Highly expressed ACSF2 may determine the butyrate metabolic preference of the rumen epithelium

Highly expressed ACSF2 may determine the butyrate metabolic preference of the rumen epithelium | 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 Highly expressed ACSF2 may determine the butyrate metabolic preference of the rumen epithelium Zan Huang, Ming-Mei Song, Jing Qin, Junru Tian, Kai Zhang, Yali Zhang, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8528676/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Ruminant epithelia preferentially catabolize butyrate to fuel ketogenesis, yet the mechanism by which the rumen epithelium establishes this preference remains unclear. Here, we identify ACSF2 as a mitochondrial acyl-CoA synthetase (ACS) that catalyzes the activation of butyrate to butyryl-CoA, thereby enabling ketogenesis and accounting for this bias. We found that ACSF2 is markedly enriched in the forestomachs across ovine organs, with expression far exceeding other ACSs in the rumen epithelium, and it is rising during postnatal establishment of fermentative function. Single-cell transcriptomics and immunostaining localize ACSF2 to the mitochondria-rich layers, where it is co-expressed in mitochondria with ketogenesis genes, notably the rate-limiting enzyme HMGCS2. Further gain- and loss-of-function experiments show that ACSF2 activates butyrate to butyryl-CoA, enhances butyrate-supported growth, and is required for efficient butyrate consumption and cell fitness under butyrate-dependent conditions. These findings define ACSF2 as a key mitochondrial gatekeeper for butyrate utilization and ketogenesis in the rumen epithelium, providing a molecular mechanism for butyrate-biased energy metabolism during rumen maturation. Biological sciences/Biochemistry/Lipids/Fatty acids Scientific community and society/Agriculture Biological sciences/Zoology/Animal physiology Full Text Additional Declarations There is NO Competing Interest. Supplementary Files PrimersgRNAandantibodylist.xlsx Table 1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-8528676","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":570060444,"identity":"3bb56576-06f8-45c7-86cd-9b3bfd7b59b0","order_by":0,"name":"Zan Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYBACAxiDn4EhAUQzNhCtRbKBZC0GByA0YS3mEsnPHn75Y5dnfLvh4WceBhvZDQeYnz3Ap8VyRpq5sQxPcrHZnQPJ0jwMacYbDrCZG+DTYnAjwUxaQoI5cduNhASglsOJGw7wsEng15L+TVrCoD5x84yE5N88DP+J0ZJjJvkhAWi4REIa0JYDRGg586ZMmuHA8cQZNxLSLOcYJBvPPMxmhl/L8fRtkj/+VCf2z8hJvvGmwk6273jzM7xaQICZB0zxJECiiZmQeiBg/AGm2A8QoXYUjIJRMApGIgAANYZKgLQWu9oAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0001-8552-533X","institution":"Nanjing Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Zan","middleName":"","lastName":"Huang","suffix":""},{"id":570060445,"identity":"34197d8d-605a-4363-a751-0308fdb9ce91","order_by":1,"name":"Ming-Mei Song","email":"","orcid":"","institution":"Nanjing Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"Ming-Mei","middleName":"","lastName":"Song","suffix":""},{"id":570060446,"identity":"de9e88a5-884a-4c87-afa8-1ae2808fb023","order_by":2,"name":"Jing Qin","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Jing","middleName":"","lastName":"Qin","suffix":""},{"id":570060447,"identity":"2ea6eaff-6bc0-44d9-81d0-116e57c7db8b","order_by":3,"name":"Junru Tian","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Junru","middleName":"","lastName":"Tian","suffix":""},{"id":570060448,"identity":"8d877096-395a-4db1-b248-7a3595e0e059","order_by":4,"name":"Kai Zhang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Kai","middleName":"","lastName":"Zhang","suffix":""},{"id":570060449,"identity":"acce5a41-98ad-4912-86d4-9dbb885d4000","order_by":5,"name":"Yali Zhang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yali","middleName":"","lastName":"Zhang","suffix":""},{"id":570060450,"identity":"3b73dafc-6cf3-4452-a089-08177f0888f0","order_by":6,"name":"Xiao'e Xiang","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Xiao'e","middleName":"","lastName":"Xiang","suffix":""},{"id":570060451,"identity":"e46159da-9814-40cc-a67d-dd610fa7393c","order_by":7,"name":"Weiyun Zhu","email":"","orcid":"https://orcid.org/0000-0001-9222-6581","institution":"Nanjing Agriculture University","correspondingAuthor":false,"prefix":"","firstName":"Weiyun","middleName":"","lastName":"Zhu","suffix":""},{"id":570060452,"identity":"8043118a-cfde-4180-9907-331ae85c47e1","order_by":8,"name":"Shengyong Mao","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shengyong","middleName":"","lastName":"Mao","suffix":""},{"id":570060453,"identity":"ce54524a-7a89-4502-bf09-63d3a1f877bb","order_by":9,"name":"Junhua Liu","email":"","orcid":"","institution":"Nanjing Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Junhua","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2026-01-06 08:29:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8528676/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8528676/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":99777610,"identity":"f8dfbeda-d054-4dcb-ae91-1f8eeba27c2b","added_by":"auto","created_at":"2026-01-08 10:13:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1644601,"visible":true,"origin":"","legend":"","description":"","filename":"ACSF2determinesthebutyratemetabolicpreferenceoftherumenepithelium.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8528676/v1/d8a95798ea822b789bcfe470.pdf"},{"id":99798565,"identity":"4b40201f-548e-4c72-a165-a32f8c32cadd","added_by":"auto","created_at":"2026-01-08 13:48:37","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":12662,"visible":true,"origin":"","legend":"","description":"","filename":"PrimersgRNAandantibodylist.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8528676/v1/65a0d3f9169da48597f0f1ff.xlsx"},{"id":99777609,"identity":"4b373881-5188-43da-8f30-0a5285d85190","added_by":"auto","created_at":"2026-01-08 10:13:11","extension":"json","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9898,"visible":true,"origin":"","legend":"","description":"","filename":"COMMSBIO260143T.json","url":"https://assets-eu.researchsquare.com/files/rs-8528676/v1/f2f3f05d30d5542b33bfc469.json"},{"id":100369220,"identity":"320987d7-461e-42c1-a9b7-9798b3bca9e9","added_by":"auto","created_at":"2026-01-16 07:58:49","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1537323,"visible":true,"origin":"","legend":"Article File","description":"","filename":"ACSF2determinesthebutyratemetabolicpreferenceoftherumenepithelium.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8528676/v1_covered_ac90fd1b-6bf7-4a11-8146-72243d5ae161.pdf"},{"id":99777607,"identity":"d9c05a1d-8e8e-4458-a2d7-42483b3df0f9","added_by":"auto","created_at":"2026-01-08 10:13:11","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":12662,"visible":true,"origin":"","legend":"Table 1","description":"","filename":"PrimersgRNAandantibodylist.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-8528676/v1/54680e67ffe249f3edf5a41e.xlsx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Highly expressed ACSF2 may determine the butyrate metabolic preference of the rumen epithelium","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8528676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8528676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Ruminant epithelia preferentially catabolize butyrate to fuel ketogenesis, yet the mechanism by which the rumen epithelium establishes this preference remains unclear. Here, we identify ACSF2 as a mitochondrial acyl-CoA synthetase (ACS) that catalyzes the activation of butyrate to butyryl-CoA, thereby enabling ketogenesis and accounting for this bias. We found that ACSF2 is markedly enriched in the forestomachs across ovine organs, with expression far exceeding other ACSs in the rumen epithelium, and it is rising during postnatal establishment of fermentative function. Single-cell transcriptomics and immunostaining localize ACSF2 to the mitochondria-rich layers, where it is co-expressed in mitochondria with ketogenesis genes, notably the rate-limiting enzyme HMGCS2. Further gain- and loss-of-function experiments show that ACSF2 activates butyrate to butyryl-CoA, enhances butyrate-supported growth, and is required for efficient butyrate consumption and cell fitness under butyrate-dependent conditions. These findings define ACSF2 as a key mitochondrial gatekeeper for butyrate utilization and ketogenesis in the rumen epithelium, providing a molecular mechanism for butyrate-biased energy metabolism during rumen maturation.","manuscriptTitle":"Highly expressed ACSF2 may determine the butyrate metabolic preference of the rumen epithelium","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-08 10:13:04","doi":"10.21203/rs.3.rs-8528676/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"582a6c1c-b405-49cb-b409-8b3f41fd5ea1","owner":[],"postedDate":"January 8th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":60657181,"name":"Biological sciences/Biochemistry/Lipids/Fatty acids"},{"id":60657182,"name":"Scientific community and society/Agriculture"},{"id":60657183,"name":"Biological sciences/Zoology/Animal physiology"}],"tags":[],"updatedAt":"2026-01-13T17:41:32+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-08 10:13:04","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8528676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8528676","identity":"rs-8528676","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}