Early expression of beta- and gamma-subunits of epithelial sodium channel during human airway development

The amiloride-sensitive epithelial Na(+) channel (ENaC) is an apical membrane protein complex involved in active Na(+) absorption and in control of fluid composition in airways. There are no data reporting the distribution of its pore-forming alpha-, beta-, and gamma-subunits in the developing human lung. With use of two different rabbit polyclonal antisera raised against beta- and gamma-ENaC, immunohistochemical localization of the channel was performed in fetal (10-35 wk) and in adult human airways. Both subunits were detected after 17 wk of gestation on the apical domain of bronchial ciliated cells, in glandular ducts, and in bronchiolar ciliated and Clara cells. After 30 wk, the distribution of beta- and gamma-subunits was similar in fetal and adult airways. In large airways, the two subunits were detected in ciliated cells, in cells lining glandular ducts, and in the serous gland cells. In the distal bronchioles, beta- and gamma-subunits were identified in ciliated and Clara cells. Ultrastructural immunogold labeling confirmed the identification of beta- and gamma-ENaC proteins in submucosal serous cells and bronchiolar Clara cells. Early expression of ENaC proteins in human fetal airways suggests that Na(+) absorption might begin significantly before birth, even if secretion is still dominant.     

Morphogenesis of the respiratory bronchiole in rhesus monkey lungs

The epithelium of the respiratory bronchiole in the adult rhesus monkey consists of two populations: a pseudostratified epithelium with basal, mucous goblet, and ciliated cells located near the pulmonary artery (PA); and a simple cuboidal epithelium composed only of nonciliated bronchiolar epithelial (or Clara) cells in areas away from the PA. This study describes the pattern of differentiation of these two epithelial populations, and their relationship to the PA and to the time of appearance of alveoli in the respiratory bronchiole of the rhesus monkey during the period of 90-125 days gestational age (DGA). These events were related to changes in the adjacent parenchyma. Dissected airways of infusion-fixed, critical-point-dried lungs were evaluated by scanning microscopy followed by light microscopy of the same airways. At 54% of gestation (90 DGA), the distal airway was lined by a mixture of ciliated and nonciliated cells. By 67% of gestation (110 DGA), the ciliated cells were confined to the epithelium over the PA. The underlying connective tissue initially was cellular containing few fibers but was fibrous by 76% of gestation (125 DGA). Alveolarization began near the most distal cartilage at 57% of gestation (95 DGA), the same period at which secondary septation occurred in the distal acinus. Thus, alveolarization occurred simultaneously in two centers: 1) the proximal centriacinar region in the vicinity of the most distal cartilage and 2) the distal lung parenchyma. The duration of centriacinar alveolarization was short, approximately 5 days.     

Natriuretic peptides stimulate steroidogenesis in the fetal rat testis

To study the regulation of fetal testicular steroidogenesis in the rat, we examined effects of members of the natriuretic peptide family, that is, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP), on testosterone production of dispersed Leydig cells of rat fetuses at Embryonic Day (E) 18.5. All three peptides stimulated testosterone production, with significant effect at concentrations > or =1 x 10(-8) mol/L of ANP, > or =1 x 10(-9) mol/L of BNP, and > or =1 x 10(-6) mol/L of CNP. Likewise, receptors for all three peptides (i.e., NPR-A, NPR-B, and NPR-C) were expressed in the fetal testis as early as E15.5. The natriuretic peptides had no effect on cAMP production by fetal Leydig cells. When tested in combination with two other peptides previously shown to stimulate fetal testicular steroidogenesis, vasoactive intestinal peptide and pituitary adenylate cyclase-stimulating polypeptide (PACAP-27), the combined effects did not differ significantly from the maximum effect with any one of the peptides alone. In conclusion, our present findings provide both functional and molecular evidences for NPR-A, NPR-B, and NPR-C in the fetal testis. Because ANP has previously been detected in fetal plasma and we now demonstrate the expression of BNP and CNP in fetal testes, these findings indicate involvement of the natriuretic peptides in endocrine and paracrine regulation during the early phase of fetal testicular steroidogenesis at E15.5--19.5 (i.e., before the onset of pituitary LH secretion).     

Immunolocalization of natriuretic peptides and their receptors in goat (Capra hircus) heart

Natriuretic peptides are structurally similar, but genetically distinct, hormones that participate in cardiovascular homeostasis by regulating blood and extracellular fluid volume and blood pressure. We investigated the distribution of natriuretic peptides and their receptors in goat (Capra hircus) heart tissue using the peroxidase-anti-peroxidase (PAP) immunohistochemical method. Strong staining of atrial natriuretic peptide (ANP) was observed in atrial cardiomyocytes, while strong staining for brain natriuretic peptide (BNP) was observed in ventricular cardiomyocytes. Slightly stronger cytoplasmic C-type natriuretic peptide (CNP) immunostaining was detected in the ventricles compared to the atria. Natriuretic peptide receptor-A (NPR-A) immunoreactivity was more prominent in the atria, while natriuretic peptide receptor-B (NPR-B) immunoreactivity was stronger in the ventricles. Cytoplasmic natriuretic peptide receptor-C (NPR-C) immunoreactivity was observed in both the atria and ventricles, although staining was more prominent in the ventricles. ANP immunoreactivity ranged from weak to strong in endothelial and vascular smooth muscle cells. Endothelial cells exhibited moderate to strong BNP immunoreactivity, while vascular smooth cells displayed weak to strong staining. Endothelial cells exhibited weak to strong cytoplasmic CNP immunoreactivity. Vascular smooth muscle cells were labeled moderately to strongly for CNP. Weak to strong cytoplasmic NPR-A immunoreactivity was found in the endothelial cells and vascular smooth muscle cells stained weakly to moderately for NPR-A. Endothelial and vascular smooth cells exhibited weak to strong cytoplasmic NPR-B immunoreactivity. Moderate to strong NPR-C immunoreactivity was observed in the endothelial and smooth muscle cells. Small gender differences in the immunohistochemical distribution of natriuretic peptides and receptors were observed. Our findings suggest that endothelial cells, vascular smooth cells and cardiomyocytes express both natriuretic peptides and their receptors.     

Renal C-type natriuretic peptide and natriuretic peptide receptor B mRNA expression are affected by water deprivation in the Spinifex Hopping mouse

This study investigated the effect of water deprivation on the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) mRNA, and the ability of NPR-B to generate cGMP in the Spinifex Hopping mouse, Notomys alexis. This rodent is a native of central and western Australia that is well adapted to survive in arid environments. Initially, CNP and NPR-B cDNAs (partial for NPR-B) were cloned and sequenced, and were shown to have high homology with those of rat and mouse. RT-PCR analysis showed CNP mRNA expression in the kidney, proximal and distal colon and small intestine, whilst NPR-B mRNA expression was found in the kidney, proximal and distal colon and the atria. Using a semi-quantitative multiplex PCR technique, the expression of renal CNP and NPR-B mRNA was determined in 7- and 14-day water-deprived hopping mice, in parallel with control hopping mice (access to water). Water deprivation significantly decreased the relative levels of CNP and NPR-B mRNA expression in both the 7- and 14-day water-deprived hopping mice, when compared to control hopping mice. In contrast, the ability of CNP to stimulate cGMP production was significantly increased after 14 days of water deprivation. This study shows that alterations in the renal CNP/NPR-B system may be an important physiological adjustment when water is scarce.     

Structural requirements of C-type natriuretic peptide for elevation of cyclic GMP in cultured vascular smooth muscle cells.

C-type natriuretic peptide (CNP), which was recently found to be a selective ligand for one of the two known natriuretic peptide receptor guanylyl cyclases (NPR-B), potently stimulates cGMP production in cultured rat vascular smooth muscle cells (VSMC) and exerts potent antiproliferative effects on the cells. To investigate the structural requirements of CNP for stimulation of cGMP accumulation via NPR-B, we prepared CNP analogs and tested them on cultured rat VSMC. Our results indicate that only the ring portion of CNP with a disulfide bond (CNP(6-22)) participates in stimulation of cGMP accumulation, especially the sequence Leu9-Lys10-Leu11 in the ring portion executes essential roles for both elevation of cGMP and selectivity of the ligand for NPR-B. We also found a good correlation between the activities of the CNP analogs for stimulation of cGMP accumulation and inhibition of DNA synthesis.     

Functional expression of components of the natriuretic peptide system in human ocular nonpigmented ciliary epithelial cells

The expression of the natriuretic peptide system in the human ocular ciliary epithelium (CE) and in cultured nonpigmented (NPE) ciliary epithelial cells was examined. By RT-PCR and DNA sequencing, we demonstrated that the CE and NPE cells express mRNA for (i) ANP; (ii) BNP; (iii) NPR-A, NPR-B, and NPR-C receptors; and (iv) the neutral endopeptidase 24.11. Radioimmunoassay results indicate that BNP is secreted by cultured NPE cells at much higher levels than ANP. NPR-A and NPR-B receptors elicited a cGMP response to ANP, BNP, and CNP, in a rank order of potency (CNP > ANP >/= BNP), indicative that the NPR-B receptor is predominant in NPE cells. A71915, an inhibitor of NPR-A activity, attenuated (65-75%) cGMP response to ANP and BNP, but not to CNP. C-ANP4-23 elicited an inhibitory effect (30-37%) on basal levels of cAMP in NPE cells and on forskolin NPE-treated cells, indicative that the NPR-C receptor is functional in these cells. PMA induced, in NPE cells, a long-term downregulation (75-85%) of NPR-C receptor mRNA, but not of NPR-A or NPR-B receptor mRNA, suggesting a differential regulation of NPR-C receptor mRNA via activation of PKC. Collectively, our data provide molecular evidence that all the components of the natriuretic peptide system with the exception of CNP are coexpressed in the ocular NPE ciliary epithelial cells, where they may function as local autocrine/paracrine modulators to influence eye pressure.     

Localization of pulmonary carbonyl reductase in guinea pig and mouse: enzyme histochemical and immunohistochemical studies.

We studied the localization of carbonyl reductase (E.C. 1.1.1.184) in guinea pig and mouse lung by enzyme histochemistry and immunohistochemistry, using antibodies against the guinea pig lung enzyme which crossreacted with the lung enzymes of both animals. Carbonyl reductase activity was detectable in the bronchiolar epithelial cells of small airways and in alveolar cells. In the immunohistochemical staining for carbonyl reductase, the reaction was strongest in the non-ciliated bronchiolar cells (Clara cells) and was weak in the ciliated cells and type II alveolar pneumocytes. Injection of a single dose of naphthalene led to significant impairment of carbonyl reductase activity and of microsomal mixed-function oxidase activities in mouse lung, with a marked decrease in both activity and immunoreactive staining in the bronchiolar epithelial cells. The results indicate that carbonyl reductase is localized primarily in the Clara cells, which are known to be sites of pulmonary drug metabolism.     

Implication of C-type natriuretic peptide-3 signaling in glycosaminoglycan synthesis and chondrocyte hypertrophy during TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells

This study investigated the involvement of CNP-3, chick homologue for human C-type natriuretic peptide (CNP), in TGF-β1 induced chondrogenic differentiation of chicken bone marrow-derived mesenchymal stem cells (MSCs). Chondrogenic differentiation of MSCs in pellet cultures was induced by TGF-β1. Chondrogenic differentiation and glycosaminoglycan synthesis were analyzed on the basis of basic histology, collagen type II expression, and Alcian blue staining. Antibodies against CNP and NPR-B were used to block their function during these processes. Results revealed that expression of CNP-3 and NPR-B in MSCs were regulated by TGF-β1 in monolayer cultures at mRNA level. In pellet cultures of MSCs, TGF-β1 successfully induced chondrogenic differentiation and glycosaminoglycan synthesis. Addition of CNP into the TGF-β1 supplemented chondrogenic differentiation medium further induced the glycosaminoglycan synthesis and hypertrophy of differentiated chondrocytes in these pellets. Pellets induced with TGF-β1 and treated with antibodies against CNP and NPR-B, did show collagen type II expression, however, Alcian blue staining showing glycosaminoglycan synthesis was significantly suppressed. In conclusion, CNP-3/NPR-B signaling may strongly be involved in synthesis of glycosaminoglycans of the chondrogenic matrix and hypertrophy of differentiated chondrocytes during TGF-β1 induced chondrogenic differentiation of MSCs.     

Reduced ability of C-type natriuretic peptide (CNP) to activate natriuretic peptide receptor B (NPR-B) causes dwarfism in lbab -/- mice

C-type natriuretic peptide (CNP) stimulates endochondrial ossification by activating the transmembrane guanylyl cyclase, natriuretic peptide receptor-B (NPR-B). Recently, a spontaneous autosomal recessive mutation that causes severe dwarfism in mice was identified. The mutant, called long bone abnormality (lbab), contains a single point mutation that converts an arginine to a glycine in a conserved coding region of the CNP gene, but how this mutation affects CNP activity has not been reported. Here, we determined that 30-fold to greater than 100-fold more CNP(lbab) was required to activate NPR-B as compared to wild-type CNP in whole cell cGMP elevation and membrane guanylyl cyclase assays. The reduced ability of CNP(lbab) to activate NPR-B was explained, at least in part, by decreased binding since 10-fold more CNP(lbab) than wild-type CNP was required to compete with [(125)I][Tyr(0)]CNP for receptor binding. Molecular modeling suggested that the conserved arginine is critical for binding to an equally conserved acidic pocket in NPR-B. These results indicate that reduced binding to and activation of NPR-B causes dwarfism in lbab(-/-) mice.     

Deletion mutants of the natriuretic peptide receptor type B: expression of cDNA, purification and characteristics of proteins

The C type natriuretic peptide (CNP) is a peptide hormone stimulating vasorelaxation and inhibiting cell proliferation. CNP activates the type B natriuretic peptide receptor (NPR-B), known as the guanylate cyclase B membrane enzyme, which results in the cGMP release. To study functional properties of NPR-B, its gene fragments were expressed in methylotrophic yeasts Pichia pastoris. Conditions were found providing for secretion of functionally active recombinant proteins NPR-Bs and NPR-B1 into the cultural medium in a yield of 25 mg/ml culture. Their specific activity was 0.97 and 0.93 mumol cGMP min-1 mg-1 protein, respectively. It was shown that NPR-B belongs to the family of Ser/Thr protein kinases and can be autophosphorylated at the serine residues.     

C-type natriuretic peptide receptor expression in pancreatic alpha cells

Atrial natriuretic peptide (ANP), brain type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) comprise a family of natriuretic peptides that mediate their biological effects through three natriuretic peptide receptor subtypes, NPR-A (ANP, BNP), NPR-B (CNP) and NPR-C (ANP, BNP, CNP). Several reports have provided evidence for the expression of ANP and specific binding sites for ANP in the pancreas. The purpose of this study was to identify the ANP receptor subtype and to localize its expression to a specific cell type in the human pancreas. NPR-C immunoreactivity, but neither ANP nor NPR-A, was detected in human islets by immunofluorescent staining. No immunostaining was observed in the exocrine pancreas or ductal structures. Double-staining revealed that NPR-C was expressed mainly in the glucagon-containing alpha cells. NPR-C mRNA and protein were detected in isolated human islets by RT-PCR and Western blot analysis, respectively. NPR-C expression was also detected by immunofluorescent staining in glucagonoma but not in insulinoma. ANP, as well as BNP and CNP, stimulated glucagon secretion from perifused human islets (1,111 ± 55% vs. basal [7.3 fmol/min]; P < 0.001). This response was mimicked by cANP(4–23), a selective agonist of NPR-C. In conclusion, the NPR-C receptor is expressed in normal and neoplastic human alpha cells. These findings suggest a role for natriuretic peptides in the regulation of glucagon secretion from human alpha cells.     

Expression of C-type natriuretic peptide and its receptor NPR-B in cardiomyocytes

C-type natriuretic peptide (CNP) was recently found in myocardium at the mRNA and protein levels, but it is not known whether cardiomyocytes are able to produce CNP. The aim of this study was to determine the expression of CNP and its specific receptor NPR-B in cardiac cells, both in vitro and ex vivo. CNP, brain natriuretic peptide (BNP) and natriuretic peptide receptor (NPR)-B mRNA expression were examined by RT-PCR in the H9c2 rat cardiac myoblast cell line, in neonatal rat primary cardiomyocytes and in human umbilical vein endothelial cells (HUVECs) as control. CNP protein expression was probed in cardiac tissue sections obtained from adult male minipigs by immunohistochemistry, and in H9c2 cells both by immunocytochemistry and by specific radioimmunoassay. The results showed that cardiac cells as well as endothelial cells were able to produce CNP. Unlike cardiomyocytes, as expected, in endothelial cells expression of BNP was not detected. NPR-B mRNA expression was found in both cell types. Production of CNP in the heart muscle cells at protein level was confirmed by radioimmunological determination (H9c2: CNP = 0.86 ± 0.083 pg/mg) and by immunocytochemistry studies. By immunostaining of tissue sections, CNP was detected in both endothelium and cardiomyocytes. Expression of CNP in cardiac cells at gene and protein levels suggests that the heart is actively involved in the production of CNP.     

C-型钠尿肽与血管损伤性疾病

C-型钠尿肽(C-type natriuretic peptide, CNP)作为钠尿肽家系的一员, 主要是由血管内皮分泌,CNP与血管平滑肌细胞钠尿肽受体-B(NPR-B)结合,激活颗粒型鸟苷酸环化酶,促进细胞内cGMP 水平升高,以旁分泌和/或自分泌方式调节循环系统功能稳态.CNP广泛分布于血管系统,尤其在内皮细胞中高表达.CNP具有利钠、利尿、调节血管张力、抑制血管平滑肌细胞迁移、增殖等作用,与高血压、动脉粥样硬化、血栓形成、冠脉成形术后再狭窄和血管钙化等多种血管损伤性疾病密切相关.     

Cloning and functional expression of the bovine natriuretic peptide receptor-B (natriuretic factor R1c subtype)

We describe the isolation of a 3,276 base pair cDNA for the bovine natriuretic peptide receptor-B (NPR-B). Expression of this clone in Cos-P cells demonstrates that it encodes an agonist-dependent guanylyl cyclase. Porcine CNP stimulates the activity of this receptor up to 200-fold with an ED₅₀ of 12±2 nM, whereas brain natriuretic peptide C-type natriuretic peptide (CNP) and atrial natriuretic factor (ANF) are less efficacious. In addition, ligand binding studies indicate that this receptor exhibits the pharmacology appropriate for the bovine NPR-B. CNP binds to Cos-P cell membranes expressing this clone with a K_d of 13±1 pM, and natriuretic peptides compete for [¹²⁵I]-CNP binding with a rank order of pCNP>pBNP>rANF. Thus, the expressed receptor-guanylyl cyclase exhibits the expected pharmacological profile for ligand binding and cyclase activation of the bovine NPR-B receptor.Abbreviations BSA bovine serum albumin - dNTP deoxynucleotide triphosphate - SDS sodium dodecyl sulfate - DEAE-dextran diethylaminoethyl-dextran - EDTA ethylenediamine tetraacetic acid - Tris Tris(hydroxymethyl)aminomethane - DMSO dimethyl sulfoxide - RP-HPLC reverse phase-high performance liquid chromatography - AMV avian myeloblastosis virus - Arg arginine - Lys lysine     

Sequencing and cardiac expression of natriuretic peptide receptor 2 (NPR-B) in Sus Scrofa

The cardiovascular actions of the C-type natriuretic peptide (CNP) are mainly mediated by the interaction with natriuretic peptide receptor-B (NPR-B). The aim of this study was to identify the sequence of NPR-B in Sus Scrofa, which is not present in GenBank, to verify the expression of NPR-B in the different cardiac chambers of normal pigs and evaluate its homology with murine and human species. Using the guanidinium thyocyanate-phenol-chloroform method, we extracted total RNA from samples obtained from heart of mouse and from the atrium, ventricle, and septum of normal pigs. Pig NPR-B mRNA was sequenced using polymerase chain reaction primers designed from mouse consensus sequences. Sus Scrofa natriuretic peptide receptor 2 mRNA, 1-396 bp, was submitted to GenBank (accession number DQ487044). The presence of NPR-B at mRNA level was detected in all the cardiac chambers; moreover, the bands obtained from pig cardiac tissue shared a 93% sequence homology with a region of the mouse NPR-B and a 95% sequence homology with Homo sapiens. Therefore, NPR-B sequencing provides a new tool to investigate the role of CNP under physiological and pathological conditions in the experimental and clinical setting.     

Natriuretic peptides cause relaxation of human esophageal mucosal muscle

Natriuretic peptides have been demonstrated to cause relaxation of the human gallbladder muscle through interaction with natriuretic peptide receptor-B (NPR-B/NPR2). Effects of natriuretic peptides in the human esophageal muscle were unknown. To investigate the effects of natriuretic peptides in the human esophagus, we measured relaxation of muscularis mucosae strips isolated from the human esophagus caused by C-type natriuretic peptide (CNP), brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and des[Gln(18), Ser(19), Gly(20), Leu(21), Gly(22)]ANP(4-23) amide (cANP(4-23)), a selective natriuretic peptide receptor-C (NPR-C) agonist. In endothelin-1 or carbachol-contracted mucosal muscle strips, CNP caused moderate, sustained and concentration-dependent relaxation. BNP caused a very mild relaxation whereas ANP and cANP(4-23) did not cause any relaxation. CNP was much more potent than BNP and ANP in causing relaxation. These suggest the existence of NPR-B mediating relaxation. The CNP-induced relaxation was not affected by tetrodotoxin or atropine in endothelin-1-contracted esophageal strips and not by tetrodotoxin in carbachol-contracted strips, indicating a direct effect of CNP on the human esophageal muscularis mucosae. Taken together, these results demonstrate that natriuretic peptides cause relaxation of the muscularis mucosae of the human esophagus and suggest that the relaxation is through interaction with NPR-B. Natriuretic peptides may play an important role in the control of human esophageal motility.     

Gene expression of C-type natriuretic peptide and of its specific receptor NPR-B in human leukocytes of healthy and heart failure subjects

C-type natriuretic peptide (CNP), a member of the family of natriuretic peptides, is synthesized and secreted from monocytes and macrophages that resulted to be a source of CNP at inflammatory sites. This suggests that special attention should be focused on the possible role of CNP in the immune system, in addition to its effects on the cardiovascular system. The aim of this study was to evaluate the possibility of measuring the mRNA expression of CNP and NPR-B, its specific receptor, in human whole blood samples of healthy (N; n=7) and heart failure (HF; n=7) subjects by Real-Time PCR (RT-PCR). Total RNA was extracted from leukocytes with QIAamp RNA Blood Kit and/or with PAXgene Blood RNA Kit. RT-PCR was performed and optimized for each primer. The experimental results were normalized with the three most stably expressed genes. CNP and NPR-B expression trend was similar in both fresh and frozen human whole blood. Significant higher levels of CNP and NPR-B mRNA expression were found in HF patients with respect to controls (CNP: N=1.23±0.33 vs. HF=6.54±2.09 p=0.027; NPR-B: N=0.85±0.23 vs. HF=5.31±1.98 p=0.04). A significant correlation between CNP and NPR-B (r=0.86, p<0.0001) was observed. Further studies are needed to clarify the pathophysiological properties of this peptide but the possibility to measure CNP and NPR-B mRNA expression in human leukocytes with a fast and easy procedure is a useful starting point for future investigation devoted to better understand the biomolecular processes associated to different diseases.     

Brain-specific natriuretic peptide receptor-B deletion attenuates high-fat diet-induced visceral and hepatic lipid deposition in mice

C-type natriuretic peptide (CNP) and its receptor, natriuretic peptide receptor-B (NPR-B), are abundantly distributed in the hypothalamus. To explore the role of central CNP/NPR-B signaling in energy regulation, we generated mice with brain-specific NPR-B deletion (BND mice) by crossing Nestin-Cre transgenic mice and mice with a loxP-flanked NPR-B locus. Brain-specific NPR-B deletion prevented body weight gain induced by a high-fat diet (HFD), and the mesenteric fat and liver weights were significantly decreased in BND mice fed an HFD. The decreased liver weight in BND mice was attributed to decreased lipid accumulation in the liver, which was confirmed by histologic findings and lipid content. Gene expression analysis revealed a significant decrease in the mRNA expression levels of CD36, Fsp27, and Mogat1 in the liver of BND mice, and uncoupling protein 2 mRNA expression was significantly lower in the mesenteric fat of BND mice fed an HFD than in that of control mice. This difference was not observed in the epididymal or subcutaneous fat. Although previous studies reported that CNP/NPR-B signaling inhibits SNS activity in rodents, SNS is unlikely to be the underlying mechanism of the metabolic phenotype observed in BND mice.Taken together, CNP/NPR-B signaling in the brain could be a central factor that regulates visceral lipid accumulation and hepatic steatosis under HFD conditions. Further analyses of the precise mechanisms will enhance our understanding of the contribution of the CNP/NPR-B system to energy regulation.