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Intestinal mucins and cholesterol uptake in vitro
34
Biochimica et Biophysics Acta 833 (1985) 34-43 Elsevier
BBA 51821
Intestinal mucins and cholesterol uptake in vitro Robert M. Mayer *, Carleton R. Treadwell, Linda L. Gallo and George V. Vahouny ** Department
ofBiochemistryThe
Key words:
George Washington University, School of Medicine and Health Sciences, 2300 Eye Street, Northwest, Washington, DC 20037 (U.S.A.)
Cholesterol
(Received
July llth,
esterification;
Intestinal
1984)
absorption;
Bile salt; Sialomucin
A mucus material, secreted by intestinal segments, with a high affinity for cholesterol, has been isolated and chemically characterized. The mucin contained 11% carbohydrate, largely as glucosamine, galactose and N-acetylneuraminic acid, and 19% lipid, of which 86% was unesterified fatty acid. The isolated material readily bound cholesterol in a stoichiometric manner. Conditions known to enhance cholesterol absorption in vivo also decreased mucin complexing to cholesterol in vitro. This association of cholesterol and intestinal surface mucin also occurred during incubations of intestinal segments with dispersed cholesterol, resulting in a high level of intestinal adsorption, with little or no cellular absorption of the sterol. However, when cholesterol was solubilized in simple or complex micelles containing bile salts, surface adsorption of cholesterol was reduced and net absorption was increased. The results suggest that surface mucin binding of cholesterol may represent at least one major diffusion limitation to cholesterol absorption in the intestine. Introduction The possible role of the mucin layer of the intestinal epithelial surface as a transport barrier to lipids was originally proposed by Favarger and Metzger [l], who suggested that cholesterol was extensively bound to surface mucoproteins prior to transfer into enterocytes and subsequent cellular transport. Glover et al. [2,3] also demonstrated extensive association of sterol with cellular proteins and suggested that this ‘binding’ precluded the efficient cellular esterification and subsequent transport of the sterol. In limited studies from this laboratory [4,5], it was reported that the uptake of unesterified cholesterol by everted intestinal segments was
* Present address: Department of Chemistry, Ohio versity, Columbus, OH, U.S.A. ** To whom correspondence should be addressed. 0005-2760/85/$03.30
State
0 1985 Elsevier Science Publishers
Uni-
B.V.
largely accounted for by the formation of an adsorption complex with surface-associated mucins. Sterol uptake was reduced by inclusion of bile salts, a phenomenon also observed by others [6-81. These data are, however, inconsistent with the acknowledged role of specific bile salts in stimulating cholesterol absorption in vivo [9,10]. More recent kinetic studies by Smithson et al. [ll] have indicated that the nutrient diffusion barrier at the intestinal surface is not entirely accounted for by the unstirred water layer [12,13], but is associated with the intestinal mucus coat. Similar conclusions have been proposed for a role of mucins in limiting diffusion of hydrogen ions across the gastric mucosa [14,15]. In the present studies, an intestinal mucin has been isolated and characterized, and the ability of the glycoprotein to bind cholesterol has been assessed. In addition, studies have been designed to determine the extent of surface adsorption of cholesterol during in vitro incubations of everted
35
intestinal sacs with various albumin-stabilized micellar preparations of cholesterol.
and
Experimental procedures Isolation
and churacterizution
of intestinul
mucin
Intestinal mucin was obtained by a modification of an earlier procedure [5]. Everted intestinal sacs (20 cm) were prepared from fasting male Wistar rats and were filled with 0.15 M phosphate buffer (pH 6.2) as the serosal fluid. These were incubated for 2 h at 37°C in 25 ml of the same buffer, rinsed with saline and discarded. The incubation solution was centrifuged at 500 x g. This procedure yielded 25530 mg of mucus material per intestine, following lyophylization and dessication over CaCIZ. For analyses of carbohydrate compositon and content, 25-mg samples of the dried preparation were hydrolyzed by refluxing in 5 ml 3 M HCI at 100°C for 12 h. The hydrolysates were neutralized with 6 M NaOH and diluted to 10 ml with distilled water. Aliquots of these hydrolysates were used for determination of all sugar components except sialic acid. These included fucose [16], total hexose [17] corrected for fucose, hexosamines [18], uranic acids [19] and sulfate [20]. Known amounts of authentic sugars were treated in the same manner to correct for procedural losses. For analysis of sialic acid, the mucin was sonicated in 0.05 M H,SO, and hydrolyzed for 1 h at 80°C in glass-stoppered tubes. Sialic acid content was determined by the procedure of Warren [21] and corrected for procedural losses using authentic Nacetylneuraminic acid. Protein was estimated by method of Lowry et al. [22]. Total ash content was determined gravimetrically following 30 min combustion of the mucin sample. Lipid extraction was carried out in 25 vol. of ethanol/diethyl ether (2: 1, v/v) as previously described [9]. Major lipid classes were separated by silicic acid column chromatography [23]. Appropriate fractions were analyzed for cholesterol [24], glyceride glycerol [25], phospholipid phosphorus [26] and unesterified fatty acids [27]. In addition, where indicated, cholesterol was precipitated as the digitonide and purified prior to analysis of cholesterol radioactivity and mass [28].
Cholesterol-mucin complex formation. Two approaches were employed for the assessment of cholesterol-mucin interactions. In both procedures, sonicated suspensions of 39 pmol [4“C]cholesterol (New England Nuclear Corp.) in 3 ml of 0.15 M phosphate buffer (pH 6.2) were mixed with 10 mg of mucin in 1 ml of phosphate buffer and incubations were carried out for 15 min at 25’C. In the first procedure, the insoluble white precipitate was separated by centrifugation at 700 x g. Control incubations included unmixed suspensions of either cholesterol or mucin. The precipitates were resuspended in 0.25 M sucrose, vortexed and layered with petroleum ether. After recentrifugation and removal of solvents, the solvent-layering procedure was repeated up to three times prior to extraction and analysis of the ‘protein-bound’ cholesterol. In the second procedure, the incubation mixture was adjusted to 0.3 M with sucrose to allow immediate flotation of unbound cholesterol and to circumvent possible trapping of cholesterol in the insoluble complex during centrifugation and the remainder of the procedure was as described above. Both approaches gave the same results and the latter was used routinely. Cholesterol uptake by intestinal segments. Everted intestinal sacs (6, 9 or 12 cm, as indicated in individual studies) were prepared from the proximal third of the small intestine of control animals and rats with biliary drainage [29] by the procedure of Wilson and Wiseman [30]. These were randomized for individual studies. Incubation media contained either albumin-stabilized or micellar preparations of cholesterol. The albuminstabilized system consisted of various amounts (lo-26 pmol) of [4-‘4C]cholesterol, sonicated (10 kHz, 15 min) in 4-10 ml 0.15 M phosphate buffer, to which was added 1 ml buffer containing 10 mg bovine serum albumin and 12 mg glucose. This was homogenized prior to use. All incubations were at 37°C under 95% 0,/5% CO,. Micellar [4-i4C]cholesterol was prepared according to the procedure of Thornton et al. [31]. This included sonication of the 0.15 M phosphate buffer containing combinations of bile salt, dioleylphosphatidylcholine and oleic acid in a molar ratio of 10 : 1, respectively, with cholesterol. Preparations were centrifuged at 30000 x g for 30
36
min and the clear infranatant was obtained. Following analyses for lipid content, 10 ml of this preparation were added to 1 ml buffer containing albumin (10 mg) and glucose (12 mg). Incubation conditions are described for individual studies. Except where indicated, results on absorption are expressed as nmol/g wet intestine. It was determined in preliminary studies that the use of [‘4C]inulin as an extracellular space marker [13] did not distinguish between surface adsorption of cholesterol and absorption of the sterol. As an alternative procedure, incubated intestinal segments were rinsed with saline and emptied of the serosal fluid. The segments were split longitudinally and the mucosa was scraped with a microscope slide. The collected material was homogenized in 5 ml 0.25 M sucrose and centrifuged at 2500 X g for 30 min. The pellet and supernatant were individually extracted in 10 vol. boiling acetone/ethanol (1 : 1, v/v) for analysis of cholesterol radioactivity.
TABLE
I
COMPOSITION MUCIN
OF
THE
ISOLATED
INTESTINAL
Analysis of individual components of the mucin (25-mg samples) is described under Experimental procedures. Figures represent means+ SE. of 15-20 analyses except where indicated. Cholesterol was 95.2% unesterified cholesterol. Constituent
f%of Total mucin
Protein Carbohydrate Composition of total carbohydrates glucosamine galactose and mannose sialic acid fucose uranic acids ash sulfate Lipid Composition of total lipid (8) unesterified fatty acids phospholipid triacylglycerols cholesterol
63.5 f 4.0 10.6 a (8) 50.7+_0.5 26.2+ 1.0 13.1 + 2.2 h 8.4*1.0 <1.4h 0.1 b 19.0*0.4
c
s5.1+ 3.7 h 7.lkO.6 3.1 kO.6 4.1 * 0.4
Results Characterization of intestinal mucin The isolation mucins, or preparations delipidated with acetone at -20°C were not solubilized in either saline or Tris/maleate buffer (pH 4.8-7.5). Sonication (10 kHz) in 5% NaCl for 5 min resulted in a turbid solution which was not clarified by centrifugation at 700 X g. This supernatant, containing 90% of the weighed protein, was chemically characterized. As shown in Table I, protein represented almost two-thirds (63.5 f 4.0%), and carbohydrate comprised about 11% of the total mass of the intestinal mucin. The carbohydrates were identified as glucosamine (50.7%), galactose and mannose (26.2%), fucose (8.4%) and N-acetylneuraminic acid (13.1%). Uranic acids comprised only 1.4% of the total carbohydrate, and sulfate was not detected even in pooled samples. The high content of lipid (19% of the total dry weight of the mucin) was largely accounted for by unesterified fatty acids. Of the small amount of cholesterol present in the material (4.1%), over 95% was unesterified. Overall, 92.5% of the dry mass of the mucin could be accounted for by analyses of the individual components.
a Represents the sum of the individual carbohydrate b Represents the mean from 3-5 determinations. ’ Total lipid represents the sum of the individual tions.
fractions. lipid frac-
Cholesterol-mucin complex formation The stoichiometry of cholesterol binding to the mucin was determined at several concentrations of mucin or cholesterol (Fig. 1). Complex formation in the presence of 3.87 pmol sonicated cholesterol was proportional to the mucin concentration (2.2 prnol cholesterol/mg mucin) within the range investigated. The correlation coefficient, calculated by a linear least-squares analysis, was determined to be 0.998. In the presence of 5.45 mg mucin, complex formation was also proportional to cholesterol concentration, attaining saturation (2.3 pmol cholesterol/mg mucin) at 13 prnol cholesterol. The slope of the curve (0.98) suggests that, at concentrations of cholesterol below saturation, there is a tight complex between the sterol and the glycoprotein. Intestinal uptake of cholesterol In the initial studies, everted intestinal cm; 0.5-0.8 g wet weight) were incubated
sacs (9 for 2 h
31
MUCIN (mg)
g E 2
12-
ts 2
lo-
0’
6-
g
6-
Fig. 2. Effects of oleic acid and bile salt concentrations on cholesterol uptake by everted intestinal segments. (A) Intestinal segments (9 cm) were incubated for 2 h at 37°C in 4 ml 0.15 M phosphate buffer (pH 6.2) containing 10 pmol cholesterol, 10 mg albumin, 12 mg glucose and increasing concentrations of olecic acid. (B) Similar incubation media contained O-100 pmol sodium taurocholate. Values represent means for two observations at each concentration.
14-
i 0’
4
=
2-
2
4
6
6
10
CHOLESTEROL
12
14
26
(fi mol)
Fig. 1. Effect of varying concentrations of cholesterol and mucin on formation of mucin-cholesterol complex. (A) Sonicated dispersions of cholesterol (38.7 pmol) in 3 ml 0.15 M phosphate buffer (pH 6.2) were incubated for 15 min at 25°C with 1 ml phosphate buffer containing increasing amounts of mucin. Methods for isolation, extraction and determination of bound cholesterol are described under Experimental procedures. Data represent means of six determinations. (B) Varying amounts of cholesterol, sonicated in 3 ml phosphate buffer, were incubated with 1 ml preparations of 5.35 mg mucin 15 min at 25’C. Data represent means from three determinations.
at 37°C in 4 ml of an albumin-stabilized medium containing 10 Fmol [4-‘4C]cholesterol, 12 mg glucose and 10 mg albumin. Approx. 8-9 pmol cholesterol/g intestine were recovered with the intestinal tissue (Fig. 2). Addition of incremental amounts of taurocholate (lo-100 pmol) to the incubation media resulted in marked decreases in
cholesterol uptake by everted sacs, attaining levels of less than 1 pmol cholesterol/g intestine at a concentration of 10 mM taurocholate (Fig. 2). Addition of incremental amounts of oleic acid to the homogenized, albumin-stabilized media (Fig. 2) also caused decreased intestinal uptake of cholesterol, attaining a level less than 1 pmol cholesterol uptake/g intestine at an oleic acid concentration of 15 mM. The reduced intestinal uptake of cholesterol from taurocholate-containing media was determined to be a direct effect on the intestine rather than an effect on the solubility and/or availability of cholesterol from micellar media. Intestinal sacs (12 cm) were preincubated for 1 h in 10 ml of the phosphate buffer media containing 5-80 prnol of taurocholate. These were removed, rinsed with buffer and transferred to the albuminstabilized media containing 10 pmol cholesterol. As shown in Fig. 3, cholesterol uptake during the second incubation period (1 h) was decreased in intestinal sacs which had been preincubated with increasing concentrations of the bile salt. Mucosal distribution of cholesterol uptake The procedure for distinguishing ‘adsorbed’ from ‘absorbed’ cholesterol by centrifugation of
6,
4.
2.
I
I
I
20
40
60
1
When sacs were incubated with albumin-stabilized cholesterol and 20 mM taurocholate, 150 nmol (1.8% of uptake) were associated with the 2500 x g supernatant in 60 min (Fig. 4). Based on these and comparable types of studies, cholesterol associated with the 2500 X g sediment was defined as surface-associated or ‘adsorbed’ cholesterol and that the supernatant was taken as cellular or ‘absorbed’ cholesterol. The time-course of cholesterol absorption by intestinal sacs incubated with albumin-stabilized media containing 26 pmol cholesterol and 20 mM taurocholate is shown in Fig. 4. The data were obtained by analysis of duplicate sacs at 30-min intervals during the course of a 2-h incubation at 37°C. Absorption of cholesterol appeared linear during the first hour and, in all subsequent studies, incubations were conducted for 1 h.
60
BILE SALT, ,umol
260 Fig 3. Effect of preincubation of intestinal segments in media containing sodium taurocholate (O-SO pmol) prior to determination of cholesterol uptake. Everted intestinal segments (12 cm) were incubated for 1 h in 10 ml 0.15 M phosphate buffer (pH 6.2) containing amounts of sodium taurocholate indicated. Segments were then transferred to 4 ml media 4 ml of containing 10 pmol of albumin-stabilized cholesterol and were incubated for an additional hour for assay of cholesterol uptake as described in the text. Values represent means for two observations.
208
156
104
mucosal homogenates at 2500 X g for 30 min was tested by various approaches. A complex of [4r4C]cholesterol and 30 mg mucin was prepared by incubation for 15 min at 25°C. Subsequent incubation of this material and sedimentation at 2500 X g for 30 min indicated that only 0.0046% of the cholesterol radioactivity was recovered in the 2500 x g supernatant. Intestinal sacs (12 cm) were incubated for 2 h at 37°C with albumin-stabilized media containing 26 pmol [4-‘4C]cholesterol. Analysis of radioactivity associated with mucosal homogenates of thoroughly rinsed sacs indicated cholesterol ‘ uptake’ to be 19 pmol, or 73% of that in the media. After centrifugation at 2500 X g for 30 min, 18.9 pm01 of cholesterol were recovered with the sedimented cell debris and mucin and only 22 nmol (0.1%) were associated with the supernatant fraction.
52
30
60 INCUBATION
90 TIME,
120
MIN
Fig. 4. Absorption of cholesterol by everted intestinal segments. Everted intestinal segments (12 cm) were incubated for periods of up to 2 h in 10 ml media containing 26 pmol [414C 1cholesterol ) 10 mg albumin, 12 mg glucose and 20 prnol sodium taurocholate. The intestinal segments were rinsed and freed of serosal fluid. The mucosa was scraped, homogenized in 5 ml buffer and centrifuged at 2500 X g for 30 min. Radioactivity in the supernatant was considered as ‘absorbed’ cholesterol. Data are expressed as nmol/g wet weight and represent means from three studies. The bars represent the range of the measurements.
39
Incubations
with micellar cholesterol
bile salt-containing media. Under all conditions, however, cholesterol absorption was improved and, when calculated as a percentage of the total cholesterol associated with intestinal mucosa, the highest levels of absorpiton occurred from bile salt-containing media. When similar incubations were conducted with intestinal sacs from 24-h bile-diverted rats (Table II), the levels of cholesterol adsorption from the albumin-stabilized and the micellar media were comparable to those observed using sacs from normal rats (compare Table II, columns 1 and 4). However, with media containing cholesterol alone, cholesterol with oleic acid or cholesterol with oleic acid and phospholipid, cholesterol adsorption accounted for essentially all of the cholesterol uptake by intestinal sacs. Under these conditions, only lo-20 nmol of the uptake was recovered in the 2500 X g supernatants of mucosal homogenates. With micellar media containing oleic acid and taurocholate, absorbed cholesterol (410 nmol) accounted for 11.4% of total cholesterol uptake by sacs or 12.8% of the cholesterol adsorbed. Incubations of sacs in complete micellar media contain-
To test the extent of adsorption and absorption of cholesterol from various micellar media, intestinal sacs (12 cm) were prepared from untreated rats and from rats which had been subjected to biliary drainage for 24 h prior to use. Incubation media contained albumin and cholesterol alone (26 pmol) or cholesterol with oleic acid (125 pmol), oleic acid and taurocholate (29 pmol), oleic acid and dioleylphosphatidylcholine (1.9 pmol) or a mixture with all components. These were sonicated and, for micellar preparations, the soluble infranatant fraction was obtained by centrifugation at 30000 X g for 30 min. Because of the variable amount of cholesterol in each preparation, all data were normalized to equal concentrations of cholesterol. As summarized in Table II, incubations of control intestinal segments with cholesterol alone for 1 h resulted in extensive adsorption of cholesterol (73%) while the amount absorbed was only 0.3% of that adsorbed. Addition of various combinations of oleic acid, phospholipid and bile salts resulted in a marked reduction in adsorbed cholesterol with the greatest effects occurring with TABLE
II
CHOLESTEROL BILE-DIVERTED
ADSORI’TlON RATS
AND
ABSORPTION
BY EVERTED
INTESTINAL
SEGMENTS
FROM
CONTROL
AND
Everted intestinal segments (12 cm) were incubated for 1 h at 37°C in 0.15 M phosphate buffer media (pH 6.2) containing 10 mg albumin and 26 f.rmol [4-14C]cholesterol and including the indicated additions. Additions included oleic acid (125 amol), dioleyl phosphatidylcholine (1.9 nmol) and/or sodium taurocholate (29 pmol). With the exception of the sonicated cholesterol control, each incubation mixture was sonicated and centrifuged at 30000 X g for 30 min. The soluble infranatant was used for incubations. After incubation the intestinal segments were rinsed and the mucosa was scarped and homogenized in 5 ml of 0.25 M sucrose. Labeled cholesterol in the 2500 x g sediment was considered as adsorbed and that in the supernatant was defined as absorbed. Data represent means from three studies. Addition to cholesterolcontaining media
Normal
Adsorption (pmol)
None Oleic acid Oleic acid + phosphatidylcholine Oleic acid + taurochoke Oleic acid + phosphatidylcholine + taurocholate
rats
Bile-diverted Absorption pm01
Adsorption % of adsorbed
(pmol)
rats Absorption p mol
% of adsorbed
18.9 9.5
0.05 0.31
0.3 3.2
19.7 9.6
0.02 0.01
0.1 0.1
8.0
0.31
3.8
5.7
0.01
0.2
3.7
0.36
9.7
3.2
0.41
12.8
3.0
0.36
12.0
2.3
0.49
21.3
40
ing fatty acid, phospholipid and bile salt resulted in the lowest levels of cholesterol uptake (2.8 pmol) but the highest levels of cholesterol absorption (490 nmol, or 17.3% of the total uptake of cholesterol). To test the relationship between cholesterol absorption from micellar media containing varying amounts of cholesterol, media (10 ml) were prepared cont~ning 125 pmol oleic acid, 1.9 pmol dioleylphosphatidylcholine, 29 pmol sodium taurocholate and varying amounts of cholesterol (1.3-26 pmol). Intestinal sacs (12 cm) were prepared from intestines of normal rats and incubated in duplicate for 1 h at 37°C. As shown in Fig. 5, cholesterol absorption as assessed by recoveries of [4-‘4CJcholesterol in the 2500 x g supernatant of mucosal homogenates was directly proportional to the media cholesterol concentration.
300
z E c
1
Bile acid structure and cholesterol absorption Based on earlier studies comparing individual bile acids and cholesterol absorption in vivo [lo], the same bile acids were tested for effects on cholesterol absorption by everted intestinal sacs. Micellar media were prepared [31] containing 200 pmol of each bile acid, 11 pmol oleic acid and 52 pmol cholesterol in 0.35 M phosphate buffer (pH 6.2). 10 ml of the 30000 X g infranatants containing 15.5-17.1 ,umol cholesterol were mixed with 1 ml buffer containing 10 mg albumin and 1 pmol glucose. Incubations with intestinal sacs from normal rats were encountered for 1 h at 37°C. As shown in Table III, cholesterol absorption from micellar media containing either dehydrocholate or deoxycholate was not better than that obtained with the sonicated, albumin-stabilized preparation of cholesterol (20 nmol or 0.1% of the cholestero1 uptake). However, with media containing either cholate or its glycine or taurine conjugates, cholesterol absorption increased dramatically, with the highest level obtained from media containing taurocholate.
250-
TABLE
III
EFFECT OF VARIOUS BILE SALTS ON CHOLESTEROL ABSOR~ION BY EVERTED INTESTINAL SACS
I 10
5 MEDIA
I 15
CHOLESTEROL.
I 20
I 25
Everted intestinal segments (12 cm) were incubated in 0.15 M phosphate buffer media (pH 6.2) containing 52 pmol [4“C]cholesterol, 71 pmol oleic acid and 200 pmol of the bile salt indicated. Prior to use, the media were sonicated and centrifuged at 30000x g for 30 min. 10 ml of the infranatant were added to 1 ml buffer containing 1 nmol glucose and 10 mg albumin. Incubations were for 1 h at 37°C under 95% 0,/S% CO,. Following incubation, the intestinal mucosa was scraped and homogenized in 5 ml 0.25 M sucrose. Labeled cholesterol in the 2500x g supernatant was considered as absorbed cholesterol. Figures represent means for three studies.
~moi
Fig. 5. Cholesterol absorption by everted intestinal segments. Intestinal segments (12 cm) were incubated in micellar media containing 1.3-26 pmol cholesterol, sodium taurocholate, phosphatidylcholine and oleic acid as described in the text. Incubations were for 60 min at 37*C. Segments were removed, rinsed and cleared of serosal media. The mucosa was scraped and homogenized in 5 ml 0.25 M sucrose. This was centrifuged at 2500x g for 30 min and the supernatant radioactivity was taken to represent absorbed cholesterol. Data for absorption are expressed as nmol/g wet weight.
Bile salt added
None Dehydrocholate Deoxycholate Cholate Gylcholate Taurocholate
Cholesterol
absorption
,umol
%
0.03 0.02 0.02 0.41 0.70 1.03
0.2 0.1 0.1 3.0 4.9 7.4
41
Discussion The obligatory role of bile and of its constituent bile acids for intestinal absorption of cholesterol has long been recognized as necessary for both micellar solubilization of intraluminal lipids [28,32-341 and for activation of the cholesterol esterase of the intestinal mucosa [28,35]. Furthermore, Swell et al. [9] have suggested that specific bile salts are required for promoting the initial transfer of cholesterol across the mucosal diffusion barrier. Westergaard and Dietschy [12] have proposed that the major diffusion barrier to cholesterol absorption in the intestine is an unstirred water layer associated with the epithelial surface. The bile salt-containing micellar phase in the intestinal luman presumably decreases the resistance of this diffusion-limiting layer [6,13] and improves sterol absorption. However, the intestinal surface also contains a variable mucus coat which has been assigned an increasing list of putative functions [36,37]. Among these are cytoprotection of mucosal cells from injury, antigenic responses, and antibacterial and antiviral activities [37]. More recently, various studies [11,14,15] have also emphasized the importance of intestinal surface mucins as a diffusion-limiting barrier in the intestinal tract. These reports, together with earlier studies from this laboratory [4,5] implying an interaction of cholesterol with the intestinal surface, prompted a reinvestigation of the possible role of mucins in limiting cholesterol absorbability. The intestinal mucin which was isolated and chemically characterized has a high content of protein relative to carbohydrate, contains no sulfated sugars and appears to represent a typical sialomucoprotein [15,38-401. The high content of lipid (19%), of which most is unesterified fatty acid, compares favorably with gastric mucin which contains 19% lipid, of which almost half is unesterified fatty acid [15]. The extractability of lipids from both gastric mucin [15] and the mucin analyzed in the present study suggests that these lipids are not covalently bound to the mucin. The isolated mucin has a strong affinity for complexing cholesterol in vitro, and both the protein alone and the mucin-cholesterol complex are readily solubilized by bile salt-containing micellar
media. The in vivo binding of cholesterol by mucin could provide an explanation for the extensive adsorption of cholesterol by everted intestinal sacs which occurs in the absence of bile salts. This type of adsorption has been observed earlier [7] using unabsorbable markers to correct for distribution in extracellular space [13]. However, it is assumed that the distributional and binding characteristics of the extracelular marker and of cholesterol are identical and are not differentially affected during perturbations of the incubation media (e.g., bile salts, fatty acids, etc.). This is probably not the case, since cholesterol interacts with both isolated and intestinal surface mucins. Thus, cholesterol ‘uptake’ by intestinal segments is markedly decreased with increasing concentrations of bile salt [7] or phospholipid [8]. This decreased uptake of cholesterol has been largely attributed to effective competition between micelles and the intestine for the sterol, and has not been considered as the effect of these micellar constituents on the binding characteristics of the surface mucins. As had been reported earlier (see Refs. 4, 5, 7 and S), increased concentrations of media bile salt or oleic acid resulted in dramatic decreases in cholesterol uptake by everted intestinal sacs. However, a similar decrease in cholesterol uptake by sacs was observed when intestinal segments were exposed to increasing concentrations of bile salts and were rinsed prior to studies on cholesterol uptake. These findings in particular suggest that the bile salt specifically altered the surface properties of the intestine rather than simply modifying the solubility characteristics of the sterol. These observations on bile salt and fatty acid inhibition of cholesterol uptake by everted intestinal sacs are, however, inconsistent with the in vivo data demonstrating stimulation of cholesterol absorption by specific bile acids [9,10]. Based on earlier observations [4,5] and those in the present study on mucin-cholesterol interactions, an approach was designed to attempt to differentiate sterol adsorption to the mucosal surface from absorbed cholesterol. This involved sedimentation of mucin-cholesterol complexes in mucosal homogenates of incubated sacs at 2500 x g for 30 min. With this approach, essentially all of the cholesterol complexed to mucin in vitro or cholesterol taken up by intestinal sacs in the ab-
42
sence of bile salts was associated with the 2500 x g pellet. This cholesterol was therefore considered to be adsorbed to the mucosal surface, and not intracellular. It is also important to note that this sterol did not redistribute between cellular organelles following homogenization of the mucosa. Thus, only cholesterol recovered in the 2500 X g supernatant was considered absorbed. This approach circumvented the use of extracellular space markers [6,13], since, except for the unabsorbability of these materials, little is known of their distributional or intestinal surface-binding characteristics. With the current approach, incubation of intestinal sacs in micellar media resulted in decreased adsorption of cholesterol, but increased amounts of cholesterol absorbed, or ‘soluble’, at 2500 X g. Absorption was found to be dependent on incubation time and to be linear with the media cholesterol concentration. The maximum levels of cholesterol absorption observed in the present studies (150-500 nmol/g intestine per h) are comparable to the calculated levels of absorption in vivo from the data of Swell et al. [41]. The importance of bile salts, and specifically cholic acid and its conjugates, was demonstrated by the use of intestinal sacts from rats deprived of bile flow for 24 h. With these preparations, surface absorption of cholesterol was decreased by mixtures of fatty acid, phospholipid and bile salt, but absorption was only improved when the incubation media contained taurocholate. These findings in vitro are in accord with those observed in vivo [9,10,34]. Furthermore, as had been demonstrated earlier in vivo [lo], significant levels of cholesterol absorption by intestinal sacs only occurred from incubation media containing cholic acid. The collective findings in the current in vitro studies are completely compaytible with those obtained in vivo, concerning the importance of specific bile salts in intestinal absorption of cholesterol. The same factors that affect the resistance of the unstirred water layer [6,12,13] also affect the extent of mucin-cholesterol interaction and in the same direction. The formation of a complex between cholesterol and intestinal mucin also has a parallel in the interaction of biliary cholesterol with a glycoprotein secreted by the gall bladder epithelium [42]. In
this organ, bile salts inhibit secretion of an epithelial glycoprotein, which complexes with cholesterol as a nucleating agent in gallstone formation [42]. Thus, interactions of cholesterol with mucins can occur and the role of these surface glycoproteins in intestinal transport of lipids in particular, and nutrients in general, requires additional consideration. Acknowledgements ‘This work was supported in part by grants from USPHS (HL-32982) and the Department of Agriculture (83-0016).
References 1 Favarger, P. and Metzger, E.F. (1952) Helv. Chim. Acta 35, 1811-1890 2 Clover, J. and Green, C. (1957) Biochem. J. 67, 308-316 3 Clover, J., Green, C. and Stainer, D.W. (1959) Biochem. J. 72, 82-87 4 Smith, A.L., Hauk, R. and Treadwell, CR. (1958) Am. J. Physiol. 193, 34-40 5 Smith, A.L. and Treadwell, CR. (1958) Am. J. Physiol. (1958) Am. J. Physiol. 195, 773-778 6 Salee, V.L. and Dietschy, J.M. (1973) J. Lipid Res. 14, 475-484 7 Thomson, A.B.R. (1980) J. Lipid Res. 21, 687-698 8 Thomson, A.B.R. (1981) Lipids 16, 881-887 9 Swell, L., Trout, E.C., Hopper, J.R., Field, H., Jr. and Treadwell, CR. (1958) Proc. Sot. Exp. Biol. Med. 98, 174-176 10 Vahouny, G.V., Gregorian, H.M. and Treadwell, CR. (1959) Proc. Sot. Exp. Biol. Med. 101, 538-540 11 Smithson, K.W., Millar, D.B., Jacobs, L.R. and Gray, G.M. (1981) Science 214, 1241-1244 H. and Dietschy, J.M. (1974) J. Clin. Invest. 12 Westergaard, 54, 718-732 13 Thomson, A.B.R. and Dietschy, J.M. (1991) in Physiology of the Gastrointestinal Tract (Johnson, L.R., ed.), pp. 1147-1220, Raven Press, New York 14 Williams, S.E. and Turnberg, L.A. (1980) Gastroenterology 79, 299-304 15 Sarosiek, J., Slomiany, A., Takgi, A. and Slomiany, B.L. (1984) Biochim. Biophys. Res. Commun. 118, 523-531 Analysis 16 Dische, Z. (1955) in Methods of Biochemical (Click, D., ed.), pp. 313-358, Academic Press, New York 17 Roe, J.H. (1955) J. Biol. Chem. 212, 335-343 18 Schloss, B. (1951) Anal. Chem. 23, 1321-1325 19 Dische, Z. (1947) J. Biol. Chem. 167, 189-198 M.Q. (1954) Anal. Chem. 26. 20 Fritz, J.S. and Freeland, 1593-1595 21 Warren, L. (1959) J. Biol. Chem. 234, 1971-1975
43
22 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 23 Hirsch, J. and Ahrens, E.H., Jr. (1958) J. Biol. Chem. 233, 311-320 24 Sperry. W.M. and Webb, M. (1950) J. Biol. Chem. 187, 97-106 25 Lambert, M. and Neish, A.C. (1950) Can. J. Res. 28. 83-89 26 Fiske, C.H. and Subbarow, F. (1925) J. Biol. Chem. 66, 3755400 27 Dole, V.P. and Meinertz, H. (1960) J. Biol. Chem. 235, 2559-2599 28 Vahouny, G.V., Borja, C.R., Mayer, R.M. and Treadwell, C.R. (1960) Anal. Biochem. 1, 371-381 29 Borja, C.R., Vahouny, G.V. and Treadwell, C.R. (1964) Am. J. Physiol. 206, 223-228 30 Wilson, T.H. and Wiseman, G. (1954) J. Physiol. 123, 116-125 31 Thornton, A.G., Vahouny, G.V. and Treadwell. C.R. (1968) Proc. Sot. Exp. Biol. Med. 127. 629-632 32 Siperstein. M.D., Chaikoff, I.L. and Reinhardt, W.O. (1952) J. Biol. Chem. 198, 111-114
33 Vahouny, G.V., Woo, C.H. and Treadwell, C.R. (1958) Am. J. Physiol. 193, 41-47 34 Treadwell, C.R. and Vahouny, G.V. (1968) in Handbook of Physiology-Alimentary Canal, pp. 1407-1438, American Physiological Society, Bethesda, MD. 35 Hyun, J., Steinberg, M., Treadwell, C.R. and Vahouny, G.V. (1971) Biochem. Biophys. Res. Commun. 44, 819-825 36 Forstner, G., Sturgess, J. and Forstner. J. (1977) Adv. Exp., Med. Biol. 89, 349-369 37 Cassidy, M.M., Lightfoot, F.G. and Vahouny, G.V. (1981) in Membrane Biophysics. Structure and Function of Epithelia (Dinno, M., ed.), pp. 97-127, Alan R. Liss, Inc., New York 38 Kornfeld, R. and Kornfeld, S. (1976) Ann. Rev. Biochem. 45, 217-237 39 Carleson, D. (1968) J. Biol. Chem. 243, 616-626 40 Bertolini, M. and Pigman, W. (1970) Carb. Res. 14, 53-63 41 Swell, L., Trout, E.C. Field, H., Jr. and Treadwell, C.R. (1959) Ann. N.Y. Acad. Sci. 72, 813-825 42 Lee, S.P., Carey, M.D. and La Mont, J.T. (1981) Science 211, 1429-1431
Biochimica et Biophysics Acta 833 (1985) 34-43 Elsevier
BBA 51821
Intestinal mucins and cholesterol uptake in vitro Robert M. Mayer *, Carleton R. Treadwell, Linda L. Gallo and George V. Vahouny ** Department
ofBiochemistryThe
Key words:
George Washington University, School of Medicine and Health Sciences, 2300 Eye Street, Northwest, Washington, DC 20037 (U.S.A.)
Cholesterol
(Received
July llth,
esterification;
Intestinal
1984)
absorption;
Bile salt; Sialomucin
A mucus material, secreted by intestinal segments, with a high affinity for cholesterol, has been isolated and chemically characterized. The mucin contained 11% carbohydrate, largely as glucosamine, galactose and N-acetylneuraminic acid, and 19% lipid, of which 86% was unesterified fatty acid. The isolated material readily bound cholesterol in a stoichiometric manner. Conditions known to enhance cholesterol absorption in vivo also decreased mucin complexing to cholesterol in vitro. This association of cholesterol and intestinal surface mucin also occurred during incubations of intestinal segments with dispersed cholesterol, resulting in a high level of intestinal adsorption, with little or no cellular absorption of the sterol. However, when cholesterol was solubilized in simple or complex micelles containing bile salts, surface adsorption of cholesterol was reduced and net absorption was increased. The results suggest that surface mucin binding of cholesterol may represent at least one major diffusion limitation to cholesterol absorption in the intestine. Introduction The possible role of the mucin layer of the intestinal epithelial surface as a transport barrier to lipids was originally proposed by Favarger and Metzger [l], who suggested that cholesterol was extensively bound to surface mucoproteins prior to transfer into enterocytes and subsequent cellular transport. Glover et al. [2,3] also demonstrated extensive association of sterol with cellular proteins and suggested that this ‘binding’ precluded the efficient cellular esterification and subsequent transport of the sterol. In limited studies from this laboratory [4,5], it was reported that the uptake of unesterified cholesterol by everted intestinal segments was
* Present address: Department of Chemistry, Ohio versity, Columbus, OH, U.S.A. ** To whom correspondence should be addressed. 0005-2760/85/$03.30
State
0 1985 Elsevier Science Publishers
Uni-
B.V.
largely accounted for by the formation of an adsorption complex with surface-associated mucins. Sterol uptake was reduced by inclusion of bile salts, a phenomenon also observed by others [6-81. These data are, however, inconsistent with the acknowledged role of specific bile salts in stimulating cholesterol absorption in vivo [9,10]. More recent kinetic studies by Smithson et al. [ll] have indicated that the nutrient diffusion barrier at the intestinal surface is not entirely accounted for by the unstirred water layer [12,13], but is associated with the intestinal mucus coat. Similar conclusions have been proposed for a role of mucins in limiting diffusion of hydrogen ions across the gastric mucosa [14,15]. In the present studies, an intestinal mucin has been isolated and characterized, and the ability of the glycoprotein to bind cholesterol has been assessed. In addition, studies have been designed to determine the extent of surface adsorption of cholesterol during in vitro incubations of everted
35
intestinal sacs with various albumin-stabilized micellar preparations of cholesterol.
and
Experimental procedures Isolation
and churacterizution
of intestinul
mucin
Intestinal mucin was obtained by a modification of an earlier procedure [5]. Everted intestinal sacs (20 cm) were prepared from fasting male Wistar rats and were filled with 0.15 M phosphate buffer (pH 6.2) as the serosal fluid. These were incubated for 2 h at 37°C in 25 ml of the same buffer, rinsed with saline and discarded. The incubation solution was centrifuged at 500 x g. This procedure yielded 25530 mg of mucus material per intestine, following lyophylization and dessication over CaCIZ. For analyses of carbohydrate compositon and content, 25-mg samples of the dried preparation were hydrolyzed by refluxing in 5 ml 3 M HCI at 100°C for 12 h. The hydrolysates were neutralized with 6 M NaOH and diluted to 10 ml with distilled water. Aliquots of these hydrolysates were used for determination of all sugar components except sialic acid. These included fucose [16], total hexose [17] corrected for fucose, hexosamines [18], uranic acids [19] and sulfate [20]. Known amounts of authentic sugars were treated in the same manner to correct for procedural losses. For analysis of sialic acid, the mucin was sonicated in 0.05 M H,SO, and hydrolyzed for 1 h at 80°C in glass-stoppered tubes. Sialic acid content was determined by the procedure of Warren [21] and corrected for procedural losses using authentic Nacetylneuraminic acid. Protein was estimated by method of Lowry et al. [22]. Total ash content was determined gravimetrically following 30 min combustion of the mucin sample. Lipid extraction was carried out in 25 vol. of ethanol/diethyl ether (2: 1, v/v) as previously described [9]. Major lipid classes were separated by silicic acid column chromatography [23]. Appropriate fractions were analyzed for cholesterol [24], glyceride glycerol [25], phospholipid phosphorus [26] and unesterified fatty acids [27]. In addition, where indicated, cholesterol was precipitated as the digitonide and purified prior to analysis of cholesterol radioactivity and mass [28].
Cholesterol-mucin complex formation. Two approaches were employed for the assessment of cholesterol-mucin interactions. In both procedures, sonicated suspensions of 39 pmol [4“C]cholesterol (New England Nuclear Corp.) in 3 ml of 0.15 M phosphate buffer (pH 6.2) were mixed with 10 mg of mucin in 1 ml of phosphate buffer and incubations were carried out for 15 min at 25’C. In the first procedure, the insoluble white precipitate was separated by centrifugation at 700 x g. Control incubations included unmixed suspensions of either cholesterol or mucin. The precipitates were resuspended in 0.25 M sucrose, vortexed and layered with petroleum ether. After recentrifugation and removal of solvents, the solvent-layering procedure was repeated up to three times prior to extraction and analysis of the ‘protein-bound’ cholesterol. In the second procedure, the incubation mixture was adjusted to 0.3 M with sucrose to allow immediate flotation of unbound cholesterol and to circumvent possible trapping of cholesterol in the insoluble complex during centrifugation and the remainder of the procedure was as described above. Both approaches gave the same results and the latter was used routinely. Cholesterol uptake by intestinal segments. Everted intestinal sacs (6, 9 or 12 cm, as indicated in individual studies) were prepared from the proximal third of the small intestine of control animals and rats with biliary drainage [29] by the procedure of Wilson and Wiseman [30]. These were randomized for individual studies. Incubation media contained either albumin-stabilized or micellar preparations of cholesterol. The albuminstabilized system consisted of various amounts (lo-26 pmol) of [4-‘4C]cholesterol, sonicated (10 kHz, 15 min) in 4-10 ml 0.15 M phosphate buffer, to which was added 1 ml buffer containing 10 mg bovine serum albumin and 12 mg glucose. This was homogenized prior to use. All incubations were at 37°C under 95% 0,/5% CO,. Micellar [4-i4C]cholesterol was prepared according to the procedure of Thornton et al. [31]. This included sonication of the 0.15 M phosphate buffer containing combinations of bile salt, dioleylphosphatidylcholine and oleic acid in a molar ratio of 10 : 1, respectively, with cholesterol. Preparations were centrifuged at 30000 x g for 30
36
min and the clear infranatant was obtained. Following analyses for lipid content, 10 ml of this preparation were added to 1 ml buffer containing albumin (10 mg) and glucose (12 mg). Incubation conditions are described for individual studies. Except where indicated, results on absorption are expressed as nmol/g wet intestine. It was determined in preliminary studies that the use of [‘4C]inulin as an extracellular space marker [13] did not distinguish between surface adsorption of cholesterol and absorption of the sterol. As an alternative procedure, incubated intestinal segments were rinsed with saline and emptied of the serosal fluid. The segments were split longitudinally and the mucosa was scraped with a microscope slide. The collected material was homogenized in 5 ml 0.25 M sucrose and centrifuged at 2500 X g for 30 min. The pellet and supernatant were individually extracted in 10 vol. boiling acetone/ethanol (1 : 1, v/v) for analysis of cholesterol radioactivity.
TABLE
I
COMPOSITION MUCIN
OF
THE
ISOLATED
INTESTINAL
Analysis of individual components of the mucin (25-mg samples) is described under Experimental procedures. Figures represent means+ SE. of 15-20 analyses except where indicated. Cholesterol was 95.2% unesterified cholesterol. Constituent
f%of Total mucin
Protein Carbohydrate Composition of total carbohydrates glucosamine galactose and mannose sialic acid fucose uranic acids ash sulfate Lipid Composition of total lipid (8) unesterified fatty acids phospholipid triacylglycerols cholesterol
63.5 f 4.0 10.6 a (8) 50.7+_0.5 26.2+ 1.0 13.1 + 2.2 h 8.4*1.0 <1.4h 0.1 b 19.0*0.4
c
s5.1+ 3.7 h 7.lkO.6 3.1 kO.6 4.1 * 0.4
Results Characterization of intestinal mucin The isolation mucins, or preparations delipidated with acetone at -20°C were not solubilized in either saline or Tris/maleate buffer (pH 4.8-7.5). Sonication (10 kHz) in 5% NaCl for 5 min resulted in a turbid solution which was not clarified by centrifugation at 700 X g. This supernatant, containing 90% of the weighed protein, was chemically characterized. As shown in Table I, protein represented almost two-thirds (63.5 f 4.0%), and carbohydrate comprised about 11% of the total mass of the intestinal mucin. The carbohydrates were identified as glucosamine (50.7%), galactose and mannose (26.2%), fucose (8.4%) and N-acetylneuraminic acid (13.1%). Uranic acids comprised only 1.4% of the total carbohydrate, and sulfate was not detected even in pooled samples. The high content of lipid (19% of the total dry weight of the mucin) was largely accounted for by unesterified fatty acids. Of the small amount of cholesterol present in the material (4.1%), over 95% was unesterified. Overall, 92.5% of the dry mass of the mucin could be accounted for by analyses of the individual components.
a Represents the sum of the individual carbohydrate b Represents the mean from 3-5 determinations. ’ Total lipid represents the sum of the individual tions.
fractions. lipid frac-
Cholesterol-mucin complex formation The stoichiometry of cholesterol binding to the mucin was determined at several concentrations of mucin or cholesterol (Fig. 1). Complex formation in the presence of 3.87 pmol sonicated cholesterol was proportional to the mucin concentration (2.2 prnol cholesterol/mg mucin) within the range investigated. The correlation coefficient, calculated by a linear least-squares analysis, was determined to be 0.998. In the presence of 5.45 mg mucin, complex formation was also proportional to cholesterol concentration, attaining saturation (2.3 pmol cholesterol/mg mucin) at 13 prnol cholesterol. The slope of the curve (0.98) suggests that, at concentrations of cholesterol below saturation, there is a tight complex between the sterol and the glycoprotein. Intestinal uptake of cholesterol In the initial studies, everted intestinal cm; 0.5-0.8 g wet weight) were incubated
sacs (9 for 2 h
31
MUCIN (mg)
g E 2
12-
ts 2
lo-
0’
6-
g
6-
Fig. 2. Effects of oleic acid and bile salt concentrations on cholesterol uptake by everted intestinal segments. (A) Intestinal segments (9 cm) were incubated for 2 h at 37°C in 4 ml 0.15 M phosphate buffer (pH 6.2) containing 10 pmol cholesterol, 10 mg albumin, 12 mg glucose and increasing concentrations of olecic acid. (B) Similar incubation media contained O-100 pmol sodium taurocholate. Values represent means for two observations at each concentration.
14-
i 0’
4
=
2-
2
4
6
6
10
CHOLESTEROL
12
14
26
(fi mol)
Fig. 1. Effect of varying concentrations of cholesterol and mucin on formation of mucin-cholesterol complex. (A) Sonicated dispersions of cholesterol (38.7 pmol) in 3 ml 0.15 M phosphate buffer (pH 6.2) were incubated for 15 min at 25°C with 1 ml phosphate buffer containing increasing amounts of mucin. Methods for isolation, extraction and determination of bound cholesterol are described under Experimental procedures. Data represent means of six determinations. (B) Varying amounts of cholesterol, sonicated in 3 ml phosphate buffer, were incubated with 1 ml preparations of 5.35 mg mucin 15 min at 25’C. Data represent means from three determinations.
at 37°C in 4 ml of an albumin-stabilized medium containing 10 Fmol [4-‘4C]cholesterol, 12 mg glucose and 10 mg albumin. Approx. 8-9 pmol cholesterol/g intestine were recovered with the intestinal tissue (Fig. 2). Addition of incremental amounts of taurocholate (lo-100 pmol) to the incubation media resulted in marked decreases in
cholesterol uptake by everted sacs, attaining levels of less than 1 pmol cholesterol/g intestine at a concentration of 10 mM taurocholate (Fig. 2). Addition of incremental amounts of oleic acid to the homogenized, albumin-stabilized media (Fig. 2) also caused decreased intestinal uptake of cholesterol, attaining a level less than 1 pmol cholesterol uptake/g intestine at an oleic acid concentration of 15 mM. The reduced intestinal uptake of cholesterol from taurocholate-containing media was determined to be a direct effect on the intestine rather than an effect on the solubility and/or availability of cholesterol from micellar media. Intestinal sacs (12 cm) were preincubated for 1 h in 10 ml of the phosphate buffer media containing 5-80 prnol of taurocholate. These were removed, rinsed with buffer and transferred to the albuminstabilized media containing 10 pmol cholesterol. As shown in Fig. 3, cholesterol uptake during the second incubation period (1 h) was decreased in intestinal sacs which had been preincubated with increasing concentrations of the bile salt. Mucosal distribution of cholesterol uptake The procedure for distinguishing ‘adsorbed’ from ‘absorbed’ cholesterol by centrifugation of
6,
4.
2.
I
I
I
20
40
60
1
When sacs were incubated with albumin-stabilized cholesterol and 20 mM taurocholate, 150 nmol (1.8% of uptake) were associated with the 2500 x g supernatant in 60 min (Fig. 4). Based on these and comparable types of studies, cholesterol associated with the 2500 X g sediment was defined as surface-associated or ‘adsorbed’ cholesterol and that the supernatant was taken as cellular or ‘absorbed’ cholesterol. The time-course of cholesterol absorption by intestinal sacs incubated with albumin-stabilized media containing 26 pmol cholesterol and 20 mM taurocholate is shown in Fig. 4. The data were obtained by analysis of duplicate sacs at 30-min intervals during the course of a 2-h incubation at 37°C. Absorption of cholesterol appeared linear during the first hour and, in all subsequent studies, incubations were conducted for 1 h.
60
BILE SALT, ,umol
260 Fig 3. Effect of preincubation of intestinal segments in media containing sodium taurocholate (O-SO pmol) prior to determination of cholesterol uptake. Everted intestinal segments (12 cm) were incubated for 1 h in 10 ml 0.15 M phosphate buffer (pH 6.2) containing amounts of sodium taurocholate indicated. Segments were then transferred to 4 ml media 4 ml of containing 10 pmol of albumin-stabilized cholesterol and were incubated for an additional hour for assay of cholesterol uptake as described in the text. Values represent means for two observations.
208
156
104
mucosal homogenates at 2500 X g for 30 min was tested by various approaches. A complex of [4r4C]cholesterol and 30 mg mucin was prepared by incubation for 15 min at 25°C. Subsequent incubation of this material and sedimentation at 2500 X g for 30 min indicated that only 0.0046% of the cholesterol radioactivity was recovered in the 2500 x g supernatant. Intestinal sacs (12 cm) were incubated for 2 h at 37°C with albumin-stabilized media containing 26 pmol [4-‘4C]cholesterol. Analysis of radioactivity associated with mucosal homogenates of thoroughly rinsed sacs indicated cholesterol ‘ uptake’ to be 19 pmol, or 73% of that in the media. After centrifugation at 2500 X g for 30 min, 18.9 pm01 of cholesterol were recovered with the sedimented cell debris and mucin and only 22 nmol (0.1%) were associated with the supernatant fraction.
52
30
60 INCUBATION
90 TIME,
120
MIN
Fig. 4. Absorption of cholesterol by everted intestinal segments. Everted intestinal segments (12 cm) were incubated for periods of up to 2 h in 10 ml media containing 26 pmol [414C 1cholesterol ) 10 mg albumin, 12 mg glucose and 20 prnol sodium taurocholate. The intestinal segments were rinsed and freed of serosal fluid. The mucosa was scraped, homogenized in 5 ml buffer and centrifuged at 2500 X g for 30 min. Radioactivity in the supernatant was considered as ‘absorbed’ cholesterol. Data are expressed as nmol/g wet weight and represent means from three studies. The bars represent the range of the measurements.
39
Incubations
with micellar cholesterol
bile salt-containing media. Under all conditions, however, cholesterol absorption was improved and, when calculated as a percentage of the total cholesterol associated with intestinal mucosa, the highest levels of absorpiton occurred from bile salt-containing media. When similar incubations were conducted with intestinal sacs from 24-h bile-diverted rats (Table II), the levels of cholesterol adsorption from the albumin-stabilized and the micellar media were comparable to those observed using sacs from normal rats (compare Table II, columns 1 and 4). However, with media containing cholesterol alone, cholesterol with oleic acid or cholesterol with oleic acid and phospholipid, cholesterol adsorption accounted for essentially all of the cholesterol uptake by intestinal sacs. Under these conditions, only lo-20 nmol of the uptake was recovered in the 2500 X g supernatants of mucosal homogenates. With micellar media containing oleic acid and taurocholate, absorbed cholesterol (410 nmol) accounted for 11.4% of total cholesterol uptake by sacs or 12.8% of the cholesterol adsorbed. Incubations of sacs in complete micellar media contain-
To test the extent of adsorption and absorption of cholesterol from various micellar media, intestinal sacs (12 cm) were prepared from untreated rats and from rats which had been subjected to biliary drainage for 24 h prior to use. Incubation media contained albumin and cholesterol alone (26 pmol) or cholesterol with oleic acid (125 pmol), oleic acid and taurocholate (29 pmol), oleic acid and dioleylphosphatidylcholine (1.9 pmol) or a mixture with all components. These were sonicated and, for micellar preparations, the soluble infranatant fraction was obtained by centrifugation at 30000 X g for 30 min. Because of the variable amount of cholesterol in each preparation, all data were normalized to equal concentrations of cholesterol. As summarized in Table II, incubations of control intestinal segments with cholesterol alone for 1 h resulted in extensive adsorption of cholesterol (73%) while the amount absorbed was only 0.3% of that adsorbed. Addition of various combinations of oleic acid, phospholipid and bile salts resulted in a marked reduction in adsorbed cholesterol with the greatest effects occurring with TABLE
II
CHOLESTEROL BILE-DIVERTED
ADSORI’TlON RATS
AND
ABSORPTION
BY EVERTED
INTESTINAL
SEGMENTS
FROM
CONTROL
AND
Everted intestinal segments (12 cm) were incubated for 1 h at 37°C in 0.15 M phosphate buffer media (pH 6.2) containing 10 mg albumin and 26 f.rmol [4-14C]cholesterol and including the indicated additions. Additions included oleic acid (125 amol), dioleyl phosphatidylcholine (1.9 nmol) and/or sodium taurocholate (29 pmol). With the exception of the sonicated cholesterol control, each incubation mixture was sonicated and centrifuged at 30000 X g for 30 min. The soluble infranatant was used for incubations. After incubation the intestinal segments were rinsed and the mucosa was scarped and homogenized in 5 ml of 0.25 M sucrose. Labeled cholesterol in the 2500 x g sediment was considered as adsorbed and that in the supernatant was defined as absorbed. Data represent means from three studies. Addition to cholesterolcontaining media
Normal
Adsorption (pmol)
None Oleic acid Oleic acid + phosphatidylcholine Oleic acid + taurochoke Oleic acid + phosphatidylcholine + taurocholate
rats
Bile-diverted Absorption pm01
Adsorption % of adsorbed
(pmol)
rats Absorption p mol
% of adsorbed
18.9 9.5
0.05 0.31
0.3 3.2
19.7 9.6
0.02 0.01
0.1 0.1
8.0
0.31
3.8
5.7
0.01
0.2
3.7
0.36
9.7
3.2
0.41
12.8
3.0
0.36
12.0
2.3
0.49
21.3
40
ing fatty acid, phospholipid and bile salt resulted in the lowest levels of cholesterol uptake (2.8 pmol) but the highest levels of cholesterol absorption (490 nmol, or 17.3% of the total uptake of cholesterol). To test the relationship between cholesterol absorption from micellar media containing varying amounts of cholesterol, media (10 ml) were prepared cont~ning 125 pmol oleic acid, 1.9 pmol dioleylphosphatidylcholine, 29 pmol sodium taurocholate and varying amounts of cholesterol (1.3-26 pmol). Intestinal sacs (12 cm) were prepared from intestines of normal rats and incubated in duplicate for 1 h at 37°C. As shown in Fig. 5, cholesterol absorption as assessed by recoveries of [4-‘4CJcholesterol in the 2500 x g supernatant of mucosal homogenates was directly proportional to the media cholesterol concentration.
300
z E c
1
Bile acid structure and cholesterol absorption Based on earlier studies comparing individual bile acids and cholesterol absorption in vivo [lo], the same bile acids were tested for effects on cholesterol absorption by everted intestinal sacs. Micellar media were prepared [31] containing 200 pmol of each bile acid, 11 pmol oleic acid and 52 pmol cholesterol in 0.35 M phosphate buffer (pH 6.2). 10 ml of the 30000 X g infranatants containing 15.5-17.1 ,umol cholesterol were mixed with 1 ml buffer containing 10 mg albumin and 1 pmol glucose. Incubations with intestinal sacs from normal rats were encountered for 1 h at 37°C. As shown in Table III, cholesterol absorption from micellar media containing either dehydrocholate or deoxycholate was not better than that obtained with the sonicated, albumin-stabilized preparation of cholesterol (20 nmol or 0.1% of the cholestero1 uptake). However, with media containing either cholate or its glycine or taurine conjugates, cholesterol absorption increased dramatically, with the highest level obtained from media containing taurocholate.
250-
TABLE
III
EFFECT OF VARIOUS BILE SALTS ON CHOLESTEROL ABSOR~ION BY EVERTED INTESTINAL SACS
I 10
5 MEDIA
I 15
CHOLESTEROL.
I 20
I 25
Everted intestinal segments (12 cm) were incubated in 0.15 M phosphate buffer media (pH 6.2) containing 52 pmol [4“C]cholesterol, 71 pmol oleic acid and 200 pmol of the bile salt indicated. Prior to use, the media were sonicated and centrifuged at 30000x g for 30 min. 10 ml of the infranatant were added to 1 ml buffer containing 1 nmol glucose and 10 mg albumin. Incubations were for 1 h at 37°C under 95% 0,/S% CO,. Following incubation, the intestinal mucosa was scraped and homogenized in 5 ml 0.25 M sucrose. Labeled cholesterol in the 2500x g supernatant was considered as absorbed cholesterol. Figures represent means for three studies.
~moi
Fig. 5. Cholesterol absorption by everted intestinal segments. Intestinal segments (12 cm) were incubated in micellar media containing 1.3-26 pmol cholesterol, sodium taurocholate, phosphatidylcholine and oleic acid as described in the text. Incubations were for 60 min at 37*C. Segments were removed, rinsed and cleared of serosal media. The mucosa was scraped and homogenized in 5 ml 0.25 M sucrose. This was centrifuged at 2500x g for 30 min and the supernatant radioactivity was taken to represent absorbed cholesterol. Data for absorption are expressed as nmol/g wet weight.
Bile salt added
None Dehydrocholate Deoxycholate Cholate Gylcholate Taurocholate
Cholesterol
absorption
,umol
%
0.03 0.02 0.02 0.41 0.70 1.03
0.2 0.1 0.1 3.0 4.9 7.4
41
Discussion The obligatory role of bile and of its constituent bile acids for intestinal absorption of cholesterol has long been recognized as necessary for both micellar solubilization of intraluminal lipids [28,32-341 and for activation of the cholesterol esterase of the intestinal mucosa [28,35]. Furthermore, Swell et al. [9] have suggested that specific bile salts are required for promoting the initial transfer of cholesterol across the mucosal diffusion barrier. Westergaard and Dietschy [12] have proposed that the major diffusion barrier to cholesterol absorption in the intestine is an unstirred water layer associated with the epithelial surface. The bile salt-containing micellar phase in the intestinal luman presumably decreases the resistance of this diffusion-limiting layer [6,13] and improves sterol absorption. However, the intestinal surface also contains a variable mucus coat which has been assigned an increasing list of putative functions [36,37]. Among these are cytoprotection of mucosal cells from injury, antigenic responses, and antibacterial and antiviral activities [37]. More recently, various studies [11,14,15] have also emphasized the importance of intestinal surface mucins as a diffusion-limiting barrier in the intestinal tract. These reports, together with earlier studies from this laboratory [4,5] implying an interaction of cholesterol with the intestinal surface, prompted a reinvestigation of the possible role of mucins in limiting cholesterol absorbability. The intestinal mucin which was isolated and chemically characterized has a high content of protein relative to carbohydrate, contains no sulfated sugars and appears to represent a typical sialomucoprotein [15,38-401. The high content of lipid (19%), of which most is unesterified fatty acid, compares favorably with gastric mucin which contains 19% lipid, of which almost half is unesterified fatty acid [15]. The extractability of lipids from both gastric mucin [15] and the mucin analyzed in the present study suggests that these lipids are not covalently bound to the mucin. The isolated mucin has a strong affinity for complexing cholesterol in vitro, and both the protein alone and the mucin-cholesterol complex are readily solubilized by bile salt-containing micellar
media. The in vivo binding of cholesterol by mucin could provide an explanation for the extensive adsorption of cholesterol by everted intestinal sacs which occurs in the absence of bile salts. This type of adsorption has been observed earlier [7] using unabsorbable markers to correct for distribution in extracellular space [13]. However, it is assumed that the distributional and binding characteristics of the extracelular marker and of cholesterol are identical and are not differentially affected during perturbations of the incubation media (e.g., bile salts, fatty acids, etc.). This is probably not the case, since cholesterol interacts with both isolated and intestinal surface mucins. Thus, cholesterol ‘uptake’ by intestinal segments is markedly decreased with increasing concentrations of bile salt [7] or phospholipid [8]. This decreased uptake of cholesterol has been largely attributed to effective competition between micelles and the intestine for the sterol, and has not been considered as the effect of these micellar constituents on the binding characteristics of the surface mucins. As had been reported earlier (see Refs. 4, 5, 7 and S), increased concentrations of media bile salt or oleic acid resulted in dramatic decreases in cholesterol uptake by everted intestinal sacs. However, a similar decrease in cholesterol uptake by sacs was observed when intestinal segments were exposed to increasing concentrations of bile salts and were rinsed prior to studies on cholesterol uptake. These findings in particular suggest that the bile salt specifically altered the surface properties of the intestine rather than simply modifying the solubility characteristics of the sterol. These observations on bile salt and fatty acid inhibition of cholesterol uptake by everted intestinal sacs are, however, inconsistent with the in vivo data demonstrating stimulation of cholesterol absorption by specific bile acids [9,10]. Based on earlier observations [4,5] and those in the present study on mucin-cholesterol interactions, an approach was designed to attempt to differentiate sterol adsorption to the mucosal surface from absorbed cholesterol. This involved sedimentation of mucin-cholesterol complexes in mucosal homogenates of incubated sacs at 2500 x g for 30 min. With this approach, essentially all of the cholesterol complexed to mucin in vitro or cholesterol taken up by intestinal sacs in the ab-
42
sence of bile salts was associated with the 2500 x g pellet. This cholesterol was therefore considered to be adsorbed to the mucosal surface, and not intracellular. It is also important to note that this sterol did not redistribute between cellular organelles following homogenization of the mucosa. Thus, only cholesterol recovered in the 2500 X g supernatant was considered absorbed. This approach circumvented the use of extracellular space markers [6,13], since, except for the unabsorbability of these materials, little is known of their distributional or intestinal surface-binding characteristics. With the current approach, incubation of intestinal sacs in micellar media resulted in decreased adsorption of cholesterol, but increased amounts of cholesterol absorbed, or ‘soluble’, at 2500 X g. Absorption was found to be dependent on incubation time and to be linear with the media cholesterol concentration. The maximum levels of cholesterol absorption observed in the present studies (150-500 nmol/g intestine per h) are comparable to the calculated levels of absorption in vivo from the data of Swell et al. [41]. The importance of bile salts, and specifically cholic acid and its conjugates, was demonstrated by the use of intestinal sacts from rats deprived of bile flow for 24 h. With these preparations, surface absorption of cholesterol was decreased by mixtures of fatty acid, phospholipid and bile salt, but absorption was only improved when the incubation media contained taurocholate. These findings in vitro are in accord with those observed in vivo [9,10,34]. Furthermore, as had been demonstrated earlier in vivo [lo], significant levels of cholesterol absorption by intestinal sacs only occurred from incubation media containing cholic acid. The collective findings in the current in vitro studies are completely compaytible with those obtained in vivo, concerning the importance of specific bile salts in intestinal absorption of cholesterol. The same factors that affect the resistance of the unstirred water layer [6,12,13] also affect the extent of mucin-cholesterol interaction and in the same direction. The formation of a complex between cholesterol and intestinal mucin also has a parallel in the interaction of biliary cholesterol with a glycoprotein secreted by the gall bladder epithelium [42]. In
this organ, bile salts inhibit secretion of an epithelial glycoprotein, which complexes with cholesterol as a nucleating agent in gallstone formation [42]. Thus, interactions of cholesterol with mucins can occur and the role of these surface glycoproteins in intestinal transport of lipids in particular, and nutrients in general, requires additional consideration. Acknowledgements ‘This work was supported in part by grants from USPHS (HL-32982) and the Department of Agriculture (83-0016).
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