The intestinal absorption of cholesterol and lipid micronutrients such as vitamin E has been shown to share some common pathways. The present study aims to further compare the uptake of cholesterol ([ 3 H]cholesterol v. 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3-ol (NBD-cholesterol)) and tocopherol in Caco-2 TC-7 cells and in mouse intestine, with special focus on the respective roles of scavenger receptor class B type I (SR-BI) and Niemann-Pick C1-like 1 (NPC1L1). Conversely to NBD-cholesterol, the uptakes of [ 3 H]cholesterol and tocopherol by Caco-2 cells were impaired by both block lipid transport-1 and ezetimibe, which inhibit SR-BI and NPC1L1, respectively. These inhibitions occurred only when cholesterol or tocopherol was delivered to cells included in micelles that contained biliary acid and at least oleic acid as a lipid. In vivo, after 2 h of digestion in mice, the uptake of the two cholesterol analogues and of tocopherol all showed distinct patterns along the duodenum-jejunum axis. [ 3 H]Cholesterol uptake, which correlated closely to NPC1L1 mRNA expression in wild-type (wt) mice, was strongly inhibited by ezetimibe. Intestinal SR-BI overexpression did not change NPC1L1 expression and led to a significant increase in [ 3 H]cholesterol uptake in the distal jejunum. Conversely, neither ezetimibe treatment nor SR-BI overexpression had an effect on NBD-cholesterol uptake. However, in contrast with SR-BI mRNA expression, tocopherol absorption increased strongly up to the distal jejunum in wt mice where it was specifically inhibited by ezetimibe, and was increased in the proximal intestine of intestinal SR-BI-overexpressing mice. Thus, cholesterol and tocopherol uptakes share common pathways in cell culture models, but display different in vivo absorption patterns associated with distinct contributions of SR-BI and NPC1L1.

ABSTRACT:Cattle represent an important source of animal-derived foodproducts; nonetheless, our knowledge about the expression of drug-metabolizing enzymes (DMEs) in present and other foodproducing animals still remains superficial, despite the obvious toxicological consequences. Breed represents an internal factor that modulates DME expression and catalytic activity. In the present work, the effect of breed upon relevant phase I and phase II DMEs was investigated at the pretranscriptional and post-translational levels in male Charolais (CH), Piedmontese (PM) and Blonde d'Aquitaine (BA) cattle. Because specific substrates for cattle have not yet been identified, the breed effect upon specific cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), or glutathione S-transferase (GST) DMEs, in terms of catalytic activity, was determined by using human marker substrates. Among P450s, benzphetamine N-demethylase, 16␤-, 6␤-, and 2␤-testosterone hydroxylase, aniline and p-nitrophenol hydroxylase, and ␣-naphthol and p-nitrophenol UGT activities were significantly higher in CH; in contrast, lower levels of CYP1A1-, CYP1A2-, CYP2B6-, CYP2C9-, CYP2C18-, CYP3A4-, and UGT1A1-like mRNAs were noticed, with CH < PM < BA as a trend. CYP2B and CYP3A mRNA results were confirmed with immunoblotting, too. As regards conjugative DMEs, UGT1A6-like mRNA levels were consistent with respective catalytic activities. Both 1-chloro-2,4-dinitrobenzene and 3,4-dichloronitrobenzene GST activities were higher in BA, and these results agreed with GSTA1-, GSTM1-, and GSTP1-like mRNA amounts. Correlation analysis between catalytic activities and mRNAs showed either significant or uneven results, depending on the substrate. These findings confirm previous data obtained in laboratory species; however, further studies are required to ascribe this behavior to pretranscriptional or post-translational phenomena.Historically, studies concerning drug-metabolizing enzyme (DME) expression and regulation in veterinary food-producing species (e.g., cattle, swine, poultry) have always been considered of lesser interest, if compared with those done in man and laboratory animals. This is rather peculiar. Basically, the DMEs comparative knowledge is useful either to extrapolate pharmacotoxicological data from one species to another or to extend veterinary drug licenses of use from major to minor or exotic species. On the other hand, farm animals are often exposed to pesticides, pollutants, or drugs (sometimes used illicitly to increase growth performances), which are potentially harmful for the animal itself and also for humans, if the consumption of animal edible tissues containing relevant amounts of residues occurs. Consequently, drug metabolism studies, performed in these animals, are of value for the evaluation of the consumer's risk (Sivapathasundaram et al., 2001, This study was supported by the Università degli Studi di Padova (Grants 60A08-8213/05, Studi sull'espressione degli enzimi farmaco-metabolizzanti nel fegato di bovini appartenenti a razze dive...

Intestinal absorption of dietary fat is a complex process mediated by enterocytes leading to lipid assembly and secretion of circulating lipoproteins as chylomicrons, vLDL and intestinal HDL (iHDL). Understanding lipid digestion is of importance knowing the correlation between excessive fat absorption and atherosclerosis. By using time-of-flight secondary ion mass spectrometry (TOF-SIMS), we illustrated a spatio-temporal localization of fat in mice duodenum, at different times of digestion after a lipid gavage, for the first time. Fatty acids progressively increased in enterocytes as well as taurocholic acid, secreted by bile and engaged in the entero-hepatic re-absorption cycle. Cytosolic lipid droplets (CLD) from enterocytes were originally purified separating chylomicron-like, intermediate droplets and smaller HDL-like. A lipidomic quantification revealed their contents in triglycerides, free and esterified cholesterol, phosphatidylcholine, sphingomyelin and ceramides but also in free fatty acids, mono- and di-acylglycerols. An acyl-transferase activity was identified and the enzyme monoacylglycerol acyl transferase 2 (MGAT2) was immunodetected in all CLD. The largest droplets was also shown to contain the microsomal triglyceride transfer protein (MTTP), the acyl-coenzyme A-cholesterol acyltransferases (ACAT) 1 and 2, hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). This highlights the fact that during the digestion of fats, enterocyte CLD contain some enzymes involved in the different stages of the metabolism of diet fatty acids and cholesterol, in anticipation of the crucial work of endoplasmic reticulum in the process. The data further underlines the dual role of chylomicrons and iHDL in fat digestion which should help to efficiently complement lipid-lowering therapy.