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Alterations in microbiome structure have been associated with a range of diseases, including type 2 diabetes mellitus. However, the underlying mechanisms for this remain poorly understood. In this study, a novel in vitro approach was utilized to investigate the interplay between gut bacteria, dietary metabolites, and metabolic dysfunction. Methods: Two representative gut bacterial species\u2014Bacteroides thetaiotaomicron and Lactobacillus fermentum\u2014were isolated from human faecal samples and subjected to controlled dietary manipulation to mimic eubiotic and dysbiotic conditions. Metabolites produced under these conditions were extracted, characterized, and quantified. To assess the functional impact of these metabolites, we utilized the INS-1 832\/3 insulinoma cell line, evaluating insulin sensitivity through glucose-stimulated insulin secretion and ERK1\/2 activation. Results: Our findings demonstrate that metabolites derived from high-carbohydrate\/high-fat diets exacerbate metabolic dysfunction, whereas those generated under high-fibre conditions significantly enhance insulin secretion and glucose-dependent ERK1\/2 activation in co-culture compared to monocultures. Conclusions: This work systematically disentangles the complex interactions between gut microbiota, diet, and disease, providing mechanistic insights into how microbial metabolites contribute to the onset of metabolic disorders.<\/jats:p>","DOI":"10.3390\/nu18020279","type":"journal-article","created":{"date-parts":[[2026,1,15]],"date-time":"2026-01-15T16:21:13Z","timestamp":1768494073000},"page":"279","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Investigating the Role of Diet-Manipulated Gut Bacteria in Pathogenesis of Type 2 Diabetes Mellitus\u2014An In Vitro Approach"],"prefix":"10.3390","volume":"18","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4069-3836","authenticated-orcid":false,"given":"Asha","family":"Guraka","sequence":"first","affiliation":[{"name":"College of Science and Engineering, University of Derby, Derby DE22 1GB, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Marie","family":"Lush","sequence":"additional","affiliation":[{"name":"College of Science and Engineering, University of Derby, Derby DE22 1GB, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4746-2416","authenticated-orcid":false,"given":"Georgios","family":"Zouganelis","sequence":"additional","affiliation":[{"name":"College of Science and Engineering, University of Derby, Derby DE22 1GB, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Joe","family":"Waldron","sequence":"additional","affiliation":[{"name":"College of Science and Engineering, University of Derby, Derby DE22 1GB, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0391-3721","authenticated-orcid":false,"given":"Subbareddy","family":"Mekapothula","sequence":"additional","affiliation":[{"name":"School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8381-2917","authenticated-orcid":false,"given":"Jinit","family":"Masania","sequence":"additional","affiliation":[{"name":"College of Science and Engineering, University of Derby, Derby DE22 1GB, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4167-1332","authenticated-orcid":false,"given":"Gareth Wynn Vaughan","family":"Cave","sequence":"additional","affiliation":[{"name":"School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6801-7245","authenticated-orcid":false,"given":"Myra Elizabeth","family":"Conway","sequence":"additional","affiliation":[{"name":"College of Science and Engineering, University of Derby, Derby DE22 1GB, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Gyanendra","family":"Tripathi","sequence":"additional","affiliation":[{"name":"School of Science and Technology, Nottingham Trent University, Nottingham NG11 8NS, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2989-9078","authenticated-orcid":false,"given":"Ali","family":"Kermanizadeh","sequence":"additional","affiliation":[{"name":"College of Science and Engineering, University of Derby, Derby DE22 1GB, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,1,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Silva, Y.P., Bernardi, A., and Frozza, R.L. 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