Metabolomics identifies novel Hnf1alpha-dependent physiological pathways in vivo

Abstract

Mutations in the HNF1A gene cause maturity-onset diabetes of the young type 3, one of the most common genetic causes of non-insulin-dependent (type 2) diabetes mellitus. Although the whole-body Hnf1a-null mouse recapitulates the low insulin levels and high blood glucose observed in human maturity-onset diabetes of the young type 3 patients, these mice also suffer from Laron dwarfism and aminoaciduria, suggesting a role for hepatocyte nuclear factor 1α (Hnf1α) in pathophysiologies distinct from non-insulin-dependent (type 2) diabetes mellitus. In an effort to identify pathways associated with inactivation of Hnf1α, an ultraperformance liquid chromatography coupled to mass spectrometry-based metabolomics study was conducted on urine samples from wild-type and Hnf1a-null mice. An increase in phenylalanine metabolites is in agreement with the known regulation of the phenylalanine hydroxylase gene by Hnf1α. This metabolomic approach also identified urinary biomarkers for three tissue-specific dysfunctions previously unassociated with Hnf1α function. 1) Elevated indolelactate coupled to decreased xanthurenic acid also indicated defects in the indole and kynurenine pathways of tryptophan metabolism, respectively. 2) An increase in the neutral amino acid proline in the urine of Hnf1a-null mice correlated with loss of renal apical membrane transporters of the Slc6a family. 3) Further investigation into the mechanism of aldosterone increase revealed an overactive adrenal gland in Hnf1a-null mice possibly due to inhibition of negative feedback regulation. Although the phenotype of the Hnf1a-null mouse is complex, metabolomics has opened the door to investigation of several physiological systems in which Hnf1α may be a critical regulatory component.

Figure 1

PCA and OPLS plots demonstrating separation of wild-type and Hnf1a-null mice. Urine samples from wild-type and Hnf1a-null mice were subjected to UPLC-ESI-QTOFMS. A and B, The MarkerLynx data matrix was then used to generate PCA (A) and OPLS (B) models with accompanying scores plots. t[1] and t[2] correspond to principal components 1 and 2, respectively. C, The OPLS model was then used to generate a loadings S-plot showing ions important to the clustering of samples. The upper right quadrant shows those ions depleted in the Hnf1a-null mouse, and the lower left quadrant shows those ions increased in Hnf1a-null mouse urine.

Figure 2

Quantification of phenylalanine and tryptophan metabolites. After confirmation by tandem mass spectrometry fragmentation pattern comparison to authentic standards, metabolite concentrations were determined by triple-quadrupole mass spectrometry and normalized to millimoles of creatinine. Significant increases in metabolite concentration in Hnf1a-null mouse urines are indicated (***, P ≤ 0.0005). KO, Knockout; WT, wild type.

Figure 3

The kynurenine pathway of tryptophan metabolism is inhibited in Hnf1a-null mice. A, Two ions decreased in Hnf1a-null mouse urine were identified as riboflavin and xanthurenic acid. Metabolite concentrations are normalized to millimoles of creatinine. B, Gene expression of selected enzymes involved in the kynurenine pathway of tryptophan metabolism was assessed in the liver and kidney by QPCR analysis. All values are normalized to β-actin expression and are represented as fold of the wild-type value for each gene analyzed. Significant changes in Hnf1a-null mice are indicated (*, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005). KO, Knockout; WT, wild type.

Figure 4

Decrease in the expression of renal apical neutral amino acid transporters in Hnf1a-null mice. A, Gene expression analysis by QPCR of several transporters important for renal reuptake of amino acids. All values are normalized to β-actin expression and are represented as fold of the wild-type value for each gene analyzed. Significant changes in Hnf1a-null mice are indicated (*, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005). B and C, Western blot analysis of kidney membrane extracts from four individual wild-type and in Hnf1a-null mice for Slc6a20 (B) and collectrin (C). Actin and calnexin were used as loading controls. KO, Knockout; WT, wild type.

Figure 5

Urinary concentrations of Slc6a transporter substrates are elevated in Hnf1a-null mice. The concentration of proline, glycine betaine, and pipecolic acid was determined by triple-quadrupole mass spectrometry in wild-type and Hnf1α-null mouse urine. Values are normalized to millimoles of creatinine. Significant changes from wild type are indicated (*, P ≤ 0.05; **, P ≤ 0.005). KO, Knockout; WT, wild type.

Figure 6

Adrenal gland exhaustion in Hnf1a-null mice. Adrenal glands from wild-type (panels A, C, and E) and Hnf1a-null (panels B, D, and F) mice were collected for histological analysis. The medulla (M), zona fasiculata (F), and zona glomerulosa (G) are indicated in panel A. Panels A–D are hematoxylin and eosin staining of formalin-fixed and paraffin-embedded sections shown at ×40 (A and B) and ×400 (C and D) magnification. Frozen sections were used for oil red O staining and shown at ×400 magnification (E and F). Panels are representative of three individual animals assessed for each genotype.

Figure 7

A, Assessment of genes involved in steroid synthesis. QPCR analysis of RNA from adrenal glands of wild-type and Hnf1a-null mice; B, expression of Sult2a1 was assessed in the liver by QPCR; C, QPCR analysis of genes involved in the hypothalamic-pituitary-adrenal gland axis. All values are normalized to β-actin expression and are represented as fold of the wild-type value for each gene analyzed. Significant changes in Hnf1a-null mice are indicated (*, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005). KO, Knockout; WT, wild type.

Figure 8

Summary of metabolomic findings in Hnf1a-null mice. Identification of biomarkers in the urine of Hnf1a-null mice enabled discovery of several altered biochemical and physiological pathways. Hyperactive adrenal glands produce excess progesterone leading to increase in aldosterone. Several amino acid pathways are altered in Hnf1a-null mice, principally those of phenylalanine and tryptophan. Reduced phenylalanine hydroxylase (Pah) enzyme inhibits conversion of phenylalanine to tyrosine, thereby causing a build-up of phenylalanine and its bacterial metabolites. Reduction in mRNA transcripts of enzymes key in the kynurenine pathway of tryptophan metabolism may lead to the observed decrease in urinary xanthurenic acid. Additionally, this blockage in the kynurenine pathway may result in increased clearance of tryptophan through the secondary indole pathway. The renal apical transporters B⁰AT2 (Slc6a15) and IMINO (Slc6a20) are decreased in Hnf1a-null mice, thereby contributing to urinary loss of proline and glycine betaine. All of these markers are excreted in the urine, an easily obtainable biofluid in which to find clinically useful biomarkers for diagnosis of disease.