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

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.

Mechanism and regulation of folate uptake by pancreatic acinar cells: effect of chronic alcohol consumption.

Folate plays an essential role in one-carbon metabolism, and a relationship exists between methyl group metabolism and pancreatic exocrine function. Little, however, is known about the mechanism(s) and regulation of folate uptake by pancreatic acinar cells and the effect of chronic alcohol use on the process. We addressed these issues using the rat-derived pancreatic acinar cell line AR42J and freshly isolated primary rat pancreatic acinar cells as models. We found [(3)H]folic acid uptake to be 1) temperature and pH dependent with a higher uptake at acidic than at neutral/alkaline pH; 2) saturable as a function of substrate concentration at both buffer pH 7.4 and 6.0; 3) inhibited by folate structural analogs and by anion transport inhibitors at both buffer pH 7.4 and 6.0; 4) trans-stimulated by unlabeled folate; 5) adaptively regulated by the prevailing extracellular folate level, and 6) inhibited by modulators of the cAMP/PKA-mediated pathway. Both the reduced folate carrier (RFC) and the proton-coupled folate transporter (PCFT) were found to be expressed in AR42J and in primary pancreatic acinar cells, as well as in native human pancreas with expression of RFC being higher than PCFT. Chronic alcohol feeding of rats (4 wk; 36% of calories from ethanol) led to a significant decrease in folate uptake by freshly isolated primary pancreatic acinar cells compared with cells from pair-fed controls; this effect was associated with a parallel decrease in the level of expression of RFC and PCFT. These studies reveal that folate uptake by pancreatic acinar cells is via a regulated carrier-mediated process which may involve RFC and PCFT. In addition, chronic alcohol feeding leads to a marked inhibition in folate uptake by pancreatic acinar cells, an effect that is associated with reduction in level of expression of RFC and PCFT.

Pancreatic beta cells and islets take up thiamin by a regulated carrier-mediated process: studies using mice and human pancreatic preparations.

Thiamin is essential for the normal function of the endocrine pancreas, but very little is known about uptake mechanism(s) and regulation by beta cells. We addressed these issues using mouse-derived pancreatic beta-TC-6 cells, and freshly isolated primary mouse and human pancreatic islets. Results showed that thiamin uptake by beta-TC-6 cells involves a pH (but not Na+)-dependent carrier-mediated process that is saturable at both the nanomolar (apparent K(m) = 37.17 +/- 9.9 nM) and micromolar (apparent K(m) = 3.26 +/- 0.86 microM) ranges, cis-inhibited by thiamin structural analogs, and trans-stimulated by unlabeled thiamin. Involvement of carrier-mediated process was also confirmed in primary mouse and human pancreatic islets. Both THTR-1 and THTR-2 were found to be expressed in these mouse and human pancreatic preparations. Maintaining beta-TC-6 cells in the presence of a high level of thiamin led to a significant (P < 0.01) decrease in thiamin uptake, which was associated with a significant downregulation in level of expression of THTR-1 and THTR-2 at the protein and mRNA levels and a decrease in transcriptional (promoter) activity. Modulators of intracellular Ca2+/calmodulin- and protein-tyrosine kinase-mediated pathways also altered thiamin uptake. Finally, confocal imaging of live beta-TC-6 cells showed that clinical mutants of THTR-1 have mixed expression phenotypes and all led to impairment in thiamin uptake. These studies demonstrate for the first time that thiamin uptake by the endocrine pancreas is carrier mediated and is adaptively regulated by the prevailing vitamin level via transcriptional mechanisms. Furthermore, clinical mutants of THTR-1 impair thiamin uptake via different mechanisms.

Involvement of Enterobacter cloacae in the mortality of the fish, Mugil cephalus.

AIMS: To identify the causative agent of the mortality in the fish, Mugil cephalus, in Muttukadu lagoon. METHODS AND RESULTS: An enteric bacterium from the kidneys of moribund fish M. cephalus, was isolated and identified as Enterobacter cloacae (MK). Mugil cephalus was experimentally infected by this isolate and was re-isolated from the kidneys of the moribund fish. Enterobacter cloacae isolates from the lagoon water (MW1, MW2 and reference strain ATCC 13047) and the reference strain were not able to induce similar pathogenesis. The putative factor imparting pathogenicity to the MK isolate was identified as a cationic molecule, which migrated towards the cathode on agarose gel electrophoresis. CONCLUSIONS: The Ent. cloacae (MK) isolate harbouring a cationic factor was the causative agent for the mortality of M. cephalus, found in Muttukadu lagoon. SIGNIFICANCE AND IMPACT OF THE STUDY: This study reveals that human enteric bacteria MK which is considered as nonpathogenic to fish, may become pathogenic to fish when it harbours this cationic factor. This cationic factor is found to be pathogenic to the fish M. cephalus leading to mortality. It was also found to be pathogenic to mice. Therefore, the shuttling of Ent. cloacae, harbouring cationic factor, between human and fish may be of human health importance.