DUSP4 activates adipocyte thermogenesis via Crtc3 dephosphorylation-dependent UCP1 expression

DUSP4 activates adipocyte thermogenesis via Crtc3 dephosphorylation-dependent UCP1 expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article DUSP4 activates adipocyte thermogenesis via Crtc3 dephosphorylation-dependent UCP1 expression Kwang-Hee Bae, Min Jeong Son, Hae Un Kook, Jaeeun Jung, Se-Jun Park, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8324434/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Obesity arises when chronic energy surplus overwhelms the storage capacity of adipocytes, resulting in systemic metabolic dysfunction. Adaptive thermogenesis, primarily mediated by uncoupling protein 1 (Ucp1) in adipocytes, serves to counteract obesity by dissipating excess energy as heat. Despite extensive studies on thermogenic regulation, the phosphatases that regulate this protective metabolic pathway remain poorly understood. Here, we identify dual-specificity phosphatase 4 (Dusp4) as a critical modulator of adipocyte thermogenesis via direct modulation of the CREB-regulated transcription coactivator 3 (Crtc3). Dusp4 -deficient mice exhibit impaired thermogenic capacity, diminished Ucp1 expression, and increased susceptibility to diet-induced obesity accompanied by severe insulin resistance. Mechanistically, Dusp4 directly dephosphorylates serine residues on Crtc3, facilitating its nuclear translocation and subsequent transcriptional activation of Ucp1. Restoration of catalytically active Dusp4 in adipocytes rescues thermogenic gene expression, reduces adiposity, and normalizes systemic glucose homeostasis in Dusp4 -knockout mice. Collectively, these findings identify Dusp4 as a key upstream phosphatase orchestrating the Crtc3-Ucp1 thermogenic axis, and suggests its potential as a therapeutic target for obesity-associated metabolic disorders. Biological sciences/Physiology/Metabolism/Metabolic diseases/Obesity Biological sciences/Physiology/Metabolism/Fat metabolism Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 INTRODUCTION Obesity has emerged as a global epidemic that is strongly associated with metabolic disorders such as type 2 diabetes, cardiovascular disease, immune dysfunction, and certain cancers. Chronic nutrient intake excess drives adipocyte hypertrophy and hyperplasia 1 – 3 , ultimately overwhelming the storage capacity of adipose tissue and disrupting systemic metabolic homeostasis 4 , 5 . In contrast, adaptive thermogenesis within adipose tissue acts as a compensatory mechanism to counteract obesity by converting surplus calories into heat 5 – 7 . Thermogenic activation, triggered by stimuli such as cold exposure or β-adrenergic agonists (e.g., CL316,243), critically depends on mitochondrial uncoupling protein 1 (Ucp1) 8 – 11 . Ucp1 uncouples oxidative phosphorylation, dissipating the proton gradient across the mitochondrial inner membrane and generating heat instead of ATP synthesis, thereby increasing energy expenditure 12 – 15 . Thus, elucidating molecular mechanisms that regulate Ucp1 expression represents a promising therapeutic strategy for combating obesity and its associated metabolic dysfunctions. Cellular signaling cascades orchestrate dynamic response to environmental and metabolic stimuli 16 – 18 , governing critical cellular functions through tightly controlled phosphorylation-dephosphorylation cycles 19 – 22 . Among these signaling regulators, dual-specificity phosphatase 4 (Dusp4) has emerged as a versatile regulator implicated in various biological processes, including cell proliferation, immune responses, cancer progression, and metabolism 23 – 27 . Dusp4 primarily exerts its function by dephosphorylating key substrates such as mitogen-activated protein kinases (MAPKs), ERK, p38, p300, and Smad4, thereby fine-tuning multiple signaling pathways and cellular outcomes 28 – 32 . However, despite extensive studies in other biological contexts, the involvement and role of Dusp4 in adipocyte metabolism 33 , 34 , particularly in thermogenic regulation, remain largely unexplored. The CREB-regulated transcription coactivator 3 (Crtc3) is highly expressed in adipocytes and regulates energy metabolism through phosphorylation-dependent nuclear translocation 35 , 36 . However, the specific phosphatases regulating the phosphorylation state of Crtc3 have yet to be clearly identified. Given its adipose-enriched expression and enzymatic specificity, we hypothesized that Dusp4 might regulate adipocyte thermogenesis by modulating Crtc3 activity, thereby promoting the transcriptional activation of Ucp1. Here, we identify Dusp4 as a critical regulator of adipocyte thermogenesis through direct dephosphorylation of Crtc3. Using genetically engineered mouse models, we demonstrate that the loss of Dusp4 impairs thermogenic gene expression, accelerates diet-induced obesity, and leads to systemic insulin resistance. Mechanistically, Dusp4 directly interacts with and dephosphorylates Crtc3, promoting its nuclear translocation and recruitment to the Ucp1 promoter. Importantly, adipose-specific restoration of Dusp4 reverses these metabolic defects, reinstating thermogenic capacity and restoring systemic metabolic health. These findings establish the Dusp4–Crtc3–Ucp1 signaling axis as a novel regulatory pathway essential for adaptive thermogenesis and highlight Dusp4 as a viable therapeutic target to combat obesity and its metabolic complications. MATERIALS AND METHODS Cell culture and differentiation Immortalized inguinal preadipocytes were kindly provided by Dr. Shingo Kajimura (UCSF, CA, USA). The inguinal preadipocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM), which contained 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C in a 5% CO 2 incubator. For differentiation, the cells were treated with isobutylmethylxanthine (IBMX), dexamethasone, insulin, and rosiglitazone for 2 days. Following differentiation induction, the medium containing insulin was replaced every 2 days. Mature inguinal adipocytes were treated with 1 µM norepinephrine (NE) (Sigma, A7257) for 5 h. Ectopic expression and knockdown of Dusp4 Mature inguinal adipocytes were induced to express Dusp4 using a lentiviral system. Lentiviruses were produced using the Lenti-X™ 293T cells (Takara, 632180) using plasmids pLVX-green-Dusp4 and the catalytically inactive Dusp4-CS mutant (Cys284Ser) 37 . After transfection, lentiviral particles were collected and filtered with 0.45-µm filters. The obtained Dusp4 and Dusp4-CS mutant lentiviruses were concentrated using the Lenti-X™ Concentrator (Clontech, 631231). To achieve ectopic expression of wild-type Dusp4 and Dusp4-CS mutant, mature inguinal adipocytes were infected with concentrated lentiviruses in the presence of polybrene (8 µg/mL). For Dusp4 knockdown, we used a sequence based on the CDS from NCBI (NM_001411584). The lentivirus vector or knockdown cassettes were co-transfected with packaging vectors pSPAX2 and pMD2.G in HEK293T cells. Quantitative real-time PCR Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific, 15596018). cDNA was synthesized from 2 µg of total RNA using 10 pmol random primers, dNTPs, reverse transcriptase, and RNasin 38,39 . The primers used for qPCR were as follows: Ucp1 , Pgc1α , Pparγ , Prdm16 , Cidea , Dio2 , Atp5 , Cox2 and Cox4 . Gene expression levels were normalized to L32 as the reference gene qPCR reactions were performed using a CFX Maestro system (Bio-Rad) and 20× EvaGreen Supermix (Solgent, 31000-B500) according to the manufacturer’s instructions. Western blot and immunoprecipitation Total proteins were extracted using NP-40 lysis buffer (1% NP-40, 2.5 mM sodium phosphate, 1 mM EDTA, and 150 mM NaCl). A quantitative analysis of total protein concentration was performed using the Bradford Protein Assay (Bio-Rad, 5000002). Cell lysates were incubated with primary antibodies for Hsp90 (Santa Cruz, 1:5,000), Dusp4 (Santa Cruz, 1:1,000), Ucp1 (Cell signaling, 1:1,000), Pgc1α (Abcam, 1:1,000), Flag (Sigma, 1:5,000), and HA (Santa Cruz, 1:5,000). Cell lysates were immunoprecipitated using Crtc3 (Cell Signaling, 1:200) and protein A/G agarose beads (CalBiochem, IP05). The protein-antibody conjugated beads complexes were washed three times with NP-40 lysis buffer. Protein samples were analyzed by western blotting. Luciferase assay The luciferase assay was performed as previously described 38,39 . The plasmid for the pGL3-Pgc1α promoter was obtained from Addgene (Addgene, #8887). HEK293 cells were co-transfected with β-galactosidase to normalize transfection efficiency. The analysis utilized plasmids for the pGL4-Ucp1 promoter region (-3.5 to 0.2 kb) and the pGL3-Pgc1α promoter region (-2.0 kb). Luciferase activity was measured 24 h after transfection using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol. Oxygen consumption rate measurement Inguinal adipocytes were differentiated in Seahorse XF24-well plates and equilibrated in Seahorse XF assay medium containing 25 mM glucose, 1 mM pyruvate, and 2 mM L-glutamine for 1 h at 37°C before measurement. Thermogenesis was activated by treatment with norepinephrine (NE; 1 µM) in the mature adipocytes. Seahorse assay was performed on inguinal adipocytes with oligomycin (3 μM), CCCP (2 μM) and rotenone/antimycin A (2/5 μM). The XF Analyzers were used to measure the O 2 consumption according to the suggested protocol (Agilent Technologies XF24) 39 . Dusp4 KO mice B6:129-Dusp tm1Jmol /J mice (MGI:4456264) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Exons 2 and 3 of the Dusp4 gene were replaced with a neomycin resistance cassette in 129-derived embryonic stem (ES) cells. This strain was maintained on a mixed C57BL/6-129 genetic background by the donating laboratory. The mixed C57BL/6-129 genetic background mice are followed by backcrossing 5 times with the C57BL/6 strain. Mice were maintained in a specific pathogen-free animal facility. All animal use was approved by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) animal care and use committee. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health). Statistical analysis All quantitative data are presented as the mean ± standard error of the mean (SEM). The statistical significance of differences between the two groups was determined by a two-tailed unpaired Student's t -test. In all statistical comparisons, p < 0.05 was considered statistically significant. RESULTS Thermogenic stimuli induce Dusp4 expression in adipose tissue To investigate whether Dusp4 is involved in adipose tissue thermogenesis, we first assessed its expression under thermogenic conditions. Indeed, thermogenic stimuli, such as cold exposure and treatment with the β-adrenergic agonist CL316,243 robustly induced thermogenic markers, including Ucp1 , in mouse inguinal adipose tissue (Fig. 1a࿚c). In parallel, both mRNA and protein levels of Dusp4 were significantly upregulated in vivo (Fig. 1 a,b). Conversely, under thermoneutral conditions (30°C), which suppress thermogenic gene expression, Dusp4 expression was markedly reduced (Fig. 1 d). Consistent with the in vivo data, cultured inguinal adipocytes treated with norepinephrine (NE) to mimic cold stimulation showed markedly increased Dusp4 mRNA and protein expression in vitro (Fig. 1 e,f). Together, these findings indicate that Dusp4 expression in adipose tissue is dynamically induced by thermogenic stimuli. Loss of Dusp4 increases susceptibility to diet-induced obesity To determine whether Dusp4 confers protection against obesity, we examined the metabolic phenotypes of Dusp4 knockout (KO) mice under normal chow diet (NCD) and high-fat diet (HFD) conditions. On an NCD, Dusp4 KO mice exhibited no significant changes in adipose tissue mass, adipocyte size, and body weight compared to wild-type (WT) controls (Fig. 2 a, Supplementary Fig. 1a,b). Although expression of thermogenic genes was modestly decreased (Fig. 2 b), key genes involved in lipid metabolism, such as lipogenesis, β-oxidation, and lipolysis, were comparable between genotypes (Supplementary Fig. 1c–e). Glucose tolerance was slightly improved, whereas insulin sensitivity remained normal (Supplementary Fig. 1f,g). Upon HFD challenge, however, Dusp4 KO mice showed markedly increased adiposity and body weight compared to WT mice (Fig. 2 c,d), along with enlarged adipocytes (Fig. 2 e). Expression of key thermogenic genes, including Ucp1 and Pgc1α , was substantially suppressed, whereas lipogenic genes such as Srebp1c , Fas , and Acc were elevated in Dusp4 KO mice (Fig. 2 f-g). While β-oxidation genes remained unchanged (Supplementary Fig. 2a), glucose tolerance and insulin sensitivity deteriorated substantially compared to WT controls (Supplementary Fig. 2b,c). Together, these results indicate that loss of Dusp4 predisposes mice to diet-induced obesity by impairing thermogenic responses. Dusp4 deficiency impairs thermogenic gene induction upon cold and β-adrenergic stimulation Reduced thermogenic capacity contributes significantly to obesity susceptibility. To elucidate the role of Dusp4 in adaptive thermogenesis, we exposed Dusp4 KO mice to cold or CL316,243 stimulation. Upon cold challenge, Dusp4 KO mice exhibited larger lipid droplets and decreased expression of key thermogenic genes such as Ucp1 and Pgc1α (Fig. 3a࿚d). Protein levels of these thermogenic factors were also substantially reduced (Fig. 3 c). Similarly, following CL316,243 administration, adipocytes from Dusp4 KO mice showed greater lipid accumulation and markedly diminished induction of thermogenic genes and proteins compared to WT controls (Fig. 3e࿚h). Collectively, these findings suggest that Dusp4 deficiency impairs adaptive thermogenesis under physiological and pharmacological β-adrenergic stimuli, thereby exacerbating obesity susceptibility. Catalytic activity of Dusp4 is essential in promoting thermogenic gene expression for adipocytes To determine whether the phosphatase activity of Dusp4 is required for adipocyte thermogenesis, we ectopically expressed wild-type Dusp4 or a catalytically inactive Dusp4-CS mutant in mature inguinal adipocytes using a lentiviral system. Treatment with NE robustly elevated Ucp1 protein and mRNA levels in cells expressing wild-type Dusp4, whereas cells expressing the inactive Dusp4-CS mutant exhibited markedly impaired induction of thermogenic gene (Fig. 4 a,b). Additionally, mitochondrial mass, visualized by MitoTracker staining, was increased by Dusp4 overexpression but remained suppressed in the Dusp4-CS mutant group (Fig. 4 c). Oxygen consumption rate, reflecting mitochondrial thermogenic activity, markedly increased in response to NE in Dusp4-overexpressing cells but was compromised in cells harboring the Dusp4-CS mutant (Fig. 4 d). Conversely, lentiviral knockdown of Dusp4 mildly reduced thermogenic gene expression (Supplementary Fig. 3a,b). These data firmly establish that catalytic activity of Dusp4 is required to enhance mitochondrial function and drive thermogenic gene expression in adipocytes. Dusp4 dephosphorylates Crtc3 to promote nuclear localization and transcriptional activation of thermogenic genes Having established a role for Dusp4 in adipocyte thermogenesis, we next explored its mechanistic basis. We hypothesized that Crtc3, a transcriptional coactivator regulated by phosphorylation-dependent nuclear translocation, may serve as a direct substrate of Dusp4. Co-immunoprecipitation assays revealed enhanced interaction between Dusp4 and Crtc3 following forskolin stimulation, which mimics cold-induced cAMP signaling (Fig. 5 a). Dusp4 overexpression decreased phosphorylation level of Crtc3, whereas the inactive Dusp4-CS mutant increased Crtc3 phosphorylation level (Fig. 5 b). Consistently, Crtc3 phosphorylation was elevated in Dusp4 KO adipose tissue (Fig. 5 c). Dusp4 overexpression promoted nuclear translocation of Crtc3, whereas the Dusp4-CS mutant impaired nuclear localization (Fig. 5 d). Nuclear-localized Crtc3 significantly enhanced luciferase activity driven by the Ucp1 and Pgc1α promoters, an effect abrogated by the Dusp4-CS mutant (Fig. 5 e,f). Thus, Dusp4 directly dephosphorylates Crtc3 to facilitate nuclear translocation and transcriptional activation of key thermogenic genes. Adipose-specific restoration of Dusp4 rescues impaired thermogenesis in vivo To test whether reintroducing Dusp4 into Dusp4 KO mice could rescue impaired thermogenesis, we delivered lentiviral Dusp4 into the inguinal adipose tissue of Dusp4 KO mice. Dusp4 reconstitution markedly reduced lipid droplet accumulation and increased the expression of Ucp1 and Pgc1α (Fig. 6a࿚d). Mechanistically, restoring Dusp4 decreased the phosphorylation of Crtc3, enhancing its nuclear localization, and restored Ucp1 expression to WT levels (Fig. 6 d). Collectively, these findings demonstrate that adipose-specific reintroduction of Dusp4 is sufficient to rescue impaired thermogenesis and improve systemic metabolic parameters in Dusp4-deficient mice, highlighting its therapeutic potential in mitigating metabolic dysfunction associated with obesity. DISCUSSION Obesity remains a global health crisis closely associated with metabolic dysfunction, yet therapeutic options targeting adipocyte thermogenesis to enhance energy expenditure are still limited. Currently available anti-obesity agents, such as the Pparγ agonists (e.g., rosiglitazone) and natural compounds such as resveratrol and DHA, partially ameliorate metabolic disorders by inducing thermogenic responses in adipocytes 40 – 44 . Nevertheless, precise molecular targets enabling robust and selective activation of adipocyte thermogenesis are largely undefined, underscoring an urgent need to identify novel regulators that can effectively combat obesity through energy dissipation 45 – 47 . In this study, we provide compelling evidence identifying Dusp4 as a previously unrecognized but essential regulator of adipocyte thermogenesis and systemic metabolic homeostasis. While several phosphatases have been implicated in metabolic regulation, the direct involvement of Dusp4 in adipocyte thermogenic gene regulation represents a novel mechanism with important implications for the treatment of obesity. Our results reveal that Dusp4 expression is selectively enriched in adipose tissue compared with other metabolic organs 23 , 45 , 48 – 51 , and notably induced under thermogenic conditions such as cold exposure and β-adrenergic stimulation. Notably, Dusp4 deficiency dramatically led to impaired adaptive thermogenic gene induction, heightened susceptibility to diet-induced obesity, and systemic insulin resistance. Conversely, adipose-specific restoration of catalytically active Dusp4 fully rescued impaired thermogenic capacity, reduced adiposity, and restored metabolic balance firmly establishing Dusp4 as a critical molecular regulator in adipocytes. Mechanistically, our findings uncover a novel signaling axis in which Dusp4 directly dephosphorylates Crtc3, thereby promoting its nuclear translocation and activation of thermogenic gene programs. While Crtc3 is known to regulate adipocyte metabolism via its phosphorylation-dependent localization 52 , 53 , the upstream phosphatases modulating its nuclear localization have remained elusive. Here, we demonstrated for the first time that Dusp4 interacts directly with Crtc3, removing inhibitory phosphates at critical serine residues, and thereby facilitating its nuclear entry and transcriptional activation at Ucp1 and Pgc1α promoters. Thus, we propose a new conceptual framework in which Dusp4 functions as a gatekeeper of the Crtc3࿚Ucp1 axis, effectively linking extracellular thermogenic cues to intracellular energy expenditure. The identification of the Dusp4࿚Crtc3 pathway significantly advances our understanding of the adipocyte thermogenic signaling network and opens promising avenues for therapeutic intervention. Given that Dusp4 is enzymatically tractable, pharmacological strategies that enhance its catalytic activity or stabilize its interaction with Crtc3 could augment thermogenesis and improve metabolic outcomes. Such interventions may offer a novel means of enhancing whole-body energy expenditure and reversing obesity-related metabolic dysfunction. Despite these significant findings, our study has several limitations warranting further exploration. First, our analysis was predominantly based on global Dusp4 KO mice; future studies utilizing adipocyte-specific or inducible Dusp4 deletion models will be necessary to delineate tissue-specific effects and minimize potential developmental compensation effects. Second, although we identified critical phosphorylation sites on Crtc3 regulated by Dusp4, comprehensive phosphoproteomic analyses might reveal additional substrates involved in the thermogenic signaling cascade. Finally, the translational relevance of our findings will require validation in human adipose tissue. Comparative studies of Dusp4 expression in lean versus obese individuals, along with therapeutic trials targeting Dusp4 activity, will be essential to assess clinical feasibility. In conclusion, we establish Dusp4 as an indispensable regulator of adipocyte thermogenesis through direct modulation of Crtc3 phosphorylation. This novel Dusp4࿚Crtc3࿚Ucp1 signaling axis provides a mechanistic foundation for adaptive thermogenesis and highlights Dusp4 as a promising therapeutic target for combating obesity and its associated metabolic disorders by enhancing energy expenditure. Declarations ACKNOWLEDGEMENTS This research was supported by the National Research Foundation of Korea grant funded by the Korean government (2021R1I1A2041463, and RS-2024-00347343) and the Korea Research Institute of Bioscience & Biotechnology (KRIBB) (KGM539241410990). AUTHOR CONTRIBUTIONS Conceptualization: M.J.S, K-.H.B and W.K.K. Funding acquisition: K-.H.B and W.K.K. Investigation: M.J.S., H.U.K., J.J., S-.J.P. and J.H.C. Methodology: Y.J.J., K-.J.O., E-.W.L., and B.S.H. Supervision: J.H.C., K-.H.B. and W.K.K. Writing: M.J.S., K-.H.B. and W.K.K. 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Cold exposure induces nuclear translocation of CRTC3 in brown adipose tissue. J. Cell. Biochem. 120, 9138–9146 (2019). Additional Declarations There is no conflict of interest Supplementary Files SupplementaryFig.docx Supplemental material GraphicAbstract.tif Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: revise 09 Feb, 2026 Review # 2 received at journal 03 Feb, 2026 Review # 1 received at journal 27 Jan, 2026 Reviewer # 2 agreed at journal 15 Jan, 2026 Reviewer # 1 agreed at journal 15 Jan, 2026 Reviewers invited by journal 15 Jan, 2026 Submission checks completed at journal 11 Dec, 2025 First submitted to journal 10 Dec, 2025 Unknown event 10 Dec, 2025 Editor assigned by journal 10 Dec, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8324434","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":575062440,"identity":"c69b282a-1ef4-4b88-b38a-8b95811b5315","order_by":0,"name":"Kwang-Hee 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Hsp90 was used as a loading control (n = 6ￚ8). \u003cstrong\u003ec\u003c/strong\u003e Expression of \u003cem\u003eUcp1\u003c/em\u003e and \u003cem\u003eDusp4\u003c/em\u003e mRNA in inguinal adipose tissue following daily intraperitoneal injection of CL316,243 (1 mg/kg) for 1 week (n = 6ￚ7). \u003cstrong\u003ed\u003c/strong\u003e Relative mRNA expression of \u003cem\u003eUcp1\u003c/em\u003e and \u003cem\u003eDusp4\u003c/em\u003e in inguinal adipose tissue from mice housed at room temperature (RT, 22°C) or thermoneutrality (TN, 30°C) for 1 week (n = 6ￚ8). \u003cstrong\u003ee, f,\u003c/strong\u003e Relative mRNA (\u003cstrong\u003ee\u003c/strong\u003e) and protein (\u003cstrong\u003ef\u003c/strong\u003e) expression of \u003cem\u003eUcp1\u003c/em\u003e and \u003cem\u003eDusp4\u003c/em\u003e in mature inguinal adipocytes treated with NE (1 µM) for 5 h. Data are presented as mean ± SEM. * \u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, ** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.005, *** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8324434/v1/9c91b4e48b0083f63117d710.png"},{"id":100728004,"identity":"3cf3c3a5-4f6c-4502-b6ad-985e0d3b8e4c","added_by":"auto","created_at":"2026-01-20 20:46:23","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":718391,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eDusp4\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003edeficiency impairs thermogenic gene induction and exacerbates diet-induced obesity. a\u003c/strong\u003eRepresentative H\u0026amp;E staining of brown (BAT), inguinal (ingWAT), and epididymal adipose tissues (epiWAT) from WT and \u003cem\u003eDusp4\u003c/em\u003e KO mice fed a normal chow diet (NCD) (n = 6ￚ7). Scale bar, 60 μm. \u003cstrong\u003eb\u003c/strong\u003e Relative mRNA levels of thermogenic genes in inguinal adipose tissue from mice fed on NCD (n=7-8). \u003cstrong\u003ec\u003c/strong\u003eRepresentative whole-body images of WT and \u003cem\u003eDusp4\u003c/em\u003eKO mice after 10 weeks of high-fat diet (HFD). \u003cstrong\u003ed\u003c/strong\u003e Body-weight curves of WT and \u003cem\u003eDusp4\u003c/em\u003eKO mice fed HFD for 10 weeks (n = 6ￚ7). \u003cstrong\u003ee\u003c/strong\u003eRepresentative H\u0026amp;E staining of brown (BAT), inguinal (ingWAT) and epididymal (epiWAT) adipose tissues from WT and \u003cem\u003eDusp4\u003c/em\u003e KO mice on HFD. Scale bar, 60 µm. \u003cstrong\u003ef, g,\u003c/strong\u003e Relative mRNA levels of thermogenic genes (\u003cstrong\u003ef\u003c/strong\u003e) and lipogenic genes (\u003cstrong\u003eg\u003c/strong\u003e) in inguinal adipose tissue after 10 weeks of HFD (n=10). Data are presented as mean ± SEM. * \u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, ** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.005, *** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8324434/v1/0ee7a95dfabca60aa02810d5.png"},{"id":100727638,"identity":"6dbf0c33-b92a-4fab-b16e-243d05abaaa3","added_by":"auto","created_at":"2026-01-20 20:41:21","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":585729,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDusp4 is required for thermogenic activation in response to cold and β-adrenergic stimulation. a\u003c/strong\u003e Representative H\u0026amp;E staining of inguinal adipose tissue from WT and \u003cem\u003eDusp4\u003c/em\u003e KO mice exposed to cold (6°C, 1 week). Scale bar, 60 µm. \u003cstrong\u003eb\u003c/strong\u003e Relative mRNA expression of \u003cem\u003eDusp4\u003c/em\u003e, \u003cem\u003eUcp1\u003c/em\u003e, and \u003cem\u003ePgc1α\u003c/em\u003e in inguinal adipose tissue following cold exposure (n = 8–10). \u003cstrong\u003ec\u003c/strong\u003e Western blot analysis of \u003cem\u003eUcp1\u003c/em\u003e and \u003cem\u003ePgc1α\u003c/em\u003e proteins in inguinal adipose tissue after cold exposure. Hsp90 was used as a loading control. \u003cstrong\u003ed\u003c/strong\u003eRelative mRNA levels of additional thermogenic genes in inguinal adipose tissue after cold exposure. \u003cstrong\u003ee\u003c/strong\u003eRepresentative H\u0026amp;E staining of inguinal adipose tissue from WT and \u003cem\u003eDusp4 \u003c/em\u003eKO mice after CL316,243 administration (1 mg/kg, 1 week). Scale bar, 60 µm. \u003cstrong\u003ef, g,\u003c/strong\u003e Relative mRNA expression of Dusp4 (\u003cstrong\u003ef\u003c/strong\u003e) and additional thermogenic genes (\u003cstrong\u003eg\u003c/strong\u003e) in inguinal adipose tissue after CL316,243 administration (n = 7). \u003cstrong\u003eh\u003c/strong\u003e Western blot analysis of \u003cem\u003eUcp1\u003c/em\u003e and \u003cem\u003ePgc1α\u003c/em\u003e proteins in inguinal adipose tissue following CL316,243 administration. Hsp90 was used as a loading control. Data are presented as mean ± SEM. * \u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, ** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.005, *** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8324434/v1/4d52004f1cae23e3380dabe2.png"},{"id":100727976,"identity":"f4d33a1a-611f-4cc8-8e23-f3997c3689e8","added_by":"auto","created_at":"2026-01-20 20:46:21","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":297063,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCatalytic activity of Dusp4 is essential for thermogenic gene activation via dephosphorylation of Crtc3. a, b,\u003c/strong\u003eRelative mRNA (\u003cstrong\u003ea\u003c/strong\u003e) and protein (\u003cstrong\u003eb\u003c/strong\u003e) expression levels of \u003cem\u003eUcp1\u003c/em\u003e in mature inguinal adipocytes transduced with the lentiviruses expressing either wild-type Dusp4 or catalytically inactive Dusp4-CS mutant, followed by NE treatment (1 µM, 5 h). \u003cstrong\u003ec\u003c/strong\u003e Representative fluorescence images of mitochondrial content (MitoTracker staining) in adipocytes transduced with Dusp4 or Dusp4ￚCS mutant lentiviruses. Note the increased mitochondrial content in DUSP4ￚexpressing cells. Scale bar, 50 µm. \u003cstrong\u003ed\u003c/strong\u003e Oxygen consumption rate (OCR) analysis in adipocytes transduced with indicated lentiviruses upon stimulation with NE (1 µM). Data are presented as mean ± SEM. *\u003cem\u003ep \u003c/em\u003e\u0026lt; 0.01, ** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.005, *** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8324434/v1/f5a18fdce35f6a07c68f83e9.png"},{"id":100727911,"identity":"ffac3ceb-290b-46b0-ab02-46e59796071d","added_by":"auto","created_at":"2026-01-20 20:45:22","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":467980,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDusp4 dephosphorylates Crtc3 to promote nuclear localization and thermogenic gene transcription. a\u003c/strong\u003eCo-immunoprecipitation (Co-IP) analysis of Dusp4 and Crtc3 in HEK293 cells treated with forskolin (10 µM, 30 min) showing enhanced interaction upon stimulation. \u003cstrong\u003eb\u003c/strong\u003e Immunoprecipitation (IP) and western blot analysis of Crtc3 phosphorylation status in mature inguinal adipocytes expressing Dusp4 or catalytically inactive Dusp4-CS. \u003cstrong\u003ec\u003c/strong\u003e Immunoprecipitation (IP) and western blot analysis of Crtc3 phosphorylation levels in inguinal adipocytes from Dusp4-deficiency mice. \u003cstrong\u003ed\u003c/strong\u003eRepresentative images of immunofluorescence staining showing nuclear localization of Crtc3 in mature adipocytes expressing Dusp4 or Dusp4-CS mutant lentivirus. Scale bar, 5 µm. \u003cstrong\u003ee, f,\u003c/strong\u003eLuciferase reporter assays driven by the \u003cem\u003eUcp1\u003c/em\u003e(\u003cstrong\u003ee\u003c/strong\u003e) and \u003cem\u003ePgc1α\u003c/em\u003e (\u003cstrong\u003ef\u003c/strong\u003e) promoters in HEK293 cells co-expressing \u003cem\u003eDusp4\u003c/em\u003e and \u003cem\u003eCrtc3\u003c/em\u003e, treated with forskolin. Data are presented as mean ± SEM. * \u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, ** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.005, *** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8324434/v1/9af016e379903dad7696aa1f.png"},{"id":100727778,"identity":"cc72324e-cd73-4933-8076-3a6224a20820","added_by":"auto","created_at":"2026-01-20 20:43:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":431823,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdipose-specific restoration of Dusp4 rescues thermogenic capacity. a\u003c/strong\u003eSchematic diagram illustrating lentivirus-mediated restoration of Dusp4 expression in ingWAT (left). Representative H\u0026amp;E and immunohistochemistry (DAB staining, anti-Ucp1) images of inguinal adipose tissue following lentivirus injection (control vs. Dusp4) and cold exposure (n = 5) (right). Scale bar, 60 µm. \u003cstrong\u003eb, c,\u003c/strong\u003e Relative mRNA levels of thermogenic genes, including \u003cem\u003eUcp1\u003c/em\u003e and \u003cem\u003ePgc1α\u003c/em\u003e, in ingWAT after Dusp4 restoration and subsequent cold exposure. \u003cstrong\u003ed\u003c/strong\u003eImmunoprecipitation followed by western blot analysis of phosphorylated serine residues (p-Ser), total \u003cem\u003eCrtc3\u003c/em\u003e, \u003cem\u003eUcp1\u003c/em\u003e, and \u003cem\u003ePgc1α\u003c/em\u003e proteins in ingWAT from virus-injected mice. Hsp90 was used as a loading control. Data are presented as mean ± SEM. * \u003cem\u003ep \u003c/em\u003e\u0026lt;0.01, ** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.005, *** \u003cem\u003ep \u003c/em\u003e\u0026lt;0.0005.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8324434/v1/e6c4d22dfca313041ef7f60c.png"},{"id":100731255,"identity":"9f4bac86-ceb9-4ff2-902c-a065a09b56a9","added_by":"auto","created_at":"2026-01-20 21:31:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3823578,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8324434/v1/19ef2ec2-5ce8-4bec-8e6a-eeeec9978880.pdf"},{"id":100727970,"identity":"b15f41e9-f59e-4bbd-a223-b95b0e37e6ce","added_by":"auto","created_at":"2026-01-20 20:46:09","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":275926,"visible":true,"origin":"","legend":"Supplemental material","description":"","filename":"SupplementaryFig.docx","url":"https://assets-eu.researchsquare.com/files/rs-8324434/v1/660429d4e6406e7138d11b14.docx"},{"id":100728020,"identity":"67803088-6fec-4031-9b31-442721a70c02","added_by":"auto","created_at":"2026-01-20 20:46:37","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":190592,"visible":true,"origin":"","legend":"","description":"","filename":"GraphicAbstract.tif","url":"https://assets-eu.researchsquare.com/files/rs-8324434/v1/8003522f97974310c9f5c3c5.tif"}],"financialInterests":"There is no conflict of interest","formattedTitle":"DUSP4 activates adipocyte thermogenesis via Crtc3 dephosphorylation-dependent UCP1 expression","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eObesity has emerged as a global epidemic that is strongly associated with metabolic disorders such as type 2 diabetes, cardiovascular disease, immune dysfunction, and certain cancers. Chronic nutrient intake excess drives adipocyte hypertrophy and hyperplasia\u003csup\u003e\u003cspan additionalcitationids=\"CR2\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e, ultimately overwhelming the storage capacity of adipose tissue and disrupting systemic metabolic homeostasis\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. In contrast, adaptive thermogenesis within adipose tissue acts as a compensatory mechanism to counteract obesity by converting surplus calories into heat\u003csup\u003e\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Thermogenic activation, triggered by stimuli such as cold exposure or β-adrenergic agonists (e.g., CL316,243), critically depends on mitochondrial uncoupling protein 1 (Ucp1)\u003csup\u003e\u003cspan additionalcitationids=\"CR9 CR10\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e. Ucp1 uncouples oxidative phosphorylation, dissipating the proton gradient across the mitochondrial inner membrane and generating heat instead of ATP synthesis, thereby increasing energy expenditure\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e. Thus, elucidating molecular mechanisms that regulate Ucp1 expression represents a promising therapeutic strategy for combating obesity and its associated metabolic dysfunctions.\u003c/p\u003e \u003cp\u003eCellular signaling cascades orchestrate dynamic response to environmental and metabolic stimuli\u003csup\u003e\u003cspan additionalcitationids=\"CR17\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e, governing critical cellular functions through tightly controlled phosphorylation-dephosphorylation cycles\u003csup\u003e\u003cspan additionalcitationids=\"CR20 CR21\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Among these signaling regulators, dual-specificity phosphatase 4 (Dusp4) has emerged as a versatile regulator implicated in various biological processes, including cell proliferation, immune responses, cancer progression, and metabolism\u003csup\u003e\u003cspan additionalcitationids=\"CR24 CR25 CR26\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Dusp4 primarily exerts its function by dephosphorylating key substrates such as mitogen-activated protein kinases (MAPKs), ERK, p38, p300, and Smad4, thereby fine-tuning multiple signaling pathways and cellular outcomes\u003csup\u003e\u003cspan additionalcitationids=\"CR29 CR30 CR31\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. However, despite extensive studies in other biological contexts, the involvement and role of Dusp4 in adipocyte metabolism\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e,\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e, particularly in thermogenic regulation, remain largely unexplored.\u003c/p\u003e \u003cp\u003eThe CREB-regulated transcription coactivator 3 (Crtc3) is highly expressed in adipocytes and regulates energy metabolism through phosphorylation-dependent nuclear translocation\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e,\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. However, the specific phosphatases regulating the phosphorylation state of Crtc3 have yet to be clearly identified. Given its adipose-enriched expression and enzymatic specificity, we hypothesized that Dusp4 might regulate adipocyte thermogenesis by modulating Crtc3 activity, thereby promoting the transcriptional activation of Ucp1.\u003c/p\u003e \u003cp\u003eHere, we identify Dusp4 as a critical regulator of adipocyte thermogenesis through direct dephosphorylation of Crtc3. Using genetically engineered mouse models, we demonstrate that the loss of Dusp4 impairs thermogenic gene expression, accelerates diet-induced obesity, and leads to systemic insulin resistance. Mechanistically, Dusp4 directly interacts with and dephosphorylates Crtc3, promoting its nuclear translocation and recruitment to the \u003cem\u003eUcp1\u003c/em\u003e promoter. Importantly, adipose-specific restoration of Dusp4 reverses these metabolic defects, reinstating thermogenic capacity and restoring systemic metabolic health. These findings establish the Dusp4\u0026ndash;Crtc3\u0026ndash;Ucp1 signaling axis as a novel regulatory pathway essential for adaptive thermogenesis and highlight Dusp4 as a viable therapeutic target to combat obesity and its metabolic complications.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cp\u003e\u003cstrong\u003eCell culture and differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmortalized inguinal preadipocytes were kindly provided by Dr. Shingo Kajimura (UCSF, CA, USA). The inguinal preadipocytes were cultured in Dulbecco's Modified Eagle's Medium (DMEM), which contained 10% fetal bovine serum and 1% penicillin–streptomycin at 37°C in a 5% CO\u003csub\u003e2\u003c/sub\u003e incubator. For differentiation, the cells were treated with isobutylmethylxanthine (IBMX), dexamethasone, insulin, and rosiglitazone for 2 days. Following differentiation induction, the medium containing insulin was replaced every 2 days. Mature inguinal adipocytes were treated with 1 µM norepinephrine (NE) (Sigma, A7257) for 5 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEctopic expression and knockdown of Dusp4\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMature inguinal adipocytes were induced to express Dusp4 using a lentiviral system. Lentiviruses were produced using the Lenti-X™ 293T cells (Takara, 632180) using plasmids pLVX-green-Dusp4 and the catalytically inactive Dusp4-CS mutant (Cys284Ser)\u003csup\u003e37\u003c/sup\u003e. After transfection, lentiviral particles were collected and filtered with 0.45-µm filters. The obtained Dusp4 and Dusp4-CS mutant lentiviruses were concentrated using the Lenti-X™ Concentrator (Clontech, 631231). To achieve ectopic expression of wild-type Dusp4 and Dusp4-CS mutant, mature inguinal adipocytes were infected with concentrated lentiviruses in the presence of polybrene (8 µg/mL). For Dusp4 knockdown, we used a sequence based on the CDS from NCBI (NM_001411584).\u0026nbsp;The lentivirus vector or knockdown cassettes were co-transfected with packaging vectors pSPAX2 and pMD2.G in HEK293T cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time PCR\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific, 15596018). cDNA was synthesized from 2 µg of total RNA using 10 pmol random primers, dNTPs, reverse transcriptase, and RNasin\u003csup\u003e38,39\u003c/sup\u003e. The primers used for qPCR were as follows: \u003cem\u003eUcp1\u003c/em\u003e, \u003cem\u003ePgc1α\u003c/em\u003e, \u003cem\u003ePparγ\u003c/em\u003e, \u003cem\u003ePrdm16\u003c/em\u003e, \u003cem\u003eCidea\u003c/em\u003e, \u003cem\u003eDio2\u003c/em\u003e, \u003cem\u003eAtp5\u003c/em\u003e, \u003cem\u003eCox2\u003c/em\u003e and \u003cem\u003eCox4\u003c/em\u003e. Gene expression levels were normalized to \u003cem\u003eL32\u0026nbsp;\u003c/em\u003eas the reference gene\u0026nbsp;qPCR reactions were performed using a CFX Maestro system (Bio-Rad) and 20× EvaGreen Supermix (Solgent, 31000-B500) according to the manufacturer’s instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot and immunoprecipitation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal proteins were extracted using NP-40 lysis buffer (1% NP-40, 2.5 mM sodium phosphate, 1\u0026nbsp;mM EDTA, and 150\u0026nbsp;mM NaCl). A quantitative analysis of total protein concentration was performed using the Bradford Protein Assay (Bio-Rad, 5000002). Cell lysates were incubated with primary antibodies for Hsp90 (Santa Cruz, 1:5,000), Dusp4 (Santa Cruz, 1:1,000), Ucp1 (Cell signaling, 1:1,000), Pgc1α (Abcam, 1:1,000), Flag (Sigma, 1:5,000), and HA (Santa Cruz, 1:5,000). Cell lysates were immunoprecipitated using Crtc3 (Cell Signaling, 1:200) and protein A/G agarose beads (CalBiochem, IP05). The protein-antibody conjugated beads complexes were washed three times with NP-40 lysis buffer. Protein samples were analyzed by western blotting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLuciferase assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe luciferase assay was performed as previously described\u003csup\u003e38,39\u003c/sup\u003e. The plasmid for the pGL3-Pgc1α promoter was obtained from Addgene (Addgene, #8887). HEK293 cells were co-transfected with β-galactosidase to normalize transfection efficiency. The analysis utilized plasmids for the pGL4-Ucp1 promoter region (-3.5 to 0.2 kb) and the pGL3-Pgc1α promoter region (-2.0 kb). Luciferase activity was measured 24 h after transfection using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOxygen consumption rate measurement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInguinal adipocytes were differentiated in Seahorse XF24-well plates and equilibrated in Seahorse XF assay medium containing 25 mM glucose, 1 mM pyruvate, and 2 mM L-glutamine for 1 h at 37°C before measurement. Thermogenesis was activated by treatment with norepinephrine (NE; 1 µM) in the mature adipocytes. Seahorse assay was performed on inguinal adipocytes with oligomycin (3 μM), CCCP (2 μM) and rotenone/antimycin A (2/5 μM). The XF Analyzers were used to measure the O\u003csub\u003e2\u003c/sub\u003e consumption according to the suggested protocol (Agilent Technologies XF24)\u003csup\u003e39\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDusp4\u003c/em\u003e\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;KO mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eB6:129-Dusp\u003cem\u003e\u003csup\u003etm1Jmol\u003c/sup\u003e\u003c/em\u003e/J mice (MGI:4456264) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). Exons 2 and 3 of the \u003cem\u003eDusp4\u003c/em\u003e gene were replaced with a neomycin resistance cassette in 129-derived embryonic stem (ES) cells. This strain was maintained on a mixed C57BL/6-129 genetic background by the donating laboratory. The mixed C57BL/6-129 genetic background mice are followed by backcrossing 5 times with the C57BL/6 strain. Mice were maintained in a specific pathogen-free animal facility. All animal use was approved by the Korea Research Institute of Bioscience and Biotechnology (KRIBB) animal care and use committee. All animal procedures were approved by the Institutional Animal Care and Use Committee of the Korea Research Institute of Bioscience and Biotechnology (KRIBB) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals (US National Institutes of Health).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll quantitative data are presented as the mean ± standard error of the mean (SEM). The statistical significance of differences between the two groups was determined by a two-tailed unpaired Student's \u003cem\u003et\u003c/em\u003e-test. In all statistical comparisons, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eThermogenic stimuli induce Dusp4 expression in adipose tissue\u003c/h2\u003e \u003cp\u003eTo investigate whether Dusp4 is involved in adipose tissue thermogenesis, we first assessed its expression under thermogenic conditions. Indeed, thermogenic stimuli, such as cold exposure and treatment with the β-adrenergic agonist CL316,243 robustly induced thermogenic markers, including \u003cem\u003eUcp1\u003c/em\u003e, in mouse inguinal adipose tissue (Fig.\u0026nbsp;1a࿚c). In parallel, both mRNA and protein levels of \u003cem\u003eDusp4\u003c/em\u003e were significantly upregulated \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea,b). Conversely, under thermoneutral conditions (30\u0026deg;C), which suppress thermogenic gene expression, Dusp4 expression was markedly reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed). Consistent with the \u003cem\u003ein vivo\u003c/em\u003e data, cultured inguinal adipocytes treated with norepinephrine (NE) to mimic cold stimulation showed markedly increased Dusp4 mRNA and protein expression \u003cem\u003ein vitro\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee,f). Together, these findings indicate that Dusp4 expression in adipose tissue is dynamically induced by thermogenic stimuli.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLoss of Dusp4 increases susceptibility to diet-induced obesity\u003c/h2\u003e \u003cp\u003eTo determine whether Dusp4 confers protection against obesity, we examined the metabolic phenotypes of \u003cem\u003eDusp4\u003c/em\u003e knockout (KO) mice under normal chow diet (NCD) and high-fat diet (HFD) conditions. On an NCD, \u003cem\u003eDusp4\u003c/em\u003e KO mice exhibited no significant changes in adipose tissue mass, adipocyte size, and body weight compared to wild-type (WT) controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea, Supplementary Fig.\u0026nbsp;1a,b). Although expression of thermogenic genes was modestly decreased (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb), key genes involved in lipid metabolism, such as lipogenesis, β-oxidation, and lipolysis, were comparable between genotypes (Supplementary Fig.\u0026nbsp;1c\u0026ndash;e). Glucose tolerance was slightly improved, whereas insulin sensitivity remained normal (Supplementary Fig.\u0026nbsp;1f,g). Upon HFD challenge, however, \u003cem\u003eDusp4\u003c/em\u003e KO mice showed markedly increased adiposity and body weight compared to WT mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec,d), along with enlarged adipocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ee). Expression of key thermogenic genes, including \u003cem\u003eUcp1\u003c/em\u003e and \u003cem\u003ePgc1α\u003c/em\u003e, was substantially suppressed, whereas lipogenic genes such as \u003cem\u003eSrebp1c\u003c/em\u003e, \u003cem\u003eFas\u003c/em\u003e, and \u003cem\u003eAcc\u003c/em\u003e were elevated in \u003cem\u003eDusp4\u003c/em\u003e KO mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ef-g). While β-oxidation genes remained unchanged (Supplementary Fig.\u0026nbsp;2a), glucose tolerance and insulin sensitivity deteriorated substantially compared to WT controls (Supplementary Fig.\u0026nbsp;2b,c). Together, these results indicate that loss of Dusp4 predisposes mice to diet-induced obesity by impairing thermogenic responses.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eDusp4 deficiency impairs thermogenic gene induction upon cold and β-adrenergic stimulation\u003c/h2\u003e \u003cp\u003eReduced thermogenic capacity contributes significantly to obesity susceptibility. To elucidate the role of Dusp4 in adaptive thermogenesis, we exposed \u003cem\u003eDusp4\u003c/em\u003e KO mice to cold or CL316,243 stimulation. Upon cold challenge, \u003cem\u003eDusp4\u003c/em\u003e KO mice exhibited larger lipid droplets and decreased expression of key thermogenic genes such as Ucp1 and Pgc1α (Fig.\u0026nbsp;3a࿚d). Protein levels of these thermogenic factors were also substantially reduced (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). Similarly, following CL316,243 administration, adipocytes from \u003cem\u003eDusp4\u003c/em\u003e KO mice showed greater lipid accumulation and markedly diminished induction of thermogenic genes and proteins compared to WT controls (Fig.\u0026nbsp;3e࿚h). Collectively, these findings suggest that \u003cem\u003eDusp4\u003c/em\u003e deficiency impairs adaptive thermogenesis under physiological and pharmacological β-adrenergic stimuli, thereby exacerbating obesity susceptibility.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCatalytic activity of Dusp4 is essential in promoting thermogenic gene expression for adipocytes\u003c/h2\u003e \u003cp\u003eTo determine whether the phosphatase activity of Dusp4 is required for adipocyte thermogenesis, we ectopically expressed wild-type Dusp4 or a catalytically inactive Dusp4-CS mutant in mature inguinal adipocytes using a lentiviral system. Treatment with NE robustly elevated Ucp1 protein and mRNA levels in cells expressing wild-type Dusp4, whereas cells expressing the inactive Dusp4-CS mutant exhibited markedly impaired induction of thermogenic gene (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea,b). Additionally, mitochondrial mass, visualized by MitoTracker staining, was increased by Dusp4 overexpression but remained suppressed in the Dusp4-CS mutant group (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Oxygen consumption rate, reflecting mitochondrial thermogenic activity, markedly increased in response to NE in Dusp4-overexpressing cells but was compromised in cells harboring the Dusp4-CS mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed). Conversely, lentiviral knockdown of Dusp4 mildly reduced thermogenic gene expression (Supplementary Fig.\u0026nbsp;3a,b). These data firmly establish that catalytic activity of Dusp4 is required to enhance mitochondrial function and drive thermogenic gene expression in adipocytes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eDusp4 dephosphorylates Crtc3 to promote nuclear localization and transcriptional activation of thermogenic genes\u003c/h2\u003e \u003cp\u003eHaving established a role for Dusp4 in adipocyte thermogenesis, we next explored its mechanistic basis. We hypothesized that Crtc3, a transcriptional coactivator regulated by phosphorylation-dependent nuclear translocation, may serve as a direct substrate of Dusp4. Co-immunoprecipitation assays revealed enhanced interaction between Dusp4 and Crtc3 following forskolin stimulation, which mimics cold-induced cAMP signaling (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). Dusp4 overexpression decreased phosphorylation level of Crtc3, whereas the inactive Dusp4-CS mutant increased Crtc3 phosphorylation level (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Consistently, Crtc3 phosphorylation was elevated in \u003cem\u003eDusp4\u003c/em\u003e KO adipose tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). Dusp4 overexpression promoted nuclear translocation of Crtc3, whereas the Dusp4-CS mutant impaired nuclear localization (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed). Nuclear-localized Crtc3 significantly enhanced luciferase activity driven by the Ucp1 and Pgc1α promoters, an effect abrogated by the Dusp4-CS mutant (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ee,f). Thus, Dusp4 directly dephosphorylates Crtc3 to facilitate nuclear translocation and transcriptional activation of key thermogenic genes.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eAdipose-specific restoration of Dusp4 rescues impaired thermogenesis\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo test whether reintroducing Dusp4 into \u003cem\u003eDusp4\u003c/em\u003e KO mice could rescue impaired thermogenesis, we delivered lentiviral Dusp4 into the inguinal adipose tissue of \u003cem\u003eDusp4\u003c/em\u003e KO mice. Dusp4 reconstitution markedly reduced lipid droplet accumulation and increased the expression of Ucp1 and Pgc1α (Fig.\u0026nbsp;6a࿚d). Mechanistically, restoring Dusp4 decreased the phosphorylation of Crtc3, enhancing its nuclear localization, and restored Ucp1 expression to WT levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Collectively, these findings demonstrate that adipose-specific reintroduction of Dusp4 is sufficient to rescue impaired thermogenesis and improve systemic metabolic parameters in Dusp4-deficient mice, highlighting its therapeutic potential in mitigating metabolic dysfunction associated with obesity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eObesity remains a global health crisis closely associated with metabolic dysfunction, yet therapeutic options targeting adipocyte thermogenesis to enhance energy expenditure are still limited. Currently available anti-obesity agents, such as the Pparγ agonists (e.g., rosiglitazone) and natural compounds such as resveratrol and DHA, partially ameliorate metabolic disorders by inducing thermogenic responses in adipocytes\u003csup\u003e\u003cspan additionalcitationids=\"CR41 CR42 CR43\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Nevertheless, precise molecular targets enabling robust and selective activation of adipocyte thermogenesis are largely undefined, underscoring an urgent need to identify novel regulators that can effectively combat obesity through energy dissipation\u003csup\u003e\u003cspan additionalcitationids=\"CR46\" citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eIn this study, we provide compelling evidence identifying Dusp4 as a previously unrecognized but essential regulator of adipocyte thermogenesis and systemic metabolic homeostasis. While several phosphatases have been implicated in metabolic regulation, the direct involvement of Dusp4 in adipocyte thermogenic gene regulation represents a novel mechanism with important implications for the treatment of obesity. Our results reveal that Dusp4 expression is selectively enriched in adipose tissue compared with other metabolic organs\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e,\u003cspan additionalcitationids=\"CR49 CR50\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e, and notably induced under thermogenic conditions such as cold exposure and β-adrenergic stimulation. Notably, Dusp4 deficiency dramatically led to impaired adaptive thermogenic gene induction, heightened susceptibility to diet-induced obesity, and systemic insulin resistance. Conversely, adipose-specific restoration of catalytically active Dusp4 fully rescued impaired thermogenic capacity, reduced adiposity, and restored metabolic balance firmly establishing Dusp4 as a critical molecular regulator in adipocytes.\u003c/p\u003e \u003cp\u003eMechanistically, our findings uncover a novel signaling axis in which Dusp4 directly dephosphorylates Crtc3, thereby promoting its nuclear translocation and activation of thermogenic gene programs. While Crtc3 is known to regulate adipocyte metabolism via its phosphorylation-dependent localization\u003csup\u003e\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e,\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u003c/sup\u003e, the upstream phosphatases modulating its nuclear localization have remained elusive. Here, we demonstrated for the first time that Dusp4 interacts directly with Crtc3, removing inhibitory phosphates at critical serine residues, and thereby facilitating its nuclear entry and transcriptional activation at Ucp1 and Pgc1α promoters. Thus, we propose a new conceptual framework in which Dusp4 functions as a gatekeeper of the Crtc3࿚Ucp1 axis, effectively linking extracellular thermogenic cues to intracellular energy expenditure.\u003c/p\u003e \u003cp\u003eThe identification of the Dusp4࿚Crtc3 pathway significantly advances our understanding of the adipocyte thermogenic signaling network and opens promising avenues for therapeutic intervention. Given that Dusp4 is enzymatically tractable, pharmacological strategies that enhance its catalytic activity or stabilize its interaction with Crtc3 could augment thermogenesis and improve metabolic outcomes. Such interventions may offer a novel means of enhancing whole-body energy expenditure and reversing obesity-related metabolic dysfunction. Despite these significant findings, our study has several limitations warranting further exploration. First, our analysis was predominantly based on global \u003cem\u003eDusp4\u003c/em\u003e KO mice; future studies utilizing adipocyte-specific or inducible \u003cem\u003eDusp4\u003c/em\u003e deletion models will be necessary to delineate tissue-specific effects and minimize potential developmental compensation effects. Second, although we identified critical phosphorylation sites on Crtc3 regulated by Dusp4, comprehensive phosphoproteomic analyses might reveal additional substrates involved in the thermogenic signaling cascade. Finally, the translational relevance of our findings will require validation in human adipose tissue. Comparative studies of Dusp4 expression in lean versus obese individuals, along with therapeutic trials targeting Dusp4 activity, will be essential to assess clinical feasibility.\u003c/p\u003e \u003cp\u003eIn conclusion, we establish Dusp4 as an indispensable regulator of adipocyte thermogenesis through direct modulation of Crtc3 phosphorylation. This novel Dusp4࿚Crtc3࿚Ucp1 signaling axis provides a mechanistic foundation for adaptive thermogenesis and highlights Dusp4 as a promising therapeutic target for combating obesity and its associated metabolic disorders by enhancing energy expenditure.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Research Foundation of Korea grant funded by the Korean government (2021R1I1A2041463, and RS-2024-00347343) and the Korea Research Institute of Bioscience \u0026amp; Biotechnology (KRIBB) (KGM539241410990).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: M.J.S, K-.H.B and W.K.K. Funding acquisition: K-.H.B and W.K.K. Investigation: M.J.S., H.U.K., J.J., S-.J.P. and J.H.C. Methodology: Y.J.J., K-.J.O., E-.W.L., and B.S.H. Supervision: J.H.C., K-.H.B. and W.K.K. Writing: M.J.S., K-.H.B. and W.K.K.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCheng, Z., et al. 6-gingerol ameliorates metabolic disorders by inhibiting hypertrophy and hyperplasia of adipocytes in high-fat-diet induced obese mice. \u003cem\u003eBiomed. Pharmacother.\u003c/em\u003e 146, 112491 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu, S., et al. Triiodothyronine (T3) promotes brown fat hyperplasia via thyroid hormone receptor alpha mediated adipocyte progenitor cell proliferation. \u003cem\u003eNat. Commun.\u003c/em\u003e 13, 3394 (2022).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou, H., Trudel, G., Alexeev, K., Thomas, J. \u0026amp; Laneuville, O. 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Biochem.\u003c/em\u003e 120, 9138\u0026ndash;9146 (2019).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8324434/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8324434/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eObesity arises when chronic energy surplus overwhelms the storage capacity of adipocytes, resulting in systemic metabolic dysfunction. Adaptive thermogenesis, primarily mediated by uncoupling protein 1 (Ucp1) in adipocytes, serves to counteract obesity by dissipating excess energy as heat. Despite extensive studies on thermogenic regulation, the phosphatases that regulate this protective metabolic pathway remain poorly understood. Here, we identify dual-specificity phosphatase 4 (Dusp4) as a critical modulator of adipocyte thermogenesis \u003cem\u003evia\u003c/em\u003e direct modulation of the CREB-regulated transcription coactivator 3 (Crtc3). \u003cem\u003eDusp4\u003c/em\u003e-deficient mice exhibit impaired thermogenic capacity, diminished Ucp1 expression, and increased susceptibility to diet-induced obesity accompanied by severe insulin resistance. Mechanistically, Dusp4 directly dephosphorylates serine residues on Crtc3, facilitating its nuclear translocation and subsequent transcriptional activation of Ucp1. Restoration of catalytically active Dusp4 in adipocytes rescues thermogenic gene expression, reduces adiposity, and normalizes systemic glucose homeostasis in \u003cem\u003eDusp4\u003c/em\u003e-knockout mice. Collectively, these findings identify Dusp4 as a key upstream phosphatase orchestrating the Crtc3-Ucp1 thermogenic axis, and suggests its potential as a therapeutic target for obesity-associated metabolic disorders.\u003c/p\u003e","manuscriptTitle":"DUSP4 activates adipocyte thermogenesis via Crtc3 dephosphorylation-dependent UCP1 expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-20 18:08:04","doi":"10.21203/rs.3.rs-8324434/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2026-02-09T06:55:00+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-02-04T02:13:28+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2026-01-27T13:10:10+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-16T00:38:17+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2026-01-15T13:26:53+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2026-01-15T12:02:36+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-11T23:53:36+00:00","index":"","fulltext":""},{"type":"submitted","content":"Experimental \u0026 Molecular Medicine","date":"2025-12-11T01:37:33+00:00","index":"","fulltext":""},{"type":"checksFailed","content":"","date":"2025-12-10T23:19:59+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-12-10T07:47:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"experimental-and-molecular-medicine","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"emm","sideBox":"Learn more about [Experimental \u0026 Molecular Medicine](http://www.nature.com/emm/)","snPcode":"12276","submissionUrl":"https://mts-emm.nature.com/cgi-bin/main.plex","title":"Experimental \u0026 Molecular Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e2ed7135-a53a-42ab-81c4-8d06b3c16642","owner":[],"postedDate":"January 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":61189426,"name":"Biological sciences/Physiology/Metabolism/Metabolic diseases/Obesity"},{"id":61189427,"name":"Biological sciences/Physiology/Metabolism/Fat metabolism"}],"tags":[],"updatedAt":"2026-02-10T11:13:18+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-20 18:08:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8324434","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8324434","identity":"rs-8324434","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}