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.

Public Health Relevance

Vitamin B1 (thiamin) is essential for normal function and health of the pancreas as it plays a critical role in normal cellular oxidative energy metabolism, ATP production, reduction of cellular oxidative stress, and in maintaining normal function/structure of mitochondria. Pancreatic cells cannot synthesize thiamin, and thus, must obtain the vitamin from circulation via transport across cell membrane. Once in the cytoplasm, free thiamin is readily converted to thiamin pyrophosphate (TPP), which is then taken up by mitochondria for utilization. Our studies during the previous funding period have focused on the entry process of thiamin into pancreatic cells, how the process is regulated, and how it is affected by chronic exposure to alcohol. Our focus in the current proposal is on the transport of TPP into pancreatic acinar mitochondria, how the process is being regulated, how the involved mitochondrial TPP transporter finds its way to mitochondria, and on which structural parts of the protein 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) (common external factors that are known to adversely affect pancreatic physiology and health) on mitochondrial TPP uptake process. Results of these investigations should provide novel and valuable information regarding physiology/cell biology of an important pancreatic acinar mitochondrial uptake process; such results may also have implication beyond the current mitochondrial carrier system. The results are also of pathophysiological relevance as they may shed light onto the consequences of exposure of pancreatic acinar cells to external factors that adversely affect pancreatic health, on mitochondrial physiology.

Agency
National Institute of Health (NIH)
Institute
National Institute on Alcohol Abuse and Alcoholism (NIAAA)
Type
Research Project (R01)
Project #
5R01AA018071-07
Application #
8806488
Study Section
Clinical, Integrative and Molecular Gastroenterology Study Section (CIMG)
Program Officer
Gao, Peter
Project Start
2009-05-01
Project End
2019-01-31
Budget Start
2015-02-01
Budget End
2016-01-31
Support Year
7
Fiscal Year
2015
Total Cost
$379,040
Indirect Cost
$94,689
Name
Southern California Institute for Research/Education
Department
Type
DUNS #
622027209
City
Long Beach
State
CA
Country
United States
Zip Code
90822
Sabui, Subrata; Subramanian, Veedamali S; Kapadia, Rubina et al. (2016) Structure-function characterization of the human mitochondrial thiamin pyrophosphate transporter (hMTPPT; SLC25A19): Important roles for Ile(33), Ser(34), Asp(37), His(137) and Lys(291). Biochim Biophys Acta 1858:1883-90
Nabokina, Svetlana M; Subramanian, Veedamali S; Said, Hamid M (2016) The human colonic thiamine pyrophosphate transporter (hTPPT) is a glycoprotein and N-linked glycosylation is important for its function. Biochim Biophys Acta 1858:866-71
Subramanian, Veedamali S; Constantinescu, Alexandru R; Benke, Paul J et al. (2016) Mutations in SLC5A6 associated with brain, immune, bone, and intestinal dysfunction in a young child. Hum Genet :
Srinivasan, Padmanabhan; Nabokina, Svetlana; Said, Hamid M (2015) Chronic alcohol exposure affects pancreatic acinar mitochondrial thiamin pyrophosphate uptake: studies with mouse 266-6 cell line and primary cells. Am J Physiol Gastrointest Liver Physiol 309:G750-8
Nabokina, Svetlana M; Ramos, Mel Brendan; Valle, Judith E et al. (2015) Regulation of basal promoter activity of the human thiamine pyrophosphate transporter SLC44A4 in human intestinal epithelial cells. Am J Physiol Cell Physiol 308:C750-7
Subramanian, Veedamali S; Ghosal, Abhisek; Kapadia, Rubina et al. (2015) Molecular Mechanisms Mediating the Adaptive Regulation of Intestinal Riboflavin Uptake Process. PLoS One 10:e0131698
Ghosal, Abhisek; Sabui, Subrata; Said, Hamid M (2015) Identification and characterization of the minimal 5'-regulatory region of the human riboflavin transporter-3 (SLC52A3) in intestinal epithelial cells. Am J Physiol Cell Physiol 308:C189-96
Ghosal, Abhisek; Jellbauer, Stefan; Kapadia, Rubina et al. (2015) Salmonella infection inhibits intestinal biotin transport: cellular and molecular mechanisms. Am J Physiol Gastrointest Liver Physiol 309:G123-31
Srinivasan, Padmanabhan; Thrower, Edwin C; Loganathan, Gopalakrishnan et al. (2015) Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells. PLoS One 10:e0143575
Srinivasan, Padmanabhan; Subramanian, Veedamali S; Said, Hamid M (2014) Mechanisms involved in the inhibitory effect of chronic alcohol exposure on pancreatic acinar thiamin uptake. Am J Physiol Gastrointest Liver Physiol 306:G631-9

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