Neuroplastin-55 is a receptor of Manf and protects against diet-induced obesity by promoting adipose browning

Significance

Promoting adipose browning to increase energy expenditure has great potential in obesity treatment. Here, we identify the membrane receptor neuroplastin-55 (Np55) as a critical regulator of this process. Np55 deletion in adipose tissue aggravates obesity and impairs energy expenditure and white adipose tissue (WAT) browning. Mechanistically, Np55 acts as the receptor of Manf and forms a complex with tumor necrosis factor receptor associated factor-2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) to activate p38 mitogen-activated protein kinase (MAPK) signaling pathway, enhancing thermogenesis in WAT. Np55 is indispensable for Manf-induced weight loss in obese mice. Thus, this study reveals the pivotal role of Np55 in maintaining metabolic homeostasis, highlighting its potential as a therapeutic target for obesity and related disorders.

Abstract

The browning of white adipose tissue (WAT) enhances thermogenesis and holds great potential in obesity treatment. Membrane receptors have been proven as essential factors during WAT browning. In this study, we found that transmembrane receptor neuroplastin-55 (Np55) was markedly upregulated in the adipose tissue of both obese individuals and mice. Fat-specific Np55 knockout (Np55^FKO) sensitized mice to high-fat diet-induced obesity and metabolic disorders, which was attributed to impaired energy expenditure and WAT browning. We identified that mesencephalic astrocyte-derived neurotrophic factor (Manf) acts as a ligand for Np55 with high binding affinity. Mechanistically, Np55 forms a complex with tumor necrosis factor receptor associated factor-2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) to activate p38 mitogen-activated protein kinase (MAPK) signaling pathway and conduct WAT browning after Manf stimulation. Additionally, we identified a single-nucleotide polymorphism (rs2470738600) in Manf compromised the binding affinity to Np55 and thermogenic capacity. Finally, we showed that Np55 is indispensable for Manf-induced thermogenesis and weight loss in obese mice. Our results underscore the pivotal role of Np55 as a receptor of Manf in maintaining metabolic homeostasis, highlighting its potential as a therapeutic target for obesity and related disorders.

Thermogenesis is a critical physiological process that enables mammals to maintain body temperature and energy homeostasis in response to environmental challenges or metabolic demands. Heat production occurs through shivering thermogenesis or nonshivering thermogenesis, which is mediated by specialized adipose tissues like brown adipose tissue (BAT) and beige adipose tissue (1, 2). While BAT persists in adult humans, its abundance is relatively low compared to beige adipose tissue, which resides within white adipose tissue (WAT), particularly in subcutaneous adipose tissue (3).

Beige adipocytes can be activated by hormonal and environmental stimuli, leading to the upregulation of key thermogenic genes, including uncoupling protein 1 (Ucp1) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1α). This process, termed “white adipose browning,” confers beige adipocytes with BAT-like characteristics, enhancing energy dissipation (2, 4). Obesity, however, disrupts this balance by promoting excessive triglyceride accumulation in adipocytes and impairing organelle function (5). Therefore, strategies to enhance adipose browning have emerged as promising therapeutic approaches for combating obesity and related metabolic syndromes (6, 7). Previously, we identified mesencephalic astrocyte-derived neurotrophic factor (Manf), a feeding-induced hepatokine, as a key regulator of subcutaneous fat browning by activating p38 MAPK signaling pathway (8). However, the specific receptor on adipocytes that mediates Manf’s thermogenic effects remains unidentified.

The p38 mitogen-activated protein kinase (MAPK) signaling is a central regulator of thermogenesis (9). This signaling cascade is initiated by upstream MAPK kinases (MKKs) and MAPK kinase kinases (MAP3Ks) (10). During thermogenic activation, MAP3Ks phosphorylate and activate MKK3/6, which subsequently phosphorylates p38α, triggering its nuclear translocation (11). Once in the nucleus, p38 phosphorylates activating transcription factor 2 (ATF2), which in turn regulates the expression of thermogenic genes. Although the downstream effectors of p38 MAPK are well characterized, the upstream regulatory mechanisms remain insufficiently elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is a pivotal activator of the MAPK cascade and has been shown to promote energy expenditure in BAT and beige adipose tissue. Canonical ASK1 activation is linked to cyclic AMP (cAMP) elevation via G protein–coupled receptor signaling (11). Nonetheless, emerging evidence indicates that ASK1 can also be activated through cAMP-independent mechanisms involving adapter proteins or alternative MAPK pathways (8).

Over the past decade, numerous studies have highlighted the crucial role of membrane receptors in regulating energy metabolism and obesity. For instance, transferrin receptor promotes white adipose browning by modulating iron uptake; its adipose-specific deletion impairs beige adipogenesis (12). The Mas1 receptor is essential for BAT thermogenesis under cold exposure, as its knockout mice exhibit defective thermoregulation (13). These findings underscore the importance of cell surface receptors in adipose tissue remodeling and thermogenesis. Recently, the membrane receptor neuroplastin (NPTN) has garnered attention for its neuronal functions. Yagi et al. demonstrated that NPTN suppresses NF-κB-mediated inflammation and apoptosis in both neuronal and fibroblast models (14). Intriguingly, a recent study has revealed that NPTN also regulates insulin secretion, suggesting potential metabolic functions (15). However, whether NPTN exerts its function in adipose tissue has never been investigated.

In this study, we demonstrate that Np55, an isoform of NPTN, plays a critical role in obesity and thermogenesis. Fat-specific Np55 knockout mice exhibit deteriorated metabolic dysfunction under high-fat diet (HFD) conditions and impaired thermogenic capacity. Mechanistically, we identify Np55 as the adipocyte receptor for Manf, facilitating adipose tissue browning. Loss of Np55 disrupts intracellular signaling transduction, impairs thermogenesis, and blunts Manf-induced weight loss. These findings position Np55 as a promising therapeutic target for obesity and metabolic syndrome.

Results

Np55 Is Upregulated in the Subcutaneous Adipose Tissue of Obese Individuals and Mice.

We examined the expression profile of NPTN in mice and found that Np55 is widely distributed and highly expressed in adipose tissue, whereas Np65 is predominantly expressed within the central nervous system (SI Appendix, Fig. S1 A and B), aligning with prior studies (16). To explore the potential role of Np55 during obesity, we first examined the expression levels of Np55 in the adipose tissue of obese patients and analyzed its correlation with body mass index (BMI). Based on specific inclusion and exclusion criteria, we enrolled 29 patients who underwent bariatric surgery and 28 healthy volunteers (SI Appendix, Table S1). We found that the mRNA expression of Np55 in the subcutaneous adipose tissue of obese patients was significantly elevated compared to healthy controls (Fig. 1A). Correlation analysis revealed that Np55 expression in subcutaneous adipose tissue was positively correlated with BMI (r = 0.3534, P = 0.007) (Fig. 1B), suggesting the Np55 level is closely linked to obesity. Next, we examined the expression levels of Np55 in the subcutaneous adipose tissue of HFD-induced and leptin-deficient (ob/ob) obese mice. Our findings indicated that the mRNA and protein expression of Np55 was significantly upregulated in the subcutaneous adipose tissue of both obese mouse models (Fig. 1 C–E). Furthermore, we isolated the stromal vascular fraction (SVF) and mature adipocytes from the subcutaneous adipose tissue to assess Np55 expression levels. The results showed that the protein level of Np55 was markedly higher in mature adipocytes compared to the SVF (Fig. 1F). When the SVF was cultured, induced, and differentiated into mature adipocytes, a significant increase in Np55 expression was observed postdifferentiation (Fig. 1G). These findings suggest that Np55 is highly expressed in adipose tissue, particularly in mature adipocytes, indicating a potential role in adipocyte function.

Fig. 1.

Fat-specific knockout of Np55 exacerbates HFD-induced obesity. (A) qRT-PCR analysis of Np55 expression in adipose tissue from obese patients and controls. (B) Correlation between Np55 expression and BMI. (C and D) qRT-PCR analysis of Np55 mRNA levels in iWAT from CD- vs. HFD-fed mice (C), and WT vs. ob/ob mice (D). (E) The protein levels of Np55 in iWAT from CD vs. HFD mice (Upper) and WT and ob/ob mice (Lower). (F and G) Western blotting of Np55 protein level in SVF and adipocytes (F), and adipocytes pre- and postinduction (G). (H) Np55^fl/fl and Np55^FKO mice were fed an HFD for 12 wk. Gross appearance (Upper) and growth curve (Lower) of Np55^fl/fl and Np55^FKO mice under an HFD. (I) Representative images of eWAT, iWAT, and BAT (Upper) and the ratio of eWAT, iWAT, and BAT weight to body weight (Lower) in Np55^fl/fl and Np55^FKO mice. (J) H&E staining of eWAT, iWAT, and BAT. (K) Adipocyte size of eWAT and iWAT. BAT, brown adipose tissue; iWAT, inguinal white adipose tissue; eWAT, epididymal white adipose tissue. (Scale bar: 50 μm.) All data are mean ± SEM. n = 5-7, *P < 0.05, **P < 0.01, ***P < 0.001.

Fat-Specific Knockout of Np55 Exacerbates Diet-Induced Obesity and Metabolic Disorders.

To investigate the role of Np55 in adipose tissue, we generated fat-specific knockout of Np55 (Np55^FKO) mice. Both Np55^FKO and control mice (Np55^fl/fl) developed normally without embryonic lethality. As expected, the protein level of Np55 was specifically reduced in adipose tissue but not in other tissues (SI Appendix, Fig. S1 C and D). On a chow diet (CD), the two groups showed no difference in body weight, liver/body weight ratio, adipose tissue/body weight ratio, or metabolic parameters such as fasting blood glucose, triglycerides (TG), and cholesterol (SI Appendix, Fig. S1 E–G). Metabolic cage study indicated similar energy expenditure in Np55^fl/fl mice and Np55^FKO mice (SI Appendix, Fig. S1H). Following 12 wk of HFD feeding, Np55^FKO mice exhibited significantly greater weight gain compared to Np55^fl/fl control (Fig. 1H). The inguinal white adipose tissue (iWAT) weight-to-body weight ratio was significantly higher in Np55^FKO mice, with larger adipocyte sizes observed by H&E staining (Fig. 1 I–K). The adipocyte sizes of epididymal white adipose tissue (eWAT) and BAT were also greater in Np55^FKO mice compared to controls (Fig. 1 J and K).

Given the established link between obesity and metabolic disorders, we next assessed systemic glucose and lipid homeostasis (17). After a 2-wk HFD challenge, body weights remained comparable between the two genotypes (SI Appendix, Fig. S2A). Np55^FKO mice displayed mild impairments in glucose and insulin tolerance (SI Appendix, Fig. S2 B and C). However, after 12 wk of HFD, Np55^FKO mice exhibited significantly impaired glucose tolerance and insulin sensitivity (Fig. 2 A and B). Np55^FKO mice also had higher fasting (Fig. 2C) and 30-min post–glucose stimulation insulin levels (SI Appendix, Fig. S2D). Serum levels of TG, cholesterol, and free fatty acids were significantly higher in Np55^FKO mice (Fig. 2D). Examination of the liver in Np55^FKO mice after HFD revealed increased liver size and weight compared to control mice (Fig. 2 E and F). H&E and Oil Red O staining indicated enhanced lipid accumulation in the livers of Np55^FKO mice (Fig. 2E). Quantitative assessment of hepatic lipid contents showed markedly elevated levels of TG, cholesterol, and free fatty acids in Np55^FKO mice (Fig. 2G).

Fig. 2.

Fat-specific Np55 ablation aggravates HFD-induced metabolic disorders. Np55^fl/fl and Np55^FKO mice were fed an HFD for 12 wk. (A) Blood glucose concentrations during glucose tolerance test (GTT). (B) Blood glucose concentrations compared to fasting (T0) during insulin tolerance test (ITT). (C) Fasting serum insulin levels. (D) Serum levels of TG, TC, and NEFA in Np55^fl/fl and Np55^FKO mice. (E) Representative image (Upper), H&E (Medium) and Oil red O staining (Lower) of the liver. (F) The liver weight of Np55^fl/fl and Np55^FKO mice after HFD. (G) Hepatic contents of TG, TC, and NEFA in Np55^fl/fl and Np55^FKO mice fed an HFD. (H) qRT-PCR analysis of mRNA levels of M1 and M2 macrophage markers in eWAT. (I) Immunofluorescent staining of F4/80 in eWAT. (Scale bar: 50 μm.) n = 5-8, Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Obesity is also associated with chronic inflammation in adipose tissue, involving proinflammatory M1 and anti-inflammatory M2 macrophages (18). After an HFD feeding, Np55^FKO mice showed significantly higher levels of M1 markers (F4/80, Cd68, Cd11b, and Cd11c) and lower levels of M2 markers (Arg1, Cd206, and Mrc1) compared to control mice (Fig. 2H). The increased macrophage infiltration in eWAT of Np55^FKO mice was further confirmed by immunofluorescence (IF) staining of F4/80 (Fig. 2I). These findings suggest that fat-specific knockout of Np55 significantly exacerbates HFD-induced obesity, insulin resistance, hepatic steatosis, and adipose tissue inflammation.

Fat-Specific Knockout of Np55 Impairs Whole-Body Energy Expenditure and iWAT Browning.

To elucidate the mechanisms underlying the exacerbation of obesity and metabolic dysregulation in fat-specific Np55 knockout mice, we kept mice in metabolic cages to monitor energy intake and expenditure. Np55^FKO mice exhibited a marked reduction in oxygen consumption and carbon dioxide production during both light and dark cycles compared to control mice (Fig. 3 A and B). The respiratory exchange ratio (RER) was comparable between Np55^FKO and Np55^fl/fl mice (SI Appendix, Fig. S2E), suggesting similar substrate utilization in both groups. Notably, the energy expenditure of Np55^FKO mice was significantly lower than Np55^fl/fl mice (Fig. 3C), despite no significant differences in locomotor activity between the two groups (SI Appendix, Fig. S2F). Both groups exhibited comparable food and water intake and consumption (Fig. 3D and SI Appendix, Fig. S2G). These results suggest that the increased fat accumulation in adipose tissue in fat-specific Np55 knockout mice is largely attributed to reduced energy expenditure.

Fig. 3.

Fat-specific Np55 ablation impairs energy expenditure and iWAT browning. Np55^fl/fl and Np55^FKO mice were fed an HFD for 12 wk. (A) Oxygen consumption and (B) carbon dioxide production of Np55^fl/fl and Np55^FKO mice. (C) Energy expenditure of Np55^fl/fl and Np55^FKO mice in an ANCOVA. (D) Average food intake. (E) Rectal temperature of 12-wk-HFD-fed Np55^fl/fl and Np55^FKO mice during RT and cold exposure. (F) qRT-PCR analysis of the mRNA expression of thermogenic genes in iWAT from Np55^fl/fl and Np55^FKO mice. (G) Western blot analysis of UCP1 and PGC-1α in iWAT from mice after long-term cold exposure. (H) Representative images of H&E and UCP1 IHC. (Scale bar: 50 μm.) n = 4-8, Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

To explore the reason for reduced energy expenditure in Np55^FKO mice, we tested the thermogenic abilities in Np55^FKO mice. HFD-diet mice were tested under either short-term (4 °C for 6 h) or long-term cold exposure (4 °C for 72 h). At room temperature (RT), there were no significant differences in thermogenic gene expressions in BAT between Np55^FKO and Np55^fl/fl mice (SI Appendix, Fig. S3A). Short-term cold exposure increased the mRNA levels of thermogenic genes in BAT in both groups without differences (SI Appendix, Fig. S3A). Consistently, the expressions of thermogenic protein and core temperature during short-term cold exposure were also comparable between the two groups (SI Appendix, Fig. S3 B and C). These results indicate that fat-specific Np55 knockout does not impair the thermogenesis of BAT.

WAT browning has been demonstrated to contribute to thermogenesis (19). We next investigated whether fat-specific knockout of Np55 affects iWAT browning. HFD-fed Np55^FKO mice exhibited a lower rectal temperature starting from the 12th h of exposure to 4 °C, with a significant decline compared to control mice (Fig. 3E). Under long-term cold exposure, the mRNA levels of thermogenic genes in iWAT of Np55^FKO mice were significantly decreased compared to controls (Fig. 3F). Correspondingly, UCP1 and PGC-1α protein levels in iWAT of Np55^FKO mice were markedly reduced compared to the controls (Fig. 3G). Immunohistochemical analysis further confirmed lower expression of UCP1 in iWAT of Np55^FKO mice (Fig. 3H). However, thermogenic gene expressions in BAT were similar between the two groups (SI Appendix, Fig. S3D). Same results were found in the iWAT of CD-fed Np55^FKO mice showing impaired thermogenic capacity compared to the controls under cold exposure (SI Appendix, Fig. S3 E–H). Thus, fat-specific knockout of Np55 leads to reduced energy expenditure through decreased browning of iWAT, thereby impairing the ability to maintain body temperature during prolonged cold exposure.

Long-term cold exposure boosts lipolysis in adipose tissue, serving as a major energy source in response to cold stimuli (20). At RT on CD, Np55^FKO mice showed slightly lower phosphorylation levels of key lipolytic proteins such as HSL, ATGL, and Plin1 in both iWAT (SI Appendix, Fig. S4A) and eWAT (SI Appendix, Fig. S4B). Under cold stimuli, Np55^FKO mice showed further reduced phosphorylation levels of HSL, ATGL, and Plin1 in both iWAT and eWAT compared to controls (SI Appendix, Fig. S4 A and B). Consistently, the glycerol release, an indicator of lipolysis, from both iWAT and eWAT was significantly decreased in Np55^FKO mice (SI Appendix, Fig. S4C) in basal and stimulated conditions. On HFD, Np55^FKO mice also showed impaired lipolytic responses (SI Appendix, Fig. S4D). These results suggest that the decreased lipolysis in Np55 knockout mice may be coordinated with the reduced thermogenesis. Notably, tyrosine hydroxylase (TH) protein levels and norepinephrine content in iWAT were comparable in Np55^FKO and Np55^fl/fl mice (SI Appendix, Fig. S5 A–C). Furthermore, β3-adrenergic receptor agonist CL316,243 treatment upregulated thermogenic gene expression in both groups. However, it cannot completely rescue the decreased thermogenesis in Np55^FKO mice (SI Appendix, Fig. S5D), indicating the impaired browning and lipolysis in fat-specific Np55 knockout mice are independent of sympathetic activity.

Np55 Acts as the Membrane Receptor for Manf to Mediate iWAT Browning via the p38 MAPK Pathway.

Our results so far support that the absence of Np55 in adipose tissue led to impaired thermogenesis. As Np55 is a transmembrane receptor, we wondered whether the impaired thermogenesis was due to Np55 knockout hindering the function of its ligand. NPTN has two isoforms: Np65 and Np55. Np65, mainly expressed in the central nervous system, is linked to the function of Manf (21). But whether Np55 is also the receptor of Manf remains undefined. Therefore, we investigated their possible relationship by detecting Np55 expression in models affecting Manf levels. We found that cold exposure induced the mRNA and protein expression of Np55 (Fig. 4A) and Manf (SI Appendix, Fig. S6 A and B). Feeding is reported to induce the liver and serum levels of Manf (SI Appendix, Fig. S6C) (8). Interestingly, the mRNA expression of Np55 in iWAT was also higher after refeeding (SI Appendix, Fig. S6D). These results suggest that the change of Np55 may coordinate with Manf under physiological conditions.

Fig. 4.

Np55 is the receptor of Manf and mediates p38 MAPK signaling pathway. (A) The mRNA (Upper) and protein expression (Lower) of Np55 in iWAT after long-term cold exposure. (B) Pull-down assay showing the interaction between Np55 and Manf. (C) Coimmunofluorescence staining of Manf (red) and Np55 (green) in primary subcutaneous adipocytes. (D) Quantification of the binding affinity between Np55 and Manf or BDNF by MST. (E) Binding curve for the binding affinity of Np55 to Manf. (F) The phosphorylated and total p38 and ATF2 in primary subcutaneous adipocytes of Np55^fl/fl and Np55^FKO mice after incubating with Manf for 30 min. (G) The expression of phosphorylated and total p38 and ATF2 in iWAT from Np55^fl/fl and Np55^FKO mice fed an HFD-diet after long-term cold exposure. (H) Schematic showing the strategy for generation of unilateral knockdown of Np55 in iWAT. (I) The phosphorylated and total p38 and ATF2 in iWAT of unilateral Np55 knockdown mice after long-term cold exposure. VH, vehicle. n = 6, *P < 0.05, **P < 0.01.

To directly test for a physical interaction, we performed immunoprecipitation-mass spectrometry (IP-MS) on primary subcutaneous adipocytes treated with recombinant Manf. The proteomic result identified Np55 as the most significantly enriched binding protein upon Manf stimulation (SI Appendix, Fig. S6E). Further pull-down assays confirmed a direct and reciprocal binding between Manf and the extracellular domain of Np55 (Fig. 4B). Fluorescence imaging analysis demonstrated a strong colocalization of Manf with Np55 in primary subcutaneous adipocytes (Fig. 4C). Microscale thermophoresis (MST) analysis revealed a nanomolar affinity between Manf and the Np55 extracellular domain (K_d = 34.61 ± 7.19 nM, Fig. 4D), whereas brain-derived neurotrophic factor (BDNF), another classic neurotrophic protein, exhibited a hundred-times lower affinity with Np55 (K_d = 1.74 ± 0.41 μM, Fig. 4D), indicating a specific binding of Np55 with Manf (Fig. 4D). Further binding assay corroborated that the extracellular segment of Np55 binds to Manf with similar affinity as the MST results (K_d = 14.53 ± 1.17 nM, Fig. 4E).

Previously, we showed that Manf promotes adipocyte browning by activating the p38 MAPK pathway (8). To functionally verify whether Np55 mediates the browning effects of Manf via the p38 MAPK signaling pathway, we isolated SVF from iWAT of both Np55^FKO and control mice. These SVFs were differentiated into mature adipocytes and subsequently treated with recombinant Manf protein for 30 min. In control adipocytes, Manf stimulation led to a significant upregulation of p38 phosphorylation and its downstream signaling factor ATF2 (Fig. 4F and SI Appendix, Fig. S6F). Conversely, the capacity of Manf to activate the p38 MAPK pathway was abolished in adipocytes derived from Np55^FKO mice (Fig. 4F and SI Appendix, Fig. S6F). These results indicated Np55 is capable of binding to Manf and mediating its metabolic function in vitro.

To confirm the function of Np55 in vivo, we initially assessed the p38 signaling pathway in the iWAT of Np55^fl/fl and Np55^FKO mice. Our analysis revealed a significant reduction in the phosphorylation levels of p38 and ATF2 in the iWAT of Np55^FKO mice (Fig. 4G). Next, in Manf transgenic mice (Manf^Tg) (8), we achieved unilateral Np55 knockdown in iWAT by delivering AAV-shNPTN with ADIPOQ promoter to one side, while the contralateral side received AAV-GFP as a control (Fig. 4H). After 4 wk on an HFD in Manf^Tg mice, a significant reduction in Np55 expression was observed at the AAV-shNPTN site in iWAT, confirming successful knockdown of Np55 (SI Appendix, Fig. S7 A and B). Long-term cold exposure resulted in increased phosphorylation of p38 and ATF2 at the GFP site in Manf^Tg mice. Conversely, phosphorylation of p38 and ATF2 at the Np55 knockdown site remained largely unchanged under cold exposure (Fig. 4I and SI Appendix, Fig. S7C). These findings suggest that Np55, when binding with Manf, activates the p38 MAPK pathway, thereby facilitating thermogenesis in iWAT.

Manf–Np55 Binding Transduces ASK1-p38 MAPK Pathway by Recruiting TRAF2.

A key question that remains unresolved is how Np55 transduces intracellular signals to activate the p38 MAPK signaling pathway following its binding with Manf. ASK1, a critical MAP3K in the p38 MAPK signaling cascade, has been shown to play a pivotal role in adipose tissue browning (Fig. 5A). As expected, we observed a substantial decrease in the phosphorylation levels of ASK1 and MEK3 in Np55^FKO mice under long-term cold exposure (Fig. 5 B and C). To further explore whether ASK1 activation occurs post-Manf stimulation, we isolated primary adipocytes from subcutaneous adipose tissues of Np55^FKO and Np55^fl/fl mice. We found that the phosphorylation of ASK1 and MEK3 was significantly increased in primary subcutaneous adipocytes from Np55^fl/fl mice, but not from Np55^FKO mice, following 30 min of Manf treatment (Fig. 5 D and E). These cellular findings were supported by in vivo experiments, where long-term cold exposure resulted in elevated phosphorylation of ASK1 and MEK3 in iWAT from Manf^Tg mice, but not in the shNPTN site of iWAT (Fig. 5 F and G).

Fig. 5.

Manf binding to Np55 transduces ASK1-p38 MAPK pathway by recruiting TRAF2. (A) Schematic showing the signaling cascade of MAPKs. (B) The phosphorylated and total ASK1 and MEK3 in iWAT from Np55^fl/fl and Np55^FKO mice after long-term cold exposure. (C) The semiquantitative analysis of phosphorylated ASK1 and MEK3. (D and E) The expression of phosphorylated and total ASK1 and MEK3 in primary subcutaneous adipocytes from Np55^fl/fl and Np55^FKO mice after incubating with Manf for 30 min. (F and G) The expression of phosphorylated and total ASK1 and MEK3 in iWAT of unilateral Np55 knockdown mice after long-term cold exposure. (H) Co-IP showing the association of TRAF2 and Np55 in HEK293T cells with or without Manf treatment (2.5 nM) for 15 min. (I) Co-IP showing the association of TRAF2, ASK1, and Np55 in HEK293T cells treated with or without Manf (2.5 nM) for 15 min. (J and K) The expression of phosphorylated and total p38 and AFT2 in 3T3L1 cells (J) and primary subcutaneous adipocytes (K) after being transfected with TRAF2 siRNA and treated with Manf for 30 min. Data are mean ± SEM. n = 6, *P < 0.05, **P < 0.01, ***P < 0.001.

The above results suggest that ASK1 mediates the activation of p38 MAPK signaling after Manf–Np55 binding. Further results confirmed that Manf-mediated thermogenesis was ASK1-dependent, as GS-4997, an ASK1 inhibitor, abolished Manf-activated signaling pathway and induction of UCP1 expression (SI Appendix, Fig. S8 A–D). Interestingly, cAMP levels were comparable between Np55^FKO mice and controls (SI Appendix, Fig. S8E), suggesting Np55 is unlikely to activate thermogenesis through the cAMP-p38 MAPK pathway.

The intracellular domain of Np65 has been reported not to directly interact with p38α to activate the MAPK cascade (22). Instead, Np65 engages with tumor necrosis factor receptor-associated factor 2 (TRAF2) to initiate the intracellular MAPK signaling pathway (22). Since Np55 shares the same intracellular domain structure as Np65 (SI Appendix, Fig. S8F), we hypothesize that Np55 might also bind to TRAF2 to transduce downstream signaling following Manf stimulation. To validate this hypothesis, we coexpressed Np55 and TRAF2 in HEK293 cells, followed by Manf stimulation. Immunoprecipitation assays showed that Np55 binds directly to TRAF2, and this interaction was significantly enhanced by Manf (Fig. 5H). To further explore the role of TRAF2 in the Manf–Np55-mediated activation of the ASK1-p38 MAPK pathway, we coexpressed Np55, TRAF2, and ASK1 in HEK293 cells and subjected them to Manf stimulation. The results indicated that Np55 does not directly bind to ASK1 (Fig. 5I). Instead, Np55 interacts with ASK1 through binding with TRAF2 (Fig. 5I). Moreover, Manf stimulation significantly increased the binding among Np55, TRAF2, and ASK1 (Fig. 5I). These findings suggest that Np55 forms a complex with TRAF2 and ASK1, and this interaction is enhanced by Manf stimulation.

To further confirm the role of TRAF2 in Manf-mediated activation of p38 MAPK pathway, both 3T3-L1 adipocytes and primary subcutaneous adipocytes were transfected with siRNA to knock down TRAF2 level according to the protocol (23) (Fig. 5 J and K). The results showed that TRAF2 knockdown significantly inhibited the phosphorylation of p38 and ATF2 upon Manf stimulation (Fig. 5 J and K). These findings underscore the critical role of TRAF2 in Manf-mediated activation of p38 MAPK signaling, namely Manf–Np55 binding transduces the ASK1-p38 MAPK cascade by recruiting TRAF2.

Mutation in Manf Abrogates Its Binding with Np55.

Splice-site variants of Manf, resulting in altered protein-coding, have been implicated in severe metabolic disorders (24, 25). However, the function of single-nucleotide polymorphisms (SNPs) in Manf remains unclear. By examining residues with the highest or lowest electrostatic binding energy in Manf (Fig. 6A), we identified five potential binding sites with Np55. Each of these sites contains SNPs (SI Appendix, Table S2), yet none have been demonstrated to possess clinical significance. We conducted site-directed mutagenesis, substituting alanine for the amino acids at these five sites, and purified those proteins. MST and pull-down assays revealed that only mutation 2 (the mutation at site 2), corresponding to SNP rs2470738600, exhibited a significantly reduced binding affinity with Np55 (Fig. 6 B and C and SI Appendix, Fig. S9A). Further investigation showed this specific SNP mutation in Manf impaired p38 MAPK signaling pathway and thermogenic capacity in adipocytes (Fig. 6 D and E and SI Appendix, Fig. S9B).

Fig. 6.

Mutation in Manf abrogates its binding with Np55. (A) Surface representation of Manf monomer residues, colored according to their electrostatic contribution to the binding energy (highest contributors in blue). Interface residues are highlighted. (B) Pull-down assay displaying the interaction of Np55 with different mutations of Manf. (C) Quantification of the binding affinity between Np55 and Manf or Mut2-Manf by MST. (D) The mRNA expression of thermogenic genes in primary subcutaneous adipocytes treated with Manf or Mut2-Manf. (E) The expression of phosphorylated and total p38 and ATF2 in primary subcutaneous adipocytes treated with Manf or different mutations of Manf. VH, vehicle. Data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Np55 Is Indispensable for Manf-Induced Thermogenesis and Weight Loss in Obese Mice.

Considering Np55 is a receptor for Manf in adipose tissue, we examined whether Np55 is indispensable for Manf-induced iWAT browning in primary subcutaneous adipocytes from Np55^FKO and control mice. Our results indicated that Np55 knockdown did not affect adipocyte differentiation because the mRNA expressions of adipocyte differentiation markers were not changed (SI Appendix, Fig. S10A). The mRNA expressions of thermogenic genes such as Ucp1, Cidea, Dio2, and Pgc1α were significantly increased after Manf stimulation (Fig. 7A). In contrast, Manf-induced thermogenic genes were abolished in Np55 knockout adipocytes (Fig. 7A). Western blot analysis further validated that Np55 deficiency compromised the protein levels of UCP1 and PGC-1α induced by Manf in primary subcutaneous adipocytes (Fig. 7B). Concordantly, seahorse analysis showed that a marked increase in oxygen consumption rate (OCR) by Manf treatment was completely blunted in Np55 knockout adipocytes (SI Appendix, Fig. S10 B and C). These in vitro findings indicate Np55 is essential for Manf-induced browning in iWAT.

Fig. 7.

Np55 is indispensable for Manf-induced thermogenesis and weight loss. (A) qRT-PCR analysis of the mRNA expression of thermogenic genes in primary subcutaneous adipocytes from Np55^fl/fl and Np55^FKO mice treated with recombinant Manf for 48 h. (B) Western blot analysis of the thermogenic proteins in primary subcutaneous adipocytes from Np55^fl/fl and Np55^FKO mice treated with recombinant Manf for 48 h. (C) Schematic representation of Manf-Fc or Fc treatment in HFD-induced Np55^fl/fl or Np55^FKO mice. (D) Change curve of body weight in Np55^fl/fl and Np55^FKO mice upon Manf-Fc/Fc administration. (E) The weight-to-body weight ratio of iWAT from Np55^fl/fl and Np55^FKO mice upon Manf-Fc/Fc administration. (F) qRT-PCR analysis of thermogenic genes in iWAT of Np55^fl/fl and Np55^FKO mice upon Manf-Fc/Fc administration. (G) Western blot of thermogenic proteins in iWAT of Np55^fl/fl and Np55^FKO mice upon Manf-Fc/Fc administration. VH, vehicle. Data are mean ± SEM, n = 5-6. *P < 0.05, **P < 0.01, ***P < 0.001.

Given that Manf has been reported as a hepatokine on weight loss (8), we further explored the necessity of Np55 in Manf-mediated weight reduction. We constructed a long-acting Manf protein (Manf-Fc) by fusing it with the IgG1 Fc fragment (8). Both Np55^FKO mice and control mice were subjected to a 12-week HFD and then treated with either Manf-Fc or Fc as illustrated in Fig. 7C. In control mice, Manf-Fc treatment significantly reduced weight gain and iWAT/body weight ratio (Fig. 7 D and E). However, these effects were not observed in Np55^FKO mice. Administration of Manf-Fc significantly upregulated the mRNA and protein levels of UCP1 and other thermogenic genes (Fig. 7 F and G and SI Appendix, Fig. S11A). This upregulation was substantially hindered in Np55^FKO mice (Fig. 7 F and G and SI Appendix, Fig. S11A). Metabolic cage data indicated Manf-Fc treatment significantly increased energy expenditure, oxygen consumption, and carbon dioxide production in Np55^fl/fl mice compared to the Fc-treated group (SI Appendix, Fig. S11 B–D). Notably, this increase was substantially abolished in Np55^FKO mice (SI Appendix, Fig. S11 B–D). Manf treatment did not affect respiratory exchange ratio, locomotor activity, and food intake in both Np55^FKO and Np55^fl/fl mice (SI Appendix, Fig. S11 E–G). These results suggested the beneficial effect of Manf-Fc on obesity and thermogenesis was abrogated in Np55 knockout mice.

In the unilateral Np55 knockdown model, Manf^Tg mice showed higher skin surface temperature at the GFP site in the inguinal area compared to WT mice, consistent with the role of Manf in promoting thermogenesis (SI Appendix, Fig. S11H). In contrast, the skin surface temperature at the AAV-shNPTN site was significantly reduced compared to the GFP site in Manf^Tg mice (SI Appendix, Fig. S11I). Additionally, the mRNA and protein levels of Ucp1 and Pgc1α were significantly increased at the GFP site in Manf^Tg mice compared to WT mice. This increase was largely abolished in the AAV-shNPTN site of Manf^Tg mice (SI Appendix, Fig. S11 J and K). Taken together, these findings demonstrate that Np55 is an essential receptor for Manf-induced thermogenesis and weight loss.

Discussion

Manf is induced by feeding in the liver, and functions as a thermogenic hepatokine to enhance energy expenditure in iWAT (8). While previous research has demonstrated that Manf exerts its effects through the p38 MAPK pathway, the specific receptor remained unsolved (8). In this study, we identify the neuroplastin isoform Np55 as the functional receptor for Manf in adipose tissue, mediating thermogenic regulation. This study establishes Np55 as a membrane receptor for Manf in adipose tissue, thereby expanding our understanding of interorgan metabolic crosstalk. Furthermore, we elucidate the regulatory mechanism of the TRAF2-ASK1-p38 MAPK signaling cascade in Manf–Np55-mediated thermogenesis. Our study paves the way for innovative strategies to address obesity and its related complications.

Although the physiological roles of NPTN have been extensively studied in the nervous system (26–28), its metabolic functions have only recently begun to be explored. Yagi et al. first reported that Manf-NPTN binding suppresses NF-κB signaling and mitigates inflammation (14). However, Kitamura et al. demonstrated that Manf-NPTN interaction activates NF-κB in pancreatic β-cells (15). These contradictory studies highlight the distinct roles of NPTN in different tissues. Our findings align with prior studies that Np65 is predominantly expressed in the central nervous system, whereas Np55 is widely distributed and highly expressed in adipose tissue (SI Appendix, Fig. S1 A and B). Using fat-specific Np55 knockout (Np55^FKO) mice, we demonstrate that adipose Np55 is indispensable for Manf-mediated thermogenesis. Specifically, Np55^FKO mice exhibit impaired subcutaneous adipocyte browning, reduced energy expenditure, and exacerbated obesity under HFD conditions. Np55 functions as the membrane receptor for Manf to mediate thermogenic function. Knockout of Np55 greatly abolished the metabolic function of Manf. In contrast, Np55^fl/fl mice retain Manf-induced thermogenic responses.

Structurally, Np55 is a truncated isoform of NPTN, lacking one immunoglobulin domain compared to Np65. Np65 binds Manf with moderate affinity (K_d ≈ 104 nM) (14). Our pull-down assays, IF, saturation binding, and MST experiments consistently demonstrated that Np55 binds Manf with significantly higher affinity, with K_d values in the single to double-digit nanomolar range (Fig. 4 D and E and SI Appendix, Fig. S8A). This establishes Np55 as the high-affinity receptor for Manf in adipocytes. Interestingly, despite their structural similarity, Np55 and Np65 display distinct ligand-binding profiles. Protein S100A9 binds to both isoforms, whereas S100A8 exclusively interacts with Np65 (22). This selectivity implies that the absence of a single Ig domain in Np55 confers unique ligand specificity. Although the precise binding interface between Manf and Np55 has yet to be fully elucidated, we have explored the potential binding site. Our findings indicate that the amino acid K118 of Manf is critical for the Manf–Np55 interaction. Furthermore, we found that the SNP of Manf (rs2470738600) not only compromised its interaction with Np55 but also affected its thermogenic capacity in adipocytes.

Thermogenesis in adipocytes is governed by a complex interplay of hormonal and neural signals (29, 30). Although Manf has been shown to enhance sympathetic outflow via hypothalamic POMC neurons (31), our findings establish a direct adipocyte-autonomous mechanism mediated by Np55. We elucidate that upon Manf binding, Np55 recruits the adaptor protein TRAF2, which facilitates the oligomerization and activation of ASK1. ASK1, in turn, phosphorylates MEK3/6, leading to the activation of p38 MAPK, a central regulator of thermogenic gene program. Notably, this pathway operates independently of the canonical cAMP/PKA signaling axis, which aligns with our prior reports on Manf (8).

The metabolic significance of Np55 is further highlighted by its structural homology to CD147, which is upregulated in visceral fat of obese individuals (32). CD147 has been identified as an important receptor regulating fatty acid and glucose metabolism (33, 34). We found that under physiological conditions such as feeding, the expression of Np55 was significantly elevated, paralleled by a marked increase in hepatic Manf secretion. This suggests that during feeding, the liver releases substantial amounts of Manf into the systemic circulation, which then acts on iWAT by binding to its receptor Np55. The binding of Manf to Np55 activates the downstream ASK1-MEK3-p38 MAPK signaling cascade and promotes thermogenesis in subcutaneous fat. Through the activation of this liver-adipose endocrine axis, the upregulation of Np55 in iWAT coordinately contributes to enhanced whole-body energy expenditure during food intake. However, under pathological conditions like obesity, we observed an intriguing phenomenon: Np55 expression was upregulated in the subcutaneous adipose tissue of both animal models and obese patients, whereas circulating levels of Manf were significantly reduced. Given the well-documented persistence of thermogenic impairment in obesity (35), this increase of Np55 may represent a compensatory adaptation aimed at preserving metabolic homeostasis. The absence of its essential ligand, Manf, likely undermines the capacity of Np55 to activate thermogenic signaling. Therefore, therapeutic strategies aimed at restoring functional activation of the Manf-Np55 axis, such as recombinant Manf and exploration of Np55 synthetic agonists, are of significant clinical importance for the treatment of obesity and related metabolic disorders.

In conclusion, our study identifies Np55 as the principal receptor for Manf in adipocytes, orchestrating a TRAF2/ASK1/p38 MAPK-dependent pathway that promotes adipose tissue browning and systemic energy expenditure. Given the decline in Manf levels during obesity, therapeutic strategies targeting the Manf-Np55 axis, such as Np55 agonists or Manf mimetics, may hold promise for combating metabolic disorders.

Materials and Methods

Animals.

All mice were maintained in a controlled environment with a 12-h light/dark cycle, constant temperature (25 °C), and free access to food and water. The Np55-floxed (Np55^fl/fl) strain was generously provided by Prof. Yuan Qionglan (Tongji University School of Medicine) (36). To generate fat-specific Np55 knockout (Np55^FKO) mice, Np55^fl/fl mice were crossed with adiponectin-Cre mice (C57BL/6J background, Jackson Laboratory). Manf transgenic (Manf^Tg) mice were engineered on a C57BL/6J background using an albumin promoter (8), with wild-type (WT) littermates used as controls. For diet-induced obesity, male mice (Manf^Tg, Np55^FKO, and controls) were fed a HFD (D12492; Research Diet) for 12 wk. For long-term cold exposure, mice were acclimated at 4 °C for 72 h with ad libitum food and water. For short-term cold exposure, mice were fasted but given water at 4 °C for 6 h. Core body temperature was monitored using a rectal probe thermometer. To avoid estrous cycle-related hormonal fluctuations in females influencing systemic glucose and lipid metabolism, our study mainly focused on male mice (37). Moreover, female mice exhibit a higher resistance to diet-induced obesity and related metabolic disorders (38). Animal experiments were conducted in accordance with institutional guidelines under protocol No. 20220823066, as approved by the Animal Care and Use Committee of Sichuan University.

IP-MS.

To identify potential adipocyte membrane receptors for Manf, we performed immunoprecipitation followed by mass spectrometry using primary subcutaneous adipocytes. Membrane fractions were isolated and incubated with recombinant Manf overnight, followed by immunoprecipitation with a high-affinity anti-Manf monoclonal antibody. The resulting immunoprecipitates were analyzed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) on an Orbitrap Astral mass spectrometer coupled to a Vanquish Neo UHPLC system (Thermo Fisher Scientific). Peptides were separated on a 50 cm Low-Load µPAC™ Neo HPLC Column (Thermo Scientific), and the acquired MS data were searched against the UniProtKB/ UniProt database for protein identification.

Manf–Np55 Binding Assay.

To assess direct binding between Manf and Np55. Recombinant Np55 protein (SinoBiological Inc.) was immobilized on a 96-well ELISA plate overnight at 4 °C. Plates were washed with PBS-T (0.1% Tween-20) and blocked with casein (Sigma) for 1 h at RT. Various concentrations of recombinant Manf protein were added and incubated at 37 °C for 3 h. After washing with 20 mM HEPES buffer (pH 7.4), bound Manf was detected using horseradish peroxidase (HRP)-conjugated anti-Manf antibody (1:1,000 dilution). Signal was developed using Ultra TMB-ELISA Substrate Solution (Abcam) and measured at 450 nm with background subtraction at 620 nm.

MST Assay.

Binding affinity (KD) was quantified using an MST assay with the Monolith NT.115 instrument (NanoTemper Technologies). Recombinant Np55 (SinoBiological) was fluorescently labeled using the MonolithTM RED-NHS Labeling Kit (NanoTemper) following the manufacturer’s instructions. Unlabeled Manf or BDNF (negative control) was serially diluted and mixed with labeled Np55. After a 30-min incubation at RT, the reaction mixtures were then loaded into standard glass capillaries (Monolith NT.115). MST measurements were performed at 40% power and MST power settings, as recommended by the manufacturer. KD values were calculated using NanoTemper Analysis Software with triplicate experiments.

Biochemical Analysis.

Blood samples were centrifuged at 6,000 rpm for 10 min at 4 °C to obtain serum. For hepatic lipid analysis, 50 mg liver tissue was homogenized in 0.33 mL PBS, followed by lipid extraction using a 2:1 (v/v) chloroform/methanol mixture. The extracted lipids were resuspended in 1% Triton X-100/ethanol solution. Serum and hepatic TG and total cholesterol (TC) were determined using commercial kits (Zhongsheng Technologies). Nonesterified fatty acids (NEFAs) were measured by Wako. Hepatic lipid levels were normalized to liver weight. Serum levels of insulin were quantified via ELISA (Protech). Glucose concentrations were determined using Zhongsheng Technologies kits. cAMP levels were quantified using an ELISA kit (cat. CEA003Ge, Uscn).

Histological and Immunohistochemical Analysis.

Tissue samples were processed using standard histological methods: 1) Fixation in 10% neutral buffered formalin; 2) Dehydration through graded ethanol series; 3) Clearing in xylene; 4) Paraffin embedding. For H&E staining, 5 μm sections were cut, deparaffinized, and rehydrated. Slices were stained with hematoxylin and eosin. For immunohistochemistry (IHC) and IF, deparaffinized and rehydrated sections underwent antigen retrieval by using heat-induced epitope methods and incubated with diluted antibody overnight, then incubated with HRP-conjugated antibody for 1 h. Oil Red O was performed on staining 8 μm frozen liver sections. Images were captured with an optical microscope (Nikon). Adipocyte size was quantified using ImageJ software.

Isolation and Differentiation of Primary Subcutaneous Adipocytes.

Primary subcutaneous SVFs were isolated and differentiated as the following protocol. Minced subcutaneous fat pads were digested with type II collagenase (37 °C, 7 min) with vigorous shaking. Digestion was halted with medium. Cell suspension was centrifuged (700 g, 10 min, 4 °C). The pellet was resuspended with medium and filtered through 100-μm mesh. All resuspensions were plated on collagen-coated 24-well plates in F12 medium supplemented with 10% FBS. After contact inhibition, SVFs were induced in a medium containing 10% FBS, 5 μg/mL insulin, 0.5 mM isobutylmethylxanthine, 1 μM dexamethasone, 1 nM triiodothyronine (T3), and 10 μM rosiglitazone. After 2 d, cells were cultured with maintenance medium containing 10% FBS, 1 nM T3, and 5 μg/mL insulin until harvesting. The medium was refreshed every 48 h. Mature primary subcutaneous adipocytes were transfected with 100 nM siRNA against TRAF2 and a nonspecific control according to Kilroy et al. (23). At 24 h posttransfection, cells were treated with 2.5 nM recombinant Manf for 30 min.

Cell Culture and Coimmunoprecipitation.

HEK293 cells were cultivated in DMEM (Gibco) supplemented with 10% FBS (Hyclone) at 37 °C with 5% CO₂. HEK293 cells were plated in 6-well plates and transiently cotransfected with human Np55 (Δcyto) vector, TRAF vector, and ASK1 vector (GENEWIZ). After 48 h of transfection, cells were lysed with NP-40 buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 0.25% sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and phosphatase/protease inhibitor cocktail (Sigma). Then 500 μg of total protein lysate was immunoprecipitated with anti-tag antibody and Protein A/G Agarose (B23202, Selleck) at 4 °C overnight. The beads containing the immunoprecipitated complex were washed three times with lysis buffer followed by denaturation in lysis buffer at 97 °C for 10 min. Precipitates were analyzed by SDS-PAGE.

AAV-Mediated Unilateral Np55 Knockdown.

AAV-shNp55 and AAV-GFP (control) were designed and produced by Shanghai Genechem Co. Ltd (titers: 5 × 10¹² vg/mL). The WT or Manf^Tg mice were anesthetized with isoflurane. An incision was made of the skin along the spine. Both lateral iWAT fat pads were exposed using blunt dissection. Each mouse received multiple site injections of 10 μL of either AAV-GFP or AAV-shNp55. Postinjection, the mice were maintained at a controlled temperature of 25 °C for 4 wk before cold exposure experiments.

Western Blotting.

Tissues or cells were homogenized in an ice-cold lysis buffer containing protease inhibitor cocktail (Selleck, B14001) at 4 °C. Lysates were centrifuged at 12,000 rpm for 10 min. Equal amounts (5 or 10 μg) of protein were loaded on SDS-PAGE gels and transferred to nitrocellulose membranes. The membranes were blocked with Casein and probed with antibodies. Antibody–antigen complexes were then detected using the LI-COR Odyssey Imaging System. All antibody details are provided in SI Appendix, Table S3.

Quantitative Real-Time PCR.

Total RNA was isolated from cells or frozen tissues by using Trizol reagent (Invitrogen). 1 μg RNA was reverse transcribed using a commercial RT kit (Takara). SYBR Green-based RT-PCR was carried out on a CFX96 Real-Time PCR Detection System (Bio-Rad). The primer sequences used for target gene amplifications are listed in SI Appendix, Table S4.

Human Adipose Tissue Samples.

Subcutaneous adipose tissue from obese patients was collected while they were undergoing bariatric surgery. Fat tissue from healthy controls was obtained from patients undergoing plastic surgery. All adipose tissue is originally considered medical waste. All human subject research protocols (Approval No. 201753, v2.0) were reviewed and approved by the Biological Sciences Ethical Committee of West China Hospital, Sichuan University. Study participants provided written informed consent prior to enrollment.

Statistical Analysis.

Data considered normalized distributions are expressed as mean ±SEM. If not normally distributed, data are presented as medians (interquartile range). Statistical comparisons were performed using Student’s t test for two-group comparisons; one-way ANOVA with post hoc testing for multiple groups; an ANCOVA for analyzing energy expenditure-body weight correlations; Pearson correlation analysis for assessing Np55–BMI relationships; 𝒳² tests for analyzing the differences in the distribution of males and females. A significance threshold of P < 0.05 was applied for all statistical tests.

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Dataset S02 (XLSX)

Dataset S03 (XLSX)

Dataset S04 (XLSX)

Dataset S05 (XLSX)

Dataset S06 (XLSX)

Dataset S07 (XLSX)

Data, Materials, and Software Availability

All study data are included in the article and/or supporting information. For the proteomics raw data, we have uploaded it to iProX database (https://www.iprox.cn/page/home.html) under accession number IPX0014183000 for public access (39).