Technical Abstract: Insulin resistance progressing to type 2 diabetes mellitus (T2DM) is marked by a broad perturbation of macronutrient intermediary metabolism. Understanding the biochemical networks that underlie metabolic homeostasis and how they associate with insulin action will help unravel diabetes etiology and should foster discovery of new biomarkers of disease risk and severity. We examined differences in plasma levels of >350 metabolites in fasted obese T2DM vs. obese non-diabetic African-American women, and utilized principal components analysis (PCA) to identify 53 metabolite components, many unique, contributing to a PC score strongly correlated with fasting HbA1c over a broad range of the latter (r=0.74; p<0.0001). In addition to many unidentified metabolites, specific metabolites that were increased significantly in T2DM subjects included certain amino acids (i.e., leucine/2-ketoisocaproate, valine, cystine, histidine), 2-hydroxybutanoate (2-HB), long-chain fatty acids, and carbohydrate derivatives. Leucine and valine concentrations significantly correlated with total acylcarnitine levels across the study cohort (and rising with increasing HbA1c). This appears to reflect a close link between abnormalities in glucose homeostasis, amino acid catabolism, and inefficiency of fuel combustion in the tricarboxylic acid (TCA) cycle. One proposed mechanism for the latter is “anaplerotic stress” concomitant with reduced amino acid-derived carbon flux to TCA cycle intermediates coupled to perturbation in cataplerosis (TCA intermediate loss).