CD36 Is Important for Chylomicron Formation and Secretion and May Mediate Cholesterol Uptake in the Proximal Intestine
2006; Elsevier BV; Volume: 131; Issue: 4 Linguagem: Inglês
10.1053/j.gastro.2006.08.012
ISSN1528-0012
AutoresAndromeda M. Nauli, Fatiha Nassir, Shuqin Zheng, Qing Yang, Chun–Min Lo, Sarah B. vonLehmden, Dana Lee, Ronald J. Jandacek, Nada A. Abumrad, Patrick Tso,
Tópico(s)Caveolin-1 and cellular processes
ResumoBackground & Aims: Studies are aimed to determine the role of CD36 in intestinal lipid absorption. Methods: Knock-out (KO) and wild-type (WT) lymph fistula mice were used to study fatty acids (FA) and cholesterol uptake, and chylomicron formation and secretion. Uptake of FA and cholesterol was studied by using sucrose polybehenate and fecal dual isotope methods, respectively. Results: The CD36 KO exhibited significant accumulation of dietary cholesterol in the intestinal lumen at the end of 6-hour lipid infusion and significant reduction of dietary cholesterol transport into the lymph. Fecal dual isotope studies, however, did not show any significant difference in cholesterol uptake, suggesting that given sufficient time, the KO intestine could compensate for the reduced cholesterol uptake observed in the acute lymph fistula studies. Recovery of dietary FA in the intestinal lumen was comparable between WT and KO, consistent with the sucrose polybehenate study. However, the KO mice accumulated more, albeit not significantly, dietary triacylglycerols in the intestine, followed by a significant reduction in lymphatic transport. The ratio of intestinal dietary triacylglycerols to FA was not higher in WT than KO, arguing against impaired lipid esterification. It is rather a deficiency in the formation and secretion of chylomicrons, as supported by the significantly less apolipoprotein B-48 and the smaller, albeit not significantly, lipoprotein particles secreted into the lymph of the KO. Conclusions: CD36 may play an important role in chylomicron formation and secretion and may also facilitate cholesterol uptake in the proximal intestine. Background & Aims: Studies are aimed to determine the role of CD36 in intestinal lipid absorption. Methods: Knock-out (KO) and wild-type (WT) lymph fistula mice were used to study fatty acids (FA) and cholesterol uptake, and chylomicron formation and secretion. Uptake of FA and cholesterol was studied by using sucrose polybehenate and fecal dual isotope methods, respectively. Results: The CD36 KO exhibited significant accumulation of dietary cholesterol in the intestinal lumen at the end of 6-hour lipid infusion and significant reduction of dietary cholesterol transport into the lymph. Fecal dual isotope studies, however, did not show any significant difference in cholesterol uptake, suggesting that given sufficient time, the KO intestine could compensate for the reduced cholesterol uptake observed in the acute lymph fistula studies. Recovery of dietary FA in the intestinal lumen was comparable between WT and KO, consistent with the sucrose polybehenate study. However, the KO mice accumulated more, albeit not significantly, dietary triacylglycerols in the intestine, followed by a significant reduction in lymphatic transport. The ratio of intestinal dietary triacylglycerols to FA was not higher in WT than KO, arguing against impaired lipid esterification. It is rather a deficiency in the formation and secretion of chylomicrons, as supported by the significantly less apolipoprotein B-48 and the smaller, albeit not significantly, lipoprotein particles secreted into the lymph of the KO. Conclusions: CD36 may play an important role in chylomicron formation and secretion and may also facilitate cholesterol uptake in the proximal intestine. The transmembrane protein CD36 is expressed in many cell types, including platelets,1Phillips D.R. Agin P.P. Platelet plasma membrane glycoproteins Evidence for the presence of nonequivalent disulfide bonds using nonreduced-reduced two-dimensional gel electrophoresis.J Biol Chem. 1977; 252: 2121-2126Abstract Full Text PDF PubMed Google Scholar monocytes,2Talle M.A. Rao P.E. Westberg E. Allegar N. Makowski M. Mittler R.S. Goldstein G. Patterns of antigenic expression on human monocytes as defined by monoclonal antibodies.Cell Immunol. 1983; 78: 83-99Crossref PubMed Scopus (129) Google Scholar capillary endothelial cells,3Knowles D.M. Tolidjian B. Marboe C. D’Agati V. Grimes M. Chess L. Monoclonal anti-human monocyte antibodies OKM1 and OKM5 possess distinctive tissue distributions including differential reactivity with vascular endothelium.J Immunol. 1984; 132: 2170-2173PubMed Google Scholar erythroblasts,4Edelman P. Vinci G. Villeval J.L. Vainchenker W. Henri A. Miglierina R. Rouger P. Reviron J. Breton-Gorius J. Sureau C. A monoclonal antibody against an erythrocyte ontogenic antigen identifies fetal and adult erythroid progenitors.Blood. 1986; 67: 56-63Crossref PubMed Google Scholar adipocytes,5Abumrad N.A. el Maghrabi M.R. Amri E.Z. Lopez E. Grimaldi P.A. Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation Homology with human CD36.J Biol Chem. 1993; 268: 17665-17668Abstract Full Text PDF PubMed Google Scholar and intestinal,6Poirier H. Degrace P. Niot I. Bernard A. Besnard P. Localization and regulation of the putative membrane fatty-acid transporter (FAT) in the small intestine Comparison with fatty acid-binding proteins (FABP).Eur J Biochem. 1996; 238: 368-373Crossref PubMed Scopus (174) Google Scholar, 7Chen M. Yang Y. Braunstein E. Georgeson K.E. Harmon C.M. Gut expression and regulation of FAT/CD36: possible role in fatty acid transport in rat enterocytes.Am J Physiol Endocrinol Metab. 2001; 281: E916-E923PubMed Google Scholar, 8Lobo M.V. Huerta L. Ruiz-Velasco N. Teixeiro E. de la Cueva P. Celdran A. Martin-Hidalgo A. Vega M.A. Bragado R. Localization of the lipid receptors CD36 and CLA-1/SR-BI in the human gastrointestinal tract: towards the identification of receptors mediating the intestinal absorption of dietary lipids.J Histochem Cytochem. 2001; 49: 1253-1260Crossref PubMed Scopus (112) Google Scholar mammary, and retinal epithelial cells.9Greenwalt D.E. Watt K.W. So O.Y. Jiwani N. PAS IV, an integral membrane protein of mammary epithelial cells, is related to platelet and endothelial cell CD36 (GP IV).Biochemistry. 1990; 29: 7054-7059Crossref PubMed Scopus (80) Google Scholar In rat intestinal epithelial cells, CD36 is localized in the apical brush border membranes of mainly the duodenum and jejunum.6Poirier H. Degrace P. Niot I. Bernard A. Besnard P. Localization and regulation of the putative membrane fatty-acid transporter (FAT) in the small intestine Comparison with fatty acid-binding proteins (FABP).Eur J Biochem. 1996; 238: 368-373Crossref PubMed Scopus (174) Google Scholar, 7Chen M. Yang Y. Braunstein E. Georgeson K.E. Harmon C.M. Gut expression and regulation of FAT/CD36: possible role in fatty acid transport in rat enterocytes.Am J Physiol Endocrinol Metab. 2001; 281: E916-E923PubMed Google Scholar Using human intestinal tissues, Lobo et al8Lobo M.V. Huerta L. Ruiz-Velasco N. Teixeiro E. de la Cueva P. Celdran A. Martin-Hidalgo A. Vega M.A. Bragado R. Localization of the lipid receptors CD36 and CLA-1/SR-BI in the human gastrointestinal tract: towards the identification of receptors mediating the intestinal absorption of dietary lipids.J Histochem Cytochem. 2001; 49: 1253-1260Crossref PubMed Scopus (112) Google Scholar localized CD36 expression along the proximal brush border membranes of the intestinal epithelium.CD36 binds to a wide range of ligands, such as native and modified lipoproteins,10Endemann G. Stanton L.W. Madden K.S. Bryant C.M. White R.T. Protter A.A. CD36 is a receptor for oxidized low density lipoprotein.J Biol Chem. 1993; 268: 11811-11816Abstract Full Text PDF PubMed Google Scholar, 11Calvo D. Gomez-Coronado D. Suarez Y. Lasuncion M.A. Vega M.A. Human CD36 is a high affinity receptor for the native lipoproteins HDL, LDL, and VLDL.J Lipid Res. 1998; 39: 777-788Abstract Full Text Full Text PDF PubMed Google Scholar anionic phospholipids (PL),12Rigotti A. Acton S.L. Krieger M. The class B scavenger receptors SR-BI and CD36 are receptors for anionic phospholipids.J Biol Chem. 1995; 270: 16221-16224Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar cholesterol,13Werder M. Han C.H. Wehrli E. Bimmler D. Schulthess G. Hauser H. Role of scavenger receptors SR-BI and CD36 in selective sterol uptake in the small intestine.Biochemistry. 2001; 40: 11643-11650Crossref PubMed Scopus (81) Google Scholar and fatty acids (FA).5Abumrad N.A. el Maghrabi M.R. Amri E.Z. Lopez E. Grimaldi P.A. Cloning of a rat adipocyte membrane protein implicated in binding or transport of long-chain fatty acids that is induced during preadipocyte differentiation Homology with human CD36.J Biol Chem. 1993; 268: 17665-17668Abstract Full Text PDF PubMed Google Scholar Accumulated evidence from studies conducted both in vitro14Ibrahimi A. Sfeir Z. Magharaie H. Amri E.Z. Grimaldi P. Abumrad N.A. Expression of the CD36 homolog (FAT) in fibroblast cells: effects on fatty acid transport.Proc Natl Acad Sci U S A. 1996; 93: 2646-2651Crossref PubMed Scopus (202) Google Scholar, 15Pohl J. Ring A. Korkmaz U. Ehehalt R. Stremmel W. FAT/CD36-mediated long-chain fatty acid uptake in adipocytes requires plasma membrane rafts.Mol Biol Cell. 2005; 16: 24-31Crossref PubMed Scopus (140) Google Scholar and in vivo16Febbraio M. Guy E. Coburn C. Knapp Jr, F.F. Beets A.L. Abumrad N.A. Silverstein R.L. The impact of overexpression and deficiency of fatty acid translocase (FAT)/CD36.Mol Cell Biochem. 2002; 239: 193-197Crossref PubMed Scopus (70) Google Scholar supports an important role for CD36 in facilitating FA uptake by adipose and muscle tissues. Its role in intestinal lipid uptake is less clear. In support of such a role are the high levels of CD36 and its expression pattern along the brush border membrane of the proximal intestine, which is typical of proteins implicated in lipid uptake.6Poirier H. Degrace P. Niot I. Bernard A. Besnard P. Localization and regulation of the putative membrane fatty-acid transporter (FAT) in the small intestine Comparison with fatty acid-binding proteins (FABP).Eur J Biochem. 1996; 238: 368-373Crossref PubMed Scopus (174) Google Scholar, 7Chen M. Yang Y. Braunstein E. Georgeson K.E. Harmon C.M. Gut expression and regulation of FAT/CD36: possible role in fatty acid transport in rat enterocytes.Am J Physiol Endocrinol Metab. 2001; 281: E916-E923PubMed Google ScholarA recent study by Drover et al17Drover V.A. Ajmal M. Nassir F. Davidson N.O. Nauli A.M. Sahoo D. Tso P. Abumrad N.A. CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the blood.J Clin Invest. 2005; 115: 1290-1297Crossref PubMed Scopus (186) Google Scholar showed that CD36 knock-out (KO) mice did not exhibit reduced intestinal FA uptake, but had impairments in lymph triacylglcyerol (TG) secretion and in clearance of blood chylomicrons. Whether or not CD36 plays a significant role in intestinal cholesterol uptake and transport in TG-rich lipoproteins in vivo remains unknown. A role in cholesterol uptake has been suggested by the report that CD36 bound cholesterol and that CD36 antibodies inhibited cholesterol uptake by brush border membranes.13Werder M. Han C.H. Wehrli E. Bimmler D. Schulthess G. Hauser H. Role of scavenger receptors SR-BI and CD36 in selective sterol uptake in the small intestine.Biochemistry. 2001; 40: 11643-11650Crossref PubMed Scopus (81) Google ScholarThe goal of this study is to further study the role of CD36 in intestinal uptake and transport of cholesterol and TG. Using the well-established conscious lymph fistula model, we explored the effect of CD36 deficiency on formation and secretion of chylomicrons. We also examined whether disruption of the CD36 gene would lead to an overall reduction in intestinal FA and cholesterol uptake by using sucrose polybehenate (SPB)18Jandacek R.J. Heubi J.E. Tso P. A novel, noninvasive method for the measurement of intestinal fat absorption.Gastroenterology. 2004; 127: 139-144Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar and fecal dual-isotope methods, respectively.Materials and MethodsMaterialsTriolein, cholesterol, egg phosphatidylcholine (PC), and sodium taurocholate (NaTC) were purchased from Sigma (St. Louis, MO). The radioactive [9,10-3H(N)] triolein (labels were on the 3 FA molecules, not on the glycerol moiety) and [4-14C] cholesterol were purchased from New England Nuclear (Boston, MA). Silica gel 60 plates were purchased from Fisher Scientific (Pittsburgh, PA) and were activated before use.AnimalsCD36 wild-type (WT) and KO mice were generated as described previously.17Drover V.A. Ajmal M. Nassir F. Davidson N.O. Nauli A.M. Sahoo D. Tso P. Abumrad N.A. CD36 deficiency impairs intestinal lipid secretion and clearance of chylomicrons from the blood.J Clin Invest. 2005; 115: 1290-1297Crossref PubMed Scopus (186) Google Scholar Animals were maintained on regular chow diet under a 12-hour light/12-hour dark cycle at the University of Cincinnati Laboratory Animal Medical Services. Only 4- to 12-month-old male animals were used in our studies.SPB MethodThe SPB method was developed and validated by Jandacek et al.18Jandacek R.J. Heubi J.E. Tso P. A novel, noninvasive method for the measurement of intestinal fat absorption.Gastroenterology. 2004; 127: 139-144Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar It is a noninvasive method for studying the uptake of FA. The animals are fed a diet containing TG and other essential nutrients. The method relies on measuring the ratio of fecal FA versus fecal nonabsorbable fat marker. In this study, the uptake of total dietary FA (mass) was measured against olestra, the nonabsorbable fat marker. Therefore, our SPB study here measured the uptake of overall dietary FA.Fecal Dual Isotope MethodTo compare intestinal cholesterol uptake between the CD36 WT and KO mice (n = 7 in each group), each mouse was first adapted in individual cage for 1 week before study. Each mouse was then gavaged with 200 μL of 20% Liposyn II intralipid (Abbott Laboratories, Chicago, IL) added with 0.35 mg cholesterol, 0.5 μCi [14C] cholesterol, and 5 μCi [3H] sitosterol. Fecal samples from each mouse were collected 24 hours postgavage. Mice were allowed to eat ad libitum (regular chow diet), and food intake was monitored for the period of 48 hours after gavage. Fecal samples (about 200 mg) were saponified by heating them at 80°C for 5 minutes in 4 mL of 0.5 N NaOH in methanol. After allowing the solution to cool, 2 mL of saturated saline and 10 mL of hexane were added, and the solution was mixed and organic layer extracted. The extracted organic layer was allowed to evaporate under a gentle stream of nitrogen. Radioactivity was determined by the scintillation counter after mixing the dried organic layer with 10 mL of scintillation fluid. Cholesterol uptake was determined by calculating the disintegration per minute (dpm) fractions of 14C and 3H from the homogenous gavage solution and the extracted fecal samples:%Uptake = ([14C/3H]gavage − [14C/3H]fecal)/[14C/3H]gavage × 100%Lymph and Duodenal CannulationIntestinal lymph ducts of anesthetized (ketamine, 80 mg/kg and xylazine, 20 mg/kg) mice were cannulated with polyvinyl chloride (PVC) tubing (inner diameter [ID], 0.20 mm; outside diameter [OD], 0.50 mm) as described by Bollman et al19Bollman J.L. Cain J.C. Grindlay J.H. Techniques for the collection of lymph from the liver, small intestine, or thoracic duct of the rat.J Lab Clin Med. 1949; 33: 1349-1352Google Scholar with the following modifications. Suture of the lymph cannula was replaced by application of cyanoacrylate glue (Krazy Glue, Itasca, IL); in addition, a PVC tube (ID, 0.5 mm; OD, 0.8 mm) was inserted into the duodenum through a fundal incision of the stomach and secured by a purse-string. Following the surgery, mice were infused with 5% glucose in saline (145 mmol/L NaCl, 4 mmol/L KCl, and 0.28 mol/L glucose) at a rate of 0.3 mL/h. The glucose/saline solution was replaced with the prepared lipid infusate the next morning.Lipid Infusate PreparationTriolein, [3H] triolein, cholesterol, [14C] cholesterol, and PC were dissolved in chloroform. The chloroform content was evaporated under a steady stream of nitrogen. The chloroform-free lipid mixture was then emulsified with 19 mmol/L NaTC in phosphate-buffered saline (PBS; [(in g) 0.958 Na2HPO4, 2.277 NaH2PO4, 6.8 NaCl, and 0.2982 KCl/L H2O] at pH 6.4.) and sonicated with 1 second on/1 second off pulse until the solution appeared homogenous. The homogeneity of the emulsion was checked by taking samples from the top, the middle, and the bottom of the emulsion and radioactivity determined. We considered the emulsion to be homogenous if the counts did not vary >1%.The lipid emulsion was infused for 6 hours. The hourly infusate contained 4 μmol triolein with a trace mass of [3H] triolein, 0.78 μmol cholesterol with a trace mass of [14C] cholesterol, 0.78 μmol egg PC, and 5.7 μmol NaTC in PBS.Collection of Lymph, Intestinal Lumen, and the Small IntestineLymph samples were collected hourly. At the end of the 6-hour infusion period, the animals were anesthetized with the ketamine–xylazine mixture, and stomach, small intestine, colon, and luminal content were removed. The small intestine with intact mucosa and muscular layers (unscraped) were then cut into 4 equal segments, and herein referred to as M1–M4 (M1 being the most proximal and M4 being the most distal). We did not scrape the mucosa because it is quite thin in the mouse and, depending on the strength one applies during scraping, there could be considerable variation from experiment to experiment in the amount of mucosal scraping. Tissue samples were homogenized using a Polytron homogenizer, and radioactivity of each sample was measured. In addition, lipid fraction of M1–M4 were immediately extracted using the Folch method20Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J Biol Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar for further lipid analysis. Lymphatic TG mass was determined using a TG kit from Randox (Crumlin, United Kingdom) as previously described.21Nauli A.M. Zheng S. Yang Q. Li R. Jandacek R. Tso P. Intestinal alkaline phosphatase release is not associated with chylomicron formation.Am J Physiol Gastrointest Liver Physiol. 2003; 284: G583-G587PubMed Google ScholarThin-Layer Chromatography Analysis of M1–M4Lipids extracted from M1–M4 were separated on silica gel 60 plates using a solvent system of petroleum ether/ethyl ether/glacial acetic acid with 25:5:1 volume ratio. After visualizing the samples and the comigrating reference standards by staining with iodine vapor, samples were scraped into scintillation vials, and 1 mL of absolute alcohol was added to scintillation liquid (Opti Fluor for aqueous samples) for counting of radioactivity.Lipoprotein Particle Size AnalysisCarbon-coated formvar film on a 400-mesh copper grid (Electron Microscopy Sciences, Hatfield, PA) was floated on a drop of the lymph sample. The grid was dried with filter paper and briefly added with 2% phosphotungstic acid (pH 6.0). For lipid-feeding lymph samples, 5- and 6-hour lymph samples were pooled and diluted with sterile water 1:4 vol:vol before being used for negative staining. Fasting lymph samples (collected 1 hour prior to lipid infusion) were not diluted and were added on grids as described. Standard beads (200 nm) were used for calibration (Duke Scientific Corp, Fremont, CA). The samples were examined and pictures were taken immediately by using the JEOL JEM-1230. The size of the lipoprotein particles was measured by using Adobe Photoshop and software from Reindeer Graphics. An average of 800 particles were sized per lymph sample. A previous pilot study showed that the manual and the digital counting methods agreed closely (data not shown).Chylomicron CompositionTo determine the lipid composition of chylomicron in the lymph, equal aliquots of lymph samples were layered under 0.15 mol/L NaCl and subjected to centrifugation for 30 minutes at 50,000 rpm in MLA-130 rotor in a table top ultracentrifuge (Beckman Instruments, Fullerton, CA). Chylomicrons were removed, and lipid composition was determined as described by using kits from Wako chemicals.Enzyme-Linked Immunosorbent Assay Analysis of ApolipoproteinsHigh-binding 96-well plates were precoated overnight at 4°C with 100 μL/well of 1:300 rabbit anti-rat apolipoprotein A-I, A-IV, or B in coating buffer (0.014 mol/L Na2CO3 and 0.035 mol/L NaHCO3, pH 9.6). Each well was then washed 3 times with PBS-T (0.5% Tween-20 in 0.01 mol/L PBS) and blocked with 270 μL of blocking buffer (1% bovine serum albumin in 0.01 mol/L PBS-T) for 2 hours at 4°C. After removing the blocking buffer, 100 μL of either standards or samples were added and incubated overnight at 4°C. Purified rat apolipoprotein A-I, A-IV, or B were used as standards (the linear range of each standard curves were determined in pilot studies). Samples were either from lymph collected 1 hour before lipid infusion (fasting) or from lymph collected during the 3rd–4th hour lipid infusion (feeding; the 3rd hour lymph and 4th hour lymph were pooled). For measuring apolipoprotein A-I, samples were diluted to 1:100; A-IV 1:100; and B 1:400 (sample dilution was determined in pilot studies). Each well was washed 3 times as before and incubated with 100 μL of 1:3000 goat anti-rat apolipoprotein A-I, A-IV, or B in blocking buffer for 2 hours at room temperature. The antibodies were washed 3 times and incubated with 100 μL of 1:500 horseradish peroxidase–linked rabbit anti-goat antibodies in blocking buffer for 1 hour at room temperature. The secondary antibodies were washed as before, and 200 μL of o-phenylenediamine dihydrochloride substrate (Sigma) were added to each well. After 15 minutes, the reaction was stopped by adding 50 μL of 3 mol/L HCl, and the absorbance was read at 490 nm by Synergy HT plate reader (BioTek, Winooski, VT). Samples were measured in triplicate.Statistical AnalysisThe data shown are mean values ± standard errors (SE). To compare groups throughout the 6-hour infusion, 2-way repeated measures analysis of variance (ANOVA) with Tukey as a posttest analysis was used. For comparison of data that have 2 independent variables, 2-way ANOVA was used. A t-test was used for the rest of the data analyses (comparing only 2 groups). Statistical analyses were performed using Sigmastat (SPSS Inc., Chicago, IL), and were considered significant if P < .05.ResultsFA UptakeThe SPB method allows for the analysis of FA uptake in mice fed ad libitum by determining the content of excreted FA relative to the content of a nonabsorbable marker in their fecal samples. Both CD36 WT (n = 4) and KO (n = 4) mice showed a 91% overall uptake of dietary FA mass (P = .91; Figure 1A), suggesting that ablation of CD36 is not sufficient to reduce overall FA uptake. (Note that because enterocytes do not take up TG but FA from digested TG, we prefer the term FA uptake to TG uptake.)Cholesterol UptakeUsing the fecal dual isotope method, we examined if the absence of CD36 caused a change in cholesterol uptake by the intestine. As depicted in Figure 1B, the cholesterol uptake of both WT and KO mice was about 85% (P = .90). These numbers agree well with those obtained by other investigators.22Howles P.N. Carter C.P. Hui D.Y. Dietary free and esterified cholesterol absorption in cholesterol esterase (bile salt-stimulated lipase) gene-targeted mice.J Biol Chem. 1996; 271: 7196-7202Crossref PubMed Scopus (165) Google Scholar Food intake of WT and KO mice did not show any significant difference during this study (data not shown).Both the SPB method and the fecal dual isotope method could not determine the rate and the site of dietary FA and cholesterol uptake and subsequent packaging into chylomicrons for secretion. Also, both methods are influenced by variation in stomach emptying. To more directly determine the uptake and lymphatic transport of TG and cholesterol, we employed the conscious lymph fistula model.Analysis of Lymphatic Transport of TG and Cholesterol by the Small IntestineTo further determine whether CD36 plays a role in TG and cholesterol transport by the small intestine, we analyzed the lymphatic output of CD36 WT and KO mice. (Note that because FA taken up by the enterocytes needs to be converted to TG for transport into chylomicrons, we prefer using the term TG transport to FA transport.) CD36 KO mice had a significant reduction in lymphatic output of both TG (Figure 2A) and cholesterol (Figure 2C). The reduction in lymphatic radioactive TG output (P < .001) was evident as early as the first hour of infusion (Figure 2A), reaching only 25% of the hourly infused TG as compared to 50% in the WT mice. The total TG mass (Figure 2B) in the lymph of the CD36 KO mice was also lower (P = .003) than that of CD36 WT mice throughout the entire 6-hour study. About 80% of the lymphatic TG mass output was calculated to be derived from the infused (exogenous/dietary) TG in both the CD36 WT and KO mice. CD36 KO mice also showed a reduction (P = .002) in lymphatic transport of the dietary cholesterol, reaching only 10% of the hourly infused load as compared with 30% for WT mice. This decrease was significant from the third hour of infusion onward (Figure 2C).Figure 2(A) [3H]-TG, (B) TG mass, and (C) [14C]-cholesterol transport into the lymph during continuous intraduodenal lipid infusion. Mice were equipped with lymph and duodenal cannulas, and were intraduodenally infused with a lipid emulsion containing labeled TG (the label was on all FA molecules of the TG) and cholesterol for a period of 6 hours. Lymph was collected hourly and analyzed. *P < .05 as determined by Tukey posttest analysis of 2-way repeated measures ANOVA. Values are means ± SE.View Large Image Figure ViewerDownload (PPT)Fate of Dietary FA and Cholesterol at the End of 6 Hours of Continuous InfusionFigure 3A and B show that very little of the dietary FA and cholesterol that were infused were being excreted because only a small amount of radioactivity was recovered from the colon of both groups of animals. Also, there was little if any reflux of infused lipids back to the stomach, judged from the recovery of radioactive counts from the lumen of the stomach from both groups of animals. There was a tendency for luminal [3H] counts to be higher in CD36 KO than in WT mice, but the difference was not statistically significant (P = .1220; Figure 3A, lumen). In contrast, lymphatic transport of TG in CD36 KO mice was significantly decreased relative to that of the WT controls (P = .0069; Figure 3A, lymph), and the CD36 KO mice also had a tendency to accumulate more of the infused radioactive TG in their intestine (P = .105) (Figure 3A, intestine). In a separate acute study, we observed a similar accumulation of labeled TG in the intestine of the CD36 KO when the emulsion was administered by gavage instead of by constant infusion into the duodenum as in our lymph fistula mice (data not shown). Thus, the tendency to accumulate dietary TG in the intestine of the KO was similar irrespective of whether TG was infused at a constant rate into the duodenum or gavaged as a single meal.Figure 3Fate of dietary TG (A) and cholesterol (B) at the end of 6-hour continuous infusion. Mice were equipped with lymph and duodenal cannulas, and were intraduodenally infused with a lipid emulsion containing labeled TG (the label was on all FA molecules of the TG) and cholesterol for a period of 6 hours. The recovery of radioactivity in the small intestine, lymph, stomach, intestinal lumen, and colon were determined at the end of the 6 hours by scintillation counter. *P < .05. Values are means ± SE.View Large Image Figure ViewerDownload (PPT)As shown in Figure 3B (lumen), significantly higher luminal radioactive cholesterol counts (P = .0073) were recovered in the null mice than the WT controls. As would be expected from the luminal radioactive cholesterol recovery, the CD36 KO had much reduced radioactive cholesterol output into the lymph (P = .0216; Figure 3B, lymph). There was no evidence for more radioactive cholesterol accumulated in the small intestine of the CD36 KO relative to the WT controls (P = .88; Figure 3B, intestine).Distribution of Radioactive TG and Cholesterol Along the Segments of the Small IntestineFigure 4A and B show the distribution of dietary TG and cholesterol, respectively, in the 4 equal length segments of the small intestine, M1–M4 (M1 being the most proximal and M4 being the most distal). The distribution of cholesterol in the small intestine of the KO did not show a typical gradual decrease from M1 to M2. The low recovery of both [3H] and [14C] counts in the M4 segments further support our conclusion that the infused materials were either taken up by the small intestine or remained in the lumen but were not excreted. Statistical analysis did not show any significant difference between the WT and KO animals.Figure 4Distribution of dietary TG (A) and cholesterol (B) along the segments of the small intestine. Mice were equipped with lymph and duodenal cannulas, and were intraduodenally infused with a lipid emulsion containing labeled TG (the label was on all FA molecules of the TG) and cholesterol for a period of 6 hours. At the end of the study, small intestines (n = 5) were harvested and divided into 4 equal length segments, from proximal to distal: M1, M2, M3, and M4. The amounts of radioactivity in these segments were determined by scintillation counter. No statistical significance was found between the WT and KO. Values are means ± SE.View Large Image Figure ViewerDownload (PPT)Thin-Layer Chromatography Analysis of the Lipid Fraction of
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