Portal de Periódicos da CAPES

A fluorescent cholesterol analog traces cholesterol absorption in hamsters and is esterified in vivo and in vitro

1999; Elsevier BV; Volume: 40; Issue: 10 Linguagem: Inglês

10.1016/s0022-2275(20)34891-4

1539-7262

Carl P. Sparrow, Sushma Patel, Joanne Baffic, Yu‐Sheng Chao, Melba Hernandez, My‐Hanh Lam, Judy Montenegro, Samuel D. Wright, Patricia A. Detmers,

Antibiotics Pharmacokinetics and Efficacy

The fluorescent cholesterol analog 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (fluoresterol) was characterized as a tool for exploring the biochemistry and cell biology of intestinal cholesterol absorption. Hamsters absorbed fluoresterol in a concentration- and time-dependent manner, with an efficiency of about 15–30% that of cholesterol. Fluoresterol absorption was blocked by compounds known to inhibit cholesterol absorption, implying that fluoresterol interacts with those elements of the normal pathway for cholesterol absorption on which the inhibitors act. Confocal microscopy of small intestinal tissue demonstrated that fluoresterol was taken up by absorptive epithelial cells and packaged into lipoprotein particles, suggesting a normal route of intracellular trafficking. Uptake of fluoresterol was confirmed by biochemical analysis of intestinal tissue, and a comparison of [3H]cholesterol and fluoresterol content in the mucosa suggested that fluoresterol moved through the enterocytes more rapidly than did cholesterol. This interpretation was supported by measurements of fluoresterol esterification in the mucosa. Four hours after hamsters were given fluoresterol and [3H]cholesterol orally, 44% of the fluoresterol in the intestinal mucosa was esterified, compared to 8% of the [3H]cholesterol. Caco-2 cells took up 2- to 5-fold more [3H]cholesterol than fluoresterol from bile acid micelles, and esterified 21–24% of the fluoresterol but only 1–4% of the [3H]cholesterol. Thus fluoresterol apparently interacts with the proteins required for cholesterol uptake, trafficking, and processing in the small intestine.—Sparrow, C. P., S. Patel, J. Baffic, Y-S. Chao, M. Hernandez, M-H. Lam, J. Montenegro, S. D. Wright, and P. A. Detmers. A fluorescent cholesterol analog traces cholesterol absorption in hamsters and is esterified in vivo and in vitro. J. Lipid Res. 1999. 40: 1747–1757. The fluorescent cholesterol analog 22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol (fluoresterol) was characterized as a tool for exploring the biochemistry and cell biology of intestinal cholesterol absorption. Hamsters absorbed fluoresterol in a concentration- and time-dependent manner, with an efficiency of about 15–30% that of cholesterol. Fluoresterol absorption was blocked by compounds known to inhibit cholesterol absorption, implying that fluoresterol interacts with those elements of the normal pathway for cholesterol absorption on which the inhibitors act. Confocal microscopy of small intestinal tissue demonstrated that fluoresterol was taken up by absorptive epithelial cells and packaged into lipoprotein particles, suggesting a normal route of intracellular trafficking. Uptake of fluoresterol was confirmed by biochemical analysis of intestinal tissue, and a comparison of [3H]cholesterol and fluoresterol content in the mucosa suggested that fluoresterol moved through the enterocytes more rapidly than did cholesterol. This interpretation was supported by measurements of fluoresterol esterification in the mucosa. Four hours after hamsters were given fluoresterol and [3H]cholesterol orally, 44% of the fluoresterol in the intestinal mucosa was esterified, compared to 8% of the [3H]cholesterol. Caco-2 cells took up 2- to 5-fold more [3H]cholesterol than fluoresterol from bile acid micelles, and esterified 21–24% of the fluoresterol but only 1–4% of the [3H]cholesterol. Thus fluoresterol apparently interacts with the proteins required for cholesterol uptake, trafficking, and processing in the small intestine.—Sparrow, C. P., S. Patel, J. Baffic, Y-S. Chao, M. Hernandez, M-H. Lam, J. Montenegro, S. D. Wright, and P. A. Detmers. A fluorescent cholesterol analog traces cholesterol absorption in hamsters and is esterified in vivo and in vitro. J. Lipid Res. 1999. 40: 1747–1757. The process by which cholesterol is absorbed in the intestine and transits in lipoprotein particles to plasma has been well studied, and the major steps in this pathway are understood. Whether from a dietary source or from lipoproteins taken up by the liver, cholesterol is solubilized in the lumen of the small intestine by bile acid micelles (1Wilson M.D. Rudel L.L. Review of cholesterol absorption with emphasis on dietary and biliary cholesterol.J. Lipid Res. 1994; 35: 943-955Google Scholar). Cholesterol is taken up from the micelles by the epithelial cells lining the surface of the intestinal villi. This process occurs predominantly in the jejunum (1Wilson M.D. Rudel L.L. Review of cholesterol absorption with emphasis on dietary and biliary cholesterol.J. Lipid Res. 1994; 35: 943-955Google Scholar), with the bile acids themselves being resorbed by specific transporters in the ileum (2Wong M.H. Oelkers P. Craddock A.L. Dawson P.A. Expression cloning and characterization of the hamster ileal sodium-dependent bile acid transporter.J. Biol. Chem. 1994; 269: 1340-1347Google Scholar). Upon entering the enterocytes, cholesterol is thought to move to the endoplasmic reticulum, where it can be esterified by ACAT and packaged into chylomicrons (1Wilson M.D. Rudel L.L. Review of cholesterol absorption with emphasis on dietary and biliary cholesterol.J. Lipid Res. 1994; 35: 943-955Google Scholar, 3Field F.J. Mathur S.N. Intestinal lipoprotein synthesis and secretion.Prog. Lipid Res. 1995; 34: 185-198Google Scholar). The newly formed lipoprotein particles exit the absorptive cells along their baso-lateral aspects and become available for uptake by the liver and other tissues. The initial interaction of cholesterol with the enterocyte brush border is one that is critical to the uptake process, and yet it is not well understood. Arguments have been presented that a specific cholesterol transporter is not required and that uptake is a passive process (4Grundy S.M. Absorption and metabolism of dietary cholesterol.Annu. Rev. Nutr. 1983; 3: 71-96Scopus (208) Google Scholar). On the other hand, the existence of very potent and specific compounds that inhibit cholesterol absorption implies that there is a specific target protein critical to the process (5DeNinno M.P. McCarthy P.A. Duplantier K.C. Eller C. Etienne J.B. Zawistoski M.P. Bangerter F.W. Chandler C.E. Morehouse L.A. Sugarman E.D. Wilkins R.W. Woody H.A. Zaccaro L.M. Steroidal glycoside cholesterol absorption inhibitors.J. Med. Chem. 1997; 40: 2547-2554Google Scholar, 6Harris W.S. Windsor S.L. Newton F.A. Gelfand R.A. Inhibition of cholesterol absorption with CP-148,623 lowers serum cholesterol in humans.Clin. Pharmacol. Ther. 1997; 61: 385-389Google Scholar, 7Van Heek M. France C.F. Compton D.S. McLeod R.L. Yumibe N.P. Alton K.B. Sybertz E.J. Davis Jr., H.R. In vivo metabolism-based discovery of a potent cholesterol absorption inhibitor, SCH58235, in the rat and Rhesus monkey through the identification of the active metabolites of SCH48461.J. Pharmacol. Exp. Ther. 1997; 283: 157-163Google Scholar). While candidate proteins have been proposed for the role of cholesterol transporter (8Hauser H. Dyer J.H. Nandy A. Vega M.A. Werder M. Bieliauskaite E. Weber F.E. Compassi S. Gemperli A. Boffelli D. Wehrli E. Schulthess G. Phillips M.C. Identification of a receptor mediating absorption of dietary cholesterol in the intestine.Biochemistry. 1998; 37: 17843-17850Google Scholar), the exact identity of such a protein has remained elusive. Cholesterol absorption and trafficking has also been studied in vitro, using cell lines that approximate the characteristics of the absorptive epithelium but that are more amenable to experimental manipulation. The most widely used in vitro model is the Caco-2 cell line (3Field F.J. Mathur S.N. Intestinal lipoprotein synthesis and secretion.Prog. Lipid Res. 1995; 34: 185-198Google Scholar), which was derived from a human intestinal tumor (9Fogh J. Wright W.C. Loveless J.D. Absence of HeLa cell contamination in 169 cell lines derived from human tumors.J. Natl. Cancer Inst. 1977; 58: 209-214Scopus (565) Google Scholar). When allowed to reach confluence and differentiate, these cells take up cholesterol from mixed micelles, and the cholesterol uptake is influenced by the composition of the micelles (10Mackay K. Starr J.R. Lawn R.M. Ellsworth J.L. Phosphatidylcholine hydrolysis is required for pancreatic cholesterol esterase- and phospholipase A2-facilitated cholesterol uptake into intestinal Caco-2 cells.J. Biol. Chem. 1997; 272: 13380-13389Google Scholar). The cholesterol that enters the Caco-2 plasma membrane is transported to the endoplasmic reticulum for packaging into lipoprotein (11Field F.J. Born E. Murthy S. Mathur S.N. Transport of cholesterol from the endoplasmic reticulum to the plasma membrane is constitutive in Caco-2 cells and differs from the transport of plasma membrane cholesterol to the endoplasmic reticulum.J. Lipid Res. 1998; 39: 333-343Google Scholar) by a process that may involve p-glycoproteins (12Metherall J.E. Li H. Waugh K. Role of multidrug resistance P-glycoproteins in cholesterol biosynthesis.J. Biol. Chem. 1996; 271: 2634-2640Google Scholar, 13Field F.J. Born E. Chen H. Murthy S. Mathur S.N. Esterification of plasma membrane cholesterol and triacylglycerol-rich lipoprotein secretion in Caco-2 cells: possible role of p-glycoprotein.J. Lipid Res. 1995; 36: 1533-1543Google Scholar). It has also been suggested that sterol carrier protein-2 participates in cholesterol trafficking (14Billheimer J.T. Reinhart M.P. Intracellular trafficking of sterols.Subcell. Biochem. 1990; 16: 301-331Google Scholar). However, many of the biochemical details of cholesterol absorption and trafficking are still not fully characterized. While the intracellular transit of cholesterol has been traced biochemically and through the use of radioactive probes, no one has directly observed the path of cholesterol absorption in the intestine. A fluorescently labeled molecule would offer the opportunity to make such observations and could prove useful for the identification of additional protein components in the cholesterol transport process. Here we demonstrate that a fluorescent cholesterol analog shares many properties with cholesterol. When given orally to hamsters, it was absorbed into plasma, and absorption was blocked by compounds that block cholesterol absorption. Further, it was esterified by cells both in vivo and in vitro, indicating that it is a substrate for ACAT. We have used this fluorescent probe to directly observe the uptake and movements of cholesterol in the small intestine of hamsters by confocal microscopy. Fluoresterol (22-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-23,24-bisnor-5-cholen-3β-ol) and its oleate ester were purchased from Molecular Probes (Eugene, OR). The structure of fluoresterol is shown in Fig. 1. [1,2-3H]cholesterol (43.5 Ci/mmol) was obtained from New England Nuclear (Boston, MA). The two cholesterol absorption inhibitors L-166,143 (Compound #51 in 5) and L-165,313 (CP-148,623 in 6) (Fig. 2) were synthesized at Merck Research Laboratories, Rahway, NJ. Solvents used in the quantitation of fluoresterol were all HPLC grade (Aldrich). Caco-2 cells were obtained from ATCC (Bethesda, MD). Cell culture supplies were obtained from Sigma (St. Louis, MO) or Gibco (Grand Island, NY). Phosphate-buffered saline (PBS) was purchased from Gibco. Liquid hamster diet was obtained from Bio-Serv (Frenchtown, NJ) as a powdered concentrate whose composition was 54% carbohydrate, 18% protein, 18% fat, 4% ash, 4% water, and 2% fiber by weight. The powder was dissolved in water at a ratio of 1 gram of diet to 3.96 ml of water.Fig. 2.Chemical structures of the cholesterol absorption inhibitors L-166,143 and L-165,313. L-166,143 ((3β,5α,25R)-3-[[4′′,6′′-bis[(2-fluorophenyl)carbamoyl]-β-d-cellobiosyl]oxy]-spirostan-11-one) was described by DeNinno et al. (5DeNinno M.P. McCarthy P.A. Duplantier K.C. Eller C. Etienne J.B. Zawistoski M.P. Bangerter F.W. Chandler C.E. Morehouse L.A. Sugarman E.D. Wilkins R.W. Woody H.A. Zaccaro L.M. Steroidal glycoside cholesterol absorption inhibitors.J. Med. Chem. 1997; 40: 2547-2554Google Scholar) as compound 51. L-165,313 (3β,5α,25R-spirostan-11-one cellobioside) was described by Harris et al. (6Harris W.S. Windsor S.L. Newton F.A. Gelfand R.A. Inhibition of cholesterol absorption with CP-148,623 lowers serum cholesterol in humans.Clin. Pharmacol. Ther. 1997; 61: 385-389Google Scholar) as CP-148,623.View Large Image Figure ViewerDownload (PPT) Male golden Syrian hamsters weighing 100–120 g were purchased from Charles River (Kingston, NY). All animals were given free access to water, fed a commercial rodent chow diet, and housed (5 per box) under a regular 12-h light/12-h dark lighting cycle. All animal study protocols were approved by the Merck Institutional Animal Care and Use Committee. Stock solutions of fluoresterol (1–2 mg/ml) were prepared in 100% ethanol with stirring at 50°C. Appropriate amounts of this ethanol solution were mixed with corn oil (Mazola) in a 25-ml screw-cap glass tube. The mixture was purged with argon, while held at 50°C in a water bath, to evaporate the ethanol. The resulting solution of fluoresterol in corn oil was orally administered either directly or after mixing with liquid hamster diet (Bio-Serv, Frenchtown, NJ). To make the mixture, fluoresterol (2.5 mg/ml) in corn oil was added to liquid diet (0.2 ml corn oil per ml liquid diet) containing 0.5% cholic acid and homogenized for approximately 60 sec in a Polytron PT 3000. In some experiments, the corn oil also contained [3H]cholesterol (200 μg and 1 μCi/hamster). Hamsters were fasted overnight and then orally dosed with fluoresterol and/or [3H]cholesterol. For studies using cholesterol absorption inhibitors, the dose of sterol was preceded by a gavage of 1 ml of 0.25% methylcellulose, with or without compounds. After gavage, the hamsters were returned to their cages and were allowed access to water but were not fed. At the indicated times, the hamsters were killed and weighed. Blood (0.5–1.0 ml) was collected by cardiac puncture and placed in 13 × 100 mm culture tubes containing 10 μl of 0.5 m EDTA. Plasma was collected after centrifugation at 10,000 g for 20 min at 4°C. In some experiments, the jejunum was collected and processed for confocal microscopy as described below. Absorption of [3H]cholesterol in the plasma was quantitated by bleaching 100-μl aliquots of plasma with 25 μl of 30% hydrogen peroxide and then measuring radioactivity in a liquid scintillation counter (15Harwood Jr., H.J. Chandler C.E. Pellarin L.D. Bangerter F.W. Wilkins R.W. Long C.A. Cosgrove P.G. Malinow M.R. Marzetta C.A. Pettini J.L. Savoy Y.E. Mayne J.T. Pharmacologic consequences of cholesterol absorption inhibition: alteration in cholesterol metabolism and reduction in plasma cholesterol concentration induced by the synthetic saponin β-tigogenin cellobioside (CP-88818; tiqueside).J. Lipid Res. 1993; 34: 377-395Google Scholar). Fluoresterol was quantitated in hamster plasma after solvent extraction using a modification of a procedure originally described by Dole (16Dole V.P. A relation between non-esterified fatty acids in plasma and the metabolism of glucose.J. Clin. Invest. 1956; 35: 150-154Google Scholar). Briefly, 50 μl of hamster plasma was mixed with 1.2 ml of isopropanol:heptane:125 mm H2SO4 80:19:16 in water. The precipitated protein was removed by centrifugation, and the supernatant was transferred to disposable polystyrene cuvettes. Fluoresterol fluorescence was measured in a Spex FluoroMax fluorimeter (excitation 465 nm, emission 535 nm). Plasma samples from hamsters never given fluoresterol were used as blanks, and fluoresterol dissolved in the solvent mixture described above was used to create a standard curve. An experiment in which a known amount of exogenous fluoresterol was added to hamster plasma samples prior to fluoresterol measurement confirmed that the solvents used extracted the fluoresterol quantitatively and that there was no significant quenching of fluoresterol by solvent-soluble plasma components. The relative absorption of [3H]cholesterol to fluoresterol was calculated as the ratio of plasma concentration of sterol to the amount of sterol given to the hamster, as follows: (dpm [3H]cholesterol/ml plasma) (dpm [3H ]cholesterol in dose)(ng fluoresterol/ml plasma) /(ng fluoresterol in dose)⋅ To prepare intestinal tissue for confocal microscopy, the jejunum was cut into pieces approximately 4–5 cm long. The pieces were cut lengthwise, then flushed and washed with several changes of cold 0.15 m NaCl to remove intestinal debris. After application of OCT embedding medium (Fisher Scientific, Springfield, NJ) to the external surface, each piece was rolled around a toothpick as an inside-out jelly roll, from the proximal to the distal end. The rolls were removed from the toothpicks, stood on end in OCT, and frozen on liquid nitrogen. Sections (30 μm) were cut in a cryostat, fixed on glass slides in 3.7% formaldehyde in PBS at ambient temperature for 5 min, washed twice (5 min each) in PBS, and mounted in ProLong antifade mounting medium (Molecular Probes, Eugene, OR). Sections were immediately observed on a Nikon Optiphot-2 microscope equipped with a Bio-Rad MRC 1024 scanning laser confocal attachment with a krypton–argon laser. Images were collected using either a 10× or 60× objective, with all settings maintained between samples in the same experiment. No autofluorescence was detectable in samples from hamsters that did not receive fluoresterol. The size of randomly selected fluoresterol droplets observed in the intestinal epithelium was quantitated using an ocular micrometer on the collected images. Mucosa tissue from hamsters dosed with both fluoresterol and [3H]cholesterol was prepared for analysis of sterol content and extent of sterol esterification. The small intestinal mucosa of each hamster was scraped from the underlying muscularis layer and suspended in 5 ml of Dulbecco's PBS. The mucosal suspension was homogenized with a Polytron PT 3000 for 60 sec at setting 14, on ice. Aliquots (100 μl) of the homogenate were counted in a liquid scintillation counter to determine total content of [3H]sterol (expressed as moles). One ml of the homogenate was extracted as described by Bligh and Dyer (17Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar), and the lower phase was analyzed for total fluoresterol content (moles). The same extract was used to determine the amount of [3H]cholesterol or fluoresterol that was esterified. Lipids in the chloroform phase were separated by silica gel TLC using a mobile phase of ether: petroleum ether:acetic acid 50:50:1. Spots corresponding to fluoresterol and its ester were quantitated by scanning the thin-layer plate with a FluorImager 575 (Molecular Dynamics). After this, spots corresponding to cholesterol and cholesteryl ester were identified by iodine staining and scraped for scintillation counting. Sterol esterification was calculated using the values obtained from the TLC plate and is expressed as % of sterol esterified. Separate micelles containing either [3H]cholesterol or fluoresterol were prepared by mixing the components in chloroform and/or methanol solution, evaporating the solvent under argon gas, and redissolving the micelles in Opti-MEM medium without serum. The micelles contained 10 mm taurocholate, 6 mm egg phosphatidylcholine, and either 200 μm [3H]cholesterol (20 Curie/mole) or 200 μm fluoresterol. Each preparation of micelles was sterile filtered, and the concentration of [3H]cholesterol or fluoresterol in the filtrate was determined. Virtually all of the [3H]cholesterol formed filterable micelles, giving a yield that was usually close to 100%. The yield of fluoresterol in micelles varied from 25–100%. By preparing the two sterols in separate micelles, we could therefore assure that an equimolar ratio could be achieved when they were mixed. The final mixed micelles were used the day of preparation. Caco-2 cells were maintained in Opti-MEM (Gibco), 5% fetal calf serum, 1% Serum-Max-3 (Sigma), 1% MEM non-essential amino acids, and 1% MEM vitamins. Caco-2 cells grown to confluence in 6-well plates were incubated with the micelles at 37°C for the indicated times, washed, and harvested by scraping. Aliquots of cell homogenate were used to directly measure total 3H content and to measure total fluoresterol fluorescence after dilution with isopropanol. Esterification of each sterol was determined by the TLC method described above. Hamster intestinal microsomes were prepared as described (18Clark S.B. Tercyk A.M. Reduced cholesterol transmucosal transport in rats with inhibited mucosal acyl coenzyme A:cholesterol acyltransferase and normal pancreatic function.J. Lipid Res. 1984; 25: 148-159Google Scholar), except that microsomes were dispersed in 100 mm potassium phosphate buffer (pH 7.4) containing 1 mm reduced glutathione and a protease inhibitor cocktail obtained from CalBiochem (San Diego, CA) (catalog #539131). These microsomes were used in ACAT assays performed exactly as described by Yang et al. (19Yang H. Cromley D. Wang H. Billheimer J.T. Sturley S.L. Functional expression of a cDNA to human acyl coenzyme A:cholesterol acyltransferase in yeast.J. Biol. Chem. 1997; 272: 3980-3985Google Scholar), except that both [3H]cholesterol and fluoresterol were present at a final concentration of 50 μg/ml each. After 2.5 min at 37°C, the reactions were terminated by addition of chloroform and methanol, lipids were extracted (17Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Google Scholar), and sterol esterification was quantitated as described above. To determine whether fluoresterol was absorbed after oral administraton, we administered fluoresterol to hamsters by gavage and followed its subsequent appearance in plasma. In an initial experiment hamsters received crystalline fluoresterol suspended in water. However, fluoresterol in plasma was hardly detectable (data not shown), suggesting that the crystals were not readily processed to a form that could be absorbed. In order to enhance the bioavailability of the fluoresterol, we dissolved it in corn oil and prepared an emulsion with a standard liquid diet to administer to hamsters by gavage. Absorption of fluoresterol from the emulsion was confirmed in a preliminary experiment to determine an appropriate concentration of fluoresterol to use for subsequent absorption and fluorescent microscopic studies. Increasing concentrations of fluoresterol were given to hamsters, and both blood and small intestines were collected after 3 h. Fluoresterol was readily detected in plasma at this time. Further, absorption was concentration-dependent and linear between 50 and 400 μg/dose (Fig. 3). Cryosectioned intestinal material revealed no detectable fluorescence at the lowest dose (50 μg), but stepwise increases in fluorescence present within the villi were apparent at the higher doses (not shown). Therefore 200–400 μg fluoresterol per hamster was chosen as an optimal dose range for further studies. Absorption of fluoresterol by hamsters was also time-dependent. Hamsters administered 400 μg fluoresterol had detectable fluoresterol in plasma by 30 min, and the concentration increased up to 60 min (Fig. 4). Between 1 and 4 h the plasma fluoresterol concentration increased more gradually, appearing to plateau. Four hours, therefore, represented the best choice of time for further studies using plasma measurements to examine absorption of fluoresterol. On the other hand, times less than 4 h appeared to present the best opportunity to monitor trafficking of fluoresterol in the hamster intestine. Two compounds that inhibit cholesterol absorption were used to verify that the structural changes in the fluorescent analog had little effect on its mechanism of uptake by intestinal epithelial cells. Hamsters were dosed with either L-166,143 or L-165,313 (see Fig. 2 for structures). The hamsters then received both fluoresterol and [3H]cholesterol delivered together in corn oil. Measurement of plasma fluoresterol and [3H]cholesterol content at 4 h showed that the more potent inhibitor, L-166,143, blocked the absorption of both fluoresterol and [3H]cholesterol to a similar extent (Fig. 5A). The second, less potent compound, L-165,313, also blocked absorption of both sterols to a similar extent, although 10-fold more than L-166,143 was required to achieve the same effect (Fig. 5B). For both compounds the dose–response curves were very similar for fluoresterol and cholesterol, suggesting that both are absorbed by the intestinal epithelium by a common mechanism. Thus fluoresterol movement into the hamster small intestine is likely to be representative of the early stages of cholesterol movement.Fig. 5.L-166,143 and L-165,313 inhibit absorption of both cholesterol and fluoresterol in a concentration-dependent manner. A: Hamsters (five per group) were given increasing doses of L-166,143, followed by a mixture of [3H]cholesterol (200 μg and 1 μCi) and fluoresterol (200 μg) in 200 μl corn oil. After 4 h, plasma was collected and both sterols were measured. In control animals that received no L-166,143, the ratio of cholesterol to fluoresterol absorption was 3.6 ± 0.4 (mean ± SEM). B: Hamsters (five per group) were given increasing doses of L-165,313 followed by a mixture of [3H]cholesterol (200 μg and 1 μCi) and fluoresterol (200 μg) in 200 μl corn oil. After 4 h, plasma was collected and both sterols were measured. In control animals receiving no L-165,313, the ratio of cholesterol to fluoresterol absorption was 7.1 ± 2.2 (mean ± SEM).View Large Image Figure ViewerDownload (PPT) Fluoresterol trafficking in vivo was followed by confocal microscopy in frozen sections of the hamster jejunum, the primary site of cholesterol absorption in the small intestine (1Wilson M.D. Rudel L.L. Review of cholesterol absorption with emphasis on dietary and biliary cholesterol.J. Lipid Res. 1994; 35: 943-955Google Scholar). Hamsters were gavaged with 400 μg fluoresterol in liquid diet and killed at times up to 4 h. Sections of jejunum from each time point were observed at low and high magnification. Control hamsters received liquid diet and corn oil only, without fluoresterol, and there was no fluorescence present in the jejunums of these hamsters (not shown). Overall fluorescence from fluoresterol in the jejunum was brightest at 2 h after gavage, offering the most information in low magnification views. Fluoresterol appeared as large, fluorescent droplets, primarily located in the apical cytoplasm of absorptive epithelial cells (Fig. 6). The droplet appearance of the fluoresterol was presumably due to the contemporaneous absorption of lipid from the corn oil that was used as a carrier for the fluoresterol. More diffuse fluorescence was also observed in the cytoplasm surrounding the droplets and the nuclei, but nuclei themselves remained unstained. The occasional goblet cell within the epithelial layer did not take up the probe (arrowheads, Fig. 6). Fluoresterol absorption occurred predominantly in the distal portion of the villi. Fluorescence was much reduced in intensity toward the base of the villi, and no fluorescence was visible within the crypts or the submucosa (bottom right of Fig. 6). Bright fluoresterol-containing dots were also visible within the lamina propria, indicating that fluoresterol was able to transit from the apical to the basal aspect of the absorptive cells and become packaged in lipoproteins. Sections from the time course of fluoresterol absorption viewed at higher magnification revealed that absorption was readily detectable as early as 30 min after gavage (Fig. 7). At this early time point, fluoresterol was visible associated with the brush border of the absorptive epithelial cells (arrowheads, Fig. 7), even in areas of the proximal half of the villi where less absorption was occurring. The fluoresterol within the apical portion of the absorptive epithelial cells was present as multiple, small, fluorescent droplets, with more and larger droplets visible in cells near the tips of the villi. Small fluorescent droplets were present between unlabeled nuclei, suggesting transit of fluoresterol to the baso-lateral membrane. The estimated size range of the droplets was 5–40 μm (n = 50), with the vast majority (80%) of the droplets falling in the 10–20 μm range. Fluoresterol was also present in the lamina propria at 30 min (arrows, Fig. 7), suggesting successful packaging of fluoresterol into lipoprotein particles at this early time. This observation was consistent with the appearance of fluoresterol in plasma by as early as 30 min. Two hours after gavage the gradient of absorption along the villar length remained, with more uptake occurring near the tips (Fig. 8A–C). In addition to the numerous small dots of fluorescence within the cytoplasm of the absorptive cells, there were also more large, fluorescent droplets present than at 30 min. The larger droplets may have resulted from coalescence of small droplets. Many small fluorescent dots were visible toward the lateral and basal aspects of the epithelial layer along the entire length of the villi (Fig. 8A, B, and C), although fluorescence was more concentrated distally than proximally (Fig. 8A, compared to 8C). Fluoresterol was also present in the lamina propria as both small and large punctate structures. This time appears to represent a period of peak flux of fluoresterol into and out of the epithelial layer. At 4 h, fluoresterol labeling in the villi was reduced to a very faint signal, although