Proinflammatory cytokines inhibit thiamin uptake by human and mouse pancreatic acinar cells: involvement of transcriptional mechanism(s)

Abstract

Thiamin (vitamin B1) plays critical roles in normal metabolism and function of all mammalian cells. Pancreatic acinar cells (PACs) import thiamin from circulation via specific carrier-mediated uptake that involves thiamin transporters-1 & -2 (THTR-1 & -2; products of SLC19A2 and SLC19A3, respectively). Our aim in this study was to investigate the effect(s) of pro-inflammatory cytokines on thiamin uptake by PACs. We used human primary (h)PACs, PAC 266-6 cells, and mice in vivo as models in the investigations. First, we examined the level of expression of THTR-1 & -2 mRNA in pancreatic tissues of patients with chronic pancreatitis and observed severe reduction in their expression compared to normal control subjects. Exposing hPACs and PAC 266-6 to pro-inflammatory cytokines (hyper IL-6, TNF-α, and IL-1β) was found to lead to a significant inhibition in thiamin uptake. Focusing on hyper-IL-6 (which also inhibited thiamin uptake by primary mouse PACs), the inhibition in thiamin uptake was found to be associated with significant reduction in THTR-1 & -2 proteins and mRNAs expression as well as in activity of the SLC19A2 and SLC19A3 promoters; it was also associated with reduction in level of expression of the transcription factor Sp1 (which is required for activity of these promoters). Finally, blocking the intracellular Stat3 signaling pathway was found to lead to a significant reversal in the inhibitory effect of hyper IL-6 on thiamin uptake by PAC 266-6. These results show that exposure of PACs to pro-inflammatory cytokines negatively impacts thiamin uptake via (at least in part) transcriptional mechanism(s).

Enterotoxigenic Escherichia coli heat-labile toxin drives enteropathic changes in small intestinal epithelia

“…An equal amount (∼25 μg) of the proteins were loaded on a NuPAGE 4-12% Bris–Tris gradient gels (Invitrogen) as previously described 83 , then blotted onto polyvinylidene difluoride (PVDF) membranes and probed with anti-SLC19A3 (1:1000; Cat# 13407-1-AP; Proteintech), or anti-SP1 (1:1000; Cat# ab124804; Abcam) antibodies and together with anti-beta actin (1:3000; Cat# sc-47778) primary antibodies. The specificity of the SLC19A3 antibodies was validated in our laboratory previously using different approaches that include overexpression of tagged protein or gene silencing 85 . Anti-SP1 antibodies were validated by the manufacturer using knockout cell lysate protein samples.…”

Hypoxia inhibits colonic uptake of the microbiota-generated forms of vitamin B1 via HIF-1α-mediated transcriptional regulation of their transporters

“…The proteins were then blotted onto polyvinylidene difluoride (PVDF) membranes and probed with anti-cTPPT (1:500), anti-THTR-1 (1:1,000), anti-THTR-2 (1: 1,000), anti-HIF-1α (1:500), or anti-HIF-2α (1:500) antibodies, and simultaneously with anti-GAPDH (1: 2,000) primary antibodies. Specificity of the anti-cTPPT, THTR-1, and THTR-2 antibodies were validated in our laboratory previously using different approaches that includes over-expression of tag-protein or gene silencing (53,54); Other antibodies were validated by either the respective company or by other investigators using knockout animals and/or gene knockdown approaches. The immune-reactive bands from the blot were then identified with corresponding antirabbit IR-800 dye (1:30,000) and anti-mouse IR-680 dye (1: 30,000) secondary antibodies incubation for 1 h at room temperature.…”

EnterotoxigenicEscherichia coliheat-labile toxin drives enteropathic changes in small intestinal epithelia

“…An equal amount (∼25 μg) of the proteins were loaded on a NuPAGE 4-12% Bris-Tris gradient gels (Invitrogen) as previously described 82 , then blotted onto polyvinylidene difluoride (PVDF) membranes and probed with anti-SLC19A3 (1:1000; Cat# 13407-1-AP; Proteintech), or anti-SP1 (1:1000; Cat# ab124804; Abcam) 9/27/22 5:07:19 PM antibodies and together with anti-beta actin (1:3000; Cat# sc-47778) primary antibodies. Specificity of the SLC19A3 antibodies was validated in our laboratory previously using different approaches that include overexpression of tagged protein or gene silencing 84 . Anti-SP1 antibodies were validated by the manufacturer using knockout cell lysate protein samples.…”