Metabolism of apical versus basolateral sn-2-monoacylglycerol and fatty acids in rodent small intestine
2008; Elsevier BV; Volume: 49; Issue: 8 Linguagem: Inglês
10.1194/jlr.m800116-jlr200
ISSN1539-7262
AutoresJudith Storch, Yin Zhou, William Lagakos,
Tópico(s)Diet, Metabolism, and Disease
ResumoThe metabolic fates of radiolabeled sn-2-monoacylglycerol (MG) and oleate (FA) in rat and mouse intestine, added in vivo to the apical (AP) surface in bile salt micelles, or to the basolateral (BL) surface via albumin-bound solution, were examined. Mucosal lipid products were quantified, and the results demonstrate a dramatic difference in the esterification patterns for both MG and FA, depending upon their site of entry into the enterocyte. For both lipids, the ratio of triacylglycerol to phospholipid (TG:PL) formed was approximately 10-fold higher for delivery at the AP relative to the BL surface. Further, a 3-fold higher level of FA oxidation was found for BL compared with AP substrate delivery. Incorporation of FA into individual PL species was also significantly different, with >2-fold greater incorporation into phosphatidylethanolamine (PE) and a 3-fold decrease in the phosphatidylcholine:PE ratio for AP- compared with BL-added lipid. Overnight fasting increased the TG:PL incorporation ratio for both AP and BL lipid addition, suggesting that metabolic compartmentation is a physiologically regulated phenomenon. These results support the existence of separate pools of TG and glycerolipid intermediates in the intestinal epithelial cell, and underscore the importance of substrate trafficking in the regulation of enterocyte lipid metabolism. The metabolic fates of radiolabeled sn-2-monoacylglycerol (MG) and oleate (FA) in rat and mouse intestine, added in vivo to the apical (AP) surface in bile salt micelles, or to the basolateral (BL) surface via albumin-bound solution, were examined. Mucosal lipid products were quantified, and the results demonstrate a dramatic difference in the esterification patterns for both MG and FA, depending upon their site of entry into the enterocyte. For both lipids, the ratio of triacylglycerol to phospholipid (TG:PL) formed was approximately 10-fold higher for delivery at the AP relative to the BL surface. Further, a 3-fold higher level of FA oxidation was found for BL compared with AP substrate delivery. Incorporation of FA into individual PL species was also significantly different, with >2-fold greater incorporation into phosphatidylethanolamine (PE) and a 3-fold decrease in the phosphatidylcholine:PE ratio for AP- compared with BL-added lipid. Overnight fasting increased the TG:PL incorporation ratio for both AP and BL lipid addition, suggesting that metabolic compartmentation is a physiologically regulated phenomenon. These results support the existence of separate pools of TG and glycerolipid intermediates in the intestinal epithelial cell, and underscore the importance of substrate trafficking in the regulation of enterocyte lipid metabolism. sn-2-Monoacylglycerol (MG) and FAs are the hydrolytic products of ingested triacylglycerol (TG), and provide a major source of calories in Western diets. FAs, in addition, provide critical building blocks for membrane biogenesis, are precursors for regulatory second messengers, and are now considered to directly modulate the expression of specific genes (1Clarke S.D. Polyunsaturated fatty acid regulation of gene transcription: a molecular mechanism to improve the metabolic syndrome.J. Nutr. 2001; 131: 1129-1132Crossref PubMed Scopus (324) Google Scholar). Certain MGs may also function outside of the traditionally appreciated lipid metabolic pathways. For example, sn-2-monoarachidonoyl is thought to act as an endogenous ligand for the cannabinoid receptors (2Dinh T.P. Carpenter D. Leslie F.M. Freund T.F. Katona I. Sensi S.L. Kathuria S. Piomelli D. Brain monoglyceride lipase participating in endocannabinoid inactivation.Proc. Natl. Acad. Sci. USA. 2002; 99: 10819-10824Crossref PubMed Scopus (1141) Google Scholar). Thus, the products of dietary fat digestion, once taken up by the intestinal enterocyte, may have diverse metabolic and cellular fates. It is known that FAs are taken up into the enterocyte across both the apical (AP) plasma membrane as well as across their basolateral (BL) plasma membranes. Further, the intracellular metabolism of FAs is highly dependent upon their site of entry into the cell. In 1975, Gangl and Ockner (3Gangl A. Ockner R.K. Intestinal metabolism of plasma free fatty acids. Intracellular compartmentation and mechanisms of control.J. Clin. Invest. 1975; 55: 803-813Crossref PubMed Scopus (94) Google Scholar) presented the intriguing finding that luminally derived and plasma-derived palmitic acid had different metabolic fates in the rat enterocyte. Plasma palmitate was primarily oxidized or incorporated into phospholipids (PLs), with relatively low incorporation into TG, whereas palmitate absorbed from the intestinal tract was mainly incorporated into TG. Similar results were shown in humans (4Gangl A. Renner F. In vivo metabolism of plasma free fatty acids by intestinal mucosa of man.Gastroenterology. 1978; 7: 847-850Abstract Full Text PDF Scopus (18) Google Scholar). Studies by Mansbach and Parthasarathy (5Mansbach II C.M. Parthasarathy S. A re-examination of the fate of glyceride-glycerol in neutral lipid absorption and transport.J. Lipid Res. 1982; 23: 1009-1019Abstract Full Text PDF PubMed Google Scholar) and Mansbach and Dowell (6Mansbach C.M. Dowell R.F. Uptake and metabolism of circulating fatty acids by rat intestine.Am. J. Physiol. 1992; 263: G927-G933Crossref PubMed Google Scholar) further suggested that there are two pools of neutral lipid in the rat enterocyte, dependent in part upon the site of entry of the FA precursors. Our previous studies in Caco-2 cells found a small increase in the ratio of TG to PL for apically compared with basolaterally administered palmitate and oleate (7Trotter P.J. Storch J. Fatty acid uptake and metabolism in a human intestinal cell line (Caco-2): comparison of apical and basolateral incubation.J. Lipid Res. 1991; 32: 293-304Abstract Full Text PDF PubMed Google Scholar, 8Ho S.Y. Delgado L. Storch J. Monoacylglycerol metabolism in human intestinal Caco-2 cells: evidence for metabolic compartmentation and hydrolysis.J. Biol. Chem. 2002; 277: 1816-1823Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar), supporting the existence of lipid metabolic polarity at the level of the intestinal cell itself. The hydrolysis of circulating TG-rich lipoproteins by lipoprotein lipase produces sn-2-MG (9Fielding B.A. Humphreys S.M. Allman R.F. Frayn K.F. Mono-, di- and triacylglycerol concentrations in human plasma: effects of heparin injection and of a high-fat meal.Clin. Chim. Acta. 1993; 216: 167-173Crossref PubMed Scopus (25) Google Scholar, 10El Maghrabi M.R. Waite M. Rudel L.L. Sisson P. Hydrolysis of monoacylglycerol in lipoprotein remnants catalyzed by the liver plasma membrane monoacylglycerol acyltransferase.J. Biol. Chem. 1978; 253: 974-981Abstract Full Text PDF PubMed Google Scholar), and MG has been shown to bind to albumin with high affinity (11Thumser A.E.A. Buckland A.G. Wilton D.C. Monoacylglycerol binding to human serum albumin: evidence that monooleoylglycerol binds at the dansylsarcosine site.J. Lipid Res. 1998; 39: 1033-1038Abstract Full Text Full Text PDF PubMed Google Scholar). In Caco-2 cells, we found that basolaterally delivered MG shows a small preferential incorporation into PL, and that AP-delivered MG shows a small preferential incorporation into TG (8Ho S.Y. Delgado L. Storch J. Monoacylglycerol metabolism in human intestinal Caco-2 cells: evidence for metabolic compartmentation and hydrolysis.J. Biol. Chem. 2002; 277: 1816-1823Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar, 12Ho S.Y. Storch J. Common mechanisms of monoacylglycerol and fatty acid uptake by human intestinal Caco-2 cells.Am. J. Physiol. 2001; 281: C1106-C1117Crossref PubMed Google Scholar). The uptake of MG across the BL surface of the intestinal epithelium has not been previously demonstrated, nor is anything known about the metabolism of plasma MG by the intestine. In the present studies, our goals were to determine: 1) whether MG is taken up into the intestinal mucosa from the circulation; 2) whether metabolic polarity for MG occurs in vivo; 3) whether, in addition to differences in the relative amount of PL formed from AP versus BL FA, there are also differences in the types of PL formed; 4) whether the degree of enterocyte lipid metabolic polarity of FA is fixed, or can be regulated by physiological state; and 5) the degree to which differential FA metabolism, dependent on entry site, occurs in the mouse, as a platform for further mechanistic studies using transgenic and knockout models. The results show not only that MG is taken up from the circulation, but also that its metabolic fate in rat and mouse intestinal mucosa is dependent on whether it is added via the dietary/AP route or via the bloodstream/BL route. In addition, the incorporation of oleate into PL species is markedly different for dietary compared with bloodstream FA. Finally, the metabolic fate of FA is substantially altered by short-term fasting, indicating regulatable mechanisms of polarized lipid metabolism in the intestinal enterocyte. Oleic acid and sn-2-monoolein were obtained from NuChek Prep, Inc. (Elysian, MN). [3H]oleic acid ([9,10-3H]oleic acid, 26.3 Ci/mmol) and [14C]oleic acid ([1-14C]oleic acid, 54 mCi/mmol) were obtained from Perkin Elmer-New England Nuclear (Stelton, CT). [3H]sn-2-monoolein (sn-2-[9,10 3H]monoolein, 40–60 Ci/mmol) was from American Radiochemical (St. Louis, MO). Authentic neutral lipid and PL standards were purchased from Doosan Serdary Research Laboratories (Toronto, Canada) and Avanti Polar Lipids (Alabaster, AL), respectively. Sodium taurocholate (TC) was purchased from Calbiochem (La Jolla, CA), and FA-free BSA was obtained from Sigma Aldrich (St. Louis, MO). TLC plates (Silica Gel G and Silica Gel K, both 250 μm, 150 Å) were obtained from Whatman (South Plainfield, NJ). All other materials were reagent grade or better. Stock solutions were prepared by drying the appropriate lipids under nitrogen and then adding 0.5% (final vol) ethanol, followed by a 1:1 mixture of mouse serum:0.85% NaCl. For experiments using rats, 1 ml of labeled lipid mixture containing 13–14 μCi of [14C]oleate (231–259 nmol), 75 μCi of [3H]oleate (2.9 nmol), or 65–107 μCi of [3H]monoolein (587–966 nmol) was administered to each animal. For mice, 150 μl of lipid solution containing 7.5 μCi of [14C]oleate (140 nmol) or [3H]monoolein (125 nmol) was used. Stock solutions were prepared by drying the appropriate lipids under nitrogen and then adding 0.5% (final vol) ethanol, followed by 10 mM sodium TC in 0.85% NaCl. For experiments using rats, 1 ml of TC micelles containing 3–4 μCi of [14C]oleate (46–69 nmol), 5 μCi [3H]oleate (4 nmol), or 2–4 μCi [3H]monoolein (26–39 nmol) was administered to each animal. For mice, 150 μl of TC micelles containing 1.5–3 μCi of [14C]oleate (28–52 nmol) or 1–2 μCi [3H]monoolein (12–28 nmol) was used. Male Sprague-Dawley rats (Taconic Farms; Germantown, NY) were used for several experiments, as indicated. The majority of the present studies were performed using C57BL/6J mice obtained from Jackson Laboratories (Bar Harbor, ME). For rats, animals weighed approximately 325 g and were 2–3 months old. Mice were used at 3–4 months of age and 25–30 g body weight. Experiments were performed in the fed state, typically between 8 AM and 11 AM when food had been present overnight, except for studies of fasting, when food was withheld as indicated prior to experimentation. Animals were fed Purina 5015 rodent chow (60% carbohydrate, 12% fat, 28% protein by kcal). This research was conducted in conformity with the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the studies were approved by the Rutgers University Animal Use Protocol Review Committee of the Laboratory Animals Services Department. Animals were anesthetized with ketamine-xylazine-ace promazine (80:100:150 mg/kg, respectively). The peritoneum was exposed for rapid access to the intestine. For administration of lipids via the bloodstream, the tail vein was used for rats, and for mice the jugular vein was exposed. A bolus of radiolabeled lipids, described above, was injected at time zero. For administration of lipids via the gastrointestinal tract, the stomach and small intestine were exposed. A small incision was made using microsurgical scissors <1 cm below the pyloric sphincter, and an 18 gauge blunted needle was inserted and secured in place with surgical string. A bolus of radiolableled lipids, described above, was injected at time zero. At exactly 2 min after either mode of lipid infusion, the intestine, from pylorus to cecum, was harvested, flushed with 60 ml ice-cold saline, and opened longitudinally. For rats, the intestine was divided into proximal and distal segments of equal length. Intestinal mucosa was scraped using a glass slide, and samples were placed immediately into polypropylene tubes in dry ice-acetone and were then placed in a −70°C freezer. To ensure that the radiolabeled lipids were not modified in the lumen or bloodstream during the 2 min experimental period, blood and intestinal contents were sampled at 2 min and extracted lipids were subjected to TLC and phosphorimager (or scintillation counting) analysis. This was especially important for the sn-2-MG, where isomerization to the sn-1-isomer could have been a concern, or lipolysis might have taken place prior to cellular uptake. It was consistently found that the administered labeled lipids remained in their intact form, i.e., the lipids taken up during 2 min of AP or BL administration were the FA and sn-2-MG species that were administered. Separation of MG isomers by TLC was performed as described previously (13Chon S.H. Zhou Y.X. Dixon J.L. Storch J. Intestinal monoacylglycerol metabolism: Developmental and nutritional regulation of monoacylglycerol lipase and monoacylglycerol acyltransferase.J. Biol. Chem. 2007; 282: 33346-33357Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). On average, ∼0.5 to 1% of the injected radiolabeled lipid was recovered in intestinal mucosa, and ∼50% was taken up into the mucosa from intraduodenal administration. Intestinal samples were homogenized by 20 strokes of a Dounce homogenizer (0.1–0.15 mm clearance) on ice, using a Wheaton overhead stirrer at 5,000 rpm on ice in 20 ml/g mucosa of 10 mM phosphate buffer containing 150 mM NaCl at pH 7.4 (PBS). Lipids were extracted using chloroform-methanol (2:1; v/v) by the method of Folch, Lees, and Sloane-Stanley (14Folch 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). Samples were diluted to 1 mg protein/ml of PBS, and all extractions were performed on 1 mg of tissue protein within 1 day following the experiment. Lipid extracts and known standards were spotted on Silica Gel G TLC plates and developed using a nonpolar solvent system (hexane-diethyl ether-acetic acid, 70:30:1; v/v) to separate the lipid classes, and the plates were dried and exposed to iodine vapors to visualize and identify the lipid spots. In the case of tritiated lipids, the spots corresponding to each lipid were scraped into scintillation vials containing 5 ml of ReadySafe scintillation fluid (Beckman Coulter; Fullerton, CA), vortexed vigorously, and counted the following day. For 14C-labeled lipids, the TLC plate was exposed to a phosphorimager screen overnight, and the percent of total lipid extract radioactivity present in each lipid class (excluding oxidation products) was analyzed using a Storm 840 phosphorimager. For FA, the amount of unincorporated label (range 30% to 50% of mucosal counts) was excluded from calculations. The one-dimensional TLC procedure described by Vaden et al. (15Vaden D.L. Gohil V.M. Gu Z. Greenberg M.L. Separation of yeast phospholipids using one-dimensional thin-layer chromatography.Anal. Biochem. 2005; 338: 162-164Crossref PubMed Scopus (75) Google Scholar) was used. Briefly, K5 silica gel plates were prewashed in chloroform-methanol (1:1; v/v). Plates were wetted by immersion in 1.8% boric acid in 100% ethanol, dried at room temperature for 5 min, and baked at 100°C for 20 min. The concentration zone was divided into several lanes by scraping parallel lines at 1.5 cm intervals. Aliquots of the total lipid extract were applied to the concentration zone lanes, allowed to dry, and developed using chloroform-ethanol-water-triethylamine (30:35:7:35; v/v). The plate was dried at room temperature in a fume hood for 30 min and then run for a second time in a different tank with the same solvent system, to achieve higher resolution (15Vaden D.L. Gohil V.M. Gu Z. Greenberg M.L. Separation of yeast phospholipids using one-dimensional thin-layer chromatography.Anal. Biochem. 2005; 338: 162-164Crossref PubMed Scopus (75) Google Scholar). Authentic PL standards were used to identify PL species. After development, radiolableled PLs were detected as above. To determine how much of the administered radioactive FA was oxidized, the method of Ontko and Jackson (16Ontko J.A. Jackson D. Factors affecting the rate of oxidation of fatty acids in animal tissues. Effect of substrate concentration, pH, and coenzyme A in rat liver preparations.J. Biol. Chem. 1964; 239: 3674-3682Abstract Full Text PDF PubMed Google Scholar) was used with minor modification. Briefly, 1 ml of 1 mg protein/ml sample homogenate was placed in a 15 ml disposable plastic tube. A 0.5 ml Eppendorf tube containing tissue paper soaked in 1 M benzethonium hydroxide was also placed in the 15 ml tube to trap 14CO2. The samples were acidified with 0.3 ml 3M perchloric acid, capped tightly, and incubated overnight at 37°C with shaking. The radioactivity of the tissue paper was determined, the acidified sample was vortexed and centrifuged at 2,800 rpm for 10 min, and radioactivity of the supernatant was determined. Total oxidation was calculated by adding total radioactivity of the supernatant (acid-soluble products, ketones, and TCA intermediates) plus tissue paper (CO2). This number was divided by the amount of radioactivity contained in 1 mg of sample to yield percent oxidation. Unless otherwise indicated, data are shown as mean ± SE. Statistical comparisons were performed using Microsoft Excel, and significance was determined by independent, two-tailed Student's t-tests, with P values of 0.05 or lower considered as significantly different. In the present studies, we examined whether sn-2-MG metabolism was dependent upon its site of entry into the absorptive enterocytes, and whether oleic acid demonstrated metabolic polarity similar to that reported for the saturated FA palmitate (3Gangl A. Ockner R.K. Intestinal metabolism of plasma free fatty acids. Intracellular compartmentation and mechanisms of control.J. Clin. Invest. 1975; 55: 803-813Crossref PubMed Scopus (94) Google Scholar). Labeled lipids were administered to anesthetized male rats as described above. Analysis of radiolabeled metabolites 2 min following administration showed that the major esterified product of apically delivered oleate was TG, whereas the major esterified product of basally delivered oleate was PL (Table 1). Thus, the TG:PL ratios differed by approximately 10-fold. In the proximal half of rat small intestine, TG:PL was 5.1 ± 1.6 for dietary oleate, and 0.53 ± 0.07 for bloodstream oleate (P < 0.01). Essentially no difference in relative incorporation of AP or BL oleate was found between proximal and distal segments of the intestine, despite the known lower absorptive capacity of the distal small intestine (17Kuksis A. Lehner R. Intestinal synthesis of triacylglycerols.In Intestinal Lipid Metabolism. C. M. Mansbach II, P. Tso, and A. Kuksis, editors. Kluwer Academic/Plenum Publishers, New York. 2001; : 185-213Google Scholar). The mass of mucosal lipid species following the 2 min administration of tracer lipids at the AP or BL poles of the epithelium were not different (data not shown).TABLE 1Metabolism of apically and basolaterally added FA in rat small intestineProximalDistalBL (n = 9)AP (n = 9)BL (n = 6)AP (n = 8)CE8.7 ± 1.712.9 ± 3.411.8 ± 3.214.4 ± 3.3TG14.9 ± 1.747.9 ± 5.9b22.1 ± 3.546.7 ± 6.0aDG18.6 ± 3.516.0 ± 3.012.6 ± 2.721.6 ± 5.6MG15.9 ± 3.09.1 ± 2.310.1 ± 0.710.0 ± 2.3PL31.9 ± 3.710.9 ± 1.5b48.7 ± 5.912.2 ± 1.4bTG:PL0.5 ± 0.15.1 ± 1.6b0.6 ± 0.13.9 ± 0.8bBL, basolateral; AP, apical; CE, cholesteryl ester; TG, triacylglycerol; DG, diacylglycerol; MG, monoacylglycerol; PL, phospholipid. Incorporation of [14C]18:1 into male rat small-intestinal mucosa 2 min after administration was determined as described in Materials and Methods. Results are means ± SE for the percent of total mucosa-associated labeled FA incorporated into each lipid species. 100% = 0.9 nmol for proximal BL sample, 24.9 nmol for proximal AP sample, 1.0 nmol for distal BL sample, and 8.9 nmol for distal AP sample (averages ± 10%).a P < 0.05, b P < 0.01 versus BL. Open table in a new tab BL, basolateral; AP, apical; CE, cholesteryl ester; TG, triacylglycerol; DG, diacylglycerol; MG, monoacylglycerol; PL, phospholipid. Incorporation of [14C]18:1 into male rat small-intestinal mucosa 2 min after administration was determined as described in Materials and Methods. Results are means ± SE for the percent of total mucosa-associated labeled FA incorporated into each lipid species. 100% = 0.9 nmol for proximal BL sample, 24.9 nmol for proximal AP sample, 1.0 nmol for distal BL sample, and 8.9 nmol for distal AP sample (averages ± 10%). a P < 0.05, b P < 0.01 versus BL. The acute metabolic fate of sn-2-monoolein was similarly examined, and the results show clearly that a dramatic metabolic compartmentation occurs depending upon the site of MG entry into the enterocyte. The results in Table 2show that the TG:PL ratio for MG metabolites in the proximal half of the rat small-intestinal mucosa was 0.44 ± 0.05 for bloodstream MG, and 2.0 ± 0.5 for luminal MG (P < 0.05). This approximately 5-fold difference reflected both a small increase in TG and, particularly, a decrease in MG incorporation into PL in the MG delivered via the AP surface (10.6 ± 0.7% for BL PL, 3.0 ± 0.2% for AP PL, P < 0.01).TABLE 2Metabolism of apically and basolaterally added MG in male rat small intestineProximalDistalBLAPBLAPCE4.6 ± 1.91.9 ± 0.53.5 ± 0.83.0 ± 1.0TG4.6 ± 0.76.1 ± 1.16.4 ± 0.710.0 ± 3.1FA27.7 ± 7.920.9 ± 7.819.0 ± 5.521.8 ± 6.7DG36.1 ± 5.956.3 ± 7.644.7 ± 4.651.4 ± 7.2MG16.5 ± 4.911.8 ± 2.613.9 ± 3.29.8 ± 1.6PL10.6 ± 0.73.0 ± 0.2b12.5 ± 2.34.1 ± 0.8aTG:PL0.4 ± 0.12.0 ± 0.5a0.5 ± 0.12.5 ± 0.6aIncorporation of [3H]sn-2-18:1 into rat small-intestinal mucosa 2 min after administration was determined as described in Materials and Methods. Results are means ± SE (n = 4 per group) for the percent of total mucosa-associated labeled MG incorporated into each lipid species. 100% = 17.1 nmol for proximal BL sample, 138.2 nmol for proximal AP sample, 18.0 nmol for distal BL sample, and 44.9 nmol for distal AP sample (averages ± 10%).a P < 0.05, b P < 0.01 versus BL. Open table in a new tab Incorporation of [3H]sn-2-18:1 into rat small-intestinal mucosa 2 min after administration was determined as described in Materials and Methods. Results are means ± SE (n = 4 per group) for the percent of total mucosa-associated labeled MG incorporated into each lipid species. 100% = 17.1 nmol for proximal BL sample, 138.2 nmol for proximal AP sample, 18.0 nmol for distal BL sample, and 44.9 nmol for distal AP sample (averages ± 10%). a P < 0.05, b P < 0.01 versus BL. We wished to establish the degree to which metabolic divergence of MG and FA metabolism occurred in mouse intestine, because our ultimate goal is to understand the underlying mechanisms and regulatory controls for this lipid compartmentation. The results in Table 3show that marked differences in relative partitioning of both substrates occur in the mouse intestine depending upon where they enter the enterocyte layer. Addition of oleate at the AP membrane resulted in more than 3-fold greater incorporation into TG than addition at the BL membrane (48.1 ± 4.2% for AP TG, 15.8 ± 1.8% for BL TG, P < 0.01). Essentially opposite results were found for oleate incorporation into PL, where approximately 3-fold greater incorporation into PL was observed for basolaterally added relative to apically added oleate (20.2 ± 1.4% for BL PL, 7.7 ± 0.6% for AP PL, P < 0.01). For monoolein, similar and somewhat greater metabolic differences were found, with 4-fold greater incorporation of the BL MG label into PL (17.7 ± 4.6%), relative to AP MG (4.6 ± 0.9%; P < 0.01), and almost 7-fold greater incorporation of AP MG label into TG (40.2 ± 7.6%), relative to basal MG (6.1 ± 1.9%; P < 0.01). To determine whether there was a gender difference in this apparent divergence in lipid trafficking, we compared the metabolism of apically and basolaterally added oleate in male and female mouse intestinal mucosa. Figure 1depicts the TG:PL ratios for FA and MG incorporation in male mice, and for FA in female mice. As noted, large differences are observed for both the FA and MG substrates in male mouse intestine. The dramatic differences in metabolic fate of dietary oleate relative to plasma-derived oleate were found in both male and female mice.TABLE 3Metabolism of apically and basolaterally added FA and MG in mouse small intestineFAMGBL (n = 27)AP (n = 24)BL (n = 6)AP (n = 5)CE10.0 ± 0.93.7 ± 0.4bCE4.0 ± 2.11.2 ± 0.3TG26.8 ± 2.368.5 ± 3.6bTG6.1 ± 1.940.2 ± 7.6bDG18.2 ± 1.29.3 ± 1.4bFA38.1 ± 6.322.9 ± 2.6MG8.1 ± 0.96.0 ± 1.6DG22.4 ± 4.816.8 ± 5.3PL37.0 ± 2.212.5 ± 1.2bMG11.6 ± 1.614.3 ± 1.6TG:PL0.8 ± 0.17.5 ± 1.3bPL17.7 ± 4.64.6 ± 0.9aTG:PL0.4 ± 0.19.0 ± 1.1bIncorporation of [14C]18:1 or [3H]sn-2-18:1 into male mouse small-intestinal mucosa 2 min after administration was determined as described in Materials and Methods. Results are means ± SE for the percent of total mucosa-associated labeled FA or MG incorporated into each lipid species. 100% = 1.2 nmol for FA BL sample, 22.4 nmol for FA AP sample, 0.2 nmol for MG BL sample, and 14.1 nmol for MG AP sample (averages ± 10%).a P < 0.05, b P < 0.01 versus BL. Open table in a new tab Incorporation of [14C]18:1 or [3H]sn-2-18:1 into male mouse small-intestinal mucosa 2 min after administration was determined as described in Materials and Methods. Results are means ± SE for the percent of total mucosa-associated labeled FA or MG incorporated into each lipid species. 100% = 1.2 nmol for FA BL sample, 22.4 nmol for FA AP sample, 0.2 nmol for MG BL sample, and 14.1 nmol for MG AP sample (averages ± 10%). a P < 0.05, b P < 0.01 versus BL. We examined whether, in addition to the quantitative difference in PL formed, there might also be a qualitative difference in the PL species in which the fatty acyl chains become enriched. Analysis of radiolabel incorporation into male mouse intestinal PLs, shown in Table 4, indicates that the incorporation of oleate into specific PLs is strongly dependent on its site of entry into the enterocyte. Whereas phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the two most-abundant labeled species for both incubation conditions, their relative distributions were considerably different. A >2-fold greater incorporation was found for apically added FA into PE relative to basolaterally added FA, with substantially decreased incorporation into PC and PI for AP FA relative to BL FA. Thus, the PC:PE ratio for BL FA was 2.2 ± 0.2, whereas for AP FA it was 0.7 ± 0.04 (P < 0.01). In female mouse intestine, similar results were observed (Table 4), where once again the largest difference appears to be in the incorporation of labeled fatty acyl chains into PE, which is more than 2-fold greater for dietary delivery than for bloodstream delivery of the 18:1 substrate (P < 0.01).TABLE 4Incorporation of apically and basolaterally added oleic acid into phospholipid species in mouse small-intestinal mucosaMaleFemaleBL (n = 14)AP (n = 13)BL (n = 4)AP (n = 4)PA4.1 ± 1.03.4 ± 0.45.5 ± 1.61.4 ± 0.7PE19.2 ± 2.042.4 ± 2.5b19.4 ± 3.846.0 ± 1.7bPG5.5 ± 1.22.7 ± 0.5a4.9 ± 2.10.5 ± 0.1PS5.7 ± 0.83.3 ± 0.84.3 ± 4.30.9 ± 0.1PI16.9 ± 2.810.7 ± 1.114.5 ± 1.08.5 ± 0.4aPC38.0 ± 2.930.3 ± 1.7a40.9 ± 5.841.2 ± 2.1SM4.6 ± 1.03.9 ± 0.85.7 ± 2.80.5 ± 0.1LPC5.8 ± 1.26.1 ± 0.85.1 ± 1.71.1 ± 0.2PC:PE2.19 ± 0.240.73 ± 0.04b2.28 ± 0.390.90 ± 0.08aIncorporation of [14C]18:1 into phospholipid species at 2 min following administration either into the duodenum (AP) or bloodstream (BL), as described in Materials and Methods. Results are means ± SE for the percent of total mucosa-associated labeled FA incorporated into each PL species. 100% = 0.23 nmol for male BL sample, 1.72 nmol for male AP sample, 0.25 nmol for female BL sample, and 1.81 nmol for female AP sample.a P < 0.05, b P < 0.01 versus BL. Open table in a new tab Incorporation of [14C]18:1 into phospholipid species at 2 min following administration either into the duodenum (AP) or bloodstream (BL), as described in Materials and Methods. Results are means ± SE for the percent of total mucosa-associated labeled FA incorporated into each PL species. 100% = 0.23 nmol for male BL sample, 1.72 nmol for male AP sample, 0.25 nmol for female BL sample, and 1.81 nmol for female AP sample. a P < 0.05, b P < 0.01 versus BL. Whereas the majority of exogenous FAs are used by the intestine for anabolic purposes, particularly in the fed state, a small fraction are also oxidized, primarily in mitochondria. We determined the percent of mucosal oleic acid that was oxidized when administered at the AP versus the
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