The global obesity epidemic highlights the need to understand the molecular mechanisms that regulate energy metabolism. Among emerging research areas, fat browning-the transformation of white adipose tissue into beige fat-has gained significant attention. This review explores the molecular pathways involved in fat browning triggered by fasting, physical exercise, and cold exposure, emphasizing both shared and distinct regulatory mechanisms. These stimuli consistently induce physiological responses such as lipolysis, mitochondrial biogenesis, and improved insulin sensitivity. Notably, PGC-1α and SIRT3 are upregulated across all three conditions, underscoring their central roles in mitochondrial function and energy metabolism and identifying them as promising therapeutic targets. In contrast, UCP1 and PRDM16 exhibit condition-specific regulation, suggesting they may not be universally essential for fat browning. In addition, the review discusses species-specific differences in brown adipose tissue (BAT) activation, particularly between rodents and humans, highlighting the challenges of translating animal model findings to human therapies. Future research should aim to develop selective pharmacological activators of PGC-1α and SIRT3 to enhance therapeutic outcomes while minimizing adverse effects. This review also proposes that integrating fasting, exercise, and cold exposure could provide innovative strategies to promote metabolic health.

Asymmetric development, in which functional differences occur between left-right symmetrical organs, is widespread in organisms, including fish and mollusks. However, the asymmetry of symmetrical sensory structures in Haliotis discus hannai, a gastropod with a sensitive sensory system, remains unknown. This study analyzed the transcriptomes of three sensory structures (eyestalks, cephalic tentacles, and epipodial tentacles) to explore potential asymmetries in this species. RNA-seq revealed functional differences in sensory ability and sperm-egg recognition between right and left eyestalks, with cephalic tentacles displaying asymmetry in cytoskeletal organization and cell cycle regulation. Epipodial tentacles showed similar asymmetries, including immune response differences. Moreover, the cAMP-protein kinase A (PKA)-CREB-binding protein (CBP) signaling pathway responded asymmetrically, with PKA responding to activators and inhibitors on both sides and CBP showing a stronger response on the right. These findings provide insights into sensory asymmetry in mollusks and guidance for further investigations of the molecular mechanisms underlying asymmetry in symmetrical organs.

Esophageal cancer is one of the most common digestive cancers in the world. Because of the limitation and resistence of the traditional chemotherapy drugs, it is important to explore new therapeutic targets and strategies for this refractory cancer. Recently, targeting deubiquitinases has emerged as a promising avenue for the development of anti-tumor drugs. However, the role and underlying mechanism of NSC632839, a broad-spectrum deubiquitinases inhibitor, in esophageal squamous cell carcinoma in vitro remain elusive.

Cell Counting Kit-8 assay, colony formation assay, EdU proliferation experiment and cell morphology observation were used to detect the effect of NSC632839 on cell growth. Flow cytometry was employed to detect cell apoptosis and cell cycle arrest. Immunoblot and immunofluorescence was used to evaluate the expression level of cell cycle-, apoptosis-, and autophagy-related proteins.

NSC632839 inhibited the proliferation of Kyse30 and Kyse450 cells. Mechanistically, NSC632839 induced the formation of multipolar spindles, and its concomitant spindle assembly checkpoint-dependent mitotic arrest, followed by CREB-Noxa-mediated apoptosis. Reversine, a classical MPS1 kinase inhibitor known for its ability to inhibit the spindle assembly checkpoint, could rescue NSC632839-induced cell cycle arrest and apoptosis. Additionally, NSC632839 could trigger pro-survival autophagy. Combination of autophagy inhibitor, CQ and BafA1, with NSC632839 could induce stronger cell proliferation inhibition and apoptosis than NSC632839 alone.

These findings provided a novel anti-cancer mechanism of NSC632839 and highlighted it as a potential anti-tumor agent for the treatment of esophageal cancer.

Earlier investigations had established that Diabetes mellitus (DM) caused significant damage in the central nervous system, bringing about diabetic encephalopathy and increasing the risk of cognitive-related problems. Nonetheless, the inherent pathophysiology of cognitive dysfunctions in DM is not well understood. The current study aimed to examine the possible influences of sacubitril/valsartan (SAC/VAL), an angiotensin receptor blocker/neprilysin inhibitor (ARNI), on cognitive dysfunction associated with streptozotocin (STZ)-induced diabetic rats. SAC/VAL and VAL treatments were initiated three days after the diabetic condition was established and continued daily for eight weeks. Normal, non-diabetic rats were reserved as a control group. Both SAC/VAL and VAL treatment in diabetic rats ameliorated diabetes induced oxidative stress as indicated by reduced malondialdehyde (MDA), increased total antioxidant capacity (TAO) in hippocampal tissue and decreased serum advanced glycation end products (AGEs), also inflammatory and apoptotic changes were observed and proved by the reduction of tumor necrosis factor alpha (TNF-α) and caspase -3 in rat hippocampus. SAC/VAL administration to diabetic rats also improved neuronal damages as reflected by restored cAMP response element-binding protein (CREB), brain derived neurotrophic factor (BDNF) and pre-synaptic phosphoproteins, synapsin I and growth associated protein-43 (GAP-43) in the hippocampus of diabetic rats. Additionally, SAC/VAL treated diabetic rats markedly reduced signs of cognitive deterioration during the Morris water maze test. Collectively, these findings suggested that SAC/VAL might play a vital role in improvement of the cognitive impairment observed in diabetic rats through antioxidant, anti-inflammatory and anti-apoptotic actions.

The interplay between systemic metabolism and immune responses is increasingly recognized as a significant factor in the dysregulation of glucose homeostasis associated with diabetes and obesity. Immune metabolites play crucial roles in mediating this crosstalk, with itaconate emerging as an important immune metabolite involved in the inflammatory response of macrophages. Recent studies have highlighted the role of itaconate as a regulator of glucose metabolism, particularly in the context of obesity, although the underlying mechanisms remain poorly understood. In this study, we identified itaconate as one of the metabolites that significantly increase in the liver during fasting compared to fed conditions. Mechanistically, we found that itaconate enhances glucagon-induced liver gluconeogenesis independently of insulin signaling. Notably, itaconate upregulates the expression of gluconeogenic genes both under basal conditions and in the presence of palmitic acid. Furthermore, our data indicate that the effects of itaconate occur independently of CREB activation. Instead, we demonstrate that these potentiating effects are mediated through the induction of nuclear factor erythroid 2-related factor 2 (NRF2). Our findings demonstrate that itaconate has a glucagon-potentiating effects in the liver, suggesting that itaconate may play a significant role in the pathogenesis of metabolic-associated liver diseases.

The gene A-kinase anchoring protein 11 (AKAP11) recently emerged as a shared risk factor between bipolar disorder and schizophrenia, driven by large-effect loss-of-function (LoF) variants. Recent research has uncovered the neurophysiological characteristics and synapse proteomics profile of Akap11-mutant mouse models. Considering the role of AKAP11 in binding cAMP-dependent protein kinase A (PKA) and mediating phosphorylation of numerous substrates, such as transcription factors and epigenetic regulators, and given that chromatin alterations have been implicated in the brains of patients with bipolar disorder and schizophrenia, it is crucial to uncover the transcriptomic and chromatin dysregulations following the heterozygous knockout of AKAP11, particularly in human neurons. This study uses genome-wide approaches to investigate such aberrations in human induced pluripotent stem cell (iPSC)-derived neurons. We show the impact of heterozygous AKAP11 LoF mutations on the gene expression landscape and profile the DNA methylation and histone acetylation modifications. Altogether we highlight the involvement of aberrant activity of intergenic and intronic enhancers, which are enriched in PBX homeobox 2 (PBX2) and Nuclear Factor-1 (NF1) known binding motifs, respectively, in transcription dysregulations of genes mainly involved in DNA-binding transcription factor activity, actin binding and cytoskeleton regulation, and cytokine receptor binding. We also show significant downregulation of pathways related to ribosome structure and function, a pathway also altered in BD and SCZ post-mortem brain tissues and heterozygous Akap11-KO mice synapse proteomics. A better understanding of the dysregulations resulting from haploinsufficiency in AKAP11 improves our knowledge of the biological roots and pathophysiology of BD and SCZ, paving the way for better therapeutic approaches.

The growth factor-mediated mitogen-activated protein kinase (MAPK) signaling pathways in cancer development have become increasingly important in the discovery of therapeutic agents for the treatment of cancer. RSK2 has been historically overlooked in studies regarding its involvement in physiology and signaling pathways related to human diseases, except for Coffin-Lowry syndrome, because it is located downstream of ERKs. For the last 25 years, the authors' laboratory has made groundbreaking discoveries regarding the role of RSK2, especially by elucidating its binding partners, signaling network, and crosstalk. RSK2 is an important emerging target for developing anticancer drugs. Nevertheless, further studies on the detailed mechanism and signaling network are necessary to avoid the unexpected effects of RSK2 inhibitors. This paper describes a new paradigm of RSK2, where it works as a signaling node to modulate diverse cellular processes, including cell proliferation and transformation, cell cycle regulation, chromatin remodeling, and immune response and inflammation regulation.

Structural and functional aspects of the hippocampus have been shown to be sensitive to the aging process, resulting in deficits in hippocampal-dependent cognition. Similarly, adult hippocampal neurogenesis (AHN), described as the generation of new neurons from neural stem cells in the hippocampus, has shown to be negatively affected by aging throughout life. Extensive research has highlighted the role of physical exercise (PE) in positively regulating hippocampal-dependent cognition and AHN. Here, by critically reviewing preclinical and clinical studies, we discuss the significance of PE in reversing age-associated changes of the hippocampus via modulation of AHN. We indicate that PE-induced changes operate on two main levels. On the first level, PE can potentially cause structural modifications of the hippocampus, and on the second level, it regulates the molecular and cellular pathways involved. These changes result in the vascular remodelling of the neurogenic niche, as well as the secretion of neurotrophic and antioxidant factors, which can in turn activate quiescent neural stem cells, while restoring their proliferation capacity and boosting their survival - features which are negatively impacted during aging. Understanding these mechanisms will allow us to identify new targets to tackle cognitive aging and improve quality of life.

Many patients with chronic obstructive pulmonary disease are susceptible to recurrent exacerbations. In this study, we hypothesized that endogenous acetylcholine (ACh) may act as a proinflammatory mediator because long-acting muscarinic receptor antagonists protect against exacerbations, which have an inflammatory basis. This possibility was explored by determining if carbachol (CCh), a stable ACh analog, was a genomic stimulus in BEAS-2B bronchial epithelial cells. The ability of CCh to interact with indacaterol (Ind), a long-acting β2-adrenoceptor agonist, was also assessed given that (1) sympathomimetic bronchodilators can promote adverse gene expression changes in airway structural cells, and (2) crosstalk between β2-adrenoceptor and Gq-coupled muscarinic receptor agonists is well described. Unlike Ind, which induced 624 unique genes, CCh was a relatively weak genomic stimulus, implying that ACh may not behave as a proinflammatory mediator as hypothesized. Nevertheless, checkerboard assays using BEAS-2B cells expressing a cAMP-response element luciferase reporter determined that CCh interacted with Ind in a supra-additive manner and that this interaction was replicated on 39 Ind-regulated genes. Functional annotation of the Ind-regulated transcriptomes identified "transcription" and "signalling" as the dominant themes, with gene ontology terms associated with "inflammation" and "immune processes" being highly represented. A comparable gene ontology signature was obtained when Ind and CCh were combined; however, the number, magnitude and duration of gene expression changes were significantly enhanced. If genomic interactions occur between a long-acting β2-adrenoceptor agonist and ACh in vivo, then they may enhance the expression of adverse-effect genes that could maintain, or even augment, features of lung pathology in chronic obstructive pulmonary disease. SIGNIFICANCE STATEMENT: Long-acting muscarinic receptor antagonists reduce exacerbation risk in chronic obstructive pulmonary disease, implying the etiology could have an inflammatory basis mediated by acetylcholine. However, in BEAS-2B cells, carbachol was a weak genomic stimulus, although it enhanced changes in indacaterol-regulated gene expression. Functional annotation of carbachol + indacaterol-regulated genes identified gene ontology terms associated with several themes, including inflammation. Interaction between a long-acting β2-adrenoceptor agonist and endogenous acetylcholine could, paradoxically, augment airway inflammation in patients with chronic obstructive pulmonary disease.

Disrupted lipogenic signaling and steatosis are key features of alcohol-associated liver disease (ALD). A-kinase anchoring protein 12 (AKAP12) is a scaffolding partner of the cAMP-dependent protein kinase, PKA that controls its spatiotemporal localization. Activation of PKA by cAMP inhibits lipogenesis and facilitates fatty acid oxidation (FAO). The goal of this work is to examine how AKAP12's PKA-anchoring ability regulates outcomes of alcohol-associated steatosis. Crosslinking proteomics identified PKA and its lipogenic substrates as interacting partners of AKAP12. Alcohol exposure diminished AKAP12's interaction with PKA regulatory subunits and PKA substrates, acetyl CoA carboxylase (ACC1), pyruvate dehydrogenase (PDHA) and adipose triglyceride lipase (ATGL). Alcohol inhibited PKA activity and increased triglyceride content in human hepatocytes. Forced expression of AKAP12 restored alcohol suppressed PKA activation and inhibited lipid accumulation, whereas silencing had the reverse effect. Since AKAP12 sustained PKA activity, we evaluated whether the AKAP12-PKA scaffold was important in lipid homeostasis. Inhibition of AKAP12-PKA interaction by CRISPR deletion of AKAP12's PKA binding domain in cultured hepatocytes or in mouse models of ALD dramatically suppressed PKA activity, enhanced ACC1 activity demonstrated by reduced inhibitory phosphorylation, increased lipid accumulation and reduced FAO in hepatocytes. Overexpression of AKAP12 in mouse livers sustained PKA activation, diminished basal and alcohol potentiated triglyceride content, and regulated inflammatory signaling altered by alcohol. Mechanistically, we discovered that alcohol enhanced the inhibitory activity of a kinase, serine/threonine-protein kinase 25 (STK25) on PKA that regulated its interaction with AKAP12. In conclusion, the AKAP12-PKA scaffold controls lipogenic signaling, disruption of which favors steatosis during ALD.

Despite advancements in radiotherapy, chemotherapy, endocrine therapy, targeted therapy, and immunotherapy, resistance to therapy remains a pervasive challenge in oncology, in part owing to tumor heterogeneity. Identifying new therapeutic targets is key to addressing this challenge because it can both diversify and enhance existing treatment options, particularly through combination regimens. The cAMP response element-binding protein (CREB) is a transcription factor involved in various biological processes. It is aberrantly activated in several aggressive cancer types, including breast cancer. Clinically, high CREB expression is associated with increased breast tumor aggressiveness and poor prognosis. Functionally, CREB promotes breast cancer cell proliferation, survival, invasion, metastasis, as well as therapy resistance by deregulating genes related to apoptosis, cell cycle, and metabolism. Targeting CREB with small molecule inhibitors has demonstrated promise in preclinical studies. This review summarizes the current understanding of CREB mechanisms and their potential as a therapeutic target. SIGNIFICANCE STATEMENT: cAMP response element-binding protein (CREB) is a master regulator of multiple biological processes, including neurodevelopment, metabolic regulation, and immune response. CREB is a putative proto-oncogene in breast cancer that regulates the cell cycle, apoptosis, and cellular migration. Preclinical development of CREB-targeting small molecules is underway.

In an obesogenic environment, such as the one we have been experiencing in recent decades, epigenetics provides answers to the relationship between hereditary and environmentally acquired patterns that have significantly contributed to the global rise in obesity prevalence. MicroRNA (miRNA) constitutes a diminutive non-coding small RNA molecule, 20 to 24 nucleotides in length, that functions as a regulator of gene regulation at the post-translational level. Circulating miRNAs (c-miRNAs) have been detected in multiple body fluids, including blood, plasma, serum, saliva, milk from breastfeeding mothers, and urine. These molecules hold significant therapeutic value and serve as extracellular biomarkers in metabolic diseases. They aid in the diagnosis and tracking of therapy responses, as well as dietary and physical habit modifications. Researchers have studied c-miRNAs as potential biomarkers for diagnosing and characterizing systemic diseases in people of all ages and backgrounds since then. These conditions encompass dyslipidemia, type 2 diabetes mellitus (T2DM), cardiovascular risk, metabolic syndrome, cardiovascular diseases, and obesity. This review therefore analyzes the usefulness of c-miRNAs as therapeutic markers over the past decades. It also provides an update on c-miRNAs associated with general obesity and overweight, as well as with the most prevalent pathologies in the adult population. It also examines the effect of different nutritional approaches and physical activity regarding the activity of miRNAs in circulation in adults with overweight or general obesity. All of this is done with the aim of evaluating their potential use as biomarkers in various research contexts related to overweight and obesity in adults.

Water homeostasis is regulated by the peptide hormone arginine vasopressin (AVP), which promotes water reabsorption in the renal collecting duct. The regulation of Aqp2 gene transcription is a key mechanism through which AVP modulates water transport as disruption of this mechanism leads to water balance disorders. Therefore, an important goal is to understand the regulatory processes that control Aqp2 gene transcription. While CREB (CREB1) has been proposed as the primary transcription factor responsible for Aqp2 transcription, recent evidence challenges this view, suggesting that other CREB-like transcription factors, including ATF1 and CREM, may play a role.

We employed the CRISPR/Cas9 gene-editing system to delete Atf1, Creb1, and Crem in mpkCCD cells, an immortalized mouse collecting duct cell line. These cell lines were then exposed to the vasopressin analog, dDAVP, to assess the role of these transcription factors in regulating Aqp2 expression. AQP2 protein levels were measured by immunoblotting and RNA-seq was used to analyze changes in Aqp2 mRNA abundance, as well as other transcriptomic changes.

Deletion of all three transcription factors (ATF1, CREB1, and CREM) led to a significant reduction in the vasopressin-induced upregulation of AQP2 protein, confirming their role in regulating Aqp2 expression. RNA-seq data showed that Aqp2 mRNA levels mirrored changes in protein abundance, supporting the idea that these transcription factors affect Aqp2 transcription. Rescue experiments in triple knockout cells showed that expressing any of the three transcription factors restored the response to vasopressin.

Our findings demonstrate that ATF1, CREB1, and CREM have redundant roles in regulating Aqp2 transcription. Based on these results and prior data, we propose that these CREB-family transcription factors may regulate Aqp2 gene transcription indirectly by controlling the expression of additional unidentified transcription factors.

The reward and novelty-related neuromodulator dopamine plays an important role in hippocampal long-term memory, which is thought to involve protein-synthesis-dependent synaptic plasticity. However, the direct effects of dopamine on protein synthesis, and the functional implications of newly synthesised proteins for synaptic plasticity, have not yet been investigated. We have previously reported that timing-dependent synaptic depression (t-LTD) can be converted into potentiation by dopamine application during synaptic stimulation (Brzosko et al., 2015) or postsynaptic burst activation (Fuchsberger et al., 2022). Here, we show that dopamine increases protein synthesis in mouse hippocampal CA1 neurons, enabling dopamine-dependent long-term potentiation (DA-LTP), which is mediated via the Ca2+-sensitive adenylate cyclase (AC) subtypes 1/8, cAMP, and cAMP-dependent protein kinase (PKA). We found that neuronal activity is required for the dopamine-induced increase in protein synthesis. Furthermore, dopamine induced a protein-synthesis-dependent increase in the AMPA receptor subunit GluA1, but not GluA2. We found that DA-LTP is absent in GluA1 knock-out mice and that it requires calcium-permeable AMPA receptors. Taken together, our results suggest that dopamine together with neuronal activity controls synthesis of plasticity-related proteins, including GluA1, which enable DA-LTP via a signalling pathway distinct from that of conventional LTP.

The experimental and conceptual knowledge in 1909 led to the discovery of the Hydra head organizer through transplantation experiments between pigmented and non-pigmented animals; a discovery followed by numerous transplantations demonstrating cross-regulation between activating and inhibiting components distributed along the body axis. This experimental work inspired mathematicians, engineers, physicists and computer scientists to develop theoretical models predicting the principles of developmental mechanisms. Today, we know that the Wnt/β-catenin/Sp5/Zic4 gene regulatory network (GRN) links organizer activity, morphogenesis and cellular identity in Hydra, with variable conformations depending on the region or epithelial layer, and varied phenotypes depending on which GRN element is misregulated. In intact animals, Wnt/β-catenin signaling acts as the head activator at the tip of the hypostome, restricted by Sp5 in the other regions of the animal. Moreover, in the tentacle ring, Sp5 and Zic4 act epistatically to support tentacle differentiation and prevent basal disc differentiation. Along the body column, Sp5 is self-repressed in the epidermis and acts as a head inhibitor along the gastrodermis. Other players modulate these activities, such as TSP and Margin/RAX apically, Notch signaling in the tentacle zone, Dkk1/2/4 and HAS-7 in the body column. In the developmental context of regeneration, cells below the amputation zone switch from repressed to locally de novo activated head organizer status, a transition driven by immediate symmetrical and asymmetrical metabolic changes that lead to gene expression regulations involving components and modulators of Wnt/β-catenin signaling, early-pulse and early-late transient both often symmetrical, together with sustained ones, specific to head regeneration.

Methyltransferase like 3 (METTL3) plays a crucial role in cardiovascular diseases, but its involvement in atrial fibrillation (AF) remains unclear. The study aims to explore the relationship between METTL3 and AF in atrial myocytes.

The protein level of METTL3 was evaluated in left atrial appendages (LAAs) from patients with persistent AF and in experimental AF models. cAMP-responsive element modulator (CREM) transgenic mice and CaCl2-acetylcholine (ACh)-injected mice were used as AF mice models. Methyltransferase like 3 was globally and atrial conditionally deleted in vivo to assess its role in AF. Confocal fluorescence microscopy was employed to examine calcium handling in atrial myocytes. Methylated RNA immunoprecipitation sequencing was performed to identify the downstream target genes of METTL3. Methyltransferase like 3 protein and RNA N6-methyladenosine (m6A) modification levels were significantly reduced in the LAAs of patients with AF and experimental AF models. Genetic inhibition of METTL3 promoted the development of AF in CREM transgenic mice and CaCl2-ACh-injected mice. Knockdown of METTL3 in atrial myocytes resulted in enhanced calcium handling. Reduced METTL3 levels increased SR Ca2+-ATPase Type 2a activity by up-regulating protocadherin gamma subfamily A, 10. Decreased METTL3 protein in atrial myocytes was attributed to down-regulation of cAMP-responsive element-binding protein 1/ubiquitin-specific peptidase 9 X-linked axis.

Our study established the pathophysiological role of METTL3 involved in the development of AF and provided a potential mechanism-based target for its treatment.

Insect gustatory receptors play a critical role in modulating feeding behaviors by detecting external nutritional cues through complex biochemical pathways. Bitter taste receptors are essential for insects to identify and avoid toxins. However, the detailed molecular and cellular mechanisms by which these receptors influence insect feeding behavior remain poorly understood. Our previous research identified the bitter taste receptor NlGr23a in the brown planthopper (BPH), which specifically binds to oxalic acid and elicits a significant feeding rejection response. In this study, using an Sf9 cell line stably expressing NlGr23a, we demonstrated that oxalic acid exposure significantly enhances phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB), a protein associated with BPH food consumption. Further analysis revealed the involvement of phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathway in facilitating CREB phosphorylation upon activation by oxalic acid-NlGr23a binding. These in vitro findings were corroborated by in vivo experiments examining the expression profiles of relevant proteins and protein kinases in BPHs fed an oxalic acid-supplemented diet. Our results elucidate the biochemical cascades triggered by oxalic acid-NlGr23a interaction, advancing our understanding of insect gustatory receptor-mediated feeding behavior modulation and potentially informing novel strategies for integrated pest management.

The existence of activating transcription factor 1 (ATF1) could be employed as a clinical marker in the context of cervical cancer development, although its specific mechanism has not been fully clarified.

To evaluate the presence of ATF1, miR-630, and myelin and lymphocyte protein 2 (MAL2) in cervical malignancies, we conducted quantitative reverse transcription polymerase chain reaction, immunohistochemistry, and Western blot assays; further studied the expansion, migration, invasion and epithelial-mesenchymal transition (EMT) of cervical carcinoma cells using colony formation assay, transwell, loss cytometry, Western blot. Chromatin immunoprecipitation (ChIP) and RNA immunoprecipitation (RIP) were used to verify that ATF1 could directly transcriptionally repress miR-630; dual luciferase reporter assay and RIP assay were employed to confirm that miR-630 targeted to repress MAL2.

In cervical cancer cases, elevated ATF1 expression and reduced miR-630 expression were detected, displaying a negative relationship between them. Inhibition of ATF1 hindered the growth, migration, infiltration, and EMT in cervical carcinoma cells, while upregulation of miR-630 mitigated the aggressive characteristics of these cells. ATF1 was found to transcriptionally repress miR-630 by TransmiR and ALGGEN prediction and ChIP validation. MicroRNA modulates gene expression and affects cancer progression, and we discovered that miR-630 regulates cancer progression by targeting and inhibiting MAL2.

ATF1, which modulates the miR-630/MAL2 pathway, affects the EMT process and cervical carcinoma cell growth and spread. Therefore, ATF1 may serve as a promising marker and treatment target for cervical malignancies intervention.

Epithelial mesenchymal transition (EMT) of lens epithelial cells (LECs) is one of the most important pathogenic mechanisms in lens fibrotic disorders, and the regulatory mechanisms of EMT have not been fully understood. Here, we demonstrate that the cAMP-response element binding protein (CREB) can regulate lens EMT in a phosphorylation-dependent and phosphorylation-independent manners with dual mechanisms. First, CREB-S133 phosphorylation is implicated in TGFβ-induced EMT of mouse LECs and also in injury-induced mouse anterior subcapsular cataract model. The interaction between CREB and p300 is necessary for CREB regulation of TGFβ-induced EMT, since inhibition of CREB-p300 interaction and p300 knockdown led to attenuated expression of mesenchymal genes. Second, S133A-CREB, a mutant mimicking constant dephosphorylation at S133, exhibits notable occupancy in the enhancers of mesenchymal genes and confers robust transcription activity on EMT genes. Introduction of R314A mutation in S133A-CREB, which abolishes the interaction between S133A-CREB and its co-activator, cAMP-regulated transcriptional co-activators led to substantial suppression of mesenchymal gene expression in mouse LECs. Taken together, our results showed that CREB regulates lens EMT in dual mechanisms and that the S133A-CREB acts as a novel transcription factor. Mechanistically, CREB interacts with p300 in a S133 phosphorylation-dependent manner to positively regulate lens EMT genes. In contrast, S133A-CREB interacts with cAMP-regulated transcriptional co-activators to confer a robust activation of lens EMT genes.

Corneal neovascularization (CNV) is a dynamically regulated process that arises due to a disruption in the equilibrium between pro-angiogenic and anti-angiogenic factors. Various cytokines are released by vascular endothelial cells and macrophages in damaged cornea, ultimately inducing CNV. The cAMP-response element-binding protein (CREB), a nuclear transcription factor, potentially impacts tumor angiogenesis by modulating the secretion of angiogenic proteins. This study aimed to assess the impact of 666-15, a potent inhibitor of CREB, on angiogenesis using human microvascular retinal endothelial cells (HMRECs), RAW 264.7 macrophage cell line and alkali-induce CNV mouse model. In vivo, the topical application of 666-15 (0.05 mg/mL) to the alkali-burn corneas led to 45% reduction in CNV. Additionally, in vitro treatment with 666-15 is effective in suppressing the migration, proliferation, and tube formation by HMRECs. Furthermore, treatment with 666-15 resulted in a down-regulation of pro-angiogenic cytokines expression, including VEGF-A, TGF-β1, b-FGF, and MMP-2 but simultaneously increasing anti-angiogenic cytokines expression, such as ADAMTS-1, Thrombospondin-1 (Tsp-1) and Tsp-2, both in alkali-burn corneas and HMRECs. And 666-15 inhibited the recruitment and the cytokines expression (VEGF-A, MMP-2, IL-1β, TNF-α, MCP-1 and MIP-1) of macrophage. Our findings revealed that 666-15 may suppress the function of endothelial cells and angiogenesis by restoring the homeostasis of pro-angiogenic stimuli, suggesting its potential as a therapeutic agent in the treatment of CNV and other angiogenesis-driven diseases.

Single-molecule imaging (SMI) is a powerful approach to quantify the spatiotemporal dynamics of transcription in living cells. Here, we describe a protocol of SMI for transcription and epigenetic factors in human cortical neurons derived from embryonic stem cells or induced pluripotent stem cells. Specifically, we detail the procedures for neural stem cell culture, gene transfer, microscopy, and data analysis. This protocol can be applied to the study of transcription dynamics in response to various cellular stimuli. For complete details on the use and execution of this protocol, please refer to Atsumi et al.1.

Soft tissue tumors with EWSR1/FUS fusion to genes encoding the cyclic adenosine monophosphate response element-binding (CREB) transcription factor family (ATF1, CREB1, and CREM) are rare and heterogeneous aggressive tumors, often found in the peritoneal cavity. Here, we report two cases of malignant epithelioid tumors with EWSR1::CREB fusion involving the kidney in females in their 30 s. Both tumors appeared as solitary masses, measuring 5.4 cm and 4.0 cm in diameter. Histologically, the tumors were similar, growing invasively with unclear boundaries and composed of epithelial cells with eosinophilic and clear cytoplasm arranged in sheets, nests, and trabeculae. Immunohistochemically, case 1 showed focal AE1/AE3 positivity, whereas case 2 was negative. Anaplastic lymphoma kinase was diffusely positive in case 1 and focally positive in case 2. Both cases were positive for epithelial membrane antigen, mucin-4, and synaptophysin. High-throughput sequencing identified EWSR1::CREM fusion in case 1, whereas fluorescence in situ hybridization detected EWSR1::CREB1 fusion in case 2. These cases expand the morphological and immunophenotypic characteristics of malignant epithelioid tumors with EWSR1::CREB fusion, highlighting the diagnostic challenges of immunohistochemistry and value of molecular testing for accurate diagnosis.

It has been shown that excitotoxicity and tau-mediated toxicities are major contributing factors to neuronal death in Alzheimer's disease (AD). The excitatory amino acid transporter 2 (EAAT2 or GLT-1), the major glutamate transporter in the brain that regulates glutamate levels synaptically and extrasynaptically, has been shown to be deficient in AD brains, leading to excitotoxicity and subsequent cell death. Similarly, buildup of neurofibrillary tangles, which consist of hyperphosphorylated tau protein, correlates with cognitive decline and neuronal atrophy in AD. However, common genes and pathways that are critical in the aforementioned toxicities have not been well elucidated. To investigate the impact of glutamate dyshomeostasis and tau accumulation on translational profiles of affected hippocampal neurons, we used mouse models of excitotoxicity and tau-mediated toxicities (GLT-1-/- and P301S, respectively) in conjunction with BAC-TRAP technology. Our data show that GLT-1 deficiency in CA3 pyramidal neurons leads to translational signatures characterized by dysregulation of pathways associated with synaptic plasticity and neuronal survival, while the P301S mutation induces changes in endocytic pathways and mitochondrial dysfunction. Finally, the commonly dysregulated pathways include impaired ion homeostasis and metabolic pathways. These common pathways may shed light on potential therapeutic targets for ameliorating glutamate and tau-mediated toxicities in AD.

O-Linked β-N-acetylglucosamine (O-GlcNAc) is an essential, dynamic monosaccharide post-translational modification (PTM) found on serine and threonine residues of thousands of nucleocytoplasmic proteins. The installation and removal of O-GlcNAc is controlled by a single pair of enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. Since its discovery four decades ago, O-GlcNAc has been found on diverse classes of proteins, playing important functional roles in many cellular processes. Dysregulation of O-GlcNAc homeostasis has been implicated in the pathogenesis of disease, including neurodegeneration, X-linked intellectual disability (XLID), cancer, diabetes, and immunological disorders. These foundational studies of O-GlcNAc in disease biology have motivated efforts to target O-GlcNAc therapeutically, with multiple clinical candidates under evaluation. In this review, we describe the characterization and biochemistry of OGT and OGA, cellular O-GlcNAc regulation, development of OGT and OGA inhibitors, O-GlcNAc in pathophysiology, clinical progress of O-GlcNAc modulators, and emerging opportunities for targeting O-GlcNAc. This comprehensive resource should motivate further study into O-GlcNAc function and inspire strategies for therapeutic modulation of O-GlcNAc.

Enhancement of neuronal plasticity by small-molecule therapeutics protects cognitive skills and also ameliorates progressive neurodegenerative pathologies like Alzheimer's disease (AD) and dementia. One such compound, a novel histone deacetylase 2 (HDAC2) inhibitor named JRM-28, was shown here to enhance dendritic strength, augment spine density, and upregulate post-synaptic neurotransmission in hippocampal neurons. The molecular basis for this effect correlates with JRM-28-induced upregulation of the transcription of cAMP response element-binding protein(CREB), induction of its transcriptional activity, and subsequent stimulation of expressions of CREB-dependent plasticity-associated genes, such as those encoding N-methyl-D-aspartate (NMDA) receptor subunit NR2A and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluR1. Specifically, JRM-28 stimulated the NMDA- and AMPA-receptor-sensitive ionotropic calcium influx in hippocampal neurons. Interestingly, JRM-28 did not induce NMDA- and AMPA-sensitive calcium influx in hippocampal neurons once the expression of CREB was knocked down by creb siRNA, suggesting the critical role of CREB in JRM-28-mediated upregulation of synaptic plasticity. Finally, JRM-28 upregulated CREB mRNA, CREB-dependent plasticity-associated markers, and ionotropic calcium influx in iPSC-derived AD human neurons, indicating its therapeutic implications in the amelioration of AD pathologies.

The effect of caffeine on the behavior and sleep patterns of zebrafish larvae, as well as its underlying mechanisms, has been a topic of great interest. This study aimed to investigate the impact of caffeine on zebrafish larval sleep/wake behavior and the expression of key regulatory genes such as cAMP-response element binding protein (CREB) and adenosine (ADA) in the sleep pathway. To begin, the study determined the optimal dose and duration of caffeine exposure, with the optimal doses found to be 31.25 μM, 62.5 μM, and 120 μM. Similarly, the optimal exposure time was established as no more than 120 h, ensuring a mortality rate of less than 10%. The confirmation of these conditions was achieved through the assessment of angiogenesis and the inflammatory reaction. As a result, the treatment time point of 24 h post-fertilization (hpf) was selected to examine the effects of caffeine on zebrafish larval sleep rhythm (48 h, with a light cycle of 14:10). Furthermore, the study analyzed the expression of clock genes (bmal1a, per1b, per2, per3, cry2), adenosine receptor genes (adora1a, adora1b, adora2aa, adora2ab, adora2b), and key regulatory factors (CREB and ADA). The research confirmed that caffeine could induce sleep pattern disorders, significantly upregulate adenosine receptor genes (adora1a, adora1b, adora2a, adora2ab, adora2b) (p < 0.05), and markedly decrease the total sleep time and sleep efficiency of the larvae. Additionally, the activity of ADA significantly increased during the exposure (p < 0.001), and the tissue-specific expression of CREB was also significantly increased, as assessed by immunofluorescence. Caffeine may regulate circadian clock genes through the ADA/ADORA/CREB pathway. These findings not only enhance our understanding of the effects of caffeine on zebrafish larvae but also provide valuable insights into the potential impact of caffeine on human behavior and sleep.

Multinucleation occurs in various types of advanced cancers and contributes to their malignant characteristics, including anticancer drug resistance. Therefore, inhibiting multinucleation can improve cancer prognosis; however, the molecular mechanisms underlying multinucleation remain elusive. Here, we introduced a genetic mutation in cervical cancer cells to induce cell fusion-mediated multinucleation. The olfactory receptor OR1N2 was heterozygously mutated in these fused cells; the same OR1N2 mutation was detected in multinucleated cells from clinical cervical cancer specimens. The mutation-induced structural change in the OR1N2 protein activated protein kinase A (PKA), which, in turn, mediated the non-canonical olfactory pathway. PKA phosphorylated and activated furin protease, resulting in the cleavage of the fusogenic protein syncytin-1. Because this cleaved form of syncytin-1, processed by furin, participates in cell fusion, furin inhibitors could suppress multinucleation and reduce surviving cell numbers after anticancer drug treatment. The improved anticancer drug efficacy indicates a promising therapeutic approach for advanced cervical cancers.

Butyric acid as a short-chain fatty acid (SCFA) is one of the key microbial metabolites of ruminants. Numerous studies indicate that butyrate is crucial in muscle growth and development, and plays an important molecular regulatory role mainly by inhibiting histone deacetylation. DNA methylation, a major epigenetic modification, is involved in cell differentiation. Butyrate, in addition to its role in acetylation modifications, can alter the DNA methylation status of cells. However, the impact of butyrate on the DNA methylation of bovine skeletal muscle satellite cells (SMSCs) remains unclear. In this study, we developed a differentiation model of SMSCs and employed RNA sequencing (RNA-seq) alongside whole genome bisulfite sequencing (WGBS) to explore the effects of butyrate treatment on DNA methylation status and its relationship with gene expression. Treatment of SMSCs with sodium butyrate (NaB) at 1.0 mM for 2 days significantly inhibited the expression of DNA methyltransferases (DNMT1, DNMT2, DNMT3A) at the mRNA and protein levels while promoting the expression of demethylases (TET1, TET2, TET3) at mRNA levels. WGBS identified 4292 differentially methylated regions (DMRs), comprising 2294 hypermethylated and 1998 hypomethylated regions. These DMRs were significantly enriched in the MAPK, cAMP, and Wnt signaling pathways, all of which are implicated in myogenesis and development. Combining RNA-seq and WGBS data revealed a total of 130 overlapping genes, including MDFIC, CREBBP, DMD, LTBP2 and KLF4. These genes are predominantly involved in regulating the FoxO, MAPK, PI3K-Akt, and Wnt signaling pathways. This study provides new insights into the effects of butyrate-mediated DNA methylation on SMSC development and enhances our understanding of butyrate as an epigenetic modifier beyond its role in acetylation.

The basic zippers (bZIPs) are one of two large eukaryotic families of transcription factors whose DNA binding domains are disordered in isolation but fold into stable α-helices upon target DNA binding. Here, we systematically disrupt pre-existing helical propensity within the DNA binding region of the homodimeric bZIP domain of cAMP-response element binding protein (CREB) using Ala-Gly scanning and examine the impact on target binding kinetics. We find that the secondary structure of the transition state strongly resembles that of the unbound state. The residue closest to the dimerization domain is largely folded within both unbound and transition states; dimerization apparently propagates additional helical propensity into the basic region. The results are consistent with electrostatically-enhanced DNA binding, followed by rapid folding from the folded zipper outwards. Fly-casting theory suggests that protein disorder can accelerate binding. Interestingly however, we did not observe higher association rate constants for mutants with lower levels of residual structure in the unbound state.

Hypertension has always posed a severe threat to people's health. Food-derived angiotensin-converting enzyme (ACE)-inhibitory peptides have the potential to both prevent and treat hypertension. In the current investigation, two ACE-inhibitory peptides (SLPQ and PYVRYL) from goat milk were studied for their endothelial effects using EA.hy926 cells. PYVRYL outperformed SLPQ, yet neither impacted cell survival below 200 μg/mL. Investigation of SLPQ's impact on EA.hy926 cell expression revealed 114 differentially expressed genes, with 65 downregulated and 49 upregulated. The genes were enriched in cytokine interactions, coagulation cascades, Hippo signaling, and ECM-receptor interaction. Decreased c-x-c motif chemokine ligand 2 (CXCL2), integrin subunit beta 2 (ITGB2), and fbj murine osteosarcoma viral oncogene homologue (FOS) expression and increased secreted phosphoprotein 1 (SPP1) expression may protect endothelial cells from inflammation. Our findings suggest that beyond ACE inhibition, SLPQ aids blood pressure control by influencing endothelial function, paving the way for its use as an antihypertensive food ingredient.

Solute carrier family 39, member 8 (SLC39A8), is a transmembrane transporter that mediates the cellular uptake of zinc, iron, and manganese (Mn). Human genetic studies document the involvement of SLC39A8 in Mn homeostasis, brain development, and function. However, the role and pathophysiological mechanisms of SLC39A8 in the central nervous system remain elusive. We generated Slc39a8 neuron-specific knockout (Slc39a8-NSKO) mice to study SLC39A8 function in neurons. The Slc39a8-NSKO mice displayed markedly decreased Mn levels in the whole brain and brain regions, especially the cerebellum. Radiotracer studies using 54Mn revealed that Slc39a8-NSKO mice had impaired brain uptake of Mn. Slc39a8-NSKO cerebellums exhibited morphological defects and abnormal dendritic arborization of Purkinje cells. Reduced neurogenesis and increased apoptotic cell death occurred in the cerebellar external granular layer of Slc39a8-NSKO mice. Brain Mn deficiency in Slc39a8-NSKO mice was associated with motor dysfunction. Unbiased RNA-Seq analysis revealed downregulation of key pathways relevant to neurodevelopment and synaptic plasticity, including cAMP signaling pathway genes. We further demonstrated that Slc39a8 was required for the optimal transcriptional response to the cAMP-mediated signaling pathway. In summary, our study highlighted the essential roles of SLC39A8 in brain Mn uptake and cerebellum development and functions.

Skin mast cells (MCs) mediate acute allergic reactions in the cutaneous environment and contribute to chronic dermatoses, including urticaria, and atopic or contact dermatitis. The cAMP response element binding protein (CREB), an evolutionarily well conserved transcription factor (TF) with over 4,000 binding sites in the genome, was recently found to form a feedforward loop with KIT, maintaining MC survival. The most selective MC function is degranulation with its acute release of prestored mediators. Herein, we asked whether CREB contributes to the expression and function of the degranulation-competent receptors FcεRI and MRGPRX2. Interference with CREB by pharmacological inhibition (CREBi, 666-15) or RNA interference only slightly affected the expression of these receptors, while KIT was strongly attenuated. Interestingly, MRGPRX2 surface expression moderately increased following CREB-knockdown, whereas MRGPRX2-dependent exocytosis simultaneously decreased. FcεRI expression and function were regulated consistently, although the effect was stronger at the functional level. Preformed MC mediators (tryptase, histamine, β-hexosaminidase) remained comparable following CREB attenuation, suggesting that granule synthesis did not rely on CREB function. Collectively, in contrast to KIT, FcεRI and MRGPRX2 moderately depend on unperturbed CREB function. Nevertheless, CREB is required to maintain MC releasability irrespective of stimulus, insinuating that CREB may operate by safeguarding the degranulation machinery. To our knowledge, CREB is the first factor identified to regulate MRGPRX2 expression and function in opposite direction. Overall, the ancient TF is an indispensable component of skin MCs, orchestrating not only survival and proliferation but also their secretory competence.

Thyroid hormone binds to specific nuclear receptors, regulating the expression of target genes, with major effects on cardiac function. Triiodothyronine (T3) increases the expression of key proteins related to calcium homeostasis, such as the sarcoplasmic reticulum calcium ATPase pump, but the detailed mechanism of gene regulation by T3 in cardiac voltage-gated calcium (Cav1.2) channels remains incompletely explored. Furthermore, the effects of T3 on Cav1.2 auxiliary subunits have not been investigated. We conducted quantitative reverse transcriptase polymerase chain reaction, Western blot, and immunofluorescence experiments in H9c2 cells derived from rat ventricular tissue, examining the effects of T3 on the expression of α1c, the principal subunit of Cav1.2 channels, and Cavβ4, an auxiliary Cav1.2 subunit that regulates gene expression. The translocation of phosphorylated cyclic adenosine monophosphate response element-binding protein (pCREB) by T3 was also examined. We found that T3 has opposite effects on these channel proteins, upregulating α1c and downregulating Cavβ4, and that it increases the nuclear translocation of pCREB while decreasing the translocation of Cavβ4. Finally, we found that overexpression of Cavβ4 represses the mRNA expression of α1c, suggesting that T3 upregulates the expression of the α1c subunit in response to a decrease in Cavβ4 subunit expression.

Here, we subjected the marine copepod Tigriopus japonicus to environmentally-relevant concentrations of microplastics (MPs) and mercury (Hg) for three generations (F0-F2) to investigate their physiological and molecular responses. Hg accumulation and phenotypic traits were measured in each generation, with multi-omics analysis conducted in F2. The results showed that MPs insignificantly impacted the copepod's development and reproduction, however, which were significantly compromised by Hg exposure. Interestingly, MPs significantly increased Hg accumulation and consequently aggravated this metal toxicity in T. japonicus, demonstrating their carrier role. Multi-omics analysis indicated that Hg pollution produced numerous toxic events, e.g., induction of apoptosis, damage to cell/organ morphogenesis, and disordered energy metabolism, ultimately resulting in retarded development and decreased fecundity. Importantly, MPs enhanced Hg toxicity mainly via increased oxidative apoptosis, compromised cell/organ morphogenesis, and energy depletion. Additionally, phosphoproteomic analysis revealed extensive regulation of the above processes, and also impaired neuron activity under combined MPs and Hg exposure. These alterations adversely affected development and reproduction of T. japonicus. Overall, our findings should offer novel molecular insights into the response of T. japonicus to long-term exposure to MPs and Hg, with a particular emphasis on the carrier role of MPs on Hg toxicity.

Leaf senescence is a complex process regulated by developmental and environmental factors, and plays a pivotal role in the development and life cycle of higher plants. Casein kinase 1 (CK1) is a highly conserved serine/threonine protein kinase in eukaryotes and functions in various cellular processes including cell proliferation, light signaling and hormone effects of plants. However, the biological function of CK1 in plant senescence remains unclear. Through systemic genetic and biochemical studies, we here characterized the function of Arabidopsis EL1-like (AEL), a CK1, in promoting leaf senescence by stimulating ethylene biosynthesis through phosphorylating transcription factor WRKY22. Seedlings lacking or overexpressing AELs presented delayed or accelerated leaf senescence, respectively. AELs interact with and phosphorylate WRKY22 at Thr57, Thr60 and Ser69 residues to enhance whose transactivation activity. Being consistent, increased or suppressed phosphorylation of WRKY22 resulted in the promoted or delayed leaf senescence. WRKY22 directly binds to promoter region and stimulates the transcription of 1-amino-cyclopropane-1-carboxylate synthase 7 gene to promote ethylene level and hence leaf senescence. Our studies demonstrated the crucial role of AEL-mediated phosphorylation in regulating ethylene biosynthesis and promoting leaf senescence by enhancing WRKY22 transactivation activity, which helps to elucidate the fine-controlled ethylene biosynthesis and regulatory network of leaf senescence.

The miRNAs regulate various biological processes in the mammalian body system. The role of miR-181a in the development, progression, and expansion of cancers is well-documented. However, the role of miR-181a in adipogenesis; lipid metabolism; obesity; and obesity-related issues such as diabetes mellitus needs to be explored. Therefore, in the present study, the literature was searched and bioinformatics tools were applied to explore the role of miR-181a in adipogenesis. The list of adipogenic and lipogenic target genes validated through different publications were extracted and compiled. The network and functional analysis of these target genes was performed through in-silico analysis. The mature sequence of miR-181a of different species were extracted from and were found highly conserved among the curated species. Additionally, we also used various bioinformatics tools such as target gene extraction from Targetscan, miRWalk, and miRDB, and the list of the target genes from these different databases was compared, and common target genes were predicted. These common target genes were further subjected to the enrichment score and KEGG pathways analysis. The enrichment score of the vital KEGG pathways of the target genes is the key regulator of adipogenesis, lipogenesis, obesity, and obesity-related syndromes in adipose tissues. Therefore, the information presented in the current review will explore the regulatory roles of miR-181a in fat tissues and its associated functions and manifestations.

The persistent use of opioids leads to profound changes in neuroplasticity of the brain, contributing to the emergence and persistence of addiction. However, chronic opioid use disrupts the delicate balance of the reward system in the brain, leading to neuroadaptations that underlie addiction. Chronic cocaine usage leads to synchronized alterations in gene expression, causing modifications in the Nucleus Accumbens (NAc), a vital part of the reward system of the brain. These modifications assist in the development of maladaptive behaviors that resemble addiction. Neuroplasticity in the context of addiction involves changes in synaptic connectivity, neuronal morphology, and molecular signaling pathways. Drug-evoked neuroplasticity in opioid addiction and withdrawal represents a complicated interaction between environmental, genetic, and epigenetic factors. Identifying specific transcriptional and epigenetic targets that can be modulated to restore normal neuroplasticity without disrupting essential physiological processes is a critical consideration. The discussion in this article focuses on the transcriptional aspects of drug-evoked neuroplasticity, emphasizing the role of key transcription factors, including cAMP response element-binding protein (CREB), ΔFosB, NF-kB, Myocyte-enhancing factor 2 (MEF2), Methyl-CpG binding protein 2 (MeCP2), E2F3a, and FOXO3a. These factors regulate gene expression and lead to the neuroadaptive changes observed in addiction and withdrawal. Epigenetic regulation, which involves modifying gene accessibility by controlling these structures, has been identified as a critical component of addiction development. By unraveling these complex molecular processes, this study provides valuable insights that may pave the way for future therapeutic interventions targeting the mechanisms underlying addiction and withdrawal.

A central aspect of type 2 diabetes is decreased functional β-cell mass. The orphan nuclear receptor Nr4a1 is critical for fuel utilization, but little is known regarding its regulation and function in the β-cell. Nr4a1 expression is decreased in type 2 diabetes rodent β-cells and type 2 diabetes patient islets. We have shown that Nr4a1-deficient mice have reduced β-cell mass and that Nr4a1 knockdown impairs glucose-stimulated insulin secretion (GSIS) in INS-1 832/13 β-cells. Here, we demonstrate that glucose concentration directly regulates β-cell Nr4a1 expression. We show that 11 mM glucose increases Nr4a1 expression in INS-1 832/13 β-cells and primary mouse islets. We show that glucose functions through the cAMP/PKA/CREB pathway to regulate Nr4a1 mRNA and protein expression. Using Nr4a1-/- animals, we show that Nr4a1 is necessary for GSIS and systemic glucose handling. Using RNA-seq, we define Nr4a1-regulated pathways in response to glucose in the mouse islet, including Glut2 expression. Our data suggest that Nr4a1 plays a critical role in the β-cells response to the fed state.NEW & NOTEWORTHY Nr4a1 has a key role in fuel metabolism and β-cell function, but its exact role is unclear. Nr4a1 expression is regulated by glucose concentration using cAMP/PKA/CREB pathway. Nr4a1 regulates Glut2, Ndufa4, Ins1, In2, Sdhb, and Idh3g expression in response to glucose treatment. These results suggest that Nr4a1 is necessary for proper insulin secretion both through glucose uptake and metabolism machinery.