Abstract: The balance between fats and carbohydrates in the human diet is still a matter of very active debate. Indeed, the processing of ordinary mixed meals involves complex processes within the lumen of the upper digestive tract for digestion, in the small intestine mucosa for absorption and resecretion, and in peripheral tissues and in the circulation for final handling. The purpose of this review is to focus on available knowledge on the interactions of digestible or indigestible carbohydrates with lipid and lipoprotein metabolism in the postprandial state. The observations made in humans after test meals are reported and interpreted in the light of recent findings on the cellular and molecular levels regarding possible interplays between carbohydrates and lipid moieties in some metabolic pathways. Digestible carbohydrates, especially readily digestible starches or fructose, have been shown to exacerbate and/or delay postprandial lipemia, whereas some fiber sources can lower it. While interactions between dietary fibers and the process of lipid digestion and absorption have been studied mainly in the last decades, recent studies have shown that dietary carbohydrate moieties (e.g., glucose) can stimulate the intestinal uptake of cholesterol and lipid resecretion. In addition to the well-known glucose/fructose transporters, a number of transport proteins have recently been involved in intestinal lipid processing, whose implications in such interactions are discussed. The potential importance of postprandial insulinemia in these processes is also evaluated in the light of recent findings. The interactions of carbohydrates and lipid moieties in the postprandial state may result from both acute and chronic effects, both at transcriptional and posttranscriptional levels.
Abstract: 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.
Abstract: A dose-dependent increase in cholesterol absorption was induced by glucose addition (0-75 mM) to the apical medium of TC7 cells, a well-characterized clone of Caco-2. The uptake into the cells and the secretion rate to the basolateral space were both enhanced by glucose and galactose. This up-regulation was suppressed by SGLT1 inhibition but not by GLUT2 inhibition. Cholesterol cell uptake was significantly decreased by PMA and increased by chelerythrine, with more pronounced changes in the presence of hexoses. Thus, the involvement of a protein kinase C signalling pathway was evidenced in the regulation processes of intestinal cholesterol absorption. In the presence of antibodies directed to hSR-BI cholesterol absorption was reduced by 40% and glucose or galactose no longer enhanced it. We suggest that glucose or galactose, through an interaction with SGLT1, activates a protein kinase C pathway that regulates the activity of one of the intestinal cholesterol transporters, namely hSR-BI.
Abstract: Evidence is now in favor of protein-facilitated mechanisms for the intestinal cholesterol absorption. Here we report that the unesterified cholesterol uptake by rat jejunal brush border membrane vesicles (BBMVs) is efficient, saturable, and protein-mediated. The human apolipoproteins biliary anionic peptide factor (APF) and A-I (apoA-I) up-regulate micellar cholesterol uptake in a dose-dependent manner, but for all tested concentrations (0.1-20 microM), the lipid-free APF was more efficient than apoA-I. This uptake stimulation was suppressed after addition of Pabs directed to the external lipid-binding domain of the CLA-1/SR-BI and reduced by Pabs directed to the external loop of CD36. Thus, CLA-1/SR-BI and to a lesser extent CD36 are involved in the regulation of intestinal cholesterol uptake. APF, the main protein bound to biliary lipids, is likely one of their physiological effectors. As APF is an unesterified cholesterol carrier, it could facilitate the intestinal absorption of biliary cholesterol.
Abstract: OBJECTIVES: In heart transplant recipients with diffuse coronary arteriopathy, we have previously demonstrated the prevalence of elevated homocysteinemia, also known as an independent risk factor for myocardial infarction and stroke. In hyperhomocysteinemic mini-pigs we also observed early detectable pathologic changes in the elastic laminae. We hypothesized that homocysteine causes premature breakdown in the arterial elastic fibers by activation of the elastolytic activities. METHODS: We examined the effect of homocysteine on elastase-like production by smooth muscle cells from sub-inguinal arteries of multi-organ donors (23.4 +/- 3.4 yr, n = 8). The freshly isolated cells were incubated for 0-72 h with homocysteine (0-250 microM), in the presence or absence of specific protease inhibitors. RESULTS: Homocysteine was devoid of a direct effect, but after 18 h incubation the elastase-like activities increased by 5-6-fold in the extracellular medium. The enzymes were characterized as serine proteases. Incubation of cells with a nucleic acid synthesis inhibitor (actinomycin D) or a protein synthesis inhibitor (cycloheximide) suppressed the enzyme induction. CONCLUSIONS: This is the first report of serine protease induction by homocysteine in vascular smooth muscle cells. The process may require protein synthesis and account for the early alterations of the arterial elastic structures in heart transplant recipients, and in other hyperhomocysteinemic patients, as well.
Abstract: In atherosclerotic mini-pigs, we attempted to determine (i) whether high-fat atherogenic diet disturbs the taurocholate transepithelial transport and incorporation in the ileal epithelium mounted in Ussing chambers, and (ii) whether these processes are sensitive to angiotensin converting enzyme (ACE) inhibitors which slow the development of vascular atherosclerosis. In atherosclerotic mini-pigs, the mucosal to serosal transepithelial fluxes were markedly lower (72% inhibition) and free diffusion was more altered than active processes. Taurocholate incorporation into enterocyte (75% inhibition) paralleled the flux reduction. The transport disturbance observed here might be explained by changes in bile salt permeability in relation to alterations of the membrane properties. Taurocholate absorption was lowered by atherogenic diet, whereas bile salts were not trapped in the enterocyte, therefore atherosclerosis-induced alterations preferentially affected the passage through the brush-border. In the ACE inhibitor treated atherosclerotic mini-pigs, perindopril and enalapril similarly inhibited serum ACE activities. Perindopril further corrected taurocholate fluxes by 50% and fully restored taurocholate incorporation. Since enalapril did not restore the atherosclerosis-induced alterations, the involvement of intestinal ACE in bile acid recycling and of an ACE inhibitor class effect on these mechanisms both remain to be ascertained.
Abstract: Previous results from our laboratory showed that a methionine-rich caseinate-based (metcas) diet induces hyperhomocysteinemia in miniature pigs. In the present study, the contribution of the ileal and jejunal methionine absorption to the dietary induced hyperhomocysteinemia was evaluated by measuring the mucosal to serosal fluxes and the enterocyte incorporation in intact intestinal epithelia mounted in Ussing chambers. For 4 mo, 20 miniature pigs were daily fed control or metcas diets, and an oral combination of an angiotensin-converting enzyme inhibitor (25 mg captopril, Cp) and diuretic (12.5 mg hydrochlorothiazide, HTZ) or placebo, ileal incorporation was higher in epithelia from miniature pigs metcas than in that from other groups. For a given transepithelial flux of methionine, i.e., a constant amount of methionine recovered in the serosal chamber, a greater enterocyte incorporation was detected. Cp-HTZ treatment corrected the diet-induced methionine trapping in intestinal epithelia but had little effect in control animals. In separate in vitro experiments, Cp added alone significantly activated methionine fluxes in epithelia from metcas-fed miniature pigs as it did in vivo, demonstrating that Cp rather than HTZ mainly contributed to the in vivo effects of the drug combination. Our results showed that the regulation of intestinal methionine absorption compensated the diet-induced hyperhomocysteinemia and that Cp-HTZ treatment altered these adaptative changes without increasing methioninemia and homocysteinemia.
Abstract: A human liver plasma membrane model for the evaluation of the specific binding and transport processes of drugs presenting high hepatic clearance such as vinca alkaloids was developed. Uptake of the two structural antitumor analogs, navelbine (NVB) and vincristine (VCR), which exhibit wide variabilities in their respective pharmacokinetic parameters and antitumor spectra, was investigated. The high yield, the enzymatic profile and the retention of physiologic transport capacities, as demonstrated by taurocholate uptake, revealed that this membrane preparation was well suited for studies of hepatic drug transport systems. For both drugs two distinct processes were observed: mainly membrane binding and transport. NVB was found to bind to the membrane vesicles more intensively than VCR, but the transport processes were almost identical. However only NVB uptake seems to involve Na(+)-dependent processes. These significant differences may be related to the respective lipophilicity of the drugs. The more lipophilic molecule (NVB) presents the highest uptake, which is presumably at the origin of its greatest distribution volume in vivo.
Abstract: The intestinal epithelial cell layer is the first major barrier to absorption encountered by xenobiotics. An understanding of the mechanism and sites of drug absorption and metabolism is thus a critical first step in developing orally active compounds. In this context human brush-border membrane vesicles obtained from multi-organ donor intestines have been purified. This model has been validated and used to investigate the duodenal absorption of drugs "in vitro", namely digitalis. It is well established that digitalis compounds present great variability in their respective "in vivo" bioavailability in human (60-90% for digoxin, 0% for ouabain). These particular characteristics prompted us to determine whether this membrane model constitutes a suitable tool in predicting the bioavailability or intestinal transport processes of these molecules. The uptake of [3H] digoxin and [3H] ouabain by membrane vesicles incubated in media of increasing osmolarities demonstrated that: i/two factors are involved in the uptake processes of digoxin: membrane binding and intravesicular transport (osmotic sensitive), ii/ for ouabain, no osmotic sensitivity was observed, indicating that no transport process occurred, but only membrane binding processes. These results are in complete agreement with the absolute bioavailability data reported for man "in vivo". This human brush-border model constitutes an interesting approach to the intestinal absorption phenomena which are known to be among the factors influencing the bioavailability of orally administered drugs.
Abstract: Fluorescence polarization of red blood cell (RBC) membranes evaluated using DPH was measured in patients with uncontrolled insulin-dependent diabetes mellitus and in controls. The effect of in vitro addition of pentoxifylline and propentofylline (10(-5) and 10(-4) M) was studied. The fluorescence polarization parameter (P) was lower in the diabetic patients (p = 0.20 +/- 0.03, n = 8) as compared to the controls (p = 0.28 +/- 0.02), n = 8), reflecting an increase in probe mobility in the hydrophobic environment in the depth of the double lipid layer. In vitro addition of pentoxifylline had no effect on the fluorescence parameter of RBC membranes from controls, whereas both concentrations of pentoxifylline studied (p = 0.24 +/- 0.03) significantly increased the fluorescence parameter of RBC membranes from diabetics. Propentofylline had no effect. These findings suggest that the active site responsible for improved membrane fluidity of RBC from diabetics is located on the methyl radical present in the pentoxifylline molecule but not in the propentofylline molecule.
Abstract: Erythrocytes from diabetic patients show abnormal rheology. Pentoxifylline, a methylxanthine, improves the abnormal deformability of diabetic erythrocytes, but its mechanism of action remains unclear. We have studied the effect of pentoxifylline on the lipid order of erythrocyte membranes from controls and patients with Type I diabetes. We studied the structural organization of membrane lipids in individual erythrocyte ghosts by fluorescence polarization using a cell sorter. Fluorescence polarization values (P) for 17 controls (P = 0.244) and 20 diabetic patients (P = 0.215) were significantly different. Pentoxifylline added in vitro had no effect on normal membranes, but significantly increased at 10(-5) mol X l-1 (P = 0.233), and normalized at 10(-4) mol X l-1 (P = 0.243), the P value of membrane ghosts from diabetics.
Abstract: Na+-K+-dependent ouabain-sensitive ATPase and Mg2+-ATPase have been assayed in the erythrocyte membranes of control subjects and in uncontrolled type I (insulin-dependent) diabetics. A decrease in Na+-K+-ATPase activity was observed in the patients that was significantly correlated with glycemia. The Mg2+-ATPase was increased moderately, and no correlation with glycemia was found. To study the in vivo effect of insulin, ATPase activities were measured in uncontrolled diabetics before and after a 24-h continuous insulin perfusion administered by means of an artificial pancreas. ATPase activities were corrected after normalization of glycemia. It therefore seems that glycemia and/or insulinemia are involved in the regulation of erythrocyte Na+-K+ ouabain-sensitive ATPase and to a lesser extent in that of Mg2+-dependent ATPase.
Abstract: Filtrability of erythrocytes obtained from uncontrolled Type 1 (insulin-dependent) diabetic patients is abnormal, but is corrected by insulin added in vivo or in vitro. As erythrocyte filtrability depends on several determinants, we chose to study a membrane property of erythrocytes from diabetic subjects. Membrane fluidity was studied by fluorescence polarization using a lipophilic probe, the diphenyl-hexatriene and the Coulter Epics V together with a laser Spectra-physics 2000. Fluorescence polarization values obtained for 31 normal subjects (0.253 +/- 0.043 SD) and 31 uncontrolled Type 1 diabetic patients (0.231 +/- 0.043 SD) were significantly different (p less than 0.01). Insulin (2.5.10(-9) mol/l) added in vitro increased the fluorescence polarization values of red cell membranes from diabetic patients (without insulin, fluorescence polarization values = 0.210 +/- 0.032 SD; with insulin, fluorescence polarization values = 0.253 +/- 0.024 SD, p less than 0.001, n = 15), but had no effect on normal membranes (without insulin fluorescence polarization values = 0.255 +/- 0.037 SD, with insulin, fluorescence polarization values = 0.251 +/- 0.026 SD; n = 12). Given a relationship between the lipid bilayer and membrane cytoskeleton proteins, this insulin-correctable abnormality of erythrocyte membrane fluidity may be an important determinant of the rheological behaviour of erythrocytes from diabetic patients.
Abstract: Treatment of female rats with ethinylestradiol at a dose of 60 micrograms/rat, daily for 21 days, produced marked changes in red blood cell lipids. Cholesterol was decreased by 22% and total phospholipids were increased by 13%, resulting in a 31% decrease in the cholesterol to phospholipid ratio. The mass distribution of phosphatidylcholine and phosphatidylethanolamine relative to total phospholipids was unchanged. Whereas control red cells incorporated preferentially fatty acids in phosphatidylcholine, ethinylestradiol stimulated their incorporation specifically in phosphatidylethanolamine, where increases occurred with palmitic acid (+75%), oleic acid (+68%) and arachidonic acid (+31%). Incorporation in phosphatidylcholine was unaffected with any of the 3 fatty acids. The stimulation of fatty acid incorporation in phosphatidylethanolamine is likely to reflect an estrogen-dependent increase in turnover rate of fatty acids in this phospholipid. Such alterations in lipid composition and fatty acid incorporation in red cell phospholipids may have significant effects on membrane function.
