Metabolomics Reveals Broad-Scale Metabolic Perturbations in Hyperglycemic Mothers during Pregnancy Running title: Metabolomics of hyperglycemic pregnancy Key terms: metabolomics; hyperglycemia; pregnancy

Objective: To characterize metabolites across the range of maternal glucose by comparing metabolomic profiles of mothers with high and low fasting plasma glucose (FPG). Research Design and Methods: We compared fasting serum from an oral glucose-tolerance test at ~28 weeks’ gestation from 67 Northern European ancestry mothers from the Hyperglycemia and Adverse Pregnancy Outcome (HAPO) Study with high (>90 percentile) FPG to 50 mothers with low (<10 percentile) FPG but comparable BMI. Metabolic data from biochemical analyses of conventional clinical metabolites, targeted mass-spectrometry (MS)-based measurement of amino acids, and non-targeted gas chromatography/MS were subjected to permetabolite analyses and collective pathway analyses using Unipathway annotation. Results: High FPG mothers had a metabolic profile consistent with insulin resistance including higher triglycerides, 3-hydroxybutyrate, and amino acids including alanine, proline and branched chain amino acids (false discovery rate (FDR)-adjusted p<0.05). Lower 1,5-anhydroglucitol in high FPG mothers suggested recent hyperglycemic excursions (FDR-adjusted p<0.05). Pathway analyses indicated differences in amino acid degradation pathways for the two groups (FDRadjusted p<0.05), consistent with population-based findings in non-pregnant populations. Exploratory analyses with newborn outcomes indicated positive associations for maternal triglycerides with neonatal sum of skinfolds and cord C-peptide and a negative association between maternal glycine and cord C-peptide (p<0.05). Conclusions: Metabolomics reveals perturbations in metabolism of major macronutrients and amino acid degradation pathways in high vs. low FPG mothers. Page 2 of 35 Diabetes Care