Hyperhomocysteinemia Induced by Methionine Excess is Effectively Suppressed by Betaine in Geese

Simple Summary Methionine is a proteogenic sulfur amino acid with a vital role in intermediary metabolism. However, excess Methionine (Met) intake is toxic, leading to hyperhomocysteinemia. Betaine supplementation effectively ameliorates biochemical abnormalities. However, a lack of genetic information hinders the understanding of the mechanisms underlying methionine excess-mediated effects and whether Bet can effectively suppress these effects in geese. This study was performed to evaluate the effects of excess methionine on growth performance, serum homocysteine levels, apoptotic rates, and Bax and Bcl-2 protein levels in geese and to study the role of betaine in relieving excess Met-induced hyperhomocysteinemia. It was found that excess methionine reduces body weight induced by myocardial apoptosis, and betaine can be used to effectively lower plasma homocysteine levels. Abstract The objective of our study was to investigate the effects of excess Methionine (Met) on the growth performance, serum homocysteine levels, apoptotic rates, and Bax and Bcl-2 protein levels in geese and to study the role of Bet (betaine) in relieving excess Met-induced hyperhomocysteinemia (HHcy). In this study, 150 healthy male 14-day-old Yangzhou geese of similar body weight were randomly distributed into three groups with five replicates per treatment and 10 geese per replicate: the control group (fed a control diet), the Met toxicity group (fed the control diet +1% Met), and the Bet detoxification group (fed the control diet +1% Met +0.2% Bet). At 28, 49, and 70 d of age, the geese in the Met toxicity group had significantly lower body weights than those in the control group (p < 0.05). The serum homocysteine levels in geese at 70 d of age in the detoxification group were significantly lower than those in the Met toxicity group (p < 0.05). Compared with the control, Met significantly increased cardiomyocyte apoptosis rates, while Bet reduced them. In conclusion, our results suggest that excess methionine reduces body weight induced by myocardial apoptosis, and Bet can be used to effectively lower plasma homocysteine levels.


Introduction
Methionine (Met) is a sulfur amino acid (SAA) with a vital role in intermediary metabolism [1,2]. Observations made in some prior studies have indicated that optimal supplementation with Met can improve growth performance and body protein synthesis in growing birds [3][4][5]. However, among the constituent amino acids of proteins, Met is toxic if consumed in excess [6]. The supplementation of voluntarily consumed food with Met levels that are four to six times the estimated requirements group received the basal diet supplemented with 1% Met. The Bet detoxification group received the diet given to the Met toxicity group supplemented with 0.2% Bet. The supplementation ratios of Met and Bet were cited by Xie et al. [6] and Setoue et al. [16]. The tolerable upper limit of dietary Met for growing ducks was less than 1.38% on the basis of the growth performance. The Met used in the feed was DL-Met. The geese were fed in separate plastic-floored pens with 2 cm 2 square holes that were laid 70 cm above the ground. All manure was cleaned at the end of the trial. All geese were fed and watered ad libitum for 56 d. Water was provided in a half-open plastic cylindrical water tank, and the feed was provided in feeders on one side of each pen. The geese were reared indoors under similar environmental conditions (temperature: 26.0 ± 3.0 • C; relative humidity (RH): 65.5 ± 5.0%; lighting period: 16 h; space allocation: 0.5 m 2 /gander).

Sample Collection and Analyses
Feed intake (FI) by pen was measured on a daily basis, and BW was recorded at 14, 28, 49, and 70 d of age. The average daily feed intake (ADFI), average daily gain (ADG), and feed-to-gain ratio (F/G) were calculated from 14 to 28 d and from 29 to 70 d of age, and mortality was recorded as it occurred.
Calculations: ADFI = feed consumption/feeding days in the whole period ADG = total weight gain/feeding days in the whole period Animals 2020, 10, 1642 4 of 11 F/G = ADFI/ADG When the geese reached 70 d of age, 2 geese from each treatment replicate (5 replicates per treatment; n = 30 geese) were randomly selected for blood collection from their wing veins.

Clinical Blood Parameters
Blood drawn from wing veins was cooled in ice water and centrifuged for 10 min at 4500 rpm to obtain plasma for the measurement of biochemical indices. The plasma was stored at −20 • C until analysis. The plasma Hcy concentrations in peripheral blood were determined according to the previously described methods of Feussner et al. [21]. The thiol compound was liberated from plasma proteins by reduction with tri-n-butyphosphine and derivatized with a thiol-specific fluorogenic marker, 7-fluoro-benzo-2-oxa-1,3-diazole-4-sulpho-nate(Shanghai Yishi Chemical Co., Ltd, Shanghai, China). The derivative was separated isocratically within 7 min by reversed-phase HPLC using a Superspher 100 RP-18 column(Guangzhou Xinghe Biotechnology Co., Ltd, Guangzhou, China) as stationary phase.

Apoptosis Assay
An annexin V apoptosis detection kit (BD Biosciences, Cat. No. 556547, Lake Franklin, New Jersey, NJ USA) was utilized to measure apoptosis in heart tissue following the manufacturer's instructions. After treatments, cardiomyocytes were washed twice with cold phosphate-buffered solution (PBS), trypsinized, and then resuspended in binding buffer at a concentration of 1 × 10 6 cells/mL. Then, aliquots of 100 µL of cell suspension (1 × 10 5 cells) were incubated with fluorescein isothiocyanate (FITC)-annexin V(Shanghai Yishi Chemical Co., Ltd, Shanghai, China) and propidium iodide for 15 min at room temperature in the dark. The apoptotic rate was analyzed using flow cytometry(Thermo Fisher Technology (China) Co., Ltd, Shanghai, China) within 1 h.

Western Blot Analysis
Total protein lysates were collected for standard immunoblot analysis. The protein concentrations were determined by bicinchoninic acid (BCA) protein assay(Thermo Fisher Technology (China) Co., Ltd, Shanghai, China). Aliquots of protein lysates (30 mg/lane) were loaded into gels for sodium dodecyl sulfide-polyacrylamide gel electrophoresis (SDS-PAGE), and the separated proteins were transferred to a polyvinylidene fluoride (PVDF) membrane(Thermo Fisher Technology (China) Co., Ltd, Shanghai, China). The membrane was blocked and incubated with Bcl-2 and Bax antibodies overnight at 4 • C. The Bcl-2 and Bax antibodies were purchased from AmyJet Scientific (Wuhai, China) (Bcl-2, Cat. No. 3033-100; Bax, Cat. No. 3032-100). The blots were washed with TBST (Sigma-Aldrich, Shanghai, China) and incubated with corresponding horseradish peroxidase-conjugated secondary antibodies (Jackson Laboratory, Bar Harbor, Maine, ME, USA). Finally, the blots were visualized with enhanced chemiluminescence and quantified by densitometry.

Statistical Analysis
The data are expressed as the means ± standard deviations (SDs) and were subjected to Kolmogorov-Smirnov (KS) testing to confirm normality. Different groups were compared using one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post hoc test with SPSS 17.0 (SPSS, Shanghai, China). p < 0.05 was considered to indicate statistical significance.

Growth Performance
The data for mortality at 70 d were transformed before analysis. Mortality was greater in the Met toxicity group than in the control group and the Bet detoxification group (p > 0.05), and there was no treatment effect between groups. The effects of Bet on the growth performance of geese with HHcy are shown in Table 2. The BWs of the geese at 28, 49, and 70 d of age were significantly lower in the Met toxicity group than in the control group (p < 0.05). There was no significant difference in BW at 70 d between the Met toxicity group and the Bet detoxification group (p > 0.05). The ADFI and ADG for geese at 14-28 d of age were significantly higher in the Met toxicity group and the Bet detoxification group than in the control group (p < 0.05).

Clinical Blood Parameters
The effects of Bet on serum biochemical indices of HHcy in geese are shown in Table 3. The Hcy levels in geese at 28 d, 49 d, and 70 d of age were significantly higher in the Met toxicity group than in the control group (p < 0.05). The Hcy levels in geese at 70 d of age in the detoxification group were significantly lower than those in the Met toxicity group (p < 0.05) but were not significantly different from those in the control group (p > 0.05). The serum Hcy level was a sensitive index for the identification of geese with HHcy. The Hcy values for the geese in the control group were all between 16 and 21 µmol/L, while the Hcy values for the geese in the Met toxicity group were all higher than 30 µmol/L.

Cardiomyocyte Apoptosis
The effects of Bet on cardiomyocyte apoptosis in geese with HHcy are shown in Figure 1. Compared with the control diet, the Met toxicity diet significantly increased cardiomyocyte apoptosis rates (p < 0.05), but the Bet detoxification diet reduced the apoptosis rates.

Cardiomyocyte Apoptosis
The effects of Bet on cardiomyocyte apoptosis in geese with HHcy are shown in Figure 1. Compared with the control diet, the Met toxicity diet significantly increased cardiomyocyte apoptosis rates (p < 0.05), but the Bet detoxification diet reduced the apoptosis rates.

Expression of Apoptosis-Related Proteins
The effects of Bet on the expression of apoptosis-related proteins in geese with HHcy are shown in Figure 2 and Table 4. The protein expression levels of Bcl-2 were significantly lower in the Met toxicity and Bet detoxification groups than in the control group (p < 0.05) ( Table 4). There were no significant differences between the control group and the other groups regarding the expression of other apoptosis-related proteins.

Discussion
Excess Met causes marked growth depression in birds, and very high levels of Met can lead to HHcy. However, several studies have suggested that dietary Bet might alleviate HHcy. In the present study, we investigated the effect of Bet on Met loading-induced elevations in plasma Hcy levels. We first established a model of HHcy in geese via Met loading. In previous Met loading studies, Met has been administered orally to humans [22][23] and rats [13].
In this study, the BWs of geese at 28 d, 49 d and 70 d of age were significantly lower in the Met toxicity group than in the control group, and Bet did not improve growth performance in geese at 28 d, 49 d and 70 d of age. In previous studies, excess levels of Met or a Met hydroxy analog have been found to be toxic and to cause growth depression in ducks [24] and broiler chickens [25]. Xue et al. [24] have also reported that excess DL-Met and excess L-Met are toxic to starter Pekin ducks and that both Met sources exert equivalent growth-depressing effects. Our results are in agreement with the

Discussion
Excess Met causes marked growth depression in birds, and very high levels of Met can lead to HHcy. However, several studies have suggested that dietary Bet might alleviate HHcy. In the present study, we investigated the effect of Bet on Met loading-induced elevations in plasma Hcy levels. We first established a model of HHcy in geese via Met loading. In previous Met loading studies, Met has been administered orally to humans [22,23] and rats [13].
In this study, the BWs of geese at 28, 49, and 70 d of age were significantly lower in the Met toxicity group than in the control group, and Bet did not improve growth performance in geese at 28, 49, and 70 d of age. In previous studies, excess levels of Met or a Met hydroxy analog have been found to be toxic and to cause growth depression in ducks [24] and broiler chickens [25]. Xue et al. [24] have also reported that excess DL-Met and excess L-Met are toxic to starter Pekin ducks and that both Met sources exert equivalent growth-depressing effects. Our results are in agreement with the findings of many previous studies on the toxicity of Met and its hydroxy analog in broilers [25]. At present, little information about Met toxicity in geese has been reported. Our findings also suggest that poultry, Animals 2020, 10, 1642 8 of 11 especially geese, are intolerant of excess Met from 14 to 70 d of age given that growth performance was continuously depressed during this timeframe. Previous studies have shown that betaine is effective in suppressing methionine-induced HHcy in humans [22] and rats [26]. It is usually assumed that dietary Bet affects Hcy metabolism by stimulating Hcy remethylation. In contrast, we did not detect any strong effect of Bet in Met-induced HHcy model in growth performance in our study. We could still see the Bet could alleviate HHcy-mediated BW reductions in geese at this later growth stage, as there was no significant difference in BW between the Bet detoxification group and the Met toxicity group at the age of 70 d. Possible explanations of the discrepancy are the difference in the time of Met loading: one-shot methionine injection vs. successive methionine feeding. Our test results are consistent with those of Setoue et al. [16].
Hcy is a nonessential, thiol-containing and potentially cytotoxic 4-carbon α-amino acid formed through the demethylation of Met during Met metabolism [26]. Traditional explanations of the mechanism of Hcy neurotoxicity have suggested that disturbances in methylation and remethylation processes play key roles. Increasing evidence supports the hypothesis that elevated total Hcy is an independent risk factor for coronary vascular and neurodegenerative disease [27]. High levels of Hcy are related to cerebrovascular disease, monoamine neurotransmitter deficiencies, and mood depression. Specifically, it has been hypothesized that the cerebrovascular disease and neurotransmitter deficiency caused by high Hcy levels lead to the depression of mood [28]. Taken together, the findings indicate that the serum Hcy level is a sensitive index for the identification of geese with HHcy (HHcy: higher than 30 µmol/L; normal: 16-21 µmol/L) and that 1.35% DL-Met supplementation can lead to HHcy in geese. Hyperhomocysteinemia (HHcy) was defined as mild HHcy (Hcy: 15-30 µmol/L), moderate HHcy (Hcy: 30-100 µmol/L), and severe HHcy (Hcy > 100 µmol/L) by measuring the level of Hcy in human blood [29,30]. The results in geese were approximately moderate HHcy in human.
Several studies have suggested that dietary Bet might be a determinant of plasma Hcy concentrations [31][32][33]. Setoue et al. [16] found that dietary supplementation with Bet at a level of 0.34% significantly reduced plasma Hcy levels in rats with HHcy. In the present study, dietary supplementation with 0.2% Bet was markedly effective in decreasing plasma Hcy concentrations in geese with Met loading-induced HHcy at 70 d of age. This finding indicates that Bet should be fed for at least one month to relieve HHcy.
The Hcy-lowering effect of Bet can most likely be ascribed to an increase in Bet-dependent remethylation owing to increased Bet availability and enhanced activity of the enzyme Bet methyltransferase in the liver and kidneys. This possibility is supported by the results of studies by Barak et al. [34] and Saarinen et al. [35], who found that Bet feeding elevates hepatic Bet pools in rats and chickens. Finkelstein et al. [36] have reported that an excessive intake of Met also enhances Bet methyltransferase activity, and Met and Hcy infusion reduce hepatic Bet concentrations [37] in rats. These findings may explain why Bet was highly effective in attenuating the increases in plasma Hcy caused by Met loading in the current study.
Apoptosis plays an essential role in maintaining cellular homeostasis during development, pathophysiological, and differentiation processes in multicellular organisms [38]. HHcy is a pathological condition characterized by an increase in the plasma concentration of total Hcy [39,40]. Numerous clinical and epidemiological studies have indicated that HHcy is an independent risk factor for atherothrombotic disease. In addition, some studies have revealed that Hcy causes endothelial cell dysfunction and induces apoptotic cell death in cell types relevant to atherothrombotic disease, including endothelial cells and smooth muscle cells [41]. In the present study, we found that the percentages of apoptotic cells and cells with necrotic morphology continued to increase as Hcy concentrations increased, although the absolute numbers were reduced in the presence of Bet. Apoptosis has been widely documented to occur in animal and human atherosclerotic lesions, and apoptotic cell death is increased in the atherosclerotic lesions of mice fed HHcy-inducing diets [15]. The data in this study indicate that Met toxicity can lead to apoptotic cell death in cardiomyocytes. From previous work, it is clear that the liver (as well as the kidneys and pancreas) is a target organ of Met toxicity [19]. Kharbanda et al. [42] found that exposure to 7-deaza-adenosine (a potent SAH hydrolase inhibitor) alone induces apoptosis in rat hepatocytes in primary cultures. We have further shown that Bet confers protection against apoptosis, as the absolute numbers of apoptotic cells were reduced in the presence of Bet. This protective effect of Bet against apoptosis has been corroborated in studies from different laboratories in which apoptosis has been induced under various conditions. Barak et al. [43] have shown that Bet decreases intracellular SAH levels by remethylating Hcy, resulting in a significant attenuation of apoptosis. Observations of elevations in intracellular SAH and increases in hepatocyte apoptosis in the context of alcoholic apoptosis have suggested Bet treatment as a modality for the prevention of this condition [44]. In addition, Bet has been effectively used as a treatment agent for patients with inherited genetic disorders related to HHcy, such as cystathionine β-synthase deficiency and methylene tetrahydrofolate reductase deficiency [43]. Although the Bet detoxification group showed no improvement in growth performance in the current study, we were particularly interested in the protection against apoptosis provided by Bet. The hearts of geese in the Bet detoxification group had lower absolute numbers of apoptotic cells than the hearts of geese in the Met toxicity group, showing that Bet administration appears to reverse the biochemical alterations associated with heart apoptosis.
Previous studies have demonstrated that the process of apoptosis is mediated by proteins in the Bcl-2 family and is accomplished through the Fas pathway or the Caspase-dependent apoptotic pathway, which relies on active mitochondrial control [45]. Once the balance between Bcl-2 and Bax is broken, the Caspase-dependent apoptotic pathway can be activated. In this study, Bcl-2 gene expression in goose cardiomyocytes was decreased in the Met toxicity group and the Bet detoxification group, which is consistent with the findings of previous studies. The results revealed that the apoptotic process was excessively activated by Met toxicity and that Bcl-2 family proteins might be involved in this process in cardiomyocytes. However, the expression of Bcl-2 in the Bet detoxification group was not obvious. The possible mechanisms need to be clarified in further studies.
Elucidation of the metabolic events linking excess Met intake to pathology and their dependence on other underlying metabolic processes and pathologic conditions will provide crucial insights into the roles of these important diet-dependent pathways in cardiovascular health and disease. Overall, our results indicated that the supplementation of feed with 1% Met decreased growth performance and increased plasma Hcy levels, indicating that excess Met can lead to HHcy. Bet was able to effectively lower fasting plasma Hcy levels and to prevent plasma Hcy levels from increasing after Met intake. Met toxicity significantly increased the apoptosis rates of cardiomyocytes (p < 0.05), but the presence of Bet reduced the apoptosis rates of these cells. The hearts of Bet-supplemented geese had reduced absolute numbers of apoptotic cells, showing that Bet administration appears to reverse the biochemical alterations associated with heart apoptosis.