Ruminococcus torques Administration Modestly Alleviates Dietary Selenium Deficiency- Induced Glucose Intolerance in Mature Female Mice

Ruminococcus torques Administration Modestly Alleviates Dietary Selenium Deficiency- Induced Glucose Intolerance in Mature Female Mice | 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 Ruminococcus torques Administration Modestly Alleviates Dietary Selenium Deficiency- Induced Glucose Intolerance in Mature Female Mice Ying-Chen Huang, Wen-Hsing Cheng This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7474743/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 11 You are reading this latest preprint version Abstract We previously found that dietary selenium (Se) deficiency and age increase the fecal abundance of Lachnospiraceae in aged telomere-humanized mice in a sexually dimorphic manner. Although Lachnospiraceae are key contributors of short-chain fatty acids to the host, different taxa within this family exert distinct effects on host physiology. Among them, Ruminococcus torques has been associated with type 2 diabetes. In the present study, we aimed to determine whether, and how, R. torques interacts with dietary Se to influence type 2 diabetes-like symptoms. Sixteen weaning female C57BL/6J mice were fed either a Se-adequate or Se-deficient diet for 26 weeks. From weeks 21 to 25, half of the mice in each dietary group received daily oral gavage of R. torques (2 × 10⁸ CFU in 0.2 mL). All mice were euthanized at week 26. Dietary Se deficiency induced glucose intolerance (13%) and insulin resistance (16%) ( P < 0.05). While R. torques administration modestly alleviated glucose intolerance in Se-deficient mice, it did not affect insulin resistance or fasting glucose levels. Se deficiency reduced the relative abundance of Lactobacillus spp ., F. prausnitzii , and Roseburia spp./E. rectale in the cecal content, and these taxa were unaffected by R. torques treatment. In contrast, Se deficiency increased the relative abundance of R. torques and E. coli in cecal samples, with E. coli levels further elevated by R. torques gavage. Notably, R. torques oral gavage 1 ) decreased SELENOP and GPX1 protein levels in the liver, but not in skeletal muscle, of Se-adequate mice; 2) increased liver GPX1 protein levels in Se-deficient mice. Altogether, R. torques administration modestly alleviates glucose intolerance and increases both liver GPX1 protein levels and cecal E. coli abundance in Se-deficient mature female mice with diabetic symptoms. Selenium deficiency glucose intolerance insulin resistance gut microbiota Ruminococcus torques Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction The Lachnospiraceae are a family of anaerobic bacteria within the Clostridiales order of the Firmicutes phylum, and include species previously classified as part of Clostridium cluster XIVa [ 1 , 2 ]. This family is abundant and dominant in the unperturbed adult gut microbiota [ 3 , 4 ]. However, only 25% of the genomes are closely related (> 97% similarity) to known species, and 28% do not match any records of existing databases [ 3 ]. Interestingly, 19% of sequences are closely related to recently isolated butyrate-producing bacteria from Clostridium clusters XIVa and IV, while 18% are related to Ruminococcus obeum and Ruminococcus torques , which are members of XIVa [ 3 ]. Ruminococcus is currently considered a polyphyletic genus, with species distributed across two distinct families: the Ruminococcaceae and the Lachnospiraceae [ 5 ]. Based on phenotypic traits, 16S rRNA gene sequence similarity [ 6 ], phylogenetic analysis, G + C content of genomic sequence, and DNA-DNA hybridization studies, the species R. torques , R. obeum , R. lactaris , R. gnavus , and R. gauvreauii are classified within the Lachnospiraceae family [ 7 ]. Representing a core component of the gut microbiota, Lachnospiraceae colonize the intestinal lumen from birth, with their species richness and relative abundance increasing with age. Lachnospiraceae have been implicated in obesity and diabetes in both humans and mouse models [ 8 – 14 ]; however, their specific role in type 2 diabetes remains unclear. We have previously shown that long-term dietary selenium (Se) deficiency induces type 2 diabetes-like symptoms in both male and female telomere-humanized mice [ 15 ], and increases the fecal abundance of Lachnospiraceae at 18 months of age in both sexes, but only in females at 24 months, based on 16 rRNA gene sequence analyses [ 16 ]. In contrast, Akkermansia muciniphila , a mucin-degrading bacterium that comprises 3–5% of the microbial community in healthy individuals [ 17 ], shows the most pronounced age-associated enrichment in response to dietary Se deficiency, but only in male telomere-humanized mice. Within the Lachnospiraceae family, R. torques is another mucin-degrading bacterium whose abundance is positively associated with irritable bowel syndrome in humans [ 18 , 19 ]. Because oral gavage of A. muciniphila alleviates type 2 diabetes-like symptoms in Se-deficient male mice, and these findings collectively suggest that mucin-degrading bacteria may play a critical role in modulating inflammatory responses at the gut mucosal surface [ 16 ]. Therefore, we aimed to investigate the effects of oral administration of R. torques on type 2 diabetes-like symptoms induced by dietary Se deficiency, as well as on symbiotic changes involving other mucin-degrading or short-chain fatty acid-producing bacteria in mature female mice. These results may also provide insights into sexually dimorphic responses in Se deficiency-induced type 2 diabetes and gut microbiota alterations. Materials and Methods Culture of Ruminococcus torques R. torques (ATCC BAA-2281) were cultured under strict anaerobic conditions using the Anoxomat III Jar system (Advanced Instruments, Norwood, MA) in modified reinforced clostridial medium following ATCC protocol. Cultures were washed and concentrated anaerobically in sterile, anaerobic PBS containing 25% (vol/vol) glycerol to a final concentration of 1 \(\:\times\:\) 10 10 CFU/mL, then immediately frozen and stored at − 80°C. Before oral administration, stocks were thawed and diluted anaerobically in sterile PBS with 2.5% glycerol to a final concentration of 1 \(\:\times\:\) 10 9 CFU/mL. Mice, diets, and treatment As shown in Fig. 1A, sixteen weaning female C57BL/6J mice were housed under conventional specific pathogen-free (SPF) conditions and fed either a Se-deficient or Se-adequate torula yeast-based purified diet for 26 weeks, as previously described previously [ 16 ]. At week 21, mice (n = 4 per group) received a daily oral gavage of either 2 \(\:\times\:\) 10 8 CFU live R. torques or a control vehicle (2.5% glycerol in 200 µL) for 4 weeks. Mice were handled aseptically in a controlled environment with 12-hour light/dark cycle (lights on from 6 p.m. to 6 a.m.), with ad libitum access to food and water. Body weight and food intake were monitored weekly. Fresh fecal samples were collected before and after R. torques treatment, snap-frozen in liquid nitrogen, and stored at − 80°C. Two days after the insulin sensitivity assay, mice were fasted for 6 hours, anesthetized with carbon dioxide, and euthanized by cardiac exsanguination. Liver, skeletal muscle, and cecal contents were collected, rapidly frozen in liquid nitrogen, and stored at − 80°C for further analyses. All procedures were approved by the Institutional Animal Care and Use Committee of Mississippi State University. Glucose tolerance and insulin sensitivity At weeks 21 and 26 of dietary intervention, mice fasted for 8 hours were intraperitoneally injected with glucose (1 g/kg body weight) or insulin (0.25 U/kg body weight) (Sigma Aldrich, St. Louis, MO). Blood glucose concentrations were measured using a glucose meter (Bayer Contour Next EZ, Ascensia Diabetes Care US, Inc., Parsippany, NJ) from a drop of tail vein samples collected at 0 (baseline), 0.25, 0.5, 1, 1.5, 2 hours post-injection. Insulin sensitivity was assessed 2 days after glucose tolerance testing. The area under the curve (AUC) was calculated to quantify glucose and insulin responses. Bacterial genomic DNA extraction and Quantitative PCR ( qPCR) analysis Bacterial DNA was extracted from cecal contents and feces using the QIAamp PowerFecal Pro DNA Kit (#51804, QIAGEN, Germantown, MD) following the manufacturer’s instructions. Universal primers targeting the V4 region of the bacterial 16S rRNA gene were used for amplification (primer sequences listed in Supplemental Table 1). qPCR was performed in 10 µL reactions using PowerUp™ SYBR™ Green Master Mix (#A25741, Applied Biosystems, Waltham, MA) on a QuantStudio 5 Real-Time PCR System (#A34322, Applied Biosystems) with the following thermal profile: 95°C for 2 min, followed by 40 cycles of 95°C for 5 sec and 60°C for 30 sec. Total bacterial load in fecal samples was quantified using the ΔCT method. Due to normalization of ΔCT values by subtraction against the control group, two-way ANOVA was not appropriate for analyzing ΔΔCT data, as a designated control could not be applied across two factors. Instead, unpaired t-tests were used for statistical analysis. Immunoblotting Tissues were homogenized in RIPA lysis buffer with protease inhibitors (# sc-24948, Santa Cruz Biotech, Dallas, TX) and centrifuged at 12,000 × g for 10 min at 4°C. Supernatants (30 µg protein per lane) were separated by 14% SDS-PAGE, then transferred to polyvinylidene difluoride membranes. Membranes were incubated overnight at 4°C with primary antibodies (listed in Supplemental Table 2), followed by HRP-conjugated secondary antibodies for 2 hours at room temperature. Signals were developed using Clarity Western ECL substrate, and images were captured and quantified using a Chemidoc-XS system with the volume tool in Image Lab Software (Bio-Rad Lab, Hercules, CA). Protein levels were normalized to albumin, β-tubulin, or total AKT. Statistical analysis Data are presented as means ± SEM. Most datasets were analyzed by two-way ANOVA followed by Tukey’s post hoc test, except for qPCR-based assays, which were evaluated using unpaired t -tests. Statistical analyses were performed using SAS (version 9.4) and GraphPad Prism (version 8.0). A significance level of α = 0.05 was used for all tests. Results Body weight and food intake Weaning female C57BL/6J mice exhibited steady increases in body weight (103–153%; P < 0.05; Fig. 1B) and food intake (56–70%; P < 0.05; Fig. 1C) over the 21-week dietary intervention. In mice receiving control oral gavage, dietary Se deficiency increased food intake (8–18%; P < 0.05) but had no effect on body weight. Daily oral gavage with R. torques resulted in a deduction in food intake in Se-deficient mice at weeks 23–26 (10–17%; P < 0.05), without affecting body weight. In R. torques -gavaged mice, dietary Se deficiency increased food intake at week 26 (13%; P < 0.05) but did not alter body weight. Changes in the abundance of specific genera by dietary Se deficiency and R. torques oral gavage in mice In Se-adequate mice, oral gavage with R. torques was associated with apparent increases (3- to 5-fold) in the abundances of R. torques , alongside decreases (52–82%; P < 0.05) in Lactobacillus spp . and Roseburia /E. rectale in the cecal and fecal contents (Supplemental Fig. 1). In mice receiving control oral gavage, dietary Se deficiency increased ( P ≤ 0.06) the abundances of R. torques (10.9-fold) and E. coli (9.5-fold), while decreasing that of Lactobacillus spp. (3.1-fold), F. prausnitzii (4.6-fold), and Roseburia /E. rectale (5.3-fold) in cecal content (Fig. 2A). A similar trend was observed in fecal content (Fig. 2B), but only Lactobacillus spp . (4.7-fold) and Roseburia /E. rectale (2.9-fold) reached statistical significance ( P < 0.05). Oral gavage with R. torques had no effect on these Se deficiency-induced alterations, except for a 1.7-fold increase ( P < 0.05) in cecal E. coli abundance. In mice receiving oral gavage with R. torques , dietary Se deficiency increased the relative abundance of E. coli in both cecal and fecal contents (4- to10-fold; P < 0.05) and decreased that of fecal Roseburia /E. rectale by 54% ( P < 0.05), but not in other taxa (supplemental Fig. 2). Modest alleviation by R. torques of dietary Se deficiency-induced glucose intolerance, but not insulin insensitivity, in conventional mice Se-deficient female mice at ~ 7 months of age exhibited glucose intolerance (Fig. 3A and B) and insulin resistance (Fig. 3C and D) over a 2-hour period following injections. However, fasting blood glucose concentrations were not affected by dietary Se deficiency or by R. torques oral gavage (Fig. 3A and C). While oral gavage with R. torques modestly alleviated (11% improvement; P < 0.05) Se deficiency-induced glucose intolerance at 0.5 h post-injection (Fig. 3A), it had no significant effect on overall glucose tolerance or insulin sensitivity across the entire time course in either the Se-deficient or Se-adequate mice. R. torques oral gavage and dietary Se deficiency differentially affect selenoprotein expression in muscle and liver We next assessed body Se status in the liver and skeletal muscle by evaluating protein expression levels of selected selenoproteins. Western blot analysis revealed that dietary Se deficiency reduced ( P < 0.05) the protein levels of GPX1 by 41% and SELENOW by 89% in muscle; whereas SELENOH and SELENOP expression remained unaffected (Fig. 5A). In the liver (Fig. 5B), Se deficiency decreased ( P < 0.05) protein levels of SELENOP by 50%, GPX1 by 86%, SELENOH by 56%, and SELENOW by 88%. Notably, the reduction in hepatic GPX1 was partially reversed by R. torques oral gavage (1.6-fold increase; P < 0.05). In Se-adequate mice, oral gavage of R. torques reduced ( P < 0.05) hepatic levels of both SELENOP and GPX1 by 27%, but had no effect on SELENOH or SELENOW in the liver, nor on any selenoprotein expression in muscle. Discussion We have previously shown that long-term dietary Se deficiency induces type 2 diabetes-like symptoms in telomere-humanized mice aged 12 and 18 months in both sexes [15], as well as middle-aged and mature wild-type male mice [16, 20]. Here, compared to wild-type male mice aged 4-7 months [16], dietary Se deficiency in age-matched females induces glucose intolerance and insulin resistance to a lesser extent. Consistent with our findings, there is evidence that females are generally more insulin-sensitive than males [21, 22], and women have decreased susceptibility to fatty acid–induced peripheral insulin resistance [23]. In rodent models, males typically exhibit more pronounced diabetes symptoms than females [24-27]. These sex-related differences in type 2 diabetes-like symptoms may be partially attributed to the actions of estrogen and testosterone. For example, decreases in estrogen and increases in testosterone levels during menopause are associated with a loss of subcutaneous fat, a gain of visceral fat, and increased insulin resistance [28]. Indeed, we have previously shown that Se status under Se deficiency is tissue-specific and dependent on sex and age [29]. In contrast to A. muciniphila [16] , oral gavage with R. torques only modestly alleviates Se deficiency-induced type 2 diabetes symptoms. An intriguing question arises: why do these two mucin-degrading bacteria have opposing impacts on Se deficiency-induced metabolic symptoms? Although some mucin-degrading bacteria, such as A. muciniphila ,are associated with health benefits, their close proximity to the intestinal epithelium may pose risks to host cells by compromising the gut barrier. It has been suggested that a reduction in cecal R. torques abundance in response to Lactobacillus acidophilus treatment may improve reproductive performance of broiler chickens by reducing the incidence of pasty vent, a stress-induced condition characterized by dry, cake-like droppings around the vent of baby chicks [30]. Supporting this observation, mucin degradation has been proposed to be pathogenic, as the loss of the protective mucus layer may increase the exposure of gastrointestinal epithelial cells to pathogens [31]. Moreover, the abundance of total mucosa-associated bacterial 16S rRNA genes is elevated in inflammatory bowel disorder, suggesting an increased availability of digestible endogenous mucus that may, in turn, sustain the growth of non-mucolytic mucosa-associated bacteria [32]. These findings highlight the need for further investigation into the role of mucin in shaping microbial community dynamics and host–microbe interactions. R. torques has been reported to be correlated with markers of insulin resistance in humans [33]. In our study, R. torques abundance was increased in mice fed a Se-deficient diet compared to those on a Se-adequate diet. However, our results indicate that oral administration of R. torques modestly improves glucose intolerance but has no effect on insulin resistance in Se-deficiency female mice. We speculate that 1) female mice may exhibit greater baseline insulin sensitivity, potentially masking any mild effects of R. torques ; 2) interactions between R. torques and host selenoproteins may counterbalance each other’s influence on type-2 diabetes pathogenesis; 3) the effects of R. torques on Se deficiency-induced type 2 diabetes may be age-dependent, becoming apparent only in middle-aged or older mice, but not in younger mice. Although a recent study using the type strain R. torques ATCC 27756 (BSL-1) demonstrated a pronounced alleviation of glucose intolerance in obese mice via oral gavage [2], we employed a non-type strain, R. torques ATCC BAA-2281 (BSL-2), and observed only a modest improvement in Se deficiency-induced glucose intolerance. In addition to the fact that ATCC BAA-2281 has been minimally characterized, we selected this strain to test its effect on type 2 diabetes-like symptoms because it is deposited in ATCC as a Lachnospiraceae bacterium. We have previously shown that dietary Se deficiency enriches the relative abundance of Lachnospiraceae in aged telomere-humanized mice in a sexually dimorphic manner [16]. To better understand the differing efficacy of these two R. torques strains in modulating glucose tolerance, future studies should consider factors such as host obesity status, strain-level genetic variation, and the underlying etiology of type 2 diabetes. Other gut bacteria may respond to R. torques oral gavage and influence outcomes related to glucose intolerance and insulin resistance in Se-deficient mice. Indeed, Se deficiency in females decreases the abundance of Lactobacillus spp. , F. prausnitzii , and Roseburia spp./E. rectale , while increasing the abundance of R. torques and E. coli in the cecal content. However, R. torques oral gavage results in increased E. coli abundance in Se-deficient mice and decreased Lactobacillus spp . and Roseburia spp./E. rectal abundance in Se-adequate mice. These findings are in contrast to those observed with A. muciniphila oral gavage, suggesting that these two mucin-degrading bacteria impact gut homeostasis and host health through distinct mechanisms. Altogether, our results show that the administration of the R. torques ATCC BAA-2281 strain only modestly alleviates glucose intolerance in Se-deficient female mice, and that changes in five other bacterial taxa display patterns distinct from those observed with A. muciniphila oral gavage in male mice [16]. Further studies are needed to deepen our understanding and provide mechanistic insight into how R. torques influences Se status and type 2 diabetes in the host, as well as how Se may prevent the early onset of type 2 diabetes in a sexually dimorphic manner. Abbreviations AKT, mouse thymoma viral protooncogene; GPX1, glutathione peroxidase 1; GPX3, glutathione peroxidase 1; Se, selenium; SELENOH, selenoprotein H; SELENOP, selenoprotein P; SELENOW, selenoprotein W; SCFA, short-chain fatty acid. Declarations Funding Supported by NIH DK117407. Author Contribution Conceptualization: WHC. Methodology: YCH and WHC. Investigation: YCH. Visualization: YCH. Supervision: WHC. Writing—review & editing: WHC. Funding acquisition: WHC. Both authors contributed to the article and approved the submitted version. References Vacca M, Celano G, Calabrese FM, Portincasa P, Gobbetti M, De Angelis M. The Controversial Role of Human Gut Lachnospiraceae. Microorganisms. 2020;8(4). doi: https://doi.org/10.3390/microorganisms8040573. Fan Y, Lyu L, Vazquez-Uribe R, Zhang W, Bongers M, Koulouktsis A, et al. Polypeptides synthesized by common bacteria in the human gut improve rodent metabolism. 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Supplementary Files SupplementalFigures12final.pdf SupplementalTablesfinal.pdf Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 29 Jan, 2026 Reviews received at journal 16 Dec, 2025 Reviews received at journal 08 Dec, 2025 Reviewers agreed at journal 05 Dec, 2025 Reviewers agreed at journal 02 Oct, 2025 Reviews received at journal 23 Sep, 2025 Reviewers agreed at journal 18 Sep, 2025 Reviewers invited by journal 18 Sep, 2025 Editor assigned by journal 27 Aug, 2025 Submission checks completed at journal 27 Aug, 2025 First submitted to journal 27 Aug, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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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-7474743","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":519633078,"identity":"24712739-2b76-4227-a42c-057bb07de2b4","order_by":0,"name":"Ying-Chen Huang","email":"","orcid":"","institution":"Mississippi State University","correspondingAuthor":false,"prefix":"","firstName":"Ying-Chen","middleName":"","lastName":"Huang","suffix":""},{"id":519633079,"identity":"6ecf6778-060f-4885-9ef7-bdf2a07f3c0c","order_by":1,"name":"Wen-Hsing Cheng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAsElEQVRIiWNgGAWjYFACHhAhIQfhsBGvxcKYh1QtFYk9RGsxOH724OOCXxLp+yVyDBg+lB0mQsuZvGTjmX0SuT1ALYwzzhGhxewGj5k0bw9Qi3SOATNvG3FazH8DtaTzgLT8JVKLGTPPD4kEsBZGYrTYn8kxluZtkDDsuf+s4GDPuXTCWiTbzxh+5vlTJ8/ec3jjgx9l1oS1gAFjG4Q+QKR6EPhDgtpRMApGwSgYeQAAHLM1pnDbtvMAAAAASUVORK5CYII=","orcid":"","institution":"Texas Woman’s University","correspondingAuthor":true,"prefix":"","firstName":"Wen-Hsing","middleName":"","lastName":"Cheng","suffix":""}],"badges":[],"createdAt":"2025-08-27 21:38:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7474743/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7474743/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":92435852,"identity":"24c2c603-c9f5-4ca8-b8c7-74d8b0ddbed5","added_by":"auto","created_at":"2025-09-29 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17:12:49","extension":"html","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":104946,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7474743/v1/fb75d8effb793bb41feee8d6.html"},{"id":92435849,"identity":"fc535776-b565-4fd1-a252-8ea488555ab0","added_by":"auto","created_at":"2025-09-29 17:04:49","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":109191,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eExperimental design, body weight, and food intake in female C57BL/6J mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSchematic diagram illustrating experimental design (A), body weight (B), and food intake (C) in 3-weeks-old female C57BL/6J mice fed either a Se-adequate or Se-deficient diet for 26 weeks. Twenty-one weeks after the start of dietary manipulation, mice received daily oral gavage with \u003cem\u003eR. torques\u003c/em\u003e or mock treatment for 4 weeks. Values are means ± SEMs (n=4). Means without sharing a common letter at a given time point differ, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. R.T., \u003cem\u003eR. torques\u003c/em\u003e; Se+, selenium-adequate diet; Se−, selenium-deficient diet; SPF, specific pathogen-free.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7474743/v1/848bf8b610eee2942372301a.png"},{"id":92435851,"identity":"1e4517bf-b6af-4a3d-9eca-a34c24db9bd1","added_by":"auto","created_at":"2025-09-29 17:04:49","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":71289,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFecal and cecal abundance of six bacteria taxa\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRelative abundance of \u003cem\u003eR. torques\u003c/em\u003e, \u003cem\u003eA. muciniphila\u003c/em\u003e, \u003cem\u003eLactobacillus spp.\u003c/em\u003e, \u003cem\u003eF. prausnitzii\u003c/em\u003e, \u003cem\u003eRoseburia/E. rectale\u003c/em\u003e, and \u003cem\u003eE. coli\u003c/em\u003e in cecal (A) and fecal (B) samples from female C57BL/6J mice fed either a Se-adequate or Se-deficient diet. \u003cem\u003eR. torques \u003c/em\u003eoral gavage was administered to Se-deficient mice (See Figure 1A for detailed experimental design). Values are means ± SEMs (n = 4). \u003cem\u003e*P\u003c/em\u003e \u0026lt; 0.05. R.T., \u003cem\u003eR. torques\u003c/em\u003e; Se+, selenium-adequate diet; Se−, selenium-deficient diet.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-7474743/v1/4c3efd71435df079015b77e2.png"},{"id":92435850,"identity":"d910997a-50fe-48d0-9323-2e80e6f9ddd2","added_by":"auto","created_at":"2025-09-29 17:04:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":113551,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eGlucose tolerance and insulin sensitivity assays in female C57BL/6J mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBlood glucose levels were measured following intraperitoneal injection of glucose (1 g/kg; A, B) or insulin (0.25 U/kg; C, D) in female C57BL/6J mice fed either a Se-adequate or Se-deficient diet and given \u003cem\u003eR. torques\u003c/em\u003e or mock oral gavage (see Figure 1A for detailed experimental design). Mice were fasted 8 hours prior to glucose or insulin injection. The average area under the curve was calculated from the data in panels A and C, with units expressed as mmol • h • L−1 (B and D). Values are means ± SEMs (n=4). Means without sharing a common letter at a given time point differ, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. AUC, average area under the curve; R.T., \u003cem\u003eR. torques\u003c/em\u003e; Se+, selenium-adequate diet; Se−, selenium-deficient diet.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-7474743/v1/79ef027f46fc738562dc3b1f.png"},{"id":92436613,"identity":"56125db0-2222-4836-9042-75b58c4fe433","added_by":"auto","created_at":"2025-09-29 17:12:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":219731,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot analysis of body Se status in female C57BL/6J mice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSelenoprotein expression in skeletal muscle (A) and liver (B) of female C57BL/6J mice fed either a Se-adequate or Se-deficient diet and given \u003cem\u003eR. torques\u003c/em\u003e or mock oral gavage (see Figure 1A for detailed experimental design). Protein levels were normalized to β-tubulin and expressed as a percentage of the Se-adequate control group. Values are means ± SEMs (n=4). Means without sharing a common letter differ, \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05. GPX1, glutathione peroxidase 1; R.T., \u003cem\u003eR. torques\u003c/em\u003e; Se+, Se-adequate diet; Se−, Se-deficient diet; SELENOH, selenoprotein H; SELENOP, selenoprotein P; SELENOW, selenoprotein W.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-7474743/v1/696a9aa6834c3c74ed2688f0.png"},{"id":92436784,"identity":"67138d1a-c3eb-4585-8e5d-98e9dd0416a5","added_by":"auto","created_at":"2025-09-29 17:20:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1181116,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7474743/v1/846067bd-35db-42cf-8be1-e742ff6d4f19.pdf"},{"id":92435854,"identity":"6795b929-6c01-43bf-af17-71fc63d5a01d","added_by":"auto","created_at":"2025-09-29 17:04:49","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":280078,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigures12final.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7474743/v1/310c5f94a174dfbb6de8f6d5.pdf"},{"id":92435856,"identity":"fb8e6cbf-3ce3-41d6-9a12-1cae70cd5966","added_by":"auto","created_at":"2025-09-29 17:04:49","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":176647,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTablesfinal.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7474743/v1/97dd6ed8ebd2dcc844fd7e25.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ruminococcus torques Administration Modestly Alleviates Dietary Selenium Deficiency- Induced Glucose Intolerance in Mature Female Mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe \u003cem\u003eLachnospiraceae\u003c/em\u003e are a family of anaerobic bacteria within the \u003cem\u003eClostridiales\u003c/em\u003e order of the \u003cem\u003eFirmicutes\u003c/em\u003e phylum, and include species previously classified as part of \u003cem\u003eClostridium\u003c/em\u003e cluster XIVa [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. This family is abundant and dominant in the unperturbed adult gut microbiota [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. However, only 25% of the genomes are closely related (\u0026gt;\u0026thinsp;97% similarity) to known species, and 28% do not match any records of existing databases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Interestingly, 19% of sequences are closely related to recently isolated butyrate-producing bacteria from \u003cem\u003eClostridium\u003c/em\u003e clusters XIVa and IV, while 18% are related to \u003cem\u003eRuminococcus obeum\u003c/em\u003e and \u003cem\u003eRuminococcus torques\u003c/em\u003e, which are members of XIVa [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. \u003cem\u003eRuminococcus\u003c/em\u003e is currently considered a polyphyletic genus, with species distributed across two distinct families: the \u003cem\u003eRuminococcaceae\u003c/em\u003e and the \u003cem\u003eLachnospiraceae\u003c/em\u003e [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Based on phenotypic traits, 16S rRNA gene sequence similarity [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], phylogenetic analysis, G\u0026thinsp;+\u0026thinsp;C content of genomic sequence, and DNA-DNA hybridization studies, the species \u003cem\u003eR. torques\u003c/em\u003e, \u003cem\u003eR. obeum\u003c/em\u003e, \u003cem\u003eR. lactaris\u003c/em\u003e, \u003cem\u003eR. gnavus\u003c/em\u003e, and \u003cem\u003eR. gauvreauii\u003c/em\u003e are classified within the \u003cem\u003eLachnospiraceae\u003c/em\u003e family [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eRepresenting a core component of the gut microbiota, \u003cem\u003eLachnospiraceae\u003c/em\u003e colonize the intestinal lumen from birth, with their species richness and relative abundance increasing with age. \u003cem\u003eLachnospiraceae\u003c/em\u003e have been implicated in obesity and diabetes in both humans and mouse models [\u003cspan additionalcitationids=\"CR9 CR10 CR11 CR12 CR13\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]; however, their specific role in type 2 diabetes remains unclear. We have previously shown that long-term dietary selenium (Se) deficiency induces type 2 diabetes-like symptoms in both male and female telomere-humanized mice [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and increases the fecal abundance of \u003cem\u003eLachnospiraceae\u003c/em\u003e at 18 months of age in both sexes, but only in females at 24 months, based on 16 rRNA gene sequence analyses [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In contrast, \u003cem\u003eAkkermansia muciniphila\u003c/em\u003e, a mucin-degrading bacterium that comprises 3\u0026ndash;5% of the microbial community in healthy individuals [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], shows the most pronounced age-associated enrichment in response to dietary Se deficiency, but only in male telomere-humanized mice. Within the \u003cem\u003eLachnospiraceae\u003c/em\u003e family, \u003cem\u003eR. torques\u003c/em\u003e is another mucin-degrading bacterium whose abundance is positively associated with irritable bowel syndrome in humans [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Because oral gavage of \u003cem\u003eA. muciniphila\u003c/em\u003e alleviates type 2 diabetes-like symptoms in Se-deficient male mice, and these findings collectively suggest that mucin-degrading bacteria may play a critical role in modulating inflammatory responses at the gut mucosal surface [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Therefore, we aimed to investigate the effects of oral administration of \u003cem\u003eR. torques\u003c/em\u003e on type 2 diabetes-like symptoms induced by dietary Se deficiency, as well as on symbiotic changes involving other mucin-degrading or short-chain fatty acid-producing bacteria in mature female mice. These results may also provide insights into sexually dimorphic responses in Se deficiency-induced type 2 diabetes and gut microbiota alterations.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cb\u003eCulture of\u003c/b\u003e \u003cb\u003eRuminococcus torques\u003c/b\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eR. torques\u003c/em\u003e (ATCC BAA-2281) were cultured under strict anaerobic conditions using the Anoxomat III Jar system (Advanced Instruments, Norwood, MA) in modified reinforced clostridial medium following ATCC protocol. Cultures were washed and concentrated anaerobically in sterile, anaerobic PBS containing 25% (vol/vol) glycerol to a final concentration of 1\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e10\u003c/sup\u003e CFU/mL, then immediately frozen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Before oral administration, stocks were thawed and diluted anaerobically in sterile PBS with 2.5% glycerol to a final concentration of 1\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e9\u003c/sup\u003e CFU/mL.\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eMice, diets, and treatment\u003c/h2\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;1A, sixteen weaning female C57BL/6J mice were housed under conventional specific pathogen-free (SPF) conditions and fed either a Se-deficient or Se-adequate torula yeast-based purified diet for 26 weeks, as previously described previously [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. At week 21, mice (n\u0026thinsp;=\u0026thinsp;4 per group) received a daily oral gavage of either 2\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\times\\:\\)\u003c/span\u003e\u003c/span\u003e10\u003csup\u003e8\u003c/sup\u003e CFU live \u003cem\u003eR. torques\u003c/em\u003e or a control vehicle (2.5% glycerol in 200 \u0026micro;L) for 4 weeks. Mice were handled aseptically in a controlled environment with 12-hour light/dark cycle (lights on from 6 p.m. to 6 a.m.), with \u003cem\u003ead libitum\u003c/em\u003e access to food and water. Body weight and food intake were monitored weekly. Fresh fecal samples were collected before and after \u003cem\u003eR. torques\u003c/em\u003e treatment, snap-frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C. Two days after the insulin sensitivity assay, mice were fasted for 6 hours, anesthetized with carbon dioxide, and euthanized by cardiac exsanguination. Liver, skeletal muscle, and cecal contents were collected, rapidly frozen in liquid nitrogen, and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for further analyses. All procedures were approved by the Institutional Animal Care and Use Committee of Mississippi State University.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eGlucose tolerance and insulin sensitivity\u003c/h3\u003e\n\u003cp\u003eAt weeks 21 and 26 of dietary intervention, mice fasted for 8 hours were intraperitoneally injected with glucose (1 g/kg body weight) or insulin (0.25 U/kg body weight) (Sigma Aldrich, St. Louis, MO). Blood glucose concentrations were measured using a glucose meter (Bayer Contour Next EZ, Ascensia Diabetes Care US, Inc., Parsippany, NJ) from a drop of tail vein samples collected at 0 (baseline), 0.25, 0.5, 1, 1.5, 2 hours post-injection. Insulin sensitivity was assessed 2 days after glucose tolerance testing. The area under the curve (AUC) was calculated to quantify glucose and insulin responses.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBacterial genomic DNA extraction and Quantitative PCR\u003c/b\u003e (\u003cb\u003eqPCR) analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBacterial DNA was extracted from cecal contents and feces using the QIAamp PowerFecal Pro DNA Kit (#51804, QIAGEN, Germantown, MD) following the manufacturer\u0026rsquo;s instructions. Universal primers targeting the V4 region of the bacterial 16S rRNA gene were used for amplification (primer sequences listed in Supplemental Table\u0026nbsp;1). qPCR was performed in 10 \u0026micro;L reactions using PowerUp\u0026trade; SYBR\u0026trade; Green Master Mix (#A25741, Applied Biosystems, Waltham, MA) on a QuantStudio 5 Real-Time PCR System (#A34322, Applied Biosystems) with the following thermal profile: 95\u0026deg;C for 2 min, followed by 40 cycles of 95\u0026deg;C for 5 sec and 60\u0026deg;C for 30 sec. Total bacterial load in fecal samples was quantified using the ΔCT method. Due to normalization of ΔCT values by subtraction against the control group, two-way ANOVA was not appropriate for analyzing ΔΔCT data, as a designated control could not be applied across two factors. Instead, unpaired t-tests were used for statistical analysis.\u003c/p\u003e\n\u003ch3\u003eImmunoblotting\u003c/h3\u003e\n\u003cp\u003eTissues were homogenized in RIPA lysis buffer with protease inhibitors (# sc-24948, Santa Cruz Biotech, Dallas, TX) and centrifuged at 12,000 \u0026times; g for 10 min at 4\u0026deg;C. Supernatants (30 \u0026micro;g protein per lane) were separated by 14% SDS-PAGE, then transferred to polyvinylidene difluoride membranes. Membranes were incubated overnight at 4\u0026deg;C with primary antibodies (listed in Supplemental Table\u0026nbsp;2), followed by HRP-conjugated secondary antibodies for 2 hours at room temperature. Signals were developed using Clarity Western ECL substrate, and images were captured and quantified using a Chemidoc-XS system with the volume tool in Image Lab Software (Bio-Rad Lab, Hercules, CA). Protein levels were normalized to albumin, β-tubulin, or total AKT.\u003c/p\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. Most datasets were analyzed by two-way ANOVA followed by Tukey\u0026rsquo;s post hoc test, except for qPCR-based assays, which were evaluated using unpaired \u003cem\u003et\u003c/em\u003e-tests. Statistical analyses were performed using SAS (version 9.4) and GraphPad Prism (version 8.0). A significance level of α\u0026thinsp;=\u0026thinsp;0.05 was used for all tests.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eBody weight and food intake\u003c/h2\u003e\u003cp\u003eWeaning female C57BL/6J mice exhibited steady increases in body weight (103\u0026ndash;153%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;1B) and food intake (56\u0026ndash;70%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;1C) over the 21-week dietary intervention. In mice receiving control oral gavage, dietary Se deficiency increased food intake (8\u0026ndash;18%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but had no effect on body weight. Daily oral gavage with \u003cem\u003eR. torques\u003c/em\u003e resulted in a deduction in food intake in Se-deficient mice at weeks 23\u0026ndash;26 (10\u0026ndash;17%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), without affecting body weight. In \u003cem\u003eR. torques\u003c/em\u003e-gavaged mice, dietary Se deficiency increased food intake at week 26 (13%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) but did not alter body weight.\u003c/p\u003e\u003cp\u003e\u003cb\u003eChanges in the abundance of specific genera by dietary Se deficiency and\u003c/b\u003e \u003cb\u003eR. torques\u003c/b\u003e \u003cb\u003eoral gavage in mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn Se-adequate mice, oral gavage with \u003cem\u003eR. torques\u003c/em\u003e was associated with apparent increases (3- to 5-fold) in the abundances of \u003cem\u003eR. torques\u003c/em\u003e, alongside decreases (52\u0026ndash;82%; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in \u003cem\u003eLactobacillus spp\u003c/em\u003e. and \u003cem\u003eRoseburia /E. rectale\u003c/em\u003e in the cecal and fecal contents (Supplemental Fig.\u0026nbsp;1). In mice receiving control oral gavage, dietary Se deficiency increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.06) the abundances of \u003cem\u003eR. torques\u003c/em\u003e (10.9-fold) and \u003cem\u003eE. coli\u003c/em\u003e (9.5-fold), while decreasing that of \u003cem\u003eLactobacillus spp.\u003c/em\u003e (3.1-fold), \u003cem\u003eF. prausnitzii\u003c/em\u003e (4.6-fold), and \u003cem\u003eRoseburia /E. rectale\u003c/em\u003e (5.3-fold) in cecal content (Fig.\u0026nbsp;2A). A similar trend was observed in fecal content (Fig.\u0026nbsp;2B), but only \u003cem\u003eLactobacillus spp\u003c/em\u003e. (4.7-fold) and \u003cem\u003eRoseburia /E. rectale\u003c/em\u003e (2.9-fold) reached statistical significance (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Oral gavage with \u003cem\u003eR. torques\u003c/em\u003e had no effect on these Se deficiency-induced alterations, except for a 1.7-fold increase (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in cecal \u003cem\u003eE. coli\u003c/em\u003e abundance. In mice receiving oral gavage with \u003cem\u003eR. torques\u003c/em\u003e, dietary Se deficiency increased the relative abundance of \u003cem\u003eE. coli\u003c/em\u003e in both cecal and fecal contents (4- to10-fold; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and decreased that of fecal \u003cem\u003eRoseburia /E. rectale\u003c/em\u003e by 54% (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), but not in other taxa (supplemental Fig.\u0026nbsp;2).\u003c/p\u003e\u003cp\u003e\u003cb\u003eModest alleviation by\u003c/b\u003e \u003cb\u003eR. torques\u003c/b\u003e \u003cb\u003eof dietary Se deficiency-induced glucose intolerance, but not insulin insensitivity, in conventional mice\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSe-deficient female mice at ~\u0026thinsp;7 months of age exhibited glucose intolerance (Fig.\u0026nbsp;3A and B) and insulin resistance (Fig.\u0026nbsp;3C and D) over a 2-hour period following injections. However, fasting blood glucose concentrations were not affected by dietary Se deficiency or by \u003cem\u003eR. torques\u003c/em\u003e oral gavage (Fig.\u0026nbsp;3A and C). While oral gavage with \u003cem\u003eR. torques\u003c/em\u003e modestly alleviated (11% improvement; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) Se deficiency-induced glucose intolerance at 0.5 h post-injection (Fig.\u0026nbsp;3A), it had no significant effect on overall glucose tolerance or insulin sensitivity across the entire time course in either the Se-deficient or Se-adequate mice.\u003c/p\u003e\u003cp\u003e\u003cb\u003eR. torques\u003c/b\u003e \u003cb\u003eoral gavage and dietary Se deficiency differentially affect selenoprotein expression in muscle and liver\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe next assessed body Se status in the liver and skeletal muscle by evaluating protein expression levels of selected selenoproteins. Western blot analysis revealed that dietary Se deficiency reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) the protein levels of GPX1 by 41% and SELENOW by 89% in muscle; whereas SELENOH and SELENOP expression remained unaffected (Fig.\u0026nbsp;5A). In the liver (Fig.\u0026nbsp;5B), Se deficiency decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) protein levels of SELENOP by 50%, GPX1 by 86%, SELENOH by 56%, and SELENOW by 88%. Notably, the reduction in hepatic GPX1 was partially reversed by \u003cem\u003eR. torques\u003c/em\u003e oral gavage (1.6-fold increase; \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In Se-adequate mice, oral gavage of \u003cem\u003eR. torques\u003c/em\u003e reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) hepatic levels of both SELENOP and GPX1 by 27%, but had no effect on SELENOH or SELENOW in the liver, nor on any selenoprotein expression in muscle.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe have previously shown that long-term dietary Se deficiency induces type 2 diabetes-like symptoms in telomere-humanized mice aged 12 and 18 months in both sexes\u0026nbsp;[15], as well as middle-aged and mature wild-type male mice [16, 20]. Here, compared to wild-type male mice aged 4-7 months [16], dietary Se deficiency in age-matched females induces glucose intolerance and insulin resistance to a lesser extent.\u0026nbsp;Consistent with our findings, there is evidence that females are generally more insulin-sensitive than males\u0026nbsp;[21, 22], and women have decreased susceptibility to fatty acid–induced peripheral insulin resistance\u0026nbsp;[23]. In rodent models, males typically exhibit more pronounced diabetes symptoms than females\u0026nbsp;[24-27]. These sex-related differences in type 2 diabetes-like symptoms may be partially attributed to the actions of estrogen and testosterone. For example, decreases in estrogen and increases in testosterone levels during menopause are associated with a loss of subcutaneous fat, a gain of visceral fat, and increased insulin resistance\u0026nbsp;[28]. Indeed, we have previously shown that Se status under Se deficiency is tissue-specific and dependent on sex and age\u0026nbsp;[29].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn contrast to \u003cem\u003eA. muciniphila\u003c/em\u003e [16]\u003cem\u003e,\u0026nbsp;\u003c/em\u003eoral gavage with \u003cem\u003eR. torques\u003c/em\u003e only modestly alleviates Se deficiency-induced type 2 diabetes symptoms. An intriguing question arises: why do these two mucin-degrading bacteria have opposing impacts on Se deficiency-induced metabolic symptoms? Although some mucin-degrading bacteria, such as \u003cem\u003eA. muciniphila\u003c/em\u003e,are associated with health benefits, their close proximity to the intestinal epithelium may pose risks to host cells by compromising the gut barrier. It has been suggested that a reduction in cecal \u003cem\u003eR. torques\u003c/em\u003e abundance in response to \u003cem\u003eLactobacillus acidophilus\u003c/em\u003e treatment may improve reproductive performance of broiler chickens by reducing the incidence of pasty vent, a stress-induced condition characterized by dry, cake-like droppings around the vent of baby chicks [30]. Supporting this observation, mucin degradation has been proposed to be pathogenic, as the loss of the protective mucus layer may increase the exposure of gastrointestinal epithelial cells to pathogens [31]. Moreover, the abundance of total mucosa-associated bacterial 16S rRNA genes is elevated in inflammatory bowel disorder, suggesting an increased availability of digestible endogenous mucus that may, in turn, sustain the growth of non-mucolytic mucosa-associated bacteria [32]. These findings highlight the need for further investigation into the role of mucin in shaping microbial community dynamics and host–microbe interactions.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eR. torques\u003c/em\u003e has been reported to be correlated with markers of insulin resistance in humans [33]. In our study, \u003cem\u003eR. torques\u003c/em\u003e abundance was increased in mice fed a Se-deficient diet compared to those on a Se-adequate diet. However, our results indicate that oral administration of \u003cem\u003eR. torques\u003c/em\u003e modestly improves glucose intolerance but has no effect on insulin resistance in Se-deficiency female mice. We speculate that \u003cem\u003e1)\u003c/em\u003e female mice may exhibit greater baseline insulin sensitivity, potentially masking any mild effects of \u003cem\u003eR. torques\u003c/em\u003e; \u003cem\u003e2)\u003c/em\u003e interactions between \u003cem\u003eR. torques\u003c/em\u003e and host selenoproteins may counterbalance each other’s influence on type-2 diabetes pathogenesis; 3) the effects of\u003cem\u003e\u0026nbsp;R. torques\u0026nbsp;\u003c/em\u003eon Se deficiency-induced type 2 diabetes may be age-dependent, becoming apparent only in middle-aged or older mice, but not in younger mice.\u003c/p\u003e\n\u003cp\u003eAlthough a recent study using the type strain \u003cem\u003eR. torques\u0026nbsp;\u003c/em\u003eATCC 27756 (BSL-1) demonstrated a pronounced alleviation of glucose intolerance in obese mice via oral gavage [2], we employed a non-type strain, \u003cem\u003eR. torques\u003c/em\u003e ATCC BAA-2281 (BSL-2), and observed only a modest improvement in Se deficiency-induced glucose intolerance. In addition to the fact that ATCC BAA-2281 has been minimally characterized, we selected this strain to test its effect on type 2 diabetes-like symptoms because it is deposited in ATCC as a \u003cem\u003eLachnospiraceae\u003c/em\u003e bacterium. We have previously shown that dietary Se deficiency enriches the relative abundance of \u003cem\u003eLachnospiraceae\u0026nbsp;\u003c/em\u003ein aged telomere-humanized mice in a sexually dimorphic manner [16]. To better understand the differing efficacy of these two \u003cem\u003eR. torques\u003c/em\u003e strains in modulating glucose tolerance, future studies should consider factors such as host obesity status, strain-level genetic variation, and the underlying etiology of type 2 diabetes.\u003c/p\u003e\n\u003cp\u003eOther gut bacteria may respond to \u003cem\u003eR. torques\u003c/em\u003e oral gavage and influence outcomes related to glucose intolerance and insulin resistance in Se-deficient mice. Indeed, Se deficiency in females decreases the abundance of \u003cem\u003eLactobacillus spp.\u003c/em\u003e, \u003cem\u003eF. prausnitzii\u003c/em\u003e, and \u003cem\u003eRoseburia spp./E. rectale\u003c/em\u003e, while increasing the abundance of \u003cem\u003eR. torques\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e in the cecal content. However, \u003cem\u003eR. torques\u003c/em\u003e oral gavage results in increased \u003cem\u003eE. coli\u003c/em\u003e abundance in Se-deficient mice and decreased \u003cem\u003eLactobacillus\u003c/em\u003e \u003cem\u003espp\u003c/em\u003e. and \u003cem\u003eRoseburia spp./E. rectal\u003c/em\u003e abundance in Se-adequate mice. These findings are in contrast to those observed with \u003cem\u003eA. muciniphila\u0026nbsp;\u003c/em\u003eoral gavage, suggesting that these two mucin-degrading bacteria impact gut homeostasis and host health through distinct mechanisms.\u003c/p\u003e\n\u003cp\u003eAltogether, our results show that the administration of the \u003cem\u003eR. torques\u003c/em\u003e ATCC BAA-2281 strain only modestly alleviates glucose intolerance in Se-deficient female mice, and that changes in five other bacterial taxa display patterns distinct from those observed with \u003cem\u003eA. muciniphila\u0026nbsp;\u003c/em\u003eoral gavage in male mice [16].\u0026nbsp;Further studies are needed to deepen our understanding and provide mechanistic insight into how \u003cem\u003eR. torques\u0026nbsp;\u003c/em\u003einfluences Se status and type 2 diabetes in the host, as well as how Se may prevent the early onset of type 2 diabetes in a sexually dimorphic manner.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAKT, mouse thymoma viral protooncogene; GPX1, glutathione peroxidase 1; GPX3, glutathione peroxidase 1; Se, selenium; SELENOH, selenoprotein H; SELENOP, selenoprotein P; SELENOW, selenoprotein W; SCFA, short-chain fatty acid.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eSupported by NIH DK117407.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: WHC. Methodology: YCH and WHC. Investigation: YCH. Visualization: YCH. Supervision: WHC. Writing\u0026mdash;review \u0026amp; editing: WHC. Funding acquisition: WHC. Both authors contributed to the article and approved the submitted version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eVacca M, Celano G, Calabrese FM, Portincasa P, Gobbetti M, De Angelis M. The Controversial Role of Human Gut Lachnospiraceae. Microorganisms. 2020;8(4). doi: https://doi.org/10.3390/microorganisms8040573.\u003c/li\u003e\n\u003cli\u003eFan Y, Lyu L, Vazquez-Uribe R, Zhang W, Bongers M, Koulouktsis A, et al. Polypeptides synthesized by common bacteria in the human gut improve rodent metabolism. Nature Microbiology. 2025;10(8):1918-39. doi: https://doi.org/10.1038/s41564-025-02064-x.\u003c/li\u003e\n\u003cli\u003eHold GL, Pryde SE, Russell VJ, Furrie E, Flint HJ. Assessment of microbial diversity in human colonic samples by 16S rDNA sequence analysis. 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Endocrine reviews. 2006;27(6):575-605. doi: https://doi.org/10.1210/er.2005-0020.\u003c/li\u003e\n\u003cli\u003eCao L, Zhang L, Zeng H, Wu RT, Wu TL, Cheng WH. Analyses of selenotranscriptomes and selenium concentrations in response to dietary selenium deficiency and age reveal common and distinct patterns by tissue and sex in telomere-dysfunctional mice. J Nutr. 2017;147(10):1858-66. doi: https://doi.org/10.3945/jn.117.247775.\u003c/li\u003e\n\u003cli\u003eDe Cesare A, Sirri F, Manfreda G, Moniaci P, Giardini A, Zampiga M, et al. Effect of dietary supplementation with Lactobacillus acidophilus D2/CSL (CECT 4529) on caecum microbioma and productive performance in broiler chickens. PloS one. 2017;12(5):e0176309. doi: https://doi.org/10.1371/journal.pone.0176309.\u003c/li\u003e\n\u003cli\u003eRuseler-van Embden JG, van Lieshout LM, Gosselink MJ, Marteau P. Inability of Lactobacillus casei strain GG, L. acidophilus, and Bifidobacterium bifidum to degrade intestinal mucus glycoproteins. 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Nutrition \u0026amp; diabetes. 2015;5(6):e159-e. doi: https://doi.org/10.1038/nutd.2015.9.\u003c/li\u003e\n\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":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Selenium deficiency, glucose intolerance, insulin resistance, gut microbiota, Ruminococcus torques ","lastPublishedDoi":"10.21203/rs.3.rs-7474743/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7474743/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eWe previously found that dietary selenium (Se) deficiency and age increase the\u003cem\u003e \u003c/em\u003efecal abundance of \u003cem\u003eLachnospiraceae\u003c/em\u003e in aged telomere-humanized mice in a sexually dimorphic manner. Although \u003cem\u003eLachnospiraceae\u003c/em\u003e are key contributors of short-chain fatty acids to the host, different taxa within this family exert distinct effects on host physiology. Among them, \u003cem\u003eRuminococcus torques\u003c/em\u003e has been associated with type 2 diabetes. In the present study, we aimed to determine whether, and how, \u003cem\u003eR. torques\u003c/em\u003e interacts with dietary Se to influence type 2 diabetes-like symptoms. Sixteen weaning female C57BL/6J mice were fed either a Se-adequate or Se-deficient diet for 26 weeks. From weeks 21 to 25, half of the mice in each dietary group received daily oral gavage of \u003cem\u003eR. torques\u003c/em\u003e (2 × 10⁸ CFU in 0.2 mL). All mice were euthanized at week 26. Dietary Se deficiency induced glucose intolerance (13%) and insulin resistance (16%) (\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05). While \u003cem\u003eR. torques \u003c/em\u003eadministration modestly alleviated glucose intolerance in Se-deficient mice, it did not affect insulin resistance or fasting glucose levels. Se deficiency reduced the relative abundance of \u003cem\u003eLactobacillus\u003c/em\u003e \u003cem\u003espp\u003c/em\u003e., \u003cem\u003eF. prausnitzii\u003c/em\u003e, and \u003cem\u003eRoseburia spp./E. rectale\u003c/em\u003e in the cecal content, and these taxa were unaffected by \u003cem\u003eR. torques \u003c/em\u003etreatment. In contrast, Se deficiency increased the relative abundance of \u003cem\u003eR. torques\u003c/em\u003e and \u003cem\u003eE. coli\u003c/em\u003e in cecal samples, with \u003cem\u003eE. coli\u003c/em\u003e levels further elevated by \u003cem\u003eR. torques \u003c/em\u003egavage. Notably, \u003cem\u003eR. torques\u003c/em\u003e oral gavage\u003cem\u003e 1\u003c/em\u003e) decreased SELENOP and GPX1 protein levels in the liver, but not in skeletal muscle, of Se-adequate mice; \u003cem\u003e2)\u003c/em\u003e increased liver GPX1 protein levels in Se-deficient mice. Altogether, \u003cem\u003eR. torques \u003c/em\u003eadministration modestly alleviates glucose intolerance and increases both liver GPX1 protein levels and cecal \u003cem\u003eE. coli\u003c/em\u003e abundance in Se-deficient mature female mice with diabetic symptoms.\u003c/p\u003e","manuscriptTitle":"Ruminococcus torques Administration Modestly Alleviates Dietary Selenium Deficiency- Induced Glucose Intolerance in Mature Female Mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-29 17:04:44","doi":"10.21203/rs.3.rs-7474743/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2026-01-29T19:31:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-16T10:24:23+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-08T19:26:54+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"102138228606024987246291168372002436713","date":"2025-12-06T02:20:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117875854364201074125803101211708958731","date":"2025-10-02T14:54:04+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-23T22:31:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"37907359168034794452041029904954495073","date":"2025-09-18T19:06:19+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-09-18T13:55:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-28T02:43:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-28T02:27:49+00:00","index":"","fulltext":""},{"type":"submitted","content":"Biological Trace Element Research","date":"2025-08-27T21:28:38+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"biological-trace-element-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bter","sideBox":"Learn more about [Biological Trace Element Research](https://www.springer.com/journal/12011)","snPcode":"12011","submissionUrl":"https://submission.nature.com/new-submission/12011/3","title":"Biological Trace Element Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"1044a423-6f71-478b-a460-a90a7bd36ee0","owner":[],"postedDate":"September 29th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-04-13T12:56:40+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-29 17:04:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7474743","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7474743","identity":"rs-7474743","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}