Zeitgeber Time Research Articles

Copper-induced diurnal hepatic toxicity is associated with Cry2 and Per1 in mice.

This study aimed to investigate diurnal variations in copper-induced hepatic toxicity and the molecular mechanisms underlying this chronotoxicity. Male C57BL/6J mice were intraperitoneally injected with copper chloride (CuCl2) at zeitgeber time 2 (ZT2) or 14 (ZT14), twice per week for 5 or 8 weeks. Seventy-two hours after the final CuCl2 injection, the mice were euthanized, and plasma samples were collected. The livers and kidneys were collected and weighed. In vitro experiments were performed to assess cell viability and fluctuations in clock gene expression levels in Hepa1-6 cells after CuCl2 treatment. We examined copper homeostasis- and apoptosis-related genes under clock genes overexpression. Repeated CuCl2 administration for 8 weeks resulted in more severe toxicity at ZT14 compared to ZT2. CuCl2 administration at ZT14 elevated plasma aspartate aminotransferase, hepatic tumor necrosis factor-α, and interleukin-6 for 5 weeks, whereas the toxic effects of CuCl2 administration at ZT2 were weaker. Moreover, CuCl2 treatment inhibited Hepa1-6 cell viability in a dose-dependent manner. We observed increased expression of three clock genes (Ciart, Cry2, and Per1) after CuCl2 treatment. Among them, overexpression of Cry2 and Per1 accelerated CuCl2-induced inhibition of Hepa1-6 cell viability. Moreover, we found that the overexpression of Cry2 and Per1 regulates cleaved caspase-3 by modulating the copper transporter genes ATP7B and CTR1. These results suggest that CuCl2-induced diurnal toxicity is associated with Cry2 and Per1 expression through the regulation of copper transporter genes in mice.

Brain histone beta-hydroxybutyrylation couples metabolism with gene expression.

Little is known about the impact of metabolic stimuli on brain tissue at a molecular level. The ketone body beta-hydroxybutyrate (BHB) can be a signaling molecule regulating gene transcription. Thus, we assessed lysine beta-hydroxybutyrylation (K-bhb) levels in proteins extracted from the cerebral cortex of mice undergoing a ketogenic metabolic challenge (48h fasting). We found that fasting enhanced K-bhb in a variety of proteins including histone H3. ChIP-seq experiments showed that K9 beta-hydroxybutyrylation of H3 (H3K9-bhb) was significantly enriched by fasting on more than 8000 DNA loci. Transcriptomic analysis showed that H3K9-bhb on enhancers and promoters correlated with active gene expression. One of the most enriched functional annotations both at the epigenetic and transcriptional level was "circadian rhythms''. Indeed, we found that the diurnal oscillation of specific transcripts was modulated by fasting at distinct zeitgebertimes both in the cortex and suprachiasmatic nucleus. Moreover, specific changes in locomotor activity daily features were observed during re-feeding after 48-h fasting. Thus, our results suggest that fasting remarkably impinges on the cerebral cortex transcriptional and epigenetic landscape, and BHB acts as a powerful epigenetic molecule in the brain through direct and specific histone marks remodeling in neural tissue cells.

Identification of salivary microRNA profiles in male mouse model of chronic sleep disorder

Chronic sleep disorders (CSD) comprise a potential risk factor for metabolic and cardiovascular diseases, obesity and stroke. Thus, the identification of biomarkers for CSD is an important step in the early prevention of metabolic dysfunctions induced by sleep dysfunction. Diagnostic saliva samples can be easily and noninvasively collected. Thus, we aimed to identify whole microRNA (miRNA) profiles of saliva in control and psychophysiologically stressed CSD mouse models and compare them at Zeitgeber time (ZT) 0 (lights on) and ZT12 (lights off). The findings of two-way ANOVA revealed that the expression of 342 and 109 salivary miRNAs was affected by CSD and the time of day, respectively. Interactions were found in 122 miRNAs among which, we identified 197 (ZT0) and 62 (ZT12) upregulated, and 40 (ZT0) and seven (ZT12) downregulated miRNAs in CSD mice. We showed that miR-30c-5p, which is elevated in the plasma of patients with hypersomnia, was upregulated in the saliva of CSD mice collected at ZT0. The miRNAs, miR-10a-5p, miR-146b-5p, miR-150-5p, and miR-25-3p are upregulated in the serum of humans with poor sleep quality, and these were also upregulated in the saliva of CSD mice collected at ZT0. The miRNAs miR-30c, miR146b-5p, miR150, and miR-25-5p are associated with cardiovascular diseases, and we found that plasma concentrations of brain natriuretic peptides were significantly increased in CSD mice. The present findings showed that salivary miRNA profiles could serve as useful biomarkers for predicting CSD.

PPARγ Acetylation Orchestrates Adipose Plasticity and Metabolic Rhythms.

Systemic glucose metabolism and insulin activity oscillate in response to diurnal rhythms and nutrient availability with the necessary involvement of adipose tissue to maintain metabolic homeostasis. However, the adipose-intrinsic regulatory mechanism remains elusive. Here, the dynamics of PPARγacetylation in adipose tissue are shown to orchestrate metabolic oscillation in daily rhythms. Acetylation of PPARγ displays a diurnal rhythm in young healthy mice, with the peak at zeitgeber time 0 (ZT0) and the trough at ZT18. This rhythmic pattern is deranged in pathological conditions such as obesity, aging, and circadian disruption. The adipocyte-specific acetylation-mimetic mutation of PPARγ K293Q (aKQ) restrains adipose plasticity during calorie restriction and diet-induced obesity, associated with proteolysis of a core circadian component BMAL1. Consistently, the rhythmicity in glucose tolerance and insulin sensitivity is altered in aKQ and the complementary PPARγ deacetylation-mimetic K268R/K293R (2KR) mouse models. Furthermore, the PPARγ acetylation-sensitive downstream target adipsin is revealed as a novel diurnal factor that destabilizes BMAL1 and mediates metabolic rhythms. These findings collectively signify that PPARγ acetylation is a hinge connecting adipose plasticity and metabolic rhythms, the two determinants of metabolic health.

Abstract 13596: Effects of Plasminogen Activator Inhibitor-1 on the Circadian Clock in Liver

Introduction: Plasminogen activator inhibitor-1 (PAI-1, encoded by Serpine1 ) plays a major role in the mammalian fibrinolytic system, but elevated PAI-1 activity contributes to other biological processes including the metabolic syndrome. Multiple studies suggest that disruption of circadian rhythms also promotes features of the metabolic syndrome including obesity, hypertension, and type 2 diabetes. While it has been long known that PAI-1 plasma levels exhibit a marked circadian variation, and that the expression of PAI-1 is strongly influenced by the clock, a reciprocal role for PAI-1 in regulating components of the clock itself has never been described. Hypothesis: We hypothesized that changes in PAI-1 activity influence the expression of core circadian clock genes and clock-controlled genes in the liver. Methods: We used two mouse models of reduced PAI-1 activity: oral PAI-1 inhibitor TM5614 (20 mg/kg/day) versus vehicle in C57BL/6J wild-type mice (n = 5 per group), and Serpine +/- heterozygous mice versus wild-type littermate control mice (n = 3 per group). Mice were maintained on a 14:10 light:dark cycle. Livers were collected between 3 and 4 hours after lights-on, which corresponds to Zeitgeber Time (ZT) 3-4. RNA was isolated for RNA-seq and downstream analyses. Results: Between ZT 3-4, we found that reduced PAI-1 activity led to marked changes in circadian gene expression in the liver. Both PAI-1 inhibition with TM5614 and PAI-1 haploinsufficiency significantly increased expression of the core circadian clock components Arntl (aka Bmal1) and Clock while significantly reducing expression of core clock components Per1, Per2, Per3, and clock output genes. Furthermore, we found that PAI-1 inhibition in aged transgenic mice overexpressing the stable variant of human SERPINE1 rescued two markers of impaired circadian function: 1) the premature onset of activity prior to lights-off, and 2) reduced feeding during the lights-off period. Conclusions: Taken together, these data support a reciprocal role for PAI-1 in regulating components of the circadian clock. Elevated PAI-1 activity may contribute to the metabolic syndrome through effects on the circadian clock and may provide a mechanistic link between PAI-1, the circadian clock, and aging.

Action potential firing rhythms in the suprachiasmatic nucleus of the diurnal grass rat, Arvicanthis niloticus

In mammals, daily physiological activities are regulated by a central circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). Recently, an increasing number of studies have used diurnal grass rats to analyze neuronal mechanisms regulating diurnal behavior. However, spontaneous action potential firing rhythms in SCN neurons have not been demonstrated clearly in diurnal grass rats. Therefore, the present study examined extracellular single-unit recordings from SCN neurons in acute hypothalamic slices of Arvicanthis niloticus (Nile grass rats). The results of this study found that circadian firing rhythms with the highest frequency occurred at dusk (6.4 Hz at zeitgeber time (ZT)10–12), while the secondary peak occurred at dawn (5.6 Hz at ZT0-2), and the lowest frequency took place in the middle of the night (3.6 Hz at ZT14-16). Locomotor activity recordings from a separate group of animals demonstrated that the Nile grass rats of the laboratory colony used in this study displayed diurnal behaviors that coincided with large crepuscular peaks under 12:12 h light–dark cycles and bimodal rhythms under constant dim red light. Thus, a positive correlation between SCN firing frequencies and locomotor activity levels was observed in the Nile grass rats. Previously, behavioral coupling of action potential firings in SCN neurons has been suggested by in vivo recordings while the present study demonstrates that the sustenance of bimodal firing rhythms in grass rat SCN neurons can last at least one day in vitro.

Diurnal Differences in Immune Response in Brain, Blood and Spleen After Focal Cerebral Ischemia in Mice.

The immune response to acute cerebral ischemia is a major factor in stroke pathobiology. Circadian biology modulates some aspects of immune response. The goal of this study is to compare key parameters of immune response during the active/awake phase versus inactive/sleep phase in a mouse model of transient focal cerebral ischemia. Mice were housed in normal or reversed light cycle rooms for 3 weeks, and then they were blindly subjected to transient focal cerebral ischemia. Flow cytometry was used to examine immune responses in blood, spleen, and brain at 3 days after ischemic onset. In blood, there were higher levels of circulating T cells in mice subjected to focal ischemia during zeitgeber time (ZT)1-3 (inactive or sleep phase) versus ZT13-15 mice (active or awake phase). In the spleen, organ weight and immune cell numbers were lower in ZT1-3 versus ZT13-15 mice. Consistent with these results, there was an increased infiltration of activated T cells into brain at ZT1-3 compared with ZT13-15. This proof-of-concept study indicates that there are significant diurnal effects on the immune response after focal cerebral ischemia in mice. Hence, therapeutic strategies focused on immune targets should be reassessed to account for the effects of diurnal rhythms and circadian biology in nocturnal rodent models of stroke.

Long-term environmental exposure of darkness induces hyperandrogenism in PCOS via melatonin receptor 1A and aromatase reduction.

Polycystic ovary syndrome (PCOS) is a common and complex disorder impairing female fertility, yet its etiology remains elusive. It is reported that circadian rhythm disruption might play a crucial role in PCOS pathologic progression. Here, in this research, we investigated the effect of environmental long-term circadian rhythm dysfunction and clarified its pathogenic mechanism in the development of PCOS, which might provide the targeted clinical strategies to patients with PCOS. Female SD rats were used to construct a circadian rhythm misalignment model with constant darkness (12/12-h dark/dark cycle), and the control group was kept under normal circadian rhythm exposure (12/12-h light/dark cycle) for 8 weeks. We measured their reproductive, endocrinal, and metabolic profiles at different zeitgeber times (ZTs). Different rescue methods, including melatonin receptor agonist and normal circadian rhythm restoration, and in vitro experiments on the KGN cell line were performed. We found that long-term darkness caused PCOS-like reproductive abnormalities, including estrous cycle disorder, polycystic ovaries, LH elevation, hyperandrogenism, and glucose intolerance. In addition, the expression of melatonin receptor 1A (Mtnr1a) in ovarian granulosa cells significantly decreased in the darkness group. Normal light/dark cycle and melatonin receptor agonist application relieved hyperandrogenism of darkness-treated rats. In vitro experiments demonstrated that decreased MTNR1A inhibited androgen receptor (AR) and CYP19A1 expression, and AR acted as an essential downstream factor of MTNR1A in modulating aromatase abundance. Overall, our finding demonstrates the significant influence of circadian rhythms on PCOS occurrence, suggests that MTNR1A and AR play vital roles in pathological progression of hyperandrogenism, and broadens current treatment strategies for PCOS in clinical practice.

Sleep Alterations in a Mouse Model of Spinocerebellar Ataxia Type 3.

Spinocerebellar ataxia type 3 (SCA3) is a neurodegenerative disorder showing progressive neuronal loss in several brain areas and a broad spectrum of motor and non-motor symptoms, including ataxia and altered sleep. While sleep disturbances are known to play pathophysiologic roles in other neurodegenerative disorders, their impact on SCA3 is unknown. Using spectrographic measurements, we sought to quantitatively characterize sleep electroencephalography (EEG) in SCA3 transgenic mice with confirmed disease phenotype. We first measured motor phenotypes in 18–31-week-old homozygous SCA3 YACMJD84.2 mice and non-transgenic wild-type littermate mice during lights-on and lights-off periods. We next implanted electrodes to obtain 12-h (zeitgeber time 0-12) EEG recordings for three consecutive days when the mice were 26–36 weeks old. EEG-based spectroscopy showed that compared to wild-type littermates, SCA3 homozygous mice display: (i) increased duration of rapid-eye movement sleep (REM) and fragmentation in all sleep and wake states; (ii) higher beta power oscillations during REM and non-REM (NREM); and (iii) additional spectral power band alterations during REM and wake. Our data show that sleep architecture and EEG spectral power are dysregulated in homozygous SCA3 mice, indicating that common sleep-related etiologic factors may underlie mouse and human SCA3 phenotypes.

The landscape of Chlamydomonas histone H3 lysine 4 methylation reveals both constant features and dynamic changes during the diurnal cycle.

Chromatin modifications are epigenetic regulatory features with major roles in various cellular events, yet they remain understudied in algae. We interrogated the genome-wide distribution pattern of mono- and trimethylated histone H3 lysine 4 (H3K4) using chromatin-immunoprecipitation followed by deep-sequencing (ChIP-seq) during key phases of the Chlamydomonas cell cycle: early G1 phase, Zeitgeber Time 1 (ZT1), when cells initiate biomass accumulation, S/M phase (ZT13) when cells are replicating DNA and undergoing mitosis, and late G0 phase (ZT23) when they are quiescent. Tri-methylated H3K4 was predominantly enriched at transcription start sites of the majority of protein coding genes (85%). The likelihood of a gene being marked by H3K4me3 correlated with it being transcribed at some point during the life cycle but not necessarily by continuous active transcription, as exemplified by early zygotic genes, which may remain transcriptionally dormant for thousands of generations between sexual cycles. The exceptions to this rule were around 120 loci, some of which encode non-poly-adenylated transcripts, such as small nuclear RNAs and replication-dependent histones that had H3K4me3 peaks only when they were being transcribed. Mono-methylated H3K4 was the default state for the vast majority of histones that were bound outside of transcription start sites and terminator regions of genes. A small fraction of the genome that was depleted of any H3 lysine 4 methylation was enriched for DNA cytosine methylation and the genes within these DNA methylation islands were poorly expressed. Besides marking protein coding genes, H3K4me3 ChIP-seq data served also as a annotation tool for validation of hundreds of long non-coding RNA genes.

The circadian clock sets a spatial-temporal window for recent thymic emigrants.

The diurnal timing system regulates multiple functions of lymphocytes in peripheral lymphoid organs. Whether T-cell development in the thymus and T-cell egress from the thymus are affected by the circadian clock is not clear. Herein, we used flow cytometry to examine the cell number and percentage of total thymocytes and various thymocyte subsets from Zeitgeber time (ZT) 1 to ZT21. CD4 and CD8 single-positive (SP) thymocytes, in particular, the mature CD4 SP4 thymocyte subset with emigration capability and P-phycoerythrin+ CD4 SP thymocytes in the perivascular space of the thymus, exhibited robust circadian oscillations. The diurnal expression of sphingosine-1-phosphate receptor-1 (S1PR1) and CCR2 on SP thymocytes and the rhythmic sphingosine-1-phosphate (S1P) and CCL2 gradient formed between peripheral blood and thymus likely promoted SP thymocyte egress in a circadian pattern. Switching the daylight cycle disturbed the rhythm of S1PR1 and CCR2 expression and subsequent thymocyte output. We further demonstrated that the core clock molecule BMAL1 had rhythmic binding of the promoters of Klf2, S1pr1 and Sphk2. Together, we elucidated the circadian dynamic characteristics of mature thymocyte egress, which coordinated with the diurnal changes in T-cell homing to the lymph nodes. The core rhythmic molecule BMAL1 likely promoted thymocyte emigration through transcriptional regulation of emigration-related molecules.

Abstract 096: Adrenal-specific KO Of The Circadian Clock Protein Bmal1 Alters Blood Pressure Rhythm

BMAL1 is a core circadian clock protein that regulates most physiological functions, including blood pressure (BP). Global BMAL1 knockout (KO) male mice exhibit reduced, non-dipping BP and loss of diurnal rhythm of renal sodium (Na) excretion. Although, BP and renal Na excretion rhythm remained intact in kidney-specific BMAL1 KO male mice. The adrenal gland synthesizes hormones which influence BP rhythm and renal electrolyte handling. The role of adrenal BMAL1 in the regulation of BP and renal function remains unknown. The goal was to test the hypothesis that adrenal BMAL1 is required for normal circadian rhythms of BP and renal Na excretion. Adrenal-specific aldosterone synthase Cre positive (AS)-BMAL1 KO male mice ( n =5) and littermate Cre negative, floxed control (CNTL) male mice ( n =6) underwent a telemetry study to measure BP. Cosinor analysis was performed to assess BP rhythms. During telemetry recordings, mice were placed in metabolic cages for 5 days on a normal salt diet. The latter 3 days, 12 h urine samples were collected before each light-dark Zeitgeber (6AM and 6PM/ Zeitgeber Time (ZT)0 and ZT12) to measure night/day patterns in renal Na excretion. Kidneys were collected from a separate cohort of mice ( n =7-8/genotype) at ZT0 and ZT12 for gene expression analysis of key Na transporters. No BMAL1-dependent changes in the 24 h averages for BP were observed. Cosinor analysis revealed significant differences in BP rhythm, however. The period (time taken for a full circadian cycle, ~24 h) was significantly shortened for BP in AS-BMAL1 KO mice vs. CNTL (23 h 31 vs 24 h 16, p =0.01). Acrophase (the time at which BP peaks) was shifted ~2 hours later in KO mice (KO ZT17 h 38 vs CNTL ZT15 h 45, p =0.002). The MESOR (midline estimating statistic of rhythm) of BP remained similar between groups (KO 133.5 mmHg vs CNTL 132.7 mmHg, p =0.904). Day/night Na excretion was not different in AS-BMAL1 KO mice vs. CNTL (ANOVA interaction p =0.168). This was consistent with a lack of genotype effects on gene expression for NCC, α, β, or γ ENaC. Significant time of day effects were observed for all 4 genes (all with p <0.001). In conclusion, BMAL1 KO in the adrenal gland altered BP rhythm, with no changes in renal Na excretion. This work is the first to investigate the role of adrenal BMAL1 on BP regulation.

Abstract 094: Bmal1 Is Necessary For Food-Based Entrainment Of Blood Pressure And Temperature Rhythms In The Rat

Blood pressure (BP) follows a circadian rhythm, changing throughout the day. This change is crucial for cardiovascular health and in patient populations, as loss of these rhythms are associated with increased mortality and morbidity. Though BP rhythms have been recognized as important, the factors that control and entrain them are not well-described. Since the molecular clock is ubiquitously expressed in all cells and required for many circadian processes, we tested the hypothesis that the molecular clock contributes to the timing of BP rhythms in rats. Male and female rats with whole body loss of brain and muscle ARNTL-like protein ( Bmal1 ) and wild type (WT) controls (Sprague-Dawley background) were instrumented with telemeters in the abdominal aorta and placed in 12:12h light dark cycle (LD) to measure BP and core body temperature (TMP). Next, animals were placed in constant darkness (DD) for four weeks and then placed on a 12-hour time restricted feeding regimen (DD-TRF) for five days. Cosinor analysis was used to analyze BP and TMP rhythms. In a standard LD cycle, systolic BP (SBP) and TMP rhythms of both Bmal1 -knockout ( Bmal1- KO) and WT rats have similar amplitudes (2-way ANOVA for SBP and TMP, both p>0.05) which peak around zeitgeber time (ZT)18. During DD, WTs have SBP and TMP rhythms peak at circadian time (CT)19, while Bmal1 -KO rats have SBP and TMP rhythms that peak at CT 12. SBP amplitude is lower in all groups during DD compared to LD (WT males: 6.0±0.7 vs 3.8±0.7 mmHg; WT females: 7.2±0.9 vs 5.6±0.3 mmHg; KO males: 5.4±0.6 vs 4.7±0.4 mmHg; KO females: 6.2±0.6 vs 5.3±0.8 mmHg; 3-way ANOVA, p<0.05 for light cycle). During DD-TRF (CT 14.5 – 2.5), SBP and TMP rhythms of WTs peak at CT22. Bmal1 -KO rats have SBP and TMP rhythms that peak during CT16. SBP amplitude is lowered in Bmal1 -KO rats during DD-TRF compared to DD (KO males: 2.0±0.7 mmHg; KO females: 3.8±0.6 mmHg) but not in WT rats (WT males: 3.8±0.3 mmHg; WT females: 5.1±0.4 mmHg; 3-way ANOVA, p<0.05 for feeding time, sex, and feeding time*genotype). These data suggest that the molecular clock component Bmal1 is necessary for food-based entrainment of SBP and temperature rhythms in the absence of a regular light cue. Food, light cycle, and the molecular clock may all contribute to the timing and amplitude of SBP and TMP rhythms.

Tip60/Kat5 may be a novel candidate histone acetyltransferase for the regulation of liver iron localization via acetylation.

Hepcidin (HAMP), an iron regulatory hormone synthesized by liver hepatocytes, works together with ferritin (FTH) and ferroportin (FPN) in regulating the storage, transport, and utilization of iron in the cell. Epigenetic mechanisms, especially acetylation, also play an important role in the regulation of iron metabolism. However, a target protein has not been mentioned yet. With this preliminary study, we investigated the effect of histone acetyltransferase TIP60 on the expression of HAMP, FTH, and FPN. In addition, how the depletion of Tip60, which regulates the circadian system, affects the daily expression of Hamp was examined at six Zeitgeber time (ZT) points. For this purpose, liver-specific Tip60 knockout mice (mutant) were produced with tamoxifen-inducible Cre/lox recombination and an iron overload model in mice was generated. While HAMP and FTH expressions decreased, FPN expression increased in the mutant group. Interestingly, there was no change in the iron content. A significant increase was observed in the expressions of HAMP, FTH, and FPN and total liver iron content in the liver tissue of the iron overload group. Since intracellular iron concentration is involved in regulating the circadian clock, temporal expression of Hamp was investigated in control and mutant groups at six ZT points. In the control group, Hamp accumulated in a circadian manner with maximal and minimal levels reaching around ZT16 and ZT8, respectively. In the mutant group, there was a significant reduction in Hamp expression in the light phase ZT0 and ZT4 and in the dark phase ZT16. These data are the first findings demonstrating a possible relationship between Tip60 and iron metabolism.

The Lack of Bmal1, a Core Clock Gene, in the Intestine Decreases Glucose Absorption in Mice.

The circadian clock network is an evolutionarily conserved system that regulates systemic metabolism, such as glucose homeostasis. Intestinal tissue is a pivotal organ for the regulation of glucose metabolism, mainly via glucose absorption into the circulation; however, the significance of the intestinal circadian clock network for glucose metabolism remains largely unclear. We herein utilized a mouse model in which Bmal1, a core clock gene, was deleted in an intestine-specific manner (Bmal1Int-/- mice) and demonstrated a rhythmic expression of Sglt1 with its peak at zeitgeber time (ZT) 10.7 ± 2.8 in control mice, whereas this was lost in Bmal1Int-/- mice. Mechanistically, chromatin immunoprecipitation analysis revealed rhythmic binding of CLOCK to the E-box elements in the Sglt1 gene in control mice; however, this was absent in Bmal1Int-/- mice. Accordingly, SGLT1 protein levels were decreased during the dark phase in Bmal1Int-/- mice and this was associated with impaired glucose absorption, leading to a decline in hepatic glycogen levels at ZT4, which was restored by ingestion of high-sucrose water. Additionally, when mice were starved from ZT0, greater expression of the lipolysis-related gene Pnpla2 was observed in adipose tissue of Bmal1Int-/- mice, and this was not noted when glycogen storage was restored by high-sucrose water prior to fasting, suggesting that higher Pnpla2 expression in Bmal1Int-/- mice was likely caused by lower glycogen storage. These results indicate that disruption of the intestinal circadian clock system impairs glucose absorption in the intestine and affects systemic glucose homeostasis.

Neutrophil circadian rhythm is associated with different outcomes of acute kidney injury due to cholesterol crystal embolism.

Cholesterol crystal (CC) embolism can cause acute tissue infarction and ischemic necrosis via triggering diffuse thrombotic angiopathy occluding arterioles and arteries. Neutrophils contribute to crystal-induced immunothrombosis as well as to ischemic necrosis-related necroinflammation. We speculated that CC embolism-induced acute kidney injury (AKI) would be circadian rhythm-dependent and associated with cyclic differences in neutrophil function. Injection of CC into the left kidney induced thrombotic angiopathy progressing starting as early as 3 h after CC injection followed by a progressive ischemic cortical necrosis and AKI at 24 h. In C57BL/6J mice, circulating CD11b+Ly6G+ neutrophils were higher during the day phase [Zeitgeber time (ZT) 0–12] compared to the dark phase (ZT12-24). In the time frame of thrombus formation at ZT13, more neutrophils were recruited into the injured kidney 24 h later compared to CC embolism at ZT5. This effect was associated with an increased circulating number of CXCR2+ neutrophils as well as an upregulated kidney adhesion molecule and chemokine expression. These findings were associated with a significant increase in kidney necrosis, and endothelial injury at ZT13. Thus, the time of day has an effect also on CC embolism-related AKI in association with the circadian rhythm of neutrophil recruitment.

Wheat alkylresorcinol increases fecal lipid excretion and suppresses feed efficiency in mice depending on time of supplementation

ObjectiveThe regular consumption of whole grains is linked to a lower likelihood of developing metabolic disorders. We previously found that chronic supplementation with wheat alkylresorcinols (ARs) prevents obesity and its associated metabolic symptoms induced by a high-fat high-sucrose diet (HFHSD) in mice. The aim of this study was to examine the time-of-day-dependence of these effects in mice. MethodsEight-wk-old male C57 BL/6 J mice were individually housed under a 12-h light/dark cycle (Zeitgeber time; ZT0, lights on; ZT12, lights off) and given access to a HFHSD from ZT12–16 (activity onset) and ZT20–24 (activity offset) to respectively represent breakfast and dinner times for 3 wk. Thereafter, the HFHSD was replaced with the same diet containing 0.4% ARs at either ZT12–16 or ZT20–24 for 8 wk. Control mice received the HFHSD without ARs at both feeding times. ResultsSupplementation with ARs significantly suppressed feed efficiency when given at breakfast, but not at dinner. ARs consumed at breakfast increased fecal lipid content and decreased the expression of Fat/Cd36 in enterocytes that enhances lipid uptake, but did not affect hepatic and blood lipid levels. The consumption of ARs at breakfast also upregulated the expression of Irs1, a key gene for insulin signaling in white adipose tissue and attenuated elevated blood leptin levels induced by the diet. This led to high scores for the homeostasis model assessment of insulin sensitivity, and the adiponectin/leptin ratio, a negative index of adipose tissue dysfunction. ConclusionsThese findings suggested that ARs ameliorate feed efficiency by decreasing dietary lipid absorption more effectively at the time of activity onset than offset. Further studies are needed to elucidate the molecular mechanism of the time-of-day-dependent effects of ARs on diet-induced metabolic disorders.

Administration time-dependent effects of poly (I:C) on antioxidant and immune responses along the diurnal time scale in zebrafish

ABSTRACT The circadian clock of vertebrates regulates many biological processes, including the immune system. This paper investigated whether responsiveness to poly (I:C), a synthetic analog of double-stranded RNA used as an immunostimulant, exhibits day/night differences in zebrafish. Fish were intraperitoneally (IP) injected with either phosphate-buffered saline (PBS) or poly (I:C) at two different time points: “Zeitgeber Time” (ZT) 4 (day) and ZT16 (night). Then, 6 h later, fish were euthanized, and tissue samples (skin, liver and kidney) were collected. A control group (intact fish) was also sampled at the same time points. The effect of poly (I:C) on the expression of antioxidant and immune genes was time-of-day-dependent, and the response was stronger following poly (I:C) administration in the day than at night. Time-dependent differences were observed for some genes in the PBS and control groups. However, these differences were tissue-specific. In liver, almost all the genes were affected by time of day. In kidney, poly (I:C) affected the expression of all the gene markers regardless of administration time. These findings highlight the importance of considering the time to administer poly (I:C) when evaluating the fish immune response.

The Change in Circadian Rhythms in P301S Transgenic Mice is Linked to Variability in Hsp70-related Tau Disaggregation.

Circadian disruption often involves a neurodegenerative disorder, such as Alzheimer’s disease or frontotemporal dementia, which are characterized by intraneuronal tau accumulations. The altered sleep pattern and diurnal rhythms in these disorders are the results of tau pathology. The circadian disturbance in reverse is thought to develop and potentially aggravate the condition. However, the underlying mechanism is not fully understood. In this study, perturbed oscillations in BMAL1, the core clock gene, were observed in P301S tau transgenic mice. Tau fractionation analysis of the hippocampus revealed profound fluctuations in soluble and insoluble tau protein levels that were in opposite directions to each other according to zeitgeber time. Interestingly, a diurnal oscillation was detected in the heat shock 70 kDa protein 1A (Hsp70) chaperone that was in-phase with soluble tau but out-of-phase with insoluble tau. Tau protein levels decreased in the soluble and insoluble fractions when Hsp70 was overexpressed in HEK293T cells. Transfection of the BMAL1 carrying vector was continual with the increase in Hsp70 expression and diminished tau protein levels, and it was effectively attenuated by the knockdown of Hsp70, suggesting that Bmal1 could modulate tau protein by Hsp70. Our results suggest that altered circadian oscillations affect tau status and solubility by modulating Hsp70 expression in an experimental model of tau pathology. These findings suggest Hsp70 as a possible pathogenic link between circadian disruption and aggravations of tau pathology.

Daily rhythm of some blood parameters in two Amazonian fish, Astronotus ocellatus and Brycon amazonicus

We evaluated the effect of the light–dark (LD) cycle on energy expenditure indicators for two species of Amazonian fish of commercial interest, oscar (Astronotus ocellatus) and matrinxã (Brycon amazonicus). Fish were exposed to a 12:12 h LD cycle, with lights on at ‘zeitgeber time’ (ZT) = 0 h. Six animals were used for blood collection every 4 h for 24 h (six time points, three in the light period and three in the dark period). Most haematological parameters exhibited daily rhythms with the acrophase at the end of the dark phase or the beginning of the light period for both species, which may be related to the greater energy demand of these species during the day. For A. ocellatus, triglycerides and total protein showed acrophases at ZT = 13:32 ± 2:45 h and ZT = 23:42 ± 1:55 h, respectively, while the other plasma parameters showed no significant daily differences. In B. amazonicus, significant rhythms were observed for glucose levels (acrophase at ZT = 04:16 ± 2:47 h), triglycerides (ZT = 6:51 ± 4:06 h) and total cholesterol (ZT = 23:22 ± 3:45 h). However, total protein and cortisol levels did not show rhythmicity in this species. Our results highlight the importance of the evaluation of biological rhythms for plasma physiological parameters related to energy expenditure in species with commercial interest and susceptible to stressful situations in farming conditions.