Development mechanisms
of fatty acid esterification in rat small intestine
YIH-FU SHIAU, C. UMSTETTER, EC, KENDALL, AND 0. KOLDOVSKY Gastrointestinal Section, Repartment of Medicine, Veterans Administration Hospital; Division of Biochemical Development and Metabolic Diseases, Department of Pediatrics, Childrens’ Hospital of Philadelphia; and University of Pennsylvania, Philadelphia, Pennsylvania
SHIAU,YIH-Fu,C. UMSTETTER, K. KENDALL, AND 0. KOLDOVSKY. Development of fatty acid esterification mechanisms in rut small intestine. Am, J. Physiol. 237(5): E399-E403, 1979 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 6( 5): E399-E403, 1979.-Fatty acid esterification was measured in fetal jejunal and ileal isografts implanted under the kidney capsules of adult host rats and compared to the age-cantrolled intestine grown in situ. Studies were conducted on the 21st, 35th, 49th, and 63rd postconceptional days, corresponding to prenatal, suckling, weaning, and weaned rats. Substantial fatty acid esterification activity was found in prenatal jejunum but not in ileum. A proximal-distal gradient of fatty acid esterification was observed in all groups grown in situ, but not in isografts, The monoglyceride pathway (MG-P) accounted for about one-third of total fatty acid esterification (TFAE) in jejunum grown in situ and remained constant through the study. In the ileum, MG-P was the major esterification pathway during the first 4 postnatal weeks, but decreased progressively after weaning to become insignificant in adult rats. TFAE fell in the jejunal isografts, whereas it increased in the ileum. MGP remained as the major pathway in the implanted, jejunum and ileum. Our studies suggest that luminal contents are probably the most important modulator for the development and maintenance of intestinal fatty acid esterification, and “fetal programming” manifested by changes in fatty acid esterification mechanisms in the isografts is less important.
monoglyceride pathway; phosphatidic acid pathway; acid esterification; jejunum; ileum; isograft
total fatty
OF FATABSORPTION inadultmammals have been extensively studied (7, 12, 15). Fat absorption requires hydrolysis of triglycerides to fatty acids and monoglycerides that are resynthesized to triglycerides within the intestinal epithelial cells, The information regarding developmental changes in fat absorption are meager despite the fact that fat constitutes the major source of energy in infant mammals including man (4). Published data has so far concentrated only on the luminal phase of absorption during the perinatal period (6, 13, 14). Little is known about intracellular events (2, 5). Intestinal fatty acid esterification in adult mammals is mediated by two distinct mechanisms: the phosphatidic acid (PA-P) and the monoglyceride pathways (MG-P) (7). Interaction between these two pathways in adult rat THEMECHANISMS
0363-6100/79/0000-0000$01.25
intestine have recently been studied in our laboratories (17). Using [U-14C]glucose and [“Hloleic acid, contribution of the PA-P and MG-P to total fatty acid esterification can be quantitated in intestinal mucosa (17). In this study, we investigated changes in fatty acid esterification during the perinatal development of the rat. Because of the known differences of various functions along the length of the small intestine (9), we have compared the development of the esterification processes in both jejunum and ileum. Using implanted fetal small intestine, isografts, the role of luminal contents in the development of the intestinal fatty acid esterification was elucidated. MATERIALS
AND
METHODS
Pregnant CDF strain inbred rats were purchased from Charles River Breeding Laboratories (Wilmington, MA) and were air shipped on the 12th postconceptional day (PCD). In our animal facilities, rats were housed individually and provided with laboratory rat chow and tap water ad libitum. These rats were separated into three groups, Group 1 consisted of two litters, each with eight offspring. Five of these rats were selected randomly on the 35th, 49th, and 63rd PCD and killed. The small intestine was divided into nine equal segments; the second segment from the ligament of Treitz (referred to as jejunum) and second segment from the ileocecal junction (referred to as ileum) were used in our studies. Group 2 consisted of 15 fetuses obtained from seven litters on the 21st PCD by cesarean section under sterile technique. The small intestine from two to three fetuses from each litter were used. The jejunum and ileum were implanted separately under both kidney capsules of the host rats (CDF strain, 150- to 200-g male rats) as described previously (3, 8) Because preliminary data showed that morphological growth of the jejunum and ileum was not different when grown in either right or left kidney capsules, we therefore consistently implanted the jejunum in the left and the ileum in the right kidney. On days 14,28, and 42 of the postimplantation period (equivalent to 35th, 49th, and 63rd PCD), five host rats were killed, and the implanted intestines were removed for fatty acid esterification measurement. Corresponding segments of the small intestine from the host rats were l
Copyright 0 1979 the American Physiological Society
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used to determine fatty acid esterification in adults. Group 3 consisted of 10 fetuses selected randomly from 17 fetuses obtained by cesarean section from two pregnant rats on the 21st PCD. Equivalent portions of the small intestine were used for fatty acid esterification studies. In this study, fatty acid esterification was measured in different age groups representing five different stages of perinatal development with substantial differences in both the quantity and quality of food intake. Stuge I: fetal small intestine (21st PCD or day before birth) exposed only to amniotic fluid. Stage 2: 35th PCD (2-wkold) represented the end of the obligatory suckling period during which milk was the only dietary intake. Stage 3: 49th PCD (4-wk-old) represented the end of weaning. These rats received both milk and rat chow. Stage 4: 63rd PCD (6-wk-old) were weaned rats that had consumed rat chow as the only dietary intake for 2 wk. Stage 5: adult rats that had consumed rat chow for longer than 3 mo. Food intake per unit body weight is higher .in the younger age groups than in the adult rats (4). Tissue preparation. Fetal small intestine (21st PCD) was opened longitudinally to allow adequate exposure to the incubation medium. Intestines from suckling (35th PCD), weaning (49th PCD), weaned (63rd PCD), and adult rats (3 mo) were everted and the intestinal rings cut, The implanted tissue was removed and excessive debris was washed free by cold normal saline. Ten to twenty milligrams of tissue was used in each incubation. Fatty acid esterification. The method used to determine the total fatty acid esterification (TFAE) and to separate the two esterification pathways has been previously reported (17). The incubation medium was made of 3.3 mM oleic acid, 1 mM 1-monoolein, 5.6 mM glucose, and 10 mM sodium taurocholate, in calcium-free phosphate buffer, pH 7.0. Tracer amounts of [“Hloleic acid and [U-14C]glucose were added. After a 40-min incubation, the tissue was rinsed and homogenized in acid saline (pH 1.0). Neutral lipids were extracted and separated by
JEJWNUM
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KOLDOVSKY
Normal developmental changes in jejunal and ileal fatty acid esterification (Fig. I)* Significant fatty acid esterifrcation was found in the fetal jejunum (45.3 t 3.3 nmol/mg protein per 40 min) that was similar to that found in the suckling rats (38.9 t 3.1 nmol/mg protein per 40 min). Fatty acid esterification peaked at the end of weaning period (84.8 + 4.5 nmol/mg protein per 40
M
IN SITU IMPLANT
. FIG. 1. Total fatty acid esterification measured in jejunum and ileum grown in situ and as isografts. The 2&t, 35th, 49th, and 63rd postconceptional days (PCD) correspond to prenatal, obligatory suckling, weaning, and weaned periods, Resuits are means t SE of 5 experiments.
II CONCEPTIONAL
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POST
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thin-layer chromatography. The triglyceride fraction was identified and scraped into a IO-ml Triton-toluene scintillation fluid and counted with a Packard scintillation counter. Quenching was corrected by the external standard method. TFAE was measured by the incorporation of [“Hloleic acid into triglyceride. The amount of fatty acid esterified by the PA-P was derived from the incorporation of [U“C]glucose into triglyceride. Because none of the 14C activity appeared in the fatty acid fraction of the resynthesized triglyceride, glucose was incorporated only into triglyceride as glycerol backbone (17). One molecule of glucose incorporated into triglyceride is equivalent to two molecules of glycerol formed, and each glycerol requires three molecules of fatty acid to form one molecule of triglyceride. Therefore, fatty acid esterification mediated by the PA-P can be derived by multiplying the amount of glucose incorporated by a factor of 6. The difference between TFAE and that mediated by the PAP represents the esterification mediated by the MG-P. Muterials. Oleic acid and I-monoolein were purchased from the Sigma Chemical Co. (St. Louis, MO). Taurocholate was purchased from Calbiochem (Levelly, CA). Radioactive tracers, [“Hloleic acid and [U-14C]glucose, were purchased from New England Nuclear Corp. (Boston, MA). Protein assay was the method reported by Lowry et al. (11). All of the results were means t SE of five rats in each group and were expressed as nanomoles oleic acid esterified per milligram protein per 40 min. Statistical analysis was performed by Student’s t test (1).
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min) and decreased subsequently in the weaned and adult rats (55.2 t 4,l and 42.3 + 3.1 nmol/mg protein per 40 min, respectively). Fatty acid esterification in weaning rats was significantly higher than that observed at any other time (P < 0.001). The ileum exhibited a different pattern of development. Fetal ileum had very little fatty acid esterification activity (0.6 t 0.5 nmol/mg protein per 40 min). During the first 2 wk of pustnatal life, there was a small but significant increase in fatty acid esterification (7.2 t 0.8 nmol/mg protein per 40 min, P < 0.001) and continued to increase to 10.5 t 0.8 nmol/mg protein per 40 min during the weaning period. A significant decrease in ileal fatty acid esterification was observed immediately after the rats were weaned (5.3 t 0.6 nmol/mg protein per 40 min, P < O.Ul), but returned to a higher level in adult rats (12.1 t 1.1 nmol/mg protein per 40 min). Fatty acid esterification activities were higher in the jejunum than in the ileum in all age groups studied. Relative contribution uf each esterification pathway in small intestine developing in situ (Fig. Z), The jejunum and ileum exhibit different patterns of esterification mechanisms during the developmental period and in adult rats. In the jejunum, the PA-P contributed 66.5 t 3.2% of TFAE and the MG-P 33.5 t 3.2%. The absolute amounts of fatty acid esterification mediated by the MGP in the 21st and 35th PCD were 11.9 -t- 1.8 and 17.7 & 1,3 nmol/mg protein per 40 min, respectively, reaching a peak on the 49th PCD (26.9 $- 2.3 nmol/mg protein per 40 min, P < 0.001) and were maintained at a constant level afterwards (15.8 t 2.0 and 15.9 -+ 1.8 nmol/mg protein per 40 min, respectively, for the 63rd PCD and adult rats). In contrast, the MG-P was the exclusive pathway for fetal ileal fatty acid esterification and is the major esterification pathway for the first 4 wk of postnatal life. After the rats were weaned, fatty acid esterification decreased significantly, and the decrease was due to diminishing MG-P-mediated esterification. When the rats reached adulthood, very little ileal esterification was mediated by
JESUNUM
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the MG-P, whereas the PA-P increased progressively over the entire developmental period. Intestinal fatty acid esterification capacity in isografts of fetal jejunum and ileum (Figs. I and 2). This study was designed to examine the development of small intestinal function in the absence of luminal contents. Our results showed that there was a significant decrease in jejunal fatty acid esterification 2 wk after implantation (2.5 t 0.2 nmol/mg protein per 40 min, p c O.OOl), and no further changes were observed. In contrast to the jejunum, the ileum showed a small but significant increase (3.1 t 0.5 nmol/mg protein per 40 min, P < 0.01) in the first 2 wk after implantation and there was little change afterward. Comparing the implanted jejunum to ileum, there was no difference in fatty acid esterification throughout the period examined. The MG-P was the major esterification pathway in the first 2 wk after implantation. Comparing jejunum developing in situ to that grown as isografts, TFAE in the isografts never exceeded that in situ. Similarily, implanted ileum has significantly less TFAE activities than that grown in situ, except that of the 63rd PCD implanted ileum. At this stage, TFAE in the implanted ileum was similar to that grown in situ and was due to a transient decrease in TFAE in the latter group. Although ileal fatty acid esterification capacity was similar in isografts and in situ at this stage, a vast difference was found in the pathways involved in fatty acid esterification. Contribution of the PA-P to TFAE in the ileum grown in situ was significantly higher than that grown as isografts (3.1 t 0.8 vs. 1.1 t 0.5 nmol/mg protein per 40 min, respectively, P C 0.05). DISCUSSION
In this study we have shown that the fetal jejunum possesses a substantial fatty acid esterification capacity, equal to that observed in adult rats, However, little fatty acid esterification was observed in the fetal ileum. During postnatal development, both the jejunum and ileum undergo significant changes in fatty acid esterification capacity. These changes do not occur in parallel in these
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FIG, 2. Contribution ide pathway (MG-P) esterification (TFAE). pressed as % of TFAE 5 experiments.
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49 63. CONCEPTIONAL
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of monoglycerto total fatty acid Results are exin means & SE of
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two regions. A proximal-distal gradient of fatty acid esterification was observed in all groups studied during normal development. Peak jejunal fatty acid esterification capacity was found during weaning and then decreased into adulthood. In contrast to the jejunum, peak ileal fatty acid esterification was found in adult rats, A transient decrease in fatty acid esterification was observed in the ileum immediately after weaning as a result of a fall in monoglyceride pathway-mediated esterification. The MG-P was almost absent in the adult ileum. In contrast to the ileum, MG-P-mediated fatty acid esterifrcation in the jejunum remained relatively constant throughout development. Using the isograft technique, Ferguson et al, (3) reported that the developmental pattern of sucrase activity in mice jejunal isografts was parallel to that occurring in situ. Administration of cortisone altered the developmental changes in both normally developing and implanted jejunum, suggesting that blood-borne factors did reach the implanted jejunum (3). Furthermore, Kendall et al. (8) reported different patterns of development of sucrase, lactase, and P-galactosidase in rat jejunal and ileal isografts, resembling changes observed in the normally developing intestine, implying that isografts exhibit intrinsically programmed disaccharidase and P-galactosidase developmental patterns. In contrast to normal developmental patterns of suerase, lactase, and P-galactosidase activities in isografts, we found that intestinal fatty acid esterifrcation in the isografted jejunum and ileum was inhibited and a normal proximal-distal gradient was abolished. The differences between the development of disaccharidase and P-galactosidase activities and that of fatty acid esterification suggest that luminal contents play in important modulatory role in the development of the latter. The patterns observed in implanted small intestine may represent intrinsic “fetal programming.” The markedly reduced intestinal fatty acid esterification in the implanted small intestine suggests that modulating factors appear to be more important than fetal programming in the development of intestinal fatty acid esterification. Our previous studies have shown that in the absence of oral intake, parenterally fed rats had a decreased intestinal fatty acid esterification (10, 16). Diversion of pancreaticobiliary secretions from the proximal small intestine further reduced fatty acid esterification, suggesting that the pancreaticobiliary secretions contained factors important in maintaining intestinal fatty acid esterification (10, 16). This study further supports the concept that the luminal contents (possibly pancreaticobiliary secretions) are important in the development and maintenance of intestinal fatty acid esterification, There are two studies in the literature dealing with the development of intestinal fatty acid esterification in perinatal periods. Cunningham and Leat (2) showed that the contribution of the monoglyceride pathway to total fatty acid esterifrcation in the jejunum did not change in fetal, suckling, and adult sheep. Our data is consistent
UMSTETTER,
KENDALL,
AND
KULDOVSKY
with theirs (2), Holtzapple et al. (5) reported that suckling rats have higher fatty acid esterifrcation activity than adult rats in contrast to our data. These differences could be due to the fact that Holtzapple et al. (5) did not use monoolein or glucose in their incubation medium. The importance of glucose and monoolein in stimulating intestinal fatty acid esterification has been previously reported (17). In the presence of adequate substrates, fatty acid, and ATP, the formation of acyl CoA is a function of acyl CoA synthetase activity, which is higher in the jejunum than the ileum in both suckling and adult rats (5). The difference in acyl CoA synthetase may correlate with the observed proximal-distal gradient of fatty acid esterification along the small intestine. Another enzyme, acyl CoA monoglyceride acyl transferase, required exclusively for monoglyceride pathway-mediated esterification, was found to have similar activity in the jejunum and ileum in the suckling period but lower activity in the ileum of adult rats (5). The acyl CoA monoglyceride acyl transferase activity along the jejunum and ileum reported by Holtzapple et al. (5) correlates well with the pattern of MG-P-mediated fatty acid esterifrcation reported in this study. The fall in acyl CoA monoglyceride acyl transferase activity in adult ileum corresponds to a significant reduction in the MG-P-mediated fatty acid esterification. In suckling rats, Holtzapple et al. (5) reported that acyl CoA monoglyceride acyl transferase activity in the jejunum was similar to that found in the ileum. One might therefore expect that esterification mediated by the monoglyceride pathway would be identical in the suckling jejunum and ileum. Our study showed that fatty acid esterification mediated by the MG-P in the suckling jejunum was significantly higher than that in the ileum. This finding can be explained by our previous report (17) that the rate-limiting factor for the MG-P is the availability of acyl CoA. Because acyl CoA synthetase activity is lower in the ileum than in the jejunum of the suckling rats, the availability of acyl CoA in the suckling ileum might be lower than in the jejunum. In conclusion, our studies demonstrate that intestinal fatty acid esterification mechanisms undergo substantial changes during development. Changes in jejunum do not parallel those of the ileum. The intrinsic fetal programming of intestinal development is less important in the development of intestinal fatty acid esterification than for disaccharidases (3, 8) and P-galactosidase (8). Luminal contents (possibly the pancreaticobiliary secretions) play the most important modulatory role in the development of intestinal fatty acid esterification. The authors are grateful to Dr. Gary Levine for his critical review of this manuscript. We also extend our appreciation to Ms. K. Conrad for her secretarial assitance. This study was supported in part by Veterans Administration Medical Research Grant (MRIS-642-7864) and in part by National Institutes of Health Research Grant AM-14531. Received
5 February
1979; accepted
in final
form
25 May
1979.
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