18 research outputs found
Biochemical and Molecular Mechanisms of Folate Transport in Rat Pancreas; Interference with Ethanol Ingestion
Folic acid is an essential nutrient that is required for one-carbon biosynthetic processes and for methylation of biomolecules. Deficiency of this micronutrient leads to disturbances in normal physiology of cell. Chronic alcoholism is well known to be associated with folate deficiency which is due, in part to folate malabsorption. The present study deals with the mechanistic insights of reduced folate absorption in pancreas during chronic alcoholism. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20% solution) orally for 3 months and the mechanisms of alcohol associated reduced folate uptake was studied in pancreas. The folate transport system in the pancreatic plasma membrane (PPM) was found to be acidic pH dependent one. The transporters proton coupled folate transporter (PCFT) and reduced folate carrier (RFC) are involved in folate uptake across PPM. The folate transporters were found to be associated with lipid raft microdomain of the PPM. Ethanol ingestion decreased the folate transport by reducing the levels of folate transporter molecules in lipid rafts at the PPM. The decreased transport efficiency of the PPM was reflected as reduced folate levels in pancreas. The chronic ethanol ingestion led to decreased pancreatic folate uptake. The decreased levels of PCFT and RFC expression in rat PPM were due to decreased association of these proteins with lipid rafts (LR) at the PPM
Western blots analysis of pancreatic tissue lysate (a) using anti RFC (58 kDa), anti PCFT (54 kDa) antibodies (b) Graph represents summary data of densitometric analysis, lane 1,2: Control; 3,4: ethanol fed.
<p>Western blot analysis of the PPMV (c) using anti RFC (58 kDa), anti PCFT (54 kDa) antibodies (d) Graph represents summary data of densitometric analysis. Data are expressed are means± SD of 4 separate experiments. Lane 1–3: Control; 4–6: ethanol fed. <sup>**</sup>p<0.01, <sup>***</sup>p<0.001 vs. Control.</p
Uptake of 5-[<sup>14</sup>C]-MethylTHF in the rat PPMV as a function of pH optimum.
<p>Uptake was measured by varying the pH of incubation buffer [100 mM NaCl, 80 mM mannitol, 10 mM HEPES, 10 mM 2-morpholinoethanesulfonic acid from 5.0 to 8.0, keeping intravesicular pH 7.4 at 0.5 µM substrate concentration for 10 sec. Each data point is mean± SD of 4 separate uptake determinations. *p<0.05, **p<0.01, ***p<0.001 vs. control.</p
HRM analysis of PCFT and RFC gene (a) upper panel PCFT lower panel RFC, upper/lower panel left side melting profile and upper panel/lower panel right normalized melting curves.
<p>Curves in green; Control, red; Ethanol fed and gray; 100% methylated. (b) Percentage change in methylation of genes in ethanol fed rats compared to control. <sup>***</sup>p<0.001 vs. Control.</p
Association of folate transporters (PCFT & RFC) proteins with lipid rafts in pancreatic apical membrane.
<p>The pancreatic plasma membrane vesicles were subjected to floatation on Optiprep density gradients, and fractions were collected from top of the gradients (fractions 1–4 represent detergent-resistant membrane). Fractions were separated by electrophoresis and analyzed by Western blotting using a) anti-PCFT (54 kDa) and b) RFC (58 kDa) antibodies. Blots were scanned, and the intensity of bands was determined by densitometric analysis. Data are means±SD of 4 separate experiments. The representative blot shown for PCFT and RFC expression as, lane 1–5: Control; lane 1′–5′: ethanol fed. <sup>*</sup>p<0.05<sup> **</sup>p<0.01, <sup>***</sup>p<0.001 vs. Control.</p
Uptake of 5-[<sup>14</sup>C]-methylTHF (0.5 µM) was measured with and without analog (5 µM folic acid & 5 µM methotrexate)/inhibitor {5 mM thymine pyrophosphate (TPP) & 25 µM hemin} in incubation buffer of pH 5.0.
<p>Bars are mean ±SD of 4 separate uptake determinations.</p
Folate uptake in pancreatic plasma membrane vesicles.
<p>Uptake of 5-[<sup>14</sup>C]-MethylTHF (0.5 µM) was measured in a buffer of pH 5.0 [100 mM NaCl, 80 mM mannitol, 10 mM HEPES, 10 mM 2-morpholinoethanesulfonic acid pH 5.0] for 10. Each point represents the mean±S.D. of four determinations. **p<0.01 vs. Control.</p
Immunohistochemical analysis of rat pancreas sections exposed to anti-PCFT and anti-RFC antibodies showing relative localization of PCFT (a&b) and RFC (c&d) protein.
<p>Figures (100x) shown are representative of each group.</p