Molecular Characterization of Folate Uptake in Pancreatic Acinar Cells

描述(由申请人提供)：叶酸是一种必需的维生素，对RNA/DNA合成、某些氨基酸(包括同型半胱氨酸)的新陈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。胰腺细胞不能从外源来源合成叶酸。由于涉及胰腺腺泡细胞叶酸摄取及其调控的细胞和分子机制尚不清楚，我们利用原代培养(AR42J)的大鼠胰腺腺泡细胞进行了初步研究，获得的证据表明参与了载体介导的叶酸摄取机制。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，以描述叶酸摄取过程的特异性、要求和动力学，并通过shRNA方法确定RFC和PCFT对载体介导的叶酸总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受底物可获得性(缺乏和过度补充)的适应性调节，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，通过使用正常的人类胰腺组织样本在RNA(原位杂交)和蛋白质(免疫组织化学)水平上检测RFC和PCFT的表达模式。慢性酒精中毒是胰腺炎和全身性叶酸缺乏的主要原因。虽然长期酒精摄入对胰腺生理的影响已经被深入研究，但对其对叶酸运输的具体影响却知之甚少。因此，在初步研究中，我们表明，与配对喂养的对照组相比，长期酒精喂养导致大鼠胰腺中RFC和PCFT mRNA水平显著降低。我们建议继续这些研究，并考察这些影响对胰腺腺泡细胞摄取叶酸的动力学、叶酸载体蛋白的表达水平以及细胞内叶酸含量的功能后果。我们还建议研究长期饮酒对这些细胞中SLC19A1和SLC46A1基因(分别编码RFC和PCFT)转录速率的影响。这些研究的结果应该提供关于胰腺腺泡细胞叶酸摄取和调节的机制以及慢性酒精消费对这些参数的影响的有价值的信息。这些知识可能有助于阐明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.