at ZT0 and 1.3 ± 0.3 vs 1.6 ± 0.1 pg/12 hrs at ZT12). In conclusion, our study shows that impairment of sodium excretion in obesity is time‐of‐day and sex‐dependent. It also shows a strong correlation between renal endothelin‐1 dysfunction and impaired Na+ excretion in obese male Sprague‐Dawley ratsSupport or Funding InformationP01 HL136267 to DMPThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.