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.

Light-induced LLPS of the CRY2/SPA1/FIO1 complex regulating mRNA methylation and chlorophyll homeostasis in Arabidopsis.

Light regulates chlorophyll homeostasis and photosynthesis via various molecular mechanisms in plants. The light regulation of transcription and protein stability of nuclear-encoded chloroplast proteins have been extensively studied, but how light regulation of mRNA metabolism affects abundance of nuclear-encoded chloroplast proteins and chlorophyll homeostasis remains poorly understood. Here we show that the blue light receptor cryptochrome 2 (CRY2) and the METTL16-type m6A writer FIONA1 (FIO1) regulate chlorophyll homeostasis in response to blue light. In contrast to the CRY2-mediated photo-condensation of the mRNA adenosine methylase (MTA), photoexcited CRY2 co-condenses FIO1 only in the presence of the CRY2-signalling protein SUPPRESSOR of PHYTOCHROME A (SPA1). CRY2 and SPA1 synergistically or additively activate the RNA methyltransferase activity of FIO1 in vitro, whereas CRY2 and FIO1, but not MTA, are required for the light-induced methylation and translation of the mRNAs encoding multiple chlorophyll homeostasis regulators in vivo. Our study demonstrates that the light-induced liquid-liquid phase separation of the photoreceptor/writer complexes is commonly involved in the regulation of photoresponsive changes of mRNA methylation, whereas the different photo-condensation mechanisms of the CRY/FIO1 and CRY/MTA complexes explain, at least partially, the writer-specific functions in plant photomorphogenesis.

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

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

Circadian disruption does not alter tumorigenesis in a mouse model of lymphoma.

Background: Disruption of natural light cycles, as experienced by shift workers, is linked to enhanced cancer incidence. Several mouse models of cancer develop more severe disease when exposed to irregular light/dark cycles, supporting the connection between circadian disruption and increased cancer risk. Cryptochrome 2 (CRY2), a repressive component of the molecular circadian clock, facilitates turnover of the oncoprotein c-MYC, one mechanism that may link the molecular clock to tumorigenesis. In Eµ-MYC mice, which express transgenic c-MYC in B cells and develop aggressive lymphomas and leukemia, global Cry2 deletion reduces survival and enhances tumor formation. Lighting conditions that mimic the disruption experienced by shift workers dampen Cry2 transcripts in peripheral tissues of C57BL/6J mice. Although it is milder than homozygous deletion of Cry2, we hypothesized that reduced Cry2 rhythmicity could alter MYC protein accumulation and contribute to enhanced cancer risk caused by circadian disruption. We tested this hypothesis in MYC-driven lymphoma. Methods: We housed Eµ-MYC mice in light-tight boxes set to either control (continuous cycles of 12-hours of light followed by 12-hours of dark, LD12:12) or chronic jetlag (eight-hour light phase advances every two to three days, CJL) lighting conditions and assessed the impact of disrupted light cycles on survival and tumor formation in Eµ-MYC mice. Results: Environmental disruption of circadian rhythms did not alter tumor location, tumor growth, or survival in Eµ-MYC mice. Conclusions: Dampened rhythms of Cry2 following disruption of circadian light exposures is milder than deletion of Cry2. The lack of phenotype caused by altered circadian gene expression in contrast to enhanced tumorigenesis caused by homozygous deletion of Cry2 suggests that CRY2 dosage impacts this model. Importantly, these findings indicate that increased cancer risk associated with circadian disruption arises from one or more mechanisms that are not recapitulated here, and may be different in distinct tumor types.

CRY1/2 regulate rhythmic CYP2A5 in mouse liver through repression of E4BP4.

CYP2A5, an enzyme responsible for metabolism of diverse drugs, displays circadian rhythms in its expression and activity. However, the underlying mechanisms are not fully established. Here we aimed to investigate a potential role of CRY1/2 (circadian clock modulators) in circadian regulation of hepatic CYP2A5. Regulatory effects of CRY1/2 on CYP2A5 were determined using Cry1-null and Cry2-null mice, and validated using AML-12, Hepa1-6 and HepG2 cells. CYP2A5 activities both in vivo and in vitro were assessed using coumarin 7-hydroxylation as a probe reaction. mRNA and protein levels were detected by qPCR and western blotting, respectively. Regulatory mechanism was studied using a combination of luciferase reporter assays, chromatin immunoprecipitation (ChIP) and co-immunoprecipitation (Co-IP). We found that ablation of Cry1 or Cry2 in mice reduced hepatic CYP2A5 expression (at both mRNA and protein levels) and blunted its diurnal rhythms. Consistently, these knockouts showed decreased CYP2A5 activity (characterised by coumarin 7-hydroxylation) and a loss of its time-dependency, as well as exacerbated coumarin-induced hepatotoxicity. Cell-based assays confirmed that CRY1/2 positively regulated CYP2A5 expression and rhythms. Based on combined luciferase reporter, ChIP and Co-IP assays, we unraveled that CRY1/2 interacted with E4BP4 protein to repress its inhibitory effect on Cyp2a5 transcription and expression. In conclusion, CRY1/2 regulate rhythmic CYP2A5 in mouse liver through repression of E4BP4. These findings advance our understanding of circadian regulation of drug metabolism and pharmacokinetics.

Potential Role of Phytochromes A and B and Cryptochrome 1 in the Adaptation of Solanum lycopersicum to UV-B Radiation.

UV-B causes both damage to the photosynthetic apparatus (PA) and the activation of specific mechanisms that protect the PA from excess energy and trigger a cascade of regulatory interactions with different photoreceptors, including phytochromes (PHYs) and cryptochromes (CRYs). However, the role of photoreceptors in plants' responses to UV-B radiation remains undiscovered. This study explores some of these responses using tomato photoreceptor mutants (phya, phyb1, phyab2, cry1). The effects of UV-B exposure (12.3 µmol (photons) m-2 s-1) on photosynthetic rates and PSII photochemical activity, the contents of photosynthetic and UV-absorbing pigments and anthocyanins, and the nonenzymatic antioxidant capacity (TEAC) were studied. The expression of key light-signaling genes, including UV-B signaling and genes associated with the biosynthesis of chlorophylls, carotenoids, anthocyanins, and flavonoids, was also determined. Under UV-B, phyab2 and cry1 mutants demonstrated a reduction in the PSII effective quantum yield and photosynthetic rate, as well as a reduced value of TEAC. At the same time, UV-B irradiation led to a noticeable decrease in the expression of the ultraviolet-B receptor (UVR8), repressor of UV-B photomorphogenesis 2 (RUP2), cullin 4 (CUL4), anthocyanidin synthase (ANT), phenylalanine ammonia-lease (PAL), and phytochrome B2 (PHYB2) genes in phyab2 and RUP2, CUL4, ANT, PAL, and elongated hypocotyl 5 (HY5) genes in the cry1 mutant. The results indicate the mutual regulation of UVR8, PHYB2, and CRY1 photoreceptors, but not PHYB1 and PHYA, in the process of forming a response to UV-B irradiation in tomato.

Transcription Repression of CRY2 via PER2 Interaction Promotes Adipogenesis.

The circadian clock is driven by a transcriptional-translational feedback loop, and cryptochrome 2 (CRY2) represses CLOCK/BMAL1-induced transcription activation. Despite the established role of clock in adipogenic regulation, whether the CRY2 repressor activity functions in adipocyte biology remains unclear. Here we identify a critical cysteine residue of CRY2 that mediates interaction with Period 2 (PER2). We further demonstrate that this mechanism is required for repressing circadian clock-controlled Wnt signaling to promote adipogenesis. CRY2 protein is enriched in white adipose depots and robustly induced by adipogenic differentiation. Via site-directed mutagenesis, we identified that a conserved CRY2 cysteine at 432 within the loop interfacing with PER2 mediates heterodimer complex formation that confers transcription repression. C432 mutation disrupted PER2 association without affecting BMAL1 binding, leading to loss of repression of clock transcription activation. In preadipocytes, whereas CRY2 enhanced adipocyte differentiation, the repression-defective C432 mutant suppressed this process. Furthermore, silencing of CRY2 attenuated, while stabilization of CRY2 by KL001 markedly augmented adipocyte maturation. Mechanistically, we show that transcriptional repression of Wnt pathway components underlies CRY2 modulation of adipogenesis. Collectively, our findings elucidate a CRY2-mediated repression mechanism that promotes adipocyte development, and implicate its potential as a clock intervention target for obesity.

A circadian clock protein cryptochrome inhibits the expression of inflammatory cytokines in Chinese mitten crab (Eriocheir sinensis).

Cryptochrome (Cry), as important flavoprotein, plays a key role in regulating the innate immune response, such as the release of inflammatory cytokines. In the present study, a cryptochrome homologue (EsCry) was identified from Chinese mitten crab Eriocheir sinensis, which contained a typical DNA photolyase domain, a FAD binding domain. The transcripts of EsCry were highly expressed at 11:00, and lowest at 3:00 within one day, while those of Interleukin enhancer binding factor (EsILF), Lipopolysaccharide-induced TNF-alpha factor (EsLITAF), Tumor necrosis factor (EsTNF) and Interleukin-16 (EsIL-16) showed a rhythm expression pattern contrary to EsCry. After EsCry was knocked down by dsEsCry injection, mRNA transcripts of Timeless (EsTim), Cycle (EsCyc), Circadian locomotor output cycles kaput (EsClock), Period (EsPer), and EsLITAF, EsTNF, EsILF, EsIL-16, as well as phosphorylation level of Dorsal significantly up-regulated. The transcripts of EsLITAF, EsTNF, EsILF, and EsIL-16 in EsCry-RNAi crabs significantly down-regulated after injection of NF-κB inhibitor. The interactions of EsCyc and EsCry, EsCyc and Dorsal were observed in vitro. These results indicated that EsCry negatively regulated the expression of the cytokine TNF and IL-16 via inhibiting their transcription factor LITAF and ILF through NF-κB signaling pathway, which provide evidences to better understand the circadian regulation mechanism of cytokine production in crabs.

An interplay between bZIP16, bZIP68, and GBF1 regulates nuclear photosynthetic genes during photomorphogenesis in Arabidopsis.

The development of a seedling into a photosynthetically active plant is a crucial process. Despite its importance, we do not fully understand the regulatory mechanisms behind the establishment of functional chloroplasts. We herein provide new insight into the early light response by identifying the function of three basic region/leucine zipper (bZIP) transcription factors: bZIP16, bZIP68, and GBF1. These proteins are involved in the regulation of key components required for the establishment of photosynthetically active chloroplasts. The activity of these bZIPs is dependent on the redox status of a conserved cysteine residue, which provides a mechanism to finetune light-responsive gene expression. The blue light cryptochrome (CRY) photoreceptors provide one of the major light-signaling pathways, and bZIP target genes overlap with one-third of CRY-regulated genes with an enrichment for photosynthesis/chloroplast-associated genes. bZIP16, bZIP68, and GBF1 were demonstrated as novel interaction partners of CRY1. The interaction between CRY1 and bZIP16 was stimulated by blue light. Furthermore, we demonstrate a genetic link between the bZIP proteins and cryptochromes as the cry1cry2 mutant is epistatic to the cry1cry2bzip16bzip68gbf1 mutant. bZIP16, bZIP68, and GBF1 regulate a subset of photosynthesis associated genes in response to blue light critical for a proper greening process in Arabidopsis.

Probing Transcriptional Crosstalk between Cryptochromes and Iron-sulfur Cluster Assembly 1 (MagR) in the Magnetoresponse of a Migratory Insect.

Many organisms can sense and respond to magnetic fields (MFs), with migratory species in particular utilizing geomagnetic field information for long-distance migration. Cryptochrome proteins (Crys) along with a highly conserved Iron-sulfur cluster assembly protein (i.e., MagR) have garnered significant attention for their involvement in magnetoresponse (including magnetoreception). However, in vivo investigations of potential transcriptional crosstalk between Crys and MagR genes have been limited. The brown planthopper, Nilaparvata lugens, is a major migratory pest insect and an emerging model for studying MF intensity-related magnetoresponse. Here, we explored in vivo transcriptional crosstalk between Crys (Cry1 and Cry2) and MagR in N. lugens. The expression of Crys and MagR were found to be sensitive to MF intensity changes as small as several micro-teslas. Knocking down MagR expression led to a significant downregulation of Cry1, but not Cry2. The knockdown of either Cry1 or Cry2 individually did not significantly affect MagR expression. However, their double knockdown resulted in significant upregulation of MagR. Our findings clearly indicate transcriptional crosstalk between MagR and Crys known to be involved in magnetoresponse. This work advances the understanding of magnetoresponse signaling and represents a key initial step towards elucidating the functional consequences of these novel in vivo interactions.

Cryptochrome 2 acetylation attenuates its antiproliferative effect in breast cancer.

Breast cancer is the most commonly diagnosed cancer, and its global impact is increasing. Its onset and progression are influenced by multiple cues, one of which is the disruption of the internal circadian clock. Cryptochrome 2 (Cry2) genetic dysregulation may lead to the development of some diseases and even tumors. In addition, post-translational modifications can alter the Cry2 function. Here, we aimed to elucidate the post-translational regulations of Cry2 and its role in breast cancer pathogenesis. We identified p300-drived acetylation as a novel Cry2 post-translational modification, which histone deacetylase 6 (HDAC6) could reverse. Furthermore, we found that Cry2 inhibits breast cancer proliferation, but its acetylation impairs this effect. Finally, bioinformatics analysis revealed that genes repressed by Cry2 in breast cancer were mainly enriched in the NF-κB pathway, and acetylation reversed this repression. Collectively, these results indicate a novel Cry2 regulation mechanism and provide a rationale for its role in breast tumorigenesis.

"Time" for obesity-related cancer: The role of the circadian rhythm in cancer pathogenesis and treatment.

The circadian rhythm is regulated by an intrinsic time-tracking system, composed both of a central and a peripheral clock, which influences the cycles of activities and sleep of an individual over 24 h. At the molecular level, the circadian rhythm begins when two basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) proteins, BMAL-1 and CLOCK, interact with each other to produce BMAL-1/CLOCK heterodimers in the cytoplasm. The BMAL-1/CLOCK target genes encode for the repressor components of the clock, cryptochrome (Cry1 and Cry2) and the Period proteins (Per1, Per2 and Per3). It has been recently demonstrated that the disruption of circadian rhythm is associated with an increased risk of developing obesity and obesity-related diseases. In addition, it has been demonstrated that the disruption of the circadian rhythm plays a key role in tumorigenesis. Further, an association between the circadian rhythm disruptions and an increased incidence and progression of several types of cancer (e.g., breast, prostate, colorectal and thyroid cancer) has been found. As the perturbation of circadian rhythm has adverse metabolic consequences (e.g., obesity) and at the same time tumor promoter functions, this manuscript has the aim to report how the aberrant circadian rhythms affect the development and prognosis of different types of obesity-related cancers (breast, prostate, colon rectal and thyroid cancer) focusing on both human studies and on molecular aspects.

Circadian rhythm-related factors of PER and CRY family genes function as novel therapeutic targets and prognostic biomarkers in lung adenocarcinoma.

The period (PER) and cryptochrome (CRY) families play critical roles in circadian rhythms. The imbalance of circadian factors may lead to the occurrence of cancer. Expressions of PER and CRY family members decrease in various cancers. Nevertheless, expression levels, genetic variations, and molecular mechanisms of PER and CRY family members in lung adenocarcinoma (LUAD) and their correlations with prognoses and immune infiltration in LUAD patients are still unclear. In this study, to identify their biological functions in LUAD development, comprehensive high-throughput techniques were applied to analyze the relationships of expressions of PER and CRY family members with genetic variations, molecular mechanisms, and immune infiltration. The present results showed that transcription levels of PER1 and CRY2 in LUAD were significantly downregulated. High expression levels of PER2, PER3, CRY1, and CRY2 indicated longer overall survival. Some cancer signaling pathways were related to PER and CRY family members, such as cell-cycle, histidine metabolism, and progesterone-mediated oocyte maturation pathways. Expressions of PER and CRY family members significantly affected the infiltration of different immune cells. In conclusion, our findings may help better understand the molecular basis of LUAD, and provide new perspectives of PER and CRY family members as novel biomarkers for LUAD.

Discovery of a small molecule that selectively destabilizes Cryptochrome 1 and enhances life span in p53 knockout mice.

Cryptochromes are negative transcriptional regulators of the circadian clock in mammals. It is not clear how reducing the level of endogenous CRY1 in mammals will affect circadian rhythm and the relation of such a decrease with apoptosis. Here, we discovered a molecule (M47) that destabilizes Cryptochrome 1 (CRY1) both in vitro and in vivo. The M47 selectively enhanced the degradation rate of CRY1 by increasing its ubiquitination and resulted in increasing the circadian period length of U2OS Bmal1-dLuc cells. In addition, subcellular fractionation studies from mice liver indicated that M47 increased degradation of the CRY1 in the nucleus. Furthermore, M47-mediated CRY1 reduction enhanced oxaliplatin-induced apoptosis in Ras-transformed p53 null fibroblast cells. Systemic repetitive administration of M47 increased the median lifespan of p53-/- mice by ~25%. Collectively our data suggest that M47 is a promising molecule to treat forms of cancer depending on the p53 mutation.

Iron-sulfur complex assembly: Potential players of magnetic induction in plants.

Iron-sulfur (Fe-S) clusters are involved in fundamental biological reactions and represent a highly regulated process involving a complex sequence of mitochondrial, cytosolic and nuclear-catalyzed protein-protein interactions. Iron-sulfur complex assembly (ISCA) scaffold proteins are involved in Fe-S cluster biosynthesis, nitrogen and sulfur metabolism. ISCA proteins are involved in abiotic stress responses and in the pigeon they act as a magnetic sensor by forming a magnetosensor (MagS) complex with cryptochrome (Cry). MagR gene exists in the genomes of humans, plants, and microorganisms and the interaction between Cry and MagR is highly conserved. Owing to the extensive presence of ISCA proteins in plants and the occurrence of homology between animal and human MagR with at least four Arabidopsis ISCAs and several ISCAs from different plant species, we believe that a mechanism similar to pigeon magnetoperception might be present in plants. We suggest that plant ISCA proteins, homologous of the animal MagR, are good candidates and could contribute to a better understanding of plant magnetic induction. We thus urge more studies in this regard to fully uncover the plant molecular mechanisms underlying MagR/Cry mediated magnetic induction and the possible coupling between light and magnetic induction.

Clock genes rescue nphp mutations in zebrafish.

The zebrafish pronephros model, using morpholino oligonucleotides (MO) to deplete target genes, has been extensively used to characterize human ciliopathy phenotypes. Recently, discrepancies between MO and genetically defined mutants have questioned this approach. We analyzed zebrafish with mutations in the nphp1-4-8 module to determine the validity of MO-based results. While MO-mediated depletion resulted in glomerular cyst and cloaca malformation, these ciliopathy-typical manifestations were observed at a much lower frequency in zebrafish embryos with defined nphp mutations. All nphp1-4-8 mutant zebrafish were viable and displayed decreased manifestations in the next (F2) generation, lacking maternal RNA contribution. While genetic compensation was further supported by the observation that nphp4-deficient mutants became partially refractory to MO-based nphp4 depletion, zebrafish embryos, lacking one nphp gene, became more sensitive to MO-based depletion of additional nphp genes. Transcriptome analysis of nphp8 mutant embryos revealed an upregulation of the circadian clock genes cry1a and cry5. MO-mediated depletion of cry1a and cry5 caused ciliopathy phenotypes in wild-type embryos, while cry1a and cry5 depletion in maternal zygotic nphp8 mutant embryos increased the frequency of glomerular cysts compared to controls. Importantly, cry1a and cry5 rescued the nephropathy-related phenotypes in nphp1, nphp4 or nphp8-depleted zebrafish embryos. Our results reveal that nphp mutant zebrafish resemble the MO-based phenotypes, albeit at a much lower frequency. Rapid adaption through upregulation of circadian clock genes seems to ameliorate the loss of nphp genes, contributing to phenotypic differences.

Hepatic GSK3ß-Dependent CRY1 Degradation Contributes to Diabetic Hyperglycemia.

Excessive hepatic glucose production (HGP) is a key factor promoting hyperglycemia in diabetes. Hepatic cryptochrome 1 (CRY1) plays an important role in maintaining glucose homeostasis by suppressing forkhead box O1 (FOXO1)-mediated HGP. Although downregulation of hepatic CRY1 appears to be associated with increased HGP, the mechanism(s) by which hepatic CRY1 dysregulation confers hyperglycemia in subjects with diabetes is largely unknown. In this study, we demonstrate that a reduction in hepatic CRY1 protein is stimulated by elevated E3 ligase F-box and leucine-rich repeat protein 3 (FBXL3)-dependent proteasomal degradation in diabetic mice. In addition, we found that GSK3ß-induced CRY1 phosphorylation potentiates FBXL3-dependent CRY1 degradation in the liver. Accordingly, in diabetic mice, GSK3ß inhibitors effectively decreased HGP by facilitating the effect of CRY1-mediated FOXO1 degradation on glucose metabolism. Collectively, these data suggest that tight regulation of hepatic CRY1 protein stability is crucial for maintaining systemic glucose homeostasis.

P2Y1 purinergic receptor identified as a diabetes target in a small-molecule screen to reverse circadian ß-cell failure.

The mammalian circadian clock drives daily oscillations in physiology and behavior through an autoregulatory transcription feedback loop present in central and peripheral cells. Ablation of the core clock within the endocrine pancreas of adult animals impairs the transcription and splicing of genes involved in hormone exocytosis and causes hypoinsulinemic diabetes. Here, we developed a genetically sensitized small-molecule screen to identify druggable proteins and mechanistic pathways involved in circadian ß-cell failure. Our approach was to generate ß-cells expressing a nanoluciferase reporter within the proinsulin polypeptide to screen 2640 pharmacologically active compounds and identify insulinotropic molecules that bypass the secretory defect in CRISPR-Cas9-targeted clock mutant ß-cells. We validated hit compounds in primary mouse islets and identified known modulators of ligand-gated ion channels and G-protein-coupled receptors, including the antihelmintic ivermectin. Single-cell electrophysiology in circadian mutant mouse and human cadaveric islets revealed ivermectin as a glucose-dependent secretagogue. Genetic, genomic, and pharmacological analyses established the P2Y1 receptor as a clock-controlled mediator of the insulinotropic activity of ivermectin. These findings identify the P2Y1 purinergic receptor as a diabetes target based upon a genetically sensitized phenotypic screen.

Circadian rhythms ­ 'inbuilt' 24-hour cycles ­ control many aspects of behaviour and physiology. In mammals, they operate in nearly all tissues, including those involved in glucose metabolism. Recent studies have shown that mice with faulty genes involved in circadian rhythms, the core clock genes, can develop diabetes. Diabetes arises when the body struggles to regulate blood sugar levels. In healthy individuals, the hormone insulin produced by beta cells in the pancreas regulates the amount of sugar in the blood. But when beta cells are faulty and do not generate sufficient insulin levels, or when insulin lacks the ability to stimulate cells to take up glucose, diabetes can develop. Marcheva, Weidemann, Taguchi et al. wanted to find out if diabetes caused by impaired clock genes could be treated by targeting pathways regulating the secretion of insulin. To do so, they tested over 2,500 potential drugs on genetically modified beta cells with faulty core clock genes. They further screened the drugs on mice with the same defect in their beta cells. Marcheva et al. identified one potential compound, the anti-parasite drug ivermectin, which was able to restore the secretion of insulin. When ivermectin was applied to both healthy mice and mice with faulty beta cells, the drug improved the control over glucose levels by activating a specific protein receptor that senses molecules important for storing and utilizing energy. The findings reveal new drug targets for treating forms of diabetes associated with deregulation of the pancreatic circadian clock. The drug screening strategy used in the study may also be applied to reveal mechanisms underlying other conditions associated with disrupted circadian clocks, including sleep loss and jetlag.

Circadian Clock Genes Are Correlated with Prognosis and Immune Cell Infiltration in Colon Adenocarcinoma.

BACKGROUND: Colon adenocarcinoma (COAD) is a malignancy with a high incidence and is associated with poor quality of life. Dysfunction of circadian clock genes and disruption of normal rhythms are associated with the occurrence and progression of many cancer types. However, studies that systematically describe the prognostic value and immune-related functions of circadian clock genes in COAD are lacking. METHODS: Genomic data obtained from The Cancer Genome Atlas (TCGA) database was analyzed for expression level, mutation status, potential biological functions, and prognostic performance of core circadian clock genes in COAD. Their correlations with immune infiltration and TMB/MSI score were analyzed by Spearman's correlation analysis. Pearson's correlation analysis was performed to analyze their associations with drug sensitivity. Lasso Cox regression analysis was performed to construct a prognosis signature. Moreover, an mRNA-miRNA-lncRNA regulatory axis was also detected by ceRNA network. RESULTS: In COAD tissues, the mRNA levels of CLOCK, CRY1, and NR1D1 were increased, while the mRNA levels of ARNTL, CRY2, PER1, PER3, and RORA were decreased. We also summarized the relative genetic mutation variation landscape. GO and KEGG pathway analyses demonstrated that these circadian clock genes were primarily correlated with the regulation of circadian rhythms and glucocorticoid receptor signaling pathways. COAD patients with high CRY2, NR1D1, and PER2 expression had worse prognosis. A prognostic model constructed based on the 9 core circadian clock genes predicted the COAD patients' overall survival with medium to high accuracy. A significant association between prognostic circadian clock genes and immune cell infiltration was found. Moreover, the lncRNA KCNQ1OT1/hsa-miRNA-32-5p/PER2/CRY2 regulatory axis in COAD was also detected through a mRNA-miRNA-lncRNA network. CONCLUSION: Our results identified CRY2, NR1D1, and PER2 as potential prognostic biomarkers for COAD patients and correlated their expression with immune cell infiltration. The lncRNA KCNQ1OT1/hsa-miRNA-32-5p/PER2/CRY2 regulatory axis was detected in COAD and might play a vital role in the occurrence and progression of COAD.