Inflammation-induced nitric oxide suppresses PPARα expression and function via downregulation of Sp1 transcriptional activity in adipocytes.

The activation of peroxisome proliferator-activated receptor alpha (PPARα), a ligand-dependent transcription factor that regulates lipid oxidation-related genes, has been employed to treat hyperlipidemia. Emerging evidence indicates that Ppara gene expression decreases in adipose tissue under obese conditions; however, the underlying molecular mechanisms remain elusive. Here, we demonstrate that nitric oxide (NO) suppresses Ppara expression by regulating its promoter activity via suppression of specificity protein 1 (Sp1) transcriptional activity in adipocytes. NO derived from lipopolysaccharide (LPS) -activated macrophages or a NO donor (NOR5) treatment, suppressed Ppara mRNA expression in 10T1/2 adipocytes. In addition, Ppara transcript levels were reduced in the white adipose tissue (WAT) in both acute and chronic inflammation mouse models; however, such suppressive effects were attenuated via a nitric oxide synthase 2 (NOS2) inhibitor. Endoplasmic reticulum (ER) stress inhibitors attenuated the NO-induced repressive effects on Ppara gene expression in 10T1/2 adipocytes. Promoter mutagenesis and chromatin immunoprecipitation assays revealed that NO decreased the Sp1 occupancy in the proximal promoter regions of the Ppara gene, which might partially result from the reduced Sp1 expression levels by NO. This study delineated the molecular mechanism that modulates Ppara gene transcription upon NO stimulation in white adipocytes, suggesting a possible mechanism for the transcriptional downregulation of Ppara in WAT under obese conditions.

8-Prenyl daidzein and 8-prenyl genistein from germinated soybean modulate inflammatory response in activated macrophages.

Soy isoflavones have been shown to have anti-inflammatory properties; however, the anti-inflammatory effects of isoflavone metabolites produced during soybean germination remain unclear. We found that the daidzein and genistein derivatives, 8-prenyl daidzein (8-PD) and 8-prenyl genistein (8-PG), demonstrated a more potent effect than daidzein and genistein on repressing inflammatory responses in macrophages. Although IkB protein levels were unaltered, 8-PD and 8-PG repressed nuclear factor kappa B (NF-κB) activation, which was associated with reduced ERK1/2, JNK, and p38 MAPK activation and suppressed mitogen- and stress-activated kinase 1 phosphorylation. Inflammatory responses induced by the medium containing hypertrophic adipocyte secretions were successfully suppressed by 8-PD and 8-PG treatment. In the ex vivo study, 8-PD and 8-PG significantly inhibited proinflammatory C-C motif chemokine ligand 2 (CCL2) secretion from the adipose tissues of mice fed a long-term high-fat diet. The data suggest that 8-PD and 8-PG could regulate macrophage activation under obesity conditions.

Mevalonate biosynthesis pathway regulates the development and survival of brown adipocytes.

The high thermogenic activity of brown adipose tissue (BAT) has received considerable attention. Here, we demonstrated the role of the mevalonate (MVA) biosynthesis pathway in the regulation of brown adipocyte development and survival. The inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), the rate-limiting enzyme in the MVA pathway and the molecular target of statins, suppressed brown adipocyte differentiation by suppressing protein geranylgeranylation-mediated mitotic clonal expansion. The development of BAT in neonatal mice exposed to statins during the fetal period was severely impaired. Moreover, statin-induced geranylgeranyl pyrophosphate (GGPP) deficiency led to the apoptosis of mature brown adipocytes. Brown adipocyte-specific Hmgcr knockout induced BAT atrophy and disrupted thermogenesis. Importantly, both genetic and pharmacological inhibition of HMGCR in adult mice induced morphological changes in BAT accompanied by an increase in apoptosis, and statin-treated diabetic mice showed worsened hyperglycemia. These findings revealed that MVA pathway-generated GGPP is indispensable for BAT development and survival.

Activation of ROS-PERK-TFEB by filbertone ameliorates neurodegenerative diseases via enhancing the autophagy-lysosomal pathway.

The molecular mechanisms underlying the pathogenesis of neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease (PD), and Huntington's disease remain enigmatic, resulting in an unmet need for therapeutics development. Here, we suggest that filbertone, a key flavor compound found in the fruits of hazel trees of the genus Corylus, can ameliorate PD via lowering the abundance of aggregated α-synuclein. We previously reported that inhibition of hypothalamic inflammation by filbertone is mediated by suppression of nuclear factor kappa-B. Here, we report that filbertone activates PERK through mitochondrial reactive oxygen species production, resulting in the increased nuclear translocation of transcription factor-EB in SH-SY5Y human neuroblastoma cells. TFEB activation by filbertone promotes the autophagy-lysosomal pathway, which in turn alleviates the accumulation of α-synuclein. We also demonstrate that filbertone prevented the loss of dopaminergic neurons in the substantia nigra and striatum of mice on high-fat diet. Filbertone treatment also reduced high-fat diet-induced α-synuclein accumulation through upregulation of the autophagy-lysosomal pathway. In addition, filbertone improved behavioral abnormalities (i.e., latency time to fall and decrease of running distance) in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced PD murine model. In conclusion, filbertone may show promise as a potential therapeutic for neurodegenerative disease.

Cognitive profiles in bipolar I disorder and associated risk factors: Using Wechsler adult intelligence scale-IV.

Background: Despite the growing evidence of cognitive impairments in bipolar disorder (BD), little work has evaluated cognitive performances utilizing the latest version of the Wechsler Intelligence Scale-IV (WAIS-IV), which is one of the most widely used neurocognitive assessments in clinical settings. Furthermore, clinical characteristics or demographic features that negatively affect the cognitive functioning of BD were not systematically compared or evaluated. Accordingly, the present study aimed to examine the cognitive profile of bipolar I disorder (BD-I) patients and associated risk factors. Methods: Participants included 45 patients, diagnosed with BD-I, current or most recent episode manic, and matching 46 healthy controls (HC). Cognitive performance was evaluated via WAIS-IV, and clinical characteristics of the BD-I group were examined via multiple self- and clinician-report questionnaires. Results: Multivariate analysis of covariance (MANCOVA) results indicated that the BD-I group demonstrated significantly poorer performance compared to the HC group in subtests and indexes that reflect working memory and processing speed abilities. Redundancy analysis revealed that overall symptom severity, manic symptom severity, and anxiety were significant predictors of cognitive performance in BD-I, while age of onset, past mood disorder history, depression severity, and impulsiveness showed comparatively smaller predictive values. Conclusion: The current study suggests cognitive deterioration in the cognitive proficiency area while generalized ability, including verbal comprehension and most of the perceptual reasoning skills, remain intact in BD-I. The identified risk factors of cognitive performance provide specific clinical recommendations for intervention and clinical decision-making.

Morin Disrupts Cytoskeleton Reorganization in Osteoclasts through an ROS/SHP1/c-Src Axis and Grants Protection from LPS-Induced Bone Loss.

Morin is a naturally occurring flavonoid with anti-inflammatory and antioxidative properties. Therefore, we hypothesized that morin may prevent inflammatory bone loss by reducing oxidative stress. To investigate the effect of morin on inflammatory bone loss, mice were injected with lipopolysaccharide (LPS). Osteoclasts (OCs) were analyzed by tartrate-resistant acid phosphatase (TRAP) staining and actin ring formation. Micro-computerized tomography analysis indicated that morin prevented LPS-induced bone loss in mice. In vivo TRAP staining indicated that morin decreased the number and surface of the OCs that were increased in LPS-treated mice. Furthermore, in vitro experiments indicated that morin decreased the number and activity of OCs upon LPS stimulation. Morin decreased actin ring-containing OCs with decreased activation of c-Src (Y416)/vav guanine nucleotide exchange factor 3/Ras-related C3 botulinum toxin substrate 1 compared with LPS alone. Morin decreased cytosolic reactive oxygen species (ROS), thus preventing the oxidation of Src homology region 2 domain-containing phosphatase 1 (SHP-1), followed by the inactivation of c-Src via direct interaction with SHP1. Conversely, SHP1 knockdown abolished the inhibitory effect of morin on OCs. Therefore, our findings suggest that morin disrupted cytoskeletal reorganization via an ROS/SHP1/c-Src axis in OCs, thereby granting protection from LPS-induced bone loss, which demonstrates its therapeutic potential against inflammatory bone loss.

7-Ketocholesterol-Induced Micro-RNA-107-5p Increases Number and Activity of Osteoclasts by Targeting MKP1.

Osteoclasts (OCs), which are responsible for bone resorption, play a critical role in cholesterol-induced bone loss and recent studies have suggested that various micro-RNAs (miRs) contribute to modulating OCs. We hypothesized that 7-ketocholesterol (7-KC), a metabolite responsible for cholesterol-induced bone loss, induces miR-107-5p, which affects OCs. Overexpression and knock-down of miR-107-5p were performed using miR-107-5p mimic and anti-miR-107-5p, respectively. The effects of miR-107-5p on OCs were analyzed by tartrate-resistant alkaline phosphatase staining, qPCR, and Western blot. MiR-107-5p was upregulated after 7-KC exposure in receptor activator of nuclear factor kappa-Β ligand-stimulated OCs. Furthermore, miR-107-5p upregulation was also observed in tibiae from an atherogenic diet-fed mice compared with mice fed with a normal diet. MiR-107-5p overexpression enhanced the area and number of OCs, whereas inhibiting the endogenous expression of miR-107-5p generated by 7-KC had the opposite effect. Among the possible candidates, mitogen-activated protein kinase phosphatase-1, a stress-responsive dual-specificity phosphatase that inactivates mitogen-activated protein kinase (MKP1), has been proven to be a target gene of miR-107-5p, as demonstrated by the direct interaction between miR-107-5p and the 3'-untranslated region of MKP1. Collectively, our findings demonstrate that 7-KC-induced miR-107-5p promotes differentiation and function of OCs by downregulating MKP1.

PERK activation by SB202190 ameliorates amyloidogenesis via the TFEB-induced autophagy-lysosomal pathway.

The protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK), a key ER stress sensor of the unfolded protein response (UPR), can confer beneficial effects by facilitating the removal of cytosolic aggregates through the autophagy-lysosome pathway (ALP). In neurodegenerative diseases, the ALP ameliorates the accumulation of intracellular protein aggregates in the brain. Transcription factor-EB (TFEB), a master regulator of the ALP, positively regulates key genes involved in the cellular degradative pathway. However, in neurons, the role of PERK activation in mitigating amyloidogenesis by ALP remains unclear. In this study, we found that SB202190 selectively activates PERK independently of its inhibition of p38 mitogen-activated protein kinase, but not inositol-requiring transmembrane kinase/endoribonuclease-1α (IRE1α) or activating transcription factor 6 (ATF6), in human neuroblastoma cells. PERK activation by SB202190 was dependent on mitochondrial ROS production and promoted Ca2+-calcineurin activation. The activation of the PERK-Ca2+-calcineurin axis by SB202190 positively affects TFEB activity to increase ALP in neuroblastoma cells. Collectively, our study reveals a novel physiological mechanism underlying ALP activation, dependent on PERK activation, for ameliorating amyloidogenesis in neurodegenerative diseases.

GSK-3ß inhibition by curcumin mitigates amyloidogenesis via TFEB activation and anti-oxidative activity in human neuroblastoma cells.

The translocation of transcription factor EB (TFEB) to the nucleus plays a pivotal role in the regulation of basic cellular processes, such as lysosome biogenesis and autophagy. Autophagy is an intracellular degradation system that delivers cytoplasmic constituents to the lysosome, which is important in maintaining cellular homeostasis during environmental stress. Furthermore, oxidative stress is a critical cause for the progression of neurodegenerative diseases. Curcumin has anti-oxidative and anti-inflammatory activities, and is expected to have potential therapeutic effects in various diseases. In this study, we demonstrated that curcumin regulated TFEB export signalling via inhibition of glycogen synthase kinase-3ß (GSK-3ß); GSK-3ß was inactivated by curcumin, leading to reduced phosphorylation of TFEB. We further showed that H2O2-induced oxidative stress was reduced by curcumin via the Nrf2/HO-1 pathway in human neuroblastoma cells. In addition, we showed that curcumin induced the degradation of amyloidogenic proteins, including amyloid-ß precursor protein and α-synuclein, through the TFEB-autophagy/lysosomal pathway. In conclusion, curcumin regulates autophagy by controlling TFEB through the inhibition of GSK-3ß, and increases antioxidant gene expression in human neuroblastoma cells. These results contribute to the development of novel cellular therapies for neurodegenerative diseases.

Cortical Volumetric Correlates of Childhood Trauma, Anxiety, and Impulsivity in Bipolar Disorder.

OBJECTIVE: More recently, attention has turned to the linkage between childhood trauma and emotional dysregulation, but the evidence in bipolar disorder (BD) is limited. To determine neurobiological relationships between childhood trauma, current anxiety, and impulsivity, we investigated cortical volumetric correlates of these clinical factors in BD. METHODS: We studied 36 patients with DSM-5 BD and 29 healthy controls. Childhood trauma, coexisting anxiety, and impulsivity were evaluated with the Korean version-Childhood Trauma Questionnaire (CTQ), the Korean version-Beck Anxiety Inventory (BAI), and the Korean version-Barratt Impulsiveness Scale (BIS). Voxel-based morphometry (VBM) was used to assess gray matter volume (GMV) alterations on the brain magnetic resonance imaging (MRI). Partial correlation analyses were conducted to examine associations between the GMV and each scale in the BD group. RESULTS: Childhood trauma, anxiety, and impulsivity were interrelated in BD. BD patients revealed significant inverse correlations between the GMV in the right precentral gyrus and CTQ scores (r=-0.609, p<0.0003); between the GMV in the left middle frontal gyrus and BAI scores (r=-0.363, p=0.044). Moreover, patients showed similar tendency of negative correlations between the GMV in the right precentral gyrus and BIS scores; between the GMV in the left middle frontal gyrus and CTQ scores. CONCLUSION: The present study provides evidence for a neural basis between childhood trauma and affect regulations in BD. The GMV alterations in multiple frontal lobe areas may represent neurobiological markers for anticipating the course of BD.

Cellular Contributors to Hypothalamic Inflammation in Obesity.

The hypothalamus is a crucial organ for the maintenance of appropriate body fat storage. Neurons in the hypothalamic arcuate nucleus (ARH) detect energy shortage or surplus via the circulating concentrations of metabolic hormones and nutrients, and then coordinate energy intake and expenditure to maintain energy homeostasis. Malfunction or loss of hypothalamic ARH neurons results in obesity. Accumulated evidence suggests that hypothalamic inflammation is a key pathological mechanism that links chronic overconsumption of a high-fat diet (HFD) with the development of obesity and related metabolic complications. Interestingly, overnutrition-induced hypothalamic inflammation occurs specifically in the ARH, where microglia initiate an inflammatory response by releasing proinflammatory cytokines and chemokines in response to excessive fatty acid flux. Upon more prolonged HFD consumption, astrocytes and perivascular macrophages become involved and sustain hypothalamic inflammation. ARH neurons are victims of hypothalamic inflammation, but they may actively participate in hypothalamic inflammation by sending quiescence or stress signals to surrounding glia. In this mini-review, we describe the current state of knowledge regarding the contributions of neurons and glia, and their interactions, to HFD-induced hypothalamic inflammation.

Reduced EGFR Level in eIF2α PhosphorylationDeficient Hepatocytes Is Responsible for Susceptibility to Oxidative Stress.

Reactive oxygen species (ROS) play a significant role in intracellular signaling and regulation, particularly when they are maintained at physiologic levels. However, excess ROS can cause cell damage and induce cell death. We recently reported that eIF2α phosphorylation protects hepatocytes from oxidative stress and liver fibrosis induced by fructose metabolism. Here, we found that hepatocyte-specific eIF2α phosphorylation-deficient mice have significantly reduced expression of the epidermal growth factor receptor (EGFR) and altered EGFR-mediated signaling pathways. EGFR-mediated signaling pathways are important for cell proliferation, differentiation, and survival in many tissues and cell types. Therefore, we studied whether the reduced amount of EGFR is responsible for the eIF2α phosphorylationdeficient hepatocytes' vulnerability to oxidative stress. ROS such as hydrogen peroxide and superoxides induce both EGFR tyrosine phosphorylation and eIF2α phosphorylation. eIF2α phosphorylation-deficient primary hepatocytes, or EGFR knockdown cells, have decreased ROS scavenging ability compared to normal cells. Therefore, these cells are particularly susceptible to oxidative stress. However, overexpression of EGFR in these eIF2α phosphorylationdeficient primary hepatocytes increased ROS scavenging ability and alleviated ROS-mediated cell death. Therefore, we hypothesize that the reduced EGFR level in eIF2α phosphorylation-deficient hepatocytes is one of critical factors responsible for their susceptibility to oxidative stress.

Cellular source of hypothalamic macrophage accumulation in diet-induced obesity.

BACKGROUND: Obese mice on a high-fat diet (HFD) display signs of inflammation in the hypothalamic arcuate nucleus (ARC), a critical area for controlling systemic energy metabolism. This has been suggested as a key mechanism of obesity-associated hypothalamic dysfunction. We reported earlier that bone marrow-derived macrophages accumulate in the ARC to sustain hypothalamic inflammation upon chronic exposure to an HFD. However, the mechanism underlying hypothalamic macrophage accumulation has remained unclear. METHODS: We investigated whether circulating monocytes or myeloid precursors contribute to hypothalamic macrophage expansion during chronic HFD feeding. To trace circulating myeloid cells, we generated mice that express green fluorescent protein (GFP) in their lysozyme M-expressing myeloid cells (LysMGFP mice). We conducted parabiosis and bone marrow transplantation experiments using these animals. Mice received an HFD for 12 or 30 weeks and were then sacrificed to analyze LysMGFP cells in the hypothalamus. Hypothalamic vascular permeability in the HFD-fed obese mice was also tested by examining the extravascular leakage of Evans blue and fluorescence-labeled albumin. The timing of LysMGFP cell entry to the hypothalamus during development was also evaluated. RESULTS: Our parabiosis and bone marrow transplantation experiments revealed a significant infiltration of circulating LysMGFP cells into the liver, skeletal muscle, choroid plexus, and leptomeninges but not in the hypothalamic ARC during chronic HFD feeding, despite increased hypothalamic vascular permeability. These results suggested that the recruitment of circulating monocytes is not a major mechanism for maintaining and expanding the hypothalamic macrophage population in diet-induced obesity. We demonstrated instead that LysMGFP cells infiltrate the hypothalamus during its development. LysMGFP cells appeared in the hypothalamic area from the late embryonic period. This cellular pool suddenly increased immediately after birth, peaked at the postnatal second week, and adopted an adult pattern of distribution after weaning. CONCLUSIONS: Bone marrow-derived macrophages mostly populate the hypothalamus in early postnatal life and may maintain their pool without significant recruitment of circulating monocytes throughout life, even under conditions of chronic HFD feeding.

Deficiency of fibroblast growth factor 21 aggravates obesity-induced atrophic responses in skeletal muscle.

BACKGROUND: Obesity-induced skeletal muscle inflammation is a major contributor of skeletal muscle loss/atrophy and is implicated in metabolic complications such as insulin resistance. Fibroblast growth factor 21 (FGF21) is known to be an important metabolic regulator with anti-inflammatory properties. However, the effect of FGF21 on skeletal muscle atrophy is unclear. In this study, we investigated the effect of FGF21 deficiency on obesity-induced skeletal muscle inflammation and atrophy in mice. RESULTS: The expression of atrophic factors (MuRF1 and Atrogin-1) was upregulated at the mRNA and/or protein levels in the skeletal muscle of FGF21-deficient obese mice compared with wild type obese control mice. This was accompanied by an increase in levels of inflammatory cytokines (TNFα and MCP-1) and a reduction in AMPK phosphorylation. FGF21 treatment markedly suppressed TNFα-mediated inflammatory and atrophic responses in cultured myotubes, and the actions of FGF21 were blunted by the AMPK inhibitor compound C. CONCLUSION: These findings suggest that FGF21 deficiency aggravates obesity-induced inflammation and atrophic responses in the skeletal muscle of obese mice, and FGF21 may protect inflammation-mediated atrophy through the AMPK pathway.

A new mouse model for noninvasive fluorescence-based monitoring of mitochondrial UCP1 expression.

Mitochondrial uncoupling protein 1 (UCP1) is well known for its thermogenic function in brown adipose tissue (BAT). Since UCP1 expends energy on thermogenesis, UCP1 activation has been considered an approach to ameliorate obesity. As a tool for uncovering yet unknown mechanisms of UCP1 activation, we generated a transgenic mouse model in which UCP1 expression levels are reflected in fluorescence derived from monomeric red fluorescent protein 1 (mRFP1). In these UCP1-mRFP1 BAC transgenic mice, fluorescence intensity mimics the change in UCP1 expression levels evoked through physiological or pharmacological stimulation. This transgenic mouse model will be useful in the search for bioactive compounds with the ability to induce UCP1 and for revealing undiscovered mechanisms of BAT activation.

Fibroblast growth factor 21 deficiency aggravates obesity-induced hypothalamic inflammation and impairs thermogenic response.

OBJECTIVE AND DESIGN: Hypothalamic inflammation is closely associated with metabolic dysregulation. Fibroblast growth factor 21 (FGF21) is known to be an important metabolic regulator with anti-inflammatory properties. In this study, we investigated the effects of FGF21 deficiency on obesity-induced hypothalamic inflammation and thermogenic responses. MATERIALS AND METHODS: FGF21-deficient mice and/or wild-type (WT) mice were fed a high-fat diet (HFD) for 12 weeks. RESULTS: FGF21-deficient mice fed an HFD showed increased levels of inflammatory cytokines compared with WT obese control, and this was accompanied by upregulation of gliosis markers in the hypothalamus. Expression of heat-shock protein 72, a marker of neuronal damage, was increased in the FGF21-deficient obese mice, and the expression of hypothalamic neuronal markers involved in anti-thermogenic or thermogenic responses was altered. Moreover, the protein level of uncoupling protein 1 and other thermogenic genes were markedly reduced in the brown adipose tissue of the FGF21-deficient obese mice. CONCLUSIONS: These findings suggest that FGF21 deficiency aggravates obesity-induced hypothalamic inflammation and neuronal injury, leading to alterations in hypothalamic neural circuits accompanied by a reduction of the thermogenic response.

Corrigendum: Interglial Crosstalk in Obesity-Induced Hypothalamic Inflammation.

[This corrects the article DOI: 10.3389/fnins.2018.00939.].

α-Cedrene protects rodents from high-fat diet-induced adiposity via adenylyl cyclase 3.

OBJECTIVE: The increasing global prevalence of obesity and its associated disorders points to an urgent need for the development of novel and effective strategies for the prevention of weight gain. Here, we investigated the potential of α-cedrene, a volatile sesquiterpene compound derived from cedarwood oil, in regulation of obesity and delineated the mechanisms involved. METHODS: For the prevention of obesity, C57BL/6 N mice were fed a high-fat diet (HFD) and were orally administered either with vehicle or α-cedrene for 8 weeks. For the therapy of obesity, obese Sprague Dawley rats, induced by a HFD for 8 weeks, were orally treated either with vehicle or α-cedrene for 12 weeks. To determine whether the action of α-cedrene was Adcy3 dependent, Adcy3 heterozygous null mice (Adcy3+/-) and wild-type controls were fed either HFD or α-cedrene supplemented HFD for 17 weeks. RESULTS: Oral α-cedrene administration prevented or reversed HFD-induced obesity and abnormal metabolic aberrations in rodents, without affecting their food intake. Downregulation of Adcy3 expression by small interfering RNA abrogated the beneficial effects of α-cedrene on the oxygen consumption rate and intracellular lipid accumulation in 3T3-L1 adipocytes. Similarly, in Adcy3+/- mice, the α-cedrene-driven suppression of body weight gain observed in wild-type mice was substantially (~50%) attenuated. Expression of thermogenic and lipid oxidation genes was increased in adipose tissues of α-cedrene-treated mice, with concomitant downregulation of adipogenic gene expression. These beneficial molecular changes elicited by α-cedrene were blunted in adipose tissues of Adcy3+/- mice. CONCLUSIONS: Our results highlight the potential of α-cedrene for antiobesity interventions and suggest that the antiobesity effect of α-cedrene is mediated by Adcy3 in adipose tissues.

Exogenous nicotinamide adenine dinucleotide regulates energy metabolism via hypothalamic connexin 43.

BACKGROUND: Nicotinamide adenine dinucleotide (NAD)-dependent deacetylase SIRT1 is an important regulator of hypothalamic neuronal function. Thus, an adequate hypothalamic NAD content is critical for maintaining normal energy homeostasis. METHODS: We investigated whether NAD supplementation increases hypothalamic NAD levels and affects energy metabolism in mice. Furthermore, we investigated the mechanisms underlying the effects of exogenous NAD on central metabolism upon entering the hypothalamus. RESULTS: Central and peripheral NAD administration suppressed fasting-induced hyperphagia and weight gain in mice. Extracellular NAD was imported into N1 hypothalamic neuronal cells in a connexin 43-dependent and CD73-independent manner. Consistent with the in vitro data, inhibition of hypothalamic connexin 43 blocked hypothalamic NAD uptake and NAD-induced anorexia. Exogenous NAD suppressed NPY and AgRP transcriptional activity, which was mediated by SIRT1 and FOXO1. CONCLUSIONS: Exogenous NAD is effectively transported to the hypothalamus via a connexin 43-dependent mechanism and increases hypothalamic NAD content. Therefore, NAD supplementation is a potential therapeutic method for metabolic disorders characterized by hypothalamic NAD depletion.