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Thiamin (vitamin B1) plays a vital role in cellular energy metabolism/ATP production. Pancreatic acinar cells (PACs) obtain thiamin from circulation and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. TPP is then taken up by the mitochondria via a carrier-mediated process that involves the mitochondrial TPP transporter (MTPPT; encoded by the gene SLC25A19). We have previously characterized different aspects of the mitochondrial carrier-mediated TPP uptake process, but nothing is known about its possible regulation at the posttranscriptional level. We address this issue in the current investigations focusing on the role of miRNAs in this regulation. First, we subjected the human (and rat) 3'-untranslated region (3'-UTR) of the SLC25A19 to three in-silico programs, and all have identified putative binding sites for miR-122-5p. Transfecting pmirGLO-hSLC25A19 3'-UTR into rat PAC AR42J resulted in a significant reduction in luciferase activity compared with cells transfected with pmirGLO-empty vector. Mutating as well as truncating the putative miR-122-5p binding sites in the hSLC25A19 3'-UTR led to abrogation of inhibition in luciferase activity in PAC AR42J. Furthermore, transfecting/transducing PAC AR42J and human primary PACs with mimic of miR-122-5p led to a significant inhibition in the level of expression of the MTPPT mRNA and protein as well as in mitochondrial carrier-mediated TPP uptake. Conversely, transfecting PAC AR42J with an inhibitor of miR-122-5p increased MTPPT expression and function. These findings show, for the first time, that expression and function of the MTPPT in PACs are subject to posttranscriptional regulation by miR-122-5p.NEW & NOTEWORTHY This study shows that the expression and function of mitochondrial TPP transporter (MTPPT) are subject to posttranscriptional regulation by miRNA-122-5p in pancreatic acinar cells.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by Amyloid-β peptide (Aβ) containing plaques and cognitive deficits. The pathophysiology of AD also involves neuroinflammation. Vitamin B1 (thiamin) is indispensable for normal cellular energy metabolism. Thiamin homeostasis is altered in AD, and its deficiency is known to aggravate AD pathology. Little, however, is known about possible alterations in level of expression of thiamin transporters-1 and -2 (THTR-1 and -2) in the brain of AD, and whether pro-inflammatory cytokines affect thiamin uptake by brain cells. We addressed these issues using brain tissue samples [prefrontal cortex (PFC) and hippocampus (HIP)] from AD patients and from 5XFAD mouse model of AD, together with cultured human neuroblastoma SH-SY5Y cells as model. Our results revealed a significantly lower expression of both THTR-1 and THTR-2 in the PFC and HIP of AD patients and 5XFAD mouse model of AD compared to appropriate normal controls. Further, we found that exposure of the SH-SY5Y cells to pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) led to a significant inhibition in thiamin uptake. Focusing on IL-1β, we found the inhibition in thiamin uptake to be time-dependent and reversible; it was also associated with a substantial reduction in expression of THTR-1 (but not THTR-2) protein and mRNA as well as a decrease in promoter activity of the SLC19A2 gene (which encodes THTR-1). Finally, using transcriptomic analysis, we found that thiamin availability in SH-SY5Y cells caused changes in the expression of genes relevant to AD pathways. These studies demonstrate, for the first time, that thiamin transport physiology/molecular biology parameters are negatively impacted in AD brain and that pro-inflammatory cytokines inhibit thiamin uptake by neuroblastoma cells. The results also support a possible role for thiamin in the pathophysiology of AD.

Hypoxia exerts profound effects on cell physiology, but its effect on colonic uptake of the microbiota-generated forms of vitamin B1 (i.e., thiamin pyrophosphate [TPP] and free thiamine) has not been described. Here, we used human colonic epithelial NCM460 cells and human differentiated colonoid monolayers as in vitro and ex vivo models, respectively, and were subjected to either chamber (1% O2, 5% CO2, and 94% N2) or chemically (desferrioxamine; 250 μM)-induced hypoxia followed by determination of different physiological-molecular parameters. We showed that hypoxia causes significant inhibition in TPP and free thiamin uptake by colonic NCM460 cells and colonoid monolayers; it also caused a significant reduction in the expression of TPP (SLC44A4) and free thiamin (SLC19A2 and SLC19A3) transporters and in activity of their gene promoters. Furthermore, hypoxia caused a significant induction in levels of hypoxia-inducible transcription factor (HIF)-1α but not HIF-2α. Knocking down HIF-1α using gene-specific siRNAs in NCM460 cells maintained under hypoxic conditions, on the other hand, led to a significant reversal in the inhibitory effect of hypoxia on TPP and free thiamin uptake as well as on the expression of their transporters. Finally, a marked reduction in level of expression of the nuclear factors cAMP responsive element-binding protein 1 and gut-enriched Krüppel-like factor 4 (required for activity of SLC44A4 and SLC19A2 promoters, respectively) was observed under hypoxic conditions. In summary, hypoxia causes severe inhibition in colonic TPP and free thiamin uptake that is mediated at least in part via HIF-1α-mediated transcriptional mechanisms affecting their respective transporters.

The human colonic thiamin pyrophosphate transporter (hcTPPT) mediates the uptake of the microbiota-generated and phosphorylated form of vitamin B1 (i.e., thiamin pyrophosphate) in the large intestine. Expression of hcTPPT along the absorptive tract is restricted to the large intestine, and the transporter is exclusively localized at the apical membrane domain of the polarized epithelial cells/colonocytes. Previous studies have characterized different physiological/pathophysiological aspects of the hcTPPT system, but nothing is currently known on whether the transporter has interacting partner(s) that affect its physiology/biology. We addressed this issue using a Y2H to screen a human colonic cDNA library and have identified three putative interactors, namely IQGAP-2, SNX-6, and DMXL-1. Focusing on IQGAP-2 (whose expression in human colonocytes is the highest), we found (using fluorescent microscopy imaging and coimmunoprecipitation approaches) the putative interactor to colocalize with hcTPPT and to directly interact with the transporter. Also, overexpressing IQGAP-2 in NCM460 cells and in human primary differentiated colonoid monolayers was found to lead to significant (P < 0.01) induction in TPP uptake, while knocking down (using gene-specific siRNAs) caused significant (P < 0.01 and P < 0.05) decrease in uptake. Furthermore, overexpressing IQGAP-2 in NCM460 cells was found to lead to a significant enhancement in hcTPPT protein stability. Finally, we found the expression of IQGAP-2 to be markedly suppressed in conditions/factors that negatively impact colonic TPP uptake. These results identify the IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and show that this interaction has physiological and biological consequences. NEW & NOTEWORTHY: This study reports on the identification of IQGAP-2 as an interacting partner with the hcTPPT in human colonocytes and how that impacts the transporter's physiology and cell biology. [ABSTRACT FROM AUTHOR]

Pluripotent stem cells are being actively studied as a cell source for regenerating damaged liver. For long-term survival of engrafting cells in the body, not only do the cells have to execute liver-specific function but also withstand the physical strains and invading pathogens. The cellular innate immune system orchestrated by the interferon (IFN) pathway provides the first line of defense against pathogens. The objective of this study is to assess the innate immune function as well as to systematically profile the IFN-induced genes during hepatic differentiation of pluripotent stem cells. To address this objective, we derived endodermal cells (day 5 post-differentiation), hepatoblast (day 15) and hepatocyte-like cells (day 21) from human embryonic stem cells (hESCs). Day 5, 15 and 21 cells were stimulated with IFN-α and subjected to IFN pathway analysis. Transcriptome analysis was carried out by RNA sequencing. The results showed that the IFN-α treatment activated STAT-JAK pathway in differentiating cells. Transcriptome analysis indicated stage specific expression of classical and non-classical IFN-stimulated genes (ISGs). Subsequent validation confirmed the expression of novel ISGs including RASGRP3, CLMP and TRANK1 by differentiated hepatic cells upon IFN treatment. Hepatitis C virus replication in hESC-derived hepatic cells induced the expression of ISGs--LAMP3, ETV7, RASGRP3, and TRANK1. The hESC-derived hepatic cells contain intact innate system and can recognize invading pathogens. Besides assessing the tissue-specific functions for cell therapy applications, it may also be important to test the innate immune function of engrafting cells to ensure adequate defense against infections and improve graft survival.

Humans and mammals obtain vitamin B1 from dietary and gut microbiota sources. A considerable amount of the microbiota-generated vitamin exists in the form of thiamine pyrophosphate (TPP), and colonocytes are capable of absorbing TPP via a specific carrier-mediated process that involves the colonic TPP transporter (cTPPT encoded by SLC44A4). Little is known about the relative contribution of the SLC44A4 transporter toward total colonic carrier-mediated TPP uptake and its role in colon physiology. To address these issues, we generated an Slc44a4 knockout (KO) mouse model (by Cre-Lox recombination) and found a near-complete inhibition in colonic carrier-mediated [3H]TPP uptake in the Slc44a4 KO compared with wild-type (WT) littermates. We also observed a significant reduction in KO mice's body weight and a shortening of their colon compared with WT. Using RNAseq and Ingenuity pathway analysis (IPA) approaches, we found that knocking out the colonic Slc44a4 led to changes in the level of expression of many genes, including upregulation in those associated with intestinal inflammation and colitis. Finally, we found that the Slc44a4 KO mice were more susceptible to the effect of the colitogenic dextran sodium sulfate (DSS) compared with WT animals, a finding that lends support to the recent prediction by multiple genome-wide association studies (GWAS) that SLC44A4 is a possible colitis susceptibility gene. In summary, the results of these investigations show that Slc44a4 is the predominant or only transporter involved in the colonic uptake of TPP, that the transporter is important for colon physiology, and that its deletion increases susceptibility to inflammation. NEW & NOTEWORTHY: This study shows that Slc44a4 is the predominant or only transport system involved in the uptake of the gut microbiota-generated thiamine pyrophosphate (TPP) in the colon and that its deletion affects colon physiology and increases its susceptibility to inflammation. [ABSTRACT FROM AUTHOR]