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Cholesterol membrane transporters scavenger receptor class B type I (SR-BI) and (cluster determinant 36) are involved in intestinal uptake of lutein and beta-carotene, 2 of the 3 main carotenoids of the human diet. The aim of this work was therefore to determine whether SR-BI and NPC1L1 (Niemann-Pick C1-like 1), another cholesterol transporter, are implicated in absorption of lycopene, the 3rd main carotenoid of the human diet. Anti-human SR-BI antibody and block lipid transport 1 (BLT1) (a chemical inhibitor of lipid transport by SR-BI) impaired up to 60% (all-E) and (5Z)-lycopene uptake (P < 0.05) by Caco-2 cell monolayers, which were used as a model of human intestinal epithelium. The involvement of SR-BI in lycopene absorption in vivo was then verified by comparing plasma lycopene concentrations in wild-type and SR-BI transgenic mice that were fed a diet enriched with 0.25 g/kg (all-E)-lycopene for 1 mo. Plasma lycopene concentrations were approximately 10-fold higher (P < 0.001) in mice overexpressing SR-BI in the intestine than in wild-type mice, confirming the involvement of SR-BI in lycopene absorption. Further experiments showed that (all-E)-lycopene did not affect SR-BI mRNA levels in Caco-2 cells or mouse intestine. In contrast to SR-BI, neither anti-human NPC1L1 antibody nor ezetimibe, used as inhibitors of lycopene uptake via NPC1L1, significantly impaired (all-E) or (5Z)-lycopene uptake by Caco-2 monolayers. Thus, the present data show that lycopene absorption is, at least in part, mediated by SR-BI but not by NPC1L1.

Naringin, the main flavanone of grapefruit, was reported to display numerous biological effects: antioxidant, hypocholesteremic, anti-atherogenic and favoring drug absorption. Naringin absorption mechanisms were studied in Caco-2 cells (TC7 clone). We investigated the possible involvement of several membrane transporters implicated in polyphenolic compounds intestinal transport (sodium-dependent glucose transporter 1, monocarboxylate transporter, multidrug-associated resistance proteins 1 and 2, and P-glycoprotein). Naringin was poorly absorbed by Caco-2 cells, according to its low value of apparent permeability coefficient (P(app) = 8.1 +/- 0.9 x 10(-8) cm/s). In the presence of verapamil, a specific inhibitor of P-glycoprotein, cellular uptake was increased by almost threefold after 5 min, and P(app) was doubled after 30 min. Our results indicated the involvement of P-glycoprotein, an ATP-driven efflux pump, capable of transporting naringin from the Caco-2 cell to the apical side. This phenomenon could explain, at least in part, the low absorption of this flavanone at the upper intestinal level.

Vascular smooth muscle cells (VSMCs) activation and hyperplasia, the important etiologic factors in atherosclerosis development, are inhibitable by drugs which donate nitric oxide (NO). We tested the hypothesis that administration of ISDN (60 mg/day), a NO donor, would inhibit development of vascular hyperplasia in atherosclerotic minipigs. VSMC proliferation and NO release by endothelial cells (ECs) were investigated in freshly isolated cells from minipigs fed an atherogenic diet with oral ISDN treatment (n = 8) or without (n = 8), or a control diet (n = 8) for 4 months. In ECs, atherosclerosis depressed by 60% NO release and suppressed the L-arginine negative regulatory feedback of the NO-synthase. Concomitantly, a 4-fold increased proliferation (PCNA labelling) and a 2.3-fold activation (PDGF-BB labelling) were observed in atherosclerotic VSMCs. Long-term treatment of atherosclerotic animals with ISDN restored NO release and regulatory processes of NO synthase from ECs, and reduced by half the prolifer...

In the murine preadipocyte cell line Ob 17, T3 is known to be necessary at an early step of adipose differentiation for the expression of late phenotypes [lipogenic enzymes such as malic enzyme, glycerol-3-phosphate dehydrogenase (GPDH), etc.] and not necessary for the expression of lipoprotein lipase (LPL), which emerges earlier, at growth arrest. These cells contain nuclear T3 receptors, which mainly belong to products of the c-erbA alpha gene and are down-regulated by T3. In this work, retinoic acid (RA) added to Ob 17 cells at growth arrest impaired morphological differentiation and the development of both late (malic enzyme and GPDH) and early (LPL) phenotypes regardless of whether T3 was added. T3 sensitized the cells to the inhibitory action of RA; the ED50 for GPDH activity was shifted from 0.5 microM to 3 nM in cells cultured with 1.5 nM T3. Later addition of RA (6 days after growth arrest) did not inhibit the differentiation. RA also brought out a marked and fast decrease in nuclear T3 receptors. This was observed whatever the stage of cell development and related to both a rapid decrease in the relative abundance of c-erbA alpha-related mRNA species and an increased disappearance rate, suggesting the involvement of pre- and posttranslational events. RA and T3 acted additively in decreasing the T3 receptor and c-erbA alpha mRNA levels. The effects of RA on T3 receptors were rapidly reversed after RA withdrawal; the reversal was large (75%) when RA was introduced at growth arrest and total when introduced later. The cell sensitivity to RA, considering the T3 receptors, was higher at growth arrest (ED50 for RA, 0.2 and 1.5 microM in assays with RA added at growth arrest and 5 days later, respectively). The results suggest intricated regulatory pathways between RA and T3 at an early step of adipose differentiation and also suggest that among different mechanisms through which RA may impair this differentiation, a decreased level of nuclear T3 receptors at an early period should play a role.

In a thyroid hormone-sensitive mouse preadipocyte cell line (Ob 17), the concentration of nuclear T3 receptors increases during differentiation in an insulin-independent manner and decreases by 50-60 p.cent after medium supplementation with physiological concentrations of T3. The down-regulation of T3 receptors implies both quantitative and qualitative changes. The preadipocyte T3 receptors were previously reported to be heterogeneous in gel filtration and in their reactivity towards rabbit antibodies raised against large erb A alpha peptides. This report shows that the receptor heterogeneity is not modified during cell development, whereas T3 mainly depletes the receptor species that are both the most retarded in gel filtration and preferentially recognized by c-erb A alpha-specific antisera. The c-erb A alpha-related T3 receptors which predominate in preadipocytes, are thus probably mainly involved in receptor depletion by T3. A similar T3 receptor half-life of 12-13 h was estimated after cycloheximide addition to cells at confluence or later in the differentiation phase with or without T3. This suggested that development, or T3, might have mainly affected the production of T3 receptors. In Northern hybridization studies, using alpha- or beta-type c-erb A cDNA probes containing the entire coding sequence, only alpha-type mRNAs were detected with a predominant band of 2.8 kbases and two fainter bands of about 5.5 and 6.0 kbases. The alpha-type mRNA abundance relative to beta-actin significantly increased during differentiation and decreased after T3 addition.

International audience; In the human intestinal content after a meal, cholesterol is dispersed in a complex mixture of emulsified droplets, vesicles, mixed micelles and precipitated material. The aim of this study was to determine the contribution of the main intestinal cholesterol transporters (NPC1L1, SR-BI) to the absorption processes, using different cholesterol-solubilizing donors. Cholesterol donors prepared with different taurocholate concentrations were added to an apical medium of differentiated TC7/Caco-2 cells. As the taurocholate concentrations increased, cholesterol donor size decreased (from 712 to 7 nm in diameter), which enhanced cholesterol absorption in a dose-dependent manner (38-fold). Two transport processes were observed: (1) absorption from large donors exhibited low-capacity transport with no noticeable transporter contribution; (2) efficient cholesterol absorption occurs from small lipid donors (≤23 nm diameter), mainly due to NPC1L1 and SR-BI involvement. In addition, bile acids significantly increased mRNA and protein expression of NPC1L1, but not of SR-BI. In conclusion, bile acids present in the intestinal lumen and the micelles enhance intestinal cholesterol transport into the cell by two different regulatory processes: by reducing the lipid donor size, so that small-size mixed micelles can more easily access brush-border membrane transporters, and by increasing the expression level of the enterocyte NPC1L1. These mechanisms could account for the important inter-individual variations observed in cholesterol intestinal absorption

The carotenoid lutein is thought to play a role in the human eye and to protect against age-related macular degeneration. Lutein transport in the human intestine has not been characterized. We examined lutein transport processes using Caco-2 TC-7 monolayers as a model for human intestinal epithelium. Purified lutein was mixed with phospholipids, lysophospholipids, cholesterol, mono-olein, oleic acid and taurocholate to obtain lutein-rich mixed micelles that mimicked those found under physiological conditions. The micelles were added to the apical side of Caco-2 TC-7 cell monolayers for 30 min or 3 h at 37 degrees C. Absorbed lutein, i.e. the sum of lutein recovered in the scraped cells and in the basolateral chamber, was quantified by HPLC. Transport rate was measured (i) as a function of time (from 15 to 60 min), (ii) as a function of micellar lutein concentration (from 1.5 to 15 microM), (iii) at 4 degrees C, (iv) in the basolateral to apical direction, (v) after trypsin pretreatment, (vi) in the presence of beta-carotene and/or lycopene, (vii) in the presence of increasing concentrations of antibody against SR-BI (scavenger receptor class B type 1) and (viii) in the presence of increasing concentrations of a chemical inhibitor of the selective transfer of lipids mediated by SR-BI, i.e. BLT1 (blocks lipid transport 1). The rate of transport of lutein as a function of time and as a function of concentration was saturable. It was significantly lower at 4 degrees C than at 37 degrees C (approx. 50%), in the basal to apical direction than in the opposite direction (approx. 85%), and after trypsin pretreatment (up to 45%). Co-incubation with beta-carotene, but not lycopene, decreased the lutein absorption rate (approx. 20%) significantly. Anti-SR-BI antibody and BLT1 significantly impaired the absorption rate (approx. 30% and 57% respectively). Overall, these results indicate that lutein absorption is, at least partly, protein-mediated and that some lutein is taken up through SR-BI.

OBJECTIVES The aim of this study was to evaluate the therapeutic effects of folic acid in the pig model of hyperhomocysteinemia. BACKGROUND We have previously shown that pigs fed a methionine-rich diet develop hyperhomocysteinemia, arterial lesions and thrombotic events. Elevated homocysteine level is an independent risk factor for atherosclerosis that can be markedly lowered with daily folic acid administration. However, it is not known whether this treatment can prevent arterial lesions. METHODS Three groups of pigs were studied: 8 control subjects received a standard diet; 8 received a methionine-rich diet for four months; 8 received a methionine-rich diet for 1 month and then the methionine-rich diet + 5 mg/day folic acid for 3 months. At month 4 after hemodynamic investigation, all the pigs were sacrificed. RESULTS Control animals developed few usual vascular streaks. All the pigs fed a methionine-rich diet without folic acid treatment developed hyperhomocysteinemia (10.3 ± 1.3 μmol/liter at basal state, 18.2 ± 2.5 μmol/liter at one month and 14.6 ± 3.8 μmol/liter at four months), hemodynamic abnormalities and diffuse arterial lesions with smooth muscle cell hyperplasia, endothelial alterations and elastic lamina dislocation. In this group, one pig died of venous thromboembolism and one of myocardial infarction. The pigs fed a methionine-rich diet + folic acid displayed similar arterial lesions and two had thrombotic events (one myocardial infarction and one pulmonary embolism), despite normalization of homocysteine levels (10.9 ± 1.3 μmol/liter at basal state, 19.5 ± 2.5 μmol/liter at one month and 11.4 ± 3.8 μmol/liter at four months). CONCLUSIONS In the pig model of hyperhomocysteinemia, 5 mg/day folic acid did not prevent arterial lesions or thrombotic events.

An experimental model of simultaneous recording of aortic pressure and flow and vascular diameter at the entrance to a limited territory of systemic circulation has been developed in the rabbit. A system of data acquisitions and treatment has also been perfected to evaluate the different parietal viscoelastic parameters of arterial flow in capacitance and resistance arteries. This model was used to study the pharmacodynamic effects of two vasodilator drugs, isosorbide dinitrate (ISDN) and diltiazem (DILT), administered alone or in association (ISDN + DILT) by slow intravenous infusion over 30 minutes at dosages chosen to induce comparable hypotension of about 10%. Under these conditions, the effects of ISDN, DILT and ISDN + DILT on resistance parameters were slightly different. The ISDN had no marked effect on mean aortic flow (Qm) apart from a slight decrease after 10 minutes infusion (-10.4 +/- 3.8%, p less than 0.05). On the other hand, DILT alone or in association with IDN was associated with a significant increase in Qm (DILT: +8.5 +/- 0.8%, p less than 0.001; ISDN + DILT: +9.7 +/- 2.1%, p less than 0.01). This explains the absence of a significant effect of ISDN on peripheral resistance (PR) or entrance impedance (Zc) of the vascular bed. Conversely, the effects of DILT and ISDN + DILT on these parameters were very marked (DILT:RP = -17.2 +/- 0.9%, p less than 0.001; Zc = -8.2 +/- 0.5%, p less than 0.001; ISDN + DILT: RP = -16 +/- 1.4%, p less than 0.001; Zc = -8.0 +/- 0.4%, p less than 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

In the murine preadipocyte cell line Ob 17, T3 is known as being necessary at an early step of adipose differentiation for the expression of late markers, but not necessary for the expression of early markers. These cells contain nuclear T3 receptors (RT3) which are products of c-erb A alpha proto oncogenes, and are down-regulated by T3. In these cells, retinoic acid (AR) inhibits the adipose differentiation and the expression of early and late markers, and T3 sensitizes the cells to the inhibitory action of AR. We also show that AR down-regulates the expression of RT3 at both pre- and post-translational levels. This inhibition is dependent on AR concentration, a peculiar sensitivity to AR being observed at an early step of development.

The nuclear T3 receptor was characterized in two T3-responsive preadipocyte cell lines cloned from the epididymal fat pads of ob/ob adult mice (ob 17 cells) and their lean counterpart (HGFu cells). Isolated nuclei from confluent or differentiating cells bound [125I]T3 to one class of high affinity sites exhibiting kinetic properties, stereospecificity, and salt extractibility of the nuclear T3 receptor. The solubilized T3 binding sites behave like the hepatic nuclear T3 receptor considering physicochemical and DNA binding properties. At confluence, no significant difference could be detected in equilibrium apparent affinity constant (Ka) and maximum binding capacity (MBC) for T3 whether nuclei were prepared from cells originating from ob/ob mice [Ka: 1.7 +/- (SE) 0.5 X 10(10) M-1; MBC: 432 +/- 29 fmol/mg DNA] or from lean mice (Ka: 1.6 +/- 0.2 X 10(10) M-1; MBC: 487 +/- 39 fmol/mg DNA). MBC values were in the range found in several T3-responsive tissues. This suggests that the primary defect in ob/ob mice is probably not at the level of the nuclear T3 receptor. Furthermore, during differentiation into adipose cells in both cell series, and roughly paralleling the amplifying effect of T3 on several lipogenic enzymes in the course of their development, the nuclear T3 receptor concentration significantly increased, attaining about twice the initial values after completion of the differentiation without any significant change in the affinity for T3.

As previously reported, preadipocytes cloned from the epididymal fat of lean or genetically obese mice (HGFu and ob 17, respectively) contain the nuclear T3 receptor. The number of receptor sites was similar in confluent cells of both lines and approximately doubled during adipocyte differentiation. T3 added to the culture medium increased triacylglycerol synthesis. T3 also increased fatty acid synthase specific activity, relative synthesis rate, and relative mRNA content (1.5- to 2.5-fold). Optimal responses were obtained at 1.5 nM. This study shows that under the same culture conditions in both cell lines, 1.5 nM T3 decreased the receptor concentration with no significant change in the affinity for T3. The receptor depletion was time dependent, rapid, stable in the presence of T3, and reversible in less than 24 h after its withdrawal. Receptor depletion was also dependent on T3 concentration and close to maximum at 1.5 nM T3 [45.1 +/- 2.7% (+/- SE) of the data values without T3; n = 14]. A linear relationship was observed between receptor occupancy by T3 and receptor loss. T4 and triiodothyroacetic acid also decreased the T3 receptor content, as expected from their own affinity for the receptor. These last two observations suggest that the receptor reduction is related to its occupancy by T3. The reported results, also observed in several other cell types, indicate that down-regulation of the nuclear T3 receptor by thyroid hormones is probably a generalized event in T3 target cells at least in vitro. Interpretation of its significance in preadipocyte cell lines requires further studies of rapid nuclear events following T3 receptor occupancy.

Polyclonal antibodies raised in rabbits against bacterially produced peptides in the C-terminal region of v-erb A or human c-erb Aα oncogenes recognize the nuclear triiodothyronine (T3) receptors in the T3-sensitive Ob 17 mouse preadipocyte cell line and not in mouse or rat liver. The results confirm the existence of different T3 receptors in different tissues. The results also suggest a heterogeneous receptor distribution within the preadipose cell line, with a predominance of c-erb Aα-type species. Antibodies raised against domain 149–227, but not against domain 245–325, impair T3 binding, suggesting a role for this domain in ligand binding.

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