scholarly journals Chronic alcohol exposure negatively impacts the physiological and molecular parameters of the renal biotin reabsorption process

2011 ◽  
Vol 300 (3) ◽  
pp. F611-F617 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Sandeep B. Subramanya ◽  
Hamid M. Said
Keyword(s):  
Transgenic Mice ◽  
Marked Reduction ◽  
Promoter Activity ◽  
Regulatory Region ◽  
Alcohol Exposure ◽  
Chronic Ethanol ◽  
Chronic Alcohol ◽  
Molecular Parameters ◽  
Ethanol Feeding ◽  
Proximal Tubular

Normal body homeostasis of biotin is critically dependent on its renal recovery by kidney proximal tubular epithelial cells, a process that is mediated by the sodium-dependent multivitamin transporter (SMVT; a product of the SLC5A6 gene). Chronic ethanol consumption interferes with the renal reabsorption process of a variety of nutrients, including water-soluble vitamins. To date, however, there is nothing known about the effect of chronic alcohol feeding on physiological and molecular parameters of the renal biotin reabsorption process. We addressed these issues using rats and transgenic mice carrying the human SLC5A6 (P1P2) 5′-regulatory region as an in vivo model systems of alcohol exposure, and cultured human renal proximal tubular epithelial HK-2 cells chronically exposed to alcohol as an in vitro model of alcohol exposure. The [3H]biotin uptake results showed that chronic ethanol feeding in rats leads to a significant inhibition in carrier-mediated biotin transport across both renal brush border and basolateral membrane domains. This inhibition was associated with a marked reduction in the level of expression of SMVT protein, mRNA, and heterogenous nuclear RNA (hnRNA). Furthermore, studies with transgenic mice carrying the SLC5A6 5′-regulatory region showed that chronic alcohol feeding leads to a significant decrease in promoter activity. Studies with HK-2 cells chronically exposed to alcohol again showed a marked reduction in carrier-mediated biotin uptake, which was associated with a significant reduction in promoter activity of the human SLC5A6 5′-regulatory region. These findings demonstrate for the first time that chronic ethanol feeding inhibits renal biotin transport and that this effect is, at least in part, being exerted at the transcriptional level.

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

2014 ◽  
Vol 307 (9) ◽  
pp. G941-G949 ◽  
Author(s):  
Padmanabhan Srinivasan ◽  
Rubina Kapadia ◽  
Arundhati Biswas ◽  
Hamid M. Said
Keyword(s):  
Transgenic Mice ◽  
Chronic Exposure ◽  
Methylation Status ◽  
Acinar Cells ◽  
Alcohol Exposure ◽  
Significant Inhibition ◽  
Pancreatic Acinar Cells ◽  
Chronic Alcohol ◽  
Wild Type ◽  
Chronic Alcohol Exposure

Chronic exposure to alcohol affects different physiological aspects of pancreatic acinar cells (PAC), but its effect on the uptake process of biotin is not known. We addressed this issue using mouse-derived pancreatic acinar 266-6 cells chronically exposed to alcohol and wild-type and transgenic mice (carrying the human SLC5A6 5′-promoter) fed alcohol chronically. First we established that biotin uptake by PAC is Na+ dependent and carrier mediated and involves sodium-dependent multivitamin transporter (SMVT). Chronic exposure of 266-6 cells to alcohol led to a significant inhibition in biotin uptake, expression of SMVT protein, and mRNA as well as in the activity of the SLC5A6 promoter. Similarly, chronic alcohol feeding of wild-type and transgenic mice carrying the SLC5A6 promoter led to a significant inhibition in biotin uptake by PAC, as well as in the expression of SMVT protein and mRNA and the activity of the SLC5A6 promoters expressed in the transgenic mice. We also found that chronic alcohol feeding of mice is associated with a significant increase in the methylation status of CpG islands predicted to be in the mouse Slc5a6 promoters and a decrease in the level of expression of transcription factor KLF-4, which plays an important role in regulating SLC5A6 promoter activity. These results demonstrate, for the first time, that chronic alcohol exposure negatively impacts biotin uptake in PAC and that this effect is exerted (at least in part) at the level of transcription of the SLC5A6 gene and may involve epigenetic/molecular mechanisms.

scholarly journals Inhibition of intestinal biotin absorption by chronic alcohol feeding: cellular and molecular mechanisms

2011 ◽  
Vol 300 (3) ◽  
pp. G494-G501 ◽  
Author(s):  
Sandeep B. Subramanya ◽  
Veedamali S. Subramanian ◽  
Jeyan S. Kumar ◽  
Robert Hoiness ◽  
Hamid M. Said
Keyword(s):  
Alcohol Use ◽  
Transgenic Mice ◽  
Animal Studies ◽  
Molecular Mechanisms ◽  
Basolateral Membrane ◽  
Regulatory Region ◽  
Significant Inhibition ◽  
Water Soluble ◽  
Rat Colon ◽  
Chronic Alcohol

The water-soluble vitamin biotin is essential for normal cellular functions and its deficiency leads to a variety of clinical abnormalities. Mammals obtain biotin from exogenous sources via intestinal absorption, a process mediated by the sodium-dependent multivitamin transporter (SMVT). Chronic alcohol use in humans is associated with a significant reduction in plasma biotin levels, and animal studies have shown inhibition in intestinal biotin absorption by chronic alcohol feeding. Little, however, is known about the cellular and molecular mechanisms involved in the inhibition in intestinal biotin transport by chronic alcohol use. These mechanisms were investigated in this study by using rats and transgenic mice carrying the human full-length SLC5A6 5′-regulatory region chronically fed alcohol liquid diets; human intestinal epithelial Caco-2 cells chronically exposed to alcohol were also used as models. The results showed chronic alcohol feeding of rats to lead to a significant inhibition in carrier-mediated biotin transport events across jejunal brush border and basolateral membrane domains. This inhibition was associated with a significant reduction in level of expression of the SMVT protein, mRNA, and heterogenous nuclear RNA. Chronic alcohol feeding also inhibited carrier-mediated biotin uptake in rat colon. Studies with transgenic mice confirmed the above findings and further showed chronic alcohol feeding significantly inhibited the activity of SLC5A6 5′-regulatory region. Finally, chronic exposure of Caco-2 cells to alcohol led to a significant decrease in the activity of both promoters P1 and P2 of the human SLC5A6 gene. These studies identify for the first time the cellular and molecular parameters of the intestinal biotin absorptive processes that are affected by chronic alcohol feeding.

scholarly journals Identification of a Cytochrome P4502E1/Bid/C1q-dependent Axis Mediating Inflammation in Adipose Tissue after Chronic Ethanol Feeding to Mice

2011 ◽  
Vol 286 (41) ◽  
pp. 35989-35997 ◽  
Author(s):  
Becky M. Sebastian ◽  
Sanjoy Roychowdhury ◽  
Hui Tang ◽  
Antoinette D. Hillian ◽  
Ariel E. Feldstein ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Alcohol Exposure ◽  
Chronic Ethanol ◽  
Adipose Tissue Inflammation ◽  
Wild Type ◽  
Complement Protein ◽  
Apoptotic Pathway ◽  
Tissue Inflammation ◽  
Cytochrome P4502e1 ◽  
Ethanol Feeding

Chronic, heavy alcohol exposure results in inflammation in adipose tissue, insulin resistance, and liver injury. Here we have identified a CYP2E1/Bid/C1q-dependent pathway that is activated in response to chronic ethanol and is required for the development of inflammation in adipose tissue. Ethanol feeding for 25 days to wild-type (C57BL/6J) mice increased expression of multiple markers of adipose tissue inflammation relative to pair-fed controls independent of increased body weight or adipocyte size. Ethanol feeding increased the expression of CYP2E1 in adipocytes, but not stromal vascular cells, in adipose tissue and Cyp2e1−/− mice were protected from adipose tissue inflammation in response to ethanol. Ethanol feeding also increased the number of TUNEL-positive nuclei in adipose tissue of wild-type mice but not in Cyp2e1−/− or Bid−/− mice. Apoptosis contributed to adipose inflammation, as the expression of multiple inflammatory markers was decreased in mice lacking the Bid-dependent apoptotic pathway. The complement protein C1q binds to apoptotic cells, facilitating their clearance and activating complement. Making use of C1q-deficient mice, we found that activation of complement via C1q provided the critical link between CYP2E1/Bid-dependent apoptosis and onset of adipose tissue inflammation in response to chronic ethanol. In summary, chronic ethanol increases CYP2E1 activity in adipose, leading to Bid-mediated apoptosis and activation of complement via C1q, finally resulting in adipose tissue inflammation. Taken together, these data identify a novel mechanism for the development of adipose tissue inflammation that likely contributes to the pathophysiological effects of ethanol.

In vitro and in vivo characterization of the minimal promoter region of the human thiamin transporter SLC19A2

2003 ◽  
Vol 285 (3) ◽  
pp. C633-C641 ◽  
Author(s):  
Jack C. Reidling ◽  
Hamid M. Said
Keyword(s):  
Transgenic Mice ◽  
Mammalian Cells ◽  
Promoter Activity ◽  
Molecular Mechanisms ◽  
Regulatory Region ◽  
Minimal Promoter ◽  
Human Tissues ◽  
Cis Elements

The molecular mechanisms involved in the regulation of thiamin transport in mammalian cells are poorly understood. Previous studies established that a human thiamin transporter, SLC19A2, plays a role in thiamin uptake in human tissues. We cloned the 5′ regulatory region of the SLC19A2 gene, identified the minimal promoter required for basal activity, and located multiple putative cis elements. To further characterize the SLC19A2 promoter, we investigated, in the present study, the role of the putative cis elements in regulating the activity of the SLC19A2 promoter in vitro and confirmed the activity of the SLC19A2 promoter in vivo. In vitro studies demonstrated that mutation of specific cis elements in the SLC19A2 minimal promoter [Gut-enriched Krupple-like factor (GKLF), nuclear factor-1 (NF-1), and stimulating protein-1 (SP-1)] led to a decrease in activity. Using electrophoretic mobility shift assays, four specific DNA/protein complexes were identified. The interacting factors were determined by oligonucleotide competition and antibody supershift analysis and shown to be GKLF, NF-1, and SP-1. Cotransfection studies of the SLC19A2 promoter with an SP-1 containing vector in Drosophila SL2 cells further confirmed a role for SP-1 in regulating SLC19A2 promoter activity. In vivo studies using transgenic mice established the functionality of the full-length and minimal SLC19A2 promoters. Furthermore, our studies revealed that the pattern of expression of the SLC19A2 promoter-Luciferase constructs in transgenic mice was similar to the reported SLC19A2 RNA expression pattern in native human tissues. The results demonstrate the importance of GKLF, NF-1, and SP-1 in regulating the activity of the SLC19A2 promoter and provide direct in vivo confirmation of promoter activity.

scholarly journals Identification and characterization of the minimal 5′-regulatory region of the human riboflavin transporter-3 (SLC52A3) in intestinal epithelial cells

2015 ◽  
Vol 308 (2) ◽  
pp. C189-C196 ◽  
Author(s):  
Abhisek Ghosal ◽  
Subrata Sabui ◽  
Hamid M. Said
Keyword(s):  
Epithelial Cells ◽  
Transgenic Mice ◽  
Promoter Activity ◽  
Intestinal Epithelial Cells ◽  
Regulatory Region ◽  
Regulatory Elements ◽  
Intestinal Epithelial ◽  
Identification And Characterization

The human riboflavin (RF) transporter-3 (product of the SLC52A3 gene) plays an important role in intestinal RF absorption. Our aims in this study were to identify the minimal 5′-regulatory region of the SLC52A3 gene and the regulatory element(s) involved in its activity in intestinal epithelial cells, as well as to confirm promoter activity and establish physiological relevance in vivo in transgenic mice. With the use of transiently transfected human intestinal epithelial HuTu 80 cells and 5′-deletion analysis, the minimal SLC52A3 promoter was found to be encoded between −199 and +8 bp (using the start of the transcription start site as position 1). Although several putative cis-regulatory elements were predicted in this region, only the stimulating protein-1 (Sp1) binding site (at position −74/−71 bp) was found to play a role in promoter activity, as indicated by mutational analysis. Binding of Sp1 to the minimal SLC52A3 promoter was demonstrated by means of EMSA and supershift assays and by chromatin immunoprecipitation analysis. Studies with Drosophila SL2 cells (which lack Sp activity) confirmed the importance of Sp1 in driving the activity of the SLC52A3 minimal promoter; they further showed that Sp3 can also do the activation. Finally, with the use of luciferase gene fusions, the activity of the cloned SLC52A3 promoter was confirmed in vivo in transgenic mice. These studies report, for the first time, on the identification and characterization of the SLC52A3 promoter and also demonstrate the importance of Sp1 in regulating its activity in intestinal epithelial cells.

scholarly journals Effect of chronic alcohol exposure on folate uptake by liver mitochondria

2012 ◽  
Vol 302 (1) ◽  
pp. C203-C209 ◽  
Author(s):  
Arundhati Biswas ◽  
Sundar Rajan Senthilkumar ◽  
Hamid M. Said
Keyword(s):  
Hepg2 Cells ◽  
Mammalian Cells ◽  
Regulatory Region ◽  
Alcohol Exposure ◽  
Significant Inhibition ◽  
Water Soluble ◽  
Transcriptional Level ◽  
Chronic Alcohol ◽  
Mitochondrial Carrier ◽  
Chronic Alcohol Exposure

Mammalian cells obtain folate, a water-soluble vitamin, from their surroundings via transport across cell membrane. Intracellular folate is compartmentalized between the cytoplasm and the mitochondria. Transport of folate from the cytoplasm into the mitochondria is via a specific carrier-mediated process involving the mitochondrial folate transporter (MFT). Chronic alcohol use negatively impacts folate homeostasis, but its effect on mitochondrial folate uptake is not clear. We addressed this issue using mitochondrial preparations isolated from the liver of rats chronically fed an alcohol liquid diet and from human liver HepG2 cells chronically exposed to alcohol. The results showed that chronic alcohol feeding of rats leads to a significant inhibition in mitochondrial carrier-mediated folate uptake. This inhibition was associated with a significant reduction in the level of expression of the MFT protein, mRNA, and heterogenous nuclear RNA (hnRNA). Similarly, chronic alcohol exposure (96 h) of HepG2 cells led to significant inhibition in mitochondrial carrier-mediated folate uptake, which was associated with a marked reduction in the level of expression of the human MFT (hMFT). To determine whether the latter effect is, in part, being exerted at the transcriptional level, we cloned the 5′-regulatory region of the human SLC25A32 gene (which encodes the hMFT) and showed that chronic alcohol exposure of HepG2 cells leads to a significant inhibition in its promoter activity. These studies show for the first time that chronic alcohol feeding/exposure leads to a significant inhibition in mitochondrial carrier-mediated folate uptake and that the inhibition is, in part, being exerted at the level of transcription of the SLC25A32 gene.

scholarly journals Uptake of ascorbic acid by pancreatic acinar cells is negatively impacted by chronic alcohol exposure

2016 ◽  
Vol 311 (1) ◽  
pp. C129-C135 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Padmanabhan Srinivasan ◽  
Hamid M. Said
Keyword(s):  
Ascorbic Acid ◽  
Vitamin C ◽  
Mammalian Cells ◽  
Marked Reduction ◽  
Acinar Cells ◽  
Alcohol Exposure ◽  
Epigenetic Mechanism ◽  
Pancreatic Acinar Cells ◽  
Chronic Alcohol ◽  
Chronic Alcohol Exposure

Vitamin C (ascorbic acid, AA) is indispensable for normal metabolism of all mammalian cells including pancreatic acinar cells (PACs). PACs obtain AA from their surroundings via transport across the cell membrane. Chronic alcohol exposure negatively affects body AA homeostasis; it also inhibits uptake of other micronutrients into PACs, but its effect on AA uptake is not clear. We examined this issue using both in vitro (266-6 cells) and in vivo (mice) models of chronic alcohol exposure. First, we determined the relative expression of the AA transporters 1 and 2 [i.e., sodium-dependent vitamin C transporter-1 (SVCT-1) and SVCT-2] in mouse and human PACs and found SVCT-2 to be the predominant transporter. Chronic exposure of 266-6 cells to alcohol significantly inhibited AA uptake and caused a marked reduction in SVCT-2 expression at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels. Similarly, chronic alcohol feeding of mice significantly inhibited AA uptake and caused a marked reduction in level of expression of the SVCT-2 protein, mRNA, and hnRNA. These findings suggest possible involvement of transcriptional mechanism(s) in mediating chronic alcohol effect on AA uptake by PACs. We also observed significant epigenetic changes (histone modifications) in the Slc23a2 gene (reduction in H3K4me3 level and an increase in H3K27me3 level) in the alcohol-exposed 266-6 cells. These findings show that chronic alcohol exposure inhibits PAC AA uptake and that the effect is mediated, in part, at the level of transcription of the Slc23a2 gene and may involve epigenetic mechanism(s).

scholarly journals Chronic alcohol feeding inhibits physiological and molecular parameters of intestinal and renal riboflavin transport

2013 ◽  
Vol 305 (5) ◽  
pp. C539-C546 ◽  
Author(s):  
Veedamali S. Subramanian ◽  
Sandeep B. Subramanya ◽  
Abhisek Ghosal ◽  
Hamid M. Said
Keyword(s):  
Intestinal Absorption ◽  
Brush Border ◽  
Basolateral Membrane ◽  
Alcohol Exposure ◽  
Significant Inhibition ◽  
Membrane Domains ◽  
Chronic Alcohol ◽  
Molecular Parameters ◽  
High Prevalence ◽  
Chronic Alcohol Exposure

Vitamin B2 (riboflavin, RF) is essential for normal human health. Mammals obtain RF from exogenous sources via intestinal absorption and prevent its urinary loss by reabsorption in the kidneys. Both of these absorptive events are carrier-mediated and involve specific RF transporters (RFVTs). Chronic alcohol consumption in humans is associated with a high prevalence of RF deficiency and suboptimal levels, but little is known about the effect of chronic alcohol exposure on physiological and molecular parameters of the intestinal and renal RF transport events. We addressed these issues using rats chronically fed an alcohol liquid diet and pair-fed controls as a model. The results showed that chronic alcohol feeding significantly inhibits carrier-mediated RF transport across the intestinal brush-border and basolateral membrane domains of the polarized enterocytes. This inhibition was associated with a parallel reduction in the expression of the rat RFVT-1 and -3 at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels. Chronic alcohol feeding also caused a significant inhibition in RF uptake in the colon. Similarly, a significant inhibition in carrier-mediated RF transport across the renal brush-border and basolateral membrane domains was observed, which again was associated with a significant reduction in the level of expression of RFVT-1 and -3 at the protein, mRNA, and hnRNA levels. These findings demonstrate that chronic alcohol exposure impairs both intestinal absorption and renal reabsorption processes of RF and that these effects are, at least in part, mediated via transcriptional mechanism(s) involving the slc52a1 and slc52a3 genes.

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