{"id":{"repo_id":"cambridge","oai_identifier":"oai:www.repository.cam.ac.uk:1810/377386"},"canonical_url":"https://search.dev.ndltd.org/etd/cambridge/oai:www.repository.cam.ac.uk:1810/377386","repository":{"repo_id":"cambridge","name":"Cambridge University","base_url":"https://api.repository.cam.ac.uk/server/oai/request"},"display":{"title":"Dynamic subcellular proteomics identifies novel regulators of adipocyte insulin action","abstract":"Insulin acts on adipocytes to control whole-body lipid and glucose metabolism. Following binding to its receptor on adipocytes, insulin activates a signalling cascade that promotes glucose uptake and inhibits lipolysis. These processes rely on changes in protein localisation. For example, insulin signalling induces the translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. However, our knowledge of spatial changes in response to insulin signalling is incomplete. In this thesis we used two orthologous proteomic methods to identify novel insulin-responsive protein translocation events in adipocytes, with the premise that protein localisation is intricately linked to protein function. By identifying new insulin-regulated protein translocation events, we also revealed novel functional regulators of the adipocyte insulin-response. First, the subcellular 3T3-L1 adipocyte proteome was mapped under baseline and insulin- stimulated (100 nM, 30 min) conditions using the Localisation of Organelle Proteins by Isotopic Tagging after Differential UltraCentrifugation (LOPIT-DC) method. Over 4000 proteins were identified, of which ∼40% were mapped to a specific organelle under each condition and ∼10% of proteins were predicted to translocate in response to insulin. Novel insulin-responsive protein translocation events identified included individual proteins (C3ORF18, SPRY4, GOLP3) and components of the endolysosomal trafficking and mTORC1 regulatory pathways. Our unbiased whole-cell proteomics approach revealed the plasma membrane as the most prominent site of insulin-responsive proteome remodelling. To further investigate these insulin- driven changes, we conducted a targeted cell-surface biotinylation proteomic study, enhancing the resolution of plasma membrane specific changes. This study identified additional novel insulin-stimulated protein translocation events (TSPAN31, SLC20A2, CD302) and confirmed translocation events identified in the LOPIT-DC study, including C3ORF18. C3ORF18, a previously uncharacterised protein was selected for further functional follow up, after been identified as insulin-responsive in both our subcellular and plasma membrane proteomics data-sets. Initial characterisation suggests this protein is required for insulin-stimulated glucose uptake, and we propose C3ORF18 may contribute to the generalised increase in endosomal trafficking observed in response to insulin. Collectively this thesis provides greater insight into the extent of subcellular protein translocation and plasma membrane proteome remodelling in response to insulin, providing new insight into adipocyte insulin action. Furthermore, we provide an accessible subcellular map as a resource for the adipocyte research community and provide a starting point for further investigation into the function of novel insulin-regulated proteins in adipocyte insulin action.","abstract_html":"Insulin acts on adipocytes to control whole-body lipid and glucose metabolism. Following binding to its receptor on adipocytes, insulin activates a signalling cascade that promotes glucose uptake and inhibits lipolysis. These processes rely on changes in protein localisation. For example, insulin signalling induces the translocation of the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane. However, our knowledge of spatial changes in response to insulin signalling is incomplete. In this thesis we used two orthologous proteomic methods to identify novel insulin-responsive protein translocation events in adipocytes, with the premise that protein localisation is intricately linked to protein function. By identifying new insulin-regulated protein translocation events, we also revealed novel functional regulators of the adipocyte insulin-response. First, the subcellular 3T3-L1 adipocyte proteome was mapped under baseline and insulin- stimulated (100 nM, 30 min) conditions using the Localisation of Organelle Proteins by Isotopic Tagging after Differential UltraCentrifugation (LOPIT-DC) method. Over 4000 proteins were identified, of which ∼40% were mapped to a specific organelle under each condition and ∼10% of proteins were predicted to translocate in response to insulin. Novel insulin-responsive protein translocation events identified included individual proteins (C3ORF18, SPRY4, GOLP3) and components of the endolysosomal trafficking and mTORC1 regulatory pathways. Our unbiased whole-cell proteomics approach revealed the plasma membrane as the most prominent site of insulin-responsive proteome remodelling. To further investigate these insulin- driven changes, we conducted a targeted cell-surface biotinylation proteomic study, enhancing the resolution of plasma membrane specific changes. This study identified additional novel insulin-stimulated protein translocation events (TSPAN31, SLC20A2, CD302) and confirmed translocation events identified in the LOPIT-DC study, including C3ORF18. C3ORF18, a previously uncharacterised protein was selected for further functional follow up, after been identified as insulin-responsive in both our subcellular and plasma membrane proteomics data-sets. Initial characterisation suggests this protein is required for insulin-stimulated glucose uptake, and we propose C3ORF18 may contribute to the generalised increase in endosomal trafficking observed in response to insulin. Collectively this thesis provides greater insight into the extent of subcellular protein translocation and plasma membrane proteome remodelling in response to insulin, providing new insight into adipocyte insulin action. Furthermore, we provide an accessible subcellular map as a resource for the adipocyte research community and provide a starting point for further investigation into the function of novel insulin-regulated proteins in adipocyte insulin action.","abstract_has_math":false,"creators":["Conway, Olivia"],"institution":"University of Cambridge","degree_name":"Doctor of Philosophy (PhD)","degree_level":"Doctoral","degree_discipline":null,"degree_department":null,"school":null,"contributors":[],"advisors":["Fazakerley, Daniel"],"committee_chairs":[],"committee_members":[],"year":2024,"date_issued":"2024-09-05","date_published":"2024-09-05","updated_at":"2026-07-24T01:33:15Z","subjects":["adipocyte","metabolism","proteomics"],"languages":["eng"],"rights":[],"rights_urls":["https://www.repository.cam.ac.uk/bitstreams/6c5fced5-0372-4a0f-b142-3b52650c4796/download","https://www.rioxx.net/licenses/all-rights-reserved/"],"identifier_entries":[]},"links":{"outbound_url":"https://doi.org/10.17863/CAM.114212","outbound_label":"DOI","outbound_source":"dc:identifier.doi"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["Fazakerley, Daniel"]},{"key":"dc:creator","label":"Author","values":["Conway, Olivia"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.issued","label":"Date","values":["2024-09-05"]},{"key":"dc:publisher.institution","label":"Dc Publisher Institution","values":["University of Cambridge"]},{"key":"dc:relation.isreferencedby.uri","label":"Dc Relation Isreferencedby URI","values":["https://www.repository.cam.ac.uk/handle/1810/377386"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"dc:type.qualificationlevel","label":"Dc Type Qualificationlevel","values":["Doctoral"]},{"key":"dc:type.qualificationname","label":"Dc Type Qualificationname","values":["Doctor of Philosophy (PhD)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["adipocyte","metabolism","proteomics"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:language","label":"Dc Language","values":["eng"]},{"key":"dc:rights","label":"Dc Rights","values":["https://www.repository.cam.ac.uk/bitstreams/6c5fced5-0372-4a0f-b142-3b52650c4796/download","https://www.rioxx.net/licenses/all-rights-reserved/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.doi","label":"DOI","values":["https://doi.org/10.17863/CAM.114212"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["https://www.repository.cam.ac.uk/bitstreams/c73b23f0-74f8-497e-ae8d-c40dbac8c3c3/download"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["Insulin acts on adipocytes to control whole-body lipid and glucose metabolism. 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First, the subcellular 3T3-L1 adipocyte proteome was mapped under baseline and insulin- stimulated (100 nM, 30 min) conditions using the Localisation of Organelle Proteins by Isotopic Tagging after Differential UltraCentrifugation (LOPIT-DC) method. Over 4000 proteins were identified, of which ∼40% were mapped to a specific organelle under each condition and ∼10% of proteins were predicted to translocate in response to insulin. Novel insulin-responsive protein translocation events identified included individual proteins (C3ORF18, SPRY4, GOLP3) and components of the endolysosomal trafficking and mTORC1 regulatory pathways. Our unbiased whole-cell proteomics approach revealed the plasma membrane as the most prominent site of insulin-responsive proteome remodelling. To further investigate these insulin- driven changes, we conducted a targeted cell-surface biotinylation proteomic study, enhancing the resolution of plasma membrane specific changes. 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Over 4000 proteins were identified, of which ∼40% were mapped to a specific organelle under each condition and ∼10% of proteins were predicted to translocate in response to insulin. Novel insulin-responsive protein translocation events identified included individual proteins (C3ORF18, SPRY4, GOLP3) and components of the endolysosomal trafficking and mTORC1 regulatory pathways. Our unbiased whole-cell proteomics approach revealed the plasma membrane as the most prominent site of insulin-responsive proteome remodelling. To further investigate these insulin- driven changes, we conducted a targeted cell-surface biotinylation proteomic study, enhancing the resolution of plasma membrane specific changes. This study identified additional novel insulin-stimulated protein translocation events (TSPAN31, SLC20A2, CD302) and confirmed translocation events identified in the LOPIT-DC study, including C3ORF18. C3ORF18, a previously uncharacterised protein was selected for further functional follow up, after been identified as insulin-responsive in both our subcellular and plasma membrane proteomics data-sets. Initial characterisation suggests this protein is required for insulin-stimulated glucose uptake, and we propose C3ORF18 may contribute to the generalised increase in endosomal trafficking observed in response to insulin. Collectively this thesis provides greater insight into the extent of subcellular protein translocation and plasma membrane proteome remodelling in response to insulin, providing new insight into adipocyte insulin action. 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