Sustained Hyperglycemia Drives Hepatic Metabolic Rewiring and Is Selectively Modulated by a Probiotic Strain in a Chicken Embryo Model of GDM

Sustained Hyperglycemia Drives Hepatic Metabolic Rewiring and Is Selectively Modulated by a Probiotic Strain in a Chicken Embryo Model of GDM | 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 Research Article Sustained Hyperglycemia Drives Hepatic Metabolic Rewiring and Is Selectively Modulated by a Probiotic Strain in a Chicken Embryo Model of GDM Sanya Boby, Jiddu Joseph, Erin Brannick, Shankumar Mooyottu, Muhammed Shafeekh Muyyarikkandy This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8831316/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 Background Gestational diabetes mellitus (GDM) exposes developing embryos to sustained hyperglycemia, yet dissecting embryo‑intrinsic mechanisms remains difficult in mammalian models due to maternal and placental influences. To directly evaluate hyperglycemia‑driven metabolic programming and probiotic modulation, we established a placenta‑independent in ovo chicken embryo model and tested the probiotic Leuconostoc pseudomesenteroides (MLS3) in live and heat‑inactivated (HI) forms. Methods and results Sustained hyperglycemia was induced by repeated air‑sac administration of D‑glucose from embryonic day (ED) 4–18, elevating circulating glucose, reducing embryonic mass, and increasing relative liver weight. Co‑administration of MLS3 attenuated systemic glycemia in a form‑dependent manner, with live MLS3 producing the greatest reduction, and preserved overall growth. Histological evaluation showed hepatic lipidosis and treatment‑specific glycogen accumulation without necroinflammation. Despite steatogenic features, hepatic SOD1 expression and malondialdehyde concentrations remained unchanged, consistent with an ED18 metabolic reliance on β‑oxidation and strong antioxidant buffering. Transcriptomic profiling revealed broad perturbations in oxidative phosphorylation, glycolysis/gluconeogenesis, and pyruvate metabolism, including robust induction of LDHA and PCK1, indicating a dual high‑flux metabolic state. Probiotic effects were highly context dependent: live MLS3 with glucose normalized TLR2/TLR4 signaling, suppressed lipogenesis, restored β‑oxidation, recalibrated ATP‑synthase stoichiometry, and markedly enhanced gluconeogenesis. HI MLS3 conferred partial glycemic benefit but maintained glycolytic and lipogenic signatures with a stress‑skewed transcriptome. Conclusion This embryo‑intrinsic model identifies a hyperglycemia‑induced dual metabolic program and demonstrates that live MLS3 elicits coordinated immunometabolic and mitochondrial rescue. These findings support strain‑informed probiotic strategies and provide a tractable platform for mechanistic studies preceding mammalian GDM models. Animal Science Endocrinology & Metabolism General Microbiology GDM Hyperglycemia chicken embryo model leaky gut metabolic reprogramming Full Text Additional Declarations The authors declare potential competing interests as follows: A provisional patent application covering the isolated probiotic strain Leuconostoc pseudomesenteroides MLS3, and its functional applications has been filed and is currently pending. The experiments described in this study were conducted using chicken embryos harvested on Embryonic Day 18 (E18). According to the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) and the Public Health Service (PHS) Policy, avian embryos are not considered 'live vertebrate animals' for the purposes of regulatory oversight until they hatch. Since all tissues were collected and experiments concluded prior to hatching (on E18), formal IACUC approval was not required for this study. 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-8831316","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":588348354,"identity":"a628e754-2508-4b83-8dd6-ee319f2b7ab8","order_by":0,"name":"Sanya Boby","email":"","orcid":"","institution":"University of Delaware","correspondingAuthor":false,"prefix":"","firstName":"Sanya","middleName":"","lastName":"Boby","suffix":""},{"id":588348355,"identity":"a653f21d-59a0-49b5-9828-1794ee3a0f23","order_by":1,"name":"Jiddu Joseph","email":"","orcid":"","institution":"University of Delaware","correspondingAuthor":false,"prefix":"","firstName":"Jiddu","middleName":"","lastName":"Joseph","suffix":""},{"id":588348356,"identity":"87ce6cae-6cf6-40db-a391-90024a66b1b3","order_by":2,"name":"Erin Brannick","email":"","orcid":"","institution":"University of Delaware","correspondingAuthor":false,"prefix":"","firstName":"Erin","middleName":"","lastName":"Brannick","suffix":""},{"id":588348357,"identity":"7d97ccb4-f114-436b-8aaa-397e5071800d","order_by":3,"name":"Shankumar Mooyottu","email":"","orcid":"","institution":"Auburn University","correspondingAuthor":false,"prefix":"","firstName":"Shankumar","middleName":"","lastName":"Mooyottu","suffix":""},{"id":588348358,"identity":"e97d5394-b0bc-4359-a9f9-1804bf1db5f7","order_by":4,"name":"Muhammed Shafeekh Muyyarikkandy","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAnElEQVRIiWNgGAWjYHACxgcMbAwyQIYB0VqYDYBaeEjSwiZBmhbd/tNp1TxlNjwM7M3bJIjSYnYjd9ttnnNpPAw8x8qI1cK77TZv22EeBokcMyK1nD+7rZi37T8Pg/wbYrUcyN3GzNt2AGgLD7FabuRulpxzLpmHjSet2IJYh2388KbMTo6f/fDGG0RpgQM20pSPglEwCkbBKMALAAaWJ+YRVYTuAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3405-5440","institution":"University of Delaware","correspondingAuthor":true,"prefix":"","firstName":"Muhammed","middleName":"Shafeekh","lastName":"Muyyarikkandy","suffix":""}],"badges":[],"createdAt":"2026-02-09 14:03:18","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":false,"conflictsOfInterestStatement":true,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":false},"doi":"10.21203/rs.3.rs-8831316/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8831316/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102369806,"identity":"9f316058-fbcc-48cc-878b-ea3e552508e1","added_by":"auto","created_at":"2026-02-11 03:36:09","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":7289209,"visible":true,"origin":"","legend":"","description":"","filename":"GDMManuscriptPreprint.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8831316/v1_covered_cbbbb310-6146-446f-bb3c-1b3a0b644788.pdf"}],"financialInterests":"\u003cp\u003eThe authors declare potential competing interests as follows: A provisional patent application covering the isolated probiotic strain Leuconostoc pseudomesenteroides MLS3, and its functional applications has been filed and is currently pending.\u003c/p\u003e\n\u003cp\u003eThe experiments described in this study were conducted using chicken embryos harvested on Embryonic Day 18 (E18). According to the guidelines set forth by the Institutional Animal Care and Use Committee (IACUC) and the Public Health Service (PHS) Policy, avian embryos are not considered 'live vertebrate animals' for the purposes of regulatory oversight until they hatch. Since all tissues were collected and experiments concluded prior to hatching (on E18), formal IACUC approval was not required for this study.\u003c/p\u003e","formattedTitle":"\u003cp\u003e\u003cstrong\u003eSustained Hyperglycemia Drives Hepatic Metabolic Rewiring and Is Selectively Modulated by a Probiotic Strain in a Chicken Embryo Model of GDM\u003c/strong\u003e\u003c/p\u003e","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"University of Delaware","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":"GDM, Hyperglycemia, chicken embryo model, leaky gut, metabolic reprogramming","lastPublishedDoi":"10.21203/rs.3.rs-8831316/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8831316/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGestational diabetes mellitus (GDM) exposes developing embryos to sustained hyperglycemia, yet dissecting embryo‑intrinsic mechanisms remains difficult in mammalian models due to maternal and placental influences. To directly evaluate hyperglycemia‑driven metabolic programming and probiotic modulation, we established a placenta‑independent \u003cem\u003ein ovo\u003c/em\u003e chicken embryo model and tested the probiotic \u003cem\u003eLeuconostoc pseudomesenteroides\u003c/em\u003e (MLS3) in live and heat‑inactivated (HI) forms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and results\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSustained hyperglycemia was induced by repeated air‑sac administration of D‑glucose from embryonic day (ED) 4–18, elevating circulating glucose, reducing embryonic mass, and increasing relative liver weight. Co‑administration of MLS3 attenuated systemic glycemia in a form‑dependent manner, with live MLS3 producing the greatest reduction, and preserved overall growth. Histological evaluation showed hepatic lipidosis and treatment‑specific glycogen accumulation without necroinflammation. Despite steatogenic features, hepatic SOD1 expression and malondialdehyde concentrations remained unchanged, consistent with an ED18 metabolic reliance on β‑oxidation and strong antioxidant buffering. Transcriptomic profiling revealed broad perturbations in oxidative phosphorylation, glycolysis/gluconeogenesis, and pyruvate metabolism, including robust induction of LDHA and PCK1, indicating a dual high‑flux metabolic state. Probiotic effects were highly context dependent: live MLS3 with glucose normalized TLR2/TLR4 signaling, suppressed lipogenesis, restored β‑oxidation, recalibrated ATP‑synthase stoichiometry, and markedly enhanced gluconeogenesis. HI MLS3 conferred partial glycemic benefit but maintained glycolytic and lipogenic signatures with a stress‑skewed transcriptome.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis embryo‑intrinsic model identifies a hyperglycemia‑induced dual metabolic program and demonstrates that live MLS3 elicits coordinated immunometabolic and mitochondrial rescue. These findings support strain‑informed probiotic strategies and provide a tractable platform for mechanistic studies preceding mammalian GDM models.\u003c/p\u003e","manuscriptTitle":"Sustained Hyperglycemia Drives Hepatic Metabolic Rewiring and Is Selectively Modulated by a Probiotic Strain in a Chicken Embryo Model of GDM","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-11 03:35:47","doi":"10.21203/rs.3.rs-8831316/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":"e58741c5-10c9-4b64-943f-94d41a111bd5","owner":[],"postedDate":"February 11th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":62592540,"name":"Animal Science"},{"id":62592541,"name":"Endocrinology \u0026 Metabolism"},{"id":62592542,"name":"General Microbiology"}],"tags":[],"updatedAt":"2026-02-11T03:35:47+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-11 03:35:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8831316","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8831316","identity":"rs-8831316","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}