Mtrr hypomorphic mutation alters liver morphology, metabolism and fuel storage in mice

Abstract

Nonalcoholic fatty liver disease (NAFLD) is associated with dietary folate deficiency and mutations in genes required for one‑carbon metabolism. However, the mechanism through which this occurs is unclear. To improve our understanding of this link, we investigated liver morphology, metabolism and fuel storage in adult mice with a hypomorphic mutation in the gene methionine synthase reductase (Mtrr ^gt ). MTRR enzyme is a key regulator of the methionine and folate cycles. The Mtrr ^gt mutation in mice was previously shown to disrupt one‑carbon metabolism and cause a wide-spectrum of developmental phenotypes and late adult-onset macrocytic anaemia. Here, we showed that livers of Mtrr ^gt/gt female mice were enlarged compared to control C57Bl/6J livers. Histological analysis of these livers revealed eosinophilic hepatocytes with decreased glycogen content, which was associated with down-regulation of genes involved in glycogen synthesis (e.g., Ugp2 and Gsk3a genes). While female Mtrr ^gt/gt livers showed evidence of reduced β-oxidation of fatty acids, there were no other associated changes in the lipidome in female or male Mtrr ^gt/gt livers compared with controls. Defects in glycogen storage and lipid metabolism often associate with disruption of mitochondrial electron transfer system activity. However, defects in mitochondrial function were not detected in Mtrr ^gt/gt livers as determined by high-resolution respirometry analysis. Overall, we demonstrated that adult Mtrr ^gt/gt female mice showed abnormal liver morphology that differed from the NAFLD phenotype and that was accompanied by subtle changes in their hepatic metabolism and fuel storage.

Keywords: 5-methyl-THF, 5-methyltetrahydofolate; Agl, amylo-alpha-1,6-glucosidase,4-alpha-glucanotransferase gene; BCA, bicinchoninic acid; Bhmt, betaine-homocysteine S-methyltransferase gene; CE, cholesteryl-ester; Cebpa, CCAAT/enhancer binding protein (C/EBP), alpha gene; Cer, ceramide; DAG, diacylglycerol; Ddit3, DNA damage inducible transcript 3 gene; ETS, electron transport system; FCCP, p-trifluoromethoxyphenyl hydrazine; FFA, free fatty acid; G6pc, glucose 6-phophastase gene; Gbe1, glycogen branching enzyme 1 gene; Glycogen; Gsk3, glycogen synthase kinase gene; Gyg, glycogenin gene; Gys2, glycogen synthase 2 gene; HOAD, 3-hydoxyacyl-CoA dehydrogenase; Hepatic fuel storage; Isca1, iron‑sulfur cluster assembly 1 gene; JO2, oxygen flux; LC-MS, liquid chromatography-mass spectrometry; LPC, lysophosphatidylcholine; Lipidomics; Liver metabolism; Mitochondrial function; Mthfr, methylenetetrahydrofolate reductase gene; Mtr, methionine synthase gene (also MS); Mtrr, methionine synthase reductase gene (also MSR); Myc, myelocytomatosis oncogene; NAFLD, non-alcoholic fatty liver disease; NASH, non-alcoholic steatohepatitis; Ndufs, NADH:ubiquinone oxidoreductase core subunit (ETS complex I) gene; OXPHOS, oxidative phosphorylation; One‑carbon metabolism; PA, phosphatidic acid; PAS, periodic acid Schiff; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIP, phosphatidylinositol phosphate(s); PL, phospholipid; PS, phosphatidylserine; RIPA, Radioimmunoprecipitation assay; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SM, sphingomyelin; TAG, triacylglycerol; Ugp2, UDP-glucose pyrophophorylase 2 gene; gt, gene-trap.

Fig. 1

Mtrr^gt/gt female mouse livers are large with increased eosinophilia in hepatocytes. (A) Relative mRNA expression of genes encoding for folate metabolism enzymes in C57Bl/6J control and Mtrr^gt/gt livers of female mice as determined by RT-qPCR analysis. N = 3–5 females per genotype. Mtrr WT, wildtype Mtrr transcript; Mtrr total, wildtype Mtrr transcript plus gene-trapped Mtrr transcript. Data is presented as fold change relative to C57Bl/6J controls (normalized to 1; mean ± sd). Independent t-tests, *p < .05, **p < .01. (B) Body mass and (C) liver mass in C57Bl/6J control and Mtrr^gt/gt male (m) and female (f) mice. (D) Liver mass as a percentage of body mass in male (m) and female (f) C57Bl/6 J and Mtrr^gt/gt mice. N = 4–8 mice per sex per genotype. Histological sections of C57Bl/6J and Mtrr^gt/gt female livers stained with (E-F) hematoxylin (blue) and eosin (pink; H&E) or (G-H) an antibody against Ki67 protein (brown; nuclei counterstained with hematoxylin [blue]). White arrowhead indicates a strongly eosinophilic hepatocyte. Black arrowheads indicate Ki67⁺ cells. Black boxes indicate regions shown in higher magnification to right. Scale bars: low magnification, 100 μm; high magnification, (E-F) 25 μm and (G-H) 12.5 μm. (I) Nuclear density in histological sections of female C57Bl/6J and Mtrr^gt/gt livers as represented the number of cells per mm². (J) Mitotic cell counts in histological sections of livers from C57Bl/6J and Mtrr^gt/gt female mice as determined by the percentage of hepatocytes that were Ki67⁺. At least four liver regions were assessed per female. N = 3 females per genotype. Independent t-test, ^§p = .0592, **p < .01. (K) Relative mRNA expression in C57Bl/6J control and Mtrr^gt/gt female livers of Ddit3, Myc, and Cebpa genes as determined by RT-qPCR analysis. N = 3–5 females per genotype. Data is presented as fold change relative to C57Bl/6J controls (normalized to 1; mean ± sd). Independent t-tests, *p < .05. Legend: Squares, males; Circles, females; black, C57Bl/6J; white, Mtrr^gt/gt.

Fig. 2

Mtrr^gt/gt livers from female mice show altered glycogen storage. Representative histological sections showing two liver regions of (A) C57Bl/6J and (B) Mtrr^gt/gt female mice stained with periodic acid-Schiff (PAS, dark pink; nuclei are counterstained with hematoxylin [blue]). Black boxes indicate regions shown in higher magnification to right. Scale bars: lowest magnification, 2.5 mm; middle magnification, 500 μm; high magnification, 50 μm. Graphs showing (C) the percentage of PAS⁺ liver cells and (D) the intensity of PAS staining (represented by the percentage of image area with PAS stain) in histological sections of C57Bl/6J livers and Mtrr^gt/gt livers of female mice. At least four liver regions were assessed per female. N = 3 females per genotype. Data is represented as mean ± sd. Independent t-test, ***p < .005. (E) Hepatic glycogen concentration in female C57Bl/6J and Mtrr^gt/gt mice. N = 8 livers per group. Data is represented as mean ± sd. Independent t-test. (F—H) Relative mRNA expression in C57Bl/6J control and Mtrr^gt/gt female livers was determined for (F) Gys2, Ugp2, Gbe1 and Gyg, (G)Gsk3a and Gsk3b and (H) Agl and G6pc as determined by RT-qPCR analysis. N = 6 livers per group. Data is presented as fold change relative to C57Bl/6J controls (normalized to 1; mean ± sd). Independent t-tests, ^§p = .0599, *p < .05, **p < .01, ***p < .001. Black circles, C57Bl/6J; white circles, Mtrr^gt/gt.

Fig. 3

Mtrr^gt/gt female mouse livers show altered fatty acid metabolism and not storage of lipids. (A) 3-hydroxyacyl-CoA dehydrogenase (HOAD) enzyme activity, (B) citrate synthase (CS) activity, and (C) HOAD activity relative to CS activity in male (m) and female (f) C57Bl/6J and Mtrr^gt/gt mouse liver. N = 4–6 livers per group, mean ± sd. Two-way ANOVA, Sidak's multiple comparison, *p < .05. (D) Total carnitine species and (E) relative levels of short-chain (C2-C5), medium-chain (C6-C12) and long-chain (≥C13) acyl-carnitines to the total carnitine pool as measured by liquid chromatography-mass spectrometry (LC-MS) in male (m) and female (f) C57Bl/6J and Mtrr^gt/gt mouse liver. (F) Total detected lipid, (G) levels of triacylglycerol (TAG), diacylglycerol (DAG) and free fatty acids (FFAs) and (H) levels of phosphatidylethanolamine (PE) and phosphatidylcholine (PC) measured through open profiling lipidomics by LC-MS in male (m) and female (f) C57Bl/6J and Mtrr^gt/gt mouse liver. See also Supplementary fig. 1. Data in panels D-H are normalized to an appropriate internal standard and sample protein concentration. N = 8 livers per group, mean ± sd. *p < .05, **p < .01, ***p < .001, Two-way ANOVA, Sidak's multiple comparison. Squares, males; Circles, females; black, C57Bl/6J; white, Mtrr^gt/gt.

Fig. 4

Mitochondrial function is unchanged in livers of Mtrr^gt/gt adult mice. (A) Relative mRNA expression of genes encoding for nuclear-encoded mitochondrial genes in male (m) and female (f) livers from C57Bl/6J control and Mtrr^gt/gt adult mice as determined by RT-qPCR analysis. N = 3–5 females per genotype. Data is presented as fold change relative to C57Bl/6J controls (normalized to 1; mean ± sd). (B-E) Mitochondrial respiratory function (JO₂) in male and female livers from C57Bl/6J and Mtrr^gt/gt adult mice. GM_L, malate and glutamate stimulate LEAK respiration through the N-pathway via complex I; GM_P, OXPHOS supported by glutamate and malate through the N-pathway via complex I; GMS_P, OXPHOS supported by glutamate, malate and succinate through the NS-pathway via complexes I and II; GMS_E, maximal ET capacity supported by malate, glutamate and succinate through the NS-pathway via complexes I and II in uncoupled mitochondria. Oxygen flux rates were normalized to (B-C) wet tissue mass or to (D-E) GMS_E to indicate mitochondrial respiratory capacity per mitochondrial unit. Squares, males; circles, females; black, C57Bl/6J; white, Mtrr^gt/gt.