ED-71 Ameliorates Bone Loss in Type 2 Diabetes Mellitus by Enhancing Osteogenesis Through Upregulation of the Circadian Rhythm Coregulator BMAL1.

Purpose: Bone loss is a common complication of type 2 diabetes mellitus (T2DM). Circadian rhythms play a significant role in T2DM and bone remodeling. Eldecalcitol (ED-71), a novel active vitamin D analog, has shown promise in ameliorating T2DM. We aimed to investigate whether the circadian rhythm coregulator BMAL1 mediates the anti-osteoporotic effect of ED-71 in T2DM and its associated mechanisms. Methods: A T2DM mouse model was established using high-fat diet (HDF) and streptozotocin (STZ) injection, and blood glucose levels were monitored weekly. HE staining, Masson staining, and Micro-CT were performed to assess the changes in bone mass. IHC staining and IF staining were used to detect osteoblast status and BMAL1 expression and RT-qPCR was applied to detect the change of oxidative stress factors. In vitro, high glucose (HG) stimulation was used to simulate the cell environment in T2DM. RT-qPCR, Western blot, IF, ALP staining and AR staining were used to detect osteogenic differentiation and SIRT1/GSK3ß signaling pathway. DCFH-DA staining was used to detect reactive oxygen species (ROS) levels. Results: ED-71 increased bone mass and promoted osteogenesis in T2DM mice. Moreover, ED-71 inhibited oxidative stress and promoted BMAL1 expression in osteoblasts The addition of STL1267, an agonist of the BMAL1 transcriptional repressor protein REV-ERB, reversed the inhibitory effect of ED-71 on oxidative stress and the promotional effect on osteogenic differentiation. In addition, ED-71 facilitated SIRT1 expression and reduced GSK3ß activity. The inhibition of SIRT1 with EX527 partially attenuated ED-71's effects, whereas the GSK3ß inhibitor LiCl further enhanced ED-71's positive effects on BMAL1 expression. Conclusion: ED-71 ameliorates bone loss in T2DM by upregulating the circadian rhythm coregulator BMAL1 and promoting osteogenesis through inhibition of oxidative stress. The SIRT1/GSK3ß signaling pathway is involved in the regulation of BMAL1.

BMAL1 alleviates sepsis-induced AKI by inhibiting ferroptosis.

BACKGROUND: The role of BMAL1 in various diseases remains unclear, particularly its impact on sepsis-induced acute kidney injury (AKI). This study aims to investigate the role of BMAL1 in sepsis-induced AKI and its potential effects on cell ferroptosis. Initially, we assessed BMAL1 expression levels in mice treated with sepsis-induced AKI (via LPS injection) and in LPS-stimulated renal tubular epithelial cells. Subsequently, we explored the correlation between BMAL1 and ferroptosis using sequencing technology, validating our findings throughout experimental approaches. To further elucidate BMAL1's specific effects on AKI-related ferroptosis, we constructed BMAL1 overexpression models in mice and cells, analysing its impact on AKI and ferroptosis both in vivo and in vitro. Furthermore, using transcriptome sequencing technology, we identified key BMAL1-regulated genes and their associated biological pathways, validating these findings through in vivo and in vitro experiments. RESULTS: Our findings indicate decreased BMAL1 expression in sepsis-induced AKI. BMAL1 overexpression effectively mitigated renal tubular injury by reducing ferroptosis levels in renal tubular epithelial cells. Using transcriptome sequencing and ChIP-qPCR technology, we identified YAP as a target of BMAL1. The overexpression of BMAL1 significantly reduced the transcriptional activity of YAP and inhibited the Hippo signalling pathway. Treatment with the Hippo inhibitor Verteporfin (VP) reversed the BMAL1-downregulation-induced damage. Additionally, our study revealed that YAP positively regulates ACSL4 gene expression and its downstream signalling pathways. CONCLUSION: This study demonstrates that BMAL1 overexpression alleviates renal tubular epithelial cell injury and ferroptosis by inhibiting YAP expression and the Hippo pathway, thereby exerting protective effects in sepsis-induced AKI. These findings underscore the therapeutic potential of targeting BMAL1 in managing sepsis-induced AKI.

[Mechanism of berberine in improving adipocytic IR by mediating BMAL1:CLOCK complex and regulating glucose and lipid metabolism].

To explore the action mechanism of berberine in improving adipocytic insulin resistance(IR) by mediating brain and muscle arnt-like 1(BMAL1): circadian locomotor output cycles kaput(CLOCK) complex and regulating glucose and lipid metabolism. After the IR-3T3-L1 adipocyte model was established by dexamethasone induction for 96 h, 0.5, 1, 5, 10, and 20 µmol·L~(-1) berberine was administered for 24 h. The glucose oxidase method and cell counting kit-8(CCK-8) were used to detect extracellular glucose content and cell viability, respectively. The triglyceride(TG) and glycerol contents were detected by enzyme colorimetry. Oil red O staining was used to detect lipid droplets, and fluorescence staining was used to detect Ca~(2+), mitochondrial structure, and reactive oxygen species(ROS). Adiponectin(ADPN), BMAL1, CLOCK, hormone-sensitive triglyceride lipase(HSL), carbohydrate-response element-binding protein(ChREBP), sterol regulatory element-binding protein 1C(SREBP-1C), peroxisome proliferator-activated receptor γ coactivator 1α(PGC1α), carnitine palmitoyl transferase 1α(CPT1α), and peroxisome proliferator-activated receptor α(PPARα) were detected by Western blot(WB). Moreover, the nuclear localization of BMAL1 was detected by immunofluorescence. In addition, 20 µmol·L~(-1) CLK8 inhibitor was added to detect glucose consumption and BMAL1/ChREBP/PPARα protein. The results showed that berberine increased glucose consumption in IR-3T3-L1 adipocytes without affecting cell viability and reduced TG content. In addition, 5 µmol·L~(-1) berberine increased glycerol content and reduced lipid droplet accumulation due to enhanced lipolysis, while 10 µmol·L~(-1) berberine did not affect glycerol content, and fewer lipid droplets were observed due to enhanced lipolysis and glycerol utilization. Berberine improved mitochondrial function by reducing intracellular Ca~(2+) and ROS in IR-3T3-L1 adipocytes and upregulated PGC1α to improve the mitochondrial structure. The results also showed that berberine elevated ADPN to increase the insulin sensitivity of IR-3T3-L1 adipocytes, upregulated peripheral rhythm-related proteins BMAL1 and CLOCK, and strengthened the nuclear localization of BMAL1. In addition, berberine increased key lipolysis protein and lipid oxidation rate-limiting enzyme CPT1α and downregulated the key protein of TG synthesis, SREBP-1C. Moreover, ChREBP and PPARα in IR-3T3-L1 adipocytes were upregula-ted. All the above results suggested that berberine may transform glucose into lipids to enhance the hypoglycemic effect. By considering that CLK8 specifically inhibited the CLOCK acylation to modify BMAL1 and form complex, the results showed that the addition of CLK8 to the berberine group reduced glucose consumption, which suggested that berberine upregulated the formation of BMAL1:CLOCK complex to improve glucose metabolism. The addition of CLK8 to the berberine group upregulated BMAL1 but downregulated ChREBP and PPARα, which suggested that berberine mediated BMAL1:CLOCK complex for the regulation of glucose and lipid metabo-lism to improve adipocytic IR.

Bmal1 upregulates ATG5 expression to promote autophagy in skin cutaneous melanoma.

BACKGROUND: Skin cutaneous melanoma (SKCM) is a highly aggressive and malignant tumor that arises from the malignant transformation of melanocytes. In light of the limitations of existing treatment modalities, there is a pressing need to identify new drug targets for SKCM. Aryl-hydrocarbon receptor nuclear translocator-like (ARNTL), also known as Bmal1, is a gene that has been linked to the onset and progression of cancer. However, its role in SKCM remains understudied. METHODS: The expression of Bmal1 mRNA and protein was detected using TCGA, GTEx, CCLE, and ULCAN databases. Moreover, survival analysis was performed to investigate the association between Bmal1 and immune invasion and gene expression in immune infiltrating cells via CIBERSORT, R programming, TIMER, Sangerbox, Kaplan-Meier. The study also explored the role of proteins associated with Bmal1 by using R programming and databases (STRING and GSEA). Both in vitro and in vivo studies were conducted to examine the potential role of Bmal1 in SKCM. RESULTS: Compared to normal tissues, the expression level of Bmal1 was significantly reduced in SKCM. Which has been associated with its poor prognosis. Similarly, its expression in SKCM was substantially correlated with immune infiltration, while biogenic analysis indicated that it could potentially influence the tumor immune microenvironment (TME) by influencing tumor-associated neutrophils (TANs). Moreover, Bmal1 overexpression suppressed the proliferation and invasion of melanoma cells and enhanced apoptosis, migration, and cell colony formation. CONCLUSION: This study concluded that Bmal1 is a novel biomarker that functions as both a diagnostic and prognostic indicator for the progression of SKCM.

Palmitic acid causes hepatocyte inflammation by suppressing the BMAL1-NAD+-SIRT2 axis.

Palmitic acid is the most abundant saturated fatty acid in circulation and causes hepatocyte toxicity and inflammation. As saturated fatty acid can also disrupt the circadian rhythm, the present work evaluated the connection between clock genes and NAD+ dependent Sirtuins in protecting hepatocytes from lipid-induced damage. Hepatocytes (immortal cells PH5CH8, hepatoma cells HepG2) treated with higher doses of palmitic acid (400-600µM) showed typical features of steatosis accompanied with growth inhibition and increased level of inflammatory markers (IL-6 IL-8, IL-1α and IL-1ß) together with decline in NAD+ levels. Palmitic acid treated hepatocytes showed significant decline in not only the protein levels of SIRT2 but also its activity as revealed by the acetylation status of its downstream targets (Tubulin and NF-ƙB). Additionally, the circadian expression of both SIRT2 and BMAL1 was inhibited in presence of palmitic acid in only the non-cancerous hepatocytes, PH5CH8 cells. Clinical specimens obtained from subjects with NASH-associated fibrosis, ranging from absent (F0) to cirrhosis (F4), showed a significant decline in levels of SIRT2 and BMAL1, especially in the cirrhotic liver. Ectopic expression of BMAL1 or activating SIRT2 by supplementation with nicotinamide riboside (precursor of NAD+) dampened the palmitic acid induced lipoinflammation and lipotoxicity more effectively in PH5CH8 cells as compared to HepG2 cells. Mechanistically, palmitic acid caused transcriptional suppression of SIRT2 by disrupting the chromatin occupancy of BMAL1 at its promoter site. Overall, the work suggested that SIRT2 is a clock-controlled gene that is transcriptionally regulated by BMAL1. In conclusion the activation of the BMAL1-NAD+-SIRT2 axis shows hepatoprotective effects by preventing lipotoxicity and dampening inflammation.

Bmal1 regulates the stemness and tumorigenesis of gliomas with the Wnt/ß-catenin signaling pathway.

BACKGROUND: The circadian rhythm gene Brain and Muscle Arnt-like1 (Bmal1) acts as a transcription factor and plays a crucial role in oncogenesis and embryonic development. Bmal1 is notably overexpressed in various tumors, including glioma. However, the precise mechanisms underlying the elevated Bmal1 expression in glioma malignancy remain unclear. METHODS: This study employed multiple databases, including The Cancer Genome Atlas (TCGA), GTEx, and cBioportal, to analyze Bmal1 mRNA expression in gliomas, evaluate its prognostic significance, investigate transcriptome alterations, identify key signaling pathways associated with Bmal1, and examine its interaction with tumor stem cells. Additionally, experimental validation was performed to confirm Bmal1's regulatory effects on glioma stem cells. RESULTS: Our analysis revealed differential Bmal1 expression across glioma grades and molecular subtypes. Moreover, Bmal1 significantly influences several tumor-related signaling pathways, notably the Mapk, Met, and Wnt pathways, and is actively involved with stem cells. A strong positive correlation was observed between Bmal1 and glioma stem cell markers, such as Nestin, Sox2, and Cd133. Experimental validation confirmed that Bmal1 promotes stem cell expansion and tumor progression via the Wnt/ß-catenin pathway. CONCLUSION: This study underscores the critical regulatory function of Bmal1 in glioma development. The interaction between Bmal1 and glioma stem cells appears to significantly impact glioma initiation and progression.

BMAL1 Deficiency Provokes Dry Mouth and Eyes by Down-regulating ITPR2/3.

Secretory glands, responsible for tears and saliva production, play essential roles in maintaining ocular and oral well-being. Disruptions in gland secretion can arise from various factors, including rhythm disturbances associated with sleep disorders. However, the underlying mechanisms governing these disruptions remain largely unexplored. We demonstrate that BMAL1, a core component of the circadian system, plays a critical role in regulating secretory gland secretion. Loss of BMAL1 induces vacuolation and atrophy phenotypes in acinar cells, subsequently leading to cell apoptosis and gland hypofunction, but does not cause Sjogren's syndrome, which is characterized by localized inflammatory cell infiltration. Mechanically, BMAL1 directly modulates the transcription of ITPR2 and ITPR3, thereby altering the secretion of Lactoferrin and Lysozyme. Restoration of ITPR2 and ITPR3 expression in Bmal1-deficient rats effectively alleviated the symptoms of lacrimal and parotid glands secretory dysfunction and significantly reduced dry mouth and dry eye conditions in rhythm-disordered rats. These findings highlight the essential role of BMAL1 in regulating salivary and lacrimal gland secretion and suggest a novel therapeutic approach for treating dry mouth and dry eyes associated with rhythm disorders.

Loss of endogenous circadian clock function in mice alters respiratory cycle timing in a time of day- and sex-specific manner.

Resting breathing and ventilatory chemoreflexes are regulated in a 24-hr manner by the endogenous circadian clock. However, it is unclear how circadian biology influences different phases of the breath-to-breath respiratory cycle which are predominantly controlled by pontomedullary regions of the brainstem. Here, we performed whole-body plethysmography during quiet wakefulness in young adult male and female mice lacking the core clock gene Brain and Muscle Arnt-like 1 (BMAL1) to determine the extent to which the molecular clock affects respiratory cycle timing and ventilatory airflow mechanics. Breath waveform analysis revealed that male BMAL1 knockout (KO) mice exhibit time of day-specific differences in inspiratory and expiratory times, total cycle length, end inspiratory pause, relaxation time, and respiratory rate compared to wild-type littermates. Notably, changes in respiratory pattern were not observed in female BMAL1 KO mice when compared to wild-type females. Additionally, BMAL1 deficiency did not disrupt overall minute ventilation or peak airflow in either sex, suggesting total ventilatory function during quiet wakefulness is preserved. Taken together, these findings indicate that genetic disruption of the circadian clock in mice elicits sex-specific changes in respiratory cycle timing.

Misalignment of Circadian Rhythms in Diet-Induced Obesity.

The biological clocks of the circadian timing system coordinate cellular and physiological processes and synchronize them with daily cycles. While the central clock in the suprachiasmatic nucleus (SCN) is mainly synchronized by the light/dark cycles, the peripheral clocks react to other stimuli, including the feeding/fasting state, nutrients, sleep-wake cycles, and physical activity. During the disruption of circadian rhythms due to genetic mutations or social and occupational obligations, incorrect arrangement between the internal clock system and environmental rhythms leads to the development of obesity. Desynchronization between the central and peripheral clocks by altered timing of food intake and diet composition leads to uncoupling of the peripheral clocks from the central pacemaker and to the development of metabolic disorders. The strong coupling of the SCN to the light-dark cycle creates a situation of misalignment when food is ingested during the "wrong" time of day. Food-anticipatory activity is mediated by a self-sustained circadian timing, and its principal component is a food-entrainable oscillator. Modifying the time of feeding alone greatly affects body weight, whereas ketogenic diet (KD) influences circadian biology, through the modulation of clock gene expression. Night-eating behavior is one of the causes of circadian disruption, and night eaters have compulsive and uncontrolled eating with severe obesity. By contrast, time-restricted eating (TRE) restores circadian rhythms through maintaining an appropriate daily rhythm of the eating-fasting cycle. The hypothalamus has a crucial role in the regulation of energy balance rather than food intake. While circadian locomotor output cycles kaput (CLOCK) expression levels increase with high-fat diet-induced obesity, peroxisome proliferator-activated receptor-alpha (PPARα) increases the transcriptional level of brain and muscle aryl hydrocarbon receptor nuclear translocator (ARNT)-like 1 (BMAL1) in obese subjects. In this context, effective timing of chronotherapies aiming to correct SCN-driven rhythms depends on an accurate assessment of the SCN phase. In fact, in a multi-oscillator system, local rhythmicity and its disruption reflects the disruption of either local clocks or central clocks, thus imposing rhythmicity on those local tissues, whereas misalignment of peripheral oscillators is due to exosome-based intercellular communication.Consequently, disruption of clock genes results in dyslipidemia, insulin resistance, and obesity, while light exposure during the daytime, food intake during the daytime, and sleeping during the biological night promote circadian alignment between the central and peripheral clocks. Thus, shift work is associated with an increased risk of obesity, diabetes, and cardiovascular diseases because of unusual eating times as well as unusual light exposure and disruption of the circadian rhythm.

Time of day-dependent alterations of ferroptosis in LPS-induced myocardial injury via Bmal-1/AKT/ Nrf2 in rat and H9c2 cell.

Background: One of the most prevalent causes of death in sepsis is sepsis-induced cardiomyopathy (SICM). Circadian disruption is involved in the progress of sepsis. However, the molecular mechanism remains unclear. Methods: Here, we built LPS-induced SICM in-vivo and in-vitro models. LPS was administrated at the particular Zeitgeber times (ZT), ZT4-ZT10-ZT16-ZT22 and ZT10-ZT22 in vivo and vitro experiments, respectively. Results: In vivo experiment, injection of LPS at ZT10 induced higher infiltration of inflammatory cells and content of intracellular Fe2+, and lower level of Glutathione peroxidase 4 (GPX4) and cardiac function than other ZTs (P < 0.05), which indicated that myocardial ferroptosis in septic rat presented a time of day-dependent manner. Bmal-1 protein and mRNA levels of injection of LPS at ZT10 were lower than those at other three ZTs (P < 0.05). The ratios of pAKT/AKT at ZT4 and ZT10 LPS injection were lower than those at ZT16 and ZT22 (P < 0.05). Nrf2 protein levels at ZT10 LPS injection were lower than those at other three ZTs (P < 0.05). These results indicated that the circadian of Bmal-1 and its downstream AKT/Nrf2 pathway in rat heart were inhibited under SICM condition. Consistent with in-vivo experiment, we found LPS could significantly reduce the expressions of Bmal-1 protein and mRNA in H9c2 cell. Up-regulation of Bmal-1 could reduce the cell death, oxidative stress, ferroptosis and activation of AKT/Nrf2 pathway at both ZT10 and ZT22 LPS administration. Conversely, its down-regulation presented opposite effects. AKT siRNA could weaken the effect of Bmal-1 pcDNA. Conclusion: Ferroptosis presented the time of day-dependent manners via Bmal-1/AKT/Nrf2 in vivo and vitro models of SICM.

Hepatic BMAL1 and HIF1α regulate a time-dependent hypoxic response and prevent hepatopulmonary-like syndrome.

The transcriptional response to hypoxia is temporally regulated, yet the molecular underpinnings and physiological implications are unknown. We examined the roles of hepatic Bmal1 and Hif1α in the circadian response to hypoxia in mice. We found that the majority of the transcriptional response to hypoxia is dependent on either Bmal1 or Hif1α, through shared and distinct roles that are daytime determined. We further show that hypoxia-inducible factor (HIF)1α accumulation upon hypoxia is temporally regulated and Bmal1 dependent. Unexpectedly, mice lacking both hepatic Bmal1 and Hif1α are hypoxemic and exhibit increased mortality upon hypoxic exposure in a daytime-dependent manner. These mice display mild liver dysfunction with pulmonary vasodilation likely due to extracellular signaling regulated kinase (ERK) activation, endothelial nitric oxide synthase, and nitric oxide accumulation in lungs, suggestive of hepatopulmonary syndrome. Our findings indicate that hepatic BMAL1 and HIF1α are key time-dependent regulators of the hypoxic response and can provide molecular insights into the pathophysiology of hepatopulmonary syndrome.

Up-regulation of BMAL1 by epigallocatechin-3-gallate improves neurological damage in SBI rats.

Brain Muscle ARNT-Like Protein 1 (BMAL1) suppresses oxidative stress in brain injury during surgery. Epigallocatechin-3-gallate (EGCG), a monomer in green tea, has been identified as an antioxidant and a potential agonist for BMAL1. In this work, the mechanism by which BMAL1 is regulated was investigated, as well as the therapeutic effect of EGCG on surgically injured rats. The pathological environment after brain injury during surgery was simulated by excising the right frontal lobe of rats. Rats received an intraperitoneal injection of EGCG immediately after surgery. Neurological scores and cerebral edema were recorded after surgery. Fluoro-Jade C staining, TUNEL staining, western blot, and lipid peroxidation analyses were conducted 3 days later. Here we show that the endogenous BMAL1 level decreased after brain injury. Postoperative administration of EGCG up-regulated the content of BMAL1 around the cerebral cortex, reduced the oxidative stress level, reduced neuronal apoptosis and the number of degenerated neurons, alleviated cerebral edema, and improved neurological scores in rats. This suggests that BMAL1 is an effective target for treating surgical brain injury, as well as that EGCG may be a promising agent for alleviating postoperative brain injury.

Effect of electroacupuncture pre-conditioning on the expression rhythm of core clock gene Bmal1 in uterine tissue of controlled hyperstimulation rats. / ç”µé’ˆé¢„å¤„ç†å¯¹ä¿ƒæŽ’åµå¤§é¼ å­å®«ç»„ç»‡æ ¸å¿ƒæ—¶é’ŸåŸºå› Bmal1表达节律的影响.

OBJECTIVES: To observe the effect of electroacupuncture (EA) pre-conditioning on the expression rhythm of clock gene Bmal1 in the uterine tissue of rats with controlled ovarian hyperstimulation(COH), so as to explore its mechanisms underlying improvement of the endometrial receptivity of ovarian superovulation during implantation. METHODS: Seventy-two female SD rats with typical estrous cycles were randomly divided into normal control, model and EA pre-conditioning (pre-EA) groups, with 24 rats in each group. The COH model was established by giving the rats with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (HCG) by intraperitoneal injection. The rats of the pre-EA group received EA stimulation (1 Hz/5 Hz, a tolerable strength) of "Guanyuan"(CV4) and "Sanyinjiao"(SP6) for 15 min each time, once daily (at 21：00 every day). After successive EA intervention during the first two estrous cycles, the modeling began in the third estrus cycle and the EA intervention was continued till the end of modeling, followed by raising the rats with superovulation induction and male rats undergoing vasoligation in one cage (1∶1). The rats during the estrum in the normal control group or those of the model group at the end of modeling were raised together with the male rats undergoing vasoligation in one cage. On the 5th day (04：00 AM) after raising in one cage, the rats in the three groups were sacrificed in six batches every 4 hours, with 4 rats in each group in each batch. The H.E. staining was used for revealing alterations of the endometrial thickness, number of glands and blood vessels and tissue histology, and ELISA employed to ascertain the contents of estradiol (E2) and progesterone (Pg) in serum. The expression rhythm of core clock gene Bmal1 ［In the present study, Zeitgeber time (ZT) is an artificially set laboratory time, i.e., ZT7 (07：00) is light on and ZT19 (19：00) is light off.］ and the expression of endometrial HoxA10 and leukemia inhibitory factor (LIF) mRNAs were detected by quantitative real-time PCR. The Western blot was employed to detect the expression levels of HoxA10 and LIF proteins. RESULTS: Findings of the clock gene Bmal1 level showed that the expression peak was at ZT12 and the valley value at ZT20 in the normal control group, and that of the peak value was at ZT20 and valley value at ZT12 in the model group, while in the pre-EA group, the peak value was at ZT8, and the valley value at ZT4. The difference of Bmal1 levels among the three groups was most significant at ZT12 (12：00), therefore, the tissue samples were taken at ZT12 in this study for comparison of the levels of different indexes among the 3 groups. Compared with the control group, the endometrial thickness, number of glands and blood vessels, HoxA10 and LIF mRNAs and proteins were significantly down-regulated (P<0.01, P<0.05), and contents of serum E2 and Pg were considerably up-regulated in the model group (P<0.01, P<0.05). Relevant to the model group, the pre-EA group had an apparent increase in the endometrial thickness, number of glands and blood vessels, and expression levels of HoxA10 and LIF mRNAs and proteins (P<0.05, P<0.01), and a marked decrease in the serum Pg (P<0.05). At the ZT12 (12：00 noon), compared with the normal control group, the mRNA level of Bmal1 was significantly decreased in the model group (P<0.01)；and compared with the model group, the level of Bmal1 mRNA was significantly increased in the pre-EA group (P<0.05). In addition, at the node of ZT16, the mRNA level of Bmal1 was significantly decreased in the model group in comparison with the normal control group (P<0.01). CONCLUSIONS: EA preconditioning can improve the endometrial receptivity during the implantation window period in rats with COH, which may be related to its functions in regulating the expression of clock gene Bmal1 in the uterine tissue and in correcting the disturbance of clock gene rhythm.

Entry of ZSWIM4 to the nucleus is crucial for its inhibition of KIT and BMAL1 in gastrointestinal stromal tumors.

BACKGROUND: Zinc finger SWIM-type containing 4 (ZSWIM4) is a zinc finger protein with its function largely uncharacterized. In this study, we aimed to investigate the role of ZSWIM4 in gastrointestinal stromal tumors (GISTs). RESULTS: We found that ZSWIM4 expression is inhibited by the predominantly mutated protein KIT in GISTs, while conversely, ZSWIM4 inhibits KIT expression and downstream signaling. Consistent with the observation, ZSWIM4 inhibited GIST cell survival and proliferation in vitro. RNA sequencing of GISTs from KITV558A/WT mice and KITV558A/WT/ZSWIM4-/- mice showed that loss of ZSWIM4 expression increases the expression of circadian clock pathway member BMAL1 which contributes to GIST cell survival and proliferation. In addition, we found that KIT signaling increases the distribution of ZSWIM4 in the nucleus of GIST cells, and which is important for its inhibition of KIT and BMAL1. In agreement with the results in vitro, the in vivo studies showed that ZSWIM4 deficiency increases the tumorigenesis of GISTs in KITV558A/WT mice. CONCLUSIONS: Taken together, our results revealed that the entry of ZSWIM4 to the nucleus is important for its inhibition of KIT and BMAL1, ultimately attenuating GIST tumorigenesis. The results provide a novel insight in the understanding of signal transduction in GISTs and lay strong theoretical basis for the advancement of GIST treatment.

Deletion of Bmal1 in aggrecan-expressing cells leads to mouse temporomandibular joint osteoarthritis.

INTRODUCTION: Articular cartilage is the major affected tissue during the development of osteoarthritis (OA) in temporomandibular joint (TMJ). The core circadian rhythm molecule Bmal1 regulates chondrocyte proliferation, differentiation and apoptosis; however, its roles in condylar cartilage function and in TMJ OA have not been fully elucidated. MATERIALS AND METHODS: TMJ OA mouse model was induced by unilateral anterior crossbite (UAC) and Bmal1 protein expression in condylar cartilage were examined by western blot analysis. To determine the role of Bmal1 in TMJ OA, we generated cartilage-specific Bmal1 conditional knockout (cKO) mice (Bmal1Agc1CreER mice) and hematoxylin and eosin staining, toluidine blue and Safranin O/fast green, immunohistochemistry, TUNEL assay, real-time PCR analysis and Western blot assay were followed. RESULTS: Bmal1 expression was reduced in condylar cartilage in a TMJ OA mouse model induced by UAC. The Bmal1 cKO mice displayed decreased cartilage matrix synthesis, reduced chondrocyte proliferation, increased chondrocyte hypertrophy and apoptosis as well as the upregulation of YAP expression in TMJ condylar cartilage. CONCLUSIONS: We demonstrated that Bmal1 was essential for TMJ tissue homeostasis and loss-of-function of Bmal1 in chondrocytes leads to the development of TMJ OA.

The impact of aerobic exercise timing on BMAL1 protein expression and antioxidant responses in skeletal muscle of mice.

It is well known that the adaptations of muscular antioxidant system to aerobic exercise depend on the frequency, intensity, duration, type of the exercise. Nonetheless, the timing of aerobic exercise, related to circadian rhythms or biological clock, may also affect the antioxidant defense system, but its impact remains uncertain. Bain and muscle ARNT-like 1 (BMAL1) is the core orchestrator of molecular clock, which can maintain cellular redox homeostasis by directly controlling the transcriptional activity of nuclear factor erythroid 2-related factor 2 (NRF2). So, our research objective was to evaluate the impacts of aerobic exercise training at various time points of the day on BMAL1 and NRF2-mediated antioxidant system in skeletal muscle. C57BL/6J mice were assigned to the control group, the group exercising at Zeitgeber Time 12 (ZT12), and the group exercising at ZT24. Control mice were not intervened, while ZT12 and ZT24 mice were trained for four weeks at the early and late time point of their active phase, respectively. We observed that the skeletal muscle of ZT12 mice exhibited higher total antioxidant capacity and lower reactive oxygen species compared to ZT24 mice. Furthermore, ZT12 mice improved the colocalization of BMAL1 with nucleus, the protein expression of BMAL1, NRF2, NAD(P)H quinone oxidoreductase 1, heme oxygenase 1, glutamate-cysteine ligase modifier subunit and glutathione reductase in comparison to those of ZT24 mice. In conclusion, the 4-week aerobic training performed at ZT12 is more effective for enhancing NRF2-mediated antioxidant responses of skeletal muscle, which may be attributed to the specific activation of BMAL1.

Comprehensive analysis of distinct circadian clock subtypes of adult diffuse glioma and their associations with clinicopathological, genetic, and epigenetic profiles.

The circadian clock (CC) has biological and clinical implications in gliomas. Most studies focused on CC effects on the tumor microenvironment and the application of chronotherapy. The present study focused on CC gene expression patterns and intracellular oncogenic activities. Glioma gene expression data were collected from The Human Cancer Genome Atlas (TCGA) project. After applying inclusion and exclusion criteria, we selected 666 patients from TCGA-GBM and TCGA-LGG projects and included important clinicopathological variables. The entire cohort was subjected to clustering analysis and divided into CC1 and CC2 subtypes based on statistical, biological, and clinical criteria. CC2 gliomas showed higher expression of BMAL1 and CRY1 and lower expression of CRY2 and PER2 (adjusted P < .001). CC2 gliomas had q higher activity of cell proliferation, metabolic reprogramming, angiogenesis, hypoxia, and many oncogenic signals (P < .001). The CC2 subtype contained a higher proportion of glioblastomas (P < .001) and had a worse prognosis (P < .001). Stratified Kaplan-Meier and multivariable Cox analyses illustrated that the CC subtype is an independent prognostic factor to clinicopathological characteristics (P < .001), genetic aberrations (P = .006), and biological processes (P < .001). Thus, this study shows statistical evidence of CC subtypes and their biological, and clinicopathological significance in adult gliomas.

Lack of Bmal1 leads to changes in rhythmicity and impairs motivation towards natural stimuli.

Maintaining proper circadian rhythms is essential for coordinating biological functions in mammals. This study investigates the effects of daily arrhythmicity using Bmal1-knockout (KO) mice as a model, aiming to understand behavioural and motivational implications. By employing a new mathematical analysis based on entropy divergence, we identified disrupted intricate activity patterns in mice derived by the complete absence of BMAL1 and quantified the difference regarding the activity oscillation's complexity. Changes in locomotor activity coincided with disturbances in circadian gene expression patterns. Additionally, we found a dysregulated gene expression profile particularly in brain nuclei like the ventral striatum, impacting genes related to reward and motivation. Further investigation revealed that arrhythmic mice exhibited heightened motivation for food and water rewards, indicating a link between circadian disruptions and the reward system. This research sheds light on how circadian clock alterations impact the gene expression regulating the reward system and how this, in turn, can lead to altered seeking behaviour and motivation for natural rewards. In summary, the present study contributes to our understanding of how reward processing is under the regulation of circadian clock machinery.

Dysregulation of the Suprachiasmatic Nucleus Disturbs the Circadian Rhythm and Aggravates Epileptic Seizures by Inducing Hippocampal GABAergic Dysfunction in C57BL/6 Mice.

The interplay between circadian rhythms and epilepsy has gained increasing attention. The suprachiasmatic nucleus (SCN), which acts as the master circadian pacemaker, regulates physiological and behavioral rhythms through its complex neural networks. However, the exact role of the SCN and its Bmal1 gene in the development of epilepsy remains unclear. In this study, we utilized a lithium-pilocarpine model to induce epilepsy in mice and simulated circadian disturbances by creating lesions in the SCN and specifically knocking out the Bmal1 gene in the SCN neurons. We observed that the pilocarpine-induced epileptic mice experienced increased daytime seizure frequency, irregular oscillations in core body temperature, and circadian gene alterations in both the SCN and the hippocampus. Additionally, there was enhanced activation of GABAergic projections from the SCN to the hippocampus. Notably, SCN lesions intensified seizure activity, concomitant with hippocampal neuronal damage and GABAergic signaling impairment. Further analyses using the Gene Expression Omnibus database and gene set enrichment analysis indicated reduced Bmal1 expression in patients with medial temporal lobe epilepsy, potentially affecting GABA receptor pathways. Targeted deletion of Bmal1 in SCN neurons exacerbated seizures and pathology in epilepsy, as well as diminished hippocampal GABAergic efficacy. These results underscore the crucial role of the SCN in modulating circadian rhythms and GABAergic function in the hippocampus, aggravating the severity of seizures. This study provides significant insights into how circadian rhythm disturbances can influence neuronal dysfunction and epilepsy, highlighting the therapeutic potential of targeting SCN and the Bmal1 gene within it in epilepsy management.

BMAL1 sex-specific effects in the neonatal mouse airway exposed to moderate hyperoxia.

Supplemental O2 (hyperoxia) is a critical intervention for premature infants (<34 weeks) but consequently is associated with development of bronchial airway hyperreactivity (AHR) and asthma. Clinical practice shifted toward the use of moderate hyperoxia (<60% O2), but risk for subsequent airway disease remains. In mouse models of moderate hyperoxia, neonatal mice have increased AHR with effects on airway smooth muscle (ASM), a cell type involved in airway tone, bronchodilation, and remodeling. Understanding mechanisms by which moderate O2 during the perinatal period initiates sustained airway changes is critical to drive therapeutic advancements toward treating airway diseases. We propose that cellular clock factor BMAL1 is functionally important in developing mouse airways. In adult mice, cellular clocks target pathways highly relevant to asthma pathophysiology and Bmal1 deletion increases inflammatory response, worsens lung function, and impacts survival outcomes. Our understanding of BMAL1 in the developing lung is limited, but our previous findings show functional relevance of clocks in human fetal ASM exposed to O2. Here, we characterize Bmal1 in our established mouse neonatal hyperoxia model. Our data show that Bmal1 KO deleteriously impacts the developing lung in the context of O2 and these data highlight the importance of neonatal sex in understanding airway disease.