Systemic alterations in the metabolome of diabetic NOD mice delineate increased oxidative stress accompanied by reduced inflammation and hypertriglyceremia

Abstract

Nonobese diabetic (NOD) mice are a commonly used model of type 1 diabetes (T1D). However, not all animals will develop overt diabetes despite undergoing similar autoimmune insult. In this study, a comprehensive metabolomic approach, consisting of gas chromatography time-of-flight (GC-TOF) mass spectrometry (MS), ultra-high-performance liquid chromatography-accurate mass quadruple time-of-flight (UHPLC-qTOF) MS and targeted UHPLC-tandem mass spectrometry-based methodologies, was used to capture metabolic alterations in the metabolome and lipidome of plasma from NOD mice progressing or not progressing to T1D. Using this multi-platform approach, we identified >1,000 circulating lipids and metabolites in male and female progressor and nonprogressor animals (n = 71). Statistical and multivariate analyses were used to identify age- and sex-independent metabolic markers, which best differentiated metabolic profiles of progressors and nonprogressors. Key T1D-associated perturbations were related with 1) increases in oxidation products glucono-δ-lactone and galactonic acid and reductions in cysteine, methionine and threonic acid, suggesting increased oxidative stress; 2) reductions in circulating polyunsaturated fatty acids and lipid signaling mediators, most notably arachidonic acid (AA) and AA-derived eicosanoids, implying impaired states of systemic inflammation; 3) elevations in circulating triacylglyercides reflective of hypertriglyceridemia; and 4) reductions in major structural lipids, most notably lysophosphatidylcholines and phosphatidylcholines. Taken together, our results highlight the systemic perturbations that accompany a loss of glycemic control and development of overt T1D.

Keywords: diabetic mice; inflammation; metabolomics; oxidative stress.

Fig. 1.

Biochemical network displaying metabolic differences between diabetic and nondiabetic NOD mice. Metabolites are connected based on biochemical relationships (orange, KEGG RPAIRS) or structural similarity (blue, Tanimoto coefficient ≥ 0.7). Metabolite size and color represent the importance (O-PLS-DA model loadings, LV1) and relative change (gray, P_adj > 0.05; green, decrease; red, increase) in diabetic vs. nondiabetic NOD mice. Shapes display metabolites' molecular classes or biochemical subdomains, and top descriptors of type 1 diabetes (T1D)-associated metabolic perturbations (Supplemental Table S1) are highlighted with thick black borders.

Fig. 2.

Correlation between body weight, fasting blood glucose, and major classes of measured metabolites and lipids. Heatmap is shown displaying hierarchical clustered Spearman correlations between animal characteristics (weight and fasting blood glucose) and major classes of measured metabolites and lipids.

Fig. 3.

Partial correlation network displaying associations between top 10% T1D-dependent metabolic perturbations. All significantly top 10% discriminant metabolites (n = 44) of the T1D-phenotype are connected based on partial correlations (P_adj ≤ 0.5, Supplemental Table S1). Edge width displays the absolute magnitude and color the direction (orange, positive; blue, negative) of the partial coefficient of correlation. Metabolite size and color represent the importance (O-PLS-DA model loadings, LV 1) and relative change (green, decrease; red, increase) in diabetic vs. nondiabetic mice. Shapes display metabolites' molecular classes or biochemical subdomains (see Fig. 4 legend).

Fig. 4.

Significantly perturbed eicosanoids (P_adj < 0.05) in the KEGG arachidonate metabolism pathway. Pathway enrichment was determined based on FDR adjusted hypergeometric t-test, P < 0.05 for KEGG pathways for Mus musculus. Figure displays relative fold changes (blue, decrease; yellow, increase) in means between diabetic and nondiabetic mice. *Values reported as means ± SD unless otherwise noted; †values reported median (minimum, maximum); ‡unpaired two-sample t-test, P ≤ 0.05.