Metabolomics reveals attenuation of the SLC6A20 kidney transporter in nonhuman primate and mouse models of type 2 diabetes mellitus

Abstract

To enhance understanding of the metabolic indicators of type 2 diabetes mellitus (T2DM) disease pathogenesis and progression, the urinary metabolomes of well characterized rhesus macaques (normal or spontaneously and naturally diabetic) were examined. High-resolution ultra-performance liquid chromatography coupled with the accurate mass determination of time-of-flight mass spectrometry was used to analyze spot urine samples from normal (n = 10) and T2DM (n = 11) male monkeys. The machine-learning algorithm random forests classified urine samples as either from normal or T2DM monkeys. The metabolites important for developing the classifier were further examined for their biological significance. Random forests models had a misclassification error of less than 5%. Metabolites were identified based on accurate masses (<10 ppm) and confirmed by tandem mass spectrometry of authentic compounds. Urinary compounds significantly increased (p < 0.05) in the T2DM when compared with the normal group included glycine betaine (9-fold), citric acid (2.8-fold), kynurenic acid (1.8-fold), glucose (68-fold), and pipecolic acid (6.5-fold). When compared with the conventional definition of T2DM, the metabolites were also useful in defining the T2DM condition, and the urinary elevations in glycine betaine and pipecolic acid (as well as proline) indicated defective re-absorption in the kidney proximal tubules by SLC6A20, a Na(+)-dependent transporter. The mRNA levels of SLC6A20 were significantly reduced in the kidneys of monkeys with T2DM. These observations were validated in the db/db mouse model of T2DM. This study provides convincing evidence of the power of metabolomics for identifying functional changes at many levels in the omics pipeline.

FIGURE 1.

MDS plots of normal and T2DM monkeys. A, optimization of the number of features used in the random forests model. Models were trained on 10, 20, 50, 75, 150, 250, 350, 500, 750, or 1000 variables. 150 of the top variables were determined to be the most informative (indicated by arrow). Errors bars represent 1 S.D. B, an MDS plot derived from the random forests proximity matrix using the top 150 variables. Normal (n = 10) and T2DM (n = 11) monkeys were classified with an accuracy of 95%. Monkey D10, whereas clinically defined as type 2 diabetic, was misclassified as normal.

FIGURE 2.

Urine concentration measurements were determined using a standard curve derived from authentic compounds. A, glycine betaine; B, citric acid; C, glucose; D, kynurenic acid; E, l-pipecolic acid; and F, l-proline. l-Pipecolic acid and l-proline were distinguished from their d-isomer by chiral chromatography. Data are plotted after normalizing by creatinine from normal (n = 9) and T2DM (n = 10) urine samples and are expressed as micromoles per millimole of creatinine. Statistical significance when comparing with the normal monkeys was determined by a Mann-Whitney test for non-normal data. Monkeys C07 (normal) and T11 (T2DM) were excluded as outliers by a Grubb test.

FIGURE 3.

Chiral chromatography was used to distinguish the d- and l-stereoisomers of pipecolic acid (A) and proline (B). The top panel in both A and B demonstrates separation and detection of a 50 μm solution containing mixtures of dl-pipecolic acid or dl-proline, respectively. The middle panel in both A and B demonstrates separation and detection of the l-stereoisomer only. The bottom panel in both A and B demonstrates detection of only the l-stereoisomer in a representative diabetic monkey urine sample.

FIGURE 4.

The Na⁺-dependent transporter SLC6A20 gene expression is reduced in kidneys from normal and T2DM monkeys. A, QPCR analysis of mRNAs encoded by SLC6A20 (4.5-fold reduction), SLC36A1 (no significant change), HNF1A (no significant change), and COLLECTRIN/TMEM27 (no significant change) in normal and T2DM monkeys. B, protein expression of SLC6A20 and FERROPORTIN from kidney membrane fractions isolated from normal and monkeys with T2DM. C, densitometry of the Western blots shown in B. Arbitrary units were calculated by dividing SLC6A20 densitometry values by those for ferroportin. ns, not significant. The r stands for “rhesus.”

FIGURE 5.

Wild-type and db/db urinary measurements of glycine betaine (A), pipecolic acid (B), proline (C), and glucose (D) at 12 (n = 3), 16 (n = 4), 20 (n = 4), and 24 weeks (n = 4) of age. Values are expressed as micromoles or millimoles per 24 h. Significance was determined using an unpaired t test. ns, not significant.

FIGURE 6.

QPCR analysis of the kidney proximal kidney transporters and nuclear receptors in kidneys from wild-type and db/db mice at 12 (n = 3), 16 (n = 4), 20 (n = 4), and 24 weeks (n = 4) of age. On average (using all time points), there was a 40% reduction in Slc6a20b gene expression in kidneys obtained from db/db mice. Hnf1a mRNA expression was significantly reduced in the db/db mouse at 20 and 24 weeks of age and Hnf1b mRNA was significantly reduced at 24 weeks. Slc65a2 mRNA was significantly up-regulated at 24 weeks of age in the db/db mouse. Significance was determined using an unpaired t test. *, p < 0.05, **, p < 0.01.

FIGURE 7.

Wild-type and db/db protein expression of (A) SLC6A20 and ACTIN in kidney membrane fractions isolated at 12-, 16-, 20-, and 24-weeks of age. B, densitometry of the Westerns blots shown in A. Arbitrary units were calculated by dividing SL6A20 densitometry values by those for ACTIN. ns, not significant. On average (using all time points), there was a 70% reduction in protein levels of SLC6A20 in kidney membrane fractions obtained from db/db mice. The m stands for “mouse.”

FIGURE 8.

Wild-type (circles) and db/db (squares) serum measurements of glycine betaine (A), pipecolic acid (B), and proline (C) at 12 (n = 3), 16 (n = 4), 20 (n = 4), and 24 weeks (n = 4) of age. Values are expressed as micromolar. Significance was determined using an unpaired t test. ns, not significant.