Excessive fat expenditure in MCT-induced heart failure rats is associated with BMAL1/REV-ERBα circadian rhythmic loop disruption.

Fat loss predicts adverse outcomes in advanced heart failure (HF). Disrupted circadian clocks are a primary cause of lipid metabolic issues, but it's unclear if this disruption affects fat expenditure in HF. To address this issue, we investigated the effects of disruption of the BMAL1/REV-ERBα circadian rhythmic loop on adipose tissue metabolism in HF.50 Wistar rats were initially divided into control (n = 10) and model (n = 40) groups. The model rats were induced with HF via monocrotaline (MCT) injections, while the control group received equivalent solvent injections. After establishing the HF model, the model group was further subdivided into four groups: normal rhythm (LD), inverted rhythm (DL), lentivirus vector carrying Bmal1 short hairpin RNA (LV-Bmal1 shRNA), and empty lentivirus vector control (LV-Control shRNA) groups, each with 10 rats. The DL subgroup was exposed to a reversed light-dark cycle of 8 h: 16 h (dark: light), while the rest adhered to normal light-dark conditions (light: dark 12 h: 12 h). Histological analyses were conducted using H&E, Oil Red O, and Picrosirius red stains to examine adipose and liver tissues. Immunohistochemical staining, RT-qPCR, and Western blotting were performed to detect markers of lipolysis, lipogenesis, and beiging of white adipose tissue (WAT), while thermogenesis indicators were detected in brown adipose tissue (BAT). The LD group rats exhibited decreased levels of BMAL1 protein, increased levels of REV-ERBα protein, and disrupted circadian circuits in adipose tissue compared to controls. Additionally, HF rats showed reduced adipose mass and increased ectopic lipid deposition, along with smaller adipocytes containing lower lipid content and fibrotic adipose tissue. In the LD group WAT, expression of ATGL, HSL, PKA, and p-PKA proteins increased, alongside elevated mRNA levels of lipase genes (Hsl, Atgl, Peripilin) and FFA ß-oxidation genes (Cpt1, acyl-CoA). Conversely, lipogenic gene expression (Scd1, Fas, Mgat, Dgat2) decreased, while beige adipocyte markers (Cd137, Tbx-1, Ucp-1, Zic-1) and UCP-1 protein expression increased. In BAT, HF rats exhibited elevated levels of PKA, p-PKA, and UCP-1 proteins, along with increased expression of thermogenic genes (Ucp-1, Pparγ, Pgc-1α) and lipid transportation genes (Cd36, Fatp-1, Cpt-1). Plasma NT-proBNP levels were higher in LD rats, accompanied by elevated NE and IL-6 levels in adipose tissue. Remarkably, morphologically, the adipocytes in the DL and LV-Bmal1 shRNA groups showed reduced size and lower lipid content, while lipid deposition in the liver was more pronounced in these groups compared to the LD group. At the gene/protein level, the BMAL1/REV-ERBα circadian loop exhibited severe disruption in LV-Bmal1 shRNA rats compared to LD rats. Additionally, there was increased expression of lipase genes, FFA ß oxidation genes, and beige adipocyte markers in WAT, as well as higher expression of thermogenic genes and lipid transportation genes in BAT. Furthermore, plasma NT-proBNP levels and adipose tissue levels of NE and IL-6 were elevated in LV-Bmal1 shRNA rats compared with LD rats. The present study demonstrates that disruption of the BMAL1/REV-ERBα circadian rhythmic loop is associated with fat expenditure in HF. This result suggests that restoring circadian rhythms in adipose tissue may help counteract disorders of adipose metabolism and reduce fat loss in HF.

Circadian Gene Variants: Effects in Overweight and Obese Pregnant Women.

Obesity and overweight are common and complex conditions influenced by multiple genetic and environmental factors. Several genetic variants located in the genes involved in clock systems and fat taste perception can affect metabolic health. In particular, the polymorphisms in CLOCK and BMAL1 genes were reported to be significantly related to cardiovascular disease, metabolic syndrome, sleep reduction, and evening preference. Moreover, genetic variants in the CD36 gene have been shown to be involved in lipid metabolism, regulation of fat intake, and body weight regulation. The aim of this study is to evaluate, for the first time, the association between variants in some candidate genes (namely, BMAL1 rs7950226 (G>A), CLOCK rs1801260 (A>G), CLOCK rs4864548 (G>A), CLOCK rs3736544 (G>A), CD36 rs1984112 (A>G), CD36 rs1761667 (G>A)) and overweight/obesity (OB) in pregnant women. A total of 163 normal-weight (NW) and 128 OB participants were included. A significant correlation was observed between A-allele in CLOCK rs4864548 and an increased risk of obesity (OR: 1.97; 95% CI 1.22-3.10, p = 0.005). In addition, we found that subjects carrying the haplotype of rs1801260-A, rs4864548-A, and rs3736544-G are likely to be overweight or obese (OR 1.47, 95% CI 1.03-2.09, p = 0.030), compared with those with other haplotypes. Moreover, a significant relation was observed between third-trimester lipid parameters and genetic variants-namely, CD36 rs1984112, CD36 rs1761667, BMAL1 rs7950226, and CLOCK rs1801260. A multivariate logistic regression model revealed that CLOCK rs4864548 A-allele carriage was a strong risk factor for obesity (OR 2.05, 95% CI 1.07-3.93, p = 0.029); on the other hand, greater adherence to Mediterranean diet (OR 0.80, 95% CI 0.65-0.98, p = 0.038) and higher HDL levels (OR 0.96, 95% CI 0.94-0.99, p = 0.021) were related to a reduced risk of obesity. Interestingly, an association between maternal CLOCK rs4864548 and neonatal birthweight was detected (p = 0.025). These data suggest a potential role of the polymorphisms in clock systems and in fat taste perception in both susceptibility to overweight/obesity and influencing the related metabolic traits in pregnant women.

Bmal1 integrates circadian function and temperature sensing in the suprachiasmatic nucleus.

Circadian regulation and temperature dependency are important orchestrators of molecular pathways. How the integration between these two drivers is achieved, is not understood. We monitored circadian- and temperature-dependent effects on transcription dynamics of cold-response protein RNA Binding Motif 3 (Rbm3). Temperature changes in the mammalian master circadian pacemaker, the suprachiasmatic nucleus (SCN), induced Rbm3 transcription and regulated its circadian periodicity, whereas the core clock gene Per2 was unaffected. Rbm3 induction depended on a full Brain And Muscle ARNT-Like Protein 1 (Bmal1) complement: reduced Bmal1 erased Rbm3 responses and weakened SCN circuit resilience to temperature changes. By focusing on circadian and temperature dependency, we highlight weakened transmission between core clock and downstream pathways as a potential route for reduced circadian resilience.

[Effects of BMAL2 on Aerobic Glycolysis and Cell Proliferation in Acute Myeloid Leukemia Cells].

OBJECTIVE: To explore the expression of basic helix-loop-helix ARNT like 2 (BMAL2) in acute myeloid leukemia (AML) patients and its correlation with prognosis, and analyze its effects on the aerobic glycolysis and proliferation of AML cells. METHODS: The expressions of BMAL2 in bone marrow mononuclear cells (BMMCs) of AML patients and normal control group were detected by RT-qPCR. The correlation of BMAL2 expression with prognosis of AML patients was analyzed using public database of National Center for Biotechnology Information (NCBI). The interfering in BMAL2 expression of HL-60 and Kasumi-1 cells was performed using lentiviral vector-mediated shRNA. Cell glucose metabolism and proliferation were detected by using glucose uptake experiment, lactate content test, CCK-8 assay and cell colony formation test. RESULTS: The expression level of BMAL2 mRNA in BMMCs of AML patients was significantly higher than normal control group (P < 0.01). The overall survival time of AML patients with high expression of BMAL2 was significantly shorter than those with low expression of BMAL2 (P < 0.05). Knockdown of BMAL2 significantly reduced glucose uptake and lactate production in AML cell line HL-60 and Kasumi-1 cells. The results of RT-PCR and Western blot showed that BMAL2 promoted aerobic glycolysis by enhancing the expression of HIF1A in AML cells, thereby promoting cell proliferation. CONCLUSION: BMAL2 is highly expressed in AML patients, and promotes aerobic glycolysis by enhancing the expression of HIF1A, thereby promoting cell proliferation.

Circadian coupling of mitochondria in a deep-diving mammal.

Regulation of mitochondrial oxidative phosphorylation is essential to match energy supply to changing cellular energy demands, and to cope with periods of hypoxia. Recent work implicates the circadian molecular clock in control of mitochondrial function and hypoxia sensing. Because diving mammals experience intermittent episodes of severe hypoxia, with diel patterning in dive depth and duration, it is interesting to consider circadian-mitochondrial interaction in this group. Here, we demonstrate that the hooded seal (Cystophora cristata), a deep-diving Arctic pinniped, shows strong daily patterning of diving behaviour in the wild. Cultures of hooded seal skin fibroblasts exhibit robust circadian oscillation of the core clock genes per2 and arntl. In liver tissue collected from captive hooded seals, expression of arntl was some 4-fold higher in the middle of the night than in the middle of the day. To explore the clock-mitochondria relationship, we measured the mitochondrial oxygen consumption in synchronized hooded seal skin fibroblasts and found a circadian variation in mitochondrial activity, with higher coupling efficiency of complex I coinciding with the trough of arntl expression. These results open the way for further studies of circadian-hypoxia interactions in pinnipeds during diving.

Activation of the PI3K/AKT signaling pathway by ARNTL2 enhances cellular glycolysis and sensitizes pancreatic adenocarcinoma to erlotinib.

BACKGROUND: Pancreatic adenocarcinoma (PC) is an aggressive malignancy with limited treatment options. The poor prognosis primarily stems from late-stage diagnosis and when the disease has become therapeutically challenging. There is an urgent need to identify specific biomarkers for cancer subtyping and early detection to enhance both morbidity and mortality outcomes. The addition of the EGFR tyrosine kinase inhibitor (TKI), erlotinib, to gemcitabine chemotherapy for the first-line treatment of patients with advanced pancreatic cancer slightly improved outcomes. However, restricted clinical benefits may be linked to the absence of well-characterized criteria for stratification and dependable biomarkers for the prediction of treatment effectiveness. METHODS AND RESULTS: We examined the levels of various cancer hallmarks and identified glycolysis as the primary risk factor for overall survival in PC. Subsequently, we developed a glycolysis-related score (GRS) model to accurately distinguish PC patients with high GRS. Through in silico screening of 4398 compounds, we discovered that erlotinib had the strongest therapeutic benefits for high-GRS PC patients. Furthermore, we identified ARNTL2 as a novel prognostic biomarker and a predictive factor for erlotinib treatment responsiveness in patients with PC. Inhibition of ARNTL2 expression reduced the therapeutic efficacy, whereas increased expression of ARNTL2 improved PC cell sensitivity to erlotinib. Validation in vivo using patient-derived xenografts (PDX-PC) with varying ARNTL2 expression levels demonstrated that erlotinib monotherapy effectively halted tumor progression in PDX-PC models with high ARNTL2 expression. In contrast, PDX-PC models lacking ARNTL2 did not respond favorably to erlotinib treatment. Mechanistically, we demonstrated that the ARNTL2/E2F1 axis-mediated cellular glycolysis sensitizes PC cells to erlotinib treatment by activating the PI3K/AKT signaling pathway. CONCLUSIONS: Our investigations have identified ARNTL2 as a novel prognostic biomarker and predictive indicator of sensitivity. These results will help to identify erlotinib-responsive cases of PC and improve treatment outcomes. These findings contribute to the advancement of precision oncology, enabling more accurate and targeted therapeutic interventions.

ß-Receptor blocker enhances the anabolic effect of PTH after osteoporotic fracture.

The autonomic nervous system plays a crucial role in regulating bone metabolism, with sympathetic activation stimulating bone resorption and inhibiting bone formation. We found that fractures lead to increased sympathetic tone, enhanced osteoclast resorption, decreased osteoblast formation, and thus hastened systemic bone loss in ovariectomized (OVX) mice. However, the combined administration of parathyroid hormone (PTH) and the ß-receptor blocker propranolol dramatically promoted systemic bone formation and osteoporotic fracture healing in OVX mice. The effect of this treatment is superior to that of treatment with PTH or propranolol alone. In vitro, the sympathetic neurotransmitter norepinephrine (NE) suppressed PTH-induced osteoblast differentiation and mineralization, which was rescued by propranolol. Moreover, NE decreased the PTH-induced expression of Runx2 but enhanced the expression of Rankl and the effect of PTH-stimulated osteoblasts on osteoclastic differentiation, whereas these effects were reversed by propranolol. Furthermore, PTH increased the expression of the circadian clock gene Bmal1, which was inhibited by NE-ßAR signaling. Bmal1 knockdown blocked the rescue effect of propranolol on the NE-induced decrease in PTH-stimulated osteoblast differentiation. Taken together, these results suggest that propranolol enhances the anabolic effect of PTH in preventing systemic bone loss following osteoporotic fracture by blocking the negative effects of sympathetic signaling on PTH anabolism.

Melatonin attenuates fentanyl - induced behavioral sensitization and circadian rhythm disorders in mice.

Melatonin is a neurohormone synthesized by the pineal gland to regulate the circadian rhythms and has proven to be effective in treating drug addiction and dependence. However, the effects of melatonin to modulate the drug-seeking behavior of fentanyl and its underlying molecular mechanism is elusive. This study was designed to investigate the effects of melatonin on fentanyl - induced behavioral sensitization and circadian rhythm disorders in mice. The accompanying changes in the expression of Brain and Muscle Arnt-Like (BMAL1), tyrosine hydroxylase (TH), and monoamine oxidase A (MAO-A) in relevant brain regions including the suprachiasmatic nucleus (SCN), nucleus accumbens (NAc), prefrontal cortex (PFC), and hippocampus (Hip) were investigated by western blot assays to dissect the mechanism by which melatonin modulates fentanyl - induced behavioral sensitization and circadian rhythm disorders. The present study suggest that fentanyl (0.05, 0.1 and 0.2 mg/kg) could induce behavioral sensitization and melatonin (30.0 mg/kg) could attenuate the behavioral sensitization and circadian rhythm disorders in mice. Fentanyl treatment reduced the expression of BMAL1 and MAO-A and increased that of TH in relevant brain regions. Furthermore, melatonin treatment could reverse the expression levels of BMAL1, MAO-A, and TH. In conclusion, our study demonstrate for the first time that melatonin has therapeutic potential for fentanyl addiction.

Circadian Rhythm Alteration of the Core Clock Genes and the Lipid Metabolism Genes Induced by High-Fat Diet (HFD) in the Liver Tissue of the Chinese Soft-Shelled Turtle (Trionyx sinensis).

Physiology disorders of the liver, as it is an important tissue in lipid metabolism, can cause fatty liver disease. The mechanism might be regulated by 17 circadian clock genes and 18 fat metabolism genes, together with a high-fat diet (HFD). Due to their rich nutritional and medicinal value, Chinese soft-shelled turtles (Trionyx sinensis) are very popular among the Chinese people. In the study, we aimed to investigate the influence of an HFD on the daily expression of both the core clock genes and the lipid metabolism genes in the liver tissue of the turtles. The two diets were formulated with 7.98% lipid (the CON group) and 13.86% lipid (the HFD group) to feed 180 juvenile turtles, which were randomly divided into two groups with three replicates per group and 30 turtles in each replicate for six weeks, and the diet experiment was administrated with a photophase regimen of a 24 h light/dark (12L:12D) cycle. At the end of the experiment, the liver tissue samples were collected from nine turtles per group every 3 h (zeitgeber time: ZT 0, 3, 6, 9, 12, 15, 18, 21 and 24) for 24 h to investigate the daily expression and correlation analysis of these genes. The results showed that 11 core clock genes [i.e., circadian locomotor output cycles kaput (Clock), brain and muscle arnt-like protein 1 and 2 (Bmal1/2), timeless (Tim), cryptochrome 1 (Cry2), period2 (Per2), nuclear factor IL-3 gene (Nfil3), nuclear receptor subfamily 1, treatment D, member 1 and 2 (Nr1d1/2) and retinoic acid related orphan receptor α/ß/γ ß and γ (Rorß/γ)] exhibited circadian oscillation, but 6 genes did not, including neuronal PAS domain protein 2 (Npas2), Per1, Cry1, basic helix-loop-helix family, member E40 (Bhlhe40), Rorα and D-binding protein (Dbp), and 16 lipid metabolism genes including fatty acid synthase (Fas), diacylglycerol acyltransferase 1 (Dgat1), 3-hydroxy-3-methylglutaryl-CoA reductase (Hmgcr), Low-density lipoprotein receptor-related protein 1-like (Ldlr1), Lipin 1 (Lipin1), Carnitine palmitoyltransferase 1A (Cpt1a), Peroxisome proliferator activation receptor α, ß and γ (Pparα/ß/γ), Sirtuin 1 (Sirt1), Apoa (Apoa1), Apolipoprotein B (Apob), Pyruvate Dehydrogenase kinase 4 (Pdk4), Acyl-CoA synthase long-chain1 (Acsl1), Liver X receptors α (Lxrα) and Retinoid X receptor, α (Rxra) also demonstrated circadian oscillations, but 2 genes did not, Scd and Acaca, in the liver tissues of the CON group. However, in the HFD group, the circadian rhythms' expressional patterns were disrupted for the eight core clock genes, Clock, Cry2, Per2, Nfil3, Nr1d1/2 and Rorß/γ, and the peak expression of Bmal1/2 and Tim showed delayed or advanced phases. Furthermore, four genes (Cry1, Per1, Dbp and Rorα) displayed no diurnal rhythm in the CON group; instead, significant circadian rhythms appeared in the HFD group. Meanwhile, the HFD disrupted the circadian rhythm expressions of seven fat metabolism genes (Fas, Cpt1a, Sirt1, Apoa1, Apob, Pdk4 and Acsl1). Meanwhile, the other nine genes in the HFD group also showed advanced or delayed expression peaks compared to the CON group. Most importantly of all, there were remarkably positive or negative correlations between the core clock genes and the lipid metabolism genes, and their correlation relationships were altered by the HFD. To sum up, circadian rhythm alterations of the core clock genes and the lipid metabolism genes were induced by the high-fat diet (HFD) in the liver tissues of T. sinensis. This result provides experimental and theoretical data for the mass breeding and production of T. sinensis in our country.

Comprehensive analysis of diel rhythmic expression of the medaka toll-like receptor gene family.

Several immune-related genes, including Toll-like receptors (TLR), are associated with circadian rhythms in mammals. However, information on the circadian rhythmic expression of TLRs in fish is limited. In this study, we aimed to analyze the regulation of diel oscillations in the expression of TLR genes in Japanese medaka (Oryzias latipes). The expression analysis revealed diel expression patterns of tlr1, tlr5m, tlr21, and clock genes (bmal1 and clock1) under a 12 h light:12 h dark cycle. The clock gene response element (E-box) was identified in the transcriptional regulatory regions of tlr1, tlr5m, and tlr21. Moreover, overexpressed bmal1 and clock1 enhanced expression levels of tlr1, tlr5m, and tlr21 in medaka embryo (OLHdrR-e3) cells. The expression of tlr1, tlr5m, and tlr21 was significantly decreased in OLHdrR-e3 after generating a bmal1 knockdown using a morpholino oligo. These results indicate the regulation of the diel rhythmic expression of several fish TLRs by clock genes.

Mucosal Genes Encoding Clock, Inflammation and Their Mutual Regulators Are Disrupted in Pediatric Patients with Active Ulcerative Colitis.

Patients with active ulcerative colitis (UC) display a misalignment of the circadian clock, which plays a vital role in various immune functions. Our aim was to characterize the expression of clock and inflammation genes, and their mutual regulatory genes in treatment-naïve pediatric patients with UC. Using the Inflammatory Bowel Disease Transcriptome and Metatranscriptome Meta-Analysis (IBD TaMMA) platform and R algorithms, we analyzed rectal biopsy transcriptomic data from two cohorts (206 patients with UC vs. 20 healthy controls from the GSE-109142 study, and 43 patients with UC vs. 55 healthy controls from the GSE-117993 study). We compared gene expression levels and correlation of clock genes (BMAL1, CLOCK, PER1, PER2, CRY1, CRY2), inflammatory genes (IκB, IL10, NFκB1, NFκB2, IL6, TNFα) and their mutual regulatory genes (RORα, RORγ, REV-ERBα, PGC1α, PPARα, PPARγ, AMPK, SIRT1) in patients with active UC and healthy controls. The clock genes BMAL1, CLOCK, PER1 and CRY1 and the inflammatory genes IκB, IL10, NFκB1, NFκB2, IL6 and TNFα were significantly upregulated in patients with active UC. The genes encoding the mutual regulators RORα, RORγ, PGC1α, PPARα and PPARγ were significantly downregulated in patients with UC. A uniform pattern of gene expression was found in healthy controls compared to the highly variable expression pattern in patients with UC. Among the healthy controls, inflammatory genes were positively correlated with clock genes and they all showed reduced expression. The difference in gene expression levels was associated with disease severity and endoscopic score but not with histological score. In patients with active UC, clock gene disruption is associated with abnormal mucosal immune response. Disrupted expression of genes encoding clock, inflammation and their mutual regulators together may play a role in active UC.

Mutations of the circadian clock genes Cry, Per, or Bmal1 have different effects on the transcribed and nontranscribed strands of cycling genes.

One important goal of circadian medicine is to apply time-of-day dosing to improve the efficacy of chemotherapy. However, limited knowledge of how the circadian clock regulates DNA repair presents a challenge to mechanism-based clinical application. We studied time-series genome-wide nucleotide excision repair in liver and kidney of wild type and three different clock mutant genotypes (Cry1-/-Cry2-/-, Per1-/-Per2-/-, and Bmal1-/-). Rhythmic repair on the nontranscribed strand was lost in all three clock mutants. Conversely, rhythmic repair of hundreds of genes on the transcribed strand (TSs) persisted in the livers of Cry1-/-Cry2-/- and Per1-/-Per2-/- mice. We identified a tissue-specific, promoter element-driven repair mode on TSs of collagen and angiogenesis genes in the absence of clock activators or repressors. Furthermore, repair on TSs of thousands of genes was altered when the circadian clock is disrupted. These data contribute to a better understanding of the regulatory role of the circadian clock on nucleotide excision repair in mammals and may be invaluable toward the design of time-aware platinum-based interventions in cancer.

Feasible diet and circadian interventions reduce in vivo progression of FLT3-ITD-positive acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) with an internal tandem duplication in the fms-like tyrosine kinase receptor 3 gene (FLT3-ITD) is associated with poor survival, and few studies have examined the impact of modifiable behaviors, such as nutrient quality and timing, in this subset of acute leukemia. METHODS: The influence of diet composition (low-sucrose and/or low-fat diets) and timing of diet were tested in tandem with anthracycline treatment in orthotopic xenograft mouse models. A pilot clinical study to test receptivity of pediatric leukemia patients to macronutrient matched foods was conducted. A role for the circadian protein, BMAL1 (brain and muscle ARNT-like 1), in effects of diet timing was studied by overexpression in FLT3-ITD-bearing AML cells. RESULTS: Reduced tumor burden in FLT3-ITD AML-bearing mice was observed with interventions utilizing low-sucrose and/or low-fat diets, or time-restricted feeding (TRF) compared to mice fed normal chow ad libitum. In a tasting study, macronutrient matched low-sucrose and low-fat meals were offered to pediatric acute leukemia patients who largely reported liking the meals. Expression of the circadian protein, BMAL1, was heightened with TRF and the low-sucrose diet. BMAL1 overexpression and treatment with a pharmacological inducer of BMAL1 was cytotoxic to FLT3-ITD AML cells. CONCLUSIONS: Mouse models for FLT3-ITD AML show that diet composition and timing slows progression of FLT3-ITD AML growth in vivo, potentially mediated by BMAL1. These interventions to enhance therapy efficacy show preliminary feasibility, as pediatric leukemia patients responded favorable to preparation of macronutrient matched meals.

A pilot study on the expression of circadian clock genes in the alveolar bone of mice with periodontitis.

This study aimed to investigate the expression of circadian clock genes in mouse alveolar bone, and the possible reasons for these changes. Fifty C57 mice were orally inoculated with P. gingivalis, establishing a model of periodontitis using healthy mice as controls. The alveolar bone of both groups was taken for micro-computed tomography scanning to measure the amount of attachment loss, and the relative expression of mRNA in each clock gene and periodontitis related inflammatory factor was detected by real-time fluorescence quantitative polymerase chain reaction (qRT-PCR). After the establishment of the mouse model, the height of alveolar bone in the periodontitis group was significantly lower than that in the normal group (p < 0.05). The relative transcriptional level of Bmal1, Per2, and Cry1 mRNA was in the circadian rhythm in the normal group (p ≤ 0.05), while in the periodontitis group, its circadian rhythm disappeared and the transcriptional level characteristics were changed. Interleukin (IL)-6, tumor necrosis factor-alpha (TNF-α), and interferon (IFN-γ) mRNA transcriptional level were elevated in the periodontitis group compared to the normal group. In conclusion, the mRNA transcriptional level of Bmal1, Per2, and Cry1 in alveolar bone of normal mice has circadian rhythm, but the rhythm disappears under the condition of periodontitis, and the cause of its occurrence may be related to inflammatory cytokines.

The rhythms of histones in regeneration: The epigenetic modifications determined by clock genes.

The evolutionary establishment of an internal biological clock is a primordial event tightly associated with a 24-h period. Changes in the circadian rhythm can affect cellular functions, including proliferation, DNA repair and redox state. Even isolated organs, tissues and cells can maintain an autonomous circadian rhythm. These cell-autonomous molecular mechanisms are driven by intracellular clock genes, such as BMAL1. Little is known about the role of core clock genes and epigenetic modifications in the skin. Our focus was to identify BMAL1-driven epigenetic modifications associated with gene transcription by mapping the acetylation landscape of histones in epithelial cells responding to injury. We explored the role of BMAL1 in epidermal wound and tissue regeneration using a loss-of-function approach in vivo. We worked with BMAL1 knockout mice and a contraction-resistance wound healing protocol, determining the histone modifications using specific antibodies to detect the acetylation levels of histones H3 and H4. We found significant differences in the acetylation levels of histones in both homeostatic and injured skin with deregulated BMAL1. The intact skin displayed varied acetylation levels of histones H3 and H4, including hyperacetylation of H3 Lys 9 (H3K9). The most pronounced changes were observed at the repair site, with notable alterations in the acetylation pattern of histone H4. These findings reveal the importance of histone modifications in response to injury and indicate that modulation of BMAL1 and its associated epigenetic events could be therapeutically harnessed to improve skin regeneration.

Pterostilbene targets the molecular oscillator RORγ to restore circadian rhythm oscillation and protect against sleep restriction induced metabolic disorders.

BACKGROUND: Considerable researches have directed toward metabolic disorders caused by sleep restriction (SR). SR-induced disruption of circadian metabolic rhythmicity is identified as an important pathophysiological mechanism. The flavonoid pterostilbene (PTE) is abundant in the traditional Chinese medicine dragon's blood with protective efficacy against obesity-related metabolic dysfunctions. Our previous study found that PTE ameliorates exercise intolerance and clock gene oscillation in the skeletal muscles subjected to SR. PURPOSE: This study aimed to explore whether PTE improves SR-induced metabolic disorders and delineate the relationship between PTE and the circadian clock. STUDY DESIGN AND METHODS: Two hundred male C57/B6J mice were kept awake for 20 h/d over five consecutive days and concurrently gavaged with 50, 100, or 200 mg/kg·bw/d PTE. Food consumption and body weight were monitored, and the metabolic status of the mice was evaluated by performing OGTT and ITT, measuring the serum lipid profiles and liver histopathology in response to SR. Daily behavior was analyzed by Clocklab™. The circadian rhythms of the liver clock genes and metabolic output genes were evaluated by cosine analysis. Binding between PTE and RORα/γ or NR1D1/2 was investigated by molecular docking. A luciferase reporter assay was used to determine the impact of PTE on Bmal1 transcription in SR-exposed mice co-transfected with Ad-BMAL1-LUC plus Ad-RORγ-mCherry or Ad-NR1D1-EGFP. RESULTS: PTE significantly ameliorated abnormal glucose and lipid metabolism (p < 0.05) in SR-exposed mice. PTE improved circadian behavior (p < 0.05) and rescued the circadian rhythm oscillation of the liver clock (p < 0.05) and metabolic output genes (p < 0.05) under SR condition. Molecular docking disclosed that PTE might interact with RORs, and PTE was found to increase Bmal1 promoter luciferase activity with RORE elements in the presence of Ad-RORγ-mCherry (p < 0.05). CONCLUSIONS: PTE may protect against SR-induced metabolic disorders by directly modulating RORγ to maintain circadian metabolic rhythm. The findings provide valuable insights into the potential use of PTE in the treatment of metabolic disorders associated with disruptions in the circadian rhythm.

Adaptive changes in BMAL2 with increased locomotion associated with the evolution of unihemispheric slow-wave sleep in mammals.

Marine mammals, especially cetaceans, have evolved a very special form of sleep characterized by unihemispheric slow-wave sleep (USWS) and a negligible amount or complete absence of rapid-eye-movement sleep; however, the underlying genetic mechanisms remain unclear. Here, we detected unique, significant selection signatures in basic helix-loop-helix ARNT like 2 (BMAL2; also called ARNTL2), a key circadian regulator, in marine mammal lineages, and identified two nonsynonymous amino acid substitutions (K204E and K346Q) in the important PER-ARNT-SIM domain of cetacean BMAL2 via sequence comparison with other mammals. In vitro assays revealed that these cetacean-specific mutations specifically enhanced the response to E-box-like enhancer and consequently promoted the transcriptional activation of PER2, which is closely linked to sleep regulation. The increased PER2 expression, which was further confirmed both in vitro and in vivo, is beneficial for allowing cetaceans to maintain continuous movement and alertness during sleep. Concordantly, the locomotor activities of zebrafish overexpressing the cetacean-specific mutant bmal2 were significantly higher than the zebrafish overexpressing the wild-type gene. Subsequently, transcriptome analyses revealed that cetacean-specific mutations caused the upregulation of arousal-related genes and the downregulation of several sleep-promoting genes, which is consistent with the need to maintain hemispheric arousal during USWS. Our findings suggest a potential close relationship between adaptive changes in BMAL2 and the remarkable adaptation of USWS and may provide novel insights into the genetic basis of the evolution of animal sleep.

The role of the SIRT1-BMAL1 pathway in regulating oxidative stress in the early development of ischaemic stroke.

Oxidative stress is the primary cause of ischaemic stroke and is closely related to circadian rhythm. However, the mechanism by which circadian rhythm regulates oxidative stress in ischaemic stroke remains elusive. The Silent Information Regulator 1 (SIRT1) controls circadian rhythm by activating the transcription of the circadian clock core protein Basic Helix-Loop-Helix ARNT Like 1 (BMAL1) through deacetylation. Studies have shown that the SIRT1-BMAL1 pathway can regulate oxidative stress. To investigate its correlation with oxidative stress, we examined the expression levels and influencing factors of SIRT1-BMAL1 at different times in ischaemic stroke patients and analyzed their clinical indexes, oxidative stress, and inflammatory factor indicators. The expression levels of oxidative stress and inflammatory factor indicators, including malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-α), SIRT1, and BMAL1, were detected in ischaemic stroke patients within 4.5 h of onset and in non-stroke patients. Patients were divided into four subgroups based on onset time: subgroup 1 (0:00-05:59); subgroup 2 (06:00-11:59); subgroup 3 (12:00-17: 59); and subgroup 4 (18:00-23:59). Our results showed higher MDA, IL-6, and TNF-α levels, and lower SOD, SIRT1, and BMAL1 levels in ischaemic stroke patients compared to control patients (P < 0.05). Among the four subgroups, the content of MDA, IL-6, and TNF-α was highest in patients with ischaemic stroke onset from subgroup 2 (06:00-11:59), while the expression levels of SOD, BMAL1, and SIRT1 were lowest in patients with ischaemic stroke in subgroup 2. Additionally, myeloperoxidase (MPO) reached the highest value showing the same trends consistent with MDA, IL-6, and TNF-ɑ and opposite trends consistent with SOD, BMAL1, and SIRT1. However, triglycerides (TGs), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), immediate blood glucose, immediate diastolic blood pressure, immediate systolic blood pressure, and homocysteine (HCY) did not show any statistically significant circadian rhythm changes (P > 0.05). Our findings suggest that the SIRT1-BMAL1 pathway may be involved in early oxidative stress in ischaemic stroke, which may be related to MPO.

Circadian clock gene expression and polymorphism in non-segmental vitiligo.

BACKGROUND: Vitiligo is an acquired and progressive mucocutaneous disease with the damage of functioning epidermal melanocytes. Metabolic syndrome is associated with inflammatory skin diseases incorporating vitiligo. The circadian dysfunction triggers the pathogenesis of metabolic diseases, so our study aimed to determine the relationship between aryl hydrocarbon receptor nuclear translocator-like gene, a ligand-activated transcription factor and sensor of environmental chemicals, expression and polymorphism with non-segmental vitiligo, as well as its effect on lipid profile. METHODS: This case-control study was handled on 50 non-segmental vitiligo patients (generalized (12) and localized type (focal; 24 and acrofacial; 14)) and 50 matched controls. Each subject was proposed for full history taking, clinical examinations, serum lipid profile, and measurement of BMAL1 gene expression in the blood, and BMAL1 rs2279287 polymorphism of DNA extract from whole blood by real time-PCR. RESULTS: We identified that total cholesterol, triglyceride, and low-density lipoprotein were significantly higher, but high-density lipoprotein was significantly lower in non-segmental vitiligo patients than in the control group. A significant increase in circadian gene expression in non-segmental vitiligo patients was observed, with more detection of the BMAL1 T/C genotype (92%) than the T/T genotype. There was a significant positive relationship between the level of the circadian gene and the vitiligo patient's age, age of onset, and VIDA Score. The level of the circadian gene at Cutoff  ≥ 1.16 can predict the prognosis of vitiligo with a sensitivity of 78%, specificity of 84%, and accuracy of 81%. CONCLUSION: The circadian gene has an active role in the progress of non-segmental vitiligo and targeting this gene could have a significant impact on its management.

BMAL1 positively correlates with genes regulating steroidogenesis in human luteinized granulosa cells.

In brief: In this study, we examined the relationship between BMAL1 expression and the genes regulating steroid biosynthesis in human luteinized granulosa cells. BMAL1 function is crucial for steroid production and proper ovarian function, highlighting the importance of circadian clock regulation in female reproductive health. Abstract: Human luteinized granulosa cells were collected to analyze circadian clock gene expression and its effect on the genes regulating steroid biosynthesis. We used siRNA to knock down the expression of BMAL1 in KGN cells. We measured the expression levels of genes regulating steroid biosynthesis and circadian clock RT-qPCR. We demonstrated that BMAL1 expression positively correlates with genes regulating steroid biosynthesis (CYP11A1, CYP19A1, STAR, and ESR2). The knockdown of BMAL1 in KGN cells revealed a significant decrease in steroid synthase expression. In contrast, when BMAL1 was overexpressed in KGN and HGL5 cells, we observed a significant increase in the expression of steroid synthases, such as CYP11A1 and CYP19A1. These results indicated that BMAL1 positively controls 17ß-estradiol (E2) secretion in granulosa cells. We also demonstrated that dexamethasone synchronization in KGN cells enhanced the rhythmic alterations in circadian clock genes. Our study suggests that BMAL1 plays a critical role in steroid biosynthesis in human luteinized granulosa cells, thereby emphasizing the importance of BMAL1 in the regulation of reproductive physiology.