Comparison of different vehicles to study the effect of tocopherols on gene expression in intestinal cells

Abstract

Recent studies have focused on the ability of tocopherols to regulate gene expression. For such experiments, the methodology used to deliver molecules to the cells is crucial and could lead to different results depending on the vehicle used. The objective of the present study was to compare commonly used tocopherol vehicles (ethanol, BSA and mixed micelles) in terms of toxicity, stabilization of tocopherols, uptake efficiency of tocopherols by cells and effect on gene expression. Lactate dehydrogenase measurements did not reveal cytotoxicity of any of the tested vehicles. Tocopherol recovery measurements showed that approximately 80% of the tocopherol was lost in ethanolic solutions, while only approximately 30% and 10% were lost in BSA and mixed micelles, respectively. After 24 h incubation, Caco-2 cell monolayers treated with mixed micelles exhibited the highest alpha-tocopherol intracellular concentrations (5-times those measured with the two other vehicles). Similar results were obtained with gamma-tocopherol. Vehicles, except mixed micelles that activate the FXR/bile acids signalling pathway, did not affect expression of nuclear receptors involved in lipid metabolism or their target genes. This study establishes mixed micelles as the best vehicle to deliver tocopherols to intestinal cell monolayers in culture.

Vitamin E decreases endogenous cholesterol synthesis and apo-AI-mediated cholesterol secretion in Caco-2 cells

“…For the delivery of tocopherol to cells, mixed micelles, which have similar lipid composition to those found in vivo [19], were prepared as previously described [13,20]. The concentration of tocopherols used in this study corresponds to the physiological concentration of tocopherols present in intestinal lumen [21].…”

“…Experiments were performed as previously described [20]. The sequences of primers used for this study are reported in Supplementary Table 1.…”

Fatty acids affect micellar properties and modulate vitamin D uptake and basolateral efflux in Caco-2 cells

“…The cDNA was synthesized from 1 μg of total RNA using random primers and Moloney murine leukemia virus reverse transcriptase. Real-time quantitative polymerase chain reaction analyses were performed using the Mx3005P Real-Time PCR System (Stratagene, La Jolla, CA) as previously described [22]. The primers used were as follows: MTTP: 5′-CATTTCCTCATATTCAGCTTCTGTT-3′ and 5′-AGCTTGTACAGCCGGTCATT-3′; NPC1L1: 5′-GGCAGACCTCCCAAGTCGA-3′ and 5′-ATCCTTGAAGGTGAGCGGG-3′; SR-BI: 5′-CGGCTCGGAGAGCGACTAC-3′ and 5′-GGGCTTATTCTCCATCATCACC-3′; ATP binding cassette A1 (ABCA1): 5′-TCCAGGCCAG-TACGGAATTC-3′ and 5′-TTCCTCGCCAAACCAGTAGG-3′; and 18S, 5′-CGCCGCTAGAGGT-GAAATTCT-3′ and 5′-CATTCTTGGCAAATGCTTTCG-3′.…”

Lycopene inhibits proinflammatory cytokine and chemokine expression in adipose tissue

“…The cDNA was synthesized from 1 μg of total RNA using random primers and Moloney murine leukemia virus reverse transcriptase. Real-time quantitative RT-PCR analyses were performed using the Mx3005P Real-Time PCR System (Stratagene, La Jolla, CA, USA) as previously described [27]. For each condition, expression was quantified in duplicate, and 18S mRNA was used as the endogenous control in the comparative cycle threshold (C T ) method [28].…”