The broad objective of this proposal is to determine whether a deficiency of folate, which is needed for the de novo synthesis of methyl groups, causes similar metabolic consequences as a deficiency of preformed methyl groups. Diets that contain no methionine and choline cause spontaneous liver cancer. Ethionine, an analogue of methionine that interferes with methylation, is a carcinogen, but when injected to choline deficient mice, ethionine rapidly causes acute pancreatitis. One explanation for the effects of methyl deficiency has been the decreased ratio of S- adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) that results. A wide variety of cellular methylation reactions are inhibited when the ratio of SAM/SAH is decreased, including the methylation of DNA which controls gene expression in eukaryotes. It has been shown by the P.I. that folate deficiency produces a decrease in the SAM/SAH ratio, in liver and pancreas, which is as great as that caused by methyl group deficiency. This SAM/SAH ratio is regulated by the enzyme, glycine N- methyltransferase (GNMT), which contains tightly bound folate. Immunohistochemical studies have shown that GNMT is concentrated in the exocrine pancreas. A high concentration of GNMT implies that methylation and folate are particularly important for certain cell types. These aspects are investigated in the specific aims which are: 1. To investigate whether folate deficiency affects pancreatic secretion in vivo, in situ and in tissue culture using the AR42J cell line. 2. To investigate whether folate deficiency affects the methylation of small molecules (creatine, phospholipids) and macromolecules (DNA). 3. To extend the immunohistochemical localization of GNMT to a variety of tissues. 4. To develop an immunohistochemical method for detecting folate within tissues. Immunohistochemical localization of folate will be modeled after the method used by the neurochemists for the localization of brain neurotransmitters. Rapid folate deficiency will be produced in rats by feeding the amino defined diet of Walzem and Clifford. Undermethylation of DNA will be evaluated by the ability to enzymatically incorporate additional methyl groups, by an altered expression of poly A+ RNA, and by an altered response of DNA to restriction endonucleases. Undermethylation of DNA in folate deficiency can affect gene expression. The ability to estimate folate levels of specific cells within tissues may provide a means of looking for localized folate deficiency as a cause of pre-neoplastic lesions in smokers and oral-contraceptive users.
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