Guanine and pregnenolone sulfate are associated with incident type 2 diabetes in two independent populations

Maria Barranco-Altirriba^1,2,3,4Minerva Granado-Casas^5,6,7,8Oscar Yanes^5,9Jordi Capellades^10,5Alexandra Junza^11,5Josep Franch-Nadal^5,6Joan Vendrell^12,5Gemma Llauradó^13,5Sergio Valdés^14,5Eva Garcia-Escobar^14,5Marcelino Bermúdez-López^15,16Jose Manuel Valdivielso¹⁶Victor-Miguel López-Lifante^17,18,19Cecilia Herrero-Alonso¹⁸Mireia Falguera^20,6Maria Belén Vilanova^21,6Ingrid Arteaga^18,22Pere Torán-Monserrat^18,23,24Alexandre Perera-LLuna^1,3,4Esmeralda Castelblanco²⁵Didac Mauricio^2,26,27,5*

• ¹B2SLab, Institut de Recerca i Innovació en Salut (IRIS), Universitat Politècnica de Catalunya, Barcelona, Spain
• ²Department of Endocrinology & Nutrition, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain
• ³Networking Biomedical Research Centre in the subject area of Bioengineering, Biomaterials and Nanomedicine (CIBER-BBN), Madrid, Spain
• ⁴Institut de Recerca Sant Joan de Deu, Barcelona, Spain
• ⁵CIBER of Diabetes and Associated Metabolic Diseases (CIBERDEM), Instituto de Salud Carlos III (ISCIII), Barcelona, Spain
• ⁶DAP-Cat Group, Research Support Unit, Institut Universitari d’Investigació en Atenció Primària Jordi Gol, Barcelona, Spain
• ⁷GESEC group, Department of Nursing and Physiotherapy, Faculty of Nursing and Physiotherapy, University of Lleida, Lleida, Spain
• ⁸Healthcare Research Group (GRECS), Institute of Biomedical Research in Lleida (IRBLleida), Lleida, Spain
• ⁹Universitat Rovira i Virgili, Department of Electronic Engineering, IISPV, Tarragona, Spain
• ¹⁰Institute of Health Research Pere Virgili (IISPV), Tarragona, Spain
• ¹¹Scientific and Technical Resources Services, Universitat Rovira i Virgili, Tarragona, Spain
• ¹²Department of Endocrinology and Nutrition, Research Unit, Institut d’Investigació Sanitària Pere Virgili (IISPV) - Hospital Universitari de Tarragona Joan XXIII, Universitat Rovira i Virgili, Tarragona, Spain
• ¹³Department of Endocrinology and Nutrition, Hospital del Mar, Hospital del Mar Medical Research Institute (IMIM), Barcelona, Spain
• ¹⁴UGC Endocrinología y Nutrición. Hospital Regional Universitario de Málaga. IBIMA-Plataforma BIONAND, Málaga, Spain
• ¹⁵Experimental Medicine department, University of Lleida, Lleida, Spain
• ¹⁶Vascular & Renal Translational Research group, Institut de Recerca Biomédica IRBLleida, Lleida, Spain
• ¹⁷Universitat Autonoma de Barcelona Facultat de Medicina, Bellaterra, Spain
• ¹⁸Unitat de Suport a la Recerca Metropolitana Nord, Institut Universitari d'Investigació en Atenció Primària Jordi Gol (IDIAP Jordi Gol), Mataró, Spain
• ¹⁹Primary Healthcare Palau-solità i Plegamans, Gerència d'Àmbit d'Atenció Primària Metropolitana Nord, Institut Català de la Salut, Barcelona, Spain
• ²⁰Institute for Biomedical Research Dr. Pifarré Foundation IRB Lleida, University of Lleida and Primary Health Care Centre Tàrrega, Gerència d’Atenció Primaria, Institut Català de la Salut, Lleida, Spain
• ²¹Primary Health Care Centre Igualada Nord, Consorci Sanitari de l'Anoia, Igualada, Spain
• ²²Primary Healthcare Center Vall del Tenes, Gerència d'Àmbit d'Atenció Primària Metropolitana Nord, Institut Català de la Salut, Barcelona, Spain
• ²³Universitat de Girona Facultat de Medicina, Girona, Spain
• ²⁴Multidisciplinary Research Group in Health and Society (GREMSAS), Institut Universitari d'Investigació en Atenció Primària Jordi Gol (IDIAPJGol), Barcelona, Spain
• ²⁵Division of Endocrinology, Metabolism and Lipid Research, Department of Medicine, Washington University School of Medicine, St. Louis, United States
• ²⁶University of Vic - Central University of Catalonia, Barcelona, Spain
• ²⁷Institut de Recerca Sant Pau, Barcelona, Spain

Background: Type 2 diabetes (T2D) is increasing its burden worldwide; therefore, research focused on its prediction and prevention is essential. Methods: We performed an untargeted metabolomics analysis using Ultra High-Performance Liquid Chromatography-Mass Spectrometry to discover metabolic biomarkers and biological pathways associated with incident T2D with a nested case-control design, followed by validation with targeted metabolomics in an independent cohort. In the discovery phase, plasma samples from 352 subjects (209 controls and 143 incident cases) were analyzed, collected with a mean (standard deviation) of 7.40 (0.76) years before they acquired the condition. Using this discovery phase cohort, six metabolites were identified using standards and were subsequently quantified in an independent validation phase cohort of 2,044 subjects (167 incident cases). Additionally, pathway enrichment was conducted in the discovery cohort. Results: Guanine, ecgonine, adenine, pregnenolone sulfate, phenyl sulfate, and citrulline were significantly associated with incident T2D in at least one of the analyses performed in the discovery phase. Among these, guanine, pregnenolone sulfate and citrulline maintained their significant associations with incident T2D in the validation cohort. Additionally, several pathways were significantly altered, with nucleotide metabolism and ABC transporter pathways among the most consistently affected. Conclusion: We identified significant associations of guanine, pregnenolone sulfate, and citrulline with incident T2D.