M-1000237 APARTADOS INDIVIDUALES:

Methionine is the main source of methyl groups that arepartitioned to synthesize various

methylated products includingcreatine, phosphatidylcholine (PC) and methylated DNA.

Whether increased methylation of one product can divert methionine from protein

synthesis or other methylation products was the aim of this experiment. We used an

excess of guanidinoacetate (GAA) to synthesize creatine to create a higher demand for

available methyl groups in normal weight (NW) (n = 10) and intrauterine growth-

restricted (IUGR) (n = 10) piglets. Anesthetized piglets (15–18d old) were intraportally

infused with either GAA or saline for 2 h. A bolus of L-[methyl-3H]methionine was intraportally infused at 1 h and hepatic metabolites were analyzed for methyl-3H incorporation 1 h later. Overall, 50-75% of label was recovered in creatine and PC with

negligible amounts in DNA. In both NW and IUGR piglets, excess GAA led to a 72-

125% increase in methyl incorporation into creatine (P < 0.05) with a concomitant

decrease by 76-86% in methyl incorporation into PC (P < 0.05) as well as a 38-41%

decrease in methyl incorporation into protein (P < 0.05), suggesting methyl groups were

limited for PC synthesis and that methionine was diverted from protein synthesis.

Compared to NW, IUGR piglets had lower methyl incorporation into PC (P < 0.05), but

not DNA or protein, suggesting IUGR affects methyl metabolism and could potentially

impact lipid metabolism. The partitioning of methionine is sensitive to methyl supply in

3.2 Introduction

Methionine is an indispensable amino acid that can be utilized either for protein synthesis

or as a methyl group donor for transmethylation reactions. In order to provide a methyl

group, methionine is first adenylated to form S-adenosylmethionine (SAM) which is

partitioned among an estimated 50 methyltransferases, including guanidinoacetate

methyltransferase (GAMT), phosphatidylethanolamine N-methyltransferase (PEMT) and

DNA methyltransferase (DNMT) (1).

Two of the most quantitatively important methylation reactions are the synthesis of

phosphatidylcholine (PC) from phosphatidylethanolamine (PE) via PEMT and the

synthesis of creatine from guanidinoacetate (GAA) via GAMT. Creatine can be

consumed in the diet while the remainder of the whole body requirement needs to be

endogenously synthesized. Suckling neonates only consume 25% of their requirement

from milk and thus need to synthesize the remaining 75% (2). Moreover, if dietary

choline is insufficient to meet PC synthesis requirements, then a growing neonate must

rely on methylation of PE to meet its PC needs. As these two reactions utilize the

majority of available methyl groups (3), it is important to understand how demand for

methyl groups by these two pathways can impact partitioning of methyl supply for all

methyltransferases, including DNMT.

DNMT transfers a methyl group to cytosine residues in CpG dinucleotide sequences,

which, especially when found in a promoter region, can regulate gene expression (4).

been shown to be susceptible to postnatal epigenetic modification in response to

environmental changes (5). Epigenetic modifications have been implicated in the

developmental origins of adult diseases hypothesis, which describes how an insult to the

developing fetus or neonate can result in a higher susceptibility to chronic diseases in

later life. This hypothesis was originally based on epidemiological studies which

demonstrated an association between low birth weight (i.e., IUGR neonates) and disease

in later life (6). Since then, it has been well established that nutritional insults early in life

can ‘program’ an organism’s metabolism, leading to adult diseases (7). Moreover, it has been demonstrated that DNA methylation patterns are sensitive to changes in dietary

methyl supply (8) and IUGR piglets have decreased levels of DNMT-1 mRNA compared

to normal birth weight piglets (9). As many other methylation reactions compete for these

methyl groups, it is important to understand how the methyl groups are partitioned

amongst the various methyltransferase reactions when supply is limited and how this

partitioning changes in IUGR neonates.

In order to study the effects of limited methyl supply on transmethylation partitioning, we

increased the demand for methyl groups required to synthesize creatine. Creatine is

synthesized via two reactions by the enzymes arginine:glycine amidinotransferase

(AGAT) and GAMT. AGAT forms GAA and ornithine in the kidney by transferring the

amidino group from arginine to glycine. GAA is then transported to the liver and

methylated via SAM to produce creatine and S-adenosyl-L-homocysteine (SAH). The

regulation of creatine synthesis is by AGAT, due to the feedback inhibitory effect of

shown in rats to be proportional to GAA availability (10). This suggests that an excess of

GAA in the liver would drive creatine synthesis and thus potentially increase the demand

for available methyl groups. Because certain infant formulas (i.e., soy-based) are void of

creatine, some neonates must rely solely on endogenous synthesis for their entire creatine

requirement, potentially impacting the available methyl supply. Because neonatal piglets

consume ~25% of their creatine needs via sow milk (2), we developed an acute model in

which the piglet must synthesize this additional creatine by infusing an equimolar amount

of GAA. By creating a high demand for methyl groups, it is possible to determine the

impact on other methylation reactions that also compete for the remaining methyl groups.

As fetal undernutrition has been shown to have a lasting impact on offspring, the IUGR

‘runt’ piglet has been established as a model to study early programming of adult diseases (11-14). Moreover, although we found no difference in the remethylation of

homocysteine to methionine via MS, we have demonstrated that IUGR piglets have

limited capacity to remethylate homocysteine to methionine via betaine:homocysteine

methyltransferase (BHMT), which could have consequences on the availability of methyl

groups for transmethylation reactions (15). The first objective of this study was to

determine whether methyl groups can become limiting in the neonate and to determine

the change in partitioning of methionine and methyl groups during high methyl demand

(HMD). The second objective was to determine if the partitioning of methyl groups under