Metabolomic profiling of plasma from middle-aged and advanced-age male mice reveals the metabolic abnormalities of carnitine biosynthesis in metallothionein gene knockout mice

Multivariate statistical analyses

Representative base peak intensity (BPI) chromatograms were obtained from plasma in electrospray ionization positive (ESI⁺) and negative (ESI^-) modes, respectively (Supplementary Figure 1A, 1B). All peaks in ESI⁺ or ESI^- were merged and imported into the SIMCA-P software for multivariate statistical analysis. To investigate global metabolism variations, we first used principal component analysis (PCA) to analyze all observations acquired in both ion modes. As shown in the PCA plot (Supplementary Figure 2), an overview of all samples in the data can be observed and exhibits an unclear grouping trend between the two groups. To eliminate any non-specific effects of the operative technique and confirm the biomarkers, partial least squares discriminant analysis (PLS-DA) and orthogonal partial least squares-discriminant analysis (OPLS-DA) were used to compare metabolic changes between the two groups. In the PLS-DA score plot (Supplementary Figure 3) or OPLS-DA score plot (Figure 1A–1D), a clear separation of the two groups was observed. Univariate analyses, including fold change (FC) analysis and t-test, were performed on a volcano plot (Supplementary Figure 4). We selected metabolites with variable importance in projection (VIP) > 1.5, FC > 2.0, and P < 0.05 as significant compounds.

Figure 1. Scatter plot of scores based on the OPLS-DA model. (A) ESI⁺ scan in 50-week-old mice; (B) ESI^- scan in 50-week-old mice; (C) ESI⁺ scan in 100-week-old mice; (D) ESI^- scan in 100-week-old mice. Magenta circles indicate MTKO mice and green circles indicate WT mice.

Quantitative comparison of metabolites in the plasma between 50-week-old MTKO and WT male mice

Identification of metabolites in the plasma

We identified 599 differential metabolites, of which 242 were detected in the positive ion mode and 357 in the negative ion mode in the plasma of 50-week-old mice. The results of the hierarchical clustering analysis (HCA) of compounds with statistically significant differences between the groups of MTKO and WT male mice are summarized in Table 1. The levels of 19 metabolites were higher, whereas those of 27 others were lower, in the plasma of MTKO mice compared to that of WT mice.

Fatty acids and their metabolites

The contents of C20 fatty acids and their metabolites, eicosanoids (eicosapentaenoic acid (EPA), P = 0.026, t-test, Table 1), arachidonic acid (P = 0.025, t-test, Table 1), 12-hydroxy-eicosatetraenoic-acid (12-HETE, P = 0.009, t-test, Table 1), 12S-hydroperoxy-5Z, 8Z, 10E, 14Z-eicosatetraenoic acid (12(S)-HPETE, P = 0.016, t-test, Table 1), prostaglandin E2 (PGE2, P = 0.010 in ESI⁺ mode and P = 0.014 in ESI^- mode, t-test, Table 1), 15-keto-13,14-dihydro-prostaglandin A2 (15-keto-13,14-dihydro-PGA2, P = 0.014, t-test, Table 1), thromboxane B2 (TXB2, P = 0.001, t-test, Table 1), were smaller, whereas that of C18 fatty acid metabolite,12,13-dihydroxy-9Z-octadecenoic acid (12,13-DHOME) was larger in MTKO mice compared to WT mice (P = 0.002, t-test, Table 1). Reduced PGE2 levels in muscles contributes to muscle atrophy in aged mice [21].

Glycolysis/gluconeogenesis and tricarboxylic acid (TCA) cycle metabolites

Levels of L-lactic acid (P = 0.006 in ESI⁺ mode and P = 0.018 in ESI^- mode, t-test, Table 1), oxoglutaric acid (P = 0.005, t-test, Table 1), glycerol 3-phosphate (P < 0.001, t-test, Table 1), and citric acid (P = 0.002, t-test, Table 1) were significantly lower in MTKO mice than in WT mice. These results may show differences in activity levels of glycolysis/gluconeogenesis and the TCA cycle between MTKO and WT mice at 50 weeks of age.

Amino acids and their metabolites

Among amino acids and their metabolites, the levels of taurine (2-aminoethane-1-sulfonic acid) and N-lactoylphenylalanine (P = 0.038, t-test, Table 1) were lower, whereas those of L-phenylalanine (P = 0.026, t-test, Table 1), L-lysine (P = 0.019, t-test, Table 1), N6,N6,N6-trimethyl-L-lysine (TML) (P = 0.035, t-test, Table 1 and Figure 2A), and histidylglutamic acid (P = 0.013, t-test, Table 1)were significantly higher in the plasma of MTKO mice than in that of WT mice. It has been reported that the plasma level of N-lactoylphenylalanine strongly correlates with plasma levels of lactate and L-phenylalanine [22]. Therefore, the low level of L-lactic acid could lead to L-phenylalanine accumulation in the plasma of MTKO mice. TML is an N-methylated compound of L-lysine. It is a component of histone proteins and an intermediate that is recognized as the precursor of L-carnitine (Figure 2E). The metabolic ability to L-lysine and TML may be weaker in MTKO mice than in WT mice. Hexadecanedioic acid, which is a long-chain dicarboxylic fatty acid, is converted to hexadecanedioylcarnitine by carnitine palmityltransferase (CPT) and is activated by mitochondrial fractions in the human liver for its degradation by β-oxidation [23]. In the plasma of MTKO mice, the concentration of hexadecanedioic acid (FC = 2.391, P = 0.001, t-test, Table 1) may be associated with the accumulation of TML as a carnitine precursor.

Figure 2. Levels of acylcarnitine species and carnitine biosynthesis intermediates in plasma of wild-type (WT) mice (white boxes) and MTKO mice (gray boxes). (A) N6,N6,N6-trimethyl-L-lysine (TML) level at 50 weeks of age; (B) Levels of L-carnitine [C0] and acylcarnitine species (L-acetylcarnitine [C2], Hexanoylcarnitine [C8], Decanoylcarnitine [C10], Tetradecanoylcarinitne [C14], 3,5-Tetradecadiencarnitine [C14:2], L-Palmitoylcarnitine [C16], 9-Hexadecenoylcarnitine [C16:1], Oleoylcarnitine [C18:1], Arachidonoylcarnitine [C20:4]) at 100 weeks of age; (C) TML level at 100 weeks of age; (D) Level of γ-butryobetaine (γ-BB), also known as 4-trimethylammoniobutanoic acid, at 100 weeks of age. The box denotes 25th and 75th percentiles; the line within the box denotes the 50th percentile; the whisker denotes standard deviation. (n = 3). *P < 0.05 and **P < 0.01. (E) Carnitine biosynthesis pathway. HTML, 3-hydroxyl-N6-trimethyl-L-lysine; TMABA, 4-trimethyl-aminobutyraldehyde; TML dioxygenase, TMLD; HTML aldolase, HTMLA; TMABA dehydrogenase, TMABADH; γ-BB dioxygenase, BBD.

Bile acids and bilirubin

The amounts of conjugated primary bile acids, glycocholic acid (P < 0.001, t-test, Table 1), taurocholic acid (P = 0.014, t-test, Table 1), and glycochenodeoxycholate-3-sulfate (P = 0.005, t-test, Table 1) were lower in MTKO mice than in WT mice. The level of taurocholic acid was correlated with different taurine levels in the plasma of both MTKO and WT mice. In contrast, the bilirubin level in the plasma of MTKO mice was markedly higher than that in the plasma of WT mice (P = 0.007 in ESI⁺ mode and P = 0.004 in ESI^- mode, t-test, Table 1). In the liver, bilirubin is conjugated with glucuronic acid by glucuronyltransferase. Conjugated bilirubin is then excreted from hepatocytes into the bile canaliculus. The capacity of several conjugation reactions may be limited in the liver of MTKO mice.

Nucleobases and nucleosides

Among nucleobases and nucleosides, the contents of xanthine (P = 0.031, t-test, Table 1) and its nucleoside xanthosine (P = 0.033 in ESI⁺ mode and P = 0.001 in ESI^- mode, t-test, Table 1) were remarkably lower in MTKO mice than in WT mice. Xanthine oxidoreductase (XOR) generates ROS, such as superoxide radicals and hydrogen peroxide, when it catalyzes the oxidation of hypoxanthine to xanthine, followed by the oxidation of xanthine to uric acid [24]. However, the ROS, which are synthesized in moderate amounts from xanthine with the catalytic activity of XOR, are essential for the appropriate functioning of many physiological pathways, thereby contributing to successful aging [25]. There is no evidence of high levels of ROS in aged MTKO mice. However, excessive lipid peroxidation, an indicator of oxidative stress, was observed in some tissues, including the heart [26], liver [27], and neurons [28] of MTKO mice treated with various anthropogenic stressors. Alterations in mitochondrial homeostasis, including a progressive decrease in ROS scavengers, cause ineffective ROS control and mediate cell stress responses towards age-dependent damage [29]. Due to the deficiency of MT as a ROS scavenger in mitochondria [30], the low level of xanthine in the plasma from MTKO mice might be the result of high utilization of xanthine to address a progressive generation of ROS during the aging process.

Other compounds

Among phospholipids, levels of lysophospholipid species were lower in the plasma of MTKO mice compared to that of WT mice (all P < 0.05, t-test, Table 1). Regarding metabolites probably derived from foods, the levels of 4-hydroxybenzaldehyde (P = 0.001, t-test, Table 1) and indole-3-carboxaldehyde (P = 0.001, t-test, Table 1) were significantly lower, whereas those of DL-2-aminooctanoic acid (P < 0.001, t-test, Table 1), dehydrochorismic acid (P = 0.012, t-test, Table 1), and hydroxystilbamidine (P = 0.002, t-test, Table 1) were significantly higher in the plasma of MTKO mice than in that of WT mice. These results may show differences in metabolism of the above-mentioned compounds from foods between MTKO and WT mice during a 6 h fast before plasma collection.

Quantitative comparison of metabolites in the plasma between MTKO and WT 100-week-old male mice

Identification of metabolites in the plasma

We identified 616 differential metabolites, of which 290 were detected in the positive ion mode and 326 in the negative ion mode in 100-week-old mouse plasma. HCA results of compounds with statistically significant differences between the MTKO and WT male groups are summarized in Table 2. The levels of 42 metabolites were higher, whereas those of 40 were lower in the plasma of MTKO mice than that in WT mice. Interestingly, many of these metabolites are involved in carnitine metabolism.

Carnitine metabolites

Levels of L-carnitine ((R)-3-hydroxy-4-(trimethylammonio) butyrate) and acylcarnitine species with different acyl chains were significantly lower in MTKO mice than in WT mice (all P < 0.05, t-test, Table 2 and Figure 2B). In contrast, TML levels in the plasma of MTKO mice were 5.33-fold greater than that in the plasma of WT mice (P = 0.001, t-test, Table 2) (Figure 2C). 4-trimethylammoniobutanoic acid, also known as γ-butryobetaine (γ-BB), is a metabolic intermediate in L-carnitine biosynthesis (Figure 2E), and its quantity in MTKO mice was less than one-tenth that in WT mice (P = 0.002, t-test, Table 2) (Figure 2D). Network analysis indicated that metabolites related to carnitine synthesis differed significantly between MTKO and WT mice (P = 0.0388, obtained from analysis of MBRole, Table 3 and Supplementary Figure 5). The level of 3-dehydrocarnitine, which is an intermediate in carnitine degradation, was 2.5-fold higher in MTKO mice than in WT mice (P = 0.004, t-test, Table 2).

Aliphatic dicarboxylic acids and fatty acid metabolites

The levels of sebacic acid, dodecanedioic acid, tetradecanedioic acid, and 3-hydroxytetradecanedioic acid, which are aliphatic dicarboxylic acids, were higher in the plasma of MTKO mice than in the plasma of WT mice (P = 0.032, 0.004, 0.003, and 0.005, respectively, t-test, Table 2). In humans, levels of sebacic acid are increased in the urine of patients with carnitine-acylcarnitine translocase deficiency, which is an inborn error of metabolism [31]. Dodecanedioic acid is a marker of hepatic CPT Iα-deficiency [32]. 3-hydroxytetradecanedioic acid is detected in the urine of patients with thanatophoric dysplasia type I with fibroblast growth factor receptor 3 gene mutation (S249C) due to incomplete fatty acid metabolism [33].

Hippuric acid, 2-methylhippuric acid, and suberylglycine are acyl glycines, which are minor fatty acid metabolites. The levels of these acyl glycines are increased in disorders associated with mitochondrial fatty acid β-oxidation. Thus, in the plasma of MTKO mice, higher levels of these minor fatty acid metabolites (all P < 0.05, t-test, Table 2) and the markers of fatty acid metabolism disorder can be ascribed to the lower levels of carnitine compounds. Levels of acyl glycines, palmitoylglycine, and oleoyl glycine were lower in the plasma of MTKO mice compared to that of WT mice (P = 0.015 and 0.029, respectively, t-test, Table 2). This may be because the plasma levels of palmitic acid and oleic acid were also lower in MTKO mice than in WT mice (P = 0.005 and 0.012, respectively, t-test, Table 2). The capacity of fatty acid catabolism and fatty acid biosynthesis in MTKO mice may be weaker than that in WT mice.

Other minor fatty acid metabolites were also identified. The level of 3-oxododecanoic acid in MTKO mice was lower (P = 0.032, t-test, Table 2), but that of its hydroxide, 3-hydroxydodecanoic acid, was higher in MTKO mice than in WT mice (P = 0.008, t-test, Table 2). This tendency was similar to that of myristic acid (P = 0.043, t-test, Table 2) and its hydroxide, 2-hydroxymyristic acid (P = 0.011, t-test, Table 2). In humans, 3-oxododecanoic acid is involved in fatty acid biosynthesis, and 3-oxododecanoic acid can be converted into 3-hydroxydodecanoic acid. 3-hydroxydodecanoic acid is a fatty acid associated with fatty acid metabolic disorders, such as medium-chain acyl CoA dehydrogenase deficiency, which shows low plasma and tissue carnitine levels [34, 35]. Levels of ricinoleic acid and mead acid, which are unsaturated n-9 fatty acid metabolites, were lower in MTKO mice (P = 0.033 and 0.020, respectively, t-test, Table 2). Levels of 13-L-hydroperoxylinoleic acid (13(S)-HPODE) and 12(S)-HPETE were greater in MTKO mice than those in WT mice (P = 0.024 and 0.037, respectively, t-test, Table 2). Further, levels of very long chain fatty acids, docosapentaenoic acid (22n-3), and docosatetraenoic acid (22n-6) were lower in MTKO mice than in WT mice (P = 0.032 and 0.016, respectively, t-test, Table 2). The levels of N-oleoyl-tyrosine and eicosapentaenoyl ethanolamide (EPEA) were lower in MTKO mice than in WT mice (P = 0.030 and 0.035, respectively, t-test, Table 2), although N-stearoyltaurine levels in MTKO mice were 6.3-fold higher than that in WT mice (P = 0.020, t-test, Table 2).

Phospholipids

Phospholipid species levels were higher, whereas lysophospholipid species were lower in the plasma of MTKO mice compared to that in the plasma of WT mice. As an exception, the levels of lysophosphatidylinositols, lysoPI (0:0/18:0), and lysoPI (20:4 (5Z, 8Z, 11Z, 14Z)/0:0) were significantly higher (FC > 26) in MTKO mice than in WT mice (P = 0.011 and 0.021, respectively, t-test, Table 2).

Metabolites of amino acids and other compounds

Other than carnitine species, various amino acid metabolites were also identified. The levels of taurine and creatine (2-(N-methylcarbamimidamido) acetic acid) in the plasma of MTKO mice were lower than those in the plasma of WT mice (P = 0.045 and P < 0.001, respectively, t-test, Table 2). Both are mainly biosynthesized in the liver and are widely distributed in various tissues, especially in the skeletal muscle, heart, brain, liver, kidney, and retina. Levels of valine metabolites (N-acetylvaline and γ-glutamylvaline (P = 0.022 and P < 0.001, respectively, t-test, Table 2)), L-phenylalanine (P = 0.002, t-test, Table 2) and its metabolite (3-phenyllactic acid, P = 0.039, t-test, Table 2), tryptophan metabolites (indole-3-propionic acid, indolelactic acid, kynurenic acid, and cyclic 6-hydroxymelatonin (P = 0.018, 0.044, 0.018, and 0.008, respectively, t-test, Table 2)), and ketoleucine (P = 0.010, t-test, Table 2) were higher in MTKO mice than in WT mice. Among ribonucleotide metabolites, levels of succinyladenosine, a precursor of AMP and ADP, were lower in MTKO mice than in WT mice (P = 0.023, t-test, Table 2). Among steroid metabolites, levels of 5α-tetrahydrocortisol and androsterone glucuronide in MTKO mice were lower than that in WT mice (P = 0.026 and P < 0.001, respectively, t-test, Table 2). Levels of some metabolites, probably derived from plant foods, such as proline betaine (P = 0.004, t-test, Table 2), protocatechuic acid (P = 0.005, t-test, Table 2), (S)-oleuropeic acid (P = 0.030, t-test, Table 2), caffeic acid 3-O-sulfate (P = 0.004, t-test, Table 2), ferulic acid 4-O-sulfate (P < 0.001, t-test, Table 2), and 2-phenylethyl β-D-glucopyranoside (P = 0.034, t-test, Table 2), and those probably derived from animal foods, such as DL-2-Aminooctanoic acid (P = 0.012, t-test, Table 2), were significantly higher in the plasma of MTKO mice compared to that in WT mice. The results may show differences in metabolism of these compounds between MTKO and WT mice during a 6 h fast before plasma collection.

Common features and properties of metabolites in plasma among 50-week- and 100-week-old mice

Compounds found in smaller quantities in the plasma of male MTKO mice compared to that of male WT mice

Levels of the common compound taurine were lower in the plasma of male MTKO mice compared to that of male WT mice at both 50 and 100 weeks of age (Tables 1, 2). Taurine has several functions, such as conjugation with bile acids, antioxidants, osmolytes, membrane stabilizers, and as a modulator of calcium signaling [36–38]. It protects the cardiovascular system [39], skeletal muscle [40, 41], retina [42], and central nervous system [43] against pathology and disease [44]. Taurine depletion shortens the lifespan of mice [45, 46]. Since rodents, unlike cats and humans, exhibit considerable biosynthetic capacity for taurine [47], MT gene deficiency would have a significant influence on the biosynthesis pathway and plasma levels of taurine in mice. Taurine levels in the plasma of MTKO mice were approximately one-third of that in WT mice at 50 weeks of age, and this level decreased in 100-week-old WT mice to approximately one-fourth of the previously observed level. This decrease in relative values with age may lead to muscular depression, and disorders of the cardiovascular system, the eye, and central nervous system in MTKO mice, and may finally reduce lifespan. Unlike taurine, creatine levels were lower in 100-week-old MTKO mice compared to WT mice. Creatine is mainly synthesized in the liver and has potential beneficial roles that are nearly as effective as taurine in cardiovascular function, muscle mass, cognitive function, and lifespan [38, 48]. The low levels may be a result of accelerated aging of the liver caused by MT deficiency.

Compounds found in larger quantities in the plasma of male MTKO mice versus that of WT mice

The common metabolites that were found to be consistently higher in the plasma of MTKO mice compared to that of WT mice at 50 and 100 weeks of age were L-phenylalanine, TML, and DL-2-aminooctanoic acid (Tables 1, 2). Phenylketonuria (PKU) is an inborn error of metabolism that results from a deficiency in phenylalanine hydroxylase (PAH), the enzyme catalyzing the conversion of phenylalanine to tyrosine. PKU results in severe hyperphenylalaninemia. The enzyme activity of PAH may decrease with age in the MTKO mice. However, similar to that of a mouse model of mild hyperphenylalaninemia (Pah^enu1 variant) [49], the level of phenylalanine in the plasma of MTKO mice may have little effect on its lifespan. DL-2-aminooctanoic acid is an α-amino fatty acid derived from food. The higher level of DL-2-aminoctanoic acid in the plasma of MTKO mice compared to WT mice may be caused by the low metabolic activity of fatty acids.

Influences of MT deficiency on the carnitine biosynthesis pathway

In animals, L-carnitine is biosynthesized from TML in four enzyme-catalyzed steps, as summarized in Figure 2E [50, 51]. The first enzyme, TML dioxygenase (TMLD), catalyzes the hydroxylation of TML to produce 3-hydroxy-TML (HTML). TMLD is encoded by the TML hydroxylase, epsilon (Tmlhe) gene in mice. In the second step, HTML aldolase (HTMLA) catalyzes the aldolytic cleavage of HTML into 4-N-trimethylaminobutyraldehyde (TMABA) and glycine. In mice, it is currently thought that threonine aldolase 1 (THA1, encoded by Tha1), and serine hydroxymethyltransferase (SHMT, encoded by Shmt) possess HTMLA activity. The third enzyme, TMABA dehydrogenase (TMABADH), catalyzes the dehydrogenation of TMABA into γ-BB. TMABADH is encoded by the aldehyde dehydrogenase 9 subfamily A1 (Aldh9a1) gene in mice. In the final step, γ-BB dioxygenase (BBD) catalyzes the hydroxylation of γ-BB into L-carnitine. BBD is encoded by the γ-BB, 2-oxoglutarate dioxygenase 1 (Bbox1) gene in mice. The highest TMLD activity is found in the kidney but is also present in the liver, skeletal muscle, heart, and brain in rats and humans [52–54]. The highest enzyme activities for subsequent processes converting HTML to L-carnitine are seen in the liver of rodents. OCTN2, which is encoded by the Slc22a5 gene, is an organic cation/carnitine transporter that controls the storage of L-carnitine [55].

The relative level of TML in the plasma of MTKO mice was 3.38-fold of that recorded in WT mice at 50 weeks of age (Table 1 and Figure 2A), and then increased to 5.33-fold of that in WT mice at 100 weeks of age (Table 2 and Figure 2C). Levels of γ-BB, L-carnitine, and acylcarnitines recorded in MTKO mice were lower than those observed in 100-week-old WT mice, unlike 50-week-old mice. These results show that the TML level in the plasma of MTKO mice was consistently higher than that observed in the plasma of WT mice, and that the lower levels of γ-BB and L-carnitine in MTKO mice compared to WT mice are dependent on aging.

In order to investigate why levels of carnitine-related metabolites in plasma differed between MTKO and WT mice and if there was a time-dependent change in their levels, we measured the mRNA expression levels of enzymes associated with the carnitine biosynthesis pathway and carnitine transporter in the liver and kidney of 50-week- and 100-week-old male mice. Quantitative reverse transcription PCR (RT-PCR) analysis showed that the mRNA level of Tmlhe in MTKO mice was about one-tenth of that in WT mice at both middle and advanced age in the kidney (Figures 3A, 4A) and liver (Figures 3B, 4B). There were statistically non-significant differences in the mRNA levels of other enzymes (Tha1 (Figures 3C, 4C), Shmt1 (Figures 3D, 4D), Shmt2 (Figures 3E, 4E), Aldh9a1 (Figures 3F, 4F), Bbox1 (Figures 3G, 4G)), and transporter (Slc22a5 (Figures 3H, 4H)) in the liver of MTKO and WT mice. Therefore, the higher level of TML in the plasma of MTKO mice at 50 weeks and 100 weeks of age would be caused by the constant lower expression level of Tmlhe in the kidney and liver of MTKO mice compared to WT mice. The low TMLD activity of the kidney and liver in MTKO mice may decrease the metabolism from TML to HTML at middle age, suggesting accumulation of TML in plasma, and eventually decrease the biosynthesis of γ-BB and L-carnitine at advanced ages.

Figure 3. mRNA expression levels of enzymes associated with carnitine biosynthesis and carnitine transporter in wild-type (WT) mice (white columns) and MTKO mice (black columns) at 50 weeks of age. (A) Tmlhe gene in kidney; (B) Tmlhe gene, (C) Tha1 gene, (D) Shmt1 gene, (E) Shmt2 gene, (F) Aldh9a1 gene, (G) Bbox1 gene, and (H) Slc22a5 gene in liver. The data are expressed as ratios to the mean values in the WT group. Values are presented as the mean ± SE (n = 3). *P < 0.05 and **P < 0.01.

Figure 4. mRNA expression levels of enzymes associated with carnitine biosynthesis and carnitine transporter in wild-type (WT) mice (white columns) and MTKO mice (black columns) at 100 weeks of age. (A) Tmlhe gene in kidney; (B) Tmlhe gene, (C) Tha1 gene, (D) Shmt1 gene, (E) Shmt2 gene, (F) Aldh9a1 gene, (G) Bbox1 gene, and (H) Slc22a5 gene in liver. The data are expressed as ratios to the mean values in the WT group. Values are presented as the mean ± SE (n = 3). *P < 0.05 and **P < 0.01.

Animals and treatment

MTKO mice (129S7/SvEvBrd-Mt1tm1Bri Mt2tm1Bri/J) and WT mice (129S1/SvImJ) were purchased from The Jackson Laboratory (Bar Harbor, ME, USA). These mice were maintained under specific pathogen-free conditions and mated only with the 129/Sv mouse strain to maintain the genetic background. The mice were housed in a temperature-controlled room at 22° C with 55 ± 5% humidity under a 12 h light/dark cycle, fed a chow diet (Oriental Yeast, Tokyo, Japan), and provided with drinking water ad libitum. The study was conducted according to the guidelines of the Japanese Ministry of Education, Culture, Sports, Science, and Technology, and approved by the Ethics Committee of Tokushima Bunri University (protocol code #16-1 and date of approval; 20th April, 2018).

Sample preparation

Before blood sampling, the mice were fasted for 6 h to reduce the immediate effects of dietary intake on the plasma constituents. The animals were sacrificed using isoflurane (FUJIFILM Wako Pure Chemical, Osaka, Japan), and each organ was removed. Whole blood was collected in a 1.5 mL tube with 60 IU sodium heparin (Nacalai Tesque, Kyoto, Japan) in 25 μL saline, and was centrifuged at 800 ×g for 10 min at 20° C. Collected organs and plasma supernatants were stored at -80° C until use. Plasma specimens were thawed and 100 μL plasma aliquots were transferred into 1.5 mL tubes, 300 μL methanol was added, and the mixture was vortexed for 30 s. All samples were stored at -40° C for 1 h. Samples were then vortexed for 30 s and centrifuged at 13,000 ×g at 4° C for 15 min. The supernatant (200 μL) and 5 μL of 140 μg/mL DL-o-chlorophenylalanine (Merck, Darmstadt, Germany) were transferred to a vial as an internal standard for LC-MS analysis.

LC-TOF-MS

Separation was performed using an Ultimate 3000 LC combined with Q Exactive MS (Thermo Fisher Scientific, Waltham, MA, USA) and screened with ESI-MS. The LC system comprised an ACQUITY UPLC HSS T3 (100 × 2.1 mm; 1.8 μm) with an Ultimate 3000 LC. The mobile phase was composed of solvent A (0.05% formic acid-water) and solvent B (acetonitrile) with a gradient elution (1-16 min, 95–5% A; 16-18 min, 5% A; 18-19 min, 5–95% A; 19-20 min, 95–95% A). The flow rate of the mobile phase was 0.3 mL/min. The column temperature was maintained at 40° C, and the sample manager temperature was set at 4° C. Mass spectrometry parameters in ESI⁺ and ESI^- mode are listed as follows: ESI⁺: Heater Temp 300° C; Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.0 kV; capillary temperature, 350° C; S-Lens RF level, 30%. ESI^-: Heater Temp 300° C, Sheath Gas Flow rate, 45 arb; Aux Gas Flow Rate, 15 arb; Sweep Gas Flow Rate, 1 arb; spray voltage, 3.2 kV; capillary temperature, 350° C; S-Lens RF level, 60%. Each sample was analyzed in triplicate.

Statistical analysis

In the metabolomic analysis, the raw data were acquired and aligned using the Compound Discover 3.0 (Thermo Fisher Scientific) based on the m/z value and retention time of the ion signals. Ions from both ESI^- and ESI⁺ were merged and imported into the SIMCA-P (version 14.1, Umetrics, Umea, Sweden) for multivariate analysis. PCA was first used as an unsupervised method for data visualization and outlier identification. Supervised regression modeling was then performed on the data set using PLS-DA or OPLS-DA to identify potential biomarkers. The significantly different metabolites (ions) between the two groups were filtered out based on VIP values (VIP > 1.5). Univariate analysis, including FC analysis and t-tests, were performed on a volcano plot. The biomarkers were filtered and confirmed by combining the results of the VIP values (VIP > 1.5), FC values (FC > 2.0), and t-test (P < 0.05).

The data are presented as means ± S.D. values for gene expression analysis. The significance of differences in the mean was determined using a t-test. The threshold of statistical significance was set at P < 0.05.

Compound identification

The chemical structures of important metabolites were identified using online databases, such as the Human Metabolome Database (http://hmdb.ca), Metlin (http://metlin.scripps.edu), and the Mass Bank (http://www.massbank.jp), using the data of accurate masses and MS/MS fragments. When necessary, further confirmation was acquired through comparisons with authentic standards, including retention times and MS/MS fragmentation patterns.

Cluster analysis

Mean values of metabolite contents from biological replicates of the MTKO and WT groups were used to calculate the metabolite ratio. After log transformation of the data, the median centered ratios were normalized. Hierarchical clustering analysis (HCA) was performed using the complete linkage algorithm of the program Cluster 3.0 (Stanford University). Metabolite ratios from two independent experiments for every significant metabolite were used for HCA.

Correlation-based network analysis of metabolites

To investigate the latent relationships of the metabolites, we constructed a correlation network diagram based on KEGG database and MBRole. All significant metabolites were imported to obtain categorical annotations, including pathways, enzyme interactions, and other biological annotations.

RNA isolation and mRNA expression analysis

Frozen organ lysates were prepared using RNAiso Plus reagent (Takara Bio, Ohtsu, Japan). After the organs were lysed, chloroform (1/5 of the volume of RNAiso Plus reagent) was added to the samples. The samples were centrifuged (11,000 × g) at 4° C for 15 min, and the supernatants were collected, to which one volume of RNA-free water and 0.7 volume of ethanol were added. The lysates were then added to the Nucleospin RNA Kit (Takara Bio) columns, and total RNA was purified according to the manufacturer’s instructions. For reverse transcription, 2 μg of RNA from each sample was reverse-transcribed using a High-Capacity cDNA RT Kit (Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. The cDNA samples were then subjected to real-time PCR using TB Green Premix Ex Taq II (Takara Bio) in a StepOnePlus Real-Time PCR system (Thermo Fisher Scientific). The selected primer sequences are listed in Supplementary Table 1. The PCR program was performed as follows: holding stage at 95° C for 20 s, followed by 40 cycles at 95° C for 3 s and 60° C for 30 s. Each sample was analyzed in triplicate. To verify specificity, melting curve analysis and agarose gel electrophoresis were performed on the real-time RT-PCR products. The standard curve method was used to calculate the PCR product level by plotting the quantification cycle values of a serial dilution of all cDNA sample mixtures on the y-axis against the logarithm of the standard sample concentrations on the x-axis. The relative amount of β-actin (Actb) mRNA as an internal control housekeeping gene was determined to compensate for variations in RT-PCR efficiency.