Arginase 1 promotes hepatic lipogenesis by regulating ERK2/PPARγ signaling in a non-canonical manner

Arginase 1 promotes hepatic lipogenesis by regulating ERK2/PPARγ signaling in a non-canonical manner | 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 Arginase 1 promotes hepatic lipogenesis by regulating ERK2/PPARγ signaling in a non-canonical manner Yujun Shi, Mingyang Shao, Xiaoyue Cao, Yuwei Chen, Zhiqi Zhu, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5630831/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 18 Feb, 2026 Read the published version in Nature Communications → Version 1 posted You are reading this latest preprint version Abstract The global incidence of obesity and its metabolic sequelae, notably metabolic dysfunction-associated steatohepatitis (MASLD), has escalated to epidemic levels. We unveil a previously unknown moonlighting role for arginase 1 (Arg1) in facilitating hepatic lipogenesis. Mice lacking hepatic Arg1 exhibited diminished lipid accumulation in both liver and adipocytes, an effect mirrored in genetically- or diet-induced obesity models following Arg1 inhibitor treatment. Mechanistically, Arg1 competes with RSK2 and Elk1 for binding to the substrate-binding pocket of extracellular signal-regulated kinase 2 (ERK2) via its S-shaped motif, thereby enhancing ERK2 ubiquitination and degradation and upregulating the AKT/mTOR/PPARγ and Elk1/c-Fos/PPARγ cascades, ultimately augmenting lipogenesis. Peptides designed to mimic the ERK2 substrate-binding pocket disrupted the Arg1-ERK2 interaction and improved metabolic profiles in obesity and MASLD models. Our findings implicate Arg1 regulates hepatic lipid metabolism via its physical interaction with ERK2, highlighting the Arg1-ERK2 interaction as a promising therapeutic target for obesity and related metabolic disorders. Biological sciences/Physiology/Metabolism/Fat metabolism Health sciences/Gastroenterology/Hepatology/Liver diseases/Non-alcoholic fatty liver disease hepatic metabolism Arg1 ERK2 PPARγ obesity Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Obesity and its associated disorders, including metabolic dysfunction-associated steatotic liver disease (MASLD), type II diabetes (T2DM), cardiovascular disease, and various cancers, have escalated to epidemic levels globally. 1 – 5 MASLD encompasses a spectrum of conditions ranging from metabolic dysfunction-associated steatohepatitis (MASH) to states that present significant risk factors for cirrhosis, hepatocellular carcinoma (HCC), and numerous systemic metabolic disturbances. 1 , 4 – 6 Recently, the U.S. Food and Drug Administration (FDA) has approved resmetirom as the first therapeutic agent specifically indicated for the treatment of MASH with fibrosis, a severe form of MASLD. 7 , 8 Additionally, several medications targeting T2DM, obesity, or both conditions concurrently have been approved, such as pioglitazone and glucagon-like peptide 1 (GLP-1) receptor agonists. 9 , 10 However, a notable gap exists in the approval of pharmacotherapies for MASLD, primarily due to the lack of understanding of the mechanisms underlying the initiation and progression of obesity and MASLD. Obesity is characterized by the excessive accumulation of adipose tissue, a condition arising from the dysregulation of pivotal signaling pathways crucial for maintaining metabolic homeostasis. 3 , 11 These pathways include phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT), AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase (MAPK). 11 – 13 In particular, the PI3K/AKT signaling pathway plays a critical role in enhancing lipid biogenesis while simultaneously inhibiting lipid degradation in adipose tissue. 11 In the liver, the PI3K/AKT pathway stimulates lipogenesis via the mammalian target of rapamycin (mTOR), which upregulates peroxisome proliferator-activated receptor gamma (PPARγ), a key transcription factor involved in adipogenesis. 14 , 15 AKT also regulates lipogenesis in the liver through a mTOR- independent pathways, and by exerting an inhibitory effect on AMPK, it further promotes lipogenesis and accumulation. 13 , 15 , 16 In the liver, the mitogen-activated protein kinase (MAPK) pathway plays a pivotal role in maintaining metabolic homeostasis by delicately modulating the activities of enzymes and transcription factors that govern both fatty acid synthesis and catabolism. 13 , 17 Notably, the MAPK and PI3K/AKT signaling pathways interact intricately within the signaling network, often mediated by shared upstream regulators such as receptor tyrosine kinases, GPCRs, and integrins. 13 , 18 The MAPK family, particularly ERK, p38, JNK, and BMK1, represents a highly conserved array of serine-threonine kinases. 19 Among them, the ERK1/ERK2 kinases have attracted garnered significant attention due to their extensive involved in diverse physiological and pathological processes. Upon activation by the Ras/Raf/MEK signaling cascade, ERK2 (also known as MAPK1) undergoes phosphorylation and becomes activated, subsequently translocating to the nucleus. 13 In the nucleus, ERK2 phosphorylates a suite of transcription factors, including Elk1, ETS, c-Fos, Jun, and Myc, thereby initiating a cascade of downstream responses. 20 – 22 The Ras/Raf/MEK/ERK signaling pathway meticulously regulates the expression of genes associated with lipid metabolism, influencing the expression levels of pivotal enzymes such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), and consequently modulating the rate of fatty acid synthesis. 23 , 24 Moreover, this pathway also impacts the β-oxidation process, exerting comprehensive control over lipid metabolism. 25 A key aspect of this pathway is its sophisticated negative feedback regulation mechanism, which not only dampens the activity of upstream components (Ras, Raf, MEK) but also establishes cross-regulatory interactions with other signaling pathways, such as the PI3K/AKT pathway. 18 This intricate regulatory framework not only ensures signal fidelity and cellular stability but also offers fresh insights into the finely tuned mechanisms governing liver lipid metabolism. PPARγ, a key member of the nuclear hormone receptor superfamily, acts as a critical transcription factor that controls the expression of numerous genes involved in lipid metabolism in both hepatocytes and adipocytes. 26 , 27 In the liver, PPARγ promotes fatty acid synthesis, enhances the uptake of free fatty acids (FFA), and fosters the accumulation of triglycerides (TG) within hepatocytes. 28 , 29 Conversely, in adipocytes, PPARγ stimulates the storage of excess lipids, thereby reducing the influx of lipids into the liver and alleviating hepatic lipid accumulation. 29 – 31 Given its critical role in modulating lipid metabolism in both cell types, PPARγ represents an attractive therapeutic target. Arginase, primarily existing as arginase 1 (Arg1) in mammals, is predominantly expressed in the cytoplasm of hepatocytes. It catalyzes the conversion of L-arginine to L-ornithine and urea, playing a vital role in the urea cycle for the detoxification of ammonia. 32 Children with congenital deficiency of Arg1 exhibit hyperargininemia, accompanied by spastic paraparesis, progressive neurological and intellectual deficits, and notably, persistent growth retardation. 33 This growth retardation may be attributed to a deficiency in polyamines and an excess of arginine. Polyamines, including putrescine, spermidine, and spermine, are synthesized from L-ornithine and are essential for cell proliferation, growth, and tissue repair. 34 , 35 Polyamines are pivotal in fat metabolism. Once secreted by cells, they interact with adipocytes, promoting adipose tissue vascularization and stimulating lipolysis. 36 Specifically, polyamines like spermidine enhance the production of cAMP, the second messenger of β-adrenergic receptors (βAR), thereby further augmenting lipolysis. Polyamines also regulate adipocyte generation by modulating the expression of key transcription factors essential for the differentiation of preadipocytes into mature adipocytes. 37 Moreover, by facilitating lipolysis and angiogenesis, polyamines help maintain the homeostasis of white adipose tissue (WAT), thereby preventing obesity and insulin resistance. 38 Elevating circulating L-arginine levels has been shown to alleviate MASH-related liver inflammation. In the alternative pathway for arginine hydrolysis, L-arginine serves as the sole substrate for both Arg1 and nitric oxide synthase (NOS). 39 NOS metabolizes L-arginine into L-citrulline and nitric oxide (NO). An imbalance between Arg1 and NOS can disrupt NO production, leading to an imbalance in reactive oxygen species (ROS), which can result in ROS-induced damage accumulation in MASLD. 40 , 41 It appears that Arg1 plays a potential role in the development of MASLD by regulating the synthesis of either polyamines or NOS, which warrants further exploration. In this study, we observed that Arg1, beyond its canonical role in L-arginine metabolism, exhibits a moonlighting function through physical interaction with the substrate-binding pocket of ERK2. This interaction subsequently induces the degradation of ERK2 protein, leading to the upregulation of PPARγ signaling and lipid accumulation in both the liver and lipocytes. Targeting the Arg1-ERK2 interaction represents a promising therapeutic strategy for ameliorating obesity and MASLD. Results 1. Disruption of Arg1 reduces lipid accumulation in hepatocytes and adipocytes To delve into the role of hepatic Arg1 in vivo in modulating hepatic lipid metabolism, we generated liver-specific Arg1 knockout mice using the Alb-Cre-loxP system ( Alb cre Arg1 fl/fl ; Figures S1A-S1D). Postnatally, the Alb cre Arg1 fl/fl mice began to display characteristic symptoms of Arg1 deficiency, including lethargy, sluggish movement, and growth retardation, culminating in a 52% mortality rate within the first four weeks (Figure S1E). Consequently, only mice that survived until eight weeks were chosen for subsequent experimentation. Despite exhibiting no significant difference in food intake (Figure S1F), the Alb cre Arg1 fl/fl mice notably had lower body weight and size compared to their same-sex littermate controls (Figures 1A and 1B). Although the hepatic lobular structure, zonation, and glycogen synthesis appeared relatively normal, as evidenced by H&E (hematoxylin and eosin) staining, immunohistochemistry (IHC) staining for glutamine synthetase (GS) and cytokeratin 19 (CK19), and periodic acid-Schiff (PAS) staining, respectively (Figure S1C), the ablation of Arg1 led to a marked decrease in liver size, weight, and hepatocyte size (highlighted by IHC staining for β-catenin) (Figures 1C and 1E). Strikingly, hepatocytes in Arg1 fl/fl mice, which retained intact Arg1 expression and served as wildtype control, contained uniformly distributed fine lipid droplets. In contrast, these lipid droplets were scarcely observed in the hepatocytes from Alb cre Arg1 fl/fl mice, as revealed by oil red O staining (Figure 1E). Furthermore, this deletion also induced mild liver injury, manifested by slightly elevated serum enzyme levels (Figure S1G). The liver primarily synthesizes TG, which are then transported into and stored in adipose tissue for future energy use. We observed that Alb cre Arg1 fl/fl mice had a significantly reduced mass of adipose tissue in the visceral adipose tissue (VAT) region, encompassing the epididymal fat, renal fat capsules, and mesenteric adipose tissue, compared to Arg1 fl/fl mice (Figure 1F). This finding was corroborated by a microcomputed tomography (micro-CT) scan, which captured abdominal fat accumulation at the level of the fourth lumbar vertebra and revealed a marked decrease in the volume of both VAT and subcutaneous adipose tissue (SAT) in the Arg1-deficient mice (Figures 1G and 1H). This was further substantiated by the weight of the collected VAT and SAT, revealing that the Arg1-deficient mice had less than one-sixth of the adipose fat and a significantly reduced ratio of fat tissue weight to body weight compared to the control mice (Figure 1I). Histologic analysis revealed that adipocytes in Alb cre Arg1 fl/fl mice were significantly smaller in size compared to those in control mice (Figure 1J). Additionally, Arg1-deficiency resulted in a notable decrease in both serum and liver tissue levels of TG and FFA (Figures 1K and 1L). Consistent with these observations, the livers of Alb cre Arg1 fl/fl mice exhibited a marked downregulation of genes involved in fatty acid synthesis, particularly those in the PPARγ pathway, including PPARγ itself and its downstream targets such as SREBP1, FASN, CD36, and AP2, as evidenced by both western blotting and quantitative PCR analysis (Figures 1M and 1N). To eliminate the toxic effect of hyperargininemia on the early development, growth and fat metabolism of mice, we established an inducible Arg1-knockout mouse model designated as TBG cre Arg1 fl/fl . This was achieved by administering AAV8-TBG-Cre via tail vein injection to adult Arg1 fl/fl mice (Figure S2A). Four weeks after injection, the TBG cre Arg1 fl/fl mice exhibited normal behavior and did not show any signs of mortality or liver damage, as evidenced by normal levels of ALT and AST (Figure S2B). Similar to the Alb cre Arg1 fl/fl mice, the TBG cre Arg1 fl/fl mice displayed a decrease in body weight, liver weight, and lipid accumulation in both the liver and adipose tissue (Figures S2C-S2J). Additionally, there was a reduction in the levels of TG and FFA in both the serum and liver (Figure S2K). Notably, the inducible deficiency of Arg1 led to a profound downregulation of the expression of PPARγ, FASN, and SREBP1 (Figure S2L). Although inducible Arg1-deficiency notably decreased serum arginine levels, they remained robustly elevated compared to baseline levels (Figure S2M). To further investigate the impact of Arg1 inhibition on lipid metabolism, we treated cultured mouse AML12 hepatocytes with arginase inhibitor S-(2-bromoethyl)-L-cysteine (BEC). The tetrahedral BEC boronate anion mimics the tetrahedral intermediate stage of the arginine hydrolysis reaction, enabling it to bind tightly to the arginase active site. 42 This ultimately results in the formation of an enzyme-inhibitor complex, thereby impeding arginase activity. AML12 hepatocytes, when treated with oleic acid (OA), exhibited a significant increase in both the number and size of lipid droplets. However, BEC treatment dramatically reduced this lipid droplet accumulation (Figure S3A). In parallel, we observed a decrease in the protein levels of PPARγ, FASN, and SREBP1 (Figures S3B). 2. Arg1 disruption protects against genetically- or diet-induced obesity and MASLD To further confirm that Arg1 deficiency, rather than hyperargininemia, is the primary cause of lipogenesis disorders, we administered BEC to db/db mice (Figure 2A). db/db mice, characterized by a Leptin mutation that leads to severe obesity, are an ideal model for studying the effects of Arg1 inhibition, as they exhibit cardioprotective benefits from L-arginine supplementation. 43 We initiated BEC treatment in db/db mice at 8 weeks of age and continued for 4 weeks. BEC treatment significantly suppressed body weight gain, resulting in a final body weight that was approximately two-thirds of that observed in untreated mice (Figure 2B and S3C). Moreover, BEC-treated mice displayed a remarkable reduction in lipid accumulation in the liver and adipose tissue, accompanied by decreased adipocyte size, reduced serum and hepatic TG and FFA levels, and downregulated expression of PPARγ, FASN, and SREBP1 (Figures 2C-2F and S3D-S3I). Notably, BEC treatment did not alter serum L-arginine levels (Figure 2G), possibly due to excessive L-arginine consumption by other tissue. To investigate whether Arg1 disruption plays a role in improving diet-induced obesity and fatty liver, we initiated a 12-week high-fat diet (HFD) regimen for both the TBG cre Arg1 fl/fl mice and Arg1 fl/fl mouse model (Figure 2H). Notably, the TBG cre Arg1 fl/fl mice demonstrated a marked reduction in body weight and liver size compared to their Arg1 fl/fl counterparts (Figures 2I and 2J; Figures S3J and S3K). Moreover, these mice displayed a significantly diminished accumulation of lipids in both the liver and adipose tissue, along with decreased serum and hepatic concentrations of TG and FFA (Figures 2K-2N, Figures S3L-S3O). In contrast to HFD-induced obesity-associated MASLD, which manifests as prominent macrovesicular steatosis in the liver along with a marked increase in both body weight and visceral fat mass (Figure 2K), mice subjected to a methionine- and choline- deficient (MCD) diet typically exhibit an earlier onset of steatosis and inflammatory cell infiltration in the liver. Paradoxically, however, this is accompanied by a decrease in body weight. To investigate this further, we administered MCD to TBG cre Arg1 fl/fl mice (Figure 2O). After four weeks of MCD administration, Arg1 fl/fl mice exhibited a drastic exacerbation of liver steatosis, characterized by an enlarged liver size, increased weight, and a yellowish, oily appearance (Figures 2P, and S3Q). This was further substantiated histologically by the presence of macrovesicular steatosis, hepatocellular ballooning, and frequent infiltration of inflammatory cells, as evidenced by an immunohistocheminstry staining for the pan-leukocyte marker CD45 (Figure 2Q). Notably, the ablation of Arg1 led to a significant improvement in liver histology (Figures 2P and 2Q). Consistent with this, serum and hepatic levels of TG and FFA were also markedly lower in TBG cre Arg1 fl/fl mice (Figure S3R). Additionally, proteins involved in lipogenesis were decreased in response to Arg1 ablation (Figure S3S). 3. Deficiency of Arg1 downregulates the AKT/mTOR/ PPARγ signaling pathway Our findings suggest that disruption of hepatic Arg1 has the potential to ameliorate genetic- or diet-induced obesity and MASLD by inhibiting the PPARγ pathway. Importantly, this effect appears to be independent of hyperargininemia and is specifically attributable to the inhibition of Arg1 itself. To further elucidate the mechanism underlying Arg1’s regulation of lipid metabolism in hepatocytes, we conducted RNA sequencing analysis on liver tissues from TBG cre Arg1 fl/fl and Arg1 fl/fl mice. The resulting heatmap revealed that genes targeted by PPARγ or those involved in adipogenesis regulation were among the most downregulated in TBG cre Arg1 fl/fl liver (Figures 3A and 3B). This observation was further corroborated by gene set-enrichment analysis (GSEA), which demonstrated a marked inhibition of PPARγ target gene expression in hepatocytes from TBG cre Arg1 fl/fl mice compared to controls (Figure 3C). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that Arg1 deficiency significantly inhibited the PI3K/AKT signaling pathways (Figure 3D). PI3K/AKT/mTOR signaling cascade plays a pivotal role in balancing lipogenesis and oxidation by modulating the PPARγ pathways, specifically via phosphorylation of mTOR, ultimately enhancing the uptake of FFA and promoting TG accumulation. 44 Consistent with the observed decrease in PPARγ expression in the TBG cre Arg1 fl/fl liver, there was a corresponding reduction in the protein levels of hepatic p-AKT, p-mTOR, as well as its downstream proteins p70-S6K and p-4E-BP (Figure 3E). These findings were further substantiated in Alb cre Arg1 fl/fl mice and db/db mice treated with BEC (Figures S4A and S4B). Additionally, knocking down the Arg1 gene in AML12 cells using a small-interfering RNA specifically targeting Arg1 (siArg1) attenuated lipid droplets and suppressed the AKT/mTOR/PPARγ signaling pathway (Figures S4C-S4E). We conducted a further analysis of mRNA sequencing data from HFD-fed Arg1 fl/fl and TBG cre Arg1 fl/fl mice. The results revealed that, compared to Arg1 fl/fl mice, HFD-induced TBG cre Arg1 fl/fl mice exhibited a marked downregulation of PPARγ pathways and adipogenesis-related genes (Figures 3F-3H, and S4F). Notably, GSEA indicated that genes involved in the oxidative damage response were suppressed in TBG cre Arg1 fl/fl mice, providing further evidence of reduced lipid accumulation in the Arg1-deficient liver (Figure 3G). Collectively, our findings present compelling evidence that the absence of Arg1 inhibits the AKT/mTOR/PPARγ signaling pathway, leading to a significant downregulation of genes crucial for lipogenesis. However, the precise mechanism underlying how Arg1 activates this signaling pathway to regulate hepatic lipid metabolism remains elusive. 4. Arg1 binds to and promotes ERK2 ubiquitination-mediated degradation To delve deeper into the underlying mechanism of Arg1’s involvement in lipid metabolism, we conducted co-immunoprecipitation (Co-IP) and mass spectrometry analysis to identify proteins that interact with Arg1. A total of 1444 peptides corresponding to 338 proteins were identified. KEGG pathway analysis indicated that the top 100 proteins, ranked by peptide spectrum match (PSM), were enriched in metabolism signaling pathways, including MAPK signaling, arginine biosynthesis, insulin signaling, the TCA cycle, and glycose metabolism (Figure 4A). Among the proteins that potentially interact with Arg1, ERK2 emerged as the most likely candidate due to several reasons: (1) Four peptides with high-scoring PSMs belonged to ERK2. Notably, the peptide with the highest affinity featured a unique substrate-binding pocket structure, consisting of Ala 187 -Thr 188 -Arg 189 -Trp 190 (or ATRW), 45,46 which serves as a versatile and flexible binding site for ERK2 and its interacting proteins (Figures 4B and 4C). The binding of these partner proteins to ERK2, including activators (such as MEK1/2), inactivators (such as MKP3), and substrates (such as RSK2 and Elk1), 47-49 occurs in a mutually exclusive manner, each mediating distinct cellular processes; (2) In the proteins detected by Co-IP, a clear band located between 35-55 kDa was observed in the strips pulled down by the Arg1 antibody. ERK2, with a molecular weight of 42 kDa, matches the size of this corresponding protein band (Figure 4A); (3) As a crucial member of the MAPK family, ERK2 plays pivotal roles in various cellular processes. By interacting with the AKT/mTOR/PPARγ signaling pathway, it exerts key regulatory effects on lipid metabolism, leading to the amelioration of steatosis in the liver. We subsequently delved into whether Arg1 directly binds to ERK2. As anticipated, the Co-IP results demonstrated that Arg1 interacts with ERK2 in liver tissue extracted from Arg1 fl/fl mice (Figure 4D). This direct binding was further validated by the Duolink in situ proximity ligation assay (PLA) in AML12 cells (Figure 4E). Furthermore, in AML12 cells transfected with Flag-tagged ERK2 plasmid, Flag pull-down and Co-IP experiments confirmed the interaction between exogenous Arg1 and Flag-ERK2 (Figure 4F). Next, we examined whether BEC, the Arg1 inhibitor, could disrupt the binding interaction between Arg1 and ERK2. In AML12 cells treated with BEC, we observed a lack of binding of Arg1 and ERK2, as evidenced by both Co-IP and Duolink in situ PLA experiments (Figures 4G and 4H). To further determine whether Arg1 promotes hepatic lipogenesis by suppressing ERK2 activity, we employed the ERK-selective inhibitor VX-11e to inhibit ERK2 activation in HFD-induced TBG cre Arg1 fl/fl mice (Figure 4I). Administration of VX-11e for 4 weeks significantly reversed the decreases in body weight, liver weight, and liver-to-body weight ratio observed in TBG cre Arg1 fl/fl mice (Figures S5A and S5B). Additionally, by augmenting the PPARγ pathway, VX-11e exacerbated hepatic steatosis and lipid accumulation in adipose tissue (Figures 4J-4N, Figure S5C-S5G). In MCD-treated TBG cre Arg1 fl/fl mice, VX-11e administration similarly impacted hepatic lipogenesis and the expression of PPARγ pathway (Figures S5H-S5N). Our findings suggest that Arg1 fosters hepatic lipogenesis in an ERK2-dependent manner. We discovered that knocking down Arg1 led to increased protein levels of ERK2 in both AML12 cells and TBG cre Arg1 fl/fl mice, without affecting the expression of ERK2 mRNA (Figures 4O and 4P, Figure S5O). Previous research has demonstrated that ERK2 can be degraded through ubiquitination. MG132, a proteasome inhibitor, promotes the accumulation of ubiquitinated proteins. 50 When AML12 cells were treated with MG132, we observed decreased accumulation of ubiquitinated ERK2 in response to Arg1-knockdown, as evidenced by in vitro ubiquitination assays (Figure 4Q). This suggests that Arg1 may enhance the degradation of ERK2 through the ubiquitin-proteasome pathway. 5. The S-shaped motif of Arg1 selectively binds to the substrate binding pocket of ERK2 We next set out to unravel the binding mechanism between Arg1 and ERK2. We expressed glutathione-S-transferase (GST)-tagged Arg1, His-tagged ERK2 mutants, and ERK2-derived amino acid fragments in E. coli . Our findings demonstrate that Arg1 is capable of directly binding to the full-length ERK2 protein without requiring any additional mediators (Figure 5A). An ERK2 mutant, harboring mutations in three peptides but retaining its critical ATRW sequence, displayed robust binding affinity to Arg1. In contrast, an ERK2 mutant carrying mutations within the critical 170-190 amino acid sequence exhibited significantly diminished binding to Arg1 (Figure 5A). Furthermore, the molecular mapping assay revealed that Arg1 interacts with the ERK2 amino acid fragment spanning residues 170-190 in E. coli (Figure 5B). The substrate-binding pocket structure, ATRW, located within the peptide sequence spanning residual 170-190 of ERK2, is integral for binding and activating enzymes such as ribosomal S6 kinase 2 (RSK2) and ETS-domain containing protein (Elk1). 47 To identify the exact site of Arg1 binding to the ERK2 substrate-binding pocket, we transfected AML12 cells with plasmids expressing various ERK2 mutations. In comparison to the non-mutated control (NC), mutations in the A, T, or R residues of ERK2 considerably diminished its binding to Arg1, except for mutations affecting the W residue. Notably, mutations in the R residue and those encompassing the entire ATRW region resulted in the most significant decrease in Arg1 binding (Figure 5C). Intriguingly, mutating the ATRW sequence in ERK2 led to a reduction in its ubiquitination (Figure 5C). Both RSK2 and Elk1 are known to bind to ERK2 via its substrate binding pocket. Based on this, we hypothesized that Arg1 would compete with them for this binding site. Our data revealed that ERK2 carrying the ATRW mutation prevented its binding to both RSK2 and Elk1 (Figure 5C). When Arg1 was knocked down, ERK2 was able to associate with a higher amount of RSK2 and Elk1, as evidenced by Co-IP experiments in AML12 cell (Figure 5D). Similarly, we observed consistent results in AML12 cells treated with Arg1 inhibitor BEC (Figure 4G). Arg1 is a metalloproteinase featuring a unique α/β fold structure and a binuclear manganese center. It is well established that three key regions within the Arg1 molecule are essential for L-arginine hydrolysis: the hydrolytic site, the L-R amino acid site (composed of Arg 21 and Asp 181 , which bind to the charged carboxyl group and the R-amino group of L-arginine, respectively), and the double hydrogen site (formed by His 101 and His 126 ). 51,52 Additionally, all mammalian arginases share a C-terminal S-shaped motif spanning residues 304-322. 53,54 The proper assembly of this motif into a homotrimer is essential for Arg1’s normal enzymatic activity, quaternary structure, and cooperative properties. Therefore, the amino acid residues His 101 and His 126 , Arg 21 and Asp 181 , as well as the sequence spanning residues 304-322, are indispensable for L-arginine binding in Arg1. We successfully synthesized the Arg1 mutants in the E. coli system. Notably, mutating the S-shaped motif (residues 304-322) in Arg1 completely abolished its binding affinity to ERK2, whereas mutations in the other regions exhibited minimal impact on binding (Figure 5E). To assess the significance of the S-shaped motif in Arg1 for hepatic lipogenesis, we administered AAV8 viruses expressing Arg1 21&181 -mut (AAV8-TBG-Arg1-mut 21&181 -His) or Arg1 304-322 -mut (AAV8-TBG-Arg1 304-322 -mut-His) into TBG cre Arg1 fl/fl mice (Figure 5F). The levels of His-tagged protein in the liver extracts from the Arg1-mutant groups were stably expressed (Figure 5G). The Arg1 21&181 -mut protein, which could bind to ERK2 but was unable to recognize arginine, partially mitigated the reduction in hepatic lipogenesis observed in TBG Cre Arg1 fl/fl mice. This was evidenced by higher liver weight, body weight, and liver-to-body weight ratios compared to those injected with a vector (Figures 5H and 5I). Furthermore, AAV8-Arg1 21&181 -mut treatment substantially increased hepatic lipid accumulation (Figure 5J), elevated TG and FFA levels in both serum and liver tissue (Figure 5K) and enhanced the PPARγ pathway in the TBG cre Arg1 fl/fl mice (Figure 5L). On the contrary, the AAV8-Arg1 304-322 -mut protein, which could not combine with ERK2, exhibited a negative effect on reversing the phenotype in response to Arg1 ablation. Interestingly, treatment with neither mutant virus significantly reduced serum arginine levels, demonstrating that a mutation in the ERK2 binding site of Arg1 also compromises its enzymatic function (Figure 5M). These data further support our hypothesis that the role of Arg1 in regulating lipogenesis is independent of its enzymatic activity, but rather depends on a physical combination with ERK2. 6 . Arg1 enhances lipogenesis in hepatocytes via AKT/mTOR/PPARγ and Elk1/c-Fos/PPARγ signaling pathways We have observed that the disruption of Arg1 leads to the suppression of the AKT/mTOR/PPARγ signaling pathway (Figures 3, and S7). However, the crosstalk between Arg1, mediated by ERK2, and the AKT/mTOR/PPARγ pathways remains elusive. In addition to decreasing EKR2 ubiquitination and degradation, we discovered that the disruption of Arg1 facilitates the nuclear translocation of ERK2 (Figures 6A and 6B). Overactivation of ERK2 has been reported to impede lipogenesis through at least two distinct mechanisms, as depicted in Figure 6C: (1) In the cytoplasm, ERK2 undergoes phosphorylation via the Ras-Raf-MEK cascade, enabling its substrate-binding pocket to bind to RSK2. This interaction initiates a negative feedback loop that suppresses the overactivation of Ras-Raf-MEK signaling, further inhibiting the AKT/mTOR signaling cascade and ultimately suppressing PPARγ. (2) Phosphorylated ERK2 translocates to the nucleus, where its substrate-binding pocket binds to Elk1, promoting the ERK2/Elk1/c-Fos signaling pathway, which in turn inhibits the PPARγ signaling. 47,49,55 Western blot results revealed that in the livers of TBG cre Arg1 fl/fl mice, Ras, p-Raf, and p-MEK were obviously decreased in the cytoplasm, whereas c-Fos and Elk1 in the nucleus were significantly elevated (Figures 6A and 6B). Furthermore, HFD-fed TBG cre Arg1 fl/fl and Arg1 fl/fl mice confirmed that Arg1 promotes hepatic lipid accumulation and adipose tissue lipid deposition through ERK2/PPARγ pathways, as elucidated by western blotting and immunohistochemistry staining in mouse liver (Figures 6D-6F). We next treated AML12 cells with ERK2 inhibitor VX-11e, ERK2 activator TBHQ, Arg1 siRNA, and Arg1 overexpression virus (OE), respectively. The results confirmed that the absence of Arg1 leads to excessive upregulation of ERK2, which in turn downregulates the PPARγ signaling pathway, thereby inhibiting hepatic lipogenesis. Importantly, this inhibition can be partially reversed by the ERK2 inhibitor. Conversely, Arg1 overexpression downregulates ERK2 protein levels and upregulates the PPARγ pathway, promoting hepatic lipogenesis. Notably, the ERK2 activator attenuates this promotional effect (Figures 6G and 6H). 7 . Peptide targeting Arg1 - ERK2 interaction improves the metabolic profile of obesity and MASLD in mice We have identified Arg1 as a promising therapeutic target for obesity and MASLD. In contrast to small-molecule drugs, peptides can be precisely engineered to target specific molecular receptors, offering enhanced selectivity. 56,57 We synthesized two peptides: one biologically derived (ERK2 170-200 -Bio peptide) and one chemically synthesized (ERK2 170-200 -Chem peptide). These peptides encompass amino acids 170 to 190 within the substrate-binding pocket of ERK2, which are essential for its interaction with Arg1. Additionally, the Arg 192 residual was included due to its importance for the conformational stability and functionality of the pocket structure. In the inactive state, the substrate-binding pocket of ERK2 is blocked by Arg 192 . However, phosphorylation of Thr 183 and Tyr 185 triggers a conformational change, enabling the pocket to become exposed and accessible for substrate recognition and binding. Our findings indicate that GST-tagged peptides effectively bind to Arg1 within AML12 cells (Figure S6A). Co-IP and Duolink in situ PLA assays revealed that these peptides prevented the binding between Arg1 and ERK2, while also increasing the binding of ERK2 to both RSK2 and Elk1 (Figures S6B and S6C). We next evaluated the efficacy of disrupting the Arg1-ERK2 interaction in vivo by administering 50 µg/kg of either the ERK2 170-200 -Bio peptide, the ERK2 170-200 -Chem peptide, or saline via daily tail injections to wild-type mice fed HFD for 4 weeks (Figure 7A). Compared to the saline group, both the ERK2 170-200 -Bio peptide and the ERK2 170-200 -Chem peptide significantly reduced body weight, liver weight, the liver-to-body weight ratio, and levels of ALT and AST (Figures 7B-7E, Figure S6D). Moreover, peptide therapy did not cause hyperargininemia (Figure S6E). Additionally, histological analysis, micro-CT imaging, and quantification of TG and FFA in both serum and liver revealed significantly lower lipid accumulation in the liver and adipose tissue of peptide-treated mice (Figures 7F-7I, Figures S6F-S6I). Most notably, peptide treatment provided significant protection against Arg1-mediated ERK2 degradation in obese mice. Specifically, it enhanced RSK2 levels in the cytoplasm, thereby inhibiting the Ras/Raf and AKT/mTOR signaling pathways. Within the nucleus, the activation of Elk1 led to a surge in c-Fos expression, ultimately suppressing the expression of PPARγ (Figures 7I and 7J). Next, we explored whether peptide injection could attenuate the progression of MCD-induced MASLD. We administered peptides or saline daily to wild-type mice for 2 weeks, concurrently with the MCD diet (Figure S7A). Our observations revealed that peptide injection improved metabolic profiles in MCD-fed mice compared to saline-treated mice (Figures S7B-S7F). These data indicate that both peptides can effectively treat MASLD by disrupting the Arg1-ERK2 interaction. Discussion This study reveals an unprecedented role of Arg1 in regulating hepatocyte lipogenesis through a non-enzymatic mechanism. Specifically, Arg1 interacts with ERK2 via its S-shaped motif, binding to the substrate-binding pocket of ERK2. This interaction not only facilitates ERK2 degradation but also inhibits its binding to RSK1 and Elk1. Consequently, this process enhances the PPARγ pathway, promoting hepatocyte lipogenesis and contributing to the development of obesity and MASLD (Figure 8). The functions of Arg1 have long been ascribed solely to its enzymatic activity. In hepatocytes, Arg1 catalyzes the conversion of free L-arginine into ornithine, a pivotal step in the urea cycle and ammonia metabolism. Arg1 also competes with NOS for L-arginine, thereby inhibiting NO production and weakening the anti-oxidative effect, particularly in hepatocytes and inflammatory cells such as macrophages and neutrophils. 39-41,58-60 Furthermore, via its enzymatic role, Arg1 exerts an indirect modulation on lipid metabolism, given that polyamines, the byproducts of arginine catabolism, are recognized inhibitors of lipogenesis. Hence, the hypothesis arose that Arg1 might inhibit lipogenesis and thus obesity and MASLD. Surprisingly, however, studies in both genetically and chemically Arg1-deficient mice revealed that Arg1 actually promotes lipogenesis and MASLD. Our results exclude the possibility that inhibiting Arg1 reduces lipogenesis via hyperargininemia. For instance, in db/db mice, suppressing Arg1 activity did not induce hyperargininemia but significantly decreased lipogenesis and obesity. Our in vitro experiments further confirmed that silencing Arg1 ameliorates the accumulation of lipid droplets in hepatocytes. In subsequent studies, we injected mice with viral vectors harboring mutations in the critical binding sites for arginine within the Arg1 enzyme. These mutations did not alleviate hyperargininemia in TBG cre Arg1 fl/fl mice but effectively inhibited lipogenesis in the liver. These findings together suggest that Arg1 plays a crucial regulatory role in lipid metabolism, independent of its canonical enzymatic function. In recent years, an increasing number of studies have highlighted the non-enzymatic functions, also termed “moonlighting functions”, of metabolic enzymes in diverse cell processes, such as signaling pathways, autophagy, mitochondrial function, and redox homeostasis regulation. 61,62 For example, phosphofructokinase 1 (PFKP) binds to the N-terminal SH2 domain of p85α, recruiting it to the plasma membrane and thereby activating PI3K, which in turn stimulates tumor cell proliferation. 63 Similarly, upon activation of growth factor receptors, pyruvate kinase M2 (PKM2) translocates to the nucleus, where it binds to c-Src-phosphorylated Y333 of β-catenin, thereby promoting the expression of glycolysis-related genes and enhancing glucose uptake and lactate production. 64 In both our in vivo and in vitro models, Arg1 ablation significantly downregulates the AKT/mTOR/PPARγ and Elk1/c-Fos/PPARγ pathways, both of which are known to promote lipogenesis, further exemplifying the influence of metabolic enzymes on physiological processes via non-enzymatic mechanisms. Utilizing a combination of various protein interaction research methods, we have unequivocally demonstrated that Arg1 physically binds to the substrate-binding pocket of ERK2. Furthermore, mutating the ERK2 substrate-binding pocket diminished the binding affinity of Arg1, RSK2, and Elk1 for ERK2. Conversely, ablating Arg1 enhanced the binding of RSK2 and Elk1 to ERK2, indicating a competitive binding relationship among these proteins for the ERK2 substrate-binding site. As a consequence, the binding of Arg1 to ERK2 not only triggers ERK2 ubiquitination and degradation but also impedes the interaction of RSK2 and Elk1 with ERK2, subsequently inhibiting their signaling pathway activation. This reduction in signaling activity, in turn, diminishes the functional activities of these molecules and ultimately enhances PPARγ activity. The substrate-binding pocket of ERK2, primarily composed of four amino acid residues (ATRW), undergoes conformational changes facilitated by neighboring amino acids, enabling the binding of specific proteins and subsequent regulation of their functional activities. Notably, this pocket structure exhibits a remarkable lack of strict selectivity with respect to amino acid sequence, phosphorylation sites, and spatial conformation at the binding site. 21,47,65-69 Competitive binding to this pocket has been documented for several proteins, including MKP3, RSK2, and Elk1. 47 Given this high flexibility and versatility, it is plausible that ERK2 can also interact with Arg1. In addition to the diversity in amino acid sequence and spatial conformation, partner proteins display various interaction patterns when binding to ERK2. For instance, both MKP3 and Elk1 bind to the ERK2 substrate-binding site via their FXFP domains; however, MKP3 also requires its non-catalytic amino-terminal segment for additional binding to ERK2 to fulfill its function. 47 Similarly, MEK1 not only binds to the ERK2 substrate-binding site but also requires binding to the CD site of ERK2 to stabilize the complex. 21,47,70 Moreover, RSK2 forms a heterodimer with ERK2 and undergoes activation through a series of phosphorylation reactions. After binding to the substrate-binding site of ERK2 and undergoing phosphorylation, RSK2 requires the additional binding of PDKl (pyruvate pehydrogenase kinase 1) to the essential docking site to achieve full activation. 67,68 We identified that the S-shaped motif within Arg1 is crucial for mediating the interaction between Arg1 and the ERK2 substrate-binding pocket. This motif is essential for the formation of functional Arg1 homotrimers. Our data demonstrate that occupation of the Arg1-arginine binding site by BEC not only disrupts the enzymatic activity of Arg1 but also impairs the ability of the S-shaped motif to bind to the ERK2 substrate-binding pocket. Likewise, viral vectors harboring Arg1 mutants in the S-shaped motif lose their capacity to hydrolyze arginine and are ineffective in alleviating hyperargininemia in TBG cre Arg1 fl/fl mice. These findings suggest that the spatial conformation of Arg1 is vital for its binding to ERK2. However, due to the technological limitations, the precise binding mechanism and conformational transitions involved in the interaction between Arg1 and ERK2 remain to be elucidated. Our study suggests that disrupting the interaction between Arg1 and ERK2 can inhibit hepatocyte lipogenesis, thereby preventing subsequent obesity and MASLD. We have designed a peptide that mimics the substrate-binding pocket of ERK2, specifically binding to the S-shaped motif of Arg1 and effectively inhibiting the Arg1-ERK2 interaction. This peptide demonstrates the expected role in modulating hepatic lipid metabolism and alleviating obesity and MASLD, without inducing significant hyperargininemia. The intrinsic physiological roles of ERK2 and Arg1 pose challenges for drug development. However, our work introduces a promising strategy for drug design targeting the Arg1-ERK2 interaction, offering a potential therapeutic option for individuals with obesity and MASLD. Declarations RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yujun Shi ( [email protected] ). Materials availability Materials used in this study are listed in the key resources table. All reagents and mice generated in this study are available from the lead contact with a completed Materials Transfer Agreement. Data availability mRNA-seq data generated in this study have been deposited at National Center for Biotechnology Information (NCBI) GenBank (accession numbers: PRJNA1186374 and PRJNA1186081) and are publicly available as of the date of publication. Microscopy data reported in this paper will be shared by the lead contact upon request. This paper does not report the original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Acknowledgments This research was supported by the Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 82200649) and the National Natural Science Foundation of China (Grant No. 82472241). We would also like to acknowledge the help of HUABIO and WZ Biosciences Inc. in providing peptides as well as virus strains. We are grateful to Shanghai Oe Biotech Co., Ltd., for providing sequencing services. Author contributions M.S. and X.C. conceived and designed the project. Y.C. performed mRNA-seq experiments and analysis. Z.Z., Y.S., Q.T., Q.X., T.M., Z.W., M.C., Y.Z., and R.Y. bred mice, performed mouse experiments, and analyzed the data. J.G., and J.Y. provided liver disease model setup, study design, and joint discussions on the results. Y.S. and M.S. interpreted the data and drafted and revised the manuscript. STAR*METHODS EXPERIMENTAL MODEL DETAILS Mice and diets The Arg1 fl/fl mice were provided by Dr. Pan Cong. Albumin-Cre transgenic mice were purchased from Shanghai Biomodel Organism Science. Liver-specific and hepatocyte-specific Arg1 knockout mice were generated by crossing the Arg1 fl/fl mice with Albumin-Cre mice ( Alb cre Arg1 fl/fl ) or by injecting 1×10 11 GC/mouse of AAV8-TBG-cre (provided by WZ Biosciences Inc.) ( TBG cre Arg1 fl/fl ), respectively. Eight-week-old male C57BL/6 mice and db/db mice were purchased from GemPharmatech, Nanjing, China. All mice were male and housed under specific pathogen-free conditions with corncob bedding. The animal care and experimental procedures were conducted in accordance with national and international laws, policies, and ethical guidelines, and were approved by the Animal Care and Use Committee of Sichuan University, which adhered to the criteria outlined in the NIH Guide for the Care and Use of Laboratory Animals. Mice were maintained on either a chow diet as a control, a 45% lard high-fat diet (HFD), or a methionine-choline-deficient diet (MCD). All diets were purchased from Readydietech Co., Ltd., Shenzhen, China. The HFD was used to establish animal models of obesity and MAFLD induced by obesity. 71 Six- to eight-week-old male mice were fed the HFD for 12 weeks. The MCD diet was used to establish the MAFLD model, 72 and 6- to 8-week-old male mice were fed the MCD diet for 2 weeks. The dose of BEC (CAS # 222638-67-7, purity: 99.28%, TargetMol, USA) was calculated based on previous studies that reported a 50% reduction in arginase activity in cells isolated from treated animals. A daily dose of 0.012 g of BEC was administered in 100 μL of water via oral gavage, to match the dosing equivalent used in studies where mice were exposed to BEC in their drinking water. Tert-butylhydroquinone (TBHQ) (CAS # 1948-33-0, purity: ≥95%, TargetMol, USA) is an ERK activator. 73 VX-11e (CAS #896720-20-0, purity: ≥98%, TargetMol, USA) is a potent, selective, and orally bioavailable inhibitor of ERK. 74 The mice were given VX-11e in their drinking water at a concentration of 0.5 mg/mL. METHOD DETAILS Tissue and blood collection Mice were sacrificed at the indicated times, and blood was collected. Tissues were collected and rapidly frozen using liquid nitrogen for long-term storage at -80°C. These frozen samples will be utilized for subsequent proteomic or immunoblot analysis. Furthermore, a portion of these tissues was fixed with 4% paraformaldehyde for 24 hours and subsequently embedded in paraffin for pathological staining analysis. In addition, a part of the liver was fixed in 4% paraformaldehyde overnight at 4°C. Afterward, it was infiltrated with 30% sucrose at 4°C overnight. The liver tissue was then embedded in Tissue-Tek OCT compound (Sakura; cat. no. 4583) for subsequent frozen sectioning. Measurement of biochemical parameters Collect serum samples and send them to the GLP laboratory at West China Hospital for testing of mouse liver function indicators, including ALT and AST. Hepatic lipid analysis The stored liver samples (100 mg) were lysed and homogenized in 2 mL of a solution containing 150 mmol/L NaCl, 0.1% Triton X-100, and 10 mmol/L Tris using a polytron homogenizer (cat. no. NS-310E; Microtec Co., Ltd., Chiba, Japan) for 1 minute at room temperature. The liver TG level was analyzed using a Tissue Triglyceride Assay Kit (Applygen Technologies, Beijing, China) and the results were obtained by the GLP laboratory at West China Hospital. The liver homogenate FFA level was determined using an FFA Detection Kit based on the ACS-ACOD method (Wako Pure Chemical Industries, Ltd.) and normalized to the protein levels, as previously described. Cell Culture The AML12 mouse liver cell line was maintained in a 1:1 mixture of DMEM and Ham's F12 Medium, supplemented with 10% fetal bovine serum, 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium, and 40 ng/mL dexamethasone. The cells were cultured at 37°C in a 5% (v/v) CO 2 atmosphere and subcultured every 3 days. MG132, a proteasome inhibitor, effectively blocks the proteolytic activity of the 26S proteasome complex, allowing ubiquitinated proteins to accumulate. The concentration of MG132 (CAS #133407-82-6, purity: 99.99%, supplied by TargetMol, USA) used in cell experiments was 10 μM, with a duration of action of 48 hours. In cell experiments, BEC was administered at a concentration of 0.31 μM, TBHQ at 5 μM, and VX-11e at 50 nM. Protein digestion and mass spectrometry The liver sample was processed to obtain the total protein extract. Subsequently, a Co-Immunoprecipitation (CoIP) experiment was conducted using an Arg1 antibody to isolate proteins specifically interacting with Arg1. The protein complex was subjected to SDS-PAGE for separation based on their molecular weights. The gel was stained with Coomassie Brilliant Blue to visualize the protein bands. Images of the stained gel were captured for documentation. Specific gel slices containing bands of interest were excised. The excised gel pieces were processed to extract the peptides, which were subsequently analyzed by LC-MS/MS by Shanghai Oe Biotech Co., Ltd. The results obtained from this analysis are presented in Supplementary Table 1. Proximity ligation assay AML12 cells were fixed using 3.7% paraformaldehyde and then blocked with the blocking solution provided by the Duolink PLA kit (Sigma-Aldrich, USA), 75 following the manufacturer's instructions. In summary, the fixed cells were incubated overnight with both anti-ERK2 and anti-Arg1 antibodies. After thorough washing to remove unbound antibodies, the cells were sequentially incubated with PLA probes, ligation solution, and amplification solution, all at 37°C. Finally, coverslips were mounted, and the resulting images were examined using a Leica TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany). Measurement of fat mass by microcomputed tomography (micro-CT) After being anaesthetized with isoflurane (1.5-2%) administered via air, the mice were positioned supine in an imaging cell where they were kept anaesthetized, warmed, and continuously monitored. Three-dimensional X-ray images were acquired using the CT component of a Quantum GX microCT Imaging System. The total acquisition time for these images was 15 minutes. The tube voltage was set at 80 kV, with a constant current of 32 mA. Measurement of fat mass by autopsy At the end of the different procedures, anaesthetized mice were sacrificed with a lethal dose of pentobarbital (120 mg/kg, intraperitoneally). Adipose tissues (visceral and subcutaneous) were harvested and weighed on a precision scale. Knockdown of Arg1 The siRNA targeting Arg1 (siArg1) was designed and synthesized by RiboBio. The target sequences utilized were: (1) GACTGAAGTGGACAGACTA; (2) CAAGCCTATTGACTACCTT; and (3) CTGGGTGACTCCCTGTATA. AML12 cells were treated with siArg1, and after a 72-hours incubation period, the cells were collected for subsequent western blot analysis. Construction of Plasmids and Viruses The plasmids encoding Flag-ERK2, Flag-ERK2 mut, and His-Arg1 were designed and synthesized by HUABIO, China. These plasmids were subsequently transfected into AML12 cells for a period of 48 hours. Following transfection, the cells were collected by centrifugation at 800 g for 10 minutes at 4°C.Additionally, plasmids for full-length mouse Arg1, mutant Arg1, full-length ERK2, and various ERK2 mapping constructs were also designed and synthesized by HUABIO, China. These plasmids were expressed and purified in E. coli. WZ Biosciences Inc. (Shandong, China) designed and synthesized recombinant AAV8 vectors carrying TBG-Arg1-mut 21&181 -His and AAV8-TBG-Arg1 304-322 -mut-His plasmids. These plasmids harbor mutations in the hydrolytic site or S-motif coding sequence of mouse Arg1, respectively. The recombinant AAV8 vectors were administered to animals via tail vein injection at a dose of 2×10 11 per animal. One-month post-injection, the samples were collected for further analysis. Peptide-drug synthesis Peptides were obtained through Chemical Peptide Synthesis or Biological Peptide Synthesis (HUABIO, China). (1) Sequence of Chemical Peptide Synthesis: 170-200：RVADPDHDHTGFLTEYVATRWYRAPEIMLNS, and the purity is greater than 85%;（2）Biological Peptide Synthesis: recombinant synthesis involves the expression of the peptide within a specialized system from an artificial gene. The PET28A vector with a His tag was constructed to transfect E. coli , and the purified protein was extracted. The main sequence was RVADPDHDHTGFLTEYVATRW YRAPEIMLNS, and the purity was greater than 85%. Mice were injected with 50 µg/kg via the tail vein every day. Oil red O staining Liver tissues or cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 10 minutes. After being washed twice with distilled water, they were immersed in 60% isopropyl alcohol for 5 minutes, and then fixed in oil red O dye solution for 20 minutes. Finally, they were washed with distilled water 2-5 times until there was no excess oil red O staining fluid, and observed and photographed under an optical microscope. Serum Amino Acids The concentration of amino acids in serum was determined by high performance liquid chromatography as previously described. 76 Histology and Immunohistochemistry Liver specimens were fixed in 10% neutral buffered formalin for a duration of 48 hours. Paraffin-embedded sections, each 4 μm thick, were prepared and subsequently subjected to a series of steps including dewaxing, rehydration, antigen retrieval, and quenching of endogenous peroxidase activity. The sections were then incubated with the appropriate primary antibody at 4°C overnight, followed by incubation with an anti-mouse/rabbit secondary antibody (Dako REAL EnVision Detection System, Glostrup, Denmark) for 1 hour at room temperature. Detection was achieved using 3,3′-diaminobenzidine (DAB) as a substrate. Standard protocols were employed for H&E and PAS staining. Western blot Liver tissues were homogenized for protein extraction. Sodium dodecyl sulfate‒polyacrylamide gel electrophoresis and immunoblotting were performed, and an electro chemiluminescent reagent was used for chemiluminescence detection. mRNA Isolation and Real-Time PCR Total mRNA was purified from 25 mg of liver tissue using an RNA Isolation kit (cat. RE-03011; Foregene, Chengdu, China). mRNA was reverse transcribed to cDNA using the iScriptcDNA Synthesis kit (cat. 179-8890; Bio-Rad, Hercules, CA). A CFX Connect Real-Time System (Bio-Rad) was used for real-time PCR. 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Proceedings of the National Academy of Sciences of the United States of America 116 , 21150-21159. https://doi.org/10.1073/pnas.1906182116 Additional Declarations There is NO Competing Interest. Supplementary Files FigureS1.tif Figure S1. Construction of Alb cre Arg1 fl/fl mice, related to Figure 1 (A) Schematic diagram of Arg1 fl/fl and Alb cre Arg1 fl/fl mice. (B-D) Genotyping (B), immunohistochemical (IHC) staining (C) or western blotting (D) showed that Alb cre Arg1 fl/fl mice were successfully constructed. Scale bars, 50 μm (H&E, GS, CK19, and PAS) and 100 μm (Arg1). (E) The survival curve of Alb cre Arg1 fl/fl mice (n = 92). (F, G) The average weekly food take (F) and serum AST (left) and ALT (right) concentrations (G) in Alb cre Arg1 fl/fl and Arg1 fl/fl mice (n = 5). All data represent the mean ± SEM. FigureS2.tif Figure S2. Short-term deletion of Arg1 inhibits lipid accumulation in hepatocytes and adipocytes, related to Figure 1 (A) Schematic diagram illustrating the process of infecting Arg1 fl/fl mice with AAV8-TBG-cre virus to generate TBG cre Arg1 fl/fl mice. (B-E) Serum AST (left) and ALT (right) concentrations (B), body weight (C), gross view (D), liver weight and the ratio of liver weight to body weight (E) of Arg1 fl/fl mice in response to AAV8-TBG-cre virus. (F) Representative liver gross view, IHC staining of Arg1, H&E (scale bar, 50 μm), and Oil Red O staining (scale bar, 50 μm) in liver sections of Arg1 fl/fl and TBG cre Arg1 fl/fl mice. (G-N) Comparison of various physiological and biochemical parameters between Arg1 fl/fl and TBG cre Arg1 fl/fl mice, including gross view of abdominal fat tissue (G), micro-CT images and quantification of the areas of SAT and VAT (H), representative gross views of adipose tissue (left) with corresponding tissue weights (right) (I), epididymal fat tissue with H&E staining and cell size measurements (J), serum and hepatic levels of TG and FFA (K), and western blot analysis of liver extracts (L). n = 5 per group. (M) Serum arginine concentration in Arg1 fl/fl , Alb cre Arg1 fl/fl , and TBG cre Arg1 fl/fl mice. FigureS3.tif Figure S3. Deletion of Arg1 or inhibition of its function protects against obesity and MASLD, related to Figure 2 (A, B) AML12 cells were transfected with siArg1 or siNC and then treated with oleic acid (OA) for 24 h. Oil Red O staining was used to detect the lipid accumulation (A). Scale bar, 25 µm. Relative protein levels related to PPARγ pathway in OA-treated AML12 cells (B). (C-I) Representative gross views of livers (C), liver weight and liver/body weight ratio (D), fat tissue area-to-total area ratio from micro-CT images (E), representative gross views (scale bar, 1 cm) (left) and weights (right) of adipose tissue (F), adipose cell size (G), serum and hepatic levels of TG and FFA (H), and western blot analysis of the PPARγ pathway (I) in the liver of db/db mice treated with or without BEC. n = 5 per group. (J-O) Representative gross views of livers (J), liver weight (left) and ratio of liver weight to body weight (right) (K), serum (left) and hepatic (right) TG and FFA content (L), fat tissue area-to-total area ratio from micro-CT images (M), gross views and weight of adipose tissue (N), size of epididymal adipocytes (O) in Arg1 fl/fl and TBG cre Arg1 fl/fl mice in response to an MCD diet (n = 5). (Q-S) Body weight (left), liver weight and ratio of liver weight to body weight (right) (Q), serum TG and FFA (left) and hepatic TG and FFA (right) content (R), relative protein levels in liver tissues (S) of Arg1 fl/fl and TBG cre Arg1 fl/fl mice in response to an MCD diet (n = 5). FigureS4.tif Figure S4. Arg1 deficiency inhibits the AKT/mTOR pathway, related to Figure 3 (A, B) Detection of AKT/mTOR proteins in Arg1 fl/fl and TBG cre Arg1 fl/fl mice (A) and in db/db mice treated with either water or BEC (B). (C) The siRNA (si1) that efficiently knocks down Arg1 has been identified and selected. (D) AML12 cells were transfected with siArg1 or siNC. Oil Red O staining was used to detect lipid accumulation in AML12 cells. (E) Representative western blot of AKT/mTOR/ PPARγ in AML12 cells transfected with siArg1 or siNC. (F) KEGG graph showing the upregulation of fatty acid metabolism in HFD-induced Arg1 fl/fl and TBG cre Arg1 fl/fl mice. FigureS5.tif Figure S5. ERK2 inhibitor VX-11e compensates for the reduction of hepatocyte lipogenesis caused by Arg1 knockout, related to Figure 4 (A-G) Gross views (A), body weight (left), liver weight and ratio of liver weight to body weight (right) (B), fat tissue area-to-total area ratio from micro-CT images (C), gross views of fat tissues and weight of SAT or VAT (D), H&E (Scale bars, 100 μm) in fat tissue sections (E) and quantification of the cell area (F), serum or hepatic TG (left) and FFA (right) content (G) of HFD-induced TBG cre Arg1 fl/fl mice in response to VX-11e treatment. (H) Schematic diagram of MCD-induced TBG cre Arg1 fl/fl mice treated with VX-11e. (I-M) Representative liver gross view (I), H&E staining (scale bar, 100 μm), and Oil Red O staining (scale bar, 50 μm) in liver sections (J), body weight (left), liver weight, and the ratio of liver weight to body weight (K), TG and FFA content in serum (L) and in liver tissue (M), and western blotting of PPARγ in liver tissues (N) from MCD-induced TBG cre Arg1 fl/fl mice treated with VX-11e. n = 5. (O) Relative mRNA levels of Arg1 and ERK2 were measured in AML12 cells transfected with siArg1 or siNC. FigureS6.tif Figure S6. Peptides Targeting the Arg1-ERK2 interaction enhances the metabolic profile of obesity and MASLD mice, related to Figure 7 (A) Direct interaction between Arg1 and peptide detected by Co-IP. (B) Direct interaction between endogenous Arg1 and ERK2 in AML12 cells treated with peptide for 12 hours, detected by in situ PLA (Scale bar, 50 μm). (C) The interactions between ERK2 and Arg1, Elk1, or RSK2 in AML12 cells treated with peptides were detected through Co-IP followed by western blot analysis. (D-I) ALT (left) and AST (right) concentrations (D), serum arginine concentration (E), quantification of the areas of SAT and VAT in micro-CT images (F), gross views and weight of SAT and VAT (G), SAT and VAT cell size (H), serum (left) and hepatic (right) TG and FFA (I) in HFD-fed TBG cre Arg1 fl/fl mice after treatment with ERK2 170-200 -Bio or ERK2 170-200 -Chem peptide. FigureS7.tif Figure S7. Peptides Targeting the Arg1-ERK2 interaction improves hepatic steatosis in MASLD mice, related to Figure 7 (A)Schematic diagram of MCD -induced MASLD models in mice treated with peptide. (B-F) Liver gross views (B), H&E (scale bar, 100 μm) and Oil Red O staining (scale bar, 100 μm) in liver sections (C), serum ALT (left) and AST (right) concentrations (D), body weight (left), liver weight, and ratio of liver weight to body weight (right) (E), serum (left) and hepatic (right) TG and FFA content (F) of MCD-induced mice in response to peptides (n = 5). KeyResourceTable.docx Key Resource Table Cite Share Download PDF Status: Published Journal Publication published 18 Feb, 2026 Read the published version in Nature Communications → Version 1 posted 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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University","correspondingAuthor":false,"prefix":"","firstName":"Junhua","middleName":"","lastName":"Gong","suffix":""},{"id":392216153,"identity":"b57f3e76-2e11-4935-8887-94156703ca70","order_by":14,"name":"Jiayin Yang","email":"","orcid":"","institution":"CAS","correspondingAuthor":false,"prefix":"","firstName":"Jiayin","middleName":"","lastName":"Yang","suffix":""}],"badges":[],"createdAt":"2024-12-12 10:51:48","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5630831/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5630831/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41467-026-69731-3","type":"published","date":"2026-02-18T05:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":73891825,"identity":"2996daae-9355-4238-a598-05154caae003","added_by":"auto","created_at":"2025-01-15 15:45:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":4892872,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArg1 ablation in hepatocytes reduces lipid accumulation in liver and adipose tissue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Gross views\u0026nbsp;of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003emice observed at 8 weeks prior to sacrifice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B-D)\u003c/strong\u003e Body weight (B), gross liver appearance \u003cstrong\u003e(C)\u003c/strong\u003e, and the liver weight \u003cstrong\u003e(D)\u003c/strong\u003e of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003emice (n = 5). Scale bar, 1 cm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eImmunohistochemistry for β-catenin was conducted to indirectly assess the expression pattern of β-catenin localized on the cell membrane (scale bar, 25 μm). The size of hepatocytes was quantified using ImageJ software. Oil Red O staining was performed on liver tissue from \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003emice (scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Representative gross views of abdominal fat of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G, H)\u003c/strong\u003e Representative micro-CT images were presented \u003cstrong\u003e(G)\u003c/strong\u003e, and the ratios of SAT and VAT areas to the total area was analyzed using ImageJ software in \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice\u003cstrong\u003e (H)\u003c/strong\u003e (n = 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e Representative gross images of SAT and VAT from \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice. Tissue weights of SAT and VAT (left) and tissue/body weight ratios of SAT and VAT (right) in \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J)\u003c/strong\u003e H\u0026amp;E staining of peri-epididymal adipose from \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003emice (scale bar, 100 μm) and quantification of adipose cell size (n = 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(K, L) \u003c/strong\u003eSerum TG (left) and FFA (right)\u003cstrong\u003e (K)\u003c/strong\u003e and hepatic levels of TG (left) and FFA (right)\u003cstrong\u003e (L)\u003c/strong\u003e in \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M) \u003c/strong\u003eWestern blot assay showing the protein levels of PPARγ, FASN, and SREBP1 in liver samples from \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice. β-actin was used as the internal control (n =3).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(N)\u003c/strong\u003e qRT-PCR analysis demonstrating the gene expression levels in the livers of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice. Normalized to β-actin (n = 3).\u003c/p\u003e\n\u003cp\u003eData with error bars are reported as the mean ± SEM.\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E, hematoxylin-eosin; TG, triglycerides; FFA, Free Fatty Acids; PPARγ, Peroxisome Proliferator-Activated Receptor γ; FASN, Fatty Acid Synthase; SREBP1, Sterol Regulatory Element Binding Protein 1.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/18067bab26d0d470137e98e0.png"},{"id":73891831,"identity":"f4933014-3a4e-444a-bbf1-eaf76a424c11","added_by":"auto","created_at":"2025-01-15 15:45:36","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":8677755,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDeletion of Arg1 or inhibition of its function protects against obesity and MASLD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic diagram of the experimental design for administering Arg1 inhibitor BEC to \u003cem\u003edb/db \u003c/em\u003emice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Body weight of \u003cem\u003edb/db\u003c/em\u003e mice induced with water or BEC from 0 to 4 weeks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Representative gross liver views, along with H\u0026amp;E stained (scale bar, 100 μm) and Oil Red O-stained (scale bar, 50 μm) liver sections, are shown for \u003cem\u003edb/db\u003c/em\u003e mice treated with either water or BEC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D-F)\u003c/strong\u003e Representative gross views of adipose tissue \u003cstrong\u003e(D)\u003c/strong\u003e, miro-CT images\u003cstrong\u003e (E)\u003c/strong\u003e, and adipose tissue H\u0026amp;E staining \u003cstrong\u003e(F)\u003c/strong\u003e for \u003cem\u003edb/db\u003c/em\u003e mice treated with either water or BEC. Scale bar, 100 μm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Serum arginine concentration in \u003cem\u003edb/db\u003c/em\u003e mice treated with either water or BEC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e Schematic diagram of HFD-induced obesity and NAFLD models in \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice infected with AAV8-TBG-cre or AAV8-GFP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I)\u003c/strong\u003e Body weight of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice, induced with HFD, monitored from 0 to 12 weeks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J, K) \u003c/strong\u003eRepresentative gross liver views \u003cstrong\u003e(J)\u003c/strong\u003e, H\u0026amp;E (scale bar, 100 μm) and Oil Red O staining (scale bar, 50 μm) in liver sections \u003cstrong\u003e(K) \u003c/strong\u003eof mice from groups described in\u003cstrong\u003e (H)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(L-N)\u003c/strong\u003e Representative gross views of adipose tissue \u003cstrong\u003e(L), \u003c/strong\u003emiro-CT images \u003cstrong\u003e(M)\u003c/strong\u003e and adipose tissue H\u0026amp;E staining \u003cstrong\u003e(N)\u003c/strong\u003e of HFD-induced \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(O)\u003c/strong\u003e Schematic diagram of MCD-induced NAFLD models in\u003cem\u003e Arg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(P, Q)\u003c/strong\u003e Representative liver image \u003cstrong\u003e(P)\u003c/strong\u003e, H\u0026amp;E (scale bar, 100 μm), Oil Red O staining (scale bar, 50 μm) and CD45 immunofluorescence staining (scale bar, 50 μm) in liver sections \u003cstrong\u003e(Q) \u003c/strong\u003ein MCD-induced\u003cem\u003e Arg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003eStatistical data represent the mean ± SEM. n=5 per group.\u003c/p\u003e\n\u003cp\u003eAAV, adeno-associated virus; HFD, high-fat diet; H\u0026amp;E, Hematoxylin and Eosin; MCD, Methionine-Choline-Deficient.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/ed795767462b0309c1d300fd.png"},{"id":73891830,"identity":"5cb0ed02-3842-4145-9511-727304ee7df2","added_by":"auto","created_at":"2025-01-15 15:45:35","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3630510,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArg1 ablation downregulates the AKT/mTOR/ PPARγ signaling pathway\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e The heatmap illustrates the differentially expressed genes between the livers of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eExpression levels of genes related to PPARγ pathway and lipogenesis metabolism are shown.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e GSEA reveals alterations in PPAR pathways within the livers of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (n = 3) (|NES| = 1.53; FDR q value \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eAlterations were observed in multiple signaling pathways, including the PI3K/AKT signaling pathway, in\u003cem\u003e Arg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eWestern blotting was used to assess the expression of key proteins in the AKT/mTOR signaling pathway in liver extracts from \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eArg1 deprivation reduced the expression of genes linked to the lipogenesis in the HFD-induced model.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G) \u003c/strong\u003eGSEA graphs depict the enrichment of oxidative damage response in the livers of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (n = 3) (|NES| = 1.43; FDR q value \u0026lt; 0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e The KEGG graph demonstrates the downregulation of PPAR pathways and lipogenesis metabolism in the HFD-induced\u003cem\u003e Arg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003ePPARγ, Peroxisome Proliferative Activated Receptor γ; GSEA, Gene Set Enrichment Analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/29c9522a1d472b9ffc4b8d55.png"},{"id":73893706,"identity":"2532664e-5a44-450f-8b2f-8e5a795693e5","added_by":"auto","created_at":"2025-01-15 16:01:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":6204041,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArg1 binds to ERK2 and facilitates the ubiquitination and degradation of ERK2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic diagram of experimental design for IP-MS analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003ePeptides of ERK2 identified by MS analysis as potential Arg1-interacting proteins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e Spatial structure model of ERK2.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D)\u003c/strong\u003e Co-immunoprecipitation of Arg1 and ERK2 in wild-type mouse liver.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e \u003cem\u003eIn situ\u003c/em\u003e PLA signals of Arg1 and ERK2 in AML12 cells treated with siArg1 or siNC, detected by \u003cem\u003ein situ \u003c/em\u003ePLA (left) and the number of red spots counted using ImageJ (right).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F) \u003c/strong\u003eCo-immunoprecipitation of Flag-ERK2 and Arg1 in AML12 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Co-immunoprecipitation of ERK2, Arg1, Elk1, and RSK2 in AML12 cells treated with or without BEC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e \u003cem\u003eIn situ \u003c/em\u003ePLA signals of Arg1 and ERK2 in AML12 cells treated with or without BEC, detected by duolink in situ PLA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I) \u003c/strong\u003eSchematic diagram of HFD-induced MASLD models in \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice treated with or without ERK2 inhibitor VX-11e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J-N)\u003c/strong\u003e Gross liver views \u003cstrong\u003e(J)\u003c/strong\u003e, H\u0026amp;E (scale bars, 100 µm) and Oil Red O staining (scale bars, 50 µm) in liver sections \u003cstrong\u003e(K)\u003c/strong\u003e, representative micro-CT images\u003cstrong\u003e (L)\u003c/strong\u003e, illustration of abdominal fat\u003cstrong\u003e (M)\u003c/strong\u003e, and relative protein levels of the PPARγ signaling pathway\u003cstrong\u003e (N)\u003c/strong\u003e in HFD-induced \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice, with or without VX-11e treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(O, P)\u003c/strong\u003e Western blotting of ERK2 in siRNA-treated AML12 cells \u003cstrong\u003e(O)\u003c/strong\u003e and \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice \u003cstrong\u003e(P)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(Q)\u003c/strong\u003e Ubiquitination of ERK2 in AML12 cells treated with or without MG132 and co-transfected with indicated siRNA.\u003c/p\u003e\n\u003cp\u003eIP-MS, immunoprecipitation and mass spectrometry; PLA, proximity ligation assay.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/af0d5764e729e0b69f6ac492.png"},{"id":73892888,"identity":"917674d2-ac2c-4a5a-9313-437c6082d4f6","added_by":"auto","created_at":"2025-01-15 15:53:37","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":5904417,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe S-shaped motif of Arg1 selectively binds to the substrate binding pocket of ERK2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic diagram of full-length ERK2 and deletion mutants (top). The interaction domains of Arg1 and ERK2 in \u003cem\u003eE. coli\u003c/em\u003e identified through molecular mapping assays (bottom).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B) \u003c/strong\u003eMapping of ERK2 regions that interact with Arg1. \u003cem\u003eE. coli\u003c/em\u003e was co-transfected with indicated constructs expressing His-Arg1 and GST-tagged peptides (bottom). Lysates from \u003cem\u003eE. coli \u003c/em\u003ewas utilized for Co-IP and western blotting with the indicated antibodies.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eAML12 cells were transfected with plasmids expressing full-length ERK2 and its mutants. Cell lysates were analyzed by Co-IP using anti-Flag M2 affinity gel.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eCo-immunoprecipitation of ERK2 with Arg1, Elk1, or RSK2 was performed in AML12 cells treated with siArg1 or siNC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Schematic diagram of full-length Arg1 and deletion mutants (top). Interaction domains of Arg1 and ERK2 in \u003cem\u003eE. coli\u003c/em\u003e identified by molecular mapping assays (bottom).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Schematic diagram of \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice transfected with Arg1\u003csup\u003e21\u0026amp;181\u003c/sup\u003e-mut virus or Arg1\u003csup\u003e304-322\u003c/sup\u003e-mut virus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Expression of His-Arg1 in \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice transfected with two types of His-Arg1 mutant virus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H-K) \u003c/strong\u003eGross views\u003cstrong\u003e (H)\u003c/strong\u003e, body weight (left), liver weight, and ratio of liver weight to body weight (right) \u003cstrong\u003e(I)\u003c/strong\u003e, representative liver gross view (left), H\u0026amp;E (scale bar, 100 μm) and Oil Red O staining (right) (scale bar, 50 μm) in liver sections of mice\u003cstrong\u003e (J)\u003c/strong\u003e, Serum and hepatic TG (left) and FFA (right) content \u003cstrong\u003e(K)\u003c/strong\u003e,\u003cstrong\u003e \u003c/strong\u003erelative protein levels of PPARγ pathway in liver tissues \u003cstrong\u003e(L)\u003c/strong\u003e of Arg1\u003csup\u003e21\u0026amp;181\u003c/sup\u003e-mut virus or Arg1\u003csup\u003e304-322\u003c/sup\u003e-mut virus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M) \u003c/strong\u003eSerum arginine concentration in \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice treated with Arg1\u003csup\u003e21\u0026amp;181\u003c/sup\u003e-mut virus or Arg1\u003csup\u003e304-322\u003c/sup\u003e-mut virus.\u003c/p\u003e\n\u003cp\u003eStatistical data represent the mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/c444b159d0bc83763316bd01.png"},{"id":73891814,"identity":"05011ab4-4bc8-4853-8cac-3bee16988d34","added_by":"auto","created_at":"2025-01-15 15:45:34","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":7320887,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eArg1 enhances lipogenesis in hepatocytes by inhibiting ERK2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-B) \u003c/strong\u003eExamination of\u0026nbsp;relative protein levels in the ERK2/RSK2 and ERK2/Elk1 signaling pathways across different experimental sconditions: liver tissue from \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice \u003cstrong\u003e(A)\u003c/strong\u003e, AML12 cells treated with siArg1 or siNC \u003cstrong\u003e(B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eDiagram illustrating that overactivation of ERK2 has been reported to suppress lipogenesis via at least two distinct mechanisms, ultimately leading to reduced hepatic lipogenesis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D, E)\u003c/strong\u003e The relative protein levels of the AKT/mTOR and ERK2/PPARγ signaling pathways were examined in liver tissue from HFD-induced\u003cem\u003e Arg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Immunohistochemical staining of Ras, p-P70-s6K, p-4E-BP-1 and c-Fos (nucleus) in liver sections from HFD-induced\u003cem\u003e Arg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice is shown. Scale bars, 25 µm.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G)\u003c/strong\u003e Western blotting analysis was conducted to assess the ERK2/RSK2 and ERK2/Elk1 signaling pathways in AML12 cells transfected with siRNA targeting Arg1 or an Arg1 overexpression (OE) plasmid for 24 hours, followed by treatment with either VX-11e or ERK2\u0026nbsp;activator TBHQ for an additional 48 hours.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H) \u003c/strong\u003eRepresentative Oil Red O staining (scale bar, 25 µm) was performed in AML12 cells transfected with either siRNA targeting Arg1 or an Arg1 overexpression (OE) plasmid for 24 hours, followed by treatment with either VX-11e or TBHQ for an additional 48 hours.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/181dd2f0b1c63e7cb4e17c56.png"},{"id":73891815,"identity":"572cec13-d616-4740-9ea9-fbccebb2830c","added_by":"auto","created_at":"2025-01-15 15:45:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":9176348,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePeptides targeting the Arg1-ERK2 interaction inhibit lipogenesis in mice with obesity and MASLD\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic diagram depicting the intravenous injection of a peptide via the tail vein in HFD-induced \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B-H) \u003c/strong\u003eBody weight\u003cstrong\u003e (B)\u003c/strong\u003e, gross views\u003cstrong\u003e (C)\u003c/strong\u003e, liver weight (left) and ratio of liver weight to body weight (right) \u003cstrong\u003e(D)\u003c/strong\u003e, gross liver morphology \u003cstrong\u003e(E)\u003c/strong\u003e, H\u0026amp;E staining (scale bar, 100 μm) and Oil red O staining (scale bar, 50 μm) of liver sections\u003cstrong\u003e (F)\u003c/strong\u003e, general diagram of abdominal fat \u003cstrong\u003e(G)\u003c/strong\u003e and micro-CT images \u003cstrong\u003e(H)\u003c/strong\u003e in HFD-fed \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice after treatment with ERK2\u003csup\u003e170-200\u003c/sup\u003e-Bio or ERK2\u003csup\u003e170-200\u003c/sup\u003e-Chem peptide.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I, J)\u003c/strong\u003e Representative H\u0026amp;E staining of peri-epididymal adipose tissue (scale bar, 100 μm), IHC staining (scale bar, 20 μm) \u003cstrong\u003e(I)\u003c/strong\u003e and western blotting of ERK2/PPARγ pathway \u003cstrong\u003e(J)\u003c/strong\u003e in liver tissues of HFD-fed \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice after treatment with ERK2\u003csup\u003e170-200\u003c/sup\u003e-Bio or ERK2\u003csup\u003e170-200\u003c/sup\u003e-Chem peptide. Statistical data presented as the mean ± SEM.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/54ac77bca72dc6e808ab4107.png"},{"id":73891822,"identity":"476b1800-cfc8-4760-bbec-30f1ce9fbe66","added_by":"auto","created_at":"2025-01-15 15:45:34","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":8648563,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/6a7a11071404775ed2b4b6ac.png"},{"id":105618270,"identity":"195eddbe-536d-44ee-83d5-70c3f3017da3","added_by":"auto","created_at":"2026-03-28 07:11:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":52836839,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/5ce56529-1bc1-4a1d-a09f-b7d9b2d95d29.pdf"},{"id":73891813,"identity":"e76789ca-51bf-496e-ae8d-ba3afbf31b2f","added_by":"auto","created_at":"2025-01-15 15:45:33","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":32660404,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S1. Construction of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eAlb\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003ecre\u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cem\u003e\u003cstrong\u003eArg1\u003c/strong\u003e\u003c/em\u003e\u003csup\u003e\u003cem\u003e\u003cstrong\u003efl/fl \u003c/strong\u003e\u003c/em\u003e\u003c/sup\u003e\u003cstrong\u003emice, related to Figure 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Schematic diagram of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl \u003c/em\u003e\u003c/sup\u003emice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B-D) \u003c/strong\u003eGenotyping \u003cstrong\u003e(B)\u003c/strong\u003e, immunohistochemical (IHC) staining \u003cstrong\u003e(C) \u003c/strong\u003eor western blotting\u003cstrong\u003e (D) \u003c/strong\u003eshowed that \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice were successfully constructed. Scale bars, 50 μm (H\u0026amp;E, GS, CK19, and PAS) and 100 μm (Arg1).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E) \u003c/strong\u003eThe survival curve of \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (n = 92).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F, G)\u003c/strong\u003e The average weekly food take \u003cstrong\u003e(F)\u003c/strong\u003e and serum AST (left) and ALT (right) concentrations \u003cstrong\u003e(G)\u003c/strong\u003e in \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice (n = 5). All data represent the mean ± SEM.\u003c/p\u003e","description":"","filename":"FigureS1.tif","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/741dcc5d8a3b63caf8f3dda1.tif"},{"id":73892879,"identity":"c27b9ace-9fa9-4765-9d7e-f49197434e77","added_by":"auto","created_at":"2025-01-15 15:53:36","extension":"tif","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":28395040,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S2. Short-term deletion of Arg1 inhibits lipid accumulation in hepatocytes and adipocytes,\u003c/strong\u003e \u003cstrong\u003erelated to Figure 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A) \u003c/strong\u003eSchematic diagram illustrating the process of infecting \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice with AAV8-TBG-cre virus to generate \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B-E)\u003c/strong\u003e Serum AST (left) and ALT (right) concentrations \u003cstrong\u003e(B)\u003c/strong\u003e, body weight \u003cstrong\u003e(C)\u003c/strong\u003e, gross view \u003cstrong\u003e(D)\u003c/strong\u003e, liver weight and the ratio of liver weight to body weight \u003cstrong\u003e(E)\u003c/strong\u003e of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice in response to AAV8-TBG-cre virus.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e Representative liver gross view, IHC staining of Arg1, H\u0026amp;E (scale bar, 50 μm), and Oil Red O staining (scale bar, 50 μm) in liver sections of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(G-N) \u003c/strong\u003eComparison of various physiological and biochemical parameters between \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice, including gross view of abdominal fat tissue\u003cstrong\u003e (G)\u003c/strong\u003e, micro-CT images and quantification of the areas of SAT and VAT\u003cstrong\u003e (H)\u003c/strong\u003e, representative gross views of adipose tissue (left) with corresponding tissue weights (right) \u003cstrong\u003e(I)\u003c/strong\u003e, epididymal fat tissue with H\u0026amp;E staining and cell size measurements\u003cstrong\u003e (J)\u003c/strong\u003e, serum and hepatic levels of TG and FFA\u003cstrong\u003e (K)\u003c/strong\u003e, and western blot analysis of liver extracts \u003cstrong\u003e(L)\u003c/strong\u003e. n = 5 per group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(M)\u003c/strong\u003e Serum arginine concentration\u003cem\u003e \u003c/em\u003ein\u003cem\u003e Arg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e, \u003cem\u003eAlb\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e, and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e","description":"","filename":"FigureS2.tif","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/181685a8708af4bfd486301a.tif"},{"id":73891849,"identity":"dd303a9a-de21-4925-9e43-28cea8bef8cc","added_by":"auto","created_at":"2025-01-15 15:45:36","extension":"tif","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":33474228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S3. Deletion of Arg1 or inhibition of its function protects against obesity and MASLD,\u003c/strong\u003e \u003cstrong\u003erelated to Figure 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B) \u003c/strong\u003eAML12 cells were transfected with siArg1 or siNC and then treated with oleic acid (OA) for 24 h. Oil Red O staining was used to detect the lipid accumulation\u003cstrong\u003e (A)\u003c/strong\u003e. Scale bar, 25 µm. Relative protein levels related to PPARγ pathway in OA-treated AML12 cells \u003cstrong\u003e(B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C-I)\u003c/strong\u003e Representative gross views of livers\u003cstrong\u003e (C)\u003c/strong\u003e, liver weight and liver/body weight ratio\u003cstrong\u003e (D)\u003c/strong\u003e, fat tissue area-to-total area ratio from micro-CT images \u003cstrong\u003e(E)\u003c/strong\u003e, representative gross views (scale bar, 1 cm) (left) and weights (right) of adipose tissue\u003cstrong\u003e (F)\u003c/strong\u003e, adipose cell size \u003cstrong\u003e(G)\u003c/strong\u003e, serum and hepatic levels of TG and FFA\u003cstrong\u003e (H)\u003c/strong\u003e, and western blot analysis of the PPARγ pathway\u003cstrong\u003e (I)\u003c/strong\u003e in the liver of \u003cem\u003edb/db\u003c/em\u003e mice treated with or without BEC. n = 5 per group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(J-O) \u003c/strong\u003eRepresentative gross views of livers \u003cstrong\u003e(J)\u003c/strong\u003e, liver weight (left) and ratio of liver weight to body weight (right)\u003cstrong\u003e (K)\u003c/strong\u003e, serum (left) and hepatic (right) TG and FFA content \u003cstrong\u003e(L)\u003c/strong\u003e, fat tissue area-to-total area ratio from micro-CT images \u003cstrong\u003e(M)\u003c/strong\u003e, gross views and weight of adipose tissue \u003cstrong\u003e(N)\u003c/strong\u003e, size of epididymal adipocytes \u003cstrong\u003e(O)\u003c/strong\u003e in \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice in response to an MCD diet (n = 5).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(Q-S)\u003c/strong\u003e Body weight (left), liver weight and ratio of liver weight to body weight (right) \u003cstrong\u003e(Q)\u003c/strong\u003e, serum TG and FFA (left) and hepatic TG and FFA (right) content\u003cstrong\u003e (R)\u003c/strong\u003e, relative protein levels in liver tissues \u003cstrong\u003e(S)\u003c/strong\u003e of \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice in response to an MCD diet (n = 5).\u003c/p\u003e","description":"","filename":"FigureS3.tif","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/2b13c088b8f27b2d1a26c298.tif"},{"id":73891829,"identity":"45559f98-9525-4def-91da-7e151f7c6ed9","added_by":"auto","created_at":"2025-01-15 15:45:35","extension":"tif","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":14876132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S4. Arg1 deficiency inhibits the AKT/mTOR pathway,\u003c/strong\u003e \u003cstrong\u003erelated to Figure 3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A, B)\u003c/strong\u003e Detection of AKT/mTOR proteins in \u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice \u003cstrong\u003e(A) \u003c/strong\u003eand in \u003cem\u003edb/db\u003c/em\u003e mice treated with either water or BEC \u003cstrong\u003e(B)\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C)\u003c/strong\u003e The siRNA (si1) that\u0026nbsp;efficiently knocks down Arg1 has been identified and selected.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D) \u003c/strong\u003eAML12 cells were transfected with siArg1 or siNC. Oil Red O staining was used to detect lipid accumulation in AML12 cells.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(E)\u003c/strong\u003e Representative western blot of AKT/mTOR/ PPARγ in AML12 cells transfected with siArg1 or siNC.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(F)\u003c/strong\u003e KEGG graph showing the upregulation of fatty acid metabolism in HFD-induced\u003cem\u003e Arg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e and \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice.\u003c/p\u003e","description":"","filename":"FigureS4.tif","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/f4f7fa87db47327ce86ef5b1.tif"},{"id":73891840,"identity":"1e07266b-a549-4abf-96d0-0f87b63e0191","added_by":"auto","created_at":"2025-01-15 15:45:36","extension":"tif","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":43819472,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S5. ERK2 inhibitor VX-11e compensates for the reduction of hepatocyte lipogenesis caused by Arg1 knockout,\u003c/strong\u003e \u003cstrong\u003erelated to Figure 4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A-G)\u003c/strong\u003e Gross views \u003cstrong\u003e(A)\u003c/strong\u003e, body weight (left), liver weight and ratio of liver weight to body weight (right) \u003cstrong\u003e(B)\u003c/strong\u003e, fat tissue area-to-total area ratio from micro-CT images\u003cstrong\u003e (C)\u003c/strong\u003e, gross views of fat tissues and weight of SAT or VAT \u003cstrong\u003e(D)\u003c/strong\u003e, H\u0026amp;E (Scale bars, 100 μm) in fat tissue sections \u003cstrong\u003e(E) \u003c/strong\u003eand quantification of the cell area \u003cstrong\u003e(F)\u003c/strong\u003e, serum or hepatic TG (left) and FFA (right) content\u003cstrong\u003e (G)\u003c/strong\u003e of HFD-induced \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice in response to VX-11e treatment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(H)\u003c/strong\u003e Schematic diagram of MCD-induced \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice treated with VX-11e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(I-M)\u003c/strong\u003e Representative liver gross view\u003cstrong\u003e (I)\u003c/strong\u003e, H\u0026amp;E staining (scale bar, 100 μm), and Oil Red O staining (scale bar, 50 μm) in liver sections \u003cstrong\u003e(J)\u003c/strong\u003e, body weight (left), liver weight, and the ratio of liver weight to body weight \u003cstrong\u003e(K)\u003c/strong\u003e, TG and FFA content in serum \u003cstrong\u003e(L)\u003c/strong\u003e and in liver tissue \u003cstrong\u003e(M)\u003c/strong\u003e, and western blotting of PPARγ in liver tissues \u003cstrong\u003e(N) \u003c/strong\u003efrom MCD-induced \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice treated with VX-11e. n = 5.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(O) \u003c/strong\u003eRelative mRNA levels of Arg1 and ERK2 were measured in AML12 cells transfected with siArg1 or siNC.\u003c/p\u003e","description":"","filename":"FigureS5.tif","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/1576c1fb97962edc83041de0.tif"},{"id":73892884,"identity":"9c42cd8c-fb6a-4be4-b161-72f9241fbb16","added_by":"auto","created_at":"2025-01-15 15:53:36","extension":"tif","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":16073428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S6. Peptides Targeting the Arg1-ERK2 interaction enhances the metabolic profile of obesity and MASLD mice,\u003c/strong\u003e \u003cstrong\u003erelated to Figure 7\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003e Direct interaction between Arg1 and peptide detected by Co-IP.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B)\u003c/strong\u003e Direct interaction between endogenous Arg1 and ERK2 in AML12 cells treated with peptide for 12 hours, detected by in situ PLA (Scale bar, 50 μm).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(C) \u003c/strong\u003eThe interactions between ERK2 and Arg1, Elk1, or RSK2 in AML12 cells treated with peptides were detected through Co-IP followed by western blot analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(D-I)\u003c/strong\u003e ALT (left) and AST (right) concentrations\u003cstrong\u003e (D)\u003c/strong\u003e, serum arginine concentration \u003cstrong\u003e(E)\u003c/strong\u003e, quantification of the areas of SAT and VAT in micro-CT images\u003cstrong\u003e (F)\u003c/strong\u003e, gross views and weight of SAT and VAT \u003cstrong\u003e(G)\u003c/strong\u003e, SAT and VAT cell size \u003cstrong\u003e(H)\u003c/strong\u003e, serum (left) and hepatic (right) TG and FFA\u003cstrong\u003e (I) \u003c/strong\u003ein HFD-fed \u003cem\u003eTBG\u003c/em\u003e\u003csup\u003e\u003cem\u003ecre\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eArg1\u003c/em\u003e\u003csup\u003e\u003cem\u003efl/fl\u003c/em\u003e\u003c/sup\u003e mice after treatment with ERK2\u003csup\u003e170-200\u003c/sup\u003e-Bio or ERK2\u003csup\u003e170-200\u003c/sup\u003e-Chem peptide.\u003c/p\u003e","description":"","filename":"FigureS6.tif","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/b78055208c8469b3e5f436e0.tif"},{"id":73891835,"identity":"6ba387a7-31da-41ff-aa08-726f74f1c8b0","added_by":"auto","created_at":"2025-01-15 15:45:36","extension":"tif","order_by":7,"title":"","display":"","copyAsset":false,"role":"supplement","size":12141124,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFigure S7. Peptides Targeting the Arg1-ERK2 interaction improves hepatic steatosis in MASLD mice,\u003c/strong\u003e \u003cstrong\u003erelated to Figure 7\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(A)\u003c/strong\u003eSchematic diagram of MCD -induced MASLD models in mice treated with peptide.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e(B-F)\u003c/strong\u003e Liver gross views \u003cstrong\u003e(B)\u003c/strong\u003e, H\u0026amp;E (scale bar, 100 μm) and Oil Red O staining (scale bar, 100 μm) in liver sections\u003cstrong\u003e (C)\u003c/strong\u003e, serum ALT (left) and AST (right) concentrations \u003cstrong\u003e(D)\u003c/strong\u003e, body weight (left), liver weight, and ratio of liver weight to body weight (right) \u003cstrong\u003e(E)\u003c/strong\u003e, serum (left) and hepatic (right) TG and FFA content\u003cstrong\u003e (F)\u003c/strong\u003e of MCD-induced mice in response to peptides (n = 5).\u003c/p\u003e","description":"","filename":"FigureS7.tif","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/152eb136f5af58927fa95e57.tif"},{"id":73892856,"identity":"608064ab-8702-43f8-8feb-067e43c0e2bf","added_by":"auto","created_at":"2025-01-15 15:53:35","extension":"docx","order_by":8,"title":"","display":"","copyAsset":false,"role":"supplement","size":48576,"visible":true,"origin":"","legend":"\u003cp\u003eKey Resource Table\u003c/p\u003e","description":"","filename":"KeyResourceTable.docx","url":"https://assets-eu.researchsquare.com/files/rs-5630831/v1/06003c705c541c19d9a36a40.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Arginase 1 promotes hepatic lipogenesis by regulating ERK2/PPARγ signaling in a non-canonical manner","fulltext":[{"header":"Introduction","content":"\u003cp\u003eObesity and its associated disorders, including metabolic dysfunction-associated steatotic liver disease (MASLD), type II diabetes (T2DM), cardiovascular disease, and various cancers, have escalated to epidemic levels globally.\u003csup\u003e\u003cspan additionalcitationids=\"CR2 CR3 CR4\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e MASLD encompasses a spectrum of conditions ranging from metabolic dysfunction-associated steatohepatitis (MASH) to states that present significant risk factors for cirrhosis, hepatocellular carcinoma (HCC), and numerous systemic metabolic disturbances.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e Recently, the U.S. Food and Drug Administration (FDA) has approved resmetirom as the first therapeutic agent specifically indicated for the treatment of MASH with fibrosis, a severe form of MASLD.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e Additionally, several medications targeting T2DM, obesity, or both conditions concurrently have been approved, such as pioglitazone and glucagon-like peptide 1 (GLP-1) receptor agonists.\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e However, a notable gap exists in the approval of pharmacotherapies for MASLD, primarily due to the lack of understanding of the mechanisms underlying the initiation and progression of obesity and MASLD.\u003c/p\u003e \u003cp\u003eObesity is characterized by the excessive accumulation of adipose tissue, a condition arising from the dysregulation of pivotal signaling pathways crucial for maintaining metabolic homeostasis.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e These pathways include phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT), AMP-activated protein kinase (AMPK), and mitogen-activated protein kinase (MAPK).\u003csup\u003e\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e In particular, the PI3K/AKT signaling pathway plays a critical role in enhancing lipid biogenesis while simultaneously inhibiting lipid degradation in adipose tissue.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e In the liver, the PI3K/AKT pathway stimulates lipogenesis via the mammalian target of rapamycin (mTOR), which upregulates peroxisome proliferator-activated receptor gamma (PPARγ), a key transcription factor involved in adipogenesis.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e AKT also regulates lipogenesis in the liver through a mTOR- independent pathways, and by exerting an inhibitory effect on AMPK, it further promotes lipogenesis and accumulation. \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eIn the liver, the mitogen-activated protein kinase (MAPK) pathway plays a pivotal role in maintaining metabolic homeostasis by delicately modulating the activities of enzymes and transcription factors that govern both fatty acid synthesis and catabolism.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e Notably, the MAPK and PI3K/AKT signaling pathways interact intricately within the signaling network, often mediated by shared upstream regulators such as receptor tyrosine kinases, GPCRs, and integrins.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e The MAPK family, particularly ERK, p38, JNK, and BMK1, represents a highly conserved array of serine-threonine kinases.\u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e Among them, the ERK1/ERK2 kinases have attracted garnered significant attention due to their extensive involved in diverse physiological and pathological processes. Upon activation by the Ras/Raf/MEK signaling cascade, ERK2 (also known as MAPK1) undergoes phosphorylation and becomes activated, subsequently translocating to the nucleus.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e In the nucleus, ERK2 phosphorylates a suite of transcription factors, including Elk1, ETS, c-Fos, Jun, and Myc, thereby initiating a cascade of downstream responses.\u003csup\u003e\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e The Ras/Raf/MEK/ERK signaling pathway meticulously regulates the expression of genes associated with lipid metabolism, influencing the expression levels of pivotal enzymes such as fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC), and consequently modulating the rate of fatty acid synthesis.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e Moreover, this pathway also impacts the β-oxidation process, exerting comprehensive control over lipid metabolism.\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e A key aspect of this pathway is its sophisticated negative feedback regulation mechanism, which not only dampens the activity of upstream components (Ras, Raf, MEK) but also establishes cross-regulatory interactions with other signaling pathways, such as the PI3K/AKT pathway.\u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e This intricate regulatory framework not only ensures signal fidelity and cellular stability but also offers fresh insights into the finely tuned mechanisms governing liver lipid metabolism.\u003c/p\u003e \u003cp\u003ePPARγ, a key member of the nuclear hormone receptor superfamily, acts as a critical transcription factor that controls the expression of numerous genes involved in lipid metabolism in both hepatocytes and adipocytes.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e In the liver, PPARγ promotes fatty acid synthesis, enhances the uptake of free fatty acids (FFA), and fosters the accumulation of triglycerides (TG) within hepatocytes.\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e,\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e Conversely, in adipocytes, PPARγ stimulates the storage of excess lipids, thereby reducing the influx of lipids into the liver and alleviating hepatic lipid accumulation.\u003csup\u003e\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e Given its critical role in modulating lipid metabolism in both cell types, PPARγ represents an attractive therapeutic target.\u003c/p\u003e \u003cp\u003eArginase, primarily existing as arginase 1 (Arg1) in mammals, is predominantly expressed in the cytoplasm of hepatocytes. It catalyzes the conversion of L-arginine to L-ornithine and urea, playing a vital role in the urea cycle for the detoxification of ammonia.\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e Children with congenital deficiency of Arg1 exhibit hyperargininemia, accompanied by spastic paraparesis, progressive neurological and intellectual deficits, and notably, persistent growth retardation.\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e This growth retardation may be attributed to a deficiency in polyamines and an excess of arginine. Polyamines, including putrescine, spermidine, and spermine, are synthesized from L-ornithine and are essential for cell proliferation, growth, and tissue repair.\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e,\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e Polyamines are pivotal in fat metabolism. Once secreted by cells, they interact with adipocytes, promoting adipose tissue vascularization and stimulating lipolysis.\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e Specifically, polyamines like spermidine enhance the production of cAMP, the second messenger of β-adrenergic receptors (βAR), thereby further augmenting lipolysis. Polyamines also regulate adipocyte generation by modulating the expression of key transcription factors essential for the differentiation of preadipocytes into mature adipocytes.\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e Moreover, by facilitating lipolysis and angiogenesis, polyamines help maintain the homeostasis of white adipose tissue (WAT), thereby preventing obesity and insulin resistance.\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eElevating circulating L-arginine levels has been shown to alleviate MASH-related liver inflammation. In the alternative pathway for arginine hydrolysis, L-arginine serves as the sole substrate for both Arg1 and nitric oxide synthase (NOS).\u003csup\u003e\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e NOS metabolizes L-arginine into L-citrulline and nitric oxide (NO). An imbalance between Arg1 and NOS can disrupt NO production, leading to an imbalance in reactive oxygen species (ROS), which can result in ROS-induced damage accumulation in MASLD.\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e It appears that Arg1 plays a potential role in the development of MASLD by regulating the synthesis of either polyamines or NOS, which warrants further exploration.\u003c/p\u003e \u003cp\u003eIn this study, we observed that Arg1, beyond its canonical role in L-arginine metabolism, exhibits a moonlighting function through physical interaction with the substrate-binding pocket of ERK2. This interaction subsequently induces the degradation of ERK2 protein, leading to the upregulation of PPARγ signaling and lipid accumulation in both the liver and lipocytes. Targeting the Arg1-ERK2 interaction represents a promising therapeutic strategy for ameliorating obesity and MASLD.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cem\u003e1. Disruption of Arg1 reduces lipid accumulation in hepatocytes and adipocytes\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo delve into the role of hepatic Arg1 \u003cem\u003ein vivo\u003c/em\u003e in modulating hepatic lipid metabolism, we generated liver-specific \u003cem\u003eArg1\u003c/em\u003e knockout mice using the Alb-Cre-loxP system (\u003cem\u003eAlb\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e; Figures S1A-S1D). Postnatally, the \u003cem\u003eAlb\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice began to display characteristic symptoms of Arg1 deficiency, including lethargy, sluggish movement, and growth retardation, culminating in a 52% mortality rate within the first four weeks (Figure S1E). Consequently, only mice that survived until eight weeks were chosen for subsequent experimentation. Despite exhibiting no significant difference in food intake (Figure S1F), the \u003cem\u003eAlb\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice notably had lower body weight and size compared to their same-sex littermate controls (Figures 1A and 1B). Although the hepatic lobular structure, zonation, and glycogen synthesis appeared relatively normal, as evidenced by H\u0026amp;E (hematoxylin and eosin) staining, immunohistochemistry (IHC) staining for glutamine synthetase (GS) and cytokeratin 19 (CK19), and periodic acid-Schiff (PAS) staining, respectively (Figure S1C), the ablation of Arg1 led to a marked decrease in liver size, weight, and hepatocyte size (highlighted by IHC staining for \u0026beta;-catenin) (Figures 1C and 1E). Strikingly, hepatocytes\u003cem\u003e\u0026nbsp;\u003c/em\u003ein\u003cem\u003e\u0026nbsp;Arg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice, which retained intact Arg1 expression and served as wildtype control, contained uniformly distributed fine lipid droplets. In contrast, these lipid droplets were scarcely observed in the hepatocytes from \u003cem\u003eAlb\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice, as revealed by oil red O staining (Figure 1E). Furthermore, this deletion also induced mild liver injury, manifested by slightly elevated serum enzyme levels (Figure S1G).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe liver primarily synthesizes TG, which are then transported into and stored in adipose tissue for future energy use. We observed that \u003cem\u003eAlb\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice had a significantly reduced mass of adipose tissue in the visceral adipose tissue (VAT) region, encompassing the epididymal fat, renal fat capsules, and mesenteric adipose tissue, compared to \u003cem\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Figure 1F). This finding was corroborated by a microcomputed tomography (micro-CT) scan, which captured abdominal fat accumulation at the level of the fourth lumbar vertebra and revealed a marked decrease in the volume of both VAT and subcutaneous\u0026nbsp;adipose tissue (SAT) in the Arg1-deficient mice (Figures 1G and 1H). This was further substantiated by the weight of the collected VAT and SAT, revealing that the Arg1-deficient mice had less than one-sixth of the adipose fat and a significantly reduced ratio of fat tissue weight to body weight compared to the control mice (Figure 1I). Histologic analysis revealed that adipocytes in \u003cem\u003eAlb\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice were significantly smaller in size compared to those in control mice (Figure 1J). Additionally, Arg1-deficiency resulted in a notable decrease in both serum and liver tissue levels of TG and FFA (Figures 1K and 1L). Consistent with these observations, the livers of \u003cem\u003eAlb\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice exhibited a marked downregulation of genes involved in fatty acid synthesis, particularly those in the PPAR\u0026gamma; pathway, including PPAR\u0026gamma; itself and its downstream targets such as SREBP1, FASN, CD36, and AP2,\u0026nbsp;as evidenced by both western blotting and quantitative PCR analysis (Figures 1M and 1N).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTo eliminate the toxic effect of hyperargininemia on the early development, growth and fat metabolism of mice, we established an inducible Arg1-knockout mouse model designated as \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e. This was achieved by administering AAV8-TBG-Cre via tail vein injection to adult \u003cem\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Figure S2A). Four weeks after injection, the \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice exhibited normal behavior and did not show any signs of mortality or liver damage, as evidenced by normal levels of ALT and AST (Figure S2B). Similar to the \u003cem\u003eAlb\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice, the \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice displayed a decrease in body weight, liver weight, and lipid accumulation in both the liver and adipose tissue (Figures S2C-S2J). Additionally, there was a reduction in the levels of TG and FFA in both the serum and liver (Figure S2K). Notably, the inducible deficiency of Arg1 led to a profound downregulation of the expression of PPAR\u0026gamma;, FASN, and SREBP1 (Figure S2L).\u003c/p\u003e\n\u003cp\u003eAlthough inducible Arg1-deficiency notably decreased serum arginine levels, they remained robustly elevated compared to baseline levels (Figure S2M). To further investigate the impact of Arg1 inhibition on lipid metabolism, we treated cultured mouse AML12 hepatocytes with arginase inhibitor S-(2-bromoethyl)-L-cysteine (BEC). The tetrahedral BEC boronate anion mimics the tetrahedral intermediate stage of the arginine hydrolysis reaction, enabling it to bind tightly to the arginase active site.\u003csup\u003e42\u003c/sup\u003e This ultimately results in the formation of an enzyme-inhibitor complex, thereby impeding arginase activity. AML12 hepatocytes, when treated with oleic acid (OA), exhibited a significant increase in both the number and size of lipid droplets. However, BEC treatment dramatically reduced this lipid droplet accumulation (Figure S3A). In parallel, we observed a decrease in the protein levels of PPAR\u0026gamma;, FASN, and SREBP1 (Figures S3B).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e2. Arg1 disruption protects against genetically- or diet-induced obesity and MASLD\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo further confirm that Arg1 deficiency, rather than hyperargininemia, is the primary cause of lipogenesis disorders, we administered BEC to \u003cem\u003edb/db\u003c/em\u003e mice (Figure 2A). \u003cem\u003edb/db\u003c/em\u003e mice, characterized by a Leptin mutation that leads to severe obesity, are an ideal model for studying the effects of Arg1 inhibition, as they exhibit cardioprotective benefits from L-arginine supplementation.\u003csup\u003e43\u003c/sup\u003e We initiated BEC treatment in \u003cem\u003edb/db\u003c/em\u003e mice at 8 weeks of age and continued for 4 weeks. BEC treatment significantly suppressed body weight gain, resulting in a final body weight that was approximately two-thirds of that observed in untreated mice (Figure 2B and S3C). Moreover, BEC-treated mice displayed a remarkable reduction in lipid accumulation in the liver and adipose tissue, accompanied by decreased adipocyte size, reduced serum and hepatic TG and FFA levels, and downregulated expression of PPAR\u0026gamma;, FASN, and SREBP1 (Figures 2C-2F and S3D-S3I). Notably, BEC treatment did not alter serum L-arginine levels (Figure 2G), possibly due to excessive L-arginine consumption by other tissue.\u003c/p\u003e\n\u003cp\u003eTo investigate whether Arg1 disruption plays a role in improving diet-induced obesity and fatty liver, we initiated a 12-week high-fat diet (HFD) regimen for both the \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice and \u003cem\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mouse model (Figure 2H). Notably, the \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice demonstrated a marked reduction in body weight and liver size compared to their \u003cem\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e counterparts (Figures 2I and 2J; Figures S3J and S3K). Moreover, these mice displayed a significantly diminished accumulation of lipids in both the liver and adipose tissue, along with decreased serum and hepatic concentrations of TG and FFA (Figures 2K-2N, Figures S3L-S3O). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast to HFD-induced obesity-associated MASLD, which manifests as prominent macrovesicular steatosis in the liver along with a marked increase in both body weight and visceral fat mass (Figure 2K), mice subjected to a methionine- and choline- deficient (MCD) diet typically exhibit an earlier onset of steatosis and inflammatory cell infiltration in the liver. Paradoxically, however, this is accompanied by a decrease in body weight. To investigate this further, we administered MCD to \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Figure 2O). After four weeks of MCD administration, \u003cem\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice exhibited a drastic exacerbation of liver steatosis, characterized by an enlarged liver size, increased weight, and a yellowish, oily appearance (Figures 2P, and S3Q). This was further substantiated histologically by the presence of macrovesicular steatosis, hepatocellular ballooning, and frequent infiltration of inflammatory cells, as evidenced by an immunohistocheminstry staining for the pan-leukocyte marker CD45 (Figure 2Q). Notably, the ablation of Arg1 led to a significant improvement in liver histology (Figures 2P and 2Q). Consistent with this, serum and hepatic levels of TG and FFA were also markedly lower in \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Figure S3R). Additionally, proteins involved in lipogenesis were decreased in response to Arg1 ablation (Figure S3S).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e3. Deficiency of Arg1 downregulates the AKT/mTOR/\u003c/em\u003e \u003cem\u003ePPAR\u0026gamma; signaling pathway\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;Our findings suggest that disruption of hepatic Arg1 has the potential to ameliorate genetic- or diet-induced obesity and MASLD by inhibiting the PPAR\u0026gamma; pathway. Importantly, this effect appears to be independent of hyperargininemia and is specifically attributable to the inhibition of Arg1 itself. To further elucidate the mechanism underlying Arg1\u0026rsquo;s regulation of lipid metabolism in hepatocytes, we conducted RNA sequencing analysis on liver tissues from \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice. The resulting heatmap revealed that genes targeted by PPAR\u0026gamma; or those involved in adipogenesis regulation were among the most downregulated in \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e liver (Figures 3A and 3B). This observation was further corroborated by gene set-enrichment analysis (GSEA), which demonstrated a marked inhibition of PPAR\u0026gamma; target gene expression in hepatocytes from \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice compared to controls (Figure 3C). Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis revealed that Arg1 deficiency significantly inhibited the PI3K/AKT signaling pathways (Figure 3D). PI3K/AKT/mTOR signaling cascade plays a pivotal role in balancing lipogenesis and oxidation by modulating the PPAR\u0026gamma; pathways, specifically via phosphorylation of mTOR, ultimately enhancing the uptake of FFA and promoting TG accumulation.\u003csup\u003e44\u003c/sup\u003e Consistent with the observed decrease in PPAR\u0026gamma; expression in the \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e liver, there was a corresponding reduction in the protein levels of hepatic p-AKT, p-mTOR, as well as its downstream proteins p70-S6K and p-4E-BP (Figure 3E).\u0026nbsp;These findings were further substantiated in \u003cem\u003eAlb\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice and \u003cem\u003edb/db\u003c/em\u003e mice treated with BEC (Figures S4A and S4B).\u0026nbsp;Additionally, knocking down\u0026nbsp;the Arg1 gene in AML12 cells using a small-interfering RNA specifically targeting Arg1 (siArg1) attenuated lipid droplets and suppressed the AKT/mTOR/PPAR\u0026gamma; signaling pathway (Figures S4C-S4E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe conducted a further analysis of mRNA sequencing data from HFD-fed \u003cem\u003eArg1\u003csup\u003efl/fl\u0026nbsp;\u003c/sup\u003e\u003c/em\u003eand \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice. The results revealed that, compared to \u003cem\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice, HFD-induced \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice exhibited a marked downregulation of PPAR\u0026gamma; pathways and adipogenesis-related genes (Figures 3F-3H, and S4F). Notably, GSEA indicated that genes involved in the oxidative damage response were suppressed in \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice, providing further evidence of reduced lipid accumulation in the Arg1-deficient liver (Figure 3G).\u003c/p\u003e\n\u003cp\u003eCollectively, our findings present compelling evidence that the absence of Arg1 inhibits the AKT/mTOR/PPAR\u0026gamma; signaling pathway, leading to a significant downregulation of genes crucial for lipogenesis. However, the precise mechanism underlying how Arg1 activates this signaling pathway to regulate hepatic lipid metabolism remains elusive.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e4. Arg1 binds to and promotes ERK2 ubiquitination-mediated degradation\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eTo delve deeper into the underlying mechanism of Arg1\u0026rsquo;s involvement in lipid metabolism, we conducted co-immunoprecipitation (Co-IP) and mass spectrometry analysis to identify proteins that interact with Arg1. A total of 1444 peptides corresponding to 338 proteins were identified. KEGG pathway analysis indicated that the top 100 proteins, ranked by peptide spectrum match (PSM), were enriched in metabolism signaling pathways, including MAPK signaling, arginine biosynthesis, insulin signaling, the TCA cycle, and glycose metabolism (Figure 4A).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAmong the proteins that potentially interact with Arg1, ERK2 emerged as the most likely candidate due to several reasons: (1) Four peptides with high-scoring PSMs belonged to ERK2. Notably, the peptide with the highest affinity featured a unique substrate-binding pocket structure, consisting of Ala\u003csup\u003e187\u003c/sup\u003e-Thr\u003csup\u003e188\u003c/sup\u003e-Arg\u003csup\u003e189\u003c/sup\u003e-Trp\u003csup\u003e190\u003c/sup\u003e (or ATRW),\u003csup\u003e45,46\u003c/sup\u003e which serves as a versatile and flexible binding site for ERK2 and its interacting proteins (Figures 4B and 4C). The binding of these partner proteins to ERK2, including activators (such as MEK1/2), inactivators (such as MKP3), and substrates (such as RSK2 and Elk1),\u003csup\u003e47-49\u003c/sup\u003e occurs in a mutually exclusive manner, each mediating distinct cellular processes; (2) In the proteins detected by Co-IP, a clear band located between 35-55 kDa was observed in the strips pulled down by the Arg1 antibody. ERK2, with a molecular weight of 42 kDa, matches the size of this corresponding protein band (Figure 4A); (3) As a crucial member of the MAPK family, ERK2 plays pivotal roles in various cellular processes. By interacting with the AKT/mTOR/PPAR\u0026gamma; signaling pathway, it exerts key regulatory effects on lipid metabolism, leading to the amelioration of steatosis in the liver.\u003c/p\u003e\n\u003cp\u003eWe subsequently delved into whether Arg1 directly binds to ERK2. As anticipated, the Co-IP results demonstrated that Arg1 interacts with ERK2 in liver tissue extracted from \u003cem\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Figure 4D). This direct binding was further validated by the Duolink \u003cem\u003ein situ\u003c/em\u003e proximity ligation assay (PLA) in AML12 cells (Figure 4E). Furthermore, in AML12 cells transfected with Flag-tagged ERK2 plasmid, Flag pull-down and Co-IP experiments confirmed the interaction between exogenous Arg1 and Flag-ERK2 (Figure 4F). Next, we examined whether BEC, the Arg1 inhibitor, could disrupt the binding interaction between Arg1 and ERK2. In AML12 cells treated with BEC, we observed a lack of binding of Arg1 and ERK2, as evidenced by both Co-IP and Duolink \u003cem\u003ein situ\u003c/em\u003e PLA experiments (Figures 4G and 4H).\u003c/p\u003e\n\u003cp\u003eTo further determine whether Arg1 promotes hepatic lipogenesis by suppressing ERK2 activity, we employed the ERK-selective inhibitor VX-11e to inhibit ERK2 activation in HFD-induced \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Figure 4I). Administration of VX-11e for 4 weeks significantly reversed the decreases in body weight, liver weight, and liver-to-body weight ratio observed in \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Figures S5A and S5B). Additionally, by augmenting the PPAR\u0026gamma; pathway, VX-11e exacerbated hepatic steatosis and lipid accumulation in adipose tissue (Figures 4J-4N, Figure S5C-S5G). In MCD-treated \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice, VX-11e administration similarly impacted hepatic lipogenesis and the expression of PPAR\u0026gamma; pathway (Figures S5H-S5N). Our findings suggest that Arg1 fosters hepatic lipogenesis in an ERK2-dependent manner.\u003c/p\u003e\n\u003cp\u003eWe discovered that knocking down Arg1 led to increased protein levels of ERK2 in both AML12 cells and \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice, without affecting the expression of ERK2 mRNA (Figures 4O and 4P, Figure S5O). Previous research has demonstrated that ERK2 can be degraded through ubiquitination. MG132, a proteasome inhibitor, promotes the accumulation of ubiquitinated proteins.\u003csup\u003e50\u003c/sup\u003e When AML12 cells were treated with MG132, we observed decreased accumulation of ubiquitinated ERK2 in response to Arg1-knockdown, as evidenced by \u003cem\u003ein vitro\u003c/em\u003e ubiquitination assays (Figure 4Q). This suggests that Arg1 may enhance the degradation of ERK2 through the ubiquitin-proteasome pathway.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e5. The S-shaped motif of Arg1 selectively binds to the substrate binding pocket of ERK2\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe next set out to unravel the binding mechanism between Arg1 and ERK2. We expressed glutathione-S-transferase (GST)-tagged Arg1, His-tagged ERK2 mutants, and ERK2-derived amino acid fragments in \u003cem\u003eE. coli\u003c/em\u003e. Our findings demonstrate that Arg1 is capable of directly binding to the full-length ERK2 protein without requiring any additional mediators (Figure 5A). An ERK2 mutant, harboring mutations in three peptides but retaining its critical ATRW sequence, displayed robust binding affinity to Arg1. In contrast, an ERK2 mutant carrying mutations within the critical 170-190 amino acid sequence exhibited significantly diminished binding to Arg1 (Figure 5A). Furthermore, the molecular mapping assay revealed that Arg1 interacts with the ERK2 amino acid fragment spanning residues 170-190 in \u003cem\u003eE. coli\u003c/em\u003e (Figure 5B).\u003c/p\u003e\n\u003cp\u003eThe substrate-binding pocket structure, ATRW, located within the peptide sequence spanning residual 170-190 of ERK2, is integral for binding and activating enzymes such as ribosomal S6 kinase 2 (RSK2) and ETS-domain containing protein (Elk1).\u003csup\u003e47\u003c/sup\u003e To identify the exact site of Arg1 binding to the ERK2 substrate-binding pocket, we transfected AML12 cells with plasmids expressing various ERK2 mutations. In comparison to the non-mutated control (NC), mutations in the A, T, or R residues of ERK2 considerably diminished its binding to Arg1, except for mutations affecting the W residue. Notably, mutations in the R residue and those encompassing the entire ATRW region resulted in the most significant decrease in Arg1 binding (Figure 5C). Intriguingly, mutating the ATRW sequence in ERK2 led to a reduction in its ubiquitination (Figure 5C).\u003c/p\u003e\n\u003cp\u003eBoth RSK2 and Elk1 are known to bind to ERK2 via its substrate binding pocket. Based on this, we hypothesized that Arg1 would compete with them for this binding site. Our data revealed that ERK2 carrying the ATRW mutation prevented its binding to both RSK2 and Elk1 (Figure 5C). When Arg1 was knocked down, ERK2 was able to associate with a higher amount of RSK2 and Elk1, as evidenced by Co-IP experiments in AML12 cell (Figure 5D).\u0026nbsp;Similarly, we observed consistent results in AML12 cells treated with Arg1 inhibitor BEC (Figure 4G).\u003c/p\u003e\n\u003cp\u003eArg1 is a metalloproteinase featuring a unique \u0026alpha;/\u0026beta; fold structure and a binuclear manganese center. It is well established that three key regions within the Arg1 molecule are essential for L-arginine hydrolysis: the hydrolytic site, the L-R amino acid site (composed of Arg\u003csup\u003e21\u0026nbsp;\u003c/sup\u003eand Asp\u003csup\u003e181\u003c/sup\u003e, which bind to the charged carboxyl group and the R-amino group of L-arginine, respectively), and the double hydrogen site (formed by His\u003csup\u003e101\u003c/sup\u003e and His\u003csup\u003e126\u003c/sup\u003e).\u003csup\u003e51,52\u003c/sup\u003e Additionally, all mammalian arginases share a C-terminal S-shaped motif spanning residues 304-322.\u003csup\u003e53,54\u003c/sup\u003e The proper assembly of this motif into a homotrimer is essential for Arg1\u0026rsquo;s normal enzymatic activity, quaternary structure, and cooperative properties. Therefore, the amino acid residues His\u003csup\u003e101\u003c/sup\u003e and His\u003csup\u003e126\u003c/sup\u003e, Arg\u003csup\u003e21\u003c/sup\u003e and Asp\u003csup\u003e181\u003c/sup\u003e, as well as the sequence spanning residues 304-322, are indispensable for L-arginine binding in Arg1. We successfully synthesized the Arg1 mutants in the \u003cem\u003eE. coli\u0026nbsp;\u003c/em\u003esystem. Notably, mutating the S-shaped motif (residues 304-322) in Arg1 completely abolished its binding affinity to ERK2, whereas mutations in the other regions exhibited minimal impact on binding (Figure 5E).\u003c/p\u003e\n\u003cp\u003eTo assess the significance of the S-shaped motif in Arg1 for hepatic lipogenesis, we administered AAV8 viruses expressing Arg1\u003csup\u003e21\u0026amp;181\u003c/sup\u003e-mut (AAV8-TBG-Arg1-mut\u003csup\u003e21\u0026amp;181\u003c/sup\u003e-His) or Arg1\u003csup\u003e304-322\u003c/sup\u003e-mut (AAV8-TBG-Arg1\u003csup\u003e304-322\u003c/sup\u003e-mut-His) into\u003cem\u003e\u0026nbsp;TBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Figure 5F). The levels of His-tagged protein in the liver extracts from the Arg1-mutant groups were stably expressed (Figure 5G). The Arg1\u003csup\u003e21\u0026amp;181\u003c/sup\u003e-mut protein, which could bind to ERK2 but was unable to recognize arginine, partially mitigated the reduction in hepatic lipogenesis observed in \u003cem\u003eTBG\u003csup\u003eCre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice. This was evidenced by higher liver weight, body weight, and liver-to-body weight ratios compared to those injected with a vector (Figures 5H and 5I). Furthermore, AAV8-Arg1\u003csup\u003e21\u0026amp;181\u003c/sup\u003e-mut treatment substantially increased hepatic lipid accumulation (Figure 5J), elevated TG and FFA levels in both serum and liver tissue (Figure 5K) and enhanced the PPAR\u0026gamma; pathway in the \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice (Figure 5L). On the contrary, the AAV8-Arg1\u003csup\u003e304-322\u003c/sup\u003e-mut protein, which could not combine with ERK2, exhibited a negative effect on reversing the phenotype in response to Arg1 ablation. Interestingly, treatment with neither mutant virus significantly reduced serum arginine levels, demonstrating that a mutation in the ERK2 binding site of Arg1 also compromises its enzymatic function (Figure 5M). These data further support our hypothesis that the role of Arg1 in regulating lipogenesis is independent of its enzymatic activity, but rather depends on a physical combination with ERK2.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e6\u003c/em\u003e\u003cem\u003e. Arg1 enhances lipogenesis in hepatocytes via AKT/mTOR/PPAR\u0026gamma; and Elk1/c-Fos/PPAR\u0026gamma; signaling pathways\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe have observed that the disruption of Arg1 leads to the suppression of the AKT/mTOR/PPAR\u0026gamma; signaling pathway (Figures 3, and S7). However, the crosstalk between Arg1, mediated by ERK2, and the AKT/mTOR/PPAR\u0026gamma; pathways remains elusive. In addition to decreasing EKR2 ubiquitination and degradation, we discovered that the disruption of Arg1 facilitates the nuclear translocation of ERK2 (Figures 6A and 6B). Overactivation of ERK2 has been reported to impede lipogenesis through at least two distinct mechanisms, as depicted in Figure 6C: (1) In the cytoplasm, ERK2 undergoes phosphorylation via the Ras-Raf-MEK cascade, enabling its substrate-binding pocket to bind to RSK2. This interaction initiates a negative feedback loop that suppresses the overactivation of Ras-Raf-MEK signaling, further inhibiting the AKT/mTOR signaling cascade and ultimately suppressing PPAR\u0026gamma;. (2) Phosphorylated ERK2 translocates to the nucleus, where its substrate-binding pocket binds to Elk1, promoting the ERK2/Elk1/c-Fos signaling pathway, which in turn inhibits the PPAR\u0026gamma; signaling.\u003csup\u003e\u0026nbsp;47,49,55\u003c/sup\u003e Western blot results revealed that in the livers of \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice, Ras, p-Raf, and p-MEK were obviously decreased in the cytoplasm, whereas c-Fos and Elk1 in the nucleus were significantly elevated (Figures 6A and 6B). Furthermore, HFD-fed \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e and \u003cem\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice confirmed that Arg1 promotes hepatic lipid accumulation and adipose tissue lipid deposition through ERK2/PPAR\u0026gamma; pathways, as elucidated by western blotting and immunohistochemistry staining in mouse liver (Figures 6D-6F).\u003c/p\u003e\n\u003cp\u003eWe next treated AML12 cells with ERK2 inhibitor VX-11e, ERK2 activator TBHQ, Arg1 siRNA, and Arg1 overexpression virus (OE), respectively. The results confirmed that the absence of Arg1 leads to excessive upregulation of ERK2, which in turn downregulates the PPAR\u0026gamma; signaling pathway, thereby inhibiting hepatic lipogenesis. Importantly, this inhibition can be partially reversed by the ERK2 inhibitor. Conversely, Arg1 overexpression downregulates ERK2 protein levels and upregulates the PPAR\u0026gamma; pathway, promoting hepatic lipogenesis. Notably, the ERK2 activator attenuates this promotional effect (Figures 6G and 6H).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e7\u003c/em\u003e\u003cem\u003e. Peptide targeting Arg1\u003c/em\u003e\u003cem\u003e-\u003c/em\u003e\u003cem\u003eERK2 interaction improves the metabolic profile of obesity and MASLD in mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eWe have identified Arg1 as a promising therapeutic target for obesity and MASLD. In contrast to small-molecule drugs, peptides can be precisely engineered to target specific molecular receptors, offering enhanced selectivity.\u003csup\u003e56,57\u003c/sup\u003e We synthesized two peptides: one biologically derived (ERK2\u003csup\u003e170-200\u003c/sup\u003e-Bio peptide) and one chemically synthesized (ERK2\u003csup\u003e170-200\u003c/sup\u003e-Chem peptide). These peptides encompass amino acids 170 to 190 within the substrate-binding pocket of ERK2, which are essential for its interaction with Arg1. Additionally, the Arg\u003csup\u003e192\u003c/sup\u003e residual was included due to its importance for the conformational stability and functionality of the pocket structure.\u0026nbsp;In the inactive state, the substrate-binding pocket of ERK2 is blocked by Arg\u003csup\u003e192\u003c/sup\u003e. However, phosphorylation of Thr\u003csup\u003e183\u0026nbsp;\u003c/sup\u003eand Tyr\u003csup\u003e185\u003c/sup\u003e triggers a conformational change, enabling the pocket to become exposed and accessible for substrate recognition and binding. Our findings indicate that GST-tagged peptides effectively bind to Arg1 within AML12 cells (Figure S6A). Co-IP and Duolink \u003cem\u003ein situ\u003c/em\u003e PLA assays revealed that these peptides prevented the binding between Arg1 and ERK2, while also increasing the binding of ERK2 to both RSK2 and Elk1 (Figures S6B and S6C).\u003c/p\u003e\n\u003cp\u003eWe next evaluated the efficacy of disrupting the Arg1-ERK2 interaction\u003cem\u003e\u0026nbsp;in vivo\u003c/em\u003e by administering 50 \u0026micro;g/kg of either the ERK2\u003csup\u003e170-200\u003c/sup\u003e-Bio peptide, the ERK2\u003csup\u003e170-200\u003c/sup\u003e-Chem peptide, or saline via daily tail injections to wild-type mice fed HFD for 4 weeks (Figure 7A). Compared to the saline group, both the ERK2\u003csup\u003e170-200\u003c/sup\u003e-Bio peptide and the ERK2\u003csup\u003e170-200\u003c/sup\u003e-Chem peptide significantly reduced body weight, liver weight, the liver-to-body weight ratio, and levels of ALT and AST (Figures 7B-7E, Figure S6D). Moreover, peptide therapy did not cause hyperargininemia (Figure S6E). Additionally, histological analysis, micro-CT imaging, and quantification\u0026nbsp;of TG and FFA in both serum and liver revealed significantly lower lipid accumulation in the liver and adipose tissue of peptide-treated mice (Figures 7F-7I, Figures S6F-S6I). Most notably, peptide treatment provided significant protection against Arg1-mediated ERK2 degradation in obese mice. Specifically, it enhanced RSK2 levels in the cytoplasm, thereby inhibiting the Ras/Raf and AKT/mTOR signaling pathways. Within the nucleus, the activation of Elk1 led to a surge in c-Fos expression, ultimately suppressing the expression of PPAR\u0026gamma; (Figures 7I and 7J).\u003c/p\u003e\n\u003cp\u003eNext, we explored whether peptide injection could attenuate the progression of MCD-induced MASLD. We administered peptides or saline daily to wild-type mice for 2 weeks, concurrently with the MCD diet (Figure S7A). Our observations revealed that peptide injection improved metabolic profiles in MCD-fed mice compared to saline-treated mice (Figures S7B-S7F). These data indicate that both peptides can effectively treat MASLD by disrupting the Arg1-ERK2 interaction.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study reveals an unprecedented role of Arg1 in regulating hepatocyte\u0026nbsp;lipogenesis\u0026nbsp;through a non-enzymatic mechanism. Specifically, Arg1 interacts with ERK2 via its S-shaped motif, binding to the substrate-binding pocket of ERK2. This interaction not only facilitates ERK2 degradation but also inhibits its binding to RSK1 and Elk1. Consequently, this process enhances the PPARγ pathway, promoting hepatocyte lipogenesis and contributing to the development of obesity and MASLD (Figure 8).\u003c/p\u003e\n\u003cp\u003eThe functions of Arg1 have long been ascribed solely to its enzymatic activity. In hepatocytes, Arg1 catalyzes the conversion of free L-arginine into ornithine, a pivotal step in the urea cycle and ammonia metabolism. Arg1 also competes with NOS for L-arginine, thereby inhibiting NO production and weakening the anti-oxidative effect, particularly in hepatocytes and inflammatory cells such as macrophages and neutrophils.\u003csup\u003e39-41,58-60\u0026nbsp;\u003c/sup\u003eFurthermore, via its enzymatic role, Arg1 exerts an indirect modulation on lipid metabolism, given that polyamines, the byproducts of arginine catabolism, are recognized inhibitors of lipogenesis. Hence, the hypothesis arose that Arg1 might inhibit lipogenesis and thus obesity and MASLD. Surprisingly, however, studies in both genetically and chemically Arg1-deficient mice revealed that Arg1 actually promotes lipogenesis and MASLD.\u003c/p\u003e\n\u003cp\u003eOur results exclude the possibility that inhibiting Arg1 reduces lipogenesis via hyperargininemia. For instance, in\u003cem\u003e\u0026nbsp;db/db\u003c/em\u003e mice, suppressing Arg1 activity did not induce hyperargininemia but significantly decreased lipogenesis and obesity. Our \u003cem\u003ein vitro\u003c/em\u003e experiments further confirmed that silencing Arg1 ameliorates the accumulation of lipid droplets in hepatocytes. In subsequent studies, we injected mice with viral vectors harboring mutations in the critical binding sites for arginine within the Arg1 enzyme. These mutations did not alleviate hyperargininemia in \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice but effectively inhibited lipogenesis in the liver. These findings together suggest that Arg1 plays a crucial regulatory role in lipid metabolism, independent of its canonical enzymatic function.\u003c/p\u003e\n\u003cp\u003eIn recent years, an increasing number of studies have highlighted the non-enzymatic functions, also termed “moonlighting functions”, of metabolic enzymes in diverse cell processes, such as signaling pathways, autophagy, mitochondrial function, and redox homeostasis regulation.\u003csup\u003e61,62\u003c/sup\u003e For example, phosphofructokinase 1 (PFKP) binds to the N-terminal SH2 domain of p85α, recruiting it to the plasma membrane and thereby activating PI3K, which in turn stimulates tumor cell proliferation.\u003csup\u003e63\u003c/sup\u003e Similarly, upon activation of growth factor receptors, pyruvate kinase M2 (PKM2) translocates to the nucleus, where it binds to c-Src-phosphorylated Y333 of β-catenin, thereby promoting the expression of glycolysis-related genes and enhancing glucose uptake and lactate production.\u003csup\u003e64\u003c/sup\u003e In both our \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e models, Arg1 ablation significantly downregulates the AKT/mTOR/PPARγ and Elk1/c-Fos/PPARγ pathways, both of which are known to promote lipogenesis, further exemplifying the influence of metabolic enzymes on physiological processes via non-enzymatic mechanisms.\u003c/p\u003e\n\u003cp\u003eUtilizing a combination of various protein interaction research methods, we have unequivocally demonstrated that Arg1 physically binds to the substrate-binding pocket of ERK2. Furthermore, mutating the ERK2 substrate-binding pocket diminished the binding affinity of Arg1, RSK2, and Elk1 for ERK2. Conversely, ablating Arg1 enhanced the binding of RSK2 and Elk1 to ERK2, indicating a competitive binding relationship among these proteins for the ERK2 substrate-binding site. As a consequence, the binding of Arg1 to ERK2 not only triggers ERK2 ubiquitination and degradation but also impedes the interaction of RSK2 and Elk1 with ERK2, subsequently inhibiting their signaling pathway activation. This reduction in signaling activity, in turn, diminishes the functional activities of these molecules and ultimately enhances PPARγ activity.\u003c/p\u003e\n\u003cp\u003eThe substrate-binding pocket of ERK2, primarily composed of four amino acid residues (ATRW), undergoes conformational changes facilitated by neighboring amino acids, enabling the binding of specific proteins and subsequent regulation of their functional activities. Notably, this pocket structure exhibits a remarkable lack of strict selectivity with respect to amino acid sequence, phosphorylation sites, and spatial conformation at the binding site.\u003csup\u003e\u0026nbsp;21,47,65-69\u003c/sup\u003e Competitive binding to this pocket has been documented for several proteins, including MKP3, RSK2, and Elk1.\u003csup\u003e47\u003c/sup\u003e Given this high flexibility and versatility, it is plausible that ERK2 can also interact with Arg1.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition to the diversity in amino acid sequence and spatial conformation, partner proteins display various interaction patterns when binding to ERK2. For instance, both MKP3 and Elk1 bind to the ERK2 substrate-binding site via their FXFP domains; however, MKP3 also requires its non-catalytic amino-terminal segment for additional binding to ERK2 to fulfill its function.\u003csup\u003e47\u003c/sup\u003e Similarly, MEK1 not only binds to the ERK2 substrate-binding site but also requires binding to the CD site of ERK2 to stabilize the complex.\u003csup\u003e21,47,70\u003c/sup\u003e Moreover, RSK2 forms a heterodimer with ERK2 and undergoes activation through a series of phosphorylation reactions. After binding to the substrate-binding site of ERK2 and undergoing phosphorylation, RSK2 requires the additional binding of PDKl (pyruvate pehydrogenase kinase 1) to the essential docking site to achieve full activation.\u003csup\u003e67,68\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWe identified that the S-shaped motif within Arg1 is crucial for mediating the interaction between Arg1 and the ERK2 substrate-binding pocket. This motif is essential for the formation of functional Arg1 homotrimers. Our data demonstrate that occupation of the Arg1-arginine binding site by BEC not only disrupts the enzymatic activity of Arg1 but also impairs the ability of the S-shaped motif to bind to the ERK2 substrate-binding pocket. Likewise, viral vectors harboring Arg1 mutants in the S-shaped motif lose their capacity to hydrolyze arginine and are ineffective in alleviating hyperargininemia in \u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice. These findings suggest that the spatial conformation of Arg1 is vital for its binding to ERK2. However, due to the technological limitations, the precise binding mechanism and conformational transitions involved in the interaction between Arg1 and ERK2 remain to be elucidated.\u003c/p\u003e\n\u003cp\u003eOur study suggests that disrupting the interaction between Arg1 and ERK2 can inhibit hepatocyte lipogenesis, thereby preventing subsequent obesity and MASLD. We have designed a peptide that mimics the substrate-binding pocket of ERK2, specifically binding to the S-shaped motif of Arg1 and effectively inhibiting the Arg1-ERK2 interaction. This peptide demonstrates the expected role in modulating hepatic lipid metabolism and alleviating obesity and MASLD, without inducing significant hyperargininemia. The intrinsic physiological roles of ERK2 and Arg1 pose challenges for drug development. However, our work introduces a promising strategy for drug design targeting the Arg1-ERK2 interaction, offering a potential therapeutic option for individuals with obesity and MASLD.\u0026nbsp;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eRESOURCE AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLead contact\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFurther information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Yujun Shi ([email protected]).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterials availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMaterials used in this study are listed in the key resources table. All reagents and mice generated in this study are available from the lead contact with a completed Materials Transfer Agreement.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003emRNA-seq data generated in this study have been deposited at National Center for Biotechnology Information (NCBI)\u0026nbsp;GenBank (accession numbers: PRJNA1186374 and PRJNA1186081) and are publicly available as of the date of publication. Microscopy data reported in this paper will be shared by the lead contact upon request.\u003c/li\u003e\n \u003cli\u003eThis paper does not report the original code.\u003c/li\u003e\n \u003cli\u003eAny additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Young Scientists Fund of the National Natural Science Foundation of China (Grant No. 82200649) and the National Natural Science Foundation of China (Grant No. 82472241). We would also like to acknowledge the help of HUABIO and WZ Biosciences Inc. in providing peptides as well as virus strains. We are grateful to Shanghai Oe Biotech Co., Ltd., for providing sequencing services.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eM.S. and X.C. conceived and designed the project. Y.C. performed mRNA-seq experiments and analysis. Z.Z., Y.S., Q.T., Q.X., T.M., Z.W., M.C., Y.Z., and R.Y. bred mice, performed mouse experiments, and analyzed the data. J.G., and J.Y. provided liver disease model setup, study design, and joint discussions on the results. Y.S. and M.S. interpreted the data and drafted and revised the manuscript.\u003c/p\u003e"},{"header":"STAR*METHODS","content":"\u003cp\u003e\u003cstrong\u003eEXPERIMENTAL MODEL DETAILS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMice and diets\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice were provided by Dr. Pan Cong. Albumin-Cre transgenic mice were purchased from Shanghai Biomodel Organism Science. Liver-specific and hepatocyte-specific Arg1 knockout mice were generated by crossing the \u003cem\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e mice with \u003cem\u003eAlbumin-Cre\u003c/em\u003e mice (\u003cem\u003eAlb\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e) or by injecting 1×10\u003csup\u003e11\u003c/sup\u003e GC/mouse of AAV8-TBG-cre (provided by WZ Biosciences Inc.) (\u003cem\u003eTBG\u003csup\u003ecre\u003c/sup\u003eArg1\u003csup\u003efl/fl\u003c/sup\u003e\u003c/em\u003e), respectively.\u003c/p\u003e\n\u003cp\u003eEight-week-old male C57BL/6 mice and\u003cem\u003e\u0026nbsp;db/db\u003c/em\u003e mice were purchased from GemPharmatech, Nanjing, China. All mice were male and housed under specific pathogen-free conditions with corncob bedding. The animal care and experimental procedures were conducted in accordance with national and international laws, policies, and ethical guidelines, and were approved by the Animal Care and Use Committee of Sichuan University, which adhered to the criteria outlined in the NIH Guide for the Care and Use of Laboratory Animals. Mice were maintained on either a chow diet as a control, a 45% lard high-fat diet (HFD), or a methionine-choline-deficient diet (MCD). All diets were purchased from Readydietech Co., Ltd., Shenzhen, China. The HFD was used to establish animal models of obesity and MAFLD induced by obesity.\u003csup\u003e71\u003c/sup\u003e Six- to eight-week-old male mice were fed the HFD for 12 weeks. The MCD diet was used to establish the MAFLD model,\u003csup\u003e72\u003c/sup\u003e and 6- to 8-week-old male mice were fed the MCD diet for 2 weeks. The dose of BEC (CAS # 222638-67-7, purity: 99.28%, TargetMol, USA) was calculated based on previous studies that reported a 50% reduction in arginase activity in cells isolated from treated animals. A daily dose of 0.012 g of BEC was administered in 100 μL of water via oral gavage, to match the dosing equivalent used in studies where mice were exposed to BEC in their drinking water. Tert-butylhydroquinone (TBHQ) (CAS # 1948-33-0, purity: ≥95%, TargetMol, USA) is an ERK activator.\u003csup\u003e73\u003c/sup\u003e VX-11e (CAS #896720-20-0, purity: ≥98%, TargetMol, USA) is a potent, selective, and orally bioavailable inhibitor of ERK.\u003csup\u003e74\u003c/sup\u003e The mice were given VX-11e in their drinking water at a concentration of 0.5 mg/mL.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMETHOD DETAILS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTissue and blood collection\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMice were sacrificed at the indicated times, and blood was collected. Tissues were collected and rapidly frozen using liquid nitrogen for long-term storage at -80°C. These frozen samples will be utilized for subsequent proteomic or immunoblot analysis. Furthermore, a portion of these tissues was fixed with 4% paraformaldehyde for 24 hours and subsequently embedded in paraffin for pathological staining analysis. In addition, a part of the liver was fixed in 4% paraformaldehyde overnight at 4°C. Afterward, it was infiltrated with 30% sucrose at 4°C overnight. The liver tissue was then embedded in Tissue-Tek OCT compound (Sakura; cat. no. 4583) for subsequent frozen sectioning.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of biochemical parameters\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCollect serum samples and send them to the GLP laboratory at West China Hospital for testing of mouse liver function indicators, including ALT and AST.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHepatic lipid analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe stored liver samples (100 mg) were lysed and homogenized in 2 mL of a solution containing 150 mmol/L NaCl, 0.1% Triton X-100, and 10 mmol/L Tris using a polytron homogenizer (cat. no. NS-310E; Microtec Co., Ltd., Chiba, Japan) for 1 minute at room temperature. The liver TG level was analyzed using a Tissue Triglyceride Assay Kit (Applygen Technologies, Beijing, China) and the results were obtained by the GLP laboratory at West China Hospital. The liver homogenate FFA level was determined using an FFA Detection Kit based on the ACS-ACOD method (Wako Pure Chemical Industries, Ltd.) and normalized to the protein levels, as previously described.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCell Culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe AML12 mouse liver cell line was maintained in a 1:1 mixture of DMEM and Ham's F12 Medium, supplemented with 10% fetal bovine serum, 5 μg/mL insulin, 5 μg/mL transferrin, 5 ng/mL selenium, and 40 ng/mL dexamethasone. The cells were cultured at 37°C in a 5% (v/v) CO\u003csub\u003e2\u003c/sub\u003e atmosphere and subcultured every 3 days. MG132, a proteasome inhibitor, effectively blocks the proteolytic activity of the 26S proteasome complex, allowing ubiquitinated proteins to accumulate. The concentration of MG132 (CAS #133407-82-6, purity: 99.99%, supplied by TargetMol, USA) used in cell experiments was 10 μM, with a duration of action of 48 hours. In cell experiments, BEC was administered at a concentration of 0.31 μM, TBHQ at 5 μM, and VX-11e at 50 nM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProtein digestion and mass spectrometry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe liver sample was processed to obtain the total protein extract. Subsequently, a Co-Immunoprecipitation (CoIP) experiment was conducted using an Arg1 antibody to isolate proteins specifically interacting with Arg1. The protein complex was subjected to SDS-PAGE for separation based on their molecular weights. The gel was stained with Coomassie Brilliant Blue to visualize the protein bands. Images of the stained gel were captured for documentation. Specific gel slices containing bands of interest were excised. The excised gel pieces were processed to extract the peptides, which were subsequently analyzed by LC-MS/MS by Shanghai Oe Biotech Co., Ltd. The results obtained from this analysis are presented in Supplementary Table 1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProximity ligation assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAML12 cells were fixed using 3.7% paraformaldehyde and then blocked with the blocking solution provided by the\u003cem\u003e\u0026nbsp;Duolink\u0026nbsp;\u003c/em\u003ePLA kit (Sigma-Aldrich, USA),\u003csup\u003e75\u003c/sup\u003e following the manufacturer's instructions. In summary, the fixed cells were incubated overnight with both anti-ERK2 and anti-Arg1 antibodies. After thorough washing to remove unbound antibodies, the cells were sequentially incubated with PLA probes, ligation solution, and amplification solution, all at 37°C. Finally, coverslips were mounted, and the resulting images were examined using a Leica TCS SP5 confocal microscope (Leica Microsystems, Mannheim, Germany).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of fat mass by microcomputed tomography (micro-CT)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter being anaesthetized with isoflurane (1.5-2%) administered via air, the mice were positioned supine in an imaging cell where they were kept anaesthetized, warmed, and continuously monitored. Three-dimensional X-ray images were acquired using the CT component of a Quantum GX microCT Imaging System. The total acquisition time for these images was 15 minutes. The tube voltage was set at 80 kV, with a constant current of 32 mA.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of fat mass by autopsy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt the end of the different procedures, anaesthetized mice were sacrificed with a lethal dose of pentobarbital (120 mg/kg, intraperitoneally). Adipose tissues (visceral and subcutaneous) were harvested and weighed on a precision scale.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eKnockdown of Arg1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe siRNA targeting Arg1 (siArg1) was designed and synthesized by RiboBio. The target sequences utilized were: (1) GACTGAAGTGGACAGACTA; (2) CAAGCCTATTGACTACCTT; and (3) CTGGGTGACTCCCTGTATA. AML12 cells were treated with siArg1, and after a 72-hours incubation period, the cells were collected for subsequent western blot analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of Plasmids and Viruses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe plasmids encoding Flag-ERK2, Flag-ERK2 mut, and His-Arg1 were designed and synthesized by HUABIO, China. These plasmids were subsequently transfected into AML12 cells for a period of 48 hours. Following transfection, the cells were collected by centrifugation at 800 g for 10 minutes at 4°C.Additionally, plasmids for full-length mouse Arg1, mutant Arg1, full-length ERK2, and various ERK2 mapping constructs were also designed and synthesized by HUABIO, China. These plasmids were expressed and purified in \u003cem\u003eE. coli.\u0026nbsp;\u003c/em\u003eWZ Biosciences Inc. (Shandong, China) designed and synthesized recombinant AAV8 vectors carrying TBG-Arg1-mut\u003csup\u003e21\u0026amp;181\u003c/sup\u003e-His and AAV8-TBG-Arg1\u003csup\u003e304-322\u003c/sup\u003e-mut-His plasmids. These plasmids harbor mutations in the hydrolytic site or S-motif coding sequence of mouse Arg1, respectively. The recombinant AAV8 vectors were administered to animals via tail vein injection at a dose of 2×10\u003csup\u003e11\u003c/sup\u003e per animal. One-month post-injection, the samples were collected for further analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePeptide-drug synthesis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePeptides were obtained through Chemical Peptide Synthesis or Biological Peptide Synthesis (HUABIO, China). (1) Sequence of Chemical Peptide Synthesis: 170-200：RVADPDHDHTGFLTEYVATRWYRAPEIMLNS, and the purity is greater than 85%;（2）Biological Peptide Synthesis: recombinant synthesis involves the expression of the peptide within a specialized system from an artificial gene. The PET28A vector with a His tag was constructed to transfect \u003cem\u003eE. coli\u003c/em\u003e, and the purified protein was extracted. The main sequence was RVADPDHDHTGFLTEYVATRW YRAPEIMLNS, and the purity was greater than 85%. Mice were injected with 50 µg/kg via the tail vein every day.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOil red O staining\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver tissues or cells were washed with phosphate-buffered saline (PBS) and fixed in 4% paraformaldehyde for 10 minutes. After being washed twice with distilled water, they were immersed in 60% isopropyl alcohol for 5 minutes, and then fixed in oil red O dye solution for 20 minutes. Finally, they were washed with distilled water 2-5 times until there was no excess oil red O staining fluid, and observed and photographed under an optical microscope.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSerum Amino Acids\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe concentration of amino acids in serum was determined by high performance liquid chromatography as previously described.\u003csup\u003e76\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistology and Immunohistochemistry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver specimens were fixed in 10% neutral buffered formalin for a duration of 48 hours. Paraffin-embedded sections, each 4 μm thick, were prepared and subsequently subjected to a series of steps including dewaxing, rehydration, antigen retrieval, and quenching of endogenous peroxidase activity. The sections were then incubated with the appropriate primary antibody at 4°C overnight, followed by incubation with an anti-mouse/rabbit secondary antibody (Dako REAL EnVision Detection System, Glostrup, Denmark) for 1 hour at room temperature. Detection was achieved using 3,3′-diaminobenzidine (DAB) as a substrate. Standard protocols were employed for H\u0026amp;E and PAS staining.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blot\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiver tissues were homogenized for protein extraction. Sodium dodecyl sulfate‒polyacrylamide gel electrophoresis and immunoblotting were performed, and an electro chemiluminescent reagent was used for chemiluminescence detection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003emRNA Isolation and Real-Time PCR\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal mRNA was purified from 25 mg of liver tissue using an RNA Isolation kit (cat. RE-03011; Foregene, Chengdu, China). mRNA was reverse transcribed to cDNA using the iScriptcDNA Synthesis kit (cat. 179-8890; Bio-Rad, Hercules, CA). A CFX Connect Real-Time System (Bio-Rad) was used for real-time PCR. The gene expression levels were normalized to gene expression.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data are presented as the means ± standard errors of the mean (SEMs). Statistical comparisons were conducted using Student's t-test with Welch's correction, which was performed by GraphPad Prism Software version 10 (San Diego, CA). A P value less than 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eYounossi Z. M. (2019). Non-alcoholic fatty liver disease - A global public health perspective. 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Proceedings of the National Academy of Sciences of the United States of America \u003cem\u003e116\u003c/em\u003e, 21150-21159. https://doi.org/10.1073/pnas.1906182116\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"hepatic metabolism, Arg1, ERK2, PPARγ, obesity","lastPublishedDoi":"10.21203/rs.3.rs-5630831/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5630831/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe global incidence of obesity and its metabolic sequelae, notably metabolic dysfunction-associated steatohepatitis (MASLD), has escalated to epidemic levels. We unveil a previously unknown moonlighting role for arginase 1 (Arg1) in facilitating hepatic lipogenesis. Mice lacking hepatic Arg1 exhibited diminished lipid accumulation in both liver and adipocytes, an effect mirrored in genetically- or diet-induced obesity models following Arg1 inhibitor treatment. Mechanistically, Arg1 competes with RSK2 and Elk1 for binding to the substrate-binding pocket of extracellular signal-regulated kinase 2 (ERK2) via its S-shaped motif, thereby enhancing ERK2 ubiquitination and degradation and upregulating the AKT/mTOR/PPARγ and Elk1/c-Fos/PPARγ cascades, ultimately augmenting lipogenesis. Peptides designed to mimic the ERK2 substrate-binding pocket disrupted the Arg1-ERK2 interaction and improved metabolic profiles in obesity and MASLD models. Our findings implicate Arg1 regulates hepatic lipid metabolism via its physical interaction with ERK2, highlighting the Arg1-ERK2 interaction as a promising therapeutic target for obesity and related metabolic disorders.\u003c/p\u003e","manuscriptTitle":"Arginase 1 promotes hepatic lipogenesis by regulating ERK2/PPARγ signaling in a non-canonical manner","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-01-15 15:45:27","doi":"10.21203/rs.3.rs-5630831/v1","editorialEvents":[],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"nature-communications","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"NCOMMS","sideBox":"Learn more about [Nature Communications](http://www.nature.com/ncomms/)","snPcode":"","submissionUrl":"https://mts-ncomms.nature.com/","title":"Nature Communications","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Communications","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"352f5fce-6d89-49c0-b5e8-37a82d98714f","owner":[],"postedDate":"January 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41807368,"name":"Biological sciences/Physiology/Metabolism/Fat metabolism"},{"id":41807369,"name":"Health sciences/Gastroenterology/Hepatology/Liver diseases/Non-alcoholic fatty liver disease"}],"tags":[],"updatedAt":"2026-03-28T07:11:08+00:00","versionOfRecord":{"articleIdentity":"rs-5630831","link":"https://doi.org/10.1038/s41467-026-69731-3","journal":{"identity":"nature-communications","isVorOnly":false,"title":"Nature Communications"},"publishedOn":"2026-02-18 05:00:00","publishedOnDateReadable":"February 18th, 2026"},"versionCreatedAt":"2025-01-15 15:45:27","video":"","vorDoi":"10.1038/s41467-026-69731-3","vorDoiUrl":"https://doi.org/10.1038/s41467-026-69731-3","workflowStages":[]},"version":"v1","identity":"rs-5630831","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5630831","identity":"rs-5630831","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}