Thiamin uptake by pancreatic acinar cells: effect of chronic alcohol feeding/exposure

Abstract

Thiamin is important for normal function of pancreatic acinar cells, but little is known about its mechanism of uptake and about the effect of chronic alcohol use on the process. We addressed these issues using freshly isolated rat primary and rat-derived cultured AR42J pancreatic acinar cells as models. Results showed thiamin uptake by both primary and cultured AR42J pancreatic acinar cells to be via a specific carrier-mediated mechanism and that both of the thiamin transporters 1 and 2 (THTR-1 and THTR-2) are expressed in these cells. Chronic alcohol feeding of rats was found to lead to a significant inhibition of carrier-mediated thiamin uptake by pancreatic acinar cells and was associated with a significant reduction in level of expression of THTR-1 and THTR-2 at the protein and mRNA levels. Chronic exposure (96 h) of AR42J cells to alcohol also led to a significant decreased carrier-mediated thiamin uptake, an effect that was associated with a significant decrease in the activity of the human SLC19A2 and SLC19A3 promoters expressed in these cells. We also examined the effect of chronic alcohol feeding of rats on level of expression of key thiamin metabolizing enzymes (thiamin phosphokinase and thiamin pyrophosphatase) as well as on level of expression of the mitochondrial thiamin pyrophosphate transporter of pancreatic acinar cells and observed a significant inhibition in all these parameters. These results demonstrate for the first time that thiamin uptake by pancreatic acinar cells is via a carrier-mediated process and that both the THTR-1 as well as THTR-2 are expressed in these cells. Also, chronic alcohol feeding/exposure inhibits thiamin uptake process and the inhibition is, at least in part, being exerted at the transcriptional level. Furthermore, chronic alcohol feeding also negatively impacts intracellular parameters of thiamin metabolism in pancreatic acinar cells.

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

“…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).…”

“…We examined the effect of chronic exposure of alcohol [50 mM, 96 h (32,44,47)] on the mouse-derived pancreatic acinar 266-6 cells on the initial rate of biotin (5 nM) uptake. The result showed a significant inhibition (P Ͻ 0.01) in carriermediated biotin uptake by cells chronically exposed to alcohol compared with control (no alcohol) cells (Fig.…”

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

“…Using SLC19A2 and SLC19A3 knockout mouse models, we have also shown that both thiamin transporters 1 and 2 (THTR-1 and THTR-2) (products of the SLC19A2 and SLC19A3 genes, respectively) are involved in the vitaminuptake process (22). We showed that chronic alcohol exposure leads to a significant inhibition in thiamin uptake (25) and, on the basis of preliminary in vitro investigations with rat-derived pancreatic acinar AR42J cells, this effect appeared to be mediated at the level of transcription of the SLC19A2 and SLC19A3 genes (25). Our aims in this study were to confirm the involvement of transcriptional mechanisms in mediating the alcohol inhibitory effects on the SLC19A2 and SLC19A3 promoters in an in vivo setting and to delineate the molecular mechanisms involved in this inhibition.…”

“…Transgenic mice carrying the full-length human SLC19A2 (Ϫ2,250 to Ϫ36) and SLC19A3 (Ϫ1,957 to ϩ59) promoters fused to the firefly luciferase reporter gene [previously generated and characterized by this laboratory (11,18)] were used (approval for their use was obtained from the Institutional Animal Care Use Committee of Long Beach VA Medical Center). Mice were fed the LieberDeCarli 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% (8)] for 4 wk as described by us recently (25). 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).…”