Identification of serum metabolites associated with risk of type 2 diabetes using a targeted metabolomic approach

Abstract

Metabolomic discovery of biomarkers of type 2 diabetes (T2D) risk may reveal etiological pathways and help to identify individuals at risk for disease. We prospectively investigated the association between serum metabolites measured by targeted metabolomics and risk of T2D in the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam (27,548 adults) among all incident cases of T2D (n = 800, mean follow-up 7 years) and a randomly drawn subcohort (n = 2,282). Flow injection analysis tandem mass spectrometry was used to quantify 163 metabolites, including acylcarnitines, amino acids, hexose, and phospholipids, in baseline serum samples. Serum hexose; phenylalanine; and diacyl-phosphatidylcholines C32:1, C36:1, C38:3, and C40:5 were independently associated with increased risk of T2D and serum glycine; sphingomyelin C16:1; acyl-alkyl-phosphatidylcholines C34:3, C40:6, C42:5, C44:4, and C44:5; and lysophosphatidylcholine C18:2 with decreased risk. Variance of the metabolites was largely explained by two metabolite factors with opposing risk associations (factor 1 relative risk in extreme quintiles 0.31 [95% CI 0.21-0.44], factor 2 3.82 [2.64-5.52]). The metabolites significantly improved T2D prediction compared with established risk factors. They were further linked to insulin sensitivity and secretion in the Tübingen Family study and were partly replicated in the independent KORA (Cooperative Health Research in the Region of Augsburg) cohort. The data indicate that metabolic alterations, including sugar metabolites, amino acids, and choline-containing phospholipids, are associated early on with a higher risk of T2D.

FIG. 1.

Two metabolite factors associated with risk of T2D. Presented is a two-dimensional factor loading plot obtained from PCA. For simple interpretation, metabolites that cluster together in the plot are related to one another. Metabolites presented in blue are associated with decreased risk of T2D, whereas metabolites presented in red are associated with increased risk of T2D. More specifically, the factor loadings represent the correlation coefficients of individual metabolites with corresponding metabolite factors and may range from −1 to 1. They were identified by PCA based on the correlation matrix of all metabolites significantly associated with risk of T2D in the EPIC-Potsdam study. An orthogonal varimax rotation was used, and two factors were retained because they accounted for >50% of the observed variance. a, acyl; aa, diacyl; ae, acyl-alkyl; C3, propionylcarnitine; Gly, glycine; H1, hexose; PC, phosphatidylcholine; Phe, phenylalanine; SM, sphingomyelin; Trp, tryptophan; Tyr, tyrosine; Val; valine; xLeu, isoleucine.

FIG. 2.

Relative contribution of metabolites to predict T2D in EPIC-Potsdam. Presented are ROC curves comparing different multivariable-adjusted models to predict T2D, including the DRS, the identified metabolites, glucose (Glc), and HbA_1c. The DRS (16) combines information on several diabetes risk factors, such as diet, lifestyle, and anthropometry, to estimate risk of developing T2D. The DRS is computed according to the following formula: DRS = (7.4 × waist circumference [cm]) − (2.4 × height [cm]) + (4.3 × age [years]) + (46 × hypertension [self-report]) + (49 × red meat [each 150 g/day]) – (9 × whole-grain bread [each 50 g/day]) – (4 × coffee [each 150 g/day]) – (20 × moderate alcohol [between 10 and 40 g/day]) – (2 × physical activity [h/week]) + (24 × former smoker) + (64 × current heavy smoker [≥ 20 cigarettes/day]). Metabolites are hexose; phenylalanine; glycine; sphingomyelin C16:1; diacyl-phosphatidylcholines C32:1, C36:1, C38:3, and C40:5; acyl-alkyl-phosphatidylcholines C34:3, C40:6, C42:5, C44:4, and C44:5; and lysophosphatidylcholine C18:2.

FIG. 3.

Examples of metabolites associated with risk of T2D. ▲Metabolites with an increased risk (hexose, phenylalanine, and diacyl-phosphatidylcholines [PCs] C32:1, C36:1, C38:3, and C40:5). *Metabolites with a decreased risk (glycine; sphingomyelin C16:1; acyl-alkyl-PCs C34:3, C40:6, C42:5, C44:4, and C44:5; and lysophosphatidylcholine C18:2). Note that the mass spectrometric assay used does not distinguish molecular lipids and sugar types among hexoses. Therefore, formulas are given for a molecule corresponding to molecular mass and composition. Positions of double bonds and chain length may vary if more than one acid residue is present. Arrows represent many reactions, and key intermediates are given in the brackets. aa, diacyl; ae, acyl-alkyl. (A high-quality color representation of this figure is available in the online issue.)