| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
|
FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online February 25, 2002 as doi:10.1096/fj.01-0564fje. |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Cell Biology and Anatomy, School of Medicine, and
*
Department of Nutrition, School of Public Health and School of Medicine, University of North Carolina, Chapel Hill, North Carolina, USA
2Correspondence: University of North Carolina, Department of Cell Biology and Anatomy, CB# 7090, Chapel Hill, NC 27599-7090, USA. E-mail: tsadler{at}med.unc.edu
SPECIFIC AIMS
The purpose of this research was to study choline metabolism in gastrulation/neurulation stage mouse embryos and to identify potential biochemical mechanisms associated with growth and developmental abnormalities previously shown to be caused by an inhibitor of choline uptake, 2-dimethylaminoethanol (DMAE), and an inhibitor of PtdCho synthesis, 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3). Chromatographic techniques, reverse-transcription polymerase chain reaction (RT-PCR), and immunoblotting were used to evaluate the choline metabolic pathways in control and inhibitor-treated cultured neurulating mouse embryos.
PRINCIPAL FINDINGS
1. In gastrulation/neurulation stage mouse embryos, choline was used primarily for phosphatidylcholine synthesis, but betaine and acetylcholine were also generated
In control embryos, high-pressure liquid (HPLC) and thin-layer chromatography (TLC) revealed that 90–95% of 14C-choline was incorporated into phosphocholine and phosphatidylcholine (PtdCho) (Fig. 1
, Fig. 2
). In embryos and visceral yolk sacs after 10 h of culture and in yolk sacs after 24 h, 70–80% of labeled choline was detected as phosphocholine and 15–25% was PtdCho. In embryos cultured 24 h, 40% of 14C-choline was in the form of phosphocholine and 57% was PtdCho. At both times, the remaining 14C-choline detected was free choline, betaine, glycerophosphocholine, sphingomyelin, and (in yolk sacs) acetylcholine. To determine whether PtdCho could also be synthesized through the phosphatidylethanolamine methyltransferase (PeMT) pathway (Fig. 3
), embryos and yolk sacs were incubated with 3H-ethanolamine. Approximately 95% of labeled ethanolamine detected in the lipid phase was in the form of phosphatidylethanolamine (PtdEtn), but no 3H label was evident in PtdCho.
|
|
|
Although betaine was found to be synthesized from 14C-choline, it was not known whether betaine could contribute methyl groups for methionine synthesis during gastrulation and neurulation (Fig. 3)
. RT-PCR showed that betaine homocysteine methyltransferase was not expressed in either yolk sac or embryonic tissue until gestational day (GD) 10, when neurulation is nearly complete.
Immunoblotting was used to determine whether choline acetyltransferase (ChAT) could be detected in GD 9, 9.5, and 10 embryos and visceral yolk sacs. In adult brain, the anti-ChAT antibody labeled protein bands of 70 and 45 kDa. However, 70 kDa ChAT was undetectable in the embryos and yolk sacs studied. Instead, in embryos, the antibody labeled a 50 kDa band. In both embryos and yolk sacs, an anti-ChAT labeled protein band of 40 kDa was observed.
2. Treatment with DMAE or ET-18-OCH3 affected accumulation of 14C-labeled choline, betaine, phosphocholine, PtdCho, and sphingomyelin
After 10 h of embryo culture with 375 µM DMAE, free 14C-choline in the embryo was reduced by 60%; in both embryos and yolk sacs, incorporation of 14C-choline into phosphocholine, PtdCho, and sphingomyelin was decreased to 25%, 35% and 50% of control values, respectively (Fig. 1)
. In DMAE-treated embryos, labeled betaine was threefold higher than in controls. In embryos and yolk sacs, treatment with DMAE resulted in reduced PtdEtn synthesized from 3H-ethanolamine.
The effects of 275 µM ET-18-OCH3 on choline uptake and metabolism were evaluated after 10 and 24 h of embryo culture (Fig. 2
, 10 h). In visceral yolk sacs after 10 or 24 h and in embryos after 24 h, treatment with ET-18-OCH3 resulted in a twofold increase in labeled phosphocholine. After 10 h of culture, PtdCho and sphingomyelin accumulation was reduced by 50% in embryos exposed to ET-18-OCH3. A trend toward reduced PtdCho (P=0.0675) and sphingomyelin (P
0.05) synthesis was observed in 24 h ET-18-OCH3-treated embryos. In visceral yolk sacs, ET-18-OCH3 treatment resulted in a 25–30% increase in PtdEtn synthesized from 3H-ethanolamine, but PtdCho synthesis through the PeMT pathway was not detected.
3. Altered levels of diacylglycerol and ceramide were observed in inhibitor-treated embryos
Since diacylglycerol (DAG) and ceramide (CER) are important cell signaling molecules, HPLC was used to investigate whether embryonic or yolk sac DAG and CER levels were altered after 24 h exposure to DMAE or ET-18-OCH3. Compared to controls, diacylglycerol concentrations were elevated by 17% in ET-18-OCH3-treated embryos. Treatment with either DMAE or ET-18-OCH3 resulted in a 15% and 25% increase, respectively, in embryonic ceramide.
CONCLUSIONS AND SIGNIFICANCE
In a rapidly growing organism such as an embryo, it is expected that PtdCho and sphingomyelin synthesis through the CDP-choline pathway (Fig. 3)
would be required for cell membrane assembly and that these molecules may have regulatory roles when hydrolyzed into signaling molecules, such as diacylglycerol and ceramide. It is also possible that additional PtdCho be synthesized through PeMT (Fig. 3)
. Our results show that the majority of choline used by cultured embryos and their visceral yolk sacs is phosphorylated to phosphocholine, which is then used for PtdCho synthesis through CDP-choline. An increase in the incorporation of 14C-choline into PtdCho after 24 h culture suggests that the CDP-choline pathway for PtdCho synthesis is still developing. Our experiments found that additional PtdCho could not be synthesized through PeMT even in the presence of choline inhibitors.
Betaine has the potential to play an important role during neurulation as a methyl donor for the production of methionine and as a means of lowering homocysteine concentration (Fig. 3)
. Recent studies show that methionine and folic acid can prevent neural tube defects such as spina bifida and exencephaly, possibly by altering methyl group metabolism and/or reducing elevated homocysteine. However, in the present study, RT-PCR analysis indicated that betaine homocysteine methyltransferase was not expressed until neural tube closure is nearly complete. Hence, choline would not be an acceptable substitute for folic acid as a methyl donor in preventing neural tube defects.
It is possible that the acetylcholine (ACh) synthesized in visceral yolk sacs could act as a growth factor or morphogen regulating early mammalian embryogenesis. Such activity has been suggested in chick and sea urchin embryos. A70 kDa ChAT was undetectable in the mouse embryos and yolk sacs studied; alternatively spliced forms of ChAT may be responsible for the synthesis of ACh observed. It has been documented that the ChAT gene generates multiple mRNA splice variants. It would be of interest to determine whether these proposed alternatively spliced forms of ChAT are active in converting choline to ACh and to determine whether ACh synthesis is altered in embryos treated with DMAE or ET-18-OCH3.
In a recent study, we showed that inhibitors of choline uptake and metabolism, DMAE and ET-18-OCH3, resulted in increased cell death and craniofacial and neural tube defects in neurulation stage mouse embryos grown in culture. The present study suggests that reduced PtdCho availability caused by decreased choline transport and/or inhibition of PtdCho synthesis through the CDP-choline pathway could be responsible for the defects (Figs. 1
2
3
). Our results with DMAE are consistent with previous reports that DMAE is a competitive inhibitor of choline uptake and transport and an inhibitor of choline kinase. DMAE caused a reduction in PtdEtn synthesis, reflecting that besides being a choline analog, it is structurally related to ethanolamine. Further investigation into the effect of DMAE on ethanolamine metabolism is necessary to understand the full impact of DMAE on embryonic development. In cell culture, ET-18-OCH3 has been shown to inhibit PtdCho synthesis at the level of CTP:phosphocholine cytidylyltransferase (Fig. 3)
. The pattern of increased phosphocholine and reduced PtdCho observed in our studies suggests that ET-18-OCH3 inhibited PtdCho synthesis in the same manner in the developing conceptus. Diacylglycerol and ceramide levels were elevated in ET-18-OCH3-treated embryos, suggesting that the effects of ET-18-OCH3 on embryonic development could reflect abnormal cell signaling.
Choline was recently determined to be an essential nutrient by the Institute of Medicine. In adults, choline deficiency results in liver damage in humans and hepatocarcinomas in rats. Pregnancy has been shown to deplete choline pools, and the demand for choline by a developing fetus could put the mother and the conceptus at risk for choline deficiency. Prenatal choline deficiency has been associated with altered spatial and temporal memory, increased apoptosis, reduced cell proliferation, and abnormal cell differentiation in the fetal hippocampus. It has been suggested that an increase in dietary choline during pregnancy may be beneficial. The current research contributes to this idea by establishing a critical role for choline, especially for PtdCho synthesis, during the early stages of organogenesis, when many birth defects are induced.
The potential teratogenic effects of DMAE are of special concern as it is currently sold as a nutrient supplement that claims to enhance acetylcholine-related functions such as memory and learning. A related molecule found in many commercial products, diethanolamine, has been shown to disrupt choline metabolism and cause tumor formation in mice. Consequently, further investigation into the effects of reduced choline availability and abnormal choline metabolism in the mother and the developing conceptus is warranted.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.01-0564fje; to cite this article, use FASEB J. (February 25, 2002) 10.1096/fj.01-0564fje 
This article has been cited by other articles:
|
|
 |
|
  G. Wu, C. Aoyama, S. G. Young, and D. E. Vance Early Embryonic Lethality Caused by Disruption of the Gene for Choline Kinase {alpha}, the First Enzyme in Phosphatidylcholine Biosynthesis J. Biol. Chem., January 18, 2008; 283(3): 1456 - 1462. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Higa, E. Gonzalez, M.C. Pustovrh, V. White, E. Capobianco, N. Martinez, and A. Jawerbaum PPAR{delta} and its activator PGI2 are reduced in diabetic embryopathy: involvement of PPAR{delta} activation in lipid metabolic and signalling pathways in rat embryo early organogenesis Mol. Hum. Reprod., February 1, 2007; 13(2): 103 - 110. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. N. Craciunescu, R. Wu, and S. H. Zeisel Diethanolamine alters neurogenesis and induces apoptosis in fetal mouse hippocampus FASEB J, August 1, 2006; 20(10): 1635 - 1640. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Kohlmeier, K.-A. da Costa, L. M. Fischer, and S. H. Zeisel Genetic variation of folate-mediated one-carbon transfer pathway predicts susceptibility to choline deficiency in humans PNAS, November 1, 2005; 102(44): 16025 - 16030. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. Wang, S. Magdaleno, I. Tabas, and S. Jackowski Early Embryonic Lethality in Mice with Targeted Deletion of the CTP:Phosphocholine Cytidylyltransferase {alpha} Gene (Pcyt1a) Mol. Cell. Biol., April 15, 2005; 25(8): 3357 - 3363. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Ulrey, L. Liu, L. G. Andrews, and T. O. Tollefsbol The impact of metabolism on DNA methylation Hum. Mol. Genet., April 15, 2005; 14(suppl_1): R139 - R147. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. H. Zeisel Nutritional Importance of Choline for Brain Development J. Am. Coll. Nutr., December 1, 2004; 23(suppl_6): 621S - 626S. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. L. Jacobs, C. Devlin, I. Tabas, and D. E. Vance Targeted Deletion of Hepatic CTP:phosphocholine Cytidylyltransferase {alpha} in Mice Decreases Plasma High Density and Very Low Density Lipoproteins J. Biol. Chem., November 5, 2004; 279(45): 47402 - 47410. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
