Abstract
The mammalian small intestine contains two related cellular retinol-binding proteins, CRBP and CRBP II, which are thought to have distinct functions. The human intestinal cell line, Caco-2, was used as a model system for testing the hypothesis that intracellular levels of these proteins directly modulate the absorption and subsequent metabolism of retinol in enterocytes. Immunoblot and Northern blot hybridization demonstrated that Caco-2 cells express CRBP II and cellular retinoic acid-binding protein I in increasing amounts as the cells become more differentiated but do not express detectable quantities of CRBP. Stably transfected cloned Caco-2 cell lines that over-express CRBP II or coexpress CRBP and CRBP II and a control cell line that contains the expression vector without an insert were established. Retinol uptake and retinyl ester synthesis were increased up to 2-fold by coexpression of CRBP or over-expression of CRBP II. No significant differences were detected in the pattern of retinyl esters synthesized from exogenous [3H]retinol. This suggests that the fatty acid pools utilized for retinol esterification were the same despite differences in the CRBP and CRBP II phenotype of the cell lines. There were no differences between apical and basolateral [3H]retinol uptake or metabolism for filter grown transfected or wild type cell monolayers. Thus, neither CRBP II nor CRBP appear to preferentially interact with luminal- or plasma-derived retinol. Notably, in a cell line which over-expressed CRBP, endogenous CRBP II was reduced by 5-10-fold compared with the wild type and control cell lines. These studies indicate that CRBP and CRBP II levels are determinants of intracellular retinol accumulation and esterification, and they suggest that CRBP-bound retinol or a metabolite can regulate the expression of CRBP II in the mammalian intestine.
Highlights
The mammalian small intestine contains two relatedase which uses the sn-1 fattyacid of phosphatidylcholine as cellular retinol-binding proteins, Cellular retinol-binding protein (CRBP) and CRBP11, the fatty acid donor [1,2,3] or by microsomal acyl-CoA:retinol which are thought to have distinct functions
Many of the fundamentalprocesses by which the mammalian small intestine assimilates retinoids and carotenhoaidvse notbeen fully characterized.Inparticularlittleis known about the mechanism of retinol absorption, the metabolism cell line that contains thexepression vector withoutan of carotenoids, the targetingof retinoids to sitesof metabolic insert were established.Retinol uptakeandretinyl processingwithintheenterocyte,thesynthesis of retinyl estesrynthesiws eriencreasedupto
Intestinal fatty acid metabolism serves as an retinol esterification were the same despite differeanpcpeasrent example of differential metabolism and trafficking in theCRBP and CRBP I1 phenotype of the cell lines. of plasma- and luminal-derived nutrients by the enterocyte
Summary
Media were changed 2 days post-plating and daily cloned into the EcoRI site of pSFFV.neo to produce SFFV.CRBP. Medium conditioned by incubation for 24 h with For construction of the CRBP I1 expression vector, SFFV.CRBPI1, 1-4-day post-confluent Caco-2 monolayers was used for the cloning rat CRBP I1 (rCRBP 11) cDNA [10] was digested with BarnHI, the of stable transfected cell lines. A control cell line, wereremoved and centrifuged at 1000 X g at 4 “C for 10 min to Caco-neo, was cloned following transfection with SFFV-neo without a cDNA insert. Preparation of CellExtracts-Caco-2 monolayers were washed with [16], and substitution of normal rabbit serum produced no specific ice-cold PBS and harvested by scraping.
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