Disruption of the circadian clock component BMAL1 elicits an endocrine adaption impacting on insulin sensitivity and liver disease.

SignificanceWhile increasing evidence associates the disruption of circadian rhythms with pathologic conditions, including obesity, type 2 diabetes, and nonalcoholic fatty liver diseases (NAFLD), the involved mechanisms are still poorly described. Here, we show that, in both humans and mice, the pathogenesis of NAFLD is associated with the disruption of the circadian clock combined with perturbations of the growth hormone and sex hormone pathways. However, while this condition protects mice from the development of fibrosis and insulin resistance, it correlates with increased fibrosis in humans. This suggests that the perturbation of the circadian clock and its associated disruption of the growth hormone and sex hormone pathways are critical for the pathogenesis of metabolic and liver diseases.

Bmal1 inhibits phenotypic transformation of hepatic stellate cells in liver fibrosis via IDH1/α-KG-mediated glycolysis.

Hepatic stellate cells (HSCs) play an important role in the initiation and development of liver fibrogenesis, and abnormal glucose metabolism is increasingly being considered a crucial factor controlling phenotypic transformation in HSCs. However, the role of the factors affecting glycolysis in HSCs in the experimental models of liver fibrosis has not been completely elucidated. In this study, we showed that glycolysis was significantly enhanced, while the expression of brain and muscle arnt-like protein-1 (Bmal1) was downregulated in fibrotic liver tissues of mice, primary HSCs, and transforming growth factor-ß1 (TGF-ß1)-induced LX2 cells. Overexpression of Bmal1 in TGF-ß1-induced LX2 cells blocked glycolysis and inhibited the proliferation and phenotypic transformation of activated HSCs. We further confirmed the protective effect of Bmal1 in liver fibrosis by overexpressing Bmal1 from hepatic adeno-associated virus 8 in mice. In addition, we also showed that the regulation of glycolysis by Bmal1 is mediated by the isocitrate dehydrogenase 1/α-ketoglutarate (IDH1/α-KG) pathway. Collectively, our results indicated that a novel Bmal1-IDH1/α-KG axis may be involved in regulating glycolysis of activated HSCs and might hence be used as a therapeutic target for alleviating liver fibrosis.

Clock proteins and training modify exercise capacity in a daytime-dependent manner.

Exercise and circadian biology are closely intertwined with physiology and metabolism, yet the functional interaction between circadian clocks and exercise capacity is only partially characterized. Here, we tested different clock mutant mouse models to examine the effect of the circadian clock and clock proteins, namely PERIODs and BMAL1, on exercise capacity. We found that daytime variance in endurance exercise capacity is circadian clock controlled. Unlike wild-type mice, which outperform in the late compared with the early part of their active phase, PERIODs- and BMAL1-null mice do not show daytime variance in exercise capacity. It appears that BMAL1 impairs and PERIODs enhance exercise capacity in a daytime-dependent manner. An analysis of liver and muscle glycogen stores as well as muscle lipid utilization suggested that these daytime effects mostly relate to liver glycogen levels and correspond to the animals' feeding behavior. Furthermore, given that exercise capacity responds to training, we tested the effect of training at different times of the day and found that training in the late compared with the early part of the active phase improves exercise performance. Overall, our findings suggest that clock proteins shape exercise capacity in a daytime-dependent manner through changes in liver glycogen levels, likely due to their effect on animals' feeding behavior.

Core circadian clock transcription factor BMAL1 regulates mammary epithelial cell growth, differentiation, and milk component synthesis.

The role the mammary epithelial circadian clock plays in gland development and lactation is unknown. We hypothesized that mammary epithelial clocks function to regulate mammogenesis and lactogenesis, and propose the core clock transcription factor BMAL1:CLOCK regulates genes that control mammary epithelial development and milk synthesis. Our objective was to identify transcriptional targets of BMAL1 in undifferentiated (UNDIFF) and lactogen differentiated (DIFF) mammary epithelial cells (HC11) using ChIP-seq. Ensembl gene IDs with the nearest transcriptional start site to ChIP-seq peaks were explored as potential targets, and represented 846 protein coding genes common to UNDIFF and DIFF cells and 2773 unique to DIFF samples. Genes with overlapping peaks between samples (1343) enriched cell-cell adhesion, membrane transporters and lipid metabolism categories. To functionally verify targets, an HC11 line with Bmal1 gene knocked out (BMAL1-KO) using CRISPR-CAS was created. BMAL1-KO cultures had lower cell densities over an eight-day growth curve, which was associated with increased (p<0.05) levels of reactive oxygen species and lower expression of superoxide dismutase 3 (Sod3). RT-qPCR analysis also found lower expression of the putative targets, prolactin receptor (Prlr), Ppara, and beta-casein (Csn2). Findings support our hypothesis and highlight potential importance of clock in mammary development and substrate transport.

Postnatal deletion of Bmal1 in mice protects against obstructive renal fibrosis via suppressing Gli2 transcription.

Circadian clock is involved in regulating most renal physiological functions, including water and electrolyte balance and blood pressure homeostasis, however, the role of circadian clock in renal pathophysiology remains largely unknown. Here we aimed to investigate the role of Bmal1, a core clock component, in the development of renal fibrosis, the hallmark of pathological features in many renal diseases. The inducible Bmal1 knockout mice (iKO) whose gene deletion occurred in adulthood were used in the study. Analysis of the urinary water, sodium and potassium excretion showed that the iKO mice exhibit abolished diurnal variations. In the model of renal fibrosis induced by unilateral ureteral obstruction, the iKO mice displayed significantly decreased tubulointerstitial fibrosis reflected by attenuated collagen deposition and mitigated expression of fibrotic markers α-SMA and fibronectin. The hedgehog pathway transcriptional effectors Gli1 and Gli2, which have been reported to be involved in the pathogenesis of renal fibrosis, were significantly decreased in the iKO mice. Mechanistically, ChIP assay and luciferase reporter assay revealed that BMAL1 bound to the promoter of and activate the transcription of Gli2, but not Gli1, suggesting that the involvement of Bmal1 in renal fibrosis was possibly mediated via Gli2-dependent mechanisms. Furthermore, treatment with TGF-ß increased Bmal1 in cultured murine proximal tubular cells. Knockdown of Bmal1 abolished, while overexpression of Bmal1 increased, Gli2 and the expression of fibrosis-related genes. Collectively, these results revealed a prominent role of the core clock gene Bmal1 in tubulointerstitial fibrosis. Moreover, we identified Gli2 as a novel target of Bmal1, which may mediate the adverse effect of Bmal1 in obstructive nephropathy.

Bmal1 promotes prostaglandin E2 synthesis by upregulating Ptgs2 transcription in response to increasing estradiol levels in day 4 pregnant mice.

Prostaglandin G/H synthase 2 (PTGS2) is a rate-limiting enzyme in prostaglandin synthesis. The present study assessed the role of the uterine circadian clock on Ptgs2 transcription in response to steroid hormones during early pregnancy. We demonstrated that the core clock genes (Bmal1, Per2, Nr1d1, and Dbp), Vegf, and Ptgs2, and their encoded proteins, have rhythmic expression in the mouse uterus from days 3.5 to 4.5 (D3.5-4.5) of pregnancy. Progesterone (P4) treatment of cultured uterus endometrial stromal cells (UESCs) isolated from mPer2Luciferase reporter gene knock-in mice on D4 induced a phase shift in PER2::LUCIFERASE oscillations. This P4-induced phase shift of PER2::LUCIFERASE oscillations was significantly attenuated by the P4 antagonist RU486. Additionally, the amplitude of PER2::LUCIFERASE oscillations was increased by estradiol (E2) treatment in the presence of P4. Consistently, the mRNA levels of clock genes (Bmal1 and Per2), Vegf, and Ptgs2 were markedly increased by E2 treatment of UESCs in the presence of P4. Treatment with E2 also promoted prostaglandin E2 (PGE2) synthesis by UESCs. Depletion of Bmal1 in UESCs by small-interfering RNA (siRNA) decreased the transcript levels of clock genes (Nr1d1 and Dbp), Vegf, and Ptgs2 compared with nonsilencing siRNA treatment. Bmal1 knockdown also inhibited PGE2 synthesis. Moreover, the mRNA expression levels of clock genes (Nr1d1 and Dbp), Vegf, and Ptgs2, and their respective proteins were significantly decreased in the uterus of Bmal1-/- mice. Thus, these data suggest that Bmal1 in mice promotes PGE2 synthesis by upregulating Ptgs2 in response to increases in E2 on D4 of pregnancy.NEW & NOTEWORTHY Rhythmic expression of Bmal1 and Ptgs2 was observed in the uterus isolated from D3.5-4.5 of pregnant mice. E2 increased the expression of Bmal1 and Ptg2 in UESCs isolated from mice on D4. The expression of Ptgs2 was significantly decreased in Bmal1-siRNA treated UESCs. Bmal1 knockdown also inhibited PGE2 synthesis. Thus, these data suggest that Bmal1 in mice promotes PGE2 synthesis by upregulating Ptgs2 in response to increases in E2 on D4 of pregnancy.

The SNARE regulator Complexin3 is a target of the cone circadian clock.

BMAL1 is a core component of the mammalian circadian clockwork. Removal of BMAL1 from the retina significantly affects visual information processing in both rod and cone pathways. To identify potential pathways and/or molecules through which BMAL1 alters signal transmission at the cone pedicle, we performed an RNA-seq differential expression analysis between cone-specific Bmal1 knockout cones (cone-Bmal1-/- ) and wild-type (WT) cones. We found 88 genes differentially expressed. Among these, Complexin3 (Cplx3), a SNARE regulator at ribbon synapses, was downregulated fivefold in the mutant cones. The purpose of this work was to determine whether BMAL1 and/or the cone clock controls CPLX3 protein expression at cone pedicles. We found that CPLX3 expression level was decreased twofold in cone-Bmal1-/- cones. Furthermore, CPLX3 expression was downregulated at night compared to the day in WT cones but remained constitutively low in mutant cones both day and night. The transcript and protein expression levels of Cplx4-the other complexin expressed in cones-were similar in WT and mutant cones; in WT cones, CPLX4 protein level did not change with the time of day. In silico analysis revealed four potential BMAL1:CLOCK binding sites upstream from exon one of Cplx3 and none upstream of exon one of Cplx4. Our results suggest that CPLX3 expression is regulated at the transcriptional level by the cone clock. The modulation of CPLX3 may be a mechanism by which the clock controls the cone synaptic transfer function to second-order cells and thereby impacts retinal signal processing during the day/night cycle.

The Cell-Autonomous Clock of VIP Receptor VPAC2 Cells Regulates Period and Coherence of Circadian Behavior.

Circadian (approximately daily) rhythms pervade mammalian behavior. They are generated by cell-autonomous, transcriptional/translational feedback loops (TTFLs), active in all tissues. This distributed clock network is coordinated by the principal circadian pacemaker, the hypothalamic suprachiasmatic nucleus (SCN). Its robust and accurate time-keeping arises from circuit-level interactions that bind its individual cellular clocks into a coherent time-keeper. Cells that express the neuropeptide vasoactive intestinal peptide (VIP) mediate retinal entrainment of the SCN; and in the absence of VIP, or its cognate receptor VPAC2, circadian behavior is compromised because SCN cells cannot synchronize. The contributions to pace-making of other cell types, including VPAC2-expressing target cells of VIP, are, however, not understood. We therefore used intersectional genetics to manipulate the cell-autonomous TTFLs of VPAC2-expressing cells. Measuring circadian behavioral and SCN rhythmicity in these temporally chimeric male mice thus enabled us to determine the contribution of VPAC2-expressing cells (∼35% of SCN cells) to SCN time-keeping. Lengthening of the intrinsic TTFL period of VPAC2 cells by deletion of the CK1εTau allele concomitantly lengthened the period of circadian behavioral rhythms. It also increased the variability of the circadian period of bioluminescent TTFL rhythms in SCN slices recorded ex vivo Abrogation of circadian competence in VPAC2 cells by deletion of Bmal1 severely disrupted circadian behavioral rhythms and compromised TTFL time-keeping in the corresponding SCN slices. Thus, VPAC2-expressing cells are a distinct, functionally powerful subset of the SCN circuit, contributing to computation of ensemble period and maintenance of circadian robustness. These findings extend our understanding of SCN circuit topology.

Core clock gene Bmal1 deprivation impairs steroidogenesis in mice luteinized follicle cells.

Luteinization is the event of corpus luteum formation, a way of follicle cells transformation and a process of steroidogenesis alteration. As the core clock gene, Bmal1 was involved in the regulation of ovulation process and luteal function afterwards. Till now, the underlying roles of luteinization played by Bmal1 remain unknown. To explore the unique role of Bmal1 in luteal steroidogenesis and its underlying pathway, we investigated the luteal hormone synthesis profile in Bmal1 knockout female mice. We found that luteal hormone synthesis was notably impaired, and phosphorylation of PI3K/NfκB pathway was significantly activated. Then, the results were verified in in vitro cultured cells, including isolated Bmal1 interference granulosa cells (GCs) and theca cells (TCs), respectively. Hormones levels of supernatant culture media and mRNA expressions of steroidogenesis-associated genes (star, Hsd3ß2, cyp19a1 in GCs, Lhcgr, star, Hsd3ß2, cyp17a1 in TCs) were mutually decreased, while the phosphorylation of PI3K/NfκB was promoted during in vitro luteinization. After PI3K specific-inhibitor LY294002 intervention, mRNA expressions of Lhcgr and Hsd3ß2 were partially rescued in Bmal1 interference TCs, together with significantly increased androstenedione and T synthesis. Further exploration in TCs demonstrated BMAL1 interacted directly but negatively with NfκB p65 (RelA), a subunit which was supposed as a mediator in Bmal1-governed PI3K signaling regulation. Taken together, we verified the novel role of Bmal1 in luteal steroidogenesis, achieving by negative interplay with RelA-mediated PI3K/NfκB pathway.

Improved locomotor recovery after contusive spinal cord injury in Bmal1-/- mice is associated with protection of the blood spinal cord barrier.

The transcription factor BMAL1/ARNTL is a non-redundant component of the clock pathway that regulates circadian oscillations of gene expression. Loss of BMAL1 perturbs organismal homeostasis and usually exacerbates pathological responses to many types of insults by enhancing oxidative stress and inflammation. Surprisingly, we observed improved locomotor recovery and spinal cord white matter sparing in Bmal1-/- mice after T9 contusive spinal cord injury (SCI). While acute loss of neurons and oligodendrocytes was unaffected, Bmal1 deficiency reduced the chronic loss of oligodendrocytes at the injury epicenter 6 weeks post SCI. At 3 days post-injury (dpi), decreased expression of genes associated with cell proliferation, neuroinflammation and disruption of the blood spinal cord barrier (BSCB) was also observed. Moreover, intraspinal extravasation of fibrinogen and immunoglobulins was decreased acutely at dpi 1 and subacutely at dpi 7. Subacute decrease of hemoglobin deposition was also observed. Finally, subacutely reduced levels of the leukocyte marker CD45 and even greater reduction of the pro-inflammatory macrophage receptor CD36 suggest not only lower numbers of those cells but also their reduced inflammatory potential. These data indicate that Bmal1 deficiency improves SCI outcome, in part by reducing BSCB disruption and hemorrhage decreasing cytotoxic neuroinflammation and attenuating the chronic loss of oligodendrocytes.

A role for alternative splicing in circadian control of exocytosis and glucose homeostasis.

The circadian clock is encoded by a negative transcriptional feedback loop that coordinates physiology and behavior through molecular programs that remain incompletely understood. Here, we reveal rhythmic genome-wide alternative splicing (AS) of pre-mRNAs encoding regulators of peptidergic secretion within pancreatic ß cells that are perturbed in Clock-/- and Bmal1-/- ß-cell lines. We show that the RNA-binding protein THRAP3 (thyroid hormone receptor-associated protein 3) regulates circadian clock-dependent AS by binding to exons at coding sequences flanking exons that are more frequently skipped in clock mutant ß cells, including transcripts encoding Cask (calcium/calmodulin-dependent serine protein kinase) and Madd (MAP kinase-activating death domain). Depletion of THRAP3 restores expression of the long isoforms of Cask and Madd, and mimicking exon skipping in these transcripts through antisense oligonucleotide delivery in wild-type islets reduces glucose-stimulated insulin secretion. Finally, we identify shared networks of alternatively spliced exocytic genes from islets of rodent models of diet-induced obesity that significantly overlap with clock mutants. Our results establish a role for pre-mRNA alternative splicing in ß-cell function across the sleep/wake cycle.

NAMPT and BMAL1 Are Independently Involved in the Palmitate-Mediated Induction of Neuroinflammation in Hypothalamic Neurons.

Obesity is a prominent metabolic disease that predisposes individuals to multiple comorbidities, including type 2 diabetes mellitus, cardiovascular diseases, and cancer. Elevated circulating levels of fatty acids contribute to the development of obesity, in part, by targeting the hypothalamus. Palmitate, the most abundant circulating saturated fatty acid, has been demonstrated to dysregulate NAMPT and circadian clock proteins, as well as induce neuroinflammation. These effects ultimately result in hypothalamic dysregulation of feeding behavior and energy homeostasis. NAMPT is the rate-limiting enzyme of the NAD+ salvage pathway and its expression is under the control of the circadian clock. NAD+ produced from NAMPT can modulate the circadian clock, demonstrating bidirectional interactions between circadian and metabolic pathways. Using NPY/AgRP-expressing mHypoE-46 neurons as well as the novel mHypoA-BMAL1-WT/F and mHypoA-BMAL1-KO/F cell lines, we studied whether there were any interactions between NAMPT and the core circadian clock protein BMAL1 in the palmitate-mediated induction of neuroinflammation. We report that palmitate altered Nampt, Bmal1, Per2 and the inflammatory genes Nf-κb, IκBα, Il-6, and Tlr4. Contrary to studies performed with peripheral tissues, the palmitate-mediated induction in Nampt was independent of BMAL1, and basal Nampt levels did not appear to exhibit rhythmic expression. Palmitate-induced downregulation of Bmal1 and Per2 was independent of NAMPT. However, NAMPT and BMAL1 were both involved in the regulation of Nf-κb, IκBα, Il-6, and Tlr4, as NAMPT inhibition resulted in the repression of basal Nf-κb and IκBα and normalized palmitate-mediated increases in Il-6, and Tlr4. On the other hand, BMAL1 deletion repressed basal Nf-κb, but increased basal Il-6. We conclude that NAMPT and BMAL1 do not interact at the transcriptional level in hypothalamic neurons, but are independently involved in the expression of inflammatory genes.

Bmal1 Deletion in Myeloid Cells Attenuates Atherosclerotic Lesion Development and Restrains Abdominal Aortic Aneurysm Formation in Hyperlipidemic Mice.

OBJECTIVE: Although the molecular components of circadian rhythms oscillate in discrete cellular components of the vasculature and many aspects of vascular function display diurnal variation, the cellular connections between the molecular clock and inflammatory cardiovascular diseases remain to be elucidated. Previously we have shown that pre- versus postnatal deletion of Bmal1 (brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1), the nonredundant core clock gene has contrasting effects on atherogenesis. Here we investigated the effect of myeloid cell Bmal1 deletion on atherogenesis and abdominal aortic aneurysm formation in mice. Approach and Results: Mice lacking Bmal1 in myeloid cells were generated by crossing Bmal1 flox/flox mice with lysozyme 2 promoter-driven Cre recombinase mice on a hyperlipidemic low-density lipoprotein receptor-deficient background and were fed on a high-fat diet to induce atherosclerosis. Atherogenesis was restrained, concomitant with a reduction of aortic proinflammatory gene expression in myeloid cell Bmal1 knockout mice. Body weight, blood pressure, blood glucose, triglycerides, and cholesterol were unaltered. Similarly, myeloid cell depletion of Bmal1 also restrained Ang II (angiotensin II) induced formation of abdominal aortic aneurysm in hyperlipidemic mice. In vitro, RNA-Seq analysis demonstrated a proinflammatory response in cultured macrophages in which there was overexpression of Bmal1. CONCLUSIONS: Myeloid cell Bmal1 deletion retards atherogenesis and restrains the formation of abdominal aortic aneurysm and may represent a potential therapeutic target for inflammatory cardiovascular diseases.

BMAL1 regulation of microglia-mediated neuroinflammation in MPTP-induced Parkinson's disease mouse model.

Dysfunction of the circadian rhythm is one of most common nonmotor symptoms in Parkinson's disease (PD), but the molecular role of the circadian rhythm in PD is unclear. We here showed that inactivation of brain and muscle ARNT-like 1 (BMAL1) in 1-methyl-4-phenyl-1,2,4,5-tetrahydropyridine (MPTP)-treated mice resulted in obvious motor functional deficit, loss of dopaminergic neurons (DANs) in the substantia nigra pars compacta (SNpc), decrease of dopamine (DA) transmitter, and increased activation of microglia and astrocytes in the striatum. Time on the rotarod or calorie consumption, and food and water intake were reduced in the Bmal1-/- mice after MPTP treatment, suggesting that absence of Bmal1 may exacerbate circadian and PD motor function. We observed a significant reduction of DANs (~35%) in the SNpc, the tyrosine hydroxylase protein level in the striatum (~60%), the DA (~22%), and 3,4-dihydroxyphenylacetic acid content (~29%), respectively, in MPTP-treated Bmal1-/- mice. Loss of Bmal1 aggravated the inflammatory reaction both in vivo and in vitro. These findings suggest that BMAL1 may play an essential role in the survival of DANs and maintain normal function of the DA signaling pathway via regulating microglia-mediated neuroinflammation in the brain.

BMAL1 coordinates energy metabolism and differentiation of pluripotent stem cells.

BMAL1 is essential for the regulation of circadian rhythms in differentiated cells and adult stem cells, but the molecular underpinnings of its function in pluripotent cells, which hold a great potential in regenerative medicine, remain to be addressed. Here, using transient and permanent loss-of-function approaches in mouse embryonic stem cells (ESCs), we reveal that although BMAL1 is dispensable for the maintenance of the pluripotent state, its depletion leads to deregulation of transcriptional programs linked to cell differentiation commitment. We further confirm that depletion of Bmal1 alters the differentiation potential of ESCs in vitro. Mechanistically, we demonstrate that BMAL1 participates in the regulation of energy metabolism maintaining a low mitochondrial function which is associated with pluripotency. Loss-of-function of Bmal1 leads to the deregulation of metabolic gene expression associated with a shift from glycolytic to oxidative metabolism. Our results highlight the important role that BMAL1 plays at the exit of pluripotency in vitro and provide evidence implicating a non-canonical circadian function of BMAL1 in the metabolic control for cell fate determination.

The molecular clock protein Bmal1 regulates cell differentiation in mouse embryonic stem cells.

Mammals optimize their physiology to the light-dark cycle by synchronization of the master circadian clock in the brain with peripheral clocks in the rest of the tissues of the body. Circadian oscillations rely on a negative feedback loop exerted by the molecular clock that is composed by transcriptional activators Bmal1 and Clock, and their negative regulators Period and Cryptochrome. Components of the molecular clock are expressed during early development, but onset of robust circadian oscillations is only detected later during embryogenesis. Here, we have used naïve pluripotent mouse embryonic stem cells (mESCs) to study the role of Bmal1 during early development. We found that, compared to wild-type cells, Bmal1-/- mESCs express higher levels of Nanog protein and altered expression of pluripotency-associated signalling pathways. Importantly, Bmal1-/- mESCs display deficient multi-lineage cell differentiation capacity during the formation of teratomas and gastrula-like organoids. Overall, we reveal that Bmal1 regulates pluripotent cell differentiation and propose that the molecular clock is an hitherto unrecognized regulator of mammalian development.

Metabolic-sensing of the skeletal muscle clock coordinates fuel oxidation.

Circadian clock confers temporal control in metabolism, with its disruption leading to the development of insulin resistance. Metabolic substrate utilization in skeletal muscle is coordinated with diurnal nutrient cycles. However, whether the molecular clock is involved in this coordination is largely unknown. Using a myocyte-selective genetic ablation mouse model of the essential clock activator Bmal1, here we identify muscle-intrinsic clock as a sensor of feeding cues to orchestrate skeletal muscle oxidation required for global nutrient flux. Bmal1 in skeletal muscle responds robustly to feeding in vivo and insulin induces its expression. Muscle Bmal1 deficiency impaired the transcriptional control of glucose metabolic pathway, resulting in markedly attenuated glucose utilization and fasting hyperglycemia. Notably, the loss of Bmal1 response to feeding abolished fasting-to-feeding metabolic fuel switch from fatty acids to glucose in skeletal muscle, leading to the activation of energy-sensing pathways for fatty acid oxidation. These altered metabolic substrate oxidations in Bmal1-deficient muscle ultimately depleted circulating lipid levels that prevented hepatic steatosis. Collectively, our findings highlight the key role of the metabolic-sensing function of skeletal muscle clock in partitioning nutrient flux between muscle and liver to maintain whole-body lipid and glucose homeostasis.

Analysis of Western diet, palmitate and BMAL1 regulation of neuropeptide Y expression in the murine hypothalamus and BMAL1 knockout cell models.

Western diets that are high in saturated fat and sugar disrupt circadian rhythms, induce weight gain, and lead to metabolic diseases including obesity. However, the mechanistic link between altered circadian rhythms and energy homeostasis remains poorly understood. In C57BL/6J mice, consuming a Western diet for 16 weeks significantly reduced food intake (at zeitgeber 12-16), in association with decreases in hypothalamic expression of the orexigenic neuropeptides, neuropeptide Y (Npy) and agouti-related peptide (AgRP). To examine the acute effects of the most prevalent saturated fatty acid in a Western diet, palmitate, and the role of the core clock gene, Bmal1, in the regulation of hypothalamic feeding neuropeptides, we used heterogeneous and clonal BMAL1 knockout (KO) immortalized hypothalamic cell lines, expressing specific neuropeptides, derived from male (M) and female (F) mice. Both mHypoA-BMAL1-KO/F and mHypoA-BMAL1-KO/M cells demonstrated a loss of circadian rhythmicity in expression of the clock gene, Per2, as compared to wild-type (control) cultures. Loss of BMAL1 also altered the time-dependent expression of Npy and proopiomelanocortin, and disrupted AgRP rhythmicity. Furthermore, palmitate increased BMAL1 binding to the Npy promotor region, and palmitate treatment (50 µM for 24 h) stimulated Npy expression in a BMAL1-dependent manner in both heterogeneous and clonal NPY-expressing female-derived cell models. The results of this study demonstrate that circadian expression of Bmal1 serves as a mechanistic link between Western diet- and palmitate-induced disruptions of the normal rhythmic patterns in hypothalamic feeding-related neuropeptides.

Dynamics at the serine loop underlie differential affinity of cryptochromes for CLOCK:BMAL1 to control circadian timing.

Mammalian circadian rhythms are generated by a transcription-based feedback loop in which CLOCK:BMAL1 drives transcription of its repressors (PER1/2, CRY1/2), which ultimately interact with CLOCK:BMAL1 to close the feedback loop with ~24 hr periodicity. Here we pinpoint a key difference between CRY1 and CRY2 that underlies their differential strengths as transcriptional repressors. Both cryptochromes bind the BMAL1 transactivation domain similarly to sequester it from coactivators and repress CLOCK:BMAL1 activity. However, we find that CRY1 is recruited with much higher affinity to the PAS domain core of CLOCK:BMAL1, allowing it to serve as a stronger repressor that lengthens circadian period. We discovered a dynamic serine-rich loop adjacent to the secondary pocket in the photolyase homology region (PHR) domain that regulates differential binding of cryptochromes to the PAS domain core of CLOCK:BMAL1. Notably, binding of the co-repressor PER2 remodels the serine loop of CRY2, making it more CRY1-like and enhancing its affinity for CLOCK:BMAL1.