A) Scoring system for identification of reporter metabolites. Each metabolite is scored based on the scores of the associated enzyme-catalyzed reactions. Each enzyme, in turn, is assigned a score based on median of the p-values of the probes representing the corresponding gene. In case of a reaction catalyzed by an enzyme complex or a set of isozymes, minimum of the p-values of the corresponding enzymes is chosen. Numbers in bold are Z-scores for each reaction, the rest of the numbers represent p-values (significance of differential expression). B) Identification of transcription factor binding motifs. For a reporter metabolite, a set of up/down regulated neighbor (enzyme-coding) genes is selected. Promoter regions, upstream of transcription start site (TSS) of each of the selected genes are assessed for the enrichment of known transcription factor (TF) binding sequence motifs.

Comparisons are colored according to the dataset; blue – Mexican-American; orange – Swedish male dataset. A) Dendrogram of reporter metabolites identified in each of the comparisons based on Jaccard distance. B) Venn diagram showing the overlap of the reporter metabolites identified in the different comparisons.

A) Motif enrichment analysis for the genes associated with reporter metabolites from the T2DM vs NGT comparison. Reporter metabolites with up-regulated neighboring gene set are shown as red circles, whereas reporter metabolites with down-regulated neighboring gene set are represented as green circles. Transcription factor binding motifs (shown as triangles) are colored according to the number of enzyme sets in which they are enriched, ranging from light yellow (enriched in few sets) to orange (enriched in as many as 6 sets). Edges are scaled according to q-values signifying the confidence of the motif enrichment. B) Venn diagram showing the overlap of transcription factor binding motifs across the comparisons of T2DM with non-T2DM cases. Comparisons are colored according to the dataset; blue – Mexican-American; orange – Swedish male dataset.

Key metabolic and regulatory pathways associated with reporter metabolites identified in this study (T2DM vs NGT and T2DM vs FH− comparisons) are shown. Metabolites in bold black font are reporter metabolites. Grey shapes and arrows represent facts/hypotheses from previous studies and are not directly based on the results from the present study. Broken lines imply indirect effect while full lines denote direct effect. Chronic overfeeding and physical inactivity increase the influx of fatty acid, which promotes β-oxidation through the activation of PPARα/δ-mediated genes, without coordinated increase in TCA cycle flux. Reporter analysis supports this idea by showing the decreased activity in TCA cycle enzymes associated with reporter metabolites. Eventually, this leads to mitochondrial accumulation of metabolic by-products of incomplete β-oxidation (acylcarnitines ROS). These stresses might lead to mitochondrial overload which together with intracellular lipid-signaling (such as DAG) molecules might trigger serine a serine/threonine (Ser/Thr) kinase (Ser/Thr) cascade initiated by nPKCs. As a result, Ser/Thr phosphorylation of insulin receptor substrate 1 (IRS-1) sites is induced, thereby inhibiting IRS-1 tyrosine phosphorylation and activation of PI 3-kinase, resulting in impeded GLUT4 translocation, reduced glucose transpor, and decreased glycogen synthesis. Increased physical activity/fasting activates PGC1α and CREB (a potent inducer of PGC-1α). These actions combat lipid stress by increasing TCA cycle flux and by coupling ligand-induced PPARα/δ activity with PGC1α-mediated remodeling of downstream metabolic pathways such as respiration and β-oxidation. CDP-choline, cytidine diphosphate choline; DAG, diacylglycerol; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; GLUT4, glucose transporter-4; GSK3, glycogen synthase kinase-3; IRE1, inositol requiring kinase-1; LC-CoAs, long-chain acyl CoAs; nPKCs, novel protein kinase Cs; PA, phosphatidate; PGC1α, PPARγ co-activator-1α; PH, pleckstrin homology domain;PI, phospatidylinositol; PIP, phospatidylinositol 4-phospate; PIP2, phosphatidylinositol 4,5-bisphospate, PIP3, phospatidylinositol 3,4,5-trisphospate; PI 3-kinase, phosphoinositol 3-kinase; PPARγ, peroxisome proliferator-activated receptor-γ; PTB, phosphotyrosine binding domain; ROS, reactive oxygen species; RXR, retinoid X receptor; SH2, src homology domain; TCA, tricarboxylic acid cycle; TF, transcription factor; CPT1, carnitine palmitoyltransferase-1; PTDETN, phosphatidylethanolamine.

M value – whole-body glucose uptake during the hyperinsulinemic euglycemic clamp, Insulin 120 min – insulin levels achieved at the two hour time point of oral glucose tolerance test.