Folate, an essential vitamin, is critical for RNA/DNA synthesis, metabolism of certain amino acids (including homocysteine), and methylation. In the pancreas, folate deficiency causes a decrease in digestive enzyme secretion and increases risk of cancer. Pancreatic cells are unable to synthesize folate, obtaining it from exogenous sources. Since cellular and molecular mechanisms involved in folate uptake and its regulation in pancreatic acinar cells are unclear, we initiated preliminary studies using primary and cultured (AR42J) rat pancreatic acinar cells obtaining evidence suggesting involvement of a carrier-mediated mechanism for folate uptake. We showed that these cells express the major folate transporters, the reduced folate carrier (RFC) and the proton coupled folate transporter (PCFT). We propose to continue studies to delineate the specificity, requirements, and kinetics of the folate uptake process and to determine the relative functional contribution of RFC and PCFT toward total carrier-mediated folate uptake via an shRNA approach. We also propose to examine if the pancreatic folate uptake process is adaptively regulated by substrate availability (deficiency and over-supplementation) and whether or not it undergoes developmental regulation. Our studies will be extended to the human situation by examining the pattern of RFC and PCFT expression at both the RNA (in situ hybridization) and protein (immunohistochemistry) levels using normal human pancreatic tissue samples. Chronic alcoholism is a leading cause of pancreatitis and systemic folate deficiency. Although the effects of chronic alcohol ingestion on pancreatic physiology has been intensely studied, little is known about its effects on folate transport specifically. Thus, in preliminary studies we showed that chronic alcohol feeding to rats leads to a significant reduction in RFC and PCFT mRNA levels in the pancreas compared to their pair-fed controls. We propose to continue these studies and to examine the functional consequences of these effects on kinetics of folate uptake by pancreatic acinar cells, level of expression of the folate carrier proteins, and on intracellular folate content. We also propose to examine the effect of chronic alcohol consumption on transcription rates of the genes SLC19A1 and SLC46A1 (encoding RFC and PCFT respectively) in these cells. Results of these investigations should provide valuable information regarding the mechanisms of folate uptake and regulation in pancreatic acinar cells and on the effect of chronic alcohol consumption on these parameters. Such kowledge may shed light into the pathogenesis of certain pancreatic diseases and may ultimately assist in the designing of effective strategies to optimize pancreatic acinar folate homeostasis. ? ? ? ?
