Effects of supplemental creatine and guanidinoacetic acid on spatial memory and the brain of weaned Yucatan miniature pigs.

The emergence of creatine as a potential cognitive enhancement supplement for humans prompted an investigation as to whether supplemental creatine could enhance spatial memory in young swine. We assessed memory performance and brain concentrations of creatine and its precursor guanidinoacetic acid (GAA) in 14-16-week-old male Yucatan miniature pigs supplemented for 2 weeks with either 200 mg/kg∙d creatine (+Cr; n = 7) or equimolar GAA (157 mg/kg∙d) (+GAA; n = 8) compared to controls (n = 14). Spatial memory tests had pigs explore distinct sets of objects for 5 min. Objects were spatially controlled, and we assessed exploration times of previously viewed objects relative to novel objects in familiar or novel locations. There was no effect of either supplementation on memory performance, but pigs successfully identified novel objects after 10 (p < 0.01) and 20 min (p < 0.01) retention intervals. Moreover, pigs recognized spatial transfers after 65 min (p < 0.05). Regression analyses identified associations between the ability to identify novel objects in memory tests and concentrations of creatine and GAA in cerebellum, and GAA in prefrontal cortex (p < 0.05). The concentration of creatine in brain regions was not influenced by creatine supplementation, but GAA supplementation increased GAA concentration in cerebellum (p < 0.05), and the prefrontal cortex of +GAA pigs had more creatine/g and less GAA/g compared to +Cr pigs (p < 0.05). Creatine kinase activity and maximal reaction velocity were also higher with GAA supplementation in prefrontal cortex (p < 0.05). In conclusion, there appears to be a relationship between memory performance and guanidino compounds in the cerebellum and prefrontal cortex, but the effects were unrelated to dietary supplementation. The cerebellum is identified as a target site for GAA accretion.

Betaine or folate can equally furnish remethylation to methionine and increase transmethylation in methionine-restricted neonates.

Methionine partitioning between protein turnover and a considerable pool of transmethylation precursors is a critical process in the neonate. Transmethylation yields homocysteine, which is either oxidized to cysteine (i.e., transsulfuration), or is remethylated to methionine by folate- or betaine- (from choline) mediated remethylation pathways. The present investigation quantifies the individual and synergistic importance of folate and betaine for methionine partitioning in neonates. To minimize whole body remethylation, 4-8-d-old piglets were orally fed an otherwise complete diet without remethylation precursors folate, betaine and choline (i.e. methyl-deplete, MD-) (n=18). Dietary methionine was reduced from 0.3 to 0.2 g/(kg∙d) on day-5 to limit methionine availability, and methionine kinetics were assessed during a gastric infusion of [13C1]methionine and [2H3-methyl]methionine. Methionine kinetics were reevaluated 2 d after pigs were rescued with either dietary folate (38 µg/(kg∙d)) (MD + F) (n=6), betaine (235 mg/(kg∙d)) (MD + B) (n=6) or folate and betaine (MD + FB) (n=6). Plasma choline, betaine, dimethylglycine (DMG), folate and cysteine were all diminished or undetectable after 7 d of methyl restriction (P<.05). Post-rescue, plasma betaine and folate concentrations responded to their provision, and homocysteine and glycine concentrations were lower (P<.05). Post-rescue, remethylation and transmethylation rates were~70-80% higher (P<.05), and protein breakdown was spared by 27% (P<.05). However, rescue did not affect transsulfuration (oxidation), plasma methionine, protein synthesis or protein deposition (P>.05). There were no differences among rescue treatments; thus betaine was as effective as folate at furnishing remethylation. Supplemental betaine or folate can furnish the transmethylation requirement during acute protein restriction in the neonate.

The dynamics of methionine supply and demand during early development.

Methionine is an indispensable amino acid that, when not incorporated into protein, is converted into the methyl donor S-adenosylmethionine as entry into the methionine cycle. Following transmethylation, homocysteine is either remethylated to reform methionine or irreversibly trans-sulfurated to form cysteine. Methionine flux to transmethylation and to protein synthesis are both high in the neonate and this review focuses on the dynamics of methionine supply and demand during early development, when growth requires expansion of pools of protein and transmethylation products such as creatine and phosphatidylcholine (PC). The nutrients folate and betaine (derived from choline) donate a methyl group during remethylation, providing an endogenous supply of methionine to meet the methionine demand. During early development, variability in the dietary supply of these methionine cycle-related nutrients can affect both the supply and the demand of methionine. For example, a greater need for creatine synthesis can limit methionine availability for protein and PC synthesis, whereas increased availability of remethylation nutrients can increase protein synthesis if dietary methionine is limiting. Moreover, changes to methyl group availability early in life can lead to permanent changes in epigenetic patterns of DNA methylation, which have been implicated in the early origins of adult disease phenomena. This review aims to summarize how changes in methyl supply and demand can affect the availability of methionine for various functions and highlights the importance of variability in methionine-related nutrients in the infant diet.

Betaine is as effective as folate at re-synthesizing methionine for protein synthesis during moderate methionine deficiency in piglets.

PURPOSE: Both folate and betaine (synthesized from choline) are nutrients used to methylate homocysteine to reform the amino acid methionine following donation of its methyl group; however, it is unclear whether both remethylation pathways are of equal importance during the neonatal period when remethylation rates are high. Methionine is an indispensable amino acid that is in high demand in neonates not only for protein synthesis, but is also particularly important for transmethylation reactions, such as creatine and phosphatidylcholine synthesis. The objective of this study was to determine whether supplementation with folate, betaine, or a combination of both can equally re-synthesize methionine for protein synthesis when dietary methionine is limiting. METHODS: Piglets were fed a low methionine diet devoid of folate, choline, and betaine, and on day 6, piglets were supplemented with either folate, betaine, or folate + betaine (n = 6 per treatment) until day 10. [1-13C]-phenylalanine oxidation was measured as an indicator of methionine availability for protein synthesis both before and after 2 days of supplementation. RESULTS: Prior to supplementation, piglets had lower concentrations of plasma folate, betaine, and choline compared to baseline with no change in homocysteine. Post-supplementation, phenylalanine oxidation levels were 20-46 % lower with any methyl donor supplementation (P = 0.006) with no difference among different supplementation groups. Furthermore, both methyl donors led to similarly lower concentrations of homocysteine following supplementation (P < 0.05). CONCLUSIONS: These data demonstrate an equal capacity for betaine and folate to remethylate methionine for protein synthesis, as indicated by lower phenylalanine oxidation.

Guanidinoacetate is more effective than creatine at enhancing tissue creatine stores while consequently limiting methionine availability in Yucatan miniature pigs.

Creatine (Cr) is an important high-energy phosphate buffer in tissues with a high energy demand such as muscle and brain and is consequently a highly consumed nutritional supplement. Creatine is synthesized via the S-adenosylmethionine (SAM) dependent methylation of guanidinoacetate (GAA) which is not regulated by a feedback mechanism. The first objective of this study was to determine the effectiveness of GAA at increasing tissue Cr stores. Because SAM is required for other methylation reactions, we also wanted to determine whether an increased creatine synthesis would lead to a lower availability of methyl groups for other methylated products. Three month-old pigs (n = 18) were fed control, GAA- or Cr-supplemented diets twice daily. On day 18 or 19, anesthesia was induced 1-3 hours post feeding and a bolus of [methyl-3H]methionine was intravenously infused. After 30 minutes, the liver was analyzed for methyl-3H incorporation into protein, Cr, phosphatidylcholine (PC) and DNA. Although both Cr and GAA led to higher hepatic Cr concentration, only supplementation with GAA led to higher levels of muscle Cr (P < 0.05). Only GAA supplementation resulted in lower methyl-3H incorporation into PC and protein as well as lower hepatic SAM concentration compared to the controls, suggesting that Cr synthesis resulted in a limited methyl supply for PC and protein synthesis (P < 0.05). Although GAA is more effective than Cr at supporting muscle Cr accretion, further research should be conducted into the long term consequences of a limited methyl supply and its effects on protein and PC homeostasis.

The nutritional burden of methylation reactions.

PURPOSE OF REVIEW: Methyl group metabolism is a metabolically demanding process that has significant nutritional implications. Methionine is required not only for protein synthesis but also as the primary source of methyl groups. However, demethylated methionine can be remethylated by methyl groups from methylneogenesis (via folate) and betaine (synthesized from choline). This review discusses the impact of methylation precursors and products on the methionine requirement. RECENT FINDINGS: Recent evidence has clearly demonstrated that transmethylation reactions can consume a significant proportion of the flux of methionine. In particular, synthesis of creatine and phosphatidylcholine consume most methyl groups and their dietary provision could spare methionine. Importantly, methionine can become limiting for protein and phosphatidylcholine synthesis when creatine synthesis is upregulated. Other research has shown that betaine and choline seem to be more effective than folate at reducing hyperhomocysteinemia and impacting cardiovascular outcomes suggesting they may be limiting. SUMMARY: It appears that methyl groups can become limiting when dietary supply is inadequate or if transmethylation reactions are upregulated. These situations can impact methionine availability for protein synthesis, which can reduce growth. The methionine requirement can likely be spared by methyl donor and methylated product supplementation.