Microbial metabolite OAA from Romboutsia ilealis controls obesity and lipid metabolism through PSMD3-mediated degradation of YTHDF2

Microbial metabolite OAA from Romboutsia ilealis controls obesity and lipid metabolism through PSMD3-mediated degradation of YTHDF2 | 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 Microbial metabolite OAA from Romboutsia ilealis controls obesity and lipid metabolism through PSMD3-mediated degradation of YTHDF2 Xin Zong, Luoyi Zhu, Liang Huang, Shuqi Liu, Shiqi Luo, Yige Li This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6512177/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 17 Mar, 2026 Read the published version in Cell Death & Differentiation → Version 1 posted 9 You are reading this latest preprint version Abstract Specific gut microbiota is critically involved in the development of metabolic diseases, particularly obesity. Through studies in diabetic patients and animal models, we identified a novel gut microbe, Romboutsia ilealis , which alleviates obesity and associated metabolic disorders by modulating intestinal lipid absorption, rather than energy expenditure. Notably, 2-oxindole-3-acetic acid (OAA), a key metabolite of R. ilealis , was identified as a crucial regulator for mediating this effect. Mechanistically, biotin-labeled OAA combined with proteomic analysis revealed a direct interaction between OAA and the 26S proteasome subunit PSMD3, which leads to destabilization of the m 6 A-binding protein YTHDF2. Further investigations showed that YTHDF2 suppresses CD36 and FABP2 expression through m 6 A modified Rxrb mRNA, thereby reducing intestinal lipid absorption. In conclusion, our findings reveal a novel mechanism by which R. ilealis and its metabolite OAA modulate obesity-related lipid accumulation through PSMD3-mediated degradation of YTHDF2, highlighting their potential as innovative prebiotic or probiotic therapies for obesity. Biological sciences/Microbiology Biological sciences/Molecular biology/Epigenetics Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Obesity is widely recognized as a major public health challenge in modern society, serving as a significant risk factor for numerous chronic diseases [ 1 , 2 ]. A growing body of research highlights that obesity significantly increases the risk of metabolic diseases, including hypertension, hypercholesterolemia, atherosclerosis, type 2 diabetes and non-alcoholic fatty liver disease [ 3 , 4 ]. Traditionally, promoting energy expenditure has been considered the main approach to alleviating obesity. However, in recent years, it has been a growing recognition that controlling energy intake, especially regulating lipids absorption, is also an effective way to combat obesity [ 5 , 6 ]. In particular, the complex regulation of lipid absorption by the small intestine, the primary site of this process, is crucial for obesity intervention [ 7 ]. As such, strategic modulation of intestinal lipids absorption is gradually emerging as a cornerstone in the development of obesity prevention and treatment strategies. Microorganisms often referred to as the body's "second genome" [ 8 ], they play a crucial role in physiological and nutritional processes, particularly in digestion, absorption and metabolism of food [ 9 , 10 ]. Extensive research strongly supports the notion that disruption of the gut microbiota promotes weight loss by impairing lipid digestion and absorption in the small intestine, an effect that can be reversed through fecal microbiota transplantation [ 11 – 13 ]. Broad metagenomic studies have shed light on the complex relationships between a variety of specific gut microorganisms and human obesity. For instance, Akkermansia muciniphila controlling diet-induced obesity and improving metabolic profiles through crosstalk with the intestinal epithelium [ 14 , 15 ]. Lactobacillus and Bacillus amyloliquefaciens have demonstrated the ability to regulate lipid absorption and exert anti-obesity effects [ 16 , 17 ]. Therefore, an in-depth exploration of the functions of specific gut microorganisms and their mechanisms of action will be helpful for the development of probiotic products targeting obesity modulation. The gut microbiota primarily regulates host metabolism through its metabolic products [ 18 ]. A correlation has been reported between bacterial-derived tryptophan metabolites, such as indole, indole propionic acid, and indole sulfate, and obesity as well as metabolic syndrome [ 19 , 20 ]. Short-chain fatty acids have also been shown to play a role in fat deposition as substrates or signaling molecules [ 21 – 23 ]. In addition, a recent study found an uncharacterized bile acid named 3-succinylated cholic acid derived from the Bacteroides uniformis attenuated metabolic dysfunction-associated steatohepatitis [ 24 ]. Evidently, identification of key metabolic products is crucial for understanding the underlying mechanisms through which specific gut microbiota regulate lipid metabolism and obesity. Mechanistically, the gut microbiota and their metabolites regulate the energy metabolism of the organism through various pathways, including modulation of gene transcription, epigenetic changes such as DNA methylation and RNA post-transcriptional modification, as well as post-translational modifications of proteins like phosphorylation and ubiquitination. For example, gut microbiota regulates small intestinal lipids absorption and metabolism by modulating the level of the circadian transcription factor NFIL3 [ 25 ]. Changes in the phosphorylation or ubiquitination levels of proteins mediated by gut microbiota-derived metabolites help mitigate diseases associated with metabolic syndrome [ 26 – 28 ]. Furthermore, high-fat diet-induced gut microbiota disruption in obese mice was accompanied by decreased levels of small intestinal RNA m 6 A modifications [ 29 ]. Therefore, the metabolic pathways and mechanisms of action vary widely among microorganisms, and different obesity-related microorganisms tend to have a unique set of mechanisms that drive lipid metabolism and deposition. In this study, we investigated the biological functions of R. ilealis in regulating intestinal lipid metabolism and its underlying mechanisms. We first clarified that R. ilealis alleviates diet-induced obesity and metabolic disorders by inhibiting intestinal lipids absorption. Further in vivo and in vitro assays confirmed that the differential metabolite 2-oxoindole-3-acetate (OAA) is a key metabolite responsible for the function of R. ilealis . Mechanistically, OAA regulates lipid absorption by targeting PSMD3, which accelerates YTHDF2 ubiquitination and reduces its stability. Collectively, this study demonstrates that R. ilealis and its metabolite OAA protect host metabolism from diet induced obesity, suggesting that R. ilealis and OAA could serve as a novel therapeutic strategy for obesity and metabolic diseases. RESULTS R. ilealis reduced diet-induced obesity and metabolic disorders Previous work from a cohort study and diet induced animal model has shown that the abundance of R. ilealis was significantly higher in diabetic cohort (Fig. S1 A) and negatively correlated to body weight, serum TG (triglycerides) and TC (total cholesterol) in mice, implying a potential anti-obesity effect (Fig. S1 B–D). To validate this effect, we evaluated the impact of oral R. ilealis in high-fat diet (HFD) induced obese mice (Fig. 1 A). Correspondingly, R. ilealis significantly reduced body weight (Fig. 1 B), which was further supported by a marked weight reduction in liver, iWAT (inguinal white adipose tissue), and eWAT (epididymal white adipose tissue) (Fig. 1 C), decreased adipocyte size (Fig. 1 D), and a significant reduction in TG, TC and HDL levels (Fig. 1 E and F, Fig. S2 A). This was accompanied by enhanced glucose tolerance and insulin sensitivity, as demonstrated by better performance in the glucose tolerance test (GTT) and insulin tolerance test (ITT) in mice oral R. ilealis (Fig. 1 G and H). Furthermore, R. ilealis treatment effectively reduced the elevated expression of pro-inflammatory cytokines in both adipose tissue and liver, and serum levels of liver enzymes ALT and AST, suggesting its protective effect in the context of obesity-related metabolic dysfunction (Fig. 1 I and J, Fig. S2 B). These results demonstrate that R. ilealis is effective in treating HFD-induced obesity and lipid metabolism disorders. R. ilealis reduced obesity by suppressing lipids absorption in intestine Obesity is the result of an integrated balance between energy expenditure and nutritional intake [ 30 ]. Food intake was monitored throughout the study, and no significant differences in daily intake were observed between the groups (Fig. 2 A). Metabolic cage assays showed that oral R. ilealis did not significantly affect energy expenditure or activity levels (Fig. 2 B and C). Additionally, results for O₂ consumption and the respiratory exchange ratio (RER) were consistent with the notion of energy expenditure (Fig. S3A and B). These results suggested that the observed effects of R. ilealis on obesity were not due to changes in food consumption, metabolic rate or physical activity. Therefore, we wonder whether R. ilealis inhibited intestinal lipids absorption to resist diet-induced obesity. To verify this hypothesis, different concentrations (10 8 and 10 9 CFU/mL) of R. ilealis were administered by gavage to mice (Fig. 2 D). 16S ribosomal RNA gene sequencing of the jejunum microbiota showed that, despite successful colonization of R. ilealis (Fig. S4), the overall microbiota composition remained largely unchanged (Fig. S5). This suggests that the observed benefits of R. ilealis on obesity were not attributable to alterations in microbiota composition. Subsequently, macroscopic examination of the blood showed a noticeable reduction in lipid viscosity in the R. ilealis -treated group compared to the HFD group (Fig. 2 E). Specifically, a significant reduction of TG concentration in both serum and jejunum corresponded to that (Fig. 2 F and G). To further assess the effect of R. ilealis on intestinal lipid absorption, Bodipy-labeled palmitic acid was used to track fatty acid uptake in the intestine. The results revealed that the R. ilealis -treated group exhibited a significant reduction in fluorescence intensity (Fig. 2 H). Similar results were also observed in Oil Red O staining (Fig. 2 I). Consistently, oral R. ilealis significantly inhibited the expression of proteins and genes related to lipids absorption and transport in the jejunum, such as CD36, FABP2 and FATP4, especially in HR group (Fig. 2 J and K). These findings suggest that R. ilealis effectively inhibit intestinal lipid absorption, thereby reducing fat accumulation in the body. R. ilealis alleviates high-fat diet-induced obesity through 2-oxoindole-3-acetate (OAA) To investigate the mechanism by which R. ilealis alleviated obesity, the fecal metabolites profile was measured. Notably, PCA and PLS-DA analyses showed significant differences in both two ion patterns (Fig. 3 A and B). We finally identified 78 significantly-upregulated metabolites and 173 significantly-downregulated metabolites ( P value 1, and | log 2 (fold change) | ≥ 1) with gavage of R. ilealis (Fig. 3 C). Among them, 21 differential metabolites were involved in lipid metabolism pathway (Fig. 3 D), ranked these features with VIP scores, and the top 10 were retained for further analysis of their role in R. ilealis -mediated lipid metabolism (Fig. 3 E). The Spearman correlation analysis showed that 5 metabolites, including 1alpha-hydroxy-25,26,27-trinorvitamin D3-24-carboxylic acid, 6-[(E)-2-(4-hydroxyphenyl)ethenyl]-4-methoxy-5,6-dihydro-2H-pyran-2-one, bufalin, 2-oxoindole-3-acetate and, 2-[(1E)-3-hydroxy-3-methylbut-1-en-1-yl]-5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol, were found to be significantly associated with R. ilealis and serum TG (Fig. 3 F and G). Among these, 2-oxoindole-3-acetate (OAA) was the most significant in modulating obesity-related metabolic pathways. OAA accelerated lipids absorption in vitro and in vivo To ascertain the effect of OAA on intestinal lipids absorption, we used Bodipy-labeled palmitic acid to track fatty acid uptake (Fig. 4 A). Treatment with OAA significantly enhanced the fluorescence intensity of Bodipy-labeled palmitic acid in intestinal cells (IPEC-J2) in a concentration-dependent manner within the 100 µM OAA concentration range (Fig. 4 B and C), with no significant impact on cell viability (Fig. S6). In line with these results, intracellular TG content was higher when OAA present (Fig. 4 D), as well the expression of CD36 and FABP2 were enhanced (Fig. 4 E and F). To independently confirm that OAA promotes lipid absorption, we further investigated its effect in vivo (Fig. 4 G). It turned out that gavage of OAA significantly increased serum TG and jejunum TG accumulation at both concentrations of 5 and 10 mg/mL (Fig. 4 H and I). In turn, the fluorescence intensity of Bodipy-labeled palmitic acid in feces was significantly reduced in mice dealing with OAA (Fig. 4 J). Moreover, Oil Red O staining and tissue fluorescence revealed enhanced lipid accumulation and fatty acids uptake, consistent with the aforementioned results (Fig. 4 K and L). Similar to the in vitro observations, OAA treatment significantly upregulated the expression of CD36 and FABP2 proteins in mice (Fig. 4 M and N). Collectively, both in vivo and in vitro assays demonstrated that OAA enhanced intestinal fatty acid absorption through the activation of lipid transport pathways. OAA enhanced lipids absorption by targeting PSMD3 To explore the mechanism of OAA on accelerating lipids absorption, OAA molecular pull-down using biotin coupling was performed (Fig. 5 A, Fig. S7A–D). Among the 440 proteins detected and annotated (Fig. 5 B), two pathways of interest, “Proteasome, subunit alpha/beta” and “Proteasome component (PCI) domain” were most significantly enriched across 15 domain pathways (Fig. 5 C), suggesting that OAA may mediate the proteasome-ubiquitin degradation. Notably, 12 proteasome-related proteins were identified, with the proteasome 26S subunit protein PSMD3, involved in protein deubiquitylation, exhibiting the highest protein score (Fig. 5 D). Using molecular docking and molecular dynamic simulations, OAA was predicted to interact with the 365th glutamine and 374th lysine of PSMD3, which are conserved across three species (Fig. 5 E), and the compound showed stronger stability than PSMD3 alone (Fig. 5 F and G). Isothermal Titration Calorimetry (ITC) titration assays indicated a heat release reaction (ΔH = -1.65 × 10 − ⁹ ± 7.83 × 10 − ¹³ cal/mol) when PSMD3 was titrated into OAA (Fig. 5 H). Furthermore, OAA stabilized PSMD3 against thermal degradation compared to DMSO in thermal shift assays (Fig. 5 I). These evidences support a model in which OAA engages PSMD3 through direct binding and interaction. Moreover, we also observed that lipids absorption in intestinal epithelial cells increased when PSMD3 was knocked down (Fig. 5 J–M). These findings suggest that OAA enhances lipids absorption by targeting PSMD3. OAA disrupted PSMD3 mediated ubiquitination of YTHDF2 to reduce stability To determine whether OAA binding to PSMD3 mechanistically drives intestinal lipid absorption, co-IP/MS was employed to map the interactome of this complex (Fig. 6 A). Utilizing pathway enrichment analysis, we discovered that the interacting proteins of PSMD3 are enriched in “mRNA binding pathway”. Given the pronounced variation in YTHDF2 levels among the m 6 A related proteins (Fig. 6 B and C), we hypothesized that PSMD3 modulates lipid absorption by functionally engaging YTHDF2. We first confirmed the interaction between PSMD3 and YTHDF2 according to co-IP (Fig. 6 D). Correspondingly, in vitro ubiquitination assays revealed that PSMD3 mediated deubiqutination of YTHDF2 (Fig. 6 E), and PSMD3 deficiency suppressed YTHDF2 expression (Fig. 6 F), suggesting a regulatory interaction between the two. Besides, the presence of OAA disrupted the PSMD3 mediated deubiqutination of YTHDF2 and finally caused to reduce the expression of YTHDF2 (Fig. 6 E and G). To examine the effect of YTHDF2 on lipids absorption, we constructed Ythdf2 knockout IPEC-J2 cell line ( Ythdf2 -KO). Consistent with our hypothesis, Ythdf2 deficiency significantly increased intestinal epithelial lipid absorption (Fig. 6 H and I) and TG accumulation (Fig. 6 J), concomitant with elevated CD36 expression (Fig. 6 K and L). YTHDF2 recognized the m 6 A of Rxrb mRNA to regulate the expression of CD36 and intestinal lipids absorption Given YTHDF2's role in binding m 6 A methylation sites on mRNA, gut lipid absorption was proposed to be regulated by m 6 A methylation through OAA–YTHDF2 signaling. We initially assessed global m 6 A methylation levels of mRNA using two independent approaches: m 6 A dot blot analysis (Fig. 7 A) and m 6 A-specific ELISA assays (Fig. 7 B). Both methods demonstrated downregulation of m 6 A methylation in the presence of OAA. To confirm our hypothesis and find targets of YTDHF2, we performed MeRIP-seq analysis on IPEC-J2 with or without OAA incubation. The results confirmed the validity of the data, demonstrating that m 6 A methylation sites exhibited a canonical distribution pattern characterized by peak enrichment adjacent to transcription start sites and stop codons (Fig. 7 C, Fig. S8). Finally, we screened 520 significantly different peaks which satisfied P 2 (Fig. 7 E). KEGG pathway enrichment analysis identified the “Lipid and Atherosclerosis” pathway as the most significantly enriched (Fig. 7 E), with Rxrb showing the most prominent m 6 A methylation changes within this pathway. Furtherly, we observed OAA treatment significantly decreased the m 6 A modification levels of Rxrb , especially in the 3' UTR region, whereas the negative control gene, Hspa1b , showed minimal changed (Fig. 7 F). This methylation repositioning consequently led to a significant elevation in RXRB protein abundance (Fig. 7 G and H), and further, the elevated of RXRB increased lipids absorption according to accelerate the expression of fatty acids transporter, while HSPA1B didn’t affect (Fig. 7 I–L). Considering Rxrb mRNA expression level increased almost 2.5 times under knock out of Ythdf2 (Fig. 7 M), we propose that RXRB was a target for regulating lipid absorption along OAA–YTHDF2 signal axis. DISCUSSION The potential of probiotics in controlling metabolic disorders and body weight has been gradually recognized, as supplementation with probiotics is considered an effective way to alleviate obesity [ 31 , 32 ]. In this study, we demonstrated that R. ilealis alleviates diet-induced obesity and metabolic disorders by inhibiting intestinal lipids absorption. Furthermore, we identified the differential metabolite OAA as a key regulator in the role of R. ilealis in intestinal lipids absorption. Mechanistically, OAA, mediated by R. ilealis , influences lipids absorption by targeting PSMD3, which accelerates the ubiquitination of YTHDF2, thereby decreasing its stability. Moreover, YTHDF2 acts as a reader of m 6 A methylation site to regulate the expression of Rxrb , which in turn affects the expression of the lipid absorption related proteins CD36 and FABP2. The findings support the use of R. ilealis as a novel probiotic strategy for treating obesity and metabolic diseases. R. ilealis , a member of the Clostridia class in the Firmicutes phylum, was first described in 2011 as showing beneficial effects on the health status of rats with acute pancreatitis [ 33 ]. Its genomic information suggests a close association with energy metabolism [ 34 ]. Analysis of diabetic patients and high-fat diet-induced obesity models revealed a negative correlation between R. ilealis and both body weight and triglyceride levels, suggesting its potential in alleviating obesity and metabolic disorders [ 35 , 36 ]. We first measured food intake and energy expenditure and found that these factors were not responsible for the alleviation of HFD-induced obesity and its associated metabolic disorders by R. ilealis . Recent study showed that M. rupellensis promotes HFD-induced obesity by enhancing intestinal lipids absorption in conventionally reared SPF mice, the same findings were obtained in germ-free mice [ 37 ]. By establishing a long-term obesity model and a short-term lipids absorption model, we demonstrated the role of R. ilealis in alleviating obesity by reducing intestinal lipids absorption. Considering the potential interference from other gut microbiota, further validating the anti-obesity effect of R. ilealis using germ-free mice would provide stronger evidence. The gut microbiota play a crucial role in host physiology by producing metabolites that act as signaling molecules and substrates for metabolic reactions in the body [ 38 , 39 ]. For instance, Clostridium bifermentans and its active metabolites are involved in the regulation of intestinal lipids absorption [ 12 ]. Escherichia coli derived acetate could promote lipid oxidation and reduce lipid accumulation in enterocytes, thereby decreasing intracellular TG levels [ 40 ]. In contrast, M. rupellensis enhances jejunal lipids absorption via degrading myo-inositol, which inhibits fatty acid transport [ 37 ]. In the study, we screened for a down-regulated differential metabolite OAA by metabolomics. OAA was first reported in maize as a metabolite formed through the oxidation of indole-3-acetic acid by 2-oxoglutarate dioxygenase [ 41 , 42 ]. It is known that indoles and their derivatives work as agonists of aromatic hydrocarbon receptors (AHR). Impairment of AHR caused by gut microbiota represents a key factor contributing to the metabolic syndrome [ 43 ]. Here, our results indicated that oral OAA could enhance lipids absorption through lipids transporter proteins CD36 and FABP2. However, the precise mechanism by which R. ilealis metabolizes OAA needs to be further elucidated. To further investigate the mechanism of OAA-promoted lipids absorption. PSMD3, which interacts with OAA, was focused by biotin coupling and molecular docking. Our results indicated that the binding of PSMD3 to OAA leads to a loss of function, and a reduction in PSMD3 expression decreases lipids absorption capability. PSMD3, also known as proteasome 26S subunit non-ATPase 3, is a component of the 26S proteasome involved in the regulation of intracellular protein modification and stability [ 44 , 45 ]. Currently, PSMD3 has been reported to be involved in biological processes such as carcinogenesis and lipid metabolism by stabilizing the expression of the protein [ 46 ]. Pisano et al. found that the expression of PSMD3 and proteasome activity were lower in monocytes from overweight/obese children [ 47 ]. We found that OAA promotes the ubiquitination level of YTHDF2 by interacting with PSMD3, leading to a reduction in its protein expression. YTHDF2 is an m 6 A reader protein that regulates mRNA stability, and it has been reported to influence lipid metabolism by modulating the stability of mRNAs of various genes [ 48 , 49 ]. Using MeRIP-seq, we clarified that Rxrb with m 6 A-modified peaks is a key regulator of OAA in modulating lipids absorption. It has been reported that RXRB, as a transcription factor of the lipid transporter protein CD36, is involved in the regulation of lipids absorption and fat deposition [ 50 – 53 ]. Consistently, our results further verified that RXRB promotes lipids absorption and TG deposition by upregulating CD36 and FABP2 expression. Taken together, we discovered that R. ilealis regulates YTHDF2 protein stability by targeting PSMD3 through its metabolite OAA, which inhibits the activity of transcription factors like CD36 involved in intestinal fatty acid absorption, thereby alleviating high-fat diet-induced obesity and metabolic disorders. These findings not only enhance our understanding of the mechanism by which the specific gut microbe R. ilealis alleviates obesity, but also provide new probiotic and prebiotic strategies for the treatment of obesity and related metabolic diseases. MATERIALS AND METHODS Animals The 4-week-old C57BL/6 male mice used for the experiment were purchased from Shanghai Slac Laboratory Animal Co Ltd (Shanghai, China). Normal diet (ND) and 60% fat-fed high-fat diet (HFD) for mice were procured from Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd (Nanjing, China), where feed formulas for ND and HFD were shown in Table S1 . Mice were housed in a barrier facility following a standard diurnal photoperiod (12-hour light/dark cycle in a controlled environment) with temperatures between 18°C and 22°C; relative humidity was between 50% and 60%. Additionally, the body weights and food intake of mice were meticulously recorded once on time for monitoring and analysis. Statistical analysis Values are reported as the mean ± SEM. Statistical analyses were performed using GraphPad Prism 5. Unpaired two-tailed Student’s t-test was used when only two groups were compared, and one-way ANOVA followed by Tukey’s multiple comparison test was used for three or more groups. P < 0.05 was considered statistically significant. Omics analysis was conducted in the same way as before [ 54 ]. Declarations DATA AVAILABILITY The data used and/or analyzed to support the findings of this study are available in the main paper or in the Supplementary Information. Sequence data supporting the results of this study have been deposited in the NCBI (SRA BioProject No: PRJNA1194970 and GEO: GSE284540). The metabolomics data has been submitted to EMBL-EBI MetaboLights database with the identifier MTBL11895 (www.ebi.ac.uk/metabolights/MTBLS11895). More information seen Supplementary Information. ACKNOWLEDGEMENTS The authors appreciate Dr. Yulan Jin of the Experimental Teaching Center, College of Animal Sciences, Zhejiang University, for facility support. We also thank Professor Tao Tu and Dr. Ruyue Dong from the State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, for their support on ITC assay. AUTHOR CONTRIBUTIONS Luoyi Zhu and Liang Huang: data acquisition and analysis and writing original manuscripts. Shuqi Liu: performed the investigations. Shiqi Luo: established the methodology. Yige Li: established the methodology. Xin Zong: conceptualized the study, edited the paper, supervised the research and managed the project. All authors contributed to the article and approved the submitted version. FUNDING This work was supported by the National Key Research and Development Project of China (Grant/Award No. 2022YFD1301500); National Natural Science Foundation of China, (Grant/Award No. 32372890). COMPETING INTERESTS The authors declare no conflict of interests. 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SupplementaryInformation.docx Supporting Information for The Romboutsia lealis and derived 2-oxindole-3-aceic acid regulates obesity-associated lipid accumulation via PSMD3-mediated YTHDF2 deubiquitination Cite Share Download PDF Status: Published Journal Publication published 17 Mar, 2026 Read the published version in Cell Death & Differentiation → Version 1 posted Editorial decision: revise 11 Jun, 2025 Review # 1 received at journal 12 May, 2025 Review # 2 received at journal 08 May, 2025 Reviewer # 2 agreed at journal 07 May, 2025 Reviewer # 1 agreed at journal 07 May, 2025 Reviewers invited by journal 02 May, 2025 Submission checks completed at journal 24 Apr, 2025 Editor assigned by journal 23 Apr, 2025 First submitted to journal 23 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6512177","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":450935461,"identity":"2a7f0a60-075a-4af3-ac04-9aeba365a019","order_by":0,"name":"Xin Zong","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIie3RMQuCQBTA8SeC04XrTfoVXghC9GVsyUXByVkJcrH9oKHP0DcwHjSJc4ODU0uL0NJQkF672hZ0/+XdcD8exwGoVD8bggVQdAfjC+J8SwBWiRxTiJ3t6Mai2j+kJUIbE5j7ZJhoebVeCLyGaVKiJioCXhfDROeBiy1SuIES9dmWALk3TIyeeEi+0ZPXFMJ44DTdFo/1RJtCOCtdTSDNBZyjU175jF9GiJ3lzp09ybYFHZtHvLRMMULkcz77CvmZbPR+l97KYSZTLqtUKtU/9gYewEFPFWx9YwAAAABJRU5ErkJggg==","orcid":"","institution":"Zhejiang university","correspondingAuthor":true,"prefix":"","firstName":"Xin","middleName":"","lastName":"Zong","suffix":""},{"id":450935462,"identity":"2e22f56e-72e1-404f-ba44-eb0a7075782e","order_by":1,"name":"Luoyi Zhu","email":"","orcid":"","institution":"Zhejiang university","correspondingAuthor":false,"prefix":"","firstName":"Luoyi","middleName":"","lastName":"Zhu","suffix":""},{"id":450935463,"identity":"9717eaa9-a74c-4763-9717-eab316859fc5","order_by":2,"name":"Liang Huang","email":"","orcid":"","institution":"Zhejiang university","correspondingAuthor":false,"prefix":"","firstName":"Liang","middleName":"","lastName":"Huang","suffix":""},{"id":450935464,"identity":"6b0d6209-51a2-4dcf-b0f3-516ab1498185","order_by":3,"name":"Shuqi Liu","email":"","orcid":"","institution":"Zhejiang university","correspondingAuthor":false,"prefix":"","firstName":"Shuqi","middleName":"","lastName":"Liu","suffix":""},{"id":450935465,"identity":"316f6a25-1cb0-48e8-92f3-b65eafffad12","order_by":4,"name":"Shiqi Luo","email":"","orcid":"","institution":"Liangzhu Laboratory, School of Medicine, Zhejiang University","correspondingAuthor":false,"prefix":"","firstName":"Shiqi","middleName":"","lastName":"Luo","suffix":""},{"id":450935466,"identity":"8914d50d-3788-4a72-a572-90fbc1944e18","order_by":5,"name":"Yige Li","email":"","orcid":"","institution":"Zhejiang university","correspondingAuthor":false,"prefix":"","firstName":"Yige","middleName":"","lastName":"Li","suffix":""}],"badges":[],"createdAt":"2025-04-23 11:37:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6512177/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6512177/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41418-026-01708-7","type":"published","date":"2026-03-17T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82349432,"identity":"1a2c3ad6-b3d8-472a-bac4-6742d3bf3619","added_by":"auto","created_at":"2025-05-09 10:47:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1667275,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eR. ilealis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003ereduced diet-induced obesity and metabolic disorders. A\u003c/strong\u003e The experiment design. The mice were randomly divided into 3 groups. During the experiment, mice in the ND (normal diet) (\u003cem\u003en\u003c/em\u003e = 12) and HFD (high fat diet) groups (\u003cem\u003en\u003c/em\u003e = 14) were gavaged with saline and mice in the HFD+R group (\u003cem\u003en\u003c/em\u003e= 14) were gavaged with \u003cem\u003eR. ilealis\u003c/em\u003e in the first week. For the next 9 weeks, the ND group consumed the standard diet, and the HFD and HFD+R groups were fed HFD until the end of the experiment. \u003cstrong\u003eB\u003c/strong\u003e Body weight curves of mice. \u003cstrong\u003eC\u003c/strong\u003e Representative general view and organ index of liver, iWAT (​inguinal white adipose tissue) and eWAT (epididymal white adipose tissue​) in mice. \u003cstrong\u003eD \u003c/strong\u003eRepresentative H\u0026amp;E-stained sections of iWAT and eWAT (Scale bar, 100 μm) and Adipose area (the average of the areas of all cells in a field of view was counted (5 sections per group)). \u003cstrong\u003eE, F \u003c/strong\u003eTG (triglyceride) levels in liver and serum of mice in each group, \u003cem\u003en =\u003c/em\u003e 6. \u003cstrong\u003eG\u003c/strong\u003e Injection glucose tolerance test and associated area under the curve (AUC) quantification. \u003cstrong\u003eH \u003c/strong\u003eInsulin tolerance test and associated area under the curve (AUC) quantification. \u003cstrong\u003eI\u003c/strong\u003e Gene expression levels of mouse eWAT relative to \u003cem\u003eβ-actin\u003c/em\u003e quantified by qPCR (\u003cem\u003en =\u003c/em\u003e 3). \u003cstrong\u003eJ\u003c/strong\u003e Gene expression levels of mouse liver relative to \u003cem\u003eβ-actin\u003c/em\u003e quantified by qPCR (\u003cem\u003en =\u003c/em\u003e 3). Data were shown as mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01 and ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Source data are provided as a Source Data file.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6512177/v1/c98e0d680246862ee9245d9b.png"},{"id":82349435,"identity":"f2e75937-9660-4f71-a2a7-558999dbcb9c","added_by":"auto","created_at":"2025-05-09 10:47:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2301547,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eR. ilealis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e reduced obesity not through increasing energy expenditure but by suppressing lipids absorption in intestine.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003eFood intake of mice in ND (normal diet), HFD (high fat diet) and HFD+\u003cem\u003eR. ilealis\u003c/em\u003e groups. \u003cstrong\u003eB, C\u003c/strong\u003e Dynamic curve (\u003cstrong\u003eB\u003c/strong\u003e) and line regression analysis (\u003cstrong\u003eC\u003c/strong\u003e) of energy expenditure in mice of ND, HFD and HFD+\u003cem\u003eR. ilealis\u003c/em\u003e groups (\u003cem\u003en\u003c/em\u003e = 3). \u003cstrong\u003eD\u003c/strong\u003e Workflow of \u003cem\u003eR. liealis \u003c/em\u003etransplant in mice. \u003cstrong\u003eE\u003c/strong\u003e Representative general view of mouse serum (\u003cem\u003en =\u003c/em\u003e 13). \u003cstrong\u003eF, G\u003c/strong\u003e Serum and jejunum TG concentrations of mice in each group (\u003cem\u003en \u003c/em\u003e= 6). \u003cstrong\u003eH \u003c/strong\u003eFluorescence quantitation of intestine in \u003cem\u003eR. ilealis \u003c/em\u003etransplanted mice after gavaging Bodipy C16. \u003cstrong\u003eI \u003c/strong\u003eRepresentative view of H\u0026amp;E-stained (Scale bar, 500px) and Oil Red O stained (Scale bar, 500px) sections from jejunum\u003cstrong\u003e. J\u003c/strong\u003e Fatty acid transport protein expression levels in mouse jejunum relative to β-actin quantified by western blot (\u003cem\u003en = \u003c/em\u003e3). \u003cstrong\u003eL\u003c/strong\u003e Relative mRNA levels of fatty acid transport protein in mouse jejunum (\u003cem\u003en = \u003c/em\u003e7). Data were shown as mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Source data are provided as a Source Data file.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6512177/v1/87457ba0281c694979150292.png"},{"id":82349436,"identity":"a5f94704-e4a4-429e-8183-c9cd3bf2be8d","added_by":"auto","created_at":"2025-05-09 10:47:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1517333,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003e2-oxoindole-3-acetate (OAA) was a target of R. ilealis in obesity resistance.\u003c/strong\u003e \u003cstrong\u003eA, B\u003c/strong\u003eComparative analysis of fecal metabolites in CON and HR groups (\u003cem\u003en = \u003c/em\u003e 5, same as below), PCA plot in positive and negative (\u003cstrong\u003eA\u003c/strong\u003e) and PLS-DA (Partial Least Squares Discriminant Analysis) plot in positive and negative (\u003cstrong\u003eB\u003c/strong\u003e), \u003cstrong\u003eC\u003c/strong\u003e Differential metabolite volcano plots of the CON and HR groups plotted with \u003cem\u003eP\u003c/em\u003e-value \u0026lt; 0.05, VIP (Variable Importance in Projection) prep OPLS-DA \u0026gt; 1, and Fold change ≥ 1 as screening conditions, \u003cstrong\u003eD\u003c/strong\u003e KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis of significantly altered metabolites in mouse feces, \u003cstrong\u003eE\u003c/strong\u003eExpression profile of top 10 VIP differential metabolites, \u003cstrong\u003eF\u003c/strong\u003e Heatmap of correlation analysis between \u003cem\u003eR. ilealis\u003c/em\u003e and top 10 differential metabolites, \u003cstrong\u003eG\u003c/strong\u003eCorrelation analysis of mouse serum TG and TC levels with top 10 differential metabolites. Data were shown as mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Source data are provided as a Source Data file.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6512177/v1/4e78990fc2c82a6a4cce8099.png"},{"id":82349434,"identity":"d7a4044f-5db3-4738-9733-b00c35d0b551","added_by":"auto","created_at":"2025-05-09 10:47:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":917871,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eValidation of OAA in regulating lipids absorption \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e. \u003cstrong\u003eA\u003c/strong\u003eIPEC-J2 cell treated with OAA and Bodipy C16 strategy. \u003cstrong\u003eB\u003c/strong\u003e The optimal concentration of OAA for promoting fatty acid absorption in intestinal epithelial cells. (\u003cem\u003en =\u003c/em\u003e 5). \u003cstrong\u003eC\u003c/strong\u003e Representative fluorescence images and relative fluorescence intensity statistics of IPEC-J2 cells treated with 25 uM OAA for 24h with Bodipy C16 incubation for 10min (\u003cem\u003en =\u003c/em\u003e 3). \u003cstrong\u003eD\u003c/strong\u003e Comparison of intracellular TG (triglyceride) levels between CON and OAA groups (\u003cem\u003en =\u003c/em\u003e 4). \u003cstrong\u003eE\u003c/strong\u003e Relative mRNA levels of fatty acid transport protein (\u003cem\u003en = \u003c/em\u003e4). \u003cstrong\u003eF\u003c/strong\u003e Lipids absorption-related protein expression levels in IPEC-J2 relative to β-actin quantified by western blot. \u003cstrong\u003eG\u003c/strong\u003e Experiment design of OAA function detection \u003cem\u003ein vivo\u003c/em\u003e. \u003cstrong\u003eH, I\u003c/strong\u003e TG levels in serum (\u003cstrong\u003eH\u003c/strong\u003e) and jejunum (\u003cstrong\u003eI\u003c/strong\u003e) of mice in each group. \u003cstrong\u003eJ\u003c/strong\u003eFluorescence intensity of Bodipy in feces. \u003cstrong\u003eK\u003c/strong\u003eRepresentative view of H\u0026amp;E-stained (Scale bar, 500 px) and Oil Red O stained (Scale bar, 200 μm) sections from jejunum. \u003cstrong\u003eL\u003c/strong\u003e Bright- and dark-field intestinal micrographs under stereo fluorescence microscope. \u003cstrong\u003eM\u003c/strong\u003e Relative mRNA levels of fatty acid transport protein (\u003cem\u003en = \u003c/em\u003e3). \u003cstrong\u003eN\u003c/strong\u003e CD36 (Cluster of Differentiation 36) and FABP2 (Fatty Acid Binding Protein 2) protein expression levels in mouse jejunum by western blot (relative to β-actin). Data were shown as mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Source data are provided as a Source Data file.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6512177/v1/421f104701947be01689fbe0.png"},{"id":82351348,"identity":"ce2cad92-d2f7-4fcc-99e2-d505a0938654","added_by":"auto","created_at":"2025-05-09 10:55:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":12320787,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOAA enhanced lipids absorption by targeting PSMD3.\u003c/strong\u003e \u003cstrong\u003eA\u003c/strong\u003e Workflow of OAA molecular pull-down/MS assay. \u003cstrong\u003eB\u003c/strong\u003e The proteins detected by MS after molecular pull-down. Axis x and size of the bubble diagram represent emPAI (exponentially modified Protein Abundance Index), axis y and color represent protein coverage. \u003cstrong\u003eC\u003c/strong\u003eTop 10 of DOMAIN pathways that the proteins enriched in. \u003cstrong\u003eD \u003c/strong\u003eProteins involved in proteasome, axis x and color of bubble diagram represent protein score. \u003cstrong\u003eE \u003c/strong\u003eMolecular docking and the residues of PSMD3 (Proteasome 26S Subunit, Non-ATPase 3) that interacted with OAA; the predicted combined position and binding free energy between OAA and PSMD3 is displayed on the left, while the interacting hydrogen and residues is shown on the right, these residues’ conservation is displayed on the bottom. \u003cstrong\u003eF, G \u003c/strong\u003eMolecular dynamics simulation, RMSF (Root Mean Square Fluctuation) (\u003cstrong\u003eF\u003c/strong\u003e) and RMSD (Root Mean Square Deviation) (\u003cstrong\u003eG\u003c/strong\u003e) were recorded to show the compound stability using PSMD3 protein alone as a negative control. \u003cstrong\u003eH \u003c/strong\u003eIsothermal Titration Calorimetry (ITC) titration of PSMD3 with OAA, PSMD3 protein: OAA = 1:10 (10μM: 100μM).\u003cstrong\u003e I \u003c/strong\u003eCETSA (Cellular Thermal Shift Assay) and its normalized intensity, \u003cem\u003en =\u003c/em\u003e 2. \u003cstrong\u003eJ \u003c/strong\u003ePSMD3 protein expression level in PSMD3 knock down IPEC-J2 cell line and its normalized intensity, \u003cem\u003en =\u003c/em\u003e4. \u003cstrong\u003eK\u003c/strong\u003e–\u003cstrong\u003eM \u003c/strong\u003eTG concentration (\u003cstrong\u003eK\u003c/strong\u003e), Bodipy fluorography and fluorescence intensity (\u003cstrong\u003eL\u003c/strong\u003e) and, relative mRNA expression of lipids absorption related genes (\u003cstrong\u003eM\u003c/strong\u003e) in the PSMD3 knock down IPEC-J2 and its negative control, \u003cem\u003en =\u003c/em\u003e 3. Data were shown as mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Source data are provided as a Source Data file.\u003c/p\u003e","description":"","filename":"OnlineFigure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6512177/v1/763a4441ed9accec8f44da09.png"},{"id":82349438,"identity":"932992a9-2d50-4e39-9df2-08967ab0e45b","added_by":"auto","created_at":"2025-05-09 10:47:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1551846,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOAA disrupted PSMD3 mediated ubiquitination of YTHDF2 to reduce stability. A \u003c/strong\u003eWorkflow of co-IP/MS. \u003cstrong\u003eB \u003c/strong\u003eProteins interacting with PSMD3 (Proteasome 26S Subunit, Non-ATPase 3)\u003cstrong\u003e \u003c/strong\u003eshown as volcano plot. \u003cstrong\u003eC \u003c/strong\u003eTop 10 GO (Gene Ontology) terms the interacting proteins (intensity \u0026gt;100, fold change \u0026gt; 2) enriched in, and the m\u003csup\u003e6\u003c/sup\u003eA methylation enzyme in the mRNA binding pathway. \u003cstrong\u003eD \u003c/strong\u003eCo-IP assay verify the interaction between YTHDF2 (YTH N6-Methyladenosine RNA Binding Protein F2) and PSMD3 proteins in IPEC-J2, input sample were made from IPECJ2 overexpressed YTHDF2-FLAG and PSMD3-MYC fusion proteins. \u003cstrong\u003eE \u003c/strong\u003eThe ubiquitin level of YTHDF2 in the PSMD3 knock down IPEC-J2 cell line or the normal IPEC-J2 dealing with 25μM OAA. \u003cstrong\u003eF, G \u003c/strong\u003eThe YTHDF2 protein expression level under the PSMD3 knock down IPEC-J2 cell line (\u003cstrong\u003eF\u003c/strong\u003e) or the normal IPEC-J2 dealing with 25μM OAA (\u003cstrong\u003eG\u003c/strong\u003e). \u003cstrong\u003eH\u003c/strong\u003e–\u003cstrong\u003eJ \u003c/strong\u003eLipids absorption (\u003cstrong\u003eH, I \u003c/strong\u003e\u003cem\u003en = 4\u003c/em\u003e), which detected by dealing with Bodipy, and total TG concentration (\u003cstrong\u003eJ\u003c/strong\u003e, \u003cem\u003en = 4\u003c/em\u003e) in the YTHDF2 knock out IPEC-J2 cell line. \u003cstrong\u003eK, L \u003c/strong\u003eRelative mRNA (\u003cstrong\u003eK\u003c/strong\u003e, \u003cem\u003en =\u003c/em\u003e 3) and proteins (\u003cstrong\u003eL\u003c/strong\u003e) expression level of lipids absorption related gene. Data were shown as mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001. Source data are provided as a Source Data file.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-6512177/v1/994b2690bd5c0af59ec9a549.png"},{"id":82349455,"identity":"85ff25ad-3fc6-47ff-9441-a27534f67de4","added_by":"auto","created_at":"2025-05-09 10:47:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":11980850,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eYTHDF2 recognized the m\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e6\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eA of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eRxrb\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e mRNA to regulate the expression of CD36 and intestinal lipids absorption. A, B \u003c/strong\u003eThe mRNA m\u003csup\u003e6\u003c/sup\u003eA level in IPEC-J2 with 25 µM OAA, detected by m\u003csup\u003e6\u003c/sup\u003eA dot plot (\u003cstrong\u003eA\u003c/strong\u003e) and m\u003csup\u003e6\u003c/sup\u003eA Elisa (\u003cstrong\u003eB\u003c/strong\u003e, \u003cem\u003en =\u003c/em\u003e 3). \u003cstrong\u003eC \u003c/strong\u003eIntensity of peaks detected by MeRIP-seq (Methylated RNA Immunoprecipitation Sequencing) in IPEC-J2 with or without 25 µM OAA. \u003cstrong\u003eD \u003c/strong\u003eDifferent m\u003csup\u003e6\u003c/sup\u003eA peaks after incubation of 25μM OAA, genes involved in Lipid and atherosclerosis pathway and genes with the most fold change is labeled. \u003cstrong\u003eE \u003c/strong\u003eKEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis of genes with significant different m\u003csup\u003e6\u003c/sup\u003eA peaks. \u003cstrong\u003eF \u003c/strong\u003eDistribution of m\u003csup\u003e6\u003c/sup\u003eA peaks across RXRB (Retinoid X Receptor Beta) and HSPA1B (Heat Shock Protein Family A (Hsp70) Member 1B). \u003cstrong\u003eG, H \u003c/strong\u003eThe proteins expression level of RXRB and HSPA1B in IPEC-J2 under dealing with 25 μM OAA. \u003cstrong\u003eI–L \u003c/strong\u003eLipids absorption level of IPEC-J2 after overexpressing RXRB and HSPA1B, meanwhile dealing with Bodipy, the lipids accumulation and Bodipy fluorescence intensity (\u003cstrong\u003eI\u003c/strong\u003e), and TG (triglyceride) concentration (\u003cstrong\u003eJ\u003c/strong\u003e,\u003cem\u003e n =\u003c/em\u003e 3), relative mRNA (\u003cstrong\u003eK\u003c/strong\u003e, \u003cem\u003en =\u003c/em\u003e 3) and protein (\u003cstrong\u003eL\u003c/strong\u003e, \u003cem\u003en =\u003c/em\u003e 2) expression level of lipids absorption related genes. \u003cstrong\u003eM \u003c/strong\u003eRelative mRNA expression level of \u003cem\u003eRxrb\u003c/em\u003e. All column diagrams were shown as mean ± SEM. *, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05; **, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01. Source data are provided as a Source Data file.\u003cstrong\u003e\u003cbr\u003e\n\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"OnlineFigure7.png","url":"https://assets-eu.researchsquare.com/files/rs-6512177/v1/a79fb285baddefad38d62fb7.png"},{"id":104785165,"identity":"55ddf382-3500-40f7-9529-91321c8ae004","added_by":"auto","created_at":"2026-03-17 08:09:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11348569,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6512177/v1/0826c8b8-920a-4182-bd02-69971dcb9e95.pdf"},{"id":82352797,"identity":"661bdeee-a027-40eb-91fa-c65d8396550f","added_by":"auto","created_at":"2025-05-09 11:03:00","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2306413,"visible":true,"origin":"","legend":"Original western blot.","description":"","filename":"supplemetarymarterials2.docx","url":"https://assets-eu.researchsquare.com/files/rs-6512177/v1/f1143ac521d6d7fbe3dab835.docx"},{"id":82349446,"identity":"e35fff21-ae30-4c87-9721-880cc7f953fd","added_by":"auto","created_at":"2025-05-09 10:47:01","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":35906755,"visible":true,"origin":"","legend":"Supporting Information for The Romboutsia lealis and derived 2-oxindole-3-aceic acid regulates obesity-associated lipid accumulation via PSMD3-mediated YTHDF2 deubiquitination","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-6512177/v1/8047b52f2eaded92600ee411.docx"}],"financialInterests":"There is no duality of interest","formattedTitle":"Microbial metabolite OAA from Romboutsia ilealis controls obesity and lipid metabolism through PSMD3-mediated degradation of YTHDF2","fulltext":[{"header":"Introduction","content":"\u003cp\u003eObesity is widely recognized as a major public health challenge in modern society, serving as a significant risk factor for numerous chronic diseases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. A growing body of research highlights that obesity significantly increases the risk of metabolic diseases, including hypertension, hypercholesterolemia, atherosclerosis, type 2 diabetes and non-alcoholic fatty liver disease [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Traditionally, promoting energy expenditure has been considered the main approach to alleviating obesity. However, in recent years, it has been a growing recognition that controlling energy intake, especially regulating lipids absorption, is also an effective way to combat obesity [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In particular, the complex regulation of lipid absorption by the small intestine, the primary site of this process, is crucial for obesity intervention [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. As such, strategic modulation of intestinal lipids absorption is gradually emerging as a cornerstone in the development of obesity prevention and treatment strategies.\u003c/p\u003e \u003cp\u003eMicroorganisms often referred to as the body's \"second genome\" [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], they play a crucial role in physiological and nutritional processes, particularly in digestion, absorption and metabolism of food [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Extensive research strongly supports the notion that disruption of the gut microbiota promotes weight loss by impairing lipid digestion and absorption in the small intestine, an effect that can be reversed through fecal microbiota transplantation [\u003cspan additionalcitationids=\"CR12\" citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Broad metagenomic studies have shed light on the complex relationships between a variety of specific gut microorganisms and human obesity. For instance, \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e controlling diet-induced obesity and improving metabolic profiles through crosstalk with the intestinal epithelium [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. \u003cem\u003eLactobacillus\u003c/em\u003e and \u003cem\u003eBacillus amyloliquefaciens\u003c/em\u003e have demonstrated the ability to regulate lipid absorption and exert anti-obesity effects [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, an in-depth exploration of the functions of specific gut microorganisms and their mechanisms of action will be helpful for the development of probiotic products targeting obesity modulation.\u003c/p\u003e \u003cp\u003eThe gut microbiota primarily regulates host metabolism through its metabolic products [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. A correlation has been reported between bacterial-derived tryptophan metabolites, such as indole, indole propionic acid, and indole sulfate, and obesity as well as metabolic syndrome [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Short-chain fatty acids have also been shown to play a role in fat deposition as substrates or signaling molecules [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In addition, a recent study found an uncharacterized bile acid named 3-succinylated cholic acid derived from the \u003cem\u003eBacteroides uniformis\u003c/em\u003e attenuated metabolic dysfunction-associated steatohepatitis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Evidently, identification of key metabolic products is crucial for understanding the underlying mechanisms through which specific gut microbiota regulate lipid metabolism and obesity.\u003c/p\u003e \u003cp\u003eMechanistically, the gut microbiota and their metabolites regulate the energy metabolism of the organism through various pathways, including modulation of gene transcription, epigenetic changes such as DNA methylation and RNA post-transcriptional modification, as well as post-translational modifications of proteins like phosphorylation and ubiquitination. For example, gut microbiota regulates small intestinal lipids absorption and metabolism by modulating the level of the circadian transcription factor NFIL3 [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Changes in the phosphorylation or ubiquitination levels of proteins mediated by gut microbiota-derived metabolites help mitigate diseases associated with metabolic syndrome [\u003cspan additionalcitationids=\"CR27\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Furthermore, high-fat diet-induced gut microbiota disruption in obese mice was accompanied by decreased levels of small intestinal RNA m\u003csup\u003e6\u003c/sup\u003eA modifications [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Therefore, the metabolic pathways and mechanisms of action vary widely among microorganisms, and different obesity-related microorganisms tend to have a unique set of mechanisms that drive lipid metabolism and deposition.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the biological functions of \u003cem\u003eR. ilealis\u003c/em\u003e in regulating intestinal lipid metabolism and its underlying mechanisms. We first clarified that \u003cem\u003eR. ilealis\u003c/em\u003e alleviates diet-induced obesity and metabolic disorders by inhibiting intestinal lipids absorption. Further \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e assays confirmed that the differential metabolite 2-oxoindole-3-acetate (OAA) is a key metabolite responsible for the function of \u003cem\u003eR. ilealis\u003c/em\u003e. Mechanistically, OAA regulates lipid absorption by targeting PSMD3, which accelerates YTHDF2 ubiquitination and reduces its stability. Collectively, this study demonstrates that \u003cem\u003eR. ilealis\u003c/em\u003e and its metabolite OAA protect host metabolism from diet induced obesity, suggesting that \u003cem\u003eR. ilealis\u003c/em\u003e and OAA could serve as a novel therapeutic strategy for obesity and metabolic diseases.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eR. ilealis\u003c/b\u003e \u003cb\u003ereduced diet-induced obesity and metabolic disorders\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrevious work from a cohort study and diet induced animal model has shown that the abundance of \u003cem\u003eR. ilealis\u003c/em\u003e was significantly higher in diabetic cohort (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA) and negatively correlated to body weight, serum TG (triglycerides) and TC (total cholesterol) in mice, implying a potential anti-obesity effect (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u0026ndash;D). To validate this effect, we evaluated the impact of oral \u003cem\u003eR. ilealis\u003c/em\u003e in high-fat diet (HFD) induced obese mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Correspondingly, \u003cem\u003eR. ilealis\u003c/em\u003e significantly reduced body weight (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB), which was further supported by a marked weight reduction in liver, iWAT (inguinal white adipose tissue), and eWAT (epididymal white adipose tissue) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC), decreased adipocyte size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), and a significant reduction in TG, TC and HDL levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE and F, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA). This was accompanied by enhanced glucose tolerance and insulin sensitivity, as demonstrated by better performance in the glucose tolerance test (GTT) and insulin tolerance test (ITT) in mice oral \u003cem\u003eR. ilealis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG and H). Furthermore, \u003cem\u003eR. ilealis\u003c/em\u003e treatment effectively reduced the elevated expression of pro-inflammatory cytokines in both adipose tissue and liver, and serum levels of liver enzymes ALT and AST, suggesting its protective effect in the context of obesity-related metabolic dysfunction (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI and J, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eB). These results demonstrate that \u003cem\u003eR. ilealis\u003c/em\u003e is effective in treating HFD-induced obesity and lipid metabolism disorders.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eR. ilealis\u003c/b\u003e \u003cb\u003ereduced obesity by suppressing lipids absorption in intestine\u003c/b\u003e\u003c/p\u003e \u003cp\u003eObesity is the result of an integrated balance between energy expenditure and nutritional intake [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Food intake was monitored throughout the study, and no significant differences in daily intake were observed between the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Metabolic cage assays showed that oral \u003cem\u003eR. ilealis\u003c/em\u003e did not significantly affect energy expenditure or activity levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and C). Additionally, results for O₂ consumption and the respiratory exchange ratio (RER) were consistent with the notion of energy expenditure (Fig. S3A and B). These results suggested that the observed effects of \u003cem\u003eR. ilealis\u003c/em\u003e on obesity were not due to changes in food consumption, metabolic rate or physical activity.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTherefore, we wonder whether \u003cem\u003eR. ilealis\u003c/em\u003e inhibited intestinal lipids absorption to resist diet-induced obesity. To verify this hypothesis, different concentrations (10\u003csup\u003e8\u003c/sup\u003e and 10\u003csup\u003e9\u003c/sup\u003e CFU/mL) of \u003cem\u003eR. ilealis\u003c/em\u003e were administered by gavage to mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). 16S ribosomal RNA gene sequencing of the jejunum microbiota showed that, despite successful colonization of \u003cem\u003eR. ilealis\u003c/em\u003e (Fig. S4), the overall microbiota composition remained largely unchanged (Fig. S5). This suggests that the observed benefits of \u003cem\u003eR. ilealis\u003c/em\u003e on obesity were not attributable to alterations in microbiota composition. Subsequently, macroscopic examination of the blood showed a noticeable reduction in lipid viscosity in the \u003cem\u003eR. ilealis\u003c/em\u003e-treated group compared to the HFD group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE). Specifically, a significant reduction of TG concentration in both serum and jejunum corresponded to that (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF and G). To further assess the effect of \u003cem\u003eR. ilealis\u003c/em\u003e on intestinal lipid absorption, Bodipy-labeled palmitic acid was used to track fatty acid uptake in the intestine. The results revealed that the \u003cem\u003eR. ilealis\u003c/em\u003e-treated group exhibited a significant reduction in fluorescence intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eH). Similar results were also observed in Oil Red O staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI). Consistently, oral \u003cem\u003eR. ilealis\u003c/em\u003e significantly inhibited the expression of proteins and genes related to lipids absorption and transport in the jejunum, such as CD36, FABP2 and FATP4, especially in HR group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ and K). These findings suggest that \u003cem\u003eR. ilealis\u003c/em\u003e effectively inhibit intestinal lipid absorption, thereby reducing fat accumulation in the body.\u003c/p\u003e \u003cp\u003e \u003cb\u003eR. ilealis\u003c/b\u003e \u003cb\u003ealleviates high-fat diet-induced obesity through 2-oxoindole-3-acetate (OAA)\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo investigate the mechanism by which \u003cem\u003eR. ilealis\u003c/em\u003e alleviated obesity, the fecal metabolites profile was measured. Notably, PCA and PLS-DA analyses showed significant differences in both two ion patterns (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA and B). We finally identified 78 significantly-upregulated metabolites and 173 significantly-downregulated metabolites (\u003cem\u003eP\u003c/em\u003e value\u0026thinsp;\u0026lt;\u0026thinsp;0.05, VIP (Variable Importance in Projection)\u0026thinsp;\u0026gt;\u0026thinsp;1, and | log\u003csub\u003e2\u003c/sub\u003e (fold change) | \u0026ge; 1) with gavage of \u003cem\u003eR. ilealis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Among them, 21 differential metabolites were involved in lipid metabolism pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD), ranked these features with VIP scores, and the top 10 were retained for further analysis of their role in \u003cem\u003eR. ilealis\u003c/em\u003e-mediated lipid metabolism (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). The Spearman correlation analysis showed that 5 metabolites, including 1alpha-hydroxy-25,26,27-trinorvitamin D3-24-carboxylic acid, 6-[(E)-2-(4-hydroxyphenyl)ethenyl]-4-methoxy-5,6-dihydro-2H-pyran-2-one, bufalin, 2-oxoindole-3-acetate and, 2-[(1E)-3-hydroxy-3-methylbut-1-en-1-yl]-5-[(E)-2-(4-hydroxyphenyl)ethenyl]benzene-1,3-diol, were found to be significantly associated with \u003cem\u003eR. ilealis\u003c/em\u003e and serum TG (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF and G). Among these, 2-oxoindole-3-acetate (OAA) was the most significant in modulating obesity-related metabolic pathways.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eOAA accelerated lipids absorption\u003c/b\u003e \u003cb\u003ein vitro\u003c/b\u003e \u003cb\u003eand\u003c/b\u003e \u003cb\u003ein vivo\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTo ascertain the effect of OAA on intestinal lipids absorption, we used Bodipy-labeled palmitic acid to track fatty acid uptake (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). Treatment with OAA significantly enhanced the fluorescence intensity of Bodipy-labeled palmitic acid in intestinal cells (IPEC-J2) in a concentration-dependent manner within the 100 \u0026micro;M OAA concentration range (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and C), with no significant impact on cell viability (Fig. S6). In line with these results, intracellular TG content was higher when OAA present (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD), as well the expression of CD36 and FABP2 were enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE and F).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo independently confirm that OAA promotes lipid absorption, we further investigated its effect \u003cem\u003ein vivo\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). It turned out that gavage of OAA significantly increased serum TG and jejunum TG accumulation at both concentrations of 5 and 10 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH and I). In turn, the fluorescence intensity of Bodipy-labeled palmitic acid in feces was significantly reduced in mice dealing with OAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eJ). Moreover, Oil Red O staining and tissue fluorescence revealed enhanced lipid accumulation and fatty acids uptake, consistent with the aforementioned results (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eK and L). Similar to the \u003cem\u003ein vitro\u003c/em\u003e observations, OAA treatment significantly upregulated the expression of CD36 and FABP2 proteins in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eM and N). Collectively, both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e assays demonstrated that OAA enhanced intestinal fatty acid absorption through the activation of lipid transport pathways.\u003c/p\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eOAA enhanced lipids absorption by targeting PSMD3\u003c/h2\u003e \u003cp\u003eTo explore the mechanism of OAA on accelerating lipids absorption, OAA molecular pull-down using biotin coupling was performed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, Fig. S7A\u0026ndash;D). Among the 440 proteins detected and annotated (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB), two pathways of interest, \u0026ldquo;Proteasome, subunit alpha/beta\u0026rdquo; and \u0026ldquo;Proteasome component (PCI) domain\u0026rdquo; were most significantly enriched across 15 domain pathways (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), suggesting that OAA may mediate the proteasome-ubiquitin degradation. Notably, 12 proteasome-related proteins were identified, with the proteasome 26S subunit protein PSMD3, involved in protein deubiquitylation, exhibiting the highest protein score (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUsing molecular docking and molecular dynamic simulations, OAA was predicted to interact with the 365th glutamine and 374th lysine of PSMD3, which are conserved across three species (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE), and the compound showed stronger stability than PSMD3 alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF and G). Isothermal Titration Calorimetry (ITC) titration assays indicated a heat release reaction (ΔH = -1.65 \u0026times; 10\u003csup\u003e\u0026minus;\u003c/sup\u003e⁹ \u0026plusmn; 7.83 \u0026times; 10\u003csup\u003e\u0026minus;\u003c/sup\u003e\u0026sup1;\u0026sup3; cal/mol) when PSMD3 was titrated into OAA (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH). Furthermore, OAA stabilized PSMD3 against thermal degradation compared to DMSO in thermal shift assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI). These evidences support a model in which OAA engages PSMD3 through direct binding and interaction. Moreover, we also observed that lipids absorption in intestinal epithelial cells increased when PSMD3 was knocked down (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eJ\u0026ndash;M). These findings suggest that OAA enhances lipids absorption by targeting PSMD3.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eOAA disrupted PSMD3 mediated ubiquitination of YTHDF2 to reduce stability\u003c/h3\u003e\n\u003cp\u003eTo determine whether OAA binding to PSMD3 mechanistically drives intestinal lipid absorption, co-IP/MS was employed to map the interactome of this complex (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA). Utilizing pathway enrichment analysis, we discovered that the interacting proteins of PSMD3 are enriched in \u0026ldquo;mRNA binding pathway\u0026rdquo;. Given the pronounced variation in YTHDF2 levels among the m\u003csup\u003e6\u003c/sup\u003eA related proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and C), we hypothesized that PSMD3 modulates lipid absorption by functionally engaging YTHDF2. We first confirmed the interaction between PSMD3 and YTHDF2 according to co-IP (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). Correspondingly, \u003cem\u003ein vitro\u003c/em\u003e ubiquitination assays revealed that PSMD3 mediated deubiqutination of YTHDF2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE), and PSMD3 deficiency suppressed YTHDF2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF), suggesting a regulatory interaction between the two. Besides, the presence of OAA disrupted the PSMD3 mediated deubiqutination of YTHDF2 and finally caused to reduce the expression of YTHDF2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo examine the effect of YTHDF2 on lipids absorption, we constructed \u003cem\u003eYthdf2\u003c/em\u003e knockout IPEC-J2 cell line (\u003cem\u003eYthdf2\u003c/em\u003e-KO). Consistent with our hypothesis, \u003cem\u003eYthdf2\u003c/em\u003e deficiency significantly increased intestinal epithelial lipid absorption (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH and I) and TG accumulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ), concomitant with elevated CD36 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK and L).\u003c/p\u003e \u003cp\u003e \u003cb\u003eYTHDF2 recognized the m\u003c/b\u003e \u003csup\u003e \u003cb\u003e6\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eA of\u003c/b\u003e \u003cb\u003eRxrb\u003c/b\u003e \u003cb\u003emRNA to regulate the expression of CD36 and intestinal lipids absorption\u003c/b\u003e\u003c/p\u003e \u003cp\u003eGiven YTHDF2's role in binding m\u003csup\u003e6\u003c/sup\u003eA methylation sites on mRNA, gut lipid absorption was proposed to be regulated by m\u003csup\u003e6\u003c/sup\u003eA methylation through OAA\u0026ndash;YTHDF2 signaling. We initially assessed global m\u003csup\u003e6\u003c/sup\u003eA methylation levels of mRNA using two independent approaches: m\u003csup\u003e6\u003c/sup\u003eA dot blot analysis (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) and m\u003csup\u003e6\u003c/sup\u003eA-specific ELISA assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Both methods demonstrated downregulation of m\u003csup\u003e6\u003c/sup\u003eA methylation in the presence of OAA. To confirm our hypothesis and find targets of YTDHF2, we performed MeRIP-seq analysis on IPEC-J2 with or without OAA incubation. The results confirmed the validity of the data, demonstrating that m\u003csup\u003e6\u003c/sup\u003eA methylation sites exhibited a canonical distribution pattern characterized by peak enrichment adjacent to transcription start sites and stop codons (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, Fig. S8). Finally, we screened 520 significantly different peaks which satisfied \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 and fold change\u0026thinsp;\u0026gt;\u0026thinsp;2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE). KEGG pathway enrichment analysis identified the \u0026ldquo;Lipid and Atherosclerosis\u0026rdquo; pathway as the most significantly enriched (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE), with \u003cem\u003eRxrb\u003c/em\u003e showing the most prominent m\u003csup\u003e6\u003c/sup\u003eA methylation changes within this pathway. Furtherly, we observed OAA treatment significantly decreased the m\u003csup\u003e6\u003c/sup\u003eA modification levels of \u003cem\u003eRxrb\u003c/em\u003e, especially in the 3' UTR region, whereas the negative control gene, \u003cem\u003eHspa1b\u003c/em\u003e, showed minimal changed (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). This methylation repositioning consequently led to a significant elevation in RXRB protein abundance (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG and H), and further, the elevated of RXRB increased lipids absorption according to accelerate the expression of fatty acids transporter, while HSPA1B didn\u0026rsquo;t affect (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI\u0026ndash;L). Considering \u003cem\u003eRxrb\u003c/em\u003e mRNA expression level increased almost 2.5 times under knock out of \u003cem\u003eYthdf2\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eM), we propose that RXRB was a target for regulating lipid absorption along OAA\u0026ndash;YTHDF2 signal axis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eThe potential of probiotics in controlling metabolic disorders and body weight has been gradually recognized, as supplementation with probiotics is considered an effective way to alleviate obesity [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In this study, we demonstrated that \u003cem\u003eR. ilealis\u003c/em\u003e alleviates diet-induced obesity and metabolic disorders by inhibiting intestinal lipids absorption. Furthermore, we identified the differential metabolite OAA as a key regulator in the role of \u003cem\u003eR. ilealis\u003c/em\u003e in intestinal lipids absorption. Mechanistically, OAA, mediated by \u003cem\u003eR. ilealis\u003c/em\u003e, influences lipids absorption by targeting PSMD3, which accelerates the ubiquitination of YTHDF2, thereby decreasing its stability. Moreover, YTHDF2 acts as a reader of m\u003csup\u003e6\u003c/sup\u003eA methylation site to regulate the expression of \u003cem\u003eRxrb\u003c/em\u003e, which in turn affects the expression of the lipid absorption related proteins CD36 and FABP2. The findings support the use of \u003cem\u003eR. ilealis\u003c/em\u003e as a novel probiotic strategy for treating obesity and metabolic diseases.\u003c/p\u003e \u003cp\u003e \u003cem\u003eR. ilealis\u003c/em\u003e, a member of the Clostridia class in the Firmicutes phylum, was first described in 2011 as showing beneficial effects on the health status of rats with acute pancreatitis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Its genomic information suggests a close association with energy metabolism [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Analysis of diabetic patients and high-fat diet-induced obesity models revealed a negative correlation between \u003cem\u003eR. ilealis\u003c/em\u003e and both body weight and triglyceride levels, suggesting its potential in alleviating obesity and metabolic disorders [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. We first measured food intake and energy expenditure and found that these factors were not responsible for the alleviation of HFD-induced obesity and its associated metabolic disorders by \u003cem\u003eR. ilealis\u003c/em\u003e. Recent study showed that \u003cem\u003eM. rupellensis\u003c/em\u003e promotes HFD-induced obesity by enhancing intestinal lipids absorption in conventionally reared SPF mice, the same findings were obtained in germ-free mice [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. By establishing a long-term obesity model and a short-term lipids absorption model, we demonstrated the role of \u003cem\u003eR. ilealis\u003c/em\u003e in alleviating obesity by reducing intestinal lipids absorption. Considering the potential interference from other gut microbiota, further validating the anti-obesity effect of \u003cem\u003eR. ilealis\u003c/em\u003e using germ-free mice would provide stronger evidence.\u003c/p\u003e \u003cp\u003eThe gut microbiota play a crucial role in host physiology by producing metabolites that act as signaling molecules and substrates for metabolic reactions in the body [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. For instance, \u003cem\u003eClostridium bifermentans\u003c/em\u003e and its active metabolites are involved in the regulation of intestinal lipids absorption [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. \u003cem\u003eEscherichia coli\u003c/em\u003e derived acetate could promote lipid oxidation and reduce lipid accumulation in enterocytes, thereby decreasing intracellular TG levels [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. In contrast, \u003cem\u003eM. rupellensis\u003c/em\u003e enhances jejunal lipids absorption via degrading myo-inositol, which inhibits fatty acid transport [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In the study, we screened for a down-regulated differential metabolite OAA by metabolomics. OAA was first reported in maize as a metabolite formed through the oxidation of indole-3-acetic acid by 2-oxoglutarate dioxygenase [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. It is known that indoles and their derivatives work as agonists of aromatic hydrocarbon receptors (AHR). Impairment of AHR caused by gut microbiota represents a key factor contributing to the metabolic syndrome [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Here, our results indicated that oral OAA could enhance lipids absorption through lipids transporter proteins CD36 and FABP2. However, the precise mechanism by which \u003cem\u003eR. ilealis\u003c/em\u003e metabolizes OAA needs to be further elucidated.\u003c/p\u003e \u003cp\u003eTo further investigate the mechanism of OAA-promoted lipids absorption. PSMD3, which interacts with OAA, was focused by biotin coupling and molecular docking. Our results indicated that the binding of PSMD3 to OAA leads to a loss of function, and a reduction in PSMD3 expression decreases lipids absorption capability. PSMD3, also known as proteasome 26S subunit non-ATPase 3, is a component of the 26S proteasome involved in the regulation of intracellular protein modification and stability [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e, \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Currently, PSMD3 has been reported to be involved in biological processes such as carcinogenesis and lipid metabolism by stabilizing the expression of the protein [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Pisano et al. found that the expression of PSMD3 and proteasome activity were lower in monocytes from overweight/obese children [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. We found that OAA promotes the ubiquitination level of YTHDF2 by interacting with PSMD3, leading to a reduction in its protein expression. YTHDF2 is an m\u003csup\u003e6\u003c/sup\u003eA reader protein that regulates mRNA stability, and it has been reported to influence lipid metabolism by modulating the stability of mRNAs of various genes [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Using MeRIP-seq, we clarified that \u003cem\u003eRxrb\u003c/em\u003e with m\u003csup\u003e6\u003c/sup\u003eA-modified peaks is a key regulator of OAA in modulating lipids absorption. It has been reported that RXRB, as a transcription factor of the lipid transporter protein CD36, is involved in the regulation of lipids absorption and fat deposition [\u003cspan additionalcitationids=\"CR51 CR52\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Consistently, our results further verified that RXRB promotes lipids absorption and TG deposition by upregulating CD36 and FABP2 expression.\u003c/p\u003e \u003cp\u003eTaken together, we discovered that \u003cem\u003eR. ilealis\u003c/em\u003e regulates YTHDF2 protein stability by targeting PSMD3 through its metabolite OAA, which inhibits the activity of transcription factors like CD36 involved in intestinal fatty acid absorption, thereby alleviating high-fat diet-induced obesity and metabolic disorders. These findings not only enhance our understanding of the mechanism by which the specific gut microbe \u003cem\u003eR. ilealis\u003c/em\u003e alleviates obesity, but also provide new probiotic and prebiotic strategies for the treatment of obesity and related metabolic diseases.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003eThe 4-week-old C57BL/6 male mice used for the experiment were purchased from Shanghai Slac Laboratory Animal Co Ltd (Shanghai, China). Normal diet (ND) and 60% fat-fed high-fat diet (HFD) for mice were procured from Jiangsu Xietong Pharmaceutical Bio-engineering Co., Ltd (Nanjing, China), where feed formulas for ND and HFD were shown in \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e. Mice were housed in a barrier facility following a standard diurnal photoperiod (12-hour light/dark cycle in a controlled environment) with temperatures between 18\u0026deg;C and 22\u0026deg;C; relative humidity was between 50% and 60%. Additionally, the body weights and food intake of mice were meticulously recorded once on time for monitoring and analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eValues are reported as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Statistical analyses were performed using GraphPad Prism 5. Unpaired two-tailed Student\u0026rsquo;s t-test was used when only two groups were compared, and one-way ANOVA followed by Tukey\u0026rsquo;s multiple comparison test was used for three or more groups. \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant. Omics analysis was conducted in the same way as before [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe data used and/or analyzed to support the findings of this study are available in the main paper or in the Supplementary Information. Sequence data supporting the results of this study have been deposited in the NCBI (SRA BioProject No: PRJNA1194970 and GEO: GSE284540). The metabolomics data has been submitted to EMBL-EBI MetaboLights database with the identifier MTBL11895 (www.ebi.ac.uk/metabolights/MTBLS11895).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMore information seen Supplementary Information. \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors appreciate Dr. Yulan Jin of the Experimental Teaching Center, College of Animal Sciences, Zhejiang University, for facility support. We also thank Professor Tao Tu and Dr. Ruyue Dong from the State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, for their support on ITC assay.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLuoyi Zhu and Liang Huang: data acquisition and analysis and writing original manuscripts. Shuqi Liu: performed the investigations. Shiqi Luo: established the methodology. Yige Li: established the methodology. Xin Zong: conceptualized the study, edited the paper, supervised the research and managed the project. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Key Research and Development Project of China (Grant/Award No. 2022YFD1301500); National Natural Science Foundation of China, (Grant/Award No. 32372890).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCOMPETING INTERESTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no conflict of interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICAL APPROVAL\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll animal experiments were conducted in accordance with the protocols approved by the Animal Protection Committee of Zhejiang University. All studies complied with applicable ethical regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cbr\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eHill AJ. Obesity in Children and the 'Myth of Psychological Maladjustment': Self-Esteem in the Spotlight. Current obesity reports 2017, 6(1): 63\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMalik VS, Hu FB. The role of sugar-sweetened beverages in the global epidemics of obesity and chronic diseases. Nature reviews Endocrinology 2022, 18(4): 205\u0026ndash;218.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePowell-Wiley TM, Poirier P, Burke LE, Despr\u0026eacute;s JP, Gordon-Larsen P, Lavie CJ, \u003cem\u003eet al.\u003c/em\u003e Obesity and Cardiovascular Disease: A Scientific Statement From the American Heart Association. 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Comparative multiomics analyses reveal the breed effect on the colonic host\u0026ndash;microbe interactions in pig. iMetaOmics 2024, 1(1): e8.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-6512177/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6512177/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eSpecific gut microbiota is critically involved in the development of metabolic diseases, particularly obesity. Through studies in diabetic patients and animal models, we identified a novel gut microbe, \u003cem\u003eRomboutsia ilealis\u003c/em\u003e, which alleviates obesity and associated metabolic disorders by modulating intestinal lipid absorption, rather than energy expenditure. Notably, 2-oxindole-3-acetic acid (OAA), a key metabolite of \u003cem\u003eR. ilealis\u003c/em\u003e, was identified as a crucial regulator for mediating this effect. Mechanistically, biotin-labeled OAA combined with proteomic analysis revealed a direct interaction between OAA and the 26S proteasome subunit PSMD3, which leads to destabilization of the m\u003csup\u003e6\u003c/sup\u003eA-binding protein YTHDF2. Further investigations showed that YTHDF2 suppresses CD36 and FABP2 expression through m\u003csup\u003e6\u003c/sup\u003eA modified \u003cem\u003eRxrb\u003c/em\u003e mRNA, thereby reducing intestinal lipid absorption. In conclusion, our findings reveal a novel mechanism by which \u003cem\u003eR. ilealis\u003c/em\u003e and its metabolite OAA modulate obesity-related lipid accumulation through PSMD3-mediated degradation of YTHDF2, highlighting their potential as innovative prebiotic or probiotic therapies for obesity.\u003c/p\u003e","manuscriptTitle":"Microbial metabolite OAA from Romboutsia ilealis controls obesity and lipid metabolism through PSMD3-mediated degradation of YTHDF2","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-09 10:46:55","doi":"10.21203/rs.3.rs-6512177/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2025-06-11T15:38:25+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-05-12T13:29:43+00:00","index":1,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-05-08T13:30:56+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-07T19:22:05+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-05-07T07:41:38+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-05-02T07:11:39+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-24T10:38:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-23T11:31:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Differentiation","date":"2025-04-23T11:31:36+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-differentiation","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cdd","sideBox":"Learn more about [Cell Death \u0026 Differentiation](http://www.nature.com/cdd/)","snPcode":"41418","submissionUrl":"https://mts-cdd.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Differentiation","twitterHandle":"@cddpress","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1927ecc6-0f49-48ed-854e-3a1f1a89696d","owner":[],"postedDate":"May 9th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":47976120,"name":"Biological sciences/Microbiology"},{"id":47976121,"name":"Biological sciences/Molecular biology/Epigenetics"}],"tags":[],"updatedAt":"2026-03-17T07:59:14+00:00","versionOfRecord":{"articleIdentity":"rs-6512177","link":"https://doi.org/10.1038/s41418-026-01708-7","journal":{"identity":"cell-death-and-differentiation","isVorOnly":false,"title":"Cell Death \u0026 Differentiation"},"publishedOn":"2026-03-17 04:00:00","publishedOnDateReadable":"March 17th, 2026"},"versionCreatedAt":"2025-05-09 10:46:55","video":"","vorDoi":"10.1038/s41418-026-01708-7","vorDoiUrl":"https://doi.org/10.1038/s41418-026-01708-7","workflowStages":[]},"version":"v1","identity":"rs-6512177","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6512177","identity":"rs-6512177","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}