Correlative and quantitative 1H NMR-based metabolomics reveals specific metabolic pathway disturbances in diabetic rats

Abstract

Type 1 diabetes was induced in Sprague-Dawley rats using streptozotocin. Rat urine samples (8 diabetic and 10 control) were analyzed by 1H nuclear magnetic resonance (NMR) spectroscopy. The derived metabolites using univariate and multivariate statistical analysis were subjected to correlative analysis. Plasma metabolites were measured by a series of bioassays. A total of 17 urinary metabolites were identified in the 1H NMR spectra and the loadings plots after principal components analysis. Diabetic rats showed significantly increased levels of glucose (P < 0.00001), alanine (P < 0.0002), lactate (P < 0.05), ethanol (P < 0.05), acetate (P < 0.05), and fumarate (P < 0.05) compared with controls. Plasma assays showed higher amounts of glucose, urea, triglycerides, and thiobarbituric acid-reacting substances in diabetic rats. Striking differences in the Pearson's correlation of the 17 NMR-detected metabolites were observed between control and diabetic rats. Detailed analysis of the altered metabolite levels and their correlations indicate a significant disturbance in the glucose metabolism and tricarboxylic acid (TCA) cycle and a contribution from gut microbial metabolism. Specific perturbed metabolic pathways include the glucose-alanine and Cori cycles, the acetate switch, and choline metabolism. Detection of the altered metabolic pathways and bacterial metabolites using this correlative and quantitative NMR-based metabolomics approach should help to further the understanding of diabetes-related mechanisms.

Figures

Fig. 1

Typical 1H NMR spectra of urine from a Sprague–Dawley control rat and an STZ-induced diabetic rat. Identified metabolites: 1, glucose; 2, alanine; 3, lactate; 4, ethanol; 5, acetate; 6, pyruvate; 7, 2-oxoglutarate; 8, dimethylglycine; 9, fumarate; 10, succinate; 11, citrate; 12, formate; 13, hippurate; 14, urea; 15, allantoin; 16, dimethylamine; 17, creatinine.

Fig. 2

(A and B) Scores plot (A) and PC1 loadings plot (B) from the PCA of 1H NMR spectra of urine from diabetic and control rats (urea and residual water peaks were removed prior to PCA). (C and D) Scores plot (C) and PC2 loadings plot (D) from the PCA of 1H NMR spectra of urine from diabetic and control rats (glucose, urea, and residual water peaks were removed prior to PCA). Intensities of the NMR variables were scaled using the total volume of urine collected over 24 h. The data were analyzed after applying Pareto scaling.

Fig. 3

Pearson's correlations of the quantities of the 17 metabolites determined from rat urine samples: (A) control; (B) diabetic. The numbers for the metabolites used are as given in Table 2. (For interpretation of the references to color in the description of this figure in the text, the reader is referred to the Web version of this article.)

Fig. 4

Schematic diagram of the disturbed metabolic pathways detected by 1H NMR urine analysis. *Tissue compartments are used to illustrate the glucose–alanine and Cori cycles. (For interpretation of the references to color in this figure and in the description of the figure in the text, the reader is referred to the Web version of this article.)