Department of Pathology and Laboratory Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0529
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ABSTRACT |
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Carboxyl ester lipase (bile salt-stimulated lipase) is a pancreatic enzyme capable of hydrolyzing esters of cholesterol and fat-soluble vitamins. It also efficiently digests triglycerides (TG) into free fatty acids and glycerol and is abundant in the milk of humans and several other species. We used the mouse as a model to test the hypothesis that milk-derived carboxyl ester lipase (CEL) digests milk TG and that without its activity milk lipids and their digestion intermediates can disrupt the intestinal epithelium of neonates. CEL protein and enzymatic activity were shown to be abundant in mouse milk. After 24-h administration of the CEL-specific inhibitor, WAY-121,751-5, the small intestines of treated and control neonates were analyzed histologically for signs of fat malabsorption and injury to their villus epithelium. In vehicle-fed controls, TG were digested and absorbed in the duodenum and jejunum, whereas, in inhibitor-fed littermates, large intracellular neutral lipid droplets accumulated in enterocytes of the ileum, resulting in damage to the villus epithelium. Similar results were observed in neonates nursed by CEL knockout females compared with heterozygous controls. The results suggest that lack of CEL activity causes incomplete digestion of milk fat and lipid accumulation by enterocytes in the ileum of neonatal mice.
infant nutrition disorders; intestinal absorption; dietary fats; small intestine; enterocolitis; bile salt-stimulated lipase
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INTRODUCTION |
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AS NEONATAL CARE HAS BECOME more sophisticated, the survival of premature and low birth weight infants has increased dramatically. As a result, there has been an increase in the incidence of intestinal disorders, especially ileal perforation and necrotizing enterocolitis (NEC) (17, 31, 49). NEC is characterized by mucosal and transmural necrosis and inflammation, usually involving the terminal ileum or colon. The inflammation and loss of mucosal integrity is often accompanied by rupture of the intestinal wall and sepsis. Despite advances in the diagnosis and treatment of this disease, NEC remains a major cause of morbidity and mortality in nurseries caring for premature and low birth weight infants (17, 25, 53).
The etiology of NEC is not clear and is likely to derive from several causes. The only consistent predisposing factors remain prematurity and enteral feeding, suggesting that the immature intestinal mucosae of these infants are unable to withstand the stress associated with processing a complex diet (31). Early observations indicated that there was a reduced incidence of NEC in babies fed breast milk as opposed to formula (2, 6, 38). Subsequent work has investigated various components of breast milk that might be responsible for its protective properties. The role of immunoglobulins in protecting against infection (6, 16, 23), the roles of cytokines and growth factors in promoting maturation of the intestinal epithelium (9), and the importance of anti-inflammatory factors (24) have been investigated with mixed results. Acetyl hydrolase activity against platelet-activating factor (PAF) has been found in milk, and its role in protecting against excessive inflammatory responses to PAF has been shown in rat models of ischemia-induced NEC (21, 22, 43-45). However, recent reports suggest that other factors in milk also protect against PAF- and ischemia-induced injury (5, 40).
Dietary fat has been shown to influence both the morphology and the function of the intestinal epithelium. Several investigators have found that the type and amount of lipid in formulas and milk significantly affect the transport properties and permeability of the neonatal intestinal epithelium and the rate at which it proliferates and matures (46, 54, 61). In a neonatal pig model of NEC, Crissinger and co-workers (12, 56, 57) showed that the degree of permeability and intestinal damage directly relates to the presence and type of fat in various neonatal formulas. Luminal lipids were also found to exacerbate intestinal injury in a rat model of NEC (4).
Carboxyl ester lipase (CEL), also called bile salt-stimulated lipase (BSSL), is an enzyme present at high levels in the milk of humans and several other species (15, 20, 47). This lipase has a broad range of potential substrates, including mono-, di-, and triacylglycerols, cholesteryl esters, vitamin esters, and lysophospholipids (reviewed in Ref. 50). CEL constitutes 1-2% of milk protein (15) and has been proposed to play an important role in neonatal nutrition, specifically in the digestion and absorption of milk fat (1, 10, 27, 30, 47). Evidence to this effect has come from studies using kittens that, when fed a commercial kitten formula, gained weight at about 50% of the rate of nursed littermates. When the formula was supplemented with human CEL, however, rates of weight gain were nearly equal between the formula-fed and nursed kittens (58).
A role for CEL in fat digestion in infants is further suggested by the findings of several investigators indicating that the pancreas is unable, at birth, to secrete sufficient lipolytic enzymes and/or to respond to the secretagogues cholecystokinin and secretin (19, 34). In premature infants especially, duodenal contents lack pancreas-derived lipolytic activity (32). In dogs, Iverson et al. (30) found that gastric lipase, although highly active in the hydrolysis of TG, did not completely digest the milk fat in the stomach. Diglycerides (DG), free fatty acids, and TG are delivered to the duodenum for further digestion. In neonates with insufficient milk- or pancreas-derived lipase activity, these lipids may transit the small intestine without being completely digested, resulting in the type of lipid-dependent damage seen in the pig models of NEC referred to previously (12, 56, 57).
Thus, in preterm and low birth weight infants, milk-derived CEL may be important not only for proper nutrition but also for protection of the immature intestinal epithelium from the deleterious effects of the otherwise incompletely digested milkfat. This study describes experiments that support this hypothesis. Inhibition or elimination of CEL in neonatal mice for 24 h followed by histological analysis of the villus epithelium revealed the accumulation of neutral lipid droplets, especially in ileal enterocytes, which resulted in cellular and structural damage to the villus epithelium. This lipid accumulation by the distal small intestine suggests that milk-derived CEL may protect the premature or low birth weight infant from damage to the small bowel mucosa.
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METHODS |
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Animals. Mice were housed and cared for by the institution's Department of Laboratory Animal Medicine and were kept in humidified rooms with a 12:12-h light-dark cycle and unrestricted access to food (Teklad LM485, Harlan Teklad, Madison, WI) and water. Mice were either bred in house (CEL knockout animals) or purchased from Taconic Farms (Germantown, NY). The CEL knockout allele was backcrossed onto the C57BL/6 genetic background for seven generations, and then pups nursed by either knockout females or heterozygous females were given the same regimen of triolein administration without inhibitor as described in Inhibitor feeding. CEL knockout mice were genotyped either by PCR or by Southern blot analysis as previously described (28).
Milk collection.
Mouse milk was collected from lactating dams by continuous oral suction
with the use of a capillary tube and flexible tubing after
anesthetizing the animals with 8 µl/g body wt of 5% avertin. After
collection, milk was diluted 1:4-1:10 with PBS and was centrifuged to separate the fat. The whey was stored at 80°C until use.
Stomach milk was isolated during dissection of neonates and was stored at
80°C until assayed. For enzyme assay, a piece of the
frozen milk was weighed, gently resuspended on ice in 10 vol of PBS, and processed as above for fresh milk.
Western blots. Milk protein was fractionated on 7.5% SDS-polyacrylamide gels. After fractionation, gels were either stained with Coomassie brilliant blue or transferred to nitrocellulose (Bio-Rad Laboratories, Melville, NY) by electroblotting essentially as described (51). CEL protein was detected with the use of immunopurified polyclonal rabbit anti-rat CEL antibody and 125I-labeled goat anti-rabbit IgG (Amersham, Arlington Heights, IL) as described (14).
CEL activity. CEL enzyme activity was assayed by measuring the release of [14C]oleate from cholesteryl-[14C]oleate (DuPont NEN, Boston, MA) in the presence of 15 mM sodium taurocholate and 100 µg/ml BSA, with a reaction time of 15 min as described (7). The amount of CEL in milk samples was determined by correlation to the hydrolytic activity from known amounts of purified rat CEL. Activity in intestinal contents from knockout and control mice was similarly assayed. Protein concentrations were determined by the method of Lowry et al. (37).
Inhibitor feeding. The CEL-specific inhibitor, WAY-121,751-5, was a kind gift from Dr. Mar-Lee McKean of Wyeth-Ayerst (Princeton, NJ). A preliminary report of the activity and specificity of this inhibitor has been published (11, 42). Inhibitor was dissolved in triolein (Sigma, St. Louis, MO) at a concentration of 40 mg/ml and was stored at room temperature. Black Swiss × C57BL/6 mouse pups were fed inhibitor at 100 µg/g body wt by oral gavage with a 22-gauge 1.5-in. feeding tube (Roboz Surgical Instrument, Rockville, MD). Control pups were fed an equal volume of vehicle (triolein) alone. Experiments were performed on 4- and 8-day-old neonatal mice, with half the pups in each litter receiving inhibitor and half receiving vehicle. Dosing was repeated every 4-5 h for 20 h beginning in the afternoon to inhibit both the ingested milk CEL and any pancreatic CEL produced endogenously. After dosing, pups were returned to the cage with their mother and allowed to nurse. Litters were monitored for signs of distress and to ascertain that all pups continued to nurse normally throughout the experiment.
Tissue analysis. Four to five hours after the last dose of inhibitor, the pups were killed by CO2 asphyxiation. Blood was collected from the thoracic cavity after the inferior vena cava was severed. Samples were allowed to clot, and serum was collected after centrifugation. The small intestine was carefully removed, rinsed briefly in ice-cold PBS, and then dissected into duodenum, jejunum, and ileum. The junction between duodenum and jejunum was defined at the ligament of Treitz, and the jejunoileal junction was defined as where the diameter of the small intestine reduces markedly. The intestines were not flushed when intended for histological analyses because preliminary experiments showed that doing so denuded the villus epithelium, especially in inhibitor-fed animals. Portions of each segment were immediately frozen in OCT compound (Miles, New Haven, CT) for preparation of frozen sections. Portions of each section were fixed overnight in 10% neutral buffered Formalin and were processed for paraffin embedding. Hematoxylin and eosin (H&E) staining of paraffin sections and oil red O staining of frozen sections were performed according to standard procedures (8).
Morphometric measurements on sections of paraffin-embedded tissues were performed using Image-Pro Plus 1.3 (Media Cybernetics, Silver Spring, MD). Outlines of complete villi from comparable sections of control and affected mice were traced and their lengths compared. In this way, increases in both villus height and diameter were taken into account. Cells per villus length were counted manually. At least 45 villi from each sample were scored. For some animals from the gene-knockout experiments, intestinal contents were collected. The small intestine was opened longitudinally, and the duodenum, jejunum, and ileum were individually washed by agitation in 0.5 ml of 10 mM sodium taurocholate in PBS. The tissue was removed by centrifugation and the supernatant assayed directly for enzyme activity as described in CEL activity or for lipid content as described in Lipid analyses.Lipid analyses. Glycerolipids present in luminal contents, as well as serum cholesterol and TG, were assayed with commercially available kits according to manufacturers' directions (Sigma or Wako Chemicals USA, Richmond, VA).
Statistics. Data were analyzed with appropriate t-tests and Excel 5.0 (Microsoft, Seattle, WA) or Prism 2.01 (Graphpad Software, San Diego, CA).
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RESULTS |
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Assay of mouse milk.
In order to establish the validity of the mouse to study the role of
milk CEL, the level of enzyme in mouse milk was determined by both
enzymatic and immunoblot analyses. Figure 1
shows the immunoblot results of 45 µg of whey protein after
fractionation on a 7.5% SDS-polyacrylamide gel. Individual samples
were probed with anti-rat CEL antiserum and
125I-labeled secondary antibody.
The protein detected has the expected molecular mass of ~68 kDa, as
seen previously for mouse CEL (28). These gels established that CEL is
present in mouse milk.
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Inhibitor feeding.
To test our hypothesis that CEL activity is important in preventing
damage to the villus epithelium by the high fat content of milk, a
CEL-specific inhibitor or vehicle alone was administered to equal
numbers of 4- and 8-day-old neonatal mice. The neonates were allowed to
nurse throughout the experiment and were observed to suckle normally,
with none showing any overt signs of distress. Four to five hours after
the last dose, the pups were killed and analyzed for effects of the
inhibitor. As summarized in Table 2, all
animals continued to gain weight, with no difference between control
and treated animals. Serum TG were measured and were found to vary
greatly between individuals, with no statistically significant differences seen between control and treated groups. Because CEL has
been implicated in the absorption of dietary cholesterol esters (28),
serum cholesterol was determined for all animals as well (Table 2).
Only minor differences were found between control and treated pups.
These analyses indicated that administration of the inhibitor did not
acutely affect serum lipid levels.
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Histological findings.
Paraffin sections (10 µm) of each segment of the small intestine were
stained with H&E and were examined for signs of injury. The effects of
the inhibitor increased with progression distally along the small
intestine. As depicted in Fig. 2,
A and
D, the duodenum was minimally
affected. However, striking differences between control and treated
animals were apparent in the jejunum (Fig. 2,
B and
E) and especially in the ileum (Fig.
2, C and
F). As shown in Fig.
2E, the jejunal enterocytes of the
villus tips of treated animals were enlarged and filled with vacuoles.
In the ileum of treated animals (Fig.
2F), a majority of the enterocytes were vacuolated, causing distortion of the enterocyte morphology and of
the villus architecture as a whole. Severe distortion of the villus
tips was evident in several cases (Fig.
2F, arrowheads). These effects were
similar in the 4-day-old and 8-day-old neonates, except that the
overall severity and the involvement of the lower jejunum were greater
in the 4-day-old pups. Figure 2,
A-F,
are from 4-day-old mice.
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DISCUSSION |
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Current work from this laboratory is aimed at determining the physiological significance of CEL in neonatal health and nutrition by utilizing genetically defined mouse models such as the gene knockout mice described previously (28). This is the first quantitative report of CEL in mouse milk. Its presence had been inferred from mRNA studies of lactating mammary glands (35, 39), and Stromqvist et al. (52) reported, but did not quantitate, endogenous milk BSSL (CEL) in transgenic animals. The amount of CEL detected in the current study (~1-7% of whey protein) shows milk enzyme levels comparable with those found in humans (0.4-4% of total protein; Ref. 26), indicating that the mouse is a valid animal model for studying the role of milk-derived CEL. The wide range in enzyme activity may be due to the lactation period of the females sampled. Indeed, higher levels were found at earlier times of lactation (compare the samples labeled d2 vs. d14 in Table 1).
The results of this study show that when CEL activity is removed from the intestinal lumen, either by chemical inhibition or by genetic ablation, dietary fat digestion and/or transport is altered such that neutral lipid droplets accumulate within the intestinal epithelium of the ileum and lower jejunum. This lipid accumulation was striking and reflected the location of enterocytes along both the horizontal and vertical axes of the small intestine. The villus damage resulting from loss of luminal CEL activity was extensive enough to cause dramatic cellular distortion and to alter the villus architecture, with indications of loss of epithelial integrity. These experiments were performed with mouse pups >4 days old. Although the overall effects were qualitatively similar at different ages, lipid accumulation and villus damage were less severe in 8-day-old pups compared with 4-day-old pups, suggesting a developmental window when CEL activity is most critical.
In experiments with CEL knockout mice, both knockout and wild-type pups
<5 days old show similar effects when nursed by a knockout dam. In
older pups (5 days old), the heterozygous pups nursed by knockout
dams are less affected by the lack of milk CEL, suggesting increased
synthesis or secretion of the enzyme by the pancreas (data not shown).
Morphometric analysis revealed, on average, ~20% increase in the
cross-sectional circumference of villi from affected areas (Table 3).
This measurement translates into ~70% increase in villus volume (v = 4/3r3) because of
lipid accumulation. Although no obvious signs of inflammation or
perforation were seen in this study, the observed dysmorphology and
disruption of integrity may predispose affected tissues to further
insults. Indeed, a striking observation during removal of tissues was
that the small intestines of the treated animals were extremely fragile
compared with controls and tended to rupture under even slight tension.
Similar fragility is found in young pups nursed by knockout dams and
correlates with increased lipid accumulation due to milk-fat load or
administered lipid.
A role for milk-derived CEL in the digestion of milk fat has been suggested by several observations. The presence of this enzyme in the milk of many species, its ability to remain active after passing through the stomach, and its efficient lipolysis of all acylglycerols is well established (18, 20, 27). In addition, several studies suggest that the secretion of lipases by the neonatal pancreas (especially that of the premature infant) is insufficient to fully digest the high fat load presented by the neonatal diet (19, 32, 33, 63). Interestingly, a recent report (36) showed reduced milk-fat digestion and absorption in neonates lacking the pancreatic lipase-related protein-2, which is expressed during the neonatal period. An intriguing possibility is that the combined activities of these two lipases is required for proper digestion of milk fat.
Dietary lipids are digested and absorbed in the duodenum and jejunum, with very little fat absorption occurring in the ileum (13). In early experiments by Bennett-Clark et al. (3, 62), when adult rats were challenged with excess luminal triolein, excess lipid reached the ileum, where it accumulated in the intestinal wall. Ileal chylomicron secretion was 40% slower than in the proximal intestine. In the present study, the high fat content of the milk (plus the triolein vehicle) may have approached the limit of the digestive and/or absorptive capacity in the neonates lacking CEL activity, thereby allowing excess lipid to reach the ileum and accumulate in the villus epithelium as seen in the former study. The weanling mice showed no such accumulation, either because the digestive capacity of their pancreatic secretions was not maximally challenged by the administered lipid and their 5% fat diet or because of increased absorptive capacity in their proximal small intestine.
An additional factor that may contribute to the phenotype seen in the present experiments is the immaturity of the distal small intestine in neonates. Maturation of the small intestine occurs in a proximal-to-distal fashion and continues well into the neonatal period (13). Thus one would predict that the most severe effects would be seen in the ileum, with less-severe alterations detected in the jejunum and very little, if any, consequence in the duodenum.
Several investigators have developed neonatal animal models to study the causes of and to develop strategies for the treatment and prevention of NEC and other gastrointestinal diseases of neonates and premature infants. Panigrahi et al. (48) have isolated adherent strains of Escherichia coli from NEC patients and have shown their ability to cause disease in rabbits. Velasquez et al. (55, 56) used perfusion of intestinal loops from neonatal piglets to show the effect of dietary lipids on villus integrity after ischemia. Other evidence suggests a role for PAF in the etiology of NEC and that milk-derived PAF acetyl hydrolase can prevent destruction of the intestinal epithelium (5, 21, 22, 29). In these models, a period of hypoxia or ischemia-reperfusion is necessary to develop the symptoms seen in human NEC. A role for luminal contents in the disease process has also recently been implicated in this latter model (4). Specifically, animals with corn oil or formula in their intestinal lumen during the ischemia-reperfusion-PAF treatment suffered greater degrees of intestinal necrosis than animals with lactose or casein in their lumen.
The experiments described in this report are in agreement with the latter study. Interestingly, the effects observed in this report were most severe in the portion of the small intestine most vulnerable to NEC. Together these studies suggest an additional mechanism by which the intestinal epithelium may become sensitized to various inflammatory or infectious agents. This mechanism involves the accumulation of excess lipid in the epithelium of the distal small intestine because of insufficient lipolytic activity and/or absorptive capacity. We propose that the CEL in breast milk supplies an essential lipolytic activity for premature infants.
These studies represent the first report of a potential physiological consequence of the absence of CEL in neonatal animals. Use of the knockout mice will permit the time course of the observed lipid sensitivity to be determined. They can also be used to test whether lack of CEL results in increased susceptibility to intestinal pathogens. These mice will also be important in crossbreeding experiments with knockout mice for other lipase genes to determine the relative roles of the different pancreatic and preduodenal lipases in lipid digestion and absorption throughout development.
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ACKNOWLEDGEMENTS |
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We thank Rital Angel, Frost Smith, and the histology core for technical assistance.
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FOOTNOTES |
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This work was supported in part by National Institutes of Health Grants HD-33926 and DK-40917.
Portions of this work have been previously published in abstract form (P. N. Howles, G. Stemmerman, C. Fenoglio-Preiser, and D.Y. Hui. Bile salt-stimulated lipase activity prevents intestinal damage in neonatal mice. FASEB J. 10: A190, 1996).
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: P. N. Howles, Dept. of Pathology, Univ. of Cincinnati College of Medicine, P.O. Box 670529, Cincinnati, OH 45267-0529 (E-mail: philip.howles{at}uc.edu).
Received 7 October 1998; accepted in final form 12 May 1999.
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