1Department of Medicine, Liver Center and Gastroenterology Division, Beth Israel Deaconess Medical Center, Harvard Medical School and Harvard Digestive Diseases Center, Boston, Massachusetts 02215; and 2Mayo Clinic College of Medicine, Department of Biochemistry and Molecular Biology and Tumor Biology Program, Scottsdale, Arizona 85259
Submitted 5 March 2004 ; accepted in final form 8 April 2004
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ABSTRACT |
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ATP-binding cassette (transporter); intestinal uptake; surface mucous coat; mucin gel; micelle; nutrition
Intestinal mucins are a heterogeneous family of O-linked glycoproteins produced and secreted from mucous cells that are present in the entire gastrointestinal tract. The hydrophilic and viscoelastic nature of mucin molecules and their tendency to interact with specific luminal components, in particular, with a high affinity for cholesterol, are of critical importance for their physiological functions. On the basis of their physical-chemical states and physiological functions, intestinal mucins are usually subdivided into two groups: epithelial and gel-forming mucins (28). It has been proposed that the epithelial mucins produced by mucin gene, (Muc)1, -3, and -4, do not seem to form aggregates and are integral membrane glycoproteins located on the apical surface of epithelial cells. The gel-forming mucins, Muc2, Muc5ac, and Muc5b, secreted by specialized intestinal mucin-producing cells, are found as ingredients of the protective mucosal surface layer.
It has been observed that mucin secretion in the small intestine is regulated by multiple mucin genes (11, 18, 21, 43), and the Muc1 gene determines the presence of a membrane-associated mucin that is abundant in the secretory epithelia such as the small intestine (4, 11). In an early study, Spicer et al. (35) found that mice deficient in Muc1 mucin appear to develop normally and are healthy and fertile as well as that these mice show a significantly slower growth rate of primary breast tumors. In cystic fibrosis, mucin accumulation is abnormally high, resulting in severe intestinal obstruction; however, disruption of the Muc1 gene significantly decreases total amounts of mucins in the gastrointestinal tract of cystic fibrosis mice (29). These observations suggest that the Muc1 gene may have an important effect on the diffusion barrier of the small intestine. To explore the contributions of individual mucin genes to intestinal cholesterol metabolism, we investigated cholesterol uptake and absorption and fatty acid uptake by the enterocyte in male mice with targeted disruption of the Muc1 gene. Our results show that the intestinal uptake and absorption of cholesterol but not fatty acid are significantly decreased in Muc1-deficient (Muc1/) mice compared with the wild-type mice, and disruption of the Muc1 gene does not impair either expression levels of the genes that encode intestinal sterol efflux transporters Abcg5 and Abcg8 and fatty acid transporter Fatp4 or normal small intestinal motility. This study also provides a basic framework for investigating how other mucin genes regulate intestinal cholesterol absorption in the mouse.
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MATERIALS AND METHODS |
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Radioisotopes [1,2-3H]cholesterol, [4-14C]cholesterol, and [9,10-3H(N)]palmitic acid were purchased from New England Nuclear Life Science Products (Boston, MA), and [5,6-3H]sitostanol was from American Radiolabeled Chemicals (St. Louis, MO). Intralipid (20%, wt/vol) was purchased from Pharmacia (Clayton, NC), and medium-chain triglyceride was from Mead Johnson (Evansville, IN).
Animals and Diets
Muc1/ mice in an FVB/NJ background were generated by targeting mutation of the Muc1 gene (35). The wild-type mice displaying normal Muc1 expression on the same FVB/NJ background were purchased from the Jackson Laboratory (Bar Harbor, ME). We studied male Muc1/ and wild-type mice at 810 wk of age. Animals were maintained in a temperature-controlled room (22 ± 1°C) with a 12:12-h light-dark cycle. Mice were fed ad libitum normal rodent chow (Harlan Teklad Laboratory Animal Diets, Madison, WI) containing trace (<0.02%) amounts of cholesterol and had unrestricted access to drinking water. All procedures were in accordance with current National Institutes of Health guidelines and were approved by the Institutional Animal Care and Use Committee of Harvard University.
Measurement of Intestinal Cholesterol Absorption
Fecal dual-isotope ratio method. Nonfasted and nonanesthetized Muc1/ and wild-type mice (n = 10 per group) were given by gavage an intragastric bolus of 150 µl medium-chain triglyceride containing a mixture of 1 µCi [14C]cholesterol and 2 µCi [3H]sitostanol. Mice were then transferred to individual cages with wire mesh bottoms where they continued to ingest the chow diet for the next 4 days. During this period, mouse feces were collected daily. The ratio of the two radiolabels in the fecal extract from the 4-day pooled feces and the dosing mixture were used for calculating the percent cholesterol absorption (45, 48).
Plasma dual-isotope ratio method. Additional groups of chow-fed mice (n = 10 per group) were injected intravenously with 2.5 µCi [3H]cholesterol in 100 µl Intralipid. Immediately, each animal was given by gavage an intragastric bolus of 1 µCi [14C]cholesterol in 150 µl medium-chain triglyceride. After the dose, mice were returned to individual cages with wire mesh bottoms where they were free to eat chow for an additional 3 days. After this procedure, the ratio of the two radiolabels in the plasma was assayed by liquid-scintillation spectrometry and the percent cholesterol absorption was calculated (45, 47, 48).
Measurement of Intestinal Uptake of Cholesterol and Fatty Acid
After anesthesia, laparotomy was performed under sterile conditions through an upper midline incision. The duodenum was cannulated with a PE-10 catheter, and the catheter was externalized through the incision and implanted subcutaneously. The abdominal incision was closed tightly with 50 sutures. After 24-h recovery from the surgery and another 12 h fasting (but with water), exactly 2 µCi [14C]cholesterol and 2 µCi [3H]palmitic acid mixed with 100 µl medium-chain triglyceride were injected into the duodenum of the nonanesthetized mice via the duodenal catheter. After the injection, the mice were allowed to move freely in the cage. Exactly 45 min after instillation, the animals were anesthetized with pentobarbital sodium. At laparotomy, the entire small intestine was removed and flushed with taurocholate buffer. After being wet-weighed, the small intestine was cut into three segments with length ratios of 1:3:2 (duodenum/jejunum/ileum). The two radiolabeled sterols were extracted and counted. The radioactivity was used to calculate intestinal cholesterol and fatty acid uptake in vivo, which is expressed as dpm·g tissue1·45 min1.
Measurement of Small Intestinal Transit Times
Small intestinal transit times in chow-fed Muc1/ and wild-type mice (n = 5 per group) were measured according to previously published methods (48). Nonabsorbable [3H]sitostanol is used as a reference marker (48). In brief, 2 µCi [3H]sitostanol in 100 µl of medium-chain triglyceride were instilled into the duodenum via a transabdominal catheter implanted surgically 24 h earlier. Exactly 30 min after instilling, mice were anesthetized with pentobarbital sodium. After the abdomen was opened, the stomach, small and large intestines, and cecum were removed carefully. The small intestine was immediately frozen with liquid nitrogen and cut into 20 segments equally with a scalpel blade. The radioisotope was extracted from individual intestinal segments, and the radioactivity was determined by liquid-scintillation spectrometry. Samples of stomach, cecum, and large intestine were also examined, but none showed appreciable radioactivity. Small intestinal transit time was evaluated by geometric center methods (48).
Quantitative Real-Time PCR Assays
Freshly harvested small intestines were flushed with ice-cold saline and cut into three segments with length ratios of 1:3:2 (duodenum/jejunum/ileum). In the middle of each intestinal segment, 1.5 cm of the duodenal, jejunal, and ileal tissues were cut off, and the tissues from four mice per group were pooled. Total RNA was extracted using RNeasy Midi (Qiagen, Valencia, CA), and reverse-transcription reaction was performed using the SuperScript II First-Strand Synthesis System (Invitrogen, Carlsbad, CA) with 5 µg total RNA and random hexamers to generate cDNA. Primer Express Software (Applied Biosystems, Foster City, CA) was used to design the primers (Table 1) based on sequence data available from GenBank. Real-time PCR assays (50) for all samples were performed in triplicate on a GeneAmp 5700 Sequence Detection System (Applied Biosystems). The real-time PCR reaction contained, in a final volume of 50 µl, 1 µl cDNA, optimized concentration (100300 µM) of each primer, 200 µM concentration of probe, and 25 µl 2x TaqMan Universal PCR Master Mix (Applied Biosystems). Relative mRNA levels were calculated using the threshold cycle of an unknown sample against a standard curve with known copy numbers. To obtain a normalized target value, the target amount was divided by the endogenous reference amount of rodent Gapdh as control.
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Fresh mucins were obtained from the small intestines of chow-fed Muc1/ and wild-type mice, and the mucins from 10 mice per group were pooled. To dissolve possible crystalline and bound lipids, mucins were suspended in 10 vol of 100 mM Tris buffer (pH 7.4) containing 10 mM taurocholate and were washed three times with 100 mM Tris buffer containing taurocholate according to published methods (46). Then, mucins were washed three times with 100 mM Tris buffer (pH 7.4) without taurocholate by centrifugation at 10,000 g for 1 h. With the use of an anti-Muc1 monoclonal antibody (CT2), Muc1 mucin levels were determined by Western blot analyses (32, 51).
Statistical Methods
All data are expressed as means ± SD. Statistically significant differences among groups of mice were assessed by Student's t-test. Analyses were performed with SuperANOVA software (Abacus Concepts, Berkeley, CA). Statistical significance was defined as a two-tailed probability of <0.05.
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RESULTS |
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Figure 1 exhibits the relative mRNA levels for Muc1, Muc2, Muc3, Muc4, Muc5ac, and Muc5b, as measured by quantitative real-time PCR, in the small intestines of Muc1/ and wild-type mice. The data are expressed relative to the level of the Muc1 transcript in the wild-type mice on the chow diet, and its mRNA expression is set at 1. Our results reveal that the wild-type mice displayed relatively high levels of Muc1, Muc2, Muc3, and Muc4 mRNAs and relatively low levels of Muc5ac and Muc5b mRNAs. Of note is that the relative mRNA expression for the Muc1 gene was absent from the small intestines of Muc1/ mice. However, the mRNA expression for other mucin genes was essentially similar between Muc1/ and wild-type mice (Fig. 1). Furthermore, compared with the wild-type mice, the absence of Muc1 protein in the small intestines of Muc1/ mice (Fig. 1, inset), as assayed by Western blot hybridization using an anti-Muc1 monoclonal antibody (32), verified complete knockout of the Muc1 gene.
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Figure 2 shows percent cholesterol absorption, as measured by two independent approaches (45, 47, 48): the plasma (Fig. 2A) and the fecal dual-isotope ratio methods (Fig. 2B) in chow-fed Muc1/ and wild-type mice. Figure 2A displays that intestinal cholesterol absorption (19 ± 3%) in Muc1/ mice is significantly (P < 0.0001) decreased compared with the wild-type mice (37 ± 5%), as measured by the plasma dual-isotope ratio method. As shown in Fig. 2, A and B, no differences in the percentages of cholesterol absorption were found between two methods in both groups of mice (range 1921% in Muc1/ mice and 3742% in the wild-type mice). Although similar amounts of food (3.84.1 g/day) were eaten by both groups of mice, calculation of total cholesterol mass absorbed by the small intestine was significantly (P < 0.01) lower in Muc1/ mice (0.150.17 mg/day) compared with the wild-type mice (
0.280.35 mg/day).
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Figure 3 displays intestinal uptake rates of [14C]cholesterol and [3H]palmitic acid in chow-fed Muc1/ and wild-type mice. Our results show that the absence of the intestinal Muc1 mucin significantly (P < 0.0001) decreased the intestinal uptake rates of radiolabeled cholesterol in Muc1/ mice compared with the wild-type mice (Fig. 3A). In contrast, the intestinal uptake rates of radiolabeled palmitic acid were essentially similar between Muc1/ and wild-type mice (Fig. 3B). This suggests that there may be different regulatory mechanisms responsible for the uptake of cholesterol and fatty acid by the small intestine.
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Figure 4 shows the relative mRNA levels for two genes that encode ATP-binding cassette (Abc) transporters Abcg5 and Abcg8 in the duodenum, jejunum, and ileum of Muc1/ and wild-type mice on the chow diet. Although expression levels of Abcg5 (Fig. 4A) and Abcg8 (Fig. 4B) in the duodenum were slightly lower than those in jejunum and ileum, the relative mRNA levels for Abcg5 and Abcg8 in the jejunum and ileum were comparable in Muc1/ mice and the wild-type mice. Moreover, the absence of the intestinal Muc1 mucin did not influence expression levels of the gene that encodes fatty acid transport protein-4 (Fatp4) in Muc1/ mice, which is essentially similar to those in the wild-type mice (Fig. 4C).
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This was evaluated by the distribution of radioactivity, at 30 min after intraduodenal instillation of radiolabeled sitostanol in medium-chain triglyceride, along the small intestines of Muc1/ and wild-type mice on the chow diet. The geometric center of radiolabeled sitostanol that is used as a marker for a measure of small intestinal transit (48) ranges from 1 to 20 (see MATERIALS AND METHODS). If a value for the geometric center is 1, it shows no transit because the entire marker remains in the duodenum. In contrast, if the geometric center is 20, it indicates very rapid transit of the entire marker into the most distal segment of the ileum. We found that the distribution of radioactivity in the small intestine was essentially similar between Muc1/ and wild-type mice, with peaks between segments 7 and 16. Furthermore, the geometric centers of the distribution profiles of radioactivities within the small intestines were identical in Muc1/ (geometric center = 10.7 ± 0.7) compared with the wild-type mice (geometric center = 10.9 ± 1.0). These results suggest that the absence of the intestinal Muc1 mucin does not impair normal small intestinal motility, and the intestinal transit time is not responsible for the difference in cholesterol absorption efficiency between two groups of mice.
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DISCUSSION |
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Before cholesterol molecules move from the bulk phase of the intestinal contents to the brush border of the enterocyte and interact with a possible cholesterol transporter(s) for uptake and subsequent transport across the apical membrane of the enterocyte, they must pass through the diffusion barrier that includes the unstirred water layer and the intestinal surface mucins. The unstirred water layer is a series of water lamellae at the interface between the bulk water phase of the lumen and the apical membrane of the enterocyte (8, 26, 38, 5255). Diffusion through the unstirred water layer is a relatively slow process for cholesterol molecules that are nearly insoluble in a pure aqueous system. Therefore, after hydrolysis of cholesteryl esters by hydrolytic enzymes, the free cholesterol together with bile salts, ionized and nonionized fatty acids, monoacylglycerides, and lysophospholipids forms mixed micelles in the small intestinal lumen (17, 36). These mixed micelles function as a concentrated reservoir and transport vehicle for cholesterol molecules across the unstirred water layer and the surface mucous coat toward the brush border of the enterocyte to facilitate uptake of monomeric cholesterol by the enterocytes.
Our current study reveals that the small intestines of the wild-type mice displayed a relatively high level of steady-state Muc1 mRNA. In comparison, complete knockout of the Muc1 gene in the small intestines of Muc1/ mice was confirmed by the absence of Muc1 mRNA and protein (Fig. 1). Furthermore, we observed that the epithelium of mouse small intestine was characterized by the expression of a unique pattern of mucin genes, consisting of relatively high levels of Muc1, Muc2, Muc3, and Muc4 mRNAs as well as relatively low levels of Muc5ac and Muc5b mRNAs. Of note is that the expression pattern of small intestinal mucin genes in the mouse is similar to that in the human (5, 6, 18, 19, 33), in whom MUC1, MUC2, MUC3, and MUC4 are the predominant mucin proteins in the small intestine as detected by Northern and Western blot analyses as well as immunohistochemical methods. Furthermore, our data exhibit that mucins in mouse small intestine are highly heterogeneous, suggesting that the mucin layer of the small intestine may be composed of multiple layers. We also found that expression levels of the small intestinal mucin genes, except the Muc1 gene, were essentially similar between Muc1/ and wild-type mice. Thus it is most likely that the diffusion barrier, i.e., the unstirred water layer and the surface mucous coat, may not be influenced by the absence of the intestinal Muc1 mucin, because the Muc1 gene determines only the presence of Muc1 mucin, an epithelial mucin. Accordingly, it is possible that there may not be a significant difference in the movement of cholesterol molecules through the diffusion barrier between Muc1/ and wild-type mice, because the transfer across this barrier is a simple diffusion process in which the rate of movement is determined by the functional thickness of the entire barrier.
The question arises as to how the lack of the intestinal Muc1 mucin significantly reduces the uptake and absorption of cholesterol. Because a thin layer of mucins covers the apical surface of the small intestinal epithelium, physiological levels of the membrane-associated mucin encoded by the Muc1 may be essential for normal expression and function of intestinal sterol transporters. To explore whether the decreased cholesterol absorption in Muc1/ mice is induced by reducing influx, accelerating efflux, or both due to the absence of the Muc1 mucin, we examined expression levels of the Abcg5 and Abcg8 genes and the intestinal uptake rates of radiolabeled cholesterol. It has been proposed that the newly identified intestinal sterol transporters ABCG5 and ABCG8 (3, 25) in humans are apical cholesterol export pumps that efflux cholesterol (and most plant sterols) from enterocytes back to the intestinal lumen and reduce its fractional absorption (56). Their expression levels in the jejunum and ileum may determine, in part, variations in the efficiency of cholesterol absorption in inbred strains of mice and play a major regulatory role in mice challenged with high dietary cholesterol (9). Our data showed that the relative mRNA expression of the genes that encode these two transporters was comparable in Muc1/ and wild-type mice, suggesting that the lack of the Muc1 mucin does not influence their gene expression or physiological function. In contrast, the fact that the intestinal uptake of radiolabeled cholesterol is significantly reduced in Muc1/ mice compared with the wild-type mice implies that a cholesterol influx transporter(s) on the apical brush border membrane of the enterocyte may be involved. More recently, Altmann et al. (2) found that targeted disruption of the NPC1L1 gene encoding the Niemann-Pick C1-like 1 protein induced a significant reduction in intestinal cholesterol absorption in mice. They (2) also observed that ezetimibe lowered cholesterol absorption by 70% in the wild-type mice, and this level of cholesterol absorption was similar to that seen in the NPC1L1 knockout mice not treated with ezetimibe. Thus it was proposed that NPC1L1 could be a sterol influx transporter in the small intestine, which may be critical for intestinal uptake of sterols. Consequently, it is imperative to investigate whether the newly identified intestinal sterol influx transporter NPC1L1 plays a pivotal role in regulating cholesterol and sitosterol absorption and in determining cholesterol absorption efficiency in healthy inbred mice. Furthermore, whether expression levels of the Muc1 gene influence physiological functions of NPC1L1 in mice requires further investigations. Although several other candidate proteins (7, 16, 23, 24, 39) have been proposed for the role of a cholesterol influx transporter(s) in cholesterol absorption efficiency, the exact identify of their physiological functions remains elusive. Nevertheless, our current results suggest that the absence of the Muc1 mucin may impair the functions of the putative apical cholesterol import pump(s) that facilitates cholesterol uptake and absorption by the enterocyte. Moreover, the deficiency of the intestinal Muc1 mucin induced a
50% decrease in cholesterol absorption efficiency in Muc1/ mice, as determined by two independent techniques (Fig. 2), strongly suggesting that there may be one or more additional pathways for cholesterol absorption.
The importance of small intestinal transit times as a determining factor of cholesterol absorption has been observed in human (31) and animal studies (40, 49). Because disruption of the cholecystokinin-1 receptor (Cck-1r) gene impairs small intestinal transit leading to prolonged small intestinal transit times, these dysfunctions of gastrointestinal motility could contribute to increased intestinal cholesterol absorption. It is most likely that longer residence of cholesterol molecules in the small intestinal lumen augments the sterol's incorporation into mixed micelles and also promotes partitioning of cholesterol monomers out of micelles for capture by the putative cholesterol influx transporter(s) (7, 16, 23, 24, 39). In the present study, using a highly accurate and precise methodology, we observed that knockout of the Muc1 gene did not impair normal small intestinal motility in mice. Furthermore, some biliary factors such as secretion rates of biliary lipids (bile salts, cholesterol, and phospholipid) and cholesterol content of bile, as well as sizes, molecular compositions, and hydrophilic-hydrophobic indexes of biliary bile salt pool, could together exert a major influence on the efficiency of intestinal cholesterol absorption (48). However, our previous studies (51) showed that targeted disruption of the Muc1 gene does not influence either biliary lipid secretion or biliary bile salt species and pool sizes in chow-fed mice. Taken together, our results reveal that these lumenal and biliary factors are not a major contributor to the reduction of cholesterol absorption in Muc1/ mice.
It has been controversial for many years how the lipid-soluble amphipathic fatty acids are transported across membranes: by the diffusion mechanism (1315, 20, 22, 41), the protein-mediated mechanism (1, 10, 12, 30, 37), or both. Fatty acid transport proteins (Fatp) are a family of transmembrane proteins that may enhance fatty acid uptake. It was reported recently that in mice, Fatp4 protein is highly expressed by the epithelial cells of the small intestine and localized to the brush border membrane of the enterocyte (37). Moreover, although targeted disruption of both Fatp4 alleles induced embryonic lethality, deletion of one allele of Fatp4 resulted in a 48% reduction of Fatp4 protein levels and a 40% reduction of fatty acid uptake by the enterocyte (12). To explore whether the absence of the Muc1 mucin influences the intestinal uptake of fatty acids, we investigated expression levels of the Fatp4 gene and the intestinal uptake of radiolabeled palmitic acid. Our data demonstrate that the intestinal uptake of palmitic acid and the relative mRNA levels for the Fatp4 gene in the small intestine were comparable in Muc1/ and wild-type mice. Because techniques are not available to measure how fatty acids diffuse passively through the diffusion barrier for uptake and subsequent transport across the apical membrane of the enterocyte in vivo, we could not study the effect of the Muc1 mucin on the diffusion mechanism of fatty acid uptake in mice. Nevertheless, our results reveal that the absence of the Muc1 mucin does not impair intestinal uptake of fatty acids in mice.
We conclude that physiological levels of the membrane-associated mucin encoded by the Muc1 gene are necessary for normal intestinal uptake and absorption of cholesterol in mice, and there are different mechanisms responsible for cholesterol and fatty acid uptake by the small intestine. Furthermore, our overall findings are consistent with the concept that cholesterol absorption is a multistep process that is regulated by multiple genes at the enterocyte level, and the absorption efficiency of cholesterol is most likely to be mainly determined by the net results between influx and efflux of intraluminal cholesterol molecules across the brush border of the enterocyte.
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GRANTS |
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ACKNOWLEDGMENTS |
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Dr. D. Q.-H. Wang is a recipient of a New Scholar Award from the Ellison Medical Foundation (19992003).
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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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