The nutrigenetics of hyperhomocysteinemia: quantitative proteomics reveals differences in the methionine cycle enzymes of gene-induced versus diet-induced hyperhomocysteinemia

Abstract

Hyperhomocysteinemia has long been associated with atherosclerosis and thrombosis and is an independent risk factor for cardiovascular disease. Its causes include both genetic and environmental factors. Although homocysteine is produced in every cell as an intermediate of the methionine cycle, the liver contributes the major portion found in circulation, and fatty liver is a common finding in homocystinuric patients. To understand the spectrum of proteins and associated pathways affected by hyperhomocysteinemia, we analyzed the mouse liver proteome of gene-induced (cystathionine beta-synthase (CBS)) and diet-induced (high methionine) hyperhomocysteinemic mice using two-dimensional difference gel electrophoresis and Ingenuity Pathway Analysis. Nine proteins were identified whose expression was significantly changed by 2-fold (p < or = 0.05) as a result of genotype, 27 proteins were changed as a result of diet, and 14 proteins were changed in response to genotype and diet. Importantly, three enzymes of the methionine cycle were up-regulated. S-Adenosylhomocysteine hydrolase increased in response to genotype and/or diet, whereas glycine N-methyltransferase and betaine-homocysteine methyltransferase only increased in response to diet. The antioxidant proteins peroxiredoxins 1 and 2 increased in wild-type mice fed the high methionine diet but not in the CBS mutants, suggesting a dysregulation in the antioxidant capacity of those animals. Furthermore, thioredoxin 1 decreased in both wild-type and CBS mutants on the diet but not in the mutants fed a control diet. Several urea cycle proteins increased in both diet groups; however, arginase 1 decreased in the CBS(+/-) mice fed the control diet. Pathway analysis identified the retinoid X receptor signaling pathway as the top ranked network associated with the CBS(+/-) genotype, whereas xenobiotic metabolism and the NRF2-mediated oxidative stress response were associated with the high methionine diet. Our results show that hyperhomocysteinemia, whether caused by a genetic mutation or diet, alters the abundance of several liver proteins involved in homocysteine/methionine metabolism, the urea cycle, and antioxidant defense.

Fig. 1.

Homocysteine metabolism in liver and kidney. In classical homocystinuria, the initial enzyme of the transsulfuration pathway, CBS (Reaction 6), is deficient. MTHF, methylenetetrahydrofolate; THF, tetrahydrofolate; DHF, dihydrofolate; MeCbl, methylcobalamin; DMG, dimethylglycine; PLP, pyridoxal 5′-phosphate.

Fig. 2.

Effect of CBS genotype and HM diet on plasma homocysteine, liver thiols, and liver nitrite/nitrate concentrations. A, tHcy was measured in CBS^+/+ and CBS^+/− mice after 14 days on a C diet or a diet supplemented with 0.5% methionine in the drinking water (HM diet). Total liver concentrations of Hcy (B), Cys (C), GSH (D), Cys-Gly (E), and nitrite/nitrate (NOx; F) were determined on tissue homogenates from the same animals. Results were normalized to protein concentration and are expressed as mean ± S.D. *, p ≤ 0.05 versus (+/+) C; n = 4–6 animals per group. Error bars indicate ± 1 S.D.

Fig. 3.

Coomassie-stained 2D gel of differentially expressed proteins. One hundred micrograms of each sample was pooled, and 750 μg of total pooled protein was run on a single 2-D gel. Following electrophoresis, the gel was fixed, stained with GelCode Blue Coomassie stain (Pierce), and imaged on the Typhoon Trio imager (GE Healthcare). The gel image was matched to the analytical gel images, and differentially regulated protein bands were identified by DeCyder software (GE Healthcare). The identified bands were cut from the gel and identified by LC-MS-MS. The positions and identifications of the differentially expressed proteins are shown in the figure, and the -fold change of each protein is given in Table I. RGN, regucalcin; FTL, ferritin light chain 1; PGLS, 6-phosphogluconolactonase; ACTB, β-actin.

Fig. 4.

Regulated canonical pathways associated with hyperhomocysteinemia. The data set of identified proteins was loaded into IPA for pathway analysis. The top canonical pathways that were significantly (p ≤ 0.05) associated with the CBS^+/− genotype are indicated by the filled black bars, those associated with the wild-type mice on the HM diet are indicated by the white bars, and the those associated with the CBS^+/− mice on the HM diet are shown by the filled gray bars. The horizontal line represents the threshold value for a significance of p ≤ 0.05.

Fig. 5.

The effect of hyperhomocysteinemia on methionine cycle proteome. A, 2-D DIGE images of the spots identified by LC-MS-MS as SAHH, BHMT, and GNMT. BHMT and GNMT co-migrated as a single spot. Quantitation of the normalized spot volumes for SAHH (B) and BHMT/GNMT (C) is shown. SAHH was significantly increased in response to CBS genotype and HM diet, whereas the BHMT/GNMT spot was increased only in the HM diet groups. The Cy3 and/or Cy5 spot volumes of each sample were normalized to the spot volume for the Cy2-labeled internal standard and are expressed as mean ± S.D. *, p ≤ 0.05 versus (+/+) C; n = 4–6 animals per group. Error bars indicate ± 1 S.D.

Fig. 6.

Validation of DIGE results using ELISA and Western blot analysis. GNMT (A) and SAHH (B) concentrations were measured by ELISA in liver homogenates from wild-type CBS^+/+ and heterozygote CBS^+/− mice after 14 days on a normal diet (C diet) or a diet supplemented with 0.5% methionine in the drinking water (HM diet). Results were normalized to protein concentration and are expressed as mean ± S.D. *, p ≤ 0.05 versus (+/+) C; n = 4–6 animals per group. In C and D, the pooled homogenates of each experimental group were electrophoresed by 12% SDS-PAGE, and BHMT and arginase were detected by Western blot, respectively. Error bars indicate ± 1 S.D.

Fig. 7.

Network analysis of CBS-regulated proteins. A, direct (solid lines) and indirect (dashed lines) protein-protein interactions between the up-regulated (gray) and down-regulated (black) hepatic proteins of the CBS^+/− mice and members of the extracellular signal-regulated protein/mitogen-activated protein kinase (ERK/MAPK) signaling cascade. Solid lines indicate simple binding associations between proteins, whereas arrowed lines indicate that the protein nearest to the arrowhead is being acted upon by the protein furthest from the arrowhead. B, the top function(s) associated with the CBS genotype (black bar), the HM diet (white bar), and CBS^+/− mice on the HM diet (gray bar). TR, thyroid hormone receptor.