GLUT2 (Slc2a2) is a key glucose transporter in pancreatic β-cells, and its reduced expression is closely linked to defective glucose-stimulated insulin secretion (GSIS) and diabetes. We previously reported that pancreatic β-cell–specific nardilysin (NRDC)-deficient mice (BetaKO) exhibit a severe diabetic phenotype with defective GSIS and reduced Slc2a2 expression in islets. However, because BetaKO mice also showed reduced MafA, a key upstream regulator of Slc2a2, along with an increased α-cell/β-cell ratio and other secondary changes that could influence GLUT2 levels, the mechanism by which NRDC regulates Slc2a2 transcription remained unclear. Here, we demonstrate that NRDC controls Slc2a2 expression in a β-cell-autonomous and MafA-independent manner. By integrating publicly available ATAC-seq and ChIP-seq datasets, we identified four active enhancer regions around the murine Slc2a2 locus, two of which are evolutionarily conserved in human islets. Luciferase assays in MIN6 β-cells revealed that NRDC selectively controls the activity of a conserved enhancer located 39 kb downstream of the Slc2a2 transcription start site. Chromatin immunoprecipitation (ChIP) and re-ChIP assays further revealed that, in MIN6 cells, NRDC occupies this enhancer and is required for efficient recruitment of ISLET1, a transcription factor upstream of Slc2a2. These findings suggest that NRDC contributes to Slc2a2 regulation in addition to MafA, highlighting multifaceted roles of NRDC in pancreatic β-cell gene regulation.

BACKGROUND AND AIMS:Recent reports have shown the development of autoantibodies in patients with atherosclerosis. The purpose of this study is to examine whether these autoantibodies are associated with the risk of incident stroke in community-based population. METHODS:We performed a nested case-control study within the Japan Public Health Center-based Prospective Study involving 32,527 Japanese individuals aged 40-69 years at baseline (1990-1993). Incident stroke was followed up until the end of 2007. We identified 38 novel autoantibody biomarkers for atherosclerosis, and the plasma levels of these autoantibodies were measured by use of amplified luminescent proximity homogeneous assay-linked immunosorbent assay methods in 371 stroke cases (203 ischemic and 168 hemorrhagic) and 371 age-, sex-, and community-matched controls. Conditional odds ratios of total, ischemic, and hemorrhagic strokes according to quartiles of each autoantibody were calculated after adjustment for major cardiovascular risk factors. RESULTS:Of the 38 the novel autoantibodies, the levels of anti-DIDO1 peptide, anti-LIMA1 peptide, anti-SH3BP5 peptide, anti-FOXJ2 peptide, anti-KIF20B protein, anti-DIDO1 protein, anti-TPM3 protein, anti-COPE protein, anti-MMP1 protein and anti-IFI16 peptide autoantibodies were significantly associated with the risk of total stroke (odds ratios of the highest versus lowest quartile ≥1.63) after adjustment for major cardiovascular risk factors. The association was generally similar but weakly observed for ischemic stroke but not for hemorrhagic stroke. CONCLUSIONS:These novel autoantibodies have the potential to predict future stroke incidence.

Nardilysin (NRDC) is a zinc metallopeptidase of the M16 family that participates in diverse biological processes, including ectodomain shedding and antigen processing. NRDC also functions as a transcriptional coregulator, and this coregulatory activity depends on its metallopeptidase catalytic function. However, small-molecule inhibitors targeting NRDC have not been well characterized, limiting pharmacological investigation of its enzymatic function. Here we established a fluorescence-quenched peptide substrate assay suitable for high-throughput screening of NRDC protease activity. Using this assay, we screened 33,305 compounds and identified 140 candidate NRDC inhibitors. Through secondary screening, orthogonal validation and structure-activity relationship analysis, three inhibitor scaffolds were identified, from which compound R5-1 was selected for further characterization. Enzyme kinetic analysis demonstrated that R5-1 inhibits NRDC activity in a non-competitive manner, indicating reduced catalytic turnover rather than directly competing with substrate binding. Structural modeling and docking analysis further suggested that R5-1 binds to a distal allosteric site in NRDC. Pharmacological inhibition of NRDC with R5-1 attenuated NRDC-dependent transcriptional repression of PGC-1α, phenocopying the effect of catalytic inactivation. Furthermore, administration of R5-1 significantly ameliorated arthritis severity in a mouse model of autoimmune arthritis. Together, these findings identify R5-1 as a small-molecule inhibitor of NRDC and provide mechanistic insights linking NRDC protease activity to transcriptional regulation and inflammatory disease.

GLUT2 ( Slc2a2 ) is a key glucose transporter in pancreatic β-cells, and its reduced expression is closely linked to defective glucose-stimulated insulin secretion (GSIS) and diabetes. We previously reported that pancreatic β-cell–specific nardilysin (NRDC)-deficient mice (BetaKO) exhibit severe diabetic phenotype with defective GSIS and reduced Slc2a2 expression in islets. However, because BetaKO mice also showed reduced MafA, a key upstream regulator of Slc2a2 , along with an increased α-cell/β-cell ratio and other secondary changes that could influence GLUT2 levels, the mechanism by which NRDC regulates Slc2a2 transcription remained unclear. Here, we demonstrate that NRDC controls Slc2a2 expression in a β-cell autonomous and MafA-independent manner. By integrating publicly available ATAC-seq and ChIP-seq datasets, we identified four active enhancer regions around the murine Slc2a2 locus, two of which are evolutionarily conserved in human islets. Luciferase assays revealed that NRDC selectively controls the activity of a conserved enhancer located 39k bp downstream of the Slc2a2 transcriptional start site. Chromatin immunoprecipitation (ChIP) and re-ChIP assays further revealed that NRDC binds to this enhancer and is required for efficient recruitment of ISLET1, a transcription factor upstream of Slc2a2 . These findings indicate that NRDC directly regulates Slc2a2 in addition to MafA, highlighting multifaceted roles of NRDC in pancreatic β-cell gene regulation.

Circadian rhythms in mammals are generated by negative feedback loops, in which CLOCK and BMAL1 bind to E-box to activate transcription of Period (Per) and Cryptochrome (Cry) and the E-box-dependent transactivation is inhibited by PER and CRY proteins. Although the core transcriptional feedback loop of the circadian clock has been well defined, how this machinery interfaces with broader nuclear regulatory systems remains incompletely understood. Here, we identify nardilysin (NRDC), a metalloendopeptidase previously implicated in nuclear transcriptional regulation and metabolic homeostasis, as an unexpected modulator of the circadian clock. NRDC deficiency led to elevated PER2 protein levels in the liver and enhanced PER2 dynamics in cell-autonomous circadian oscillators, and was accompanied by a significant shortening of behavioral rhythms in mice. Biochemical analyses demonstrated that NRDC selectively associates with PER2 and CRY2 and antagonizes PER2-mediated repression of CLOCK-BMAL1-dependent transcription. Genome-wide chromatin immunoprecipitation analyses reveal that NRDC is enriched at promoter-proximal E-box-containing regions, frequently co-localizing with CLOCK binding sites. Together, these findings uncover a previously unrecognized link between circadian timing and protease-based nuclear regulation, positioning NRDC as a critical modulator of PER2 function and circadian period determination.

Obesity is a critical global health issue and a major risk factor for type 2 diabetes mellitus (T2DM). However, molecular mechanism by which obesity leads to T2DM has not been fully elucidated. Here, we investigated the role of adipocyte nardilysin (NRDC) in insulin sensitivity using adipocyte-specific NRDC-knockout mice (Adipo-KO). Despite no difference in body weight, Adipo-KO demonstrated enhanced glucose tolerance and insulin sensitivity exclusively under high fat diet (HFD) conditions. Epididymal white adipose tissue (eWAT) in Adipo-KO showed the decreased crown-like structures and F4/80-positive cells. Consistently, insulin-stimulated Akt phosphorylation was elevated in the eWAT of HFD-fed Adipo-KO and 3T3-L1 adipocytes with NRDC knockdown. In addition, NRDC knockdown led to a decrease in cellular oxygen consumption rate and hypoxia-induced HIF1α expression in 3T3-L1 adipocytes. The augmented Akt phosphorylation by NRDC knockdown was reversed by daprodustat, which stabilizes HIF1α, suggesting that the effect of NRDC on insulin sensitivity is mediated by HIF1α. Upstream analysis of the differentially expressed genes (DEGs) in NRDC knockdown 3T3-L1 adipocytes revealed that PPARγ is enriched in the promoters of the DEGs. We also confirmed the complex formation of NRDC and PPARγ. In conclusion, NRDC in adipocyte regulates insulin sensitivity through HIF1α and PPARγ.

Inflammation is closely associated with cerebrovascular diseases, cardiovascular diseases, diabetes, and cancers, and it is accompanied by the development of autoantibodies in the early stage of inflammation-related diseases. Hence, it is meaningful to discover novel antibody biomarkers targeting inflammation-related diseases. In this study, Jumonji C-domain-containing 6 (JMJD6) was identified by the serological identification of antigens through recombinant cDNA expression cloning. In particular, JMJD6 is an antigen recognized in serum IgG from patients with unstable angina pectoris (a cardiovascular disease). Then, the serum antibody levels were examined using an amplified luminescent proximity homogeneous assay-linked immunosorbent assay and a purified recombinant JMJD6 protein as an antigen. We observed elevated levels of serum anti-JMJD6 antibodies (s-JMJD6-Abs) in patients with inflammation-related diseases such as ischemic stroke, acute myocardial infarction (AMI), diabetes mellitus (DM), and cancers (including esophageal cancer, EC; gastric cancer; lung cancer; and mammary cancer), compared with the levels in healthy donors. The s-JMJD6-Ab levels were closely associated with some inflammation indicators, such as C-reactive protein and intima-media thickness (an atherosclerosis index). A better postoperative survival status of patients with EC was observed in the JMJD6-Ab-positive group than in the negative group. An immunohistochemical analysis showed that JMJD6 was highly expressed in the inflamed mucosa of esophageal tissues, esophageal carcinoma tissues, and atherosclerotic plaques. Hence, JMJD6 autoantibodies may reflect inflammation, thereby serving as a potential biomarker for diagnosing specific inflammation-related diseases, including stroke, AMI, DM, and cancers, and for prediction of the prognosis in patients with EC.

Atherosclerosis is associated with vascular diseases, diabetes, and cancers. The Jumonji C-domain-containing 6 (JMJD6) gene plays an important role in diverse biological processes. In addition, autoantibodies are reportedly useful in the early diagnosis and treatment of many diseases. Here, JMJD6 was identified by serological identification of antigens by recombinant cDNA expression cloning as an antigen recognized by serum IgG from patients with unstable angina pectoris. Then, we performed amplified luminescent proximity homogeneous assay-linked immunosorbent assay using purified recombinant antigens and found elevated levels of serum anti-JMJD6 antibodies (s-JMJD6-Abs) in patients with atherosclerosis-related diseases including ischemic stroke, acute myocardial infarction (AMI), diabetes mellitus (DM), and cancers including esophageal cancer (EC), gastric cancer, lung cancer, and mammary cancer, compared with those in healthy donors. The s-JMJD6-Ab levels were closely associated with hypertension, habitual smoking, and the intima-media thickness, an atherosclerosis index. The better postoperative survival status was shown in patients with EC from the JMJD6-Ab-positive group than the negative group, especially from JMJD6-Ab-positive/PD-L1-negative group and JMJD6-Ab-positive/p53-Ab-negative group than JMJD6-Ab-negative/PD-L1-positive group and JMJD6-Ab-negative/p53-Ab-positive group, respectively. Hence, JMJD6 autoantibodies may reflect atherosclerosis severity, thereby a potential biomarker for atherosclerosis-related diseases including ischemic stroke, AMI, DM, and cancers and for predicting EC prognosis.

Background Data on the incidence, timing, and severity of myocardial damage after anthracycline-based chemotherapy (AC) in Japanese patients with breast cancer are limited. Method We evaluated cancer therapy-related cardiac dysfunction (CTRCD) in Japanese women with breast cancer (n = 51) after the first AC according to the definitions of the 2022 European Society of Cardiology onco-cardiology guideline, including assessment of high-sensitivity troponin I (TnI) and B-type natriuretic peptide (BNP) levels. Results CTRCD was detected in 67 % of the patients (3.9 %, 7.8 %, 9.8 %, 43 %, 37 %, 22 %, 20 %, and 9.8 % of patients at 1 week and 1, 2, 3, 6, 9, 12, and 15 months post-AC, respectively) without significant left ventricular ejection fraction reduction (<50 %) and heart failure. Elevated TnI levels (>26 pg/mL) were found in 43 % of patients, and elevated BNP levels (≥35 pg/mL) were observed in 22 % of patients during the follow-up period. Conclusions Approximately two-thirds of the Japanese patients in this study experienced CTRCD, which was frequently observed at 3 or 6 months post-AC. However, all patients with CTRCD were diagnosed with mild asymptomatic CTRCD. Although, these patients were diagnosed with mild asymptomatic CTRCD, careful long-term follow-up will be required.

BackgroundSeveral hypotheses regarding the pathomechanisms of schizophrenia have been proposed. If schizophrenia is a unitary disease, then these pathological processes must be linked; however, if such links do not exist, schizophrenia may best be considered a group of disorders. Only a few studies have examined the relationships among these pathomechanisms. Herein, we examined the relationships among deficient myelination, NMDA receptor hypofunction, and metabolic dysregulation by measuring various plasma markers and examining their correlations.MethodsPlasma samples were collected from 90 patients with schizophrenia and 68 healthy controls. Concentrations of nardilysin (N-arginine dibasic convertase, NRDC), a positive regulator of myelination, the NMDA receptor co-agonist D-serine and glycine, various additional amino acids related to NMDA receptor transmission (glutamate, glutamine, and L-serine), and homocysteine (Hcy), were measured. Concentrations were compared using independent samples t-test or logistic regression, and associations were evaluated using Pearson's correlation coefficients.ResultsPlasma glycine (t = 2.05, p = 0.042), L-serine (t = 2.25, p = 0.027), and homocysteine (t = 3.71, p < 0.001) concentrations were significantly higher in patients with schizophrenia compared to those in healthy controls. Logistic regression models using age, sex, smoking status, glutamine, glutamate, glycine, L-serine, D-serine, homocysteine, and NRDC as independent variables revealed significantly lower plasma D-serine (p = 0.024) and NRDC (p = 0.028), but significantly higher L-serine (p = 0.024) and homocysteine (p = 0.001) in patients with schizophrenia. Several unique correlations were found between NMDA receptor-related amino acids and NRDC in patients with schizophrenia compared to those in healthy controls, while no correlations were found between plasma homocysteine and other markers. No associations were found between plasma marker concentrations and disease status or cognitive function in patients with schizophrenia, except for a significant correlation between plasma glycine and full intelligence quotient.ConclusionReduced myelination and NMDA receptor hypofunction may be related to pathological mechanisms in schizophrenia, while homocysteine dysregulation appears to be an independent pathological process. These results suggest that schizophrenia may be a group of disorders with unique or partially overlapping etiologies.

Acute coronary syndrome (ACS) includes myocardial infarction (MI) and unstable angina (UA). MI is defined by elevated necrosis markers, preferably high-sensitivity cardiac troponins (hs-cTn). However, it takes hours for cTn to become elevated after coronary occlusion; therefore, difficulties are associated with diagnosing early post-onset MI or UA. The aim of this prospective cohort study was to examine the diagnostic ability of serum nardilysin (NRDC) for the early detection of ACS. This study consisted of two sequential cohorts, the Phase I cohort, 435 patients presenting to the emergency room (ER) with chest pain, and the Phase II cohort, 486 patients with chest pain who underwent coronary angiography. The final diagnosis was ACS in 155 out of 435 patients (35.6%) in the phase I and 418 out of 486 (86.0%) in the phase II cohort. Among 680 patients who presented within 24 h of onset, 466 patients (68.5%) were diagnosed with ACS. Serum NRDC levels were significantly higher in patients with ACS than in those without ACS. The sensitivity of NRDC in patients who presented within 6 h after the onset was higher than that of hsTnI, and the AUC of NRDC within 1 h of the onset was higher than that of hsTnI (0.718 versus 0.633). Among hsTnI-negative patients (300 of 680 patients: 44.1%), 136 of whom (45.3%) were diagnosed with ACS, the sensitivity and the NPV of NRDC were 73.5 and 65.7%, respectively. When measured in combination with hsTnI, NRDC plays auxiliary roles in the early diagnosis of ACS.

Blood pressure is a crucial physiological parameter and its abnormalities can cause a variety of health problems. We have previously reported that mice with systemic deletion of nardilysin (NRDC), an M16 family metalloprotease, exhibit hypotension. In this study, we aimed to clarify the role of NRDC in vascular smooth muscle cells (VSMC) by generating VSMC-specific Nrdc knockout (VSMC-KO) mice. Our findings reveal that VSMC-KO mice also exhibit hypotension. Aortas isolated from VSMC-KO mice exhibited a weakened contractile response to phenylephrine, accompanied by reduced phosphorylation of myosin light chain 2 and decreased rhoA expression. VSMC isolated from VSMC-KO aortas showed a reduced increase in intracellular Ca2+ concentration induced by α-stimulants. These findings suggest that NRDC in VSMC regulates vascular contraction and blood pressure by modulating Ca2+ dynamics.

Nardilysin (NRDC) is a multifunctional protein required for maintaining homeostasis in various cellular and tissue contexts. However, its role in hematopoietic stem cells (HSCs) remains unclear. Here, through the conditional deletion of NRDC in hematopoietic cells, we demonstrate that NRDC is required for HSCs expansion in vitro and the reconstitution of hematopoiesis in vivo after transplantation. We found NRDC-deficient HSCs lose their self-renewal ability and display a preferential bias to myeloid differentiation in response to replication stress. Transcriptome data analysis revealed the upregulation of heat shock response-related genes in NRDC-deficient HSCs. Additionally, we observed increased protein synthesis in cultured NRDC-deficient HSCs. Thus, loss of NRDC may cause the inability to control protein synthesis in response to replication induced protein stress, leading to the impaired HSC self-renewal ability. This highlights a novel model of action of NRDC specifically in HSCs.

Nardilysin (N-Arginine Dibasic Convertase: NRDC) is a metalloendopeptidase belonging to the M16 family, which enzymatically cleaves on the N-terminal side of the arginine residue at a dibasic site. Although prior studies identified Dynorphin-A, somatostatin-28, α-neoendorphin, and glucagon as in vitro substrates for NRDC, but its in vivo substrates remain elusive.

IntroductionAutoantibodies against inflammatory cytokines may be used for the prevention of atherosclerosis. Preclinical studies consider colony-stimulating factor 2 (CSF2) as an essential cytokine with a causal relationship to atherosclerosis and cancer. We examined the serum anti-CSF2 antibody levels in patients with atherosclerosis or solid cancer. MethodsWe measured the serum anti-CSF2 antibody levels via amplified luminescent proximity homogeneous assay-linked immunosorbent assay based on the recognition of recombinant glutathione S-transferase-fused CSF2 protein or a CSF2-derived peptide as the antigen. ResultsThe serum anti-CSF2 antibody (s-CSF2-Ab) levels were significantly higher in patients with acute ischemic stroke (AIS), acute myocardial infarction (AMI), diabetes mellitus (DM), and chronic kidney disease (CKD) compared with healthy donors (HDs). In addition, the s-CSF2-Ab levels were associated with intima-media thickness and hypertension. The analyzes of samples obtained from a Japan Public Health Center-based prospective study suggested the utility of s-CSF2-Ab as a risk factor for AIS. Furthermore, the s-CSF2-Ab levels were higher in patients with esophageal, colorectal, gastric, and lung cancer than in HDs but not in those with mammary cancer. In addition, the s-CSF2-Ab levels were associated with unfavorable postoperative prognosis in colorectal cancer (CRC). In CRC, the s-CSF2-Ab levels were more closely associated with poor prognosis in patients with p53-Ab-negative CRC despite the lack of significant association of the anti-p53 antibody (p53-Ab) levels with the overall survival. ConclusionS-CSF2-Ab was useful for the diagnosis of atherosclerosis-related AIS, AMI, DM, and CKD and could discriminate poor prognosis, especially in p53-Ab-negative CRC.

Autoimmune hepatitis (AIH) is a refractory inflammatory disease that causes progressive liver damage. While it has been suggested that regulatory T cells (Treg) have a significant role on the pathophysiology of AIH, the precise mechanism how AIH is controlled by Treg is still elusive.

Nardilysin (NRDC) is a metalloprotease of the M16 family, which has multiple functions such as enhancing the ectodomain shedding of membrane proteins in the extracellular space and regulating transcription in the nucleus. NRDC-deficient mice (Nrdc-/-) show various phenotypes such as hypomyelination, hypothermia, and bradycardia. In the present study, we have focused on the role of NRDC in regulating heart rate and obtained the following results: (1) The intrinsic heart rate, determined by pharmacological blocking of the autonomic nervous system, was significantly reduced in Nrdc-/-; (2) Funny (If) current and T-type calcium current were significantly reduced in isolated Nrdc-/- sinus node cells; (3) Messenger RNA levels of Cav3.1 and HCN1/4, ion channels involved in sinus automaticity, were markedly decreased in Nrdc-/- hearts; (4) Gene knockdown of NRDC in primary rat cardiomyocytes reduced HCN1/4 mRNA levels; (5) Chromatin immunoprecipitation PCR showed NRDC binding to the promoter regions of Cav3.1 and HCN1/4; (6) Reintroduction of wild-type NRDC, but not the enzymatically inactive mutant of NRDC (E>A mutant), into NRDC-deficient cells restored HCN1 mRNA expression; (7) NRDC-E>A mutant knock in mice showed bradycardia and significantly reduced intrinsic heart rate, suggesting that NRDC enzyme activity is important for the control of heart rate. Together, our results indicate that NRDC in cardiomyocyte controls heart rate through the transcriptional regulation of ion channels critical for sinus automaticity.

Brown adipose tissue (BAT) is a major organ responsible for diet-induced thermogenesis, a phenomenon which converts excess energy intake into heat. Previous studies suggested that hepatocytes regulate diet-induced thermogenesis in response to changes in nutritional status. However, it is not clear how hepatocytes are involved in diet-induced thermogenesis.

Nardilysin (NRDC; N-arginine dibasic convertase) is a metalloprotease of the M16 family. We have shown that NRDC has multiple functions such as an enhancer of ectodomain shedding of membrane-anchored protein and transcriptional coregulator. NRDC-deficient mice (Nrdc-/-) show wide range of phenotypes such as hypomyelination, hypothermia, abnormal sympathetic innervation to hearts, hypotension, and bradycardia. The purpose of this study is to explore a role of NRDC in the regulation of heart rate. To this end, we have revealed the following points: (1) Intrinsic heart rate, obtained by pharmacological blocking of autonomic nervous system, was significantly reduced in Nrdc-/- compared with that of wild-type mice; (2) Messenger RNA levels of Cav3.1 and HCN1/4, ion channels responsible for sinus automaticity, were significantly reduced In Nrdc-/- hearts; (3) Funny (If) current and T-type Ca current were markedly reduced in Nrdc-/- sinus node cells; (4) Gene knockdown of NRDC in primary rat cardiomyocyte reduced mRNA levels of HCN1/4; (5) NRDC binding to the promoter region of Cav3.1 and HCN1/4 was revealed by chromatin immunoprecipitation-PCR analysis, suggesting the direct involvement of NRDC in transcriptional regulation of these ion channels; (6) Reintroduction of wild-type NRDC, but not the enzymatic inactive mutant of NRDC (E>A mutant) into NRDC-deficient cells restored the HCN1 mRNA expression, suggesting the important role of NRDC enzyme activity in the transcriptional regulation; (7) NRDC-E>A mutant knock in mice showed bradycardia and significantly reduced intrinsic heart rate. Together, our results indicate that NRDC in cardiomyocyte controls heart rate through the transcriptional regulation of ion channels critical for sinus automaticity.

Brown adipose tissue (BAT) dissipates chemical energy as heat through uncoupling protein 1 (UCP1). The induction of mitochondrial reactive oxygen species (ROS) in BAT was recently identified as a mechanism that supports UCP1-dependent thermogenesis. We previously demonstrated that nardilysin (NRDC) plays critical roles in body temperature homeostasis. Global NRDC-deficient (Nrdc–/–) mice show hypothermia due to a lower set point for body temperature, whereas BAT thermogenesis at room temperature (RT) is enhanced mainly to compensate for poor thermal insulation. To examine the primary role of NRDC in BAT thermogenesis, we generated adipocyte-specific NRDC-deficient (Adipo-KO) mice by mating Nrdc floxed (Nrdcflox/flox) mice with adiponectin-Cre mice. Adipo-KO mice showed hyperthermia at both RT and thermoneutrality. They were also more cold-tolerant than Nrdcflox/flox mice. However, UCP1 mRNA levels were significantly lower in Adipo-KO BAT at RT, thermoneutrality, and 4 °C, whereas no significant differences were observed in UCP1 protein levels at RT and 4 °C. We examined the protein stability of UCP1 using the cycloheximide chase assay and found that NRDC negatively regulated its stability via the ubiquitin–proteasome pathway. NRDC may be also involved in ROS-mediated in vivo thermogenesis because the inhibitory effects of N-acetyl cysteine, an ROS scavenger, on β3 agonist-induced thermogenesis were stronger in Adipo-KO mice. Collectively, the present results demonstrate that NRDC in BAT controls adaptive thermogenesis and body temperature homeostasis possibly via the regulation of UCP1 protein stability and ROS levels.