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Institut National de la Santé et de la Recherche Médicale Unité 683 (T.A., S.K., D.D., S.G., K.H., T.L.), Faculté de Médecine Xavier Bichat, and Department of Gynaecology (D.M.-C.), Hôpital Bichat-Claude Bernard, Paris 75018, France
Address all correspondence and requests for reprints to: Thérèse Lehy, D.Sci, Ph.D., Institut National de la Santé et de la Recherche Médicale Unité 683, Faculté de Médecine Xavier Bichat, 16 rue Henri Huchard, BP 416, Paris, Cedex 18, F-75870 France. E-mail: tlehy{at}bichat.inserm.fr.
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Abstract |
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Objective: The objective of the study was to investigate the temporal and spatial appearance of Ob-Rb in the human digestive tract and leptin in the stomach.
Design: The esophagus, stomach, and intestine samples of 7- to 24-wk-old human fetuses and adult mucosae were studied by RT-PCR, immunohistochemistry, and Western blot. Leptin was measured by RIA in amniotic fluids at 16–33 wk gestation.
Results: All mucosae expressed Ob-Rb (mRNA and/or protein) between 7 and 9 wk gestation. Leptin protein appeared by 8 wk in the gastric mucosa, whereas leptin mRNA was detected around 11 wk. Leptin levels in amniotic fluids were significantly higher during the second than during the third trimester. Overall, Ob-Rb immunoreactivity was higher in young fetuses, during the period corresponding to the formation of gastric buds and primitive intestinal crypts and the beginning of differentiation of epithelial cell types, than in the oldest. Leptin added to culture medium of gastrointestinal explants from 10- to 12-wk-old fetuses appeared to affect DNA synthesis as compared with controls, indicating that leptin receptor functionality was developing.
Conclusions: The strong expression of leptin, in amniotic fluid when fetuses begin swallowing then in the gastric mucosa, and the early presence of Ob-Rb in mucosae suggest a possible role for leptin, exerted endoluminally and in a paracrine pathway, in the developmental process (growth and/or maturation) of the human digestive tract.
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Introduction |
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In regard to the human gastrointestinal tract, leptin protein has been described in the fundic mucosa, mainly in pepsinogen-secreting cells (12, 13, 14) but also in some parietal cells, particularly in the microcanaliculus (13, 14) and more rarely in endocrine cells (12). Ob-Rb isoform has been detected in the gastric (13, 14), small intestinal (15), and colonic mucosae (16, 17) of adult human. Leptin is implicated in the regulation of several gastrointestinal functions such as the control of acid, gastrin, and somatostatin secretions in the stomach; cholecystokinin secretion and dipeptide; and glucose epithelial transports in the small intestine as well as cell proliferation in the small and large intestine (17, 18, 19, 20, 21, 22, 23).
There is accumulating evidence that leptin might be involved in the embryonic and fetal growth of mammals (24). In vitro, leptin promotes the development of mouse preimplantation embryos (25). In human, newborn weight at birth is significantly correlated with the umbilical cord blood leptin concentration, whereas growth retardation is associated with low fetal leptin levels (26, 27). In murine fetuses leptin is detected in the liver at different stages of development (28) and in human fetuses in adipose tissue at 20–38 wk gestation (29). Ob-Rb isoform is expressed and functional in pancreatic islets of rat fetuses (30). However, to date, there is no information concerning leptin ontogenesis in the human stomach and that of Ob-Rb leptin receptor in the digestive tract of any species. Moreover, although leptin exerts various actions on the adult gastrointestinal tract, no data exist about its potential roles in the functional development of digestive organs during the human gestation. In the rat, however, it was recently shown that leptin significantly stimulates glucagon-like peptide-1 secretion by fetal intestinal cell cultures expressing Ob-Rb leptin receptor (31).
So in the present study, we examined the ontogenic pattern of expression and dynamic changes in localization of the Ob-Rb receptor in different mucosae of the digestive tract in function of gestational ages of human fetuses. We investigated concomitantly the ontogenic profile of leptin in the fetal stomach. Additionally, leptin was measured in amniotic fluids at various gestational ages. Finally, we examined the effect of exogenous leptin on the DNA synthesis of cultured human fetal explants of gastrointestinal tissues.
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Materials and Methods |
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Twenty-nine human fetuses (fetal age 7–24 wk) were obtained after voluntary, spontaneous, or therapeutic abortions. Their developmental age was determined from probable fertilization dates and confirmed either by foot-length measurement for the youngest or crown-rump measurement for the oldest fetuses. Fetal tissues were used in accordance with the requirements of the Human Research Committee of Hospital Bichat-Claude Bernard, Paris. Specimens of anatomically recognizable esophagus, entire stomach, and small and large intestine were removed and processed for histological procedures. Those with visible autolysis were excluded from the study. From 9 wk gestation, stomachs were opened and cut into adjacent fragments from the cardia to the pylorus. For the oldest fetuses, the proximal duodenum, jejunum, and distal ileum were individualized in the small intestine and the cecum, ascending colon, descending colon, and rectum in the large intestine. Two fixatives were used for each segment of the digestive tract, i.e. adjacent samples were fixed the ones in Bouin’s fluid and the others in aldehydic fixatives [10% neutral formalin for 24 h at room temperature or 4% paraformaldehyde in PBS (pH 7.4) for 24 h at 4 C]. They were then embedded in paraplast and cut in 4-µm-thick sections. Fresh tissues from several fetuses were immediately frozen in liquid nitrogen and stored at –80 C. Table 1
shows the distribution of fetus ages and the number of samples examined for the different segments of the digestive tract. Biopsies from normal adult digestive tract were obtained during endoscopy and also studied.
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Bouin-, formalin-, or paraformaldehyde-fixed sections were incubated overnight at 4 C with either a goat polyclonal antibody raised against the C-terminal fragment (1146–1165) of human long form of leptin receptor (C-20; Santa Cruz Biotechnology, Santa Cruz, CA) (15) or a rabbit polyclonal antibody raised against human leptin (BioVendor Laboratory Medicine Inc., Palackeho, Czech Republic). The two antibodies were diluted 1:50. Thereafter sections were incubated with the corresponding biotinylated secondary antibody diluted 1:200, and finally with the avidin-biotin complex diluted 1:100 (ABC Vectastain kit; Vector Laboratories, Burlingame, CA). The peroxidase activity was revealed in brown by diaminobenzidine and nuclei were counterstained with Mayer’s hemalum. Gastric, duodenal, and colonic tissues from adult humans served as positive controls for Ob-Rb immunoreactivity. Adult human fundic tissue served as positive control for leptin immunoreactivity. Negative controls were obtained by omitting primary antibodies. Overnight preincubation of primary antibodies with 50 µg of the Ob-Rb blocking peptide (reference, sc-1832 P; Santa Cruz Biotechnology) or human leptin recombinant (R&D Systems, Abingdon, UK) per milliliter of diluted antiserum abolished the signal for leptin receptor or leptin, respectively.
Western immunoblotting
Frozen tissues were homogenized at 4 C in a lysis buffer. Proteins were solubilized in Laemni buffer under reducing conditions, separated by 7.5% SDS-PAGE for Ob-R or 12.5% PAGE for leptin with equal amounts (40 µg) loaded onto each lane and transferred to nitrocellulose sheets. They were probed with the leptin or leptin receptor antibodies diluted 1:200 and then with the corresponding secondary antibody diluted 1:2000 followed by the Vectastain ABC kit. Immune complexes were detected by enhanced chemiluminescence (Amersham, les Ulis, France). The specificity of Ob-Rb detection was checked by preincubation of the diluted antibody with 10 µg/ml of Ob-Rb blocking peptide. Adult human fundic, antral, and rectal mucosae served as positive tissue controls. The relative loading of protein samples was assessed after reprobing the membranes with a monoclonal 
-tubulin antibody (Sigma-Aldrich, Saint-Quentin-Fallavier, France).
RNA extraction and RT-PCR
Total RNA from frozen tissues was extracted using Trizol-Reagent (Gibco BRL, Eragny, France). First-strand cDNA was prepared from 5 µg of total RNA according to the manufacturer’s procedure (InVitrogen, Cergy-Pontoise, France) using murine reverse transcriptase. The specific cDNA primers for the Ob-Rb gene were forward 5'-GCCAAC-AACTGTGGTCTCTC-3' and reverse 5'-AGAGAAGCACTTGGTGACTG-3', giving a 246-bp amplicon. The expected size for amplicon was 246 bp. Two sets of primers were used for Ob gene. For the first set, primers were forward 5'-CCAAGATGGACCAGACACTG-3' and reverse 5'-GCCACCAGTCTGTGGAGTA-3', giving a 219-bp amplicon. The second were 5'-GATGACACCAAAACCCTCATC-3' (position 141 up to 162) and reverse 5'-GGCCACCACCTCTGTGGAGTA-3' (position 474 up to 494), giving a 353-pb amplicon. PCR was carried out for 40 cycles. For Ob-R and the first Ob primers, cycles comprised denaturation at 95 C for 1 min, annealing at 62 C for 30 sec, and extension at 72 C for 2 min. Amplification was terminated by a final extension step at 72 C for 10 min. For the second Ob primers, the three steps were at 94 C, 49 C, and 72 C for 40 sec each. For negative controls, the reaction was performed without reverse transcriptase. For positive controls, we used glyceraldehyde-3-phosphate dehydrogenase primers (32). Amplicons were separated by electrophorese in a 2% agarose gel, stained with ethidium bromide, and visualized under UV light. Adult human fundic mucosa and lipoma served as positive tissue controls.
Leptin RIA
Amniotic fluids from 11 pregnant women at 16–33 gestational weeks were collected at the time of amniocentesis, centrifuged, and supernatants stored at –20 C. Measurement of leptin in supernatants was assessed using a RIA kit for multispecies leptin from Linco Research Inc. (St. Charles, MO).
Culture of fetal tissues and evaluation of DNA synthesis
Tissues of two fetuses, 10 and 12 wk old, obtained after voluntary abortion, were rinsed in saline and processed for organ culture within 30 min after we got them: stomach, small intestine, and colon for the oldest fetus and small intestine only for the youngest. Adjacent explants, approximately 5 x 5 mm for the stomach and colon and 7–8 mm long for the small intestine, were immersed in Leibovitz L-15 medium containing 40 µg/ml nystatin and 40 µg/ml gentamicin (33). They were cultured in organ tissue culture dishes (Falcon, Los Angeles, CA) in the same serum-free medium at 37 C in a humidified atmosphere containing 5% CO2 for 26 h in the absence or presence of leptin (100 ng/ml). Culture medium with or without leptin was renewed after 21 h, and 2 µCi of 3H-thymidine (specific activity, 83 Ci/mmol; Amersham, Orsay, France) were added per milliliter of culture medium for a 5-h period. Explants were then rinsed in saline, frozen in liquid nitrogen, and kept at –80 C until DNA extraction performed with phenol. DNA content was determined and 3H-thymidine incorporation into DNA measured in a ß-scintillation counter and expressed as disintegrations per minute per 50 µg DNA.
Statistical analysis
Results were given as means ± 1 SEM. Statistical differences between groups were analyzed using when relevant the t test or the nonparametric Mann-Whitney U test for independent populations. The level of significance was set at P < 0.05.
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Results |
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We agree with the temporal sequence of anatomical development of the human gastrointestinal tract already described (34).
Esophagus
At 8 gestational weeks, a strong immunoreactivity was seen in all epithelial layers of the multilayered and vacuolated esophageal epithelium (Fig. 1A). At 10 wk, the epithelium becomes ciliated and Ob-Rb expression predominated on the membranes, with an enhancement of intensity in focal areas of the superficial cell layer (Fig. 1B). In the oldest fetuses, the immunolabeling had a more diffuse localization in the cytoplasm and its intensity decreased with age, as in adult (Fig. 1C).
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At 7 wk gestation, it was histologically difficult to differentiate the stomach from the proximal small intestine; the gastrointestinal epithelium showed a faint immunoreactivity for Ob-Rb. At 8 wk, the stomach could be anatomically individualized, and the presence of the receptor was clearly established in the multilayered gastric epithelium (Fig. 1D
). By 9–9.5 wk, Ob-Rb immunolabeling was weak to moderate in the surface cells of the simple undifferentiated epithelium and more marked in gastric buds (data not shown). At the beginning of the second trimester, fundic and antral regions are identifiable. Expression of Ob-Rb was moderate to strong on basolateral membranes and in the basal cytoplasm of mucous epithelial cells lining the luminal surface and pits of the differentiated fundic and antral mucosae. It was also localized in fundic glandular cells distributed on the lower two thirds of the mucosal thickness, most cells resembling parietal cells by their shape and size (Fig. 1, E and F). In those cells, microcanaliculi exhibited immunolabeling as in adult stomach. Moreover, the signal tended to diminish with fetal age.
Small intestine
Ob-Rb immunolabeling appeared between 7.5 and 9 wk in the intestinal epithelium still locally multilayered of primitive villi (Fig. 1G). It was clearly established at 10–11 wk when primitive crypts are developing. In all fetuses, the apical membranes of villi epithelial cells showed the strongest immunostaining, but the signal was also present on basolateral membranes. By 17 wk, well-defined crypts and villi are formed, similar to that of the adult small intestine. Ob-Rb immunoreactivity showed the same localization pattern as in the first stages (data not shown).
Large intestine
At 9–9.5 wk, the earliest time point studied, the epithelium still locally multilayered displayed a faint Ob-Rb immunolabeling in the cytoplasm of epithelial cells and a moderate to strong one on the membranes, especially apical membranes (Fig. 1H). By 17–18 wk and onward, Ob-Rb expression was moderate in both the cytoplasm and basolateral membranes of crypt cells (Fig. 1I) and strong on the apical membrane of colonocytes lining the colonic lumen (Fig. 1J). There was no noticeable variation in the staining pattern between the different segments of the large intestine. No definite variation in the signal intensity was noted as the gestational age increased.
Overall, for the leptin receptor, all fixatives gave the same distribution and comparable immunostaining intensity. However, in the small and large intestine, the signal was seen predominantly on membranes after aldehydic tissue fixation and was more diffuse in the cytoplam after Bouin’s fixation, as already reported (15).
Figure 2, A–F, illustrates Ob-Rb immunostaining in the different mucosae of the digestive tract of adult for comparison.
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Only some fetal tissues were available. Ob-Rb mRNA transcripts were detected in the human fetal stomach at 7.5 wk and in small intestine at 9–11 wk gestation (Fig. 3A). Analysis of immunoblots revealed a 120-kDa protein band in the stomach, small intestine, and colon of fetuses as in adult gastrointestinal tract. This band corresponds to the long Ob-Rb isoform (Fig. 3B).
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RT-PCR analysis did not reveal leptin mRNA in the fetal intestine at the different ages tested or in the stomach of a 7.5-wk-old fetus, which however expressed Ob-Rb mRNA (Fig. 3C). Ob mRNA was detected in the stomach of an 11.5-wk-old fetus and, as expected, in the adult human fundic mucosa and lipoma (Fig. 3D). Analysis of immunoblots indicated the presence of a 16-kDa protein band corresponding to leptin at 8 wk (Fig. 3E). Confirming these data, leptin immunostaining was negative in the stomach at 7 wk (Fig. 4A), whereas there was a clear-cut although weak leptin signal in the multilayered gastric epithelium at 8 wk (Fig. 4B). At 9.5 wk, strong immunoreactivity was evident in the bottom of developing gastric buds, particularly in the apical part of cells. (Fig. 4C). From 15 wk onward, immunostaining was essentially located in cells of the bottom of fundic glands, a site similar to that of peptic chief cells (Fig. 4D). The labeling was specific and abolished by prior absorption of leptin antibody with human leptin recombinant (Fig. 4E).
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Mean human leptin level in amniotic fluid during the period of pregnancy corresponding to 16–33 wk gestation was 9.2 ± 1.9 ng/ml. However, it was significantly higher during the second trimester (13.8 ± 3.2 ng/ml, range 4–23 ng/ml, n = 5) than during the third trimester (5.3 ± 0.9 ng/ml, range 2.7–9 ng/ml, n = 6; P = 0.05, U test).
Leptin influences the DNA synthesis in fetal gastrointestinal explants
Due to the small amount of tissues collected, a single dose of leptin was tested. The radioisotope incorporation in these explants was weak enough, probably reflecting some tissue degradation. Nevertheless, these preliminary results indicated that functionality of leptin receptors was developing in the young fetuses because mean values of DNA synthesis were affected in explants cultured with leptin, with a reduction of the DNA precursor incorporation from 15 to 33.5%, as compared with their paired control explants (Fig. 5).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We provide the first analysis of leptin and Ob-Rb expression during the morphogenesis of the digestive tract in human fetuses. We have shown that Ob-Rb transcripts were early detected, for instance at 7.5 wk in the stomach. The long Ob-Rb isoform was detected all along the mucosae of the fetal digestive tract as it was in adult. We investigated the time course of Ob-Rb protein immunostaining and noted its dynamics changes in localization and variation in intensity according to the gestational age. In the differentiated gastric mucosa from around 15 wk, the signal was mainly located on basal membranes of surface epithelial cells and in parietal cells, which is consistent with the localization shown herein and previously in adult human stomach (13, 14). In small and large intestine, Ob-Rb immunostaining, although present in the cytoplasm and on basolateral membranes, was seen predominantly on apical membranes of enterocytes and colonocytes. This is also in agreement with the localization in adult intestine described herein and previously (15, 16, 17). Overall, immunoreactivity for Ob-Rb was higher in 8- to 11-wk-old fetuses, during the time period of morphogenesis corresponding to the formation of gastric buds and primitive intestinal crypts and to the beginning of differentiation of various epithelial cell types, than in the oldest fetuses.
We and others have detected leptin mRNA in the fundic mucosa of adult human (12, 13, 14) and rat (11). To date, leptin appearance in the gastric mucosa has been reported during the sole perinatal period in the rat (35). In this animal, levels of leptin in the gastric mucosa were very weak in prenatal stages and raised steadily after birth, the more marked increase coinciding with the change from suckling to a solid diet. The diffuse immunolabeling along the fundic glands appeared only at weaning time (35). In the rat, gastrointestinal organogenesis begins around 80% gestation; by contrast, in human, it begins early, before 25% of gestation (36). So the stomach could be a source of leptin in the human fetus. We did not detect the leptin transcript in the fetal stomach at 7.5 wk, whereas Ob-Rb mRNA was found at that time; this observation may imply that the receptor is expressed some days before leptin. Indeed, the onset of leptin synthesis in the stomach takes place around the 11th wk. However, whereas no leptin immunostaining was seen in the gastric mucosa at 7.5 wk, this protein was expressed by immunoblot and immunostaining at 8 wk, although the signal was weak (see Figs. 3 and 4). In older fetuses, the leptin signal was strongly increased and essentially found in cells located in the bottom of fundic glands, a site corresponding to the localization of pepsinogen-secreting cells. These observations are in agreement with previous reports in normal adult human stomach (12, 13, 14). In the youngest fetuses, the discrepancy between the temporal appearance of leptin mRNA and protein in the stomach could be explained by a rapid turnover of small numbers of mRNA copies preventing transcripts from being detected in our experimental conditions. Leptin mRNA could also be less stable and therefore easily degraded. Alternatively, immunodetected gastric leptin may reflect the binding to the target cell receptors of leptin from another source, blood or amniotic fluid.
Potential functional implications
Leptin has been found in breast milk (8, 9), and in suckling animals it appears to influence the maturation of digestive tissues (8, 35, 37). Leptin is also presumed to play a role in the developmental process of fetus (5, 10). Components of human amniotic fluid such as growth factors and hormones are important mediators of the functional gastrointestinal development (38). They can be transported across the fetal epithelium by endocytic process (39). Thus, in the fetal rabbit, the reduction in gastric weight and acid secretion induced by esophageal ligation was reversed by gastric infusion of bovine amniotic fluid or epidermal growth factor (40). In humans, these substances can exert influences as early as 10 wk gestation, when fetuses begin swallowing amniotic fluid (36).
Leptin is a component of human amniotic fluid. In the present work, noticeable leptin amounts were measured in amniotic fluid of women, roughly comparable with those previously reported (6, 10). In addition, we found that leptin levels were significantly higher during the second than the third trimester. Therefore, the early presence in the fetal life of leptin receptors in human digestive mucosae leaves open the possibility of a new role for leptin in the developmental process of the digestive tract. Indeed, because leptin exerts specific regulatory actions in the adult digestive tract (17, 18, 19, 20, 21, 22, 23), some of them by luminal route (20, 21), we may suggest that leptin is to some degree involved in the growth and/or functional development of fetal digestive organs. Besides possible endocrine effects exerted through circulating fetal leptin (29, 41, 42), this action in the digestive tract may be exerted through a luminal pathway when leptin is mainly supplied by amniotic fluid in the youngest fetuses; all the more so leptin receptors are predominantly expressed on apical membranes of intestine. Additionally, it may be exerted through a paracrine pathway when leptin is expressed in the stomach as fetal age increases. Due to the difficulty to obtain anatomically recognizable tissues with a good apparent quality and in a suitable time delay, the effect of leptin was examined in only some human fetal gastrointestinal explants. However, our preliminary findings would rather indicate a tendency for leptin to reduce DNA synthesis in those explants. Such an inhibitory effect on cell proliferation has also been observed in human fetal jejunum and colon with epithelial growth factor, a growth factor known as a mitogen (33, 43).
Obviously, further studies should be carried out to confirm the present data and establish whether leptin truly influences the development of the human digestive organs. If so, because no abnormality of the digestive tract in leptin-deficient ob-ob animals has been so far described, it may be suggested that other factors exert a compensatory role during fetal life.
In conclusion, our present work shows for the first time the early temporal appearance of Ob-Rb in mucosae of the human digestive tract and the nearly concomitant appearance of leptin in the gastric mucosa. These data, keeping in mind the high levels of leptin in amniotic fluids, especially during the second trimester of gestation, therefore in contact with mucosa when swallowing begins, point out a possible influence of the leptin-Ob-Rb complex in the growth and/or maturation of digestive organs in human fetuses.
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Acknowledgments |
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Footnotes |
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First Published Online September 6, 2005
Abbreviation: Ob-R, Leptin receptor.
Received July 7, 2005.
Accepted August 30, 2005.
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