Novel biomarkers for pre‐diabetes identified by metabolomics

Abstract

Article25 September 2012Open Access Novel biomarkers for pre-diabetes identified by metabolomics Rui Wang-Sattler Corresponding Author Rui Wang-Sattler Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Zhonghao Yu Zhonghao Yu Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Christian Herder Christian Herder German Diabetes Center, Institute for Clinical Diabetology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany Search for more papers by this author Ana C Messias Ana C Messias Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Anna Floegel Anna Floegel Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany Search for more papers by this author Ying He Ying He Shanghai Center for Bioinformation Technology, Shanghai, China Key Lab of Systems Biology, Bioinformatics Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Katharina Heim Katharina Heim Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Monica Campillos Monica Campillos Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Christina Holzapfel Christina Holzapfel Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Else Kroener-Fresenius-Center for Nutritional Medicine, University Hospital ‘Klinikum rechts der Isar’, Technische Universität München, Munich, Germany Search for more papers by this author Barbara Thorand Barbara Thorand Institute of Epidemiology II, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Harald Grallert Harald Grallert Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Tao Xu Tao Xu Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Erik Bader Erik Bader Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Cornelia Huth Cornelia Huth Institute of Epidemiology II, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Kirstin Mittelstrass Kirstin Mittelstrass Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Angela Döring Angela Döring Institute of Epidemiology I, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Christa Meisinger Christa Meisinger Institute of Epidemiology II, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Christian Gieger Christian Gieger Institute of Genetic Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Cornelia Prehn Cornelia Prehn Genome Analysis Center, Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Werner Roemisch-Margl Werner Roemisch-Margl Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Maren Carstensen Maren Carstensen German Diabetes Center, Institute for Clinical Diabetology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany Search for more papers by this author Lu Xie Lu Xie Shanghai Center for Bioinformation Technology, Shanghai, China Search for more papers by this author Hisami Yamanaka-Okumura Hisami Yamanaka-Okumura Department of Clinical Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan Search for more papers by this author Guihong Xing Guihong Xing Benxi Diabetes Clinic, Benxi Central Hospital, Benxi, China Search for more papers by this author Uta Ceglarek Uta Ceglarek Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany Search for more papers by this author Joachim Thiery Joachim Thiery Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany Search for more papers by this author Guido Giani Guido Giani German Diabetes Center, Institute of Biometrics and Epidemiology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany Search for more papers by this author Heiko Lickert Heiko Lickert Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Xu Lin Xu Lin Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yixue Li Yixue Li Shanghai Center for Bioinformation Technology, Shanghai, China Key Lab of Systems Biology, Bioinformatics Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Heiner Boeing Heiner Boeing Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany Search for more papers by this author Hans-Georg Joost Hans-Georg Joost Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany Search for more papers by this author Martin Hrabé de Angelis Martin Hrabé de Angelis Genome Analysis Center, Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany Chair of Experimental Genetics, Technische Universität München, Munich, Germany Search for more papers by this author Wolfgang Rathmann Wolfgang Rathmann German Diabetes Center, Institute of Biometrics and Epidemiology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany Search for more papers by this author Karsten Suhre Karsten Suhre Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Faculty of Biology, Ludwig-Maximilians-Universität, Planegg-Martinsried, Germany Department of Physiology and Biophysics, Weill Cornell Medical College in Qatar (WCMC-Q), Doha, Qatar Search for more papers by this author Holger Prokisch Holger Prokisch Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Annette Peters Annette Peters Institute of Epidemiology II, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Thomas Meitinger Thomas Meitinger Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany Department of Metabolic Diseases, University Hospital Düsseldorf, Düsseldorf, Germany Search for more papers by this author Michael Roden Michael Roden German Diabetes Center, Institute for Clinical Diabetology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany Klinikum rechts der Isar, Technische Universität München, Munich, Germany Search for more papers by this author H-Erich Wichmann H-Erich Wichmann Institute of Epidemiology I, Helmholtz Zentrum München, Neuherberg, Germany Institute of Medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians-Universität, Munich, Germany Search for more papers by this author Tobias Pischon Tobias Pischon Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany Molecular Epidemiology Group, Max Delbrueck Center for Molecular Medicine (MDC), Berlin-Buch, Germany Search for more papers by this author Jerzy Adamski Jerzy Adamski Genome Analysis Center, Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany Chair of Experimental Genetics, Technische Universität München, Munich, Germany Search for more papers by this author Thomas Illig Thomas Illig Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Hannover Unified Biobank, Hannover Medical School, Hannover, Germany Search for more papers by this author Rui Wang-Sattler Corresponding Author Rui Wang-Sattler Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Zhonghao Yu Zhonghao Yu Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Christian Herder Christian Herder German Diabetes Center, Institute for Clinical Diabetology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany Search for more papers by this author Ana C Messias Ana C Messias Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Anna Floegel Anna Floegel Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany Search for more papers by this author Ying He Ying He Shanghai Center for Bioinformation Technology, Shanghai, China Key Lab of Systems Biology, Bioinformatics Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Katharina Heim Katharina Heim Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Monica Campillos Monica Campillos Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Christina Holzapfel Christina Holzapfel Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Else Kroener-Fresenius-Center for Nutritional Medicine, University Hospital ‘Klinikum rechts der Isar’, Technische Universität München, Munich, Germany Search for more papers by this author Barbara Thorand Barbara Thorand Institute of Epidemiology II, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Harald Grallert Harald Grallert Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Tao Xu Tao Xu Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Erik Bader Erik Bader Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Cornelia Huth Cornelia Huth Institute of Epidemiology II, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Kirstin Mittelstrass Kirstin Mittelstrass Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Angela Döring Angela Döring Institute of Epidemiology I, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Christa Meisinger Christa Meisinger Institute of Epidemiology II, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Christian Gieger Christian Gieger Institute of Genetic Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Cornelia Prehn Cornelia Prehn Genome Analysis Center, Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Werner Roemisch-Margl Werner Roemisch-Margl Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Maren Carstensen Maren Carstensen German Diabetes Center, Institute for Clinical Diabetology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany Search for more papers by this author Lu Xie Lu Xie Shanghai Center for Bioinformation Technology, Shanghai, China Search for more papers by this author Hisami Yamanaka-Okumura Hisami Yamanaka-Okumura Department of Clinical Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan Search for more papers by this author Guihong Xing Guihong Xing Benxi Diabetes Clinic, Benxi Central Hospital, Benxi, China Search for more papers by this author Uta Ceglarek Uta Ceglarek Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany Search for more papers by this author Joachim Thiery Joachim Thiery Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany Search for more papers by this author Guido Giani Guido Giani German Diabetes Center, Institute of Biometrics and Epidemiology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany Search for more papers by this author Heiko Lickert Heiko Lickert Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Xu Lin Xu Lin Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Yixue Li Yixue Li Shanghai Center for Bioinformation Technology, Shanghai, China Key Lab of Systems Biology, Bioinformatics Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China Search for more papers by this author Heiner Boeing Heiner Boeing Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany Search for more papers by this author Hans-Georg Joost Hans-Georg Joost Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany Search for more papers by this author Martin Hrabé de Angelis Martin Hrabé de Angelis Genome Analysis Center, Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany Chair of Experimental Genetics, Technische Universität München, Munich, Germany Search for more papers by this author Wolfgang Rathmann Wolfgang Rathmann German Diabetes Center, Institute of Biometrics and Epidemiology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany Search for more papers by this author Karsten Suhre Karsten Suhre Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany Faculty of Biology, Ludwig-Maximilians-Universität, Planegg-Martinsried, Germany Department of Physiology and Biophysics, Weill Cornell Medical College in Qatar (WCMC-Q), Doha, Qatar Search for more papers by this author Holger Prokisch Holger Prokisch Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Annette Peters Annette Peters Institute of Epidemiology II, Helmholtz Zentrum München, Neuherberg, Germany Search for more papers by this author Thomas Meitinger Thomas Meitinger Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany Department of Metabolic Diseases, University Hospital Düsseldorf, Düsseldorf, Germany Search for more papers by this author Michael Roden Michael Roden German Diabetes Center, Institute for Clinical Diabetology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany Klinikum rechts der Isar, Technische Universität München, Munich, Germany Search for more papers by this author H-Erich Wichmann H-Erich Wichmann Institute of Epidemiology I, Helmholtz Zentrum München, Neuherberg, Germany Institute of Medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians-Universität, Munich, Germany Search for more papers by this author Tobias Pischon Tobias Pischon Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany Molecular Epidemiology Group, Max Delbrueck Center for Molecular Medicine (MDC), Berlin-Buch, Germany Search for more papers by this author Jerzy Adamski Jerzy Adamski Genome Analysis Center, Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany Chair of Experimental Genetics, Technische Universität München, Munich, Germany Search for more papers by this author Thomas Illig Thomas Illig Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany Hannover Unified Biobank, Hannover Medical School, Hannover, Germany Search for more papers by this author Author Information Rui Wang-Sattler 1,‡, Zhonghao Yu1,‡, Christian Herder2,‡, Ana C Messias3,‡, Anna Floegel4, Ying He5,6, Katharina Heim7, Monica Campillos8, Christina Holzapfel1,9, Barbara Thorand10, Harald Grallert1, Tao Xu1, Erik Bader1, Cornelia Huth10, Kirstin Mittelstrass1, Angela Döring11, Christa Meisinger10, Christian Gieger12, Cornelia Prehn13, Werner Roemisch-Margl8, Maren Carstensen2, Lu Xie5, Hisami Yamanaka-Okumura14, Guihong Xing15, Uta Ceglarek16, Joachim Thiery16, Guido Giani17, Heiko Lickert18, Xu Lin19, Yixue Li5,6, Heiner Boeing4, Hans-Georg Joost4, Martin Hrabé de Angelis13,20, Wolfgang Rathmann17, Karsten Suhre8,21,22, Holger Prokisch7, Annette Peters10, Thomas Meitinger7,23, Michael Roden2,24, H-Erich Wichmann11,25, Tobias Pischon4,26, Jerzy Adamski13,20 and Thomas Illig1,27 1Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany 2German Diabetes Center, Institute for Clinical Diabetology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany 3Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany 4Department of Epidemiology, German Institute of Human Nutrition Potsdam-Rehbruecke, Nuthetal, Germany 5Shanghai Center for Bioinformation Technology, Shanghai, China 6Key Lab of Systems Biology, Bioinformatics Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China 7Institute of Human Genetics, Helmholtz Zentrum München, Neuherberg, Germany 8Institute of Bioinformatics and Systems Biology, Helmholtz Zentrum München, Neuherberg, Germany 9Else Kroener-Fresenius-Center for Nutritional Medicine, University Hospital ‘Klinikum rechts der Isar’, Technische Universität München, Munich, Germany 10Institute of Epidemiology II, Helmholtz Zentrum München, Neuherberg, Germany 11Institute of Epidemiology I, Helmholtz Zentrum München, Neuherberg, Germany 12Institute of Genetic Epidemiology, Helmholtz Zentrum München, Neuherberg, Germany 13Genome Analysis Center, Institute of Experimental Genetics, Helmholtz Zentrum München, Neuherberg, Germany 14Department of Clinical Nutrition, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan 15Benxi Diabetes Clinic, Benxi Central Hospital, Benxi, China 16Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Leipzig, Germany 17German Diabetes Center, Institute of Biometrics and Epidemiology, Leibniz Center for Diabetes Research at Heinrich Heine University, Düsseldorf, Germany 18Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany 19Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China 20Chair of Experimental Genetics, Technische Universität München, Munich, Germany 21Faculty of Biology, Ludwig-Maximilians-Universität, Planegg-Martinsried, Germany 22Department of Physiology and Biophysics, Weill Cornell Medical College in Qatar (WCMC-Q), Doha, Qatar 23Department of Metabolic Diseases, University Hospital Düsseldorf, Düsseldorf, Germany 24Klinikum rechts der Isar, Technische Universität München, Munich, Germany 25Institute of Medical Informatics, Biometry and Epidemiology, Ludwig-Maximilians-Universität, Munich, Germany 26Molecular Epidemiology Group, Max Delbrueck Center for Molecular Medicine (MDC), Berlin-Buch, Germany 27Hannover Unified Biobank, Hannover Medical School, Hannover, Germany ‡These authors contributed equally to this work *Corresponding author. Research Unit of Molecular Epidemiology, Helmholtz Zentrum München, 85764 Munich-Neuherberg, Germany. Tel.:+49 89 3187 3978; Fax:+49 89 3187 2428; E-mail: [email protected] Molecular Systems Biology (2012)8:615https://doi.org/10.1038/msb.2012.43 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Type 2 diabetes (T2D) can be prevented in pre-diabetic individuals with impaired glucose tolerance (IGT). Here, we have used a metabolomics approach to identify candidate biomarkers of pre-diabetes. We quantified 140 metabolites for 4297 fasting serum samples in the population-based Cooperative Health Research in the Region of Augsburg (KORA) cohort. Our study revealed significant metabolic variation in pre-diabetic individuals that are distinct from known diabetes risk indicators, such as glycosylated hemoglobin levels, fasting glucose and insulin. We identified three metabolites (glycine, lysophosphatidylcholine (LPC) (18:2) and acetylcarnitine) that had significantly altered levels in IGT individuals as compared to those with normal glucose tolerance, with P-values ranging from 2.4 × 10−4 to 2.1 × 10−13. Lower levels of glycine and LPC were found to be predictors not only for IGT but also for T2D, and were independently confirmed in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam cohort. Using metabolite–protein network analysis, we identified seven T2D-related genes that are associated with these three IGT-specific metabolites by multiple interactions with four enzymes. The expression levels of these enzymes correlate with changes in the metabolite concentrations linked to diabetes. Our results may help developing novel strategies to prevent T2D. Synopsis A targeted metabolomics approach was used to identify candidate biomarkers of pre-diabetes. The relevance of the identified metabolites is further corroborated with a protein-metabolite interaction network and gene expression data. Three metabolites (glycine, lysophosphatidylcholine (LPC) (18:2) and acetylcarnitine C2) were found with significantly altered levels in pre-diabetic individuals compared with normal controls. Lower levels of glycine and LPC (18:2) were found to predict risks for pre-diabetes and type 2 diabetes (T2D). Seven T2D-related genes (PPARG, TCF7L2, HNF1A, GCK, IGF1, IRS1 and IDE) are functionally associated with the three identified metabolites. The unique combination of methodologies, including prospective population-based and nested case–control, as well as cross-sectional studies, was essential for the identification of the reported biomarkers. Introduction Type 2 diabetes (T2D) is defined by increased blood glucose levels due to pancreatic β-cell dysfunction and insulin resistance without evidence for specific causes, such as autoimmune destruction of pancreatic β-cells (Krebs et al, 2002; Stumvoll et al, 2005; Muoio and Newgard, 2008). A state of pre-diabetes (i.e., impaired fasting glucose (IFG) and/or impaired glucose tolerance (IGT)) with only slightly elevated blood glucose levels may precede T2D for years (McGarry, 2002; Tabak et al, 2012). The development of diabetes in pre-diabetic individuals can be prevented or delayed by dietary changes and increased physical activity (Tuomilehto et al, 2001; Knowler et al, 2002). However, no specific biomarkers that enable prevention have been reported. Metabolomics studies allow metabolites involved in disease mechanisms to be discovered by monitoring metabolite level changes in predisposed individuals compared with healthy ones (Shaham et al, 2008; Newgard et al, 2009; Zhao et al, 2010; Pietilainen et al, 2011; Rhee et al, 2011; Wang et al, 2011; Cheng et al, 2012; Goek et al, 2012). Altered metabolite levels may serve as diagnostic biomarkers and enable preventive action. Previous cross-sectional metabolomics studies of T2D were either based on small sample sizes (Shaham et al, 2008; Wopereis et al, 2009; Zhao et al, 2010; Pietilainen et al, 2011) or did not consider the influence of common risk factors of T2D (Newgard et al, 2009). Recently, based on prospective nested case–control studies with relative large samples (Rhee et al, 2011; Wang et al, 2011), five branched-chain and aromatic amino acids were identified as predictors of T2D (Wang et al, 2011). Here, using various comprehensive large-scale approaches, we measured metabolite concentration profiles (Yu et al, 2012) in the population-based (Holle et al, 2005; Wichmann et al, 2005) Cooperative Health Research in the Region of Augsburg (KORA) baseline (survey 4 (S4)) and follow-up (F4) studies (Rathmann et al, 2009; Meisinger et al, 2010; Jourdan et al, 2012). The results of these cross-sectional and prospective studies allowed us to (i) reliably identify candidate biomarkers of pre-diabetes and (ii) build metabolite–protein networks to understand diabetes-related metabolic pathways. Results Study participants Individuals with known T2D were identified by physician-validated self-reporting (Rathmann et al, 2010) and excluded from our analysis, to avoid potential influence from anti-diabetic medication with non-fasting participants and individuals with missing values (Figure 1A). Based on both fasting and 2-h glucose values (i.e., 2 h post oral 75 g glucose load), individuals were defined according to the WHO diagnostic criteria to have normal glucose tolerance (NGT), isolated IFG (i-IFG), IGT or newly diagnosed T2D (dT2D) (WHO, 1999; Rathmann et al, 2009; Meisinger et al, 2010; Supplementary Table S1). The sample sets include 91 dT2D patients and 1206 individuals with non-T2D, including 866 participants with NGT, 102 with i-IFG and 238 with IGT, in the cross-sectional KORA S4 (Figure 1A; study characteristics are shown in Table I). Of the 1010 individuals in a fasting state who participated at baseline and follow-up surveys (Figure 1B, study characteristics of the KORA F4 survey are shown in Supplementary Table S2), 876 of them were non-diabetic at baseline. Out of these, about 10% developed T2D (i.e., 91 incident T2D) (Figure 1C). From the 641 individuals with NGT at baseline, 18% developed IGT (i.e., 118 incident IGT) 7 years later (Figure 1D). The study characteristics of the prospective KORA S4→F4 are shown in Table II. Figure 1.Population description. Metabolomics screens in the KORA cohort, at baseline S4 (A), overlapped between S4 and F4 (B) and prospective (C, D). Participant numbers are shown. Normal glucose tolerance (NGT), isolated impaired fasting glucose (i-IFG), impaired glucose tolerance (IGT), type 2 diabetes mellitus (T2D) and newly diagnosed T2D (dT2D). Non-T2D individuals include NGT, i-IFG and IGT participants. Download figure Download PowerPoint Table 1. Characteristics of the KORA S4 cross-sectional study sample Clinical and laboratory parameters NGT i-IFG IGT dT2D N 866 102 238 91 Age (years) 63.5±5.5 64.1±5.2 65.2±5.2 65.9±5.4 Sex (female) (%) 52.2 30.4 44.9 41.8 BMI (kg/m2) 27.7±4.1 29.2±4 29.6±4.1 30.2±3.9 Physical activity (% >1 h per week) 46.7 35.3 39.9 36.3 Alcohol intakea (%) 20.2 20.5 25.2 24.2 Current smoker (%) 14.8 10.8 10.9 23.1 Systolic BP (mm Hg) 131.7±18.9 138.9±17.9 140.7±19.8 146.8±21.5 HDL cholesterol (mg/dl) 60.5±16.4 55.7±15.9 55.7±15.1 50.0±15.8 LDL cholesterol (mg/dl) 154.5±39.8 152.1±37.7 155.2±38.6 146.1±44.6 Triglycerides (mg/dl) 120.7±68.3 145.0±96.0 146.6±80.0 170.6±107.1 HbA1c (%) 5.56±0.33 5.62±0.33 5.66±0.39 6.21±0.83 Fasting glucose (mg/dl) 95.6±7.1 114.2±3.7 104.5±9.7 133.2±31.7 2-h Glucose (mg/dl) 102.1±21.0 109.3±18.7 163.4±16.4 232.1±63.7 Fasting insulin (μU/ml) 10.48±7.28 16.26±9.67 13.92±9.53 17.70±12.61 NGT, normal glucose tolerance; i-IFG, isolated impaired fasting glucose; IGT, impaired glucose tolerance; dT2D, newly diagnosed type 2 diabetes; BP, blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein. Percentages of individuals or means±s.d. are given for each variable and each group (NGT, i-IFG, IGT and dT2D). a ⩾20 g/day for women; ⩾40 g/day for men. Table 2. Characteristics of the KORA S4→F4 prospective study samples NGT at baseline (n=589) Non-T2D at baseline (n=876) Remained NGT at follow-up Developed IGT at follow-up Remained Non-T2D at follow-up Developed T2D at follow-up N 471 118 785 91 Age (years) 62.4±5.4 63.9±5.5 62.9±5.4 65.5±5.2 Sex (female) (%) 52.2 55.9 50.8 34.1 BMI (kg/m2) 27.2±3.8 28.2±3.9 27.9±4 30.2±3.6 Physical activity (% >1 h per week) 52.9 43.2 52.2 58.2 Alcohol intakea (%) 19.9 20.3 20.6 19.8 Smoker (%) 14.6 9.3 12.0 14.3 Systolic BP (mm Hg) 129.6±18.2 134.2±18.7 132.4±18.6 137.8±19 HDL cholesterol (mg/dl) 61.3±16.8 58.9±16.2 60.0±16.5 51.9±12.4 LDL cholesterol (mg/dl) 153.9±38.4 156.9±42.7 154.5±39.5 157.7±41.6 Triglycerides (mg/dl) 118.1±63.9 129.5±79.0 125.0±70.0 151.2±74.2 HbA1c (%) 5.54±0.33 5.59±0.34 5.6±0.3 5.8±0.4 Fasting glucose (mg/dl) 94.7±6.9 96.6±7.1 97.7±8.8 106.1±10.1 2-h Glucose (mg/dl) 98.2±20.5 109.9±16.8 109.3±28 145.9±32.3 Fasting insulin (μU/ml) 9.91±6.48 11