Methionine Source and Level Modulate Gut pH, Amino Acid Transporters and Metabolism Related Genes in Broiler Chickens.

This study determined the effects of two methionine (Met) sources at three total sulfur amino acids (TSAA) to lysine ratios (TSAA/Lys) on gut pH, digestive enzyme activity, amino acid transporter expression, and Met metabolism of broilers. The birds were randomly assigned to a 2 × 3 factorial arrangement with Met sources (dl-Met and dl-2-hydroxy-4-(methylthio)-butanoic acid (OH-Met)) and TSAA/Lys (0.58, 0.73, and 0.88) from 1 to 21 days. The results demonstrated that dl-Met and OH-Met supported the same growth performance, but high TSAA/Lys ratio reduced the feed intake and body weight (P < 0.05). OH-Met reduced the crop chyme pH and enhanced the jejunal lipase activity (P < 0.05). ATB0,+ expression decreased with increased dl-Met levels in the duodenum; the low TSAA/Lys ratio induced a stronger mRNA expression of basolateral Met transporters. OH-Met resulted in an increase of cystathionine ß-synthase expression in the liver and a decrease in serum homocysteine levels at middle TSAA/Lys ratio compared with dl-Met treatment (P < 0.05). In conclusion, two Met sources support the same growth, but OH-Met acidified the crop chyme. The investigated transporter transcripts differed significantly along the small intestine. At the middle TSAA/Lys ratio, OH-Met showed a higher metabolic tendency of the trans-sulfuration pathway compared with dl-Met.

A systematic review of metabolism of methionine sources in animals: One parameter does not convey a comprehensive story.

The goal of this review article, based on a systematic literature search, is to critically assess the state of knowledge and experimental methodologies used to delineate the conversion and metabolism of the 2 methionine (Met) sources DL-methionine (DL-Met) and DL-2-hydroxy-4-(methylthio) butanoic acid (HMTBa). The difference in the chemical structures of HMTBa and DL-Met indicates that these molecules are absorbed and metabolized differently in animals. This review explores the methodologies used to describe the 2-step enzymatic conversion of the 3 enantiomers (D-HMTBa, L-HMTBa and D-Met) to L-Met, as well as the site of conversion at the organ and tissue levels. Extensive work was published documenting the conversion of HMTBa and D-Met into L-Met and, consequently, the incorporation into protein using a variety of in vitro techniques, such as tissue homogenates, cell lines, primary cell lines, and everted gut sacs of individual tissues. These studies illustrated the role of the liver, kidney, and intestine in the conversion of Met precursors into L-Met. A combination of in vivo studies using stable isotopes and infusions provided evidence of the wide conversion of HMTBa to L-Met by all tissues and how some tissues are net users of HMTBa, whereas others are net secreters of L-Met derived from HMTBa. Conversion of D-Met to L-Met in organs other than the liver and kidney is poorly documented. The methodology cited in the literature to determine conversion efficiency ranged from measurements of urinary, fecal, and respiratory excretion to plasma concentration and tissue incorporation of isotopes after intraperitoneal and oral infusions. Differences observed between these methodologies reflect differences in the metabolism of Met sources rather than differences in conversion efficiency. The factors affecting conversion efficiency are explored in this paper and are mostly associated with extreme dietary conditions, such as noncommercial crystalline diets that are very deficient in total sulfur amino acids with respect to requirements. Implications in the diversion of the 2 Met sources toward transsulfuration over transmethylation pathways are discussed. The strengths and weaknesses of some methodologies used are discussed in this review. From this review, it can be concluded that due to the inherent differences in conversion and metabolism of the 2 Met sources, the experimental methodologies (e.g., selecting different organs at different time points or using diets severely deficient in Met and cysteine) can impact the conclusions of the study and may explain the apparent divergences of conclusion found in the literature. It is recommended when conducting studies or reviewing the literature to properly select the experimental models that allow for differences in how the 2 Met precursors are converted to L-Met and metabolized by the animal to enable a proper comparison of their bioefficacy.

Improving pork quality traits by a short-term dietary hydroxy methionine supplementation at levels above growth requirements in finisher pigs.

The effects of dietary methionine (Met) supplies above growth requirements on tissue biology and pork quality were studied. At 70 kg, 45 crossbred pigs were fed a control (CONT) diet adequate in Met (0.22% Met) up to 105 kg. For the last 14 days before slaughter, pigs were fed with the CONT diet or with diets where the Met level was increased to Met3 (0.66% Met) or Met5 (1.10% Met). Growth performance and carcass composition did not change with the treatment. Pigs fed the Met5 treatment displayed lower TBARS and higher glutathione levels (P ≤ .05), along with higher ultimate pH (P < .01) and lower drip, lightness and hue (P ≤ .10) in the longissimus muscle, compared to the CONT and Met3 pigs. Extra-dietary Met improved ham's technological quality in the Met3 and Met5 groups (P ≤ .05). Thus, dietary Met supplementation improves pork quality without impairing growth or carcass traits.

mTORC1 signaling-associated protein synthesis in porcine mammary glands was regulated by the local available methionine depending on methionine sources.

Mechanistic target of rapamycin complex1 (mTORC1) activation and protein synthesis varied with methionine sources; however, the related mechanisms are largely unknown. Porcine mammary epithelial cells (PMEC) and mammary tissue slices (MTS) were used to test whether methionine precursors differ in providing the available methionine and thus differ in mTORC1 signaling-associated protein synthesis. PMEC with methionine deprivation for 8 h and MTS from lactating sows were cultured for 24 and 2 h, respectively, with treatment media without methionine (negative control, NC) or supplemented with 0.6 mM (for PMEC) and 0.1 mM (for MTS) of L-methionine (L-MET), D-methionine (D-MET), DL-2-hydroxy-4-(methylthio) butyric acid (HMTBA), or keto-methyl(thio)butanoic acid (KMB). The measurements included: phosphorylation of mTORC1 signaling, fractional protein synthesis rate (FSR), amino acids (AA) profile, and enzyme activities. Compared with the NC treatment, activated mTORC1 signaling as manifested by higher (P < 0.05) protein abundance of phosphorylated-S6 Kinase 1 (P-S6K1) and phosphorylated-4E-binding Protein 1 (P-4E-BP1) in PMEC and MTS, and increased protein synthesis as indicated by higher (P < 0.05) FSR in MTS occurred in L-MET and HMTBA treatments rather than in D-MET treatment. Compared with the NC treatment, methionine concentration and ratio of methionine to lysine in MTS increased (P < 0.05) in L-MET and HMTBA treatments but not in D-MET treatment, and activities of enzymes responsible for conversion of D-MET and HMTBA to keto-methionine in mammary tissues were about 10 and 50%, respectively, of that in liver. Taken together, mTORC1 signaling-associated protein synthesis in porcine mammary glands was regulated by the local available methionine depending on methionine sources.

Methionine and cysteine deficiencies altered proliferation rate and time-course differentiation of porcine preadipose cells.

Methionine (Met) is an essential sulfur amino acid (AA) limiting growth and is the precursor of cysteine (Cys), the rate-limiting factor in the synthesis of glutathione, and the main intracellular non-enzymatic antioxidant. This study aimed at determining the effects of limited supplies in Met and(or) Cys in early aspects of adipose tissue development and oxidative stress in differentiated adipocytes. Incremental reductions in Met (70, 40, and 0 µM) were compared with Met 100 µM (control dose) in porcine preadipocytes cultured in media without or with Cys (250 µM). In Cys-deprived media, both the absence (0 µM) and the lowest dose of Met (40 µM) reduced preadipocyte proliferation. Adding Cys in media only partly compensated for this decrease. On the opposite, mild Met deficiency (70 µM) did not alter preadipocyte proliferation in media without or with Cys. Strong Met deficiency (40 µM) also reduced differentiation and lipid accumulation into preadipose cells. Mild Met deficiency also reduced preadipocyte differentiation when Cys was present in the culture media, whereas in Cys-deprived media, percent of differentiated cell was similar and intracellular lipid content was slightly higher at Met 70 µM than at Met 100 µM. Finally, incremental reductions in Met in media with or without Cys lowered reactive oxygen species (ROS) production by differentiated cells. These results demonstrate the strong dependency of porcine adipogenesis to sulfur AA supplies. Strong Met deficiency decreases both proliferation and differentiation, whereas mild deficiency only alters differentiation.

Splanchnic tissues respond differently when piglets are offered a diet 30 % deficient in total sulfur amino acid for 10 days.

PURPOSE: A deficient total sulfur amino acid (TSAA) supply has been reported to differently affect the amino acid composition of tissues, but limited information is available about its effects on the morphology and metabolic properties of splanchnic tissues. METHODS: The amino acid composition, protein metabolism, glutathione concentration of the liver, proximal and distal jejunum, ileum and kidneys, and intestinal architecture were compared in 42-day-old piglets pair-fed either a diet deficient (TSAA-; 28 % deficiency) or sufficient (TSAA+) in TSAA for 10 days. RESULTS: The supply of TSAA had no effect on tissue weights, but influenced the amino acid composition in a tissue-dependent manner. Compared with animals receiving diet TSAA+, the concentrations of Met and Ser were higher in liver protein of TSAA- animals while the Cys concentration in protein was lower in the liver but higher in the distal jejunum. The TSAA supply had no effect on protein synthesis and proteolytic activities of tissues. Villus width and surface, and crypt surface were lower in the proximal jejunum of TSAA- versus TSAA+ pigs. Crypt surface in the ileum of TSAA- pigs was higher. Pigs receiving diet TSAA- had lower GSH and GSSG concentrations in the liver and proximal jejunum, but the GSH/GSSG ratio was decreased only in the liver. CONCLUSIONS: A greater nutritional priority appears to be given to splanchnic tissues so that its growth and protein metabolism can be maintained when the TSAA supply is limiting. The amino acid composition, glutathione status, and intestinal mucosa architecture are affected in a tissue-dependent manner.

Skeletal muscles respond differently when piglets are offered a diet 30% deficient in total sulfur amino acid for 10 days.

PURPOSE: Although amino acids (AA) are required for growth, little is known about the effect of a deficient AA supply on the composition and the contractile and metabolic properties of skeletal muscles. METHODS: Protein metabolism, oxidative catabolism, glutathione system, and fiber-type composition of the longissimus (LM), rhomboideus (RM), and semitendinous (SM) muscles were compared between 42-day-old piglets pair-fed for 10 days either with a diet with a 28% deficient supply of total sulfur AA (TSAA-) or with a diet with a sufficient supply of total sulfur AA (TSAA+). RESULTS: The relative weight, protein mass, and protein synthesis of LM were 10-32% lower in TSAA- pigs compared with TSAA+ pigs, while RM and SM were not affected by the TSAA supply. The TSAA supply affected the AA composition of muscles. Concentrations of Met and branched-chain AA were, respectively, 7 and 3% lower in TSAA- pigs compared with TSAA+ pigs. The His concentration was 30% higher in LM and SM in TSAA- pigs compared with TSAA+ pigs and unaffected in RM. The activity of citrate synthase was 14% higher in all three muscles of TSAA- pigs. In these pigs, the ß-hydroxy-acyl-CoA dehydrogenase activity was 20% higher in RM compared with TSAA+ pigs while that of lactate dehydrogenase was 21% lower in LM. Total and reduced glutathione concentrations were more than 70% greater in RM than in LM or SM, and these concentrations were approximately 10% lower in TSAA- pigs than in TSAA+ pigs. CONCLUSIONS: Results of this study indicate that a TSAA deficiency affects muscle properties in a muscle-dependent manner increasing the oxidative capacity of RM and reducing growth and glycolytic metabolism of LM.

Differences in plasma metabolomics between sows fed DL-methionine and its hydroxy analogue reveal a strong association of milk composition and neonatal growth with maternal methionine nutrition.

The aim of the present study was to determine whether increased consumption of methionine as DL-methionine (DLM) or its hydroxy analogue DL-2-hydroxy-4-methylthiobutanoic acid (HMTBA) could benefit milk synthesis and neonatal growth. For this purpose, eighteen cross-bred (Landrace × Yorkshire) primiparous sows were fed a control (CON), DLM or HMTBA diet (n 6 per diet) from 0 to 14 d post-partum. At postnatal day 14, piglets in the HMTBA group had higher body weight (P= 0·02) than those in the CON group, tended (P= 0·07) to be higher than those in the DLM group, and had higher (P< 0·05) mRNA abundance of jejunal fatty acid-binding protein 2, intestinal than those in the CON and DLM groups. Compared with the CON diet-fed sows, milk protein, non-fat solid, and lysine, histidine and ornithine concentrations decreased in the DLM diet-fed sows (P< 0·05), and milk fat, lactose, and cysteine and taurine concentrations increased in the HMTBA diet-fed sows (P< 0·05). Plasma homocysteine and urea N concentrations that averaged across time were increased (P< 0·05) in sows fed the DLM diet compared with those fed the CON diet. Metabolomic results based on ¹H NMR spectroscopy revealed that consumption of the HMTBA and DLM diets increased (P< 0·05) both sow plasma methionine and valine levels; however, consumption of the DLM diet led to lower (P< 0·05) plasma levels of lysine, tyrosine, glucose and acetate and higher (P< 0·05) plasma levels of citrate, lactate, formate, glycerol, myo-inositol and N-acetyl glycoprotein in sows. Collectively, neonatal growth and milk synthesis were regulated by dietary methionine levels and sources, which resulted in marked alterations in amino acid, lipid and glycogen metabolism.

Changes in plasma amino acid profiles, growth performance and intestinal antioxidant capacity of piglets following increased consumption of methionine as its hydroxy analogue.

The aim of the present study was to determine whether early weaning-induced growth retardation could be attenuated by increased consumption of methionine as DL-methionine (DLM) or DL-2-hydroxy-4-methylthiobutyrate (HMTBA) in both lactating sows and weaned piglets. Therefore, diets containing DLM and HMTBA at 25% of the total sulphur-containing amino acids (AA) present in the control (CON) diet were fed to lactating sows and weaned piglets and their responses were evaluated. Compared with the CON diet-fed sows, the HMTBA diet-fed sows exhibited a tendency (P<0·10) towards higher plasma taurine concentrations and the DLM diet-fed sows had higher (P<0·05) plasma taurine concentrations, but lower (P<0·05) isoleucine concentrations. Suckling piglets in the HMTBA treatment group had higher (P<0·05) intestinal reduced glutathione (GSH) content, lower (P<0·05) oxidised glutathione (GSSG):GSH ratio, and higher (P<0·05) plasma cysteine and glutathione peroxidase (GPx) activity than those in the CON and DLM treatment groups. The feed intake (P<0·05) and body weight of piglets averaged across post-weaning (PW) days were higher (P< 0·05) in the HMTBA treatment group than in the DLM treatment group and were higher (P<0·05) and tended (P<0·10) to be higher, respectively, in the HMTBA treatment group than in the CON treatment group. Increased (P<0·05) GSSG content and GSSG:GSH ratio and down-regulated (P<0·05) expression of nutrient transport genes were observed in the jejunum of piglets on PW day 7 than on PW day 0. On PW day 14, the HMTBA diet-fed piglets had higher (P<0·05) intestinal GSH content than the CON diet-fed piglets and higher (P<0·05) plasma GPx activity, villus height and goblet cell numbers than the CON diet- and DLM diet-fed piglets. In conclusion, early weaning-induced growth retardation appears to be attenuated through changes in plasma AA profiles and elevation of growth performance and intestinal antioxidant capacity in piglets following increased consumption of methionine as HMTBA.

The methionine precursor DL-2-hydroxy-(4-methylthio)butanoic acid protects intestinal epithelial barrier function.

DL-2-hydroxy-(4-methylthio)butanoic acid (HMTBA) is a source of dietary methionine (Met) that is widely used in poultry nutrition. We have previously shown that HMTBA is preferentially diverted to the transsulfuration pathway, which gives antioxidant metabolites such as taurine and glutathione. Therefore, here we hypothesize that this Met source can protect epithelial barrier function in an in vitro model of intestinal inflammation of Caco-2 cells. The results show that HMTBA prevents the increase in paracellular permeability induced by H2O2 or tumour necrosis factor-α. This effect can be attributed to the increased production of taurine and reduced glutathione. Similar results were obtained for DL-Met, although the protective role of the amino acid was less pronounced than that of the hydroxy analogue. In conclusion, the diversion to the transsulfuration pathway means that this Met precursor is of greater value than previously thought, due to its capacity to improve intestinal homeostasis and the quality of poultry products destined for human consumption.

Effects of dietary protein content and 2-hydroxy-4-methylthiobutanoic acid or DL-methionine supplementation on performance and oxidative status of broiler chickens.

Besides its typical role as an amino acid in protein synthesis, methionine is an important intermediate in methylation reactions. In addition, it can also be converted to cysteine and hence plays a role in the defence against oxidative stress. The present study was conducted to investigate further the role of DL-methionine (DLM) and its hydroxy analogue, DL-2-hydroxy-4-methylthiobutanoic acid (DL-HMTBA), on zootechnical performance and oxidative status of broiler chickens. Male broiler chickens were reared on two diets differing in crude protein (CP) content (low-protein, 18·3 % v. high-protein, 23·2 % CP) and were supplemented either with 0·25 % DLM or 0·25 % DL-HMTBA. Reducing the dietary protein content resulted in an impaired body weight gain (P < 0·0001). However, supplementation of DL-HMTBA to the low-protein diet partially alleviated these negative effects (P = 0·0003). This latter phenomenon could be explained by the fact that chickens fed DL-HMTBA-supplemented diets displayed a better antioxidant status as reflected in lower lipid peroxidation probably as a consequence of their higher hepatic concentrations of total and reduced glutathione compared with their DLM counterparts. On the other hand, within the high protein levels, uric acid might be an important antioxidant to explain the lower lipid peroxidation of high-protein DL-HMTBA-supplemented chickens. Hepatic methionine sulfoxide reductase-A gene expression was not significantly affected by the dietary treatments. In conclusion, the present study indicates that there are interactions between dietary protein content and supplementation of methionine analogues with respect to broiler performance and antioxidant status, also suggesting a causal link between these traits.

Methionine deprivation regulates the S6K1 pathway and protein synthesis in avian QM7 myoblasts without activating the GCN2/eIF2 alpha cascade.

Amino acids modulate mRNA translation through the 70 kDa ribosomal protein S6 kinase (S6K1) and the general control nondepressible 2 protein kinase (GCN2)/eukaryotic initiation factor 2 alpha eIF2 alpha pathways. The aim of the present study was therefore to explore the signaling cascades potentially modulated by methionine availability in quail muscle QM7 myoblasts using media providing all other amino acids. Methionine deprivation caused a lower S6K1 phosphorylation compared with control (Ctl) cells. Supplying the methionine-deprived media with L- and DL-methionine isomers restored S6K1 phosphorylation to the levels observed in Ctl cells. Methionine also regulated downstream S6K1 targets (i.e. ribosomal protein S6 and eukaryotic elongation factor 2), modulated translation preinitiation complex (PIC) assembly, and stimulated protein synthesis. Replacing the lacking methionine with D-methionine or its hydroxyanalog [2-hydroxy-(4-methylthio) butanoic acid] did not restore S6K1 activation or protein synthesis. Conversely, the S6K1 pathway was activated by a methionine precursor, the ketoanalog of methionine. Methionine availability regulated the GCN2/eIF2 alpha pathway. However, our results indicate that methionine deprivation led to lower protein synthesis without activating eIF2 alpha phosphorylation, a process known to limit the formation of the 43S PIC. Using the amino acid alcohol methioninol did not decrease S6K1 phosphorylation or activity and did not alter the regulation of protein synthesis by methionine. These findings suggest that methionine exerts an effect on S6K1 signaling and protein synthesis in avian QM7 myoblasts through a mechanism partly independent of the global regulation via tRNA charging.

Effect of pH on L- and D-methionine uptake across the apical membrane of Caco-2 cells.

The transport systems involved in intestinal methionine (Met) absorption are described as Na(+)-dependent and Na(+)-independent mechanisms. However, since recent studies have suggested the importance of the H(+) gradient as a driving force for intestinal nutrient absorption, the aim of the present work was to test whether Met transport across the apical membrane of Caco-2 cells is affected by extracellular pH. The results show that l- and d-Met uptake was increased by lowering extracellular pH from 7.4 to 5.5, in both the presence and absence of Na(+). Cis-inhibition experiments revealed that inhibition of l-Met transport by 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid (BCH) or l-lysine (l-Lys) was higher at a pH of 5.5. Moreover, the BCH-insensitive component was not affected by pH, whereas the l-Lys-insensitive component was increased by lowering extracellular pH, thus suggesting the participation of system L. The contribution of another mechanism, sensitive to both BCH and l-Lys, was also considered. The inhibition obtained with taurine (Tau) was also higher at a pH of 5.5, thus suggesting the involvement of system B(0,+) on pH-stimulated component. As for d-Met uptake, the results showed higher inhibition with l-Lys and Tau at a pH of 5.5 and no effect on the l-Lys- or Tau-insensitive component. In conclusion, Met transport across the apical membrane of Caco-2 cells is increased by low extracellular pH as the result of the stimulation of two transport systems functionally identified with systems L and B(0,+) for l-Met and with system B(0,+) for d-Met.

Mechanisms through which sulfur amino acids control protein metabolism and oxidative status.

Amino acids regulate protein synthesis and breakdown (i.e., protein turnover) and consequently protein deposition, which corresponds to the balance between the two processes. Elucidating the mechanisms involved in such regulation is important from fundamental and applied points of view since it can provide a basis to optimize amino acid requirements and to control protein mass, body composition and so forth. Amino acids, which have long been considered simply as precursors of protein synthesis, are now recognized to exert other significant influences; that is, they are precursors of essential molecules, act as mediators or signal molecules and affect numerous functions. For example, amino acids act as mediators of metabolic pathways in the same manner as certain hormones. Thus, they modulate the activity of intracellular protein kinases involved in the regulation of metabolic pathways such as mRNA translation. We provide here an overview of the roles of amino acids as regulators of protein metabolism, by focusing particularly on sulfur amino acids. The potential importance of methionine as a "nutrient signal" is discussed in the light of recent findings. Emphasis is also placed on mechanisms controlling oxidative status since sulfur amino acids are involved in the synthesis of intracellular antioxidants (glutathione, taurine etc.) and in the methionine sulfoxide reductase antioxidant system.