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 #
5R01AA018071-10
Application #
9412397
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
2018-02-01
Budget End
2019-01-31
Support Year
10
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Southern California Institute for Research/Education
Department
Type
DUNS #
622027209
City
Long Beach
State
CA
Country
United States
Zip Code
90822
Subramanian, Veedamali S; Constantinescu, Alexandru R; Benke, Paul J et al. (2017) Mutations in SLC5A6 associated with brain, immune, bone, and intestinal dysfunction in a young child. Hum Genet 136:253-261
Nabokina, Svetlana M; Ramos, Mel Brendan; Said, Hamid M (2016) Mechanism(S) Involved in the Colon-Specific Expression of the Thiamine Pyrophosphate (Tpp) Transporter. PLoS One 11:e0149255
Srinivasan, Padmanabhan; Thrower, Edwin C; Gorelick, Fred S et al. (2016) Inhibition of pancreatic acinar mitochondrial thiamin pyrophosphate uptake by the cigarette smoke component 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Am J Physiol Gastrointest Liver Physiol 310:G874-83
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
Subramanian, Veedamali S; Srinivasan, Padmanabhan; Said, Hamid M (2016) Uptake of ascorbic acid by pancreatic acinar cells is negatively impacted by chronic alcohol exposure. Am J Physiol Cell Physiol 311:C129-35
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
Udhayabanu, Tamilarasan; Subramanian, Veedamali S; Teafatiller, Trevor et al. (2016) SLC52A2 [p.P141T] and SLC52A3 [p.N21S] causing Brown-Vialetto-Van Laere Syndrome in an Indian patient: First genetically proven case with mutations in two riboflavin transporters. Clin Chim Acta 462:210-214
Sassoon, Catherine S; Zhu, Ercheng; Fang, Liwei et al. (2016) Inhibition of Intestinal Thiamin Transport in Rat Model of Sepsis. Crit Care Med 44:e875-81
Moradi, Hamid; Said, Hamid M (2016) Functional thiamine deficiency in end-stage renal disease: malnutrition despite ample nutrients. Kidney Int 90:252-254
Subramanian, Veedamali S; Kapadia, Rubina; Ghosal, Abhisek et al. (2015) Identification of residues/sequences in the human riboflavin transporter-2 that is important for function and cell biology. Nutr Metab (Lond) 12:13

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