Physiology/cell biology of pancreatic acinar mitochondrial vitamin B1 uptake

描述（由申请人提供）：维生素B1（硫胺素）对胰腺细胞的正常功能/健康是必不可少的，因为它在氧化能量代谢、ATP产生和维持正常细胞氧化还原状态中起着关键作用。低细胞内硫胺素水平导致急性能量衰竭和氧化应激；它还会导致线粒体功能受损。在器官水平上，胰腺含有高水平的硫胺素，缺乏这种维生素会导致其功能受损。胰腺不能内源性合成硫胺素，因此，必须从循环中获得维生素。本研究项目的总体目标是全面了解硫胺素进入胰腺腺泡细胞及其主要代谢物硫胺素焦磷酸(TPP；它们占细胞总硫胺素的约85-90%，仅在细胞质中产生)进入线粒体（一种含有和利用约90%细胞硫胺素的细胞器），这些过程如何被调节，以及它们如何受到已知对正常胰腺生理/健康不利的常见外部因素的影响。在之前的资助期内，研究主要集中在硫胺素通过细胞膜转运到胰腺细胞上。我们目前的研究重点是TPP转运到胰腺腺泡细胞的线粒体，特别强调这一过程是如何在转录和转录后水平上被调控的，所涉及的线粒体TPP转运体（MTPPT； SLC25A19基因的产物）是如何靶向线粒体的，以及MTPPT的哪些结构特征对其功能很重要。我们还旨在研究长期暴露于酒精和香烟烟雾的特定成分（CS）（已知对胰腺健康不利的外部因素）对摄取过程的影响。因此，在新的初步研究中，我们克隆了SLC25A19基因的5'调控区，确定了基础活性所需的最小启动子，并确定了一些可能需要的顺式调控元件。我们还获得证据表明，胰腺腺泡线粒体TPP摄取过程在维生素缺乏症中通过转录机制上调，该过程也可能受到特定细胞内调节途径的调节。此外，还获得了MTPPT如何靶向线粒体的信息。此外，有证据表明，胰腺腺泡细胞长期暴露于酒精和CS的特定成分会导致胰腺腺泡线粒体TPP摄取过程的显著抑制，并且这种影响是在SLC25A19转录水平上介导的。基于这些初步发现，我们在本提案中的工作假设是胰腺腺泡线粒体TPP摄取过程是一个受调节的事件，这种调节发生在转录和转录后水平，这种独特的序列决定了MTPPT靶向线粒体，并且胰腺腺泡细胞长期暴露于酒精和CS的特定成分中会损害TPP摄取。为了验证这一假设，提出了三个具体目标。这些研究结果有望为重要的胰腺腺泡线粒体摄取过程的生理学/细胞生物学提供有价值的信息，这些信息也可能适用于其他线粒体摄取系统。此外，这些结果具有病理生理学相关性，因为它们可能揭示了胰腺腺泡细胞暴露于对胰腺健康产生不利影响的常见外部因素对线粒体生理学的影响。

DESCRIPTION (provided by applicant): Vitamin B1 (thiamin) is indispensable for normal function/health of pancreatic cells due to its critical role in oxidative energy metabolism, ATP production, and in maintaining normal cellular redox state. Low intracellular level of thiamin leads to acute energy failure and oxidative stress; it is also known to lead to impairment in the function of mitochondria. At the organ level, the pancreas contains high levels of thiamin, and deficiency of this vitamin leads to impairment in its functions. The pancreas cannot synthesis thiamin endogenously, and thus, must obtain the vitamin from circulation. The overall goal of this research program has been focused on developing a comprehensive understanding of the molecular mechanisms involved in thiamin entry into pancreatic acinar cells and the subsequent transport (compartmentalization) of its major metabolite thiamin pyrophosphate (TPP; which represent ~ 85-90% of total cellular thiamin and which is generated exclusively in the cytoplasm) into mitochondria (an organelle that contains and utilizes ~ 90% of cellular thiamin), how these process are regulated, and how they are affected by exposure to common external factors that are known to adversely affect normal pancreatic physiology/health. Studies during the previous funding period have focused exclusively on thiamin transport into pancreatic cells across cell membrane. Our focus in the current proposal is on transport of TPP into mitochondria of pancreatic acinar cells with special emphasis on how the process is being regulated at the transcriptional and post-transcriptional levels, how the involved mitochondrial TPP transporter (MTPPT; product of the SLC25A19 gene) is targeted to mitochondria, and which structural features of MTPPT are important for its function. We also aim at investigating the effect of chronic exposure to alcohol and to specific components of cigarette smoke (CS) (external factors known to adversely affect pancreatic health) on the uptake process. Thus, in new preliminary studies, we have cloned the 5'-regulatory region of the SLC25A19 gene, determined the minimal promoter needed for basal activity, and identified a number of putative cis- regulatory elements that may be needed for its activity. We also obtained evidence to show that the pancreatic acinar mitochondrial TPP uptake process is up-regulated in thiamin deficiency via transcriptional mechanism(s), that the process is also under possible regulation by specific intracellular regulatory pathway(s). Furthermore, information was obtained on how MTPPT is targeted to mitochondria. Moreover evidence were obtained to show that chronic exposure of pancreatic acinar cells to alcohol and to specific components of CS leads to a significant inhibition in pancreatic acinar mitochondrial TPP uptake process, and that the effect is mediated at the level of SLC25A19 transcription. Based on these preliminary findings, our working hypothesis in this proposal is that the pancreatic acinar mitochondrial TPP uptake process is a regulated event, that this regulation occurs at both transcriptional and post-transcriptional levels, that unique sequence(s) dictated targeting of MTPPT to mitochondria, and that chronic exposure of pancreatic acinar cells to alcohol and to specific components of CS impairs TPP uptake. Three specific aims are proposed to test this hypothesis. Results of these investigations are expected to provide valuable information regarding physiology/cell biology of an important pancreatic acinar mitochondrial uptake process, information that may also be applicable to other mitochondrial uptake systems. Furthermore, the results are of pathophysiological relevance as they may shed light onto the consequences of exposure of pancreatic acinar cells to common external factors that adversely affect pancreatic health, on mitochondrial physiology.