Molecular Characterization of Folate Uptake in Pancreatic Acinar Cells

描述（由申请人提供）：叶酸是一种必需维生素，对RNA/DNA合成、某些氨基酸（包括同型半胱氨酸）的代谢和甲基化至关重要。在胰腺中，叶酸缺乏会导致消化酶分泌减少，增加患癌症的风险。胰腺细胞不能合成叶酸，只能从外源性来源获得。由于叶酸摄取及其在胰腺腺泡细胞中的调节所涉及的细胞和分子机制尚不清楚，我们使用原代和培养的（AR 42 J）大鼠胰腺腺泡细胞进行了初步研究，获得了表明叶酸摄取涉及载体介导机制的证据。我们发现这些细胞表达主要的叶酸转运蛋白，还原叶酸载体（RFC）和质子偶联叶酸转运蛋白（PCFT）。我们建议继续研究，以描绘特异性，要求和叶酸摄取过程的动力学，并确定RFC和PCFT通过shRNA方法对总载体介导的叶酸摄取的相对功能贡献。我们还建议检查胰腺叶酸摄取过程是否适应性地调节底物的可用性（不足和过度补充），以及它是否经历发育调节。我们的研究将扩展到人类的情况下，通过检查RFC和PCFT的表达模式在RNA（原位杂交）和蛋白质（免疫组织化学）水平使用正常人胰腺组织样本。慢性酒精中毒是胰腺炎和系统性叶酸缺乏症的主要原因。虽然长期酒精摄入对胰腺生理的影响已被深入研究，但很少有人知道它对叶酸转运的影响。因此，在初步研究中，我们表明，长期酒精喂养大鼠导致胰腺中RFC和PCFT mRNA水平显着降低相比，其配对喂养的控制。我们建议继续这些研究，并检查这些影响的功能后果，对胰腺腺泡细胞的叶酸摄取动力学，叶酸载体蛋白的表达水平，和细胞内叶酸含量。我们还建议研究慢性酒精消耗对这些细胞中基因SLC 19 A1和SLC 46 A1（分别编码RFC和PCFT）转录速率的影响。这些调查的结果应该提供有价值的信息，关于叶酸的摄取和调节胰腺腺泡细胞的机制和慢性酒精消费对这些参数的影响。这种知识可能会揭示某些胰腺疾病的发病机制，并最终可能有助于设计有效的策略来优化胰腺腺泡叶酸稳态。

DESCRIPTION (provided by applicant): 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.