Mechanisms involved in the inhibitory effect of chronic alcohol exposure on pancreatic acinar thiamin uptake

Abstract

Srinivasan P, Subramanian VS, Said HM. Mechanisms involved in the inhibitory effect of chronic alcohol exposure on pancreatic acinar thiamin uptake. Am J Physiol Gastrointest Liver Physiol 306: G631-G639, 2014. First published February 13, 2014 doi:10.1152/ajpgi.00420.2013.-Pancreatic acinar cells (PAC) obtain thiamin from the circulation via a carrier-mediated process that involves thiamin transporters 1 and 2 (THTR-1 and THTR-2; products of SLC19A2 and SLC19A3, respectively). Chronic alcohol exposure of PAC inhibits thiamin uptake, and, on the basis of in vitro studies, this inhibition appears to be transcriptionally mediated. The aim of this study was to confirm the involvement of a transcriptional mechanism in mediating the chronic alcohol effect in in vivo settings and to delineate the molecular mechanisms involved. Using transgenic mice carrying full-length SLC19A2 and SLC19A3 promoters, we found that chronic alcohol feeding led to a significant reduction in the activity of SLC19A2 and SLC19A3 promoters (as well as in thiamin uptake and expression of THTR-1 and -2). Similar findings were seen in 266-6 cells chronically exposed to alcohol in vitro. In the latter studies, the alcohol inhibitory effect was found to be mediated via the minimal SLC19A2 and SLC19A3 promoters and involved the cis-regulatory elements stimulating protein 1 (SP1)/gut-enriched Kruppel-like factor and SP1-GG-box and SP1/GC, respectively. Chronic alcohol exposure of PAC also led to a significant reduction in the expression of the SP1 transcription factor, which upon correction (via expression) led to the prevention of alcohol inhibitory effects on not only the activity of SLC19A2 and SLC19A3 promoters but also on the expression of THTR-1 and -2 mRNA and thiamin uptake. These results demonstrate that the inhibitory effect of chronic alcohol exposure on physiological/ molecular parameters of thiamin uptake by PAC is mediated via specific cis-regulatory elements in SLC19A2 and SLC19A3 minimal promoters. SLC19A2; SLC19A3; alcohol feeding; thiamin transport; cis-regulatory elements THIAMIN (VITAMIN B1) IS ESSENTIAL for normal metabolic reactions and the health of all cell types, including pancreatic acinar cells (PAC). The vitamin acts as a cofactor for multiple enzymes (transketolase, pyruvate dehydrogenase, ␣-ketoglutarate dehydrogenase, and branched-chain ketoacid dehydrogenase); these enzymes participate in a variety of metabolic reactions related to oxidative energy (sugar) metabolism, ATP production (1, 26), and reduction of cellular oxidative stress (3,14). Thus it is not surprising that cellular thiamin deficiency leads to acute energy failure and to a propensity for oxidative stress (2,3,5,14,26); such a deficiency also leads to functional and structural abnormalities in mitochondria (2).The pancreas is a complex organ with important exocrine and endocrine functions. A variety of disease conditions and factors affect the health of this vital organ, leading to significant morbidity and mortality. The pancreas maintains a high level...

Chronic alcohol exposure inhibits biotin uptake by pancreatic acinar cells: possible involvement of epigenetic mechanisms

“…Mouse primary PAC were isolated from the pancreas of adult mice by the Worthington collagenase type IV (Lakewood, NJ) digestion method as described previously (42)(43)(44). Isolated cells were suspended in K-R buffer, and uptake was performed within 1 h of isolation.…”

“…Mouse-derived pancreatic acinar 266-6 cells were chronically (96 h) exposed to alcohol [50 mM; a concentration that mimics the level of alcohol found in blood of chronic alcoholics (10)] in DMEM growth medium containing 10% FBS as described before (32,44). After 96 h of exposure biotin uptake was measured at 37°C in K-R buffer and then measured for radioactive content by using a scintillation counter as described above.…”

“…Wild-type and transgenic mice carrying the full-length human SLC5A6 promoter fused to the firefly luciferase reporter gene, previously generated and characterized by this laboratory (37), were used (approval for their use was obtained from the Institutional Animal Care Use Committee of the Long Beach VA Medical Center). Mice were fed Lieber-DeCarli ethanol-liquid diet (Dyets, Bethlehem, PA) [ethanol provided 25% of total ingested calories and was introduced gradually; calories were increased by 5% every day until we achieved 25% (25)] for 4 wk as described by us recently (44,46). Control littermates (sex-matched that had similar basal firefly luciferase mRNA expression) were pair-fed with the same liquid diet but without ethanol (maltose-dextrin replaced ethanol isocalorically).…”

“…Whole cell lysate prepared from primary mouse PAC and 266-6 cells chronically fed/exposed to alcohol and their respective controls were used for Western blot analysis as described previously (44,47). The cells were suspended in 200 l of RIPA buffer (Sigma) according to manufacturer's protocol and supplemented with protease inhibitor cocktail (Roche).…”

“…The coding region of mice Slc5a6 gene and ARPO, were PCR amplified using gene-specific primers (Table 1) for quantitative PCR study. Quantitative PCR conditions were same as described previously (44,47). The data were normalized to ARPO and then quantified by a relative relationship method (26).…”