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LIVER AND BILIARY TRACT
Pediatric Gastroenterology, Department of Pediatrics, Groningen University Institute for Drug Exploration, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
Submitted 22 March 2005 ; accepted in final form 20 December 2005
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ABSTRACT |
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biliary phospholipid availability; lymphatic lipoprotein size; essential fatty acid
95% of biliary PL, provide the main source for CM surface coating (14, 19, 22), and the supply of bile PC to the intestine increases the synthesis of apolipoprotein B48, the apolipoprotein needed for CM formation (7, 16). Additionally, the high essential fatty acid (EFA) content of biliary PC may be required for the maintenance of normal intestinal mucosal membrane composition and function (8, 24). Reduced availability of PC for hepatic very-low-density lipoprotein (VLDL) assembly in rats has been associated with decreased VLDL secretion and with the assembly of relatively large VLDL particles (26). It is well established that fat absorption and intestinal lipoprotein secretion are strongly impaired in situations of disturbed bile formation, such as cholestasis. A study (25) in rats with interrupted enterohepatic circulation by means of permanent bile diversion demonstrated a substantial lipid accumulation in intestinal mucosal cells. The administration of bile salts to these bile-diverted rats partially restored lymphatic lipid transfer, but only when both bile salts and biliary PL were supplemented was lymphatic lipid transport fully reinstated. Yet, a direct relationship between biliary PL availability and intestinal lipoprotein size has not been established.
We (33) recently characterized fat absorption in two mouse models with altered biliary PL secretion. Essential fatty acid (EFA) deficiency (EFAD) increases biliary PL secretion by 80%, in conjunction with a decrease by 30–40% in dietary lipid absorption. In multidrug resistance protein 2 (Mdr2)-deficient (Mdr2–/–) mice, in which biliary PL secretion is absent (23), plasma appearance of enterally administered lipid is delayed and lipid accumulates in enterocytes. Quantitatively, however, lipid absorption is unaffected in Mdr2–/– mice (28).
In the present study, we investigated whether and to what extent quantitative or qualitative alterations in biliary PL secretion affect CM secretion into lymph. CM size and composition were measured after mesenteric lymph duct cannulation using the aforementioned mouse models with altered intraluminal biliary PL availability, i.e., Mdr2–/– mice (no biliary PL secretion), EFAD mice (increased biliary PL secretion), and corresponding control mice. Our results show that the absence of biliary PL secretion in mice is accompanied by production of intestinal lipoproteins of increased size and decreased PL content, whereas EFAD, associated with increased biliary PL secretion, has the opposite effect.
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MATERIALS AND METHODS |
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Mice homozygous for disruption of Mdr2 and P-glycoprotein (Pgp) (Mdr2–/– mice) and wild-type Mdr2+/+ mice with a FVB background were obtained from the breeding colony at the Central Animal Facility, Academic Medical Center, Amsterdam, The Netherlands (9). For dietary induction of EFAD in mice, male wild-type FVB mice were obtained from Harlan (Horst, The Netherlands). All mice were 8 wk old, weighed 25–35 g, and were housed in a light-controlled (lights on 6 AM to 6 PM) and temperature-controlled (21°C) facility. Mice were allowed tap water and chow ad libitum until 2 h before lymph duct cannulation, because overnight fasting strongly decreased the success rate of the cannulation procedure, attributable to low visibility of the mesenteric lymph duct. All cannulation procedures were performed between 8 AM and noon. The experimental protocols were approved by the Ethics Committee for Animal Experiments, Faculty of Medical Sciences, University of Groningen, Groningen, The Netherlands.
Experimental Diets
The diets used for induction of EFAD in mice by us in present and previous studies and by others (17, 31–33) are usually high-fat diets. In contrast, in studies characterizing the Mdr2–/– mouse, standard laboratory low-fat chow is most frequently employed. To allow our results to be compared with previous murine studies on EFAD and on biliary PL deficiency, we chose to use high-fat diet for the EFAD and EFA-sufficent (EFAS) mice and standard laboratory low-fat chow for the experiments with Mdr2–/– mice. The standard laboratory low-fat chow (RMH-B, Arie Blok; Woerden, The Netherlands) contained 14 energy% fat. The high-fat EFAD diet contained 34 energy% fat and had the following fatty acid composition (in mol%): 41.4 palmitic acid (C16:0), 47.9 stearic acid (C18:0), 7.7 oleic acid (C18:1n-9), and 3 linoleic acid (C18:2n-6). An isocaloric EFAS diet was used as control diet, containing 37 energy% fat with 32.1 mol% C16:0, 5.5 mol% C18:0, 32.2 mol% C18:1n-9, and 30.2 mol% C18:2n-6 [custom synthesis, diet nos. 4141.08 (EFAD) and 4141.07 (EFAS), Arie Blok]. The parenteral lipid emulsion that was used for intraduodenal bolus administration (20% Intralipid, Fresenius Kabi; Hertogenbosch, The Netherlands) was mixed with glucose (3.3%) and NaCl (0.3%) in a 50:50 (vol:vol) ratio, and a 200-µl bolus of this mixture contained 2.4 nmol PL. This amount of PL constitutes <0.2% of luminal biliary PL secreted by EFAD, EFAS, and Mdr2+/+ mice during the 4-h lymph collection period. Inevitably, for Mdr2–/– mice, the percentage of administrated PL compared with biliary PL was up to 40% of bile PL, but again <0.2% of available bile PL in the intestinal lumen of Mdr2+/+ mice.
Experimental Procedures
Induction of EFAD in mice. All mice were fed standard laboratory chow from weaning. For the induction of EFAD, wild-type FVB mice were fed the EFAD diet for 8 wk. A control group of FVB mice was fed the isocaloric EFAS diet for 8 wk. This method for the induction of EFAD was previously applied in mice and characterized by our group (17, 31, 33).
Mesenteric lymph duct cannulation in mice. Cannulation of the mesenteric lymph duct was performed according to procedures described by Wang et al. (29, 30). After a 2-h fast, mice were anesthetized with halothane-NO2 and dorsally arched over a cotton cylinder for optimal visualization of the mesenteric lymph duct. After extra-abdominal displacement of the intestine, the common mesenteric lymph duct was exposed by removal of surrounding tissues and membranes using a blunt mini-kocher. A 0.305 x 0.635-mm (inner diameter x outer diameter) silicone catheter was introduced through the abdominal wall and positioned parallel to the lymph duct. Subsequently, the catheter was carefully inserted into a small incision in the lymphatic duct and fixated by drops of tissue glue at the junction of lymph duct and catheter. A subgroup of mice subsequently received an intraduodenal 200-µl bolus of a parenteral lipid emulsion (20% Intralipid) mixed with glucose (3.3%) and NaCl (0.3%) [50:50 (vol/vol)] after the collection of a 30-min baseline lymph sample. The lipid bolus was administered by puncture of the intestinal wall with a 25-gauge Sterican needle (Braun; Melsungen, Germany) after previous ligation of the duodenum immediately distal to the pylorus to prevent regurgitation into the stomach. After the intestine was repositioned, the abdominal incision was closed with 8-10 sutures, and mice were placed in restrainer cages in a 37°C incubator. Analgesia was maintained with an intraperitoneal injection of 0.1 mg/kg buprenorfine (Temgesic). Lymph was collected by gravity into EDTA-containing microtubes in 30-min fractions for up to 4 h after cannulation of the mesenteric lymph duct.
Analytical Techniques
Determination of lipoprotein size and composition. Lymphatic lipoprotein size and volume distribution profiles were analyzed within 6 h after lymph collection by dynamic light-scattering techniques using a Nicomp model 370 submicron particle analyzer (Nicomp Particle Sizing Systems; Santa Barbara, CA). Particle diameters were calculated from the volume distribution patterns provided by the analyzer. The lymphatic CM fraction (density = <1.006) was isolated after the collected lymph was complemented with a 1.006 g/ml NaCl solution containing 0.02% NaN3 to a final volume of 1 ml and subsequent centrifugation at 40,000 rpm at 4°C in an Optima TM LX table top centrifuge (Beckman Instruments; Palo Alto, CA) for 15 min. The top layer containing the CM fraction was isolated by tube slicing, and the volume was recorded by weight. The remaining lymph solution was again complemented with 1.006 g/ml NaCl to a final volume of 1 ml and centrifuged at 120,000 rpm for 1 h and 40 min for isolation of the VLDL fraction.
Lipoprotein lipid concentrations were measured using commercially available assay kits from Roche (Mannheim, Germany) for TG and total cholesterol and from WAKO Chemicals (Neuss, Germany) for PL.
Calculations and Statistics
All results are presented as means ± SD for the number of animals indicated. Data were statistically analyzed using Student's t-test or ANOVA test with a post hoc Bonferroni correction. The level of significance was set at P < 0.05. Analyses were performed using SPSS for Windows software (SPSS; Chicago, IL).
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RESULTS |
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DISCUSSION |
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Several conditions can alter biliary PL secretion: EFAD in mice is associated with decreased EFA contents of biliary PL and profoundly increased bile flow, whereas Mdr2-Pgp deficiency is associated with a virtual absence of biliary PL secretion. We previously demonstrated that in both murine models, the postprandial plasma appearance of enterally administered lipids is decreased. In EFAD mice, this is combined with decreased net intestinal fat absorption compared with EFAS controls, as determined by the fecal fat balance. In Mdr2–/– mice, however, net intestinal fat absorption is only marginally affected, indicating that despite postprandial hypolipidemia, dietary fat is eventually almost quantitatively absorbed during deficiency of biliary PL. In the present study, we applied these two in vivo models to investigate the relationship between the biliary PL secretion rate and the size and composition of lipoproteins produced by the intestine.
Our data indicate that in the absence of biliary PL secretion, significantly larger lipoproteins are secreted into lymph. The secretion of large lymphatic lipoproteins could be deduced from several independent observations: from particle size determination by dynamic light-scattering techniques, from the relatively increased TG and decreased PL and cholesterol concentrations in lymph of Mdr2–/– mice, and from the calculated lymphatic lipoprotein core-to-surface ratio. Our results on altered intestinal CM formation during biliary PL scarcity in mice are in line with those of Ahn et al. (1), who reported on decreased lymphatic PL and TG output and an increased lymphatic TG-to-PL ratio in zinc-deficient rats, possibly due to the limited supply of biliary PL to enterocytes during zinc deficiency. The lipoproteins secreted by Mdr2–/– mice were continuously larger during active fat absorption, but, strikingly, in nonfasted mice, the size difference was only significant during the first 2 h of lymph cannulation. Because mice had access to chow ad libitum on the night before the lymph cannulation experiments, this phenomenon could refer to a delay in intestinal CM formation or transport into lymph (and subsequently into the plasma compartment) in Mdr2–/– mice, as previously postulated (28). Possibly, the large CM assembled during intraluminal PL deficiency enter the lymph more slowly than during sufficient intestinal PL availability. This speculation, combined with the fact that there is no quantitative lipid malabsorption in Mdr2–/– mice, supports the concept that the amount of intraluminal bile PL is important for the rate of but not for the net intestinal absorption of dietary lipid.
Remarkably, cholesterol was virtually absent in intestinal lipoproteins secreted by Mdr2–/– mice, possibly related to the fact that biliary cholesterol secretion is strongly reduced in these animals. Voshol et al. (27, 28) previously reported on reduced intestinal cholesterol absorption in Mdr2–/– mice, which was postulated to result from increased intestinal de novo cholesterol synthesis combined with accelerated enterocyte desquamation due to exposure to detergent lipid-free bile. However, Kruit et al. (13) recently reported that fractional cholesterol absorption is unimpaired in Mdr2–/– mice. The application of different methods for quantifying cholesterol absorption, i.e., the plasma dual-isotope method and the fecal-dual isotope method, respectively, probably explains the discrepancy between these studies. The reduction in plasma high-density lipoprotein (HDL)-cholesterol levels noted in both reports is conceivably related to altered CM composition and secretion in Mdr2–/– mice, because a major part of HDL is thought to be derived from excess CM surface material (PL and cholesterol) shed during the lipolysis process.
The intestinal requirement of PL for production of CM may be comparable to that of the liver for the assembly and secretion of VLDL. Verkade et al. (26) and Yao and Vance (35) demonstrated in choline-deprived rats that during hepatic PC scarcity, fewer but larger VLDL particles are secreted from the liver. We (31) recently observed that EFAD mice secrete larger VLDL particles from the liver than controls, possibly secondary to hepatic PC depletion resulting from increased biliary PL secretion rates.
Animal models for increased biliary PL secretion are relatively rare. Because the profoundly augmented biliary PL secretion rate in EFAD mice affects hepatic lipoprotein size and is associated with dietary fat malabsorption, we considered the EFAD mouse an intriguing model to further study the potentially organ-specific effects of PL availability on intestinal lipoprotein production. Our data demonstrate that EFAD in mice is associated with lymphatic secretion of considerably smaller CM compared with EFAS controls, as determined by three different particle size estimation techniques. Because EFAD mice secrete larger hepatic VLDL particles than EFAS controls, secretion of smaller lipoproteins is apparently not an intrinsic feature of EFAD. Rather, EFAD differentially affects lipoprotein size in the liver and intestine, with PL availability as the major determinant of lipoprotein size.
Our data are in accordance with the results of Amate et al. (2), who demonstrated that dietary EFA-rich PL administration to piglets resulted in the production of lymphatic lipoproteins with significantly smaller diameters compared with piglets fed EFA-rich TG.
Our present data do not allow conclusions on quantitative lipid absorption due to the highly variable lymph flow rates in these studies. Possibly, continuous enteral lipid administration could overcome this limitation of our murine lymph cannulation model in future experiments.
Although the Mdr2–/– and EFAD mouse models corresponded well regarding postprandial hypolipidemia, they profoundly differed with respect to overall intestinal lipid absorption as determined by fat balance. In EFAD mice, lymphatic TG concentrations were reduced by almost 70%, combined with an abundant lymphatic PL output. In Mdr2–/– mice, in accordance with the profoundly impaired biliary PL availability, lymphatic PL content was twofold lower than lymphatic TG content, supporting the concept that the difference in bile PL (and cholesterol) contents between these two models strongly affect lymphatic dietary fat output.
In Mdr2–/– mice, plasma lipoprotein appearance was delayed, but net intestinal lipid absorption was unaffected as determined by the 72-h fat balance (28). Thus lack of biliary PL seems to impair the kinetics of dietary fat absorption, but compensatory mechanisms apparently exist that account for still almost quantitative fat uptake, such as recruitment of more distal parts of the small intestine for absorption or use of endogenously synthesized PL in enterocytes. EFAD in mice, on the other hand, induces fat malabsorption both in Mdr2+/+ and Mdr2–/– mice, i.e., irrespective of the presence of absence of biliary PL secretion (33). This suggests that during EFAD, intracellular events involved in CM formation or secretion by enterocytes, with EFA-depleted intracellular and plasma membranes, are defective and apparently cannot be compensated for, resulting in fat malabsorption. EFA depletion of biliary PL does not seem to affect net dietary fat absorption, because, although EFAD in mice (33) [as in rats (3, 4)] profoundly decreases EFA acyl chains of bile PL, fat absorption is equally impaired in EFAD Mdr2+/+ and Mdr2–/– mice. In the present study, we chose not to investigate EFAD Mdr2–/– mice, because these animals do not differ in their absence of biliary PL secretion compared with EFAS Mdr2–/– mice.
Baseline lymphatic CM from EFAS mice were significantly larger than those from Mdr2+/+ mice (Figs. 1, 3, and 5B), which can be attributed to the fact that the EFAD and EFAS diets were high-fat diets (34 energy% fat) and the Mdr2+/+ and Mdr2–/– mice were fed standard chow (14 energy% fat).
Hayashi and Tso (10) demonstrated that the number of secreted intestinal lipoproteins remains relatively constant during active lipid absorption, whereas lipoprotein size increases.
Obviously, in Mdr2 deficiency, an increased TG-to-PL ratio is a highly favorable means to maintain TG packaging into CM during lack of surface coat material. In addition, up to 20% of lipoprotein PL is thought to be derived from de novo synthesis (19), and it could be speculated that in Mdr2–/– mice, intestinal PL also partially compensate for the lack of biliary PL for CM surface coating.
Not only biliary but also enterocytic PL are markedly EFA depleted during EFAD (11, 12, 18). The rapid intestinal cellular turnover rate of enterocytes, which is even faster during EFAD (5), renders the membranes of the intestinal mucosa particularly sensitive to altered intraluminal fatty acid availability. Structural membrane modifications, such as an increased degree of PL fatty acid saturation, affect the fluidity of intestinal membranes and may subsequently result in functional alterations of membrane enzymes and transporters, thus impairing intracellular events involved in CM formation or secretion.
The deviant size and composition of lipoproteins secreted during altered biliary PL secretion (like in EFAD or biliary PL deficiency) may not only affect the rate of plasma appearance of dietary TG but also intravascular CM metabolism. Smaller CM have a lower affinity for lipoprotein lipase (LPL) than large ones, and there is competition between small CM and VLDL for available LPL on the capillary endothelium (34). Quarford and Goodman (20) and Chajek-Shaul et al. (6) demonstrated that plasma clearance of large CM, or CM remnants, is substantially more rapid than that of small particles. Similar findings were described by Martins et al. (15), who reported that particle size and number are major determinants of plasma lipoprotein clearance rates. Altered acyl chain composition on the CM surface (derived from EFA-depleted biliary PL) also affects CM clearance (21); CM with saturated PL are cleared more slowly than CM with a high content of polyunsaturated fatty acids in surface PL. Thus, by affecting lipoprotein size and clearance rates, biliary PL may affect plasma and hepatic lipid levels (31). Notwithstanding these circumstantial indications, it should be realized that our study does not allow firm conclusions on whether CM metabolism is indeed affected during altered biliary PL secretion (like EFAD or biliary PL deficiency).
Our data are compatible with the concept that the size of intestinal CM, secreted into lymph under basal conditions or during active lipid absorption, is inversely related to the quantity of biliary PL secretion and that altered lymphatic lipoprotein size is not necessarily related to net dietary fat absorption as determined by the fat balance.
For clinical implications, it might be of interest to know that for cholestatic patients, in contrast to lack of bile salts in the intestinal lumen, intraluminal PL deficiency per se does not necessarily have negative repercussions on dietary fat absorption. On the other hand, a possible consequence of cholestasis, that is, EFAD, does compromise fat absorption. To specify the clinical effects of small lymphatic lipoproteins synthesized during increased bile PL secretion in EFAD, additional studies would be required, yet prevention or treatment of EFAD in cholestatic patients could contribute to improved dietary fat absorption and subsequent metabolism.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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-linolenic acid (C18:3n-6), linoleic acid (C18:2n-6), oleic acid (C18:1n-9), and arachidonic acid (C20:4n-6) are expressed as mol% of total fatty acids. Data represent means ± SD of 7 mice/group. *P < 0.001 for differences between EFAD and EFAS mice. B: lymphatic lipoprotein size (nm), determined by dynamic light scattering, in lymph of EFAD mice and EFAS controls during active lipid absorption. Mesenteric lymph was collected, and lipoprotein size was measured in 30-min fractions before and for 3.5 h after intraduodenal lipid bolus administration. Data represent means ± SD of 6–9 mice/group. *P < 0.05 for differences between EFAD and EFAS mice.