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Protein restriction inhibits gastric cell proliferation during rat postnatal growth in parallel to ghrelin changes
Nutrition 28 (2012) 707–712
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Basic nutritional investigation
Protein restriction inhibits gastric cell proliferation during rat postnatal growth in parallel to ghrelin changes Ariane Kasai M.S., Patrıcia Gama Ph.D. *, Eliana Parisi Alvares Ph.D. ~o Paulo, Sa ~o Paulo, Brazil Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sa
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Article history: Received 11 April 2011 Accepted 11 October 2011
Objective: Gastric development depends directly on the proliferation and differentiation of epithelial cells, and these processes are controlled by multiple elements, such as diet, hormones, and growth factors. Protein restriction affects gastrointestinal functions, but its effects on gastric growth are not fully understood. Methods: The present study evaluated cell proliferation in the gastric epithelia of rats subjected to protein restriction since gestation. Because ghrelin is increasingly expressed from the fetal to the weaning stages and might be part of growth regulation, its distribution in the stomach of rats was investigated at 14, 30, and 50 d old. Results: Although the protein restriction at 8% increased the intake of food and body weight, the body mass was lower (P < 0.05). The stomach and intestine were also smaller but increased proportionately throughout treatment. Cell proliferation was estimated through DNA synthesis and metaphase indices, and lower rates (P < 0.05) were detected at the different ages. The inhibition was concomitant with a larger number of ghrelin-immunolabeled cells at 30 and 50 d postnatally. Conclusion: Protein restriction impairs cell proliferation in the gastric epithelium, and a ghrelin upsurge under this condition is parallel to lower gastric and body growth rates. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Protein restriction Stomach Cell proliferation Ghrelin Rat
Introduction Gastric epithelial cells begin to differentiate before birth but are not fully mature until the end of the third postnatal week [1, 2]. During this period, changes in the dietary pattern and feeding conditions can disturb epithelial cell proliferation, differentiation, and death, which can affect the growth of gastric glands [3–5]. Accordingly, protein restriction during pregnancy and lactation decreases body and stomach weights and decreases the small intestinal length in weanling rats [6]. However, few studies have addressed the effects of undernutrition on gastric histophysiology. Majumdar [7] reported that deficient feeding during the first 2 wk postnatally retards the growth concomitant with lower gastrin levels. In addition, greater mucous and prostaglandin secretions [8] and even lower pH [9] have been observed in adult rats subjected to protein restriction during development.
This study was supported by FAPESP 2004/10807-5. Ariane Kasai, M.S., was the recipient of a CNPq fellowship. * Corresponding author: Tel.: þ55-11-3091-7303; fax: þ55-11-3091-7402. E-mail address: [email protected] (P. Gama). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.10.003
The ontogenesis of the gastrointestinal tract is coordinated by a complex interaction of hormones, growth factors, milk-borne molecules, luminal microbes, and genetic programming [10–13]. Among these elements, ghrelin, a peptide first described as a natural ligand for the growth hormone (GH) secretagogue receptor [14], increases from birth to the fourth postnatal week in rats [15]. This orexigenic hormone is synthesized mainly in the gastric mucosa, in which endocrine cells express and modify the molecules through the action of ghrelin O-acyl-transferase [16]. Although this intracellular event is required for the production of acylated peptide, the des-acylated form is dominant in the blood, and the mechanism that regulates the differential release remains unknown [17]. Ghrelin levels can be influenced by age, dietary pattern, and feeding condition. In adult rats, plasma ghrelin was found to increase after the intake of a low-protein diet [18], calorie restriction [19], and fasting [20]. In pups, although similar observations were made, the content of ghrelin in the stomach decreased in fasted neonatal rats, indicating depletion from the gastric mucosa into the blood [21]. Functionally, ghrelin triggers GH release [14], induces food intake [17], and, for epithelial kinetics, reverses the apoptotic
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response induced by lipids in pancreatic b-cells [22] or by fasting in the intestinal mucosa [23]. Different studies have suggested that ghrelin acts through the paracrine pathways and the endocrine route [24]. Considering the importance of the interaction between feeding and hormones in the control of gastrointestinal postnatal development, we evaluated the effects of protein restriction on gastric cell proliferation and ghrelin distribution through the suckling, postweaning, and early adulthood stages. Material and methods Animals and protein restriction Wistar rats from the animal colony of the Department of Cell and Develop~o Paulo (Sa ~o mental Biology, Institute of Biomedical Sciences, University of Sa Paulo, SP, Brazil) were used according to the protocol reviewed and approved by the ethical committee on animal experimentation (CEEA no. 82/2005). Animals were maintained at 22 C under 12-h light/12-h dark schedules. Rats (200–250 g of body weight) were mated, and pregnancy was confirmed by checking the presence of spermatozoa in vaginal smears. Pregnant rats were randomly separated into two groups: control rats were fed an AIN-93G [25] normal-protein diet stria e Come rcio Ltda., Sa ~o Paulo, SP, Brazil) containing 20% casein (Rhoster Indu and protein-restricted (PR) rats were fed an AIN-93G isocaloric PR diet containing stria e Come rcio Ltda.; Table 1). All animals were fed an 8% casein (Rhoster Indu amount of chow larger than the daily average consumption previously calculated in the laboratory. The same diet offered throughout gestation was used after delivery and weaning, which was set at 21 d. Litters were culled to eight pups around the third day. During the gestational and lactation periods for dams and after the onset of weaning for pups, the food intake was calculated daily by subtracting the amount of chow left on the top cover and bed from the amount offered. The body weight was registered throughout the experimentation period. Water was offered ad libitum. Because previous studies have shown that fasting alters the kinetics of the gastric epithelium and the expression of different growth factors [3,10,12], the animals used in the present protocols were not prevented from eating.
cells were counted inside the proliferative compartment of the gland in randomly chosen fields using an ocular grid (Zeiss Integration Eyepiece I Kpl 8, Zeiss, Hamburg, Germany) at 800 magnification. At 14 d, the proliferative compartment in the corpus region of the stomach contains the entire gland, whereas at 30 d it covers the isthmus and neck glandular areas [3]. Only longitudinally sectioned areas of the gastric gland were used. The MI was reported as the number of metaphase cells per total number of cells counted multiplied by 100. In addition to the evaluation of the MI, we studied the DNA synthesis index (SI). Animals were injected intraperitoneally with bromodeoxyuridine (BrDU; Sigma, St. Louis, MO, USA; 100 mg/kg of body weight) 1 h before the sacrifice. Briefly, after being cleared of paraffin and rehydrated, the gastric sections were immersed in phosphate buffered saline (PBS; pH 7.4) 5 mmol/L, and the endogenous peroxidase was blocked with 0.5% H2O2 in methanol for 10 min. After being washed with water and PBS, the sections were incubated with 0.1% pepsin (Merck, Darmstadt, Germany) in 0.1 N HCl for 20 min in a water bath at 37 C with subsequent rinsing in PBS. The monoclonal anti-BrDU antibody (GE Healthcare, Buckinghamshire, UK) and PBS were applied to the sections at 1:100 (overnight at 4 C). Slides were washed with PBS and then incubated with the secondary antibody conjugated with peroxidase (GE Healthcare) for 1 h at room temperature (RT). BrDU immunolabeling was detected by covering sections with 0.05% 3,30 -diaminobenzidine tetrahydrochloride in PBS containing H2O2. All slides were counterstained with Mayer hematoxylin. Negative controls were performed through substitution of the primary antibody by PBS. The SI was determined according to the same criteria described for MI. The SI was reported as the number of BrDU-labeled nuclei per total number of epithelial nuclei counted multiplied by 100. Ghrelin detection in gastric mucosa
Rats 14, 30, and 50 d old were anesthetized intraperitoneally with a 1:1 (v/v) mixture of ketamine and xylazine (Anasedan and Dopalen, Vetbrands, S~ ao Paulo, SP, Brazil) at 0.5 mL/100 g of body weight. The stomach was opened and rinsed with saline, and serosal side was dried and weighed before fixation, which was performed in 10 % formalin for immunohistochemistry or in Bouin liquid for morphologic and metaphase index (MI) studies. Samples were embedded in paraffin wax. The small intestine was collected in all experiments and the length was measured.
Sections were cleared of paraffin, rehydrated with PBS containing 0.1% (v/v) Triton X-100 (10 min at RT), washed with PBS, and incubated with 0.5% (v/v) H2O2 in methanol for 10 min to block the endogenous peroxidase activity. Then the slides were washed in water and PBS and incubated with 0.05% pepsin (Merck) in 0.1 N HCl for 20 min in a water bath at 37 C for antigen retrieval. Subsequently, sections were washed in PBS containing 0.1% (w/v) bovine serum albumin (10 min) and non-specific binding was blocked with 20% (v/v) goat serum (1 h at RT). Tissue sections were incubated overnight at 4 C with polyclonal chicken anti-ghrelin antibody at 1:100 (Abcam, Cambridge, MA, USA). After washing with PBS and incubating with a biotinylated secondary antibody (2 h at RT; Jackson ImmunoResearch Laboratories, West Grove, PA, USA), the sections were treated with a streptavidin–peroxidase complex (Jackson ImmunoResearch Laboratories) for 1 h at RT. Peroxidase activity was developed by 3,30 diaminobenzidine tetrahydrochloride (Sigma) as described earlier. Negative control samples were incubated with goat serum. Sections were analyzed under a light microscope (Nikon) at 800 magnification. The presence and localization of ghrelin-labeled cells in the corpus of the stomach were observed and the number of immunostained cells was estimated in the microscopic fields. Approximately 30 fields per animal were studied. Results were obtained as the number of labeled cells per field.
Cell proliferation determination
Plasma ghrelin detection
To estimate the MI in the gastric epithelium, each animal received vincristine ao Paulo, SP, Brazil) 0.5 mg/kg of body sulfate (Zodiac Produtos Farmaceuticos, S~ weight intraperitoneally 2 h before euthanasia. After the collection and histologic procedures, non-serial 5-mm sections from the stomach wall were obtained and stained with hematoxylin and eosin. The MI was assessed by counting the number of cells arrested at metaphase under light microscopy (Nikon, Tokyo, Japan). Approximately 2500 epithelial
Blood was collected by puncture from the abdominal aorta, immediately transferred to polypropylene microtubes containing ethylenediaminetetraacetic acid (1 mg/mL) and aprotinin (0.6 TIU/mL), and centrifuged (14 000 rpm at 4 C for 15 min). Plasma samples were stored at 80 C until assayed. Ghrelin was measured using a commercial enzymatic immunoassay kit (Phoenix Pharmaceuticals, Burlingame, CA, USA) according to the manufacturer’s instructions. The sensitivity of the assay was 0.1 ng/mL. The intra- and interassay coefficients of variation reported were lower than 5% and lower than 14%, respectively.
Tissue collection
ˇ
Table 1 Composition of isocaloric (control) and protein-restricted diets* Ingredients (%)
Control
Protein restrictiony
Casein (protein) Cornstarch Dextrinized cornstarch Sucrose Soybean oil Cellulose–fiber AIN-93G mineral mix AIN-93 vitamin mix L-Cystine Choline bitartrate tert-Butyl-hydroquinone
20.00 39.75 13.20 10.00 7.00 5.00 3.50 1.00 0.30 0.25 0.0014
8.00 51.75 13.20 10.00 7.00 5.00 3.50 1.00 0.30 0.25 0.0014
* y
Prepared according the AIN-93G [25]. Isocaloric diet (3.8 kcal/g).
Statistical analysis Results were reported as mean standard deviation, and GraphPad Prism 5.1 (GraphPad Prism Software, Inc., San Diego, CA, USA) was used for statistical purposes. The data were analyzed by analysis of variance followed by the Tukey test, and statistical significance level was considered at P < 0.05.
Results Food intake did not differ between dams fed the normal control diet and those receiving the PR chow. Similarly, protein restriction did not influence feeding after the onset of weaning (data not shown). However, when the intake was adjusted for body weight (grams of chow per gram body of weight), the diet consumption was significantly higher in the PR rats (P < 0.05;
A. Kasai et al. / Nutrition 28 (2012) 707–712
Fig. 1B). It should be noted that this ratio was not considered before weaning, because throughout the suckling period, milk was the only source of nutrients and the parameter was not accessible. Also, when the different ages were compared, the intake did not change in the control or PR group. Body weight was registered for the dams during pregnancy and lactation. After starting treatment, the control and PR dams showed 29.5% and 15.2% weight gains, respectively. When pups and developing animals were considered, a lower body weight was observed in the PR group compared with the control group feeding (P < 0.05; Fig. 1A). Of note, we registered a difference on the fifth postnatal day; although the control pups progressively gained mass (P < 0.05), PR rats grew at a much lower rate. Stomach weight and intestinal length were recorded and these organs were significantly smaller in the PR group (P < 0.05; Fig. 1C, D), except for the animals at 14 d, when treatment did not exert a significant response. Also, the stomach and intestines grew proportionately through the period evaluated in the control and PR rats. We should mention that we did not record gross morphologic changes in the histologic organization of these organs. Cell proliferation was estimated in the gastric mucosa of pups and developing rats under protein restriction to determine whether the decrease of the organ might be a consequence of a lower cell division and was correlated to ghrelin production. We found that protein restriction changed the extension of the proliferative compartment in the gastric epithelium. It is known that proliferating cells are distributed in the entire gland until weaning, after which they are restricted to the isthmus/neck region [3]. Protein restriction disturbed the compartment by spreading BrDU-labeled cells along the gland (Fig. 2A–E). Moreover, protein restriction decreased the DNA SI (P < 0.05) compared with the control group during the suckling, postweaning, and early adulthood stages (Fig. 2F). In addition, a major IS decrease was recorded at 30 d, which might be caused
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by the high index detected in control rats and by the low and unchanged indices obtained for the PR group. These results were corroborated by the MI determined for 14- and 30-d-old rats (Table 2). To investigate the distribution of ghrelin-producing cells during protein restriction, we used immunohistochemistry to label the gastric epithelium and the enzymatic immunoassay to measure plasma ghrelin. First, ghrelin immunolabeling was more restricted to the basal region of the gland, and we confirmed that the hormone was confined to the cytoplasm of the endocrine cells (Fig. 3A–E). Second, protein restriction triggered different effects on the number of ghrelin-labeled cells depending on the age studied. Accordingly, although we did not record changes at 14 d, the population of ghrelin-positive cells significantly increased at 30 and 50 d in the gastric epithelium of PR rats (P < 0.05; Fig. 3F). Moreover, the effect was stronger at 30 d compared with 50 d (P < 0.05). The concentration of plasma ghrelin was lower at 14 d that at 30 d. Similarly to what was described earlier, protein restriction did not influence the levels of ghrelin at 14 d, but it significantly increased the hormonal concentration at 30 d (Table 2).
Discussion Different studies have reported the effects of protein restriction on body growth and metabolism, although only a few have aimed to describe to consequences of a low-protein diet on gastric development. In the present study, we compared the effects of regular feeding with those of protein restriction on the gastric mucosa from pregnancy to early adulthood in rats. Interestingly, the present results indicated for the first time that protein restriction significantly inhibited epithelial cell proliferation in the gastric mucosa throughout the developmental
Fig. 1. Growth and food intake evaluation in rats subjected to protein restriction (black bars) or fed normal protein levels (white bars) throughout gestation and postnatal development. (A) Body weight (grams) at 14, 30, and 50 d postnatally. (B) Food intake/body weight registered after the onset of weaning. (C) Stomach weight and (D) intestinal length throughout development. Values are presented as mean SD (n ¼ 5 animals/group). Experiments were repeated in duplicate. * P < 0.05 compared with controls; # P < 0.05 compared to the younger group on the same treatment.
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Fig. 2. Cell proliferation in the gastric epithelium of rats subjected to protein restriction throughout gestation and postnatal development. (A–E) Distribution of bromodeoxyuridine-labeled cells in the gastric gland of control animals at 14 d (A) and 50 d (D) and protein-restricted rats at 14 d (B) and 50 d (E). (C) Negative control. Original magnifications 40 in A–C and 20 in D and E. (F) The IS obtained for the control (white bars) and protein-restricted (black bars) groups. Values are presented as mean SD (n ¼ 4/group). * P < 0.05 versus control; # P < 0.05 versus the younger group on the same treatment. b, base region in the mucosa; f, foveola; g, gland; i, isthmus region in the mucosa; n, neck region in the mucosa; SI, DNA synthesis index.
period in parallel with an increasing density of ghrelinproducing cells in the stomach. Consistent with previous observations, we found that protein restriction markedly decreased body weight, despite the increased food intake. Weaver et al. [6] showed that protein restriction during the gestation and lactation stages induces
Table 2 Metaphase index and plasma ghrelin levels after protein restriction 14 d old Control
30 d old PR
Control
PR
Metaphase index (%) 1.76 0.16 1 0.04* 2.2 0.37 0.73 0.1* Plasma ghrelin (ng/mL) 0.84 0.22 0.85 0.34 1.97 0.06 2.68 0.3* PR, protein restriction * P < 0.05 compared with control at the same age.
a growth deficit throughout development and the consequences can be recorded in adults, even when protein levels are recovered after weaning. Using different feeding models, Young et al. [26] and Desai et al. [27] also described a long-lasting effect on growth retardation in malnourished pups, which includes the development of digestive organs. In the present study, we recorded decreased stomach mass and intestinal length in animals subjected to protein restriction, but these grew proportionately to aging, which corroborates a recent study that showed that long-term dietary restriction can induce the increase of gut size [19]. Interestingly, we also observed that the effect of protein restriction was more severe to the stomach than to the intestine, and such a difference might represent an adaptive mechanism to guarantee the absorption of nutrients from the diet. Because this function is essential to maintaining metabolism and growth,
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Fig. 3. Ghrelin distribution in the gastric epithelium of rats subjected to protein restriction throughout gestation and postnatal development. (A–E) Distribution of ghrelinlabeled cells in the gastric gland of control animals at 14 d (A) and 30 d (D) and protein-restricted rats at 14 d (B) and 30 d (E). (C) Negative control. Original magnifications 40 in A–C, 20 in D and E; 100 in inset in B. (F) Number of ghrelin-producing cells per field obtained for the control (white bars) and protein-restricted (black bars) groups. Values are presented as mean SD (n ¼ 4). * P < 0.05 versus control; # P < 0.05 versus younger group on the same treatment.
most studies have focused on the response of the small intestinal epithelium to dietary changes and reported that protein restriction inhibits cell proliferation in the mucosa [28–30]. We focused on the stomach, and our results demonstrated that epithelial renewal was decreased in rats subjected to protein restriction in the suckling, postweaning, and early adulthood stages. The decrease of cell proliferation in the gastric epithelium has been detected under other different feeding conditions, such as fasting [1,3,4], indicating that the presence and type of food might interfere with epithelial kinetics. Curiously, when ages were compared, a proliferative peak at 30 d in regularly fed rats and a constant rate of DNA synthesis during protein restriction were noted, which indicated a more profound response in 30-d-old animals. In addition, we observed that protein restriction altered the proliferative compartment, which is defined as the isthmus/neck region of the gland [31], by spreading the dividing cells outside this area. The molecular events behind
such changes should be investigated, but other studies have reported the influence of dietary pattern on the distribution of proliferative cells [4,5]. The epithelial cell proliferation in the gastrointestinal tract is regulated by the interaction of multiple agents, which include special feeding conditions, hormones, and growth factors [5,10, 12]. As mentioned earlier, the gastric epithelium was responsive to long-term protein restriction, which permanently affected stomach growth. Because ghrelin is a hormone involved in the control of food intake and its effects on cellular turnover have been recently uncovered in digestive organs [22–24], we evaluated the density of ghrelin-producing cells in the gastric mucosa. We found that protein restriction promoted different effects on this endocrine cell population depending on the developmental stage. Accordingly, at suckling, we did not detect changes, whereas in weaned and young adult rats we found an increase of ghrelin-immunoreactive cells. It is important to note
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that that a peak was observed at 30 d. Previous reports have consistently shown that food and calorie restrictions induce higher expressions of ghrelin O-acyl-transferase and ghrelin mRNA levels after short-term treatment [32,33], and that such a spike is followed by a decrease after months of dietary deprivation [19]. The effects of increased ghrelin can be diverse on the organs, as recently reviewed [34]. Our results indicated that gastric cell proliferation was inhibited by protein restriction in parallel with a high production of ghrelin, especially in weaned rats. Although few studies have evaluated the action of this molecule as a hormone or paracrine factor on cell kinetics, there is a consensus about its role in apoptosis suppression in the intestinal mucosa [23] and pancreatic cells [22]. In the stomach, however, a dual age-dependent effect has been described, and Warzecha et al. [35] demonstrated that ghrelin can inhibit or stimulate cell proliferation when administered to suckling or peripubertal rats, respectively. In another study, Ceranowicz et al. [24] showed that ghrelin accelerated the healing of gastric ulcers by increasing DNA synthesis in adult animals, and they postulated that this response should be indirect and mediated by GH and insulin-like growth factor-1. Comparatively, we agree with the idea that ghrelin might be differentially correlated to cell proliferation in the gastric epithelium, and that variation could reflect a change in the sensibility or functionality during development. However, because we observed constant low proliferative indices in the gastric epithelium of PR rats, other molecules could interact with ghrelin or alter its action, and the possible candidates for these functions are GH secretagogue receptor, leptin, corticosterone, and growth factors [5,12,36,37]. Altogether, our results indicated a correlation among protein restriction, inhibition of gastric cell proliferation, and high ghrelin levels, suggesting that the permanent situation of malnutrition might interfere not only with metabolism but also with cellular kinetics in the gastric mucosa, which ultimately would affect stomach growth. Acknowledgments The authors thank Cruz Alberto Mendoza Rigonati for technical assistance. References [1] Alvares EP. Extensive networks of TMPase positive basal lysosomes are present in fetal rat gastric epithelium before overt differentiation. J Submicrosc Cytol Pathol 1994;26:515–23. [2] Zhu L, Hatakeyama J, Zhang B, Makdisi J, Ender C, Forte JG. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin. Am J Physiol Gastrointest Liver Physiol 2009;296:G185–95. [3] Alvares EP, Gama P. Fasting enhances cell proliferation of gastric epithelium during the suckling period in rats. Braz J Med Biol Res 1993;26:869–73. [4] Gama P, Alvares EP. Early weaning and prolonged nursing induce changes in cell proliferation in the gastric epithelium of developing rats. J Nutr 2000;130:2594–8. [5] Osaki L, Figueiredo PM, Alvares EP, Gama P. EGFR is involved in the control of gastric cell proliferation through the activation of MAPK and Src signalling pathways in early-weaned rats. Cell Prolif 2011;44:174–82. [6] Weaver LT, Desai M, Austin S, Arthur HM, Lucas A, Hales CN. Effects of protein restriction in early life on growth and function of the gastrointestinal tract of the rat. J Pediatr Gastroenterol Nutr 1998;27:553–9. [7] Majumdar AP. Postnatal undernutrition: effect of epidermal growth factor on growth and function of the gastrointestinal tract in rats. J Pediatr Gastroenterol Nutr 1984;3:618–25. [8] Paula AC, Gracioso JS, Toma W, Bezerra R, Saad MA, De Lucca IM, et al. Is gastric ulceration different in normal and malnourished rats? Br J Nutr 2005;93:47–52. [9] Montoya CA, Leterme P, Lalles JP. A protein-free diet alters small intestinal architecture and digestive enzyme activities in rats. Reprod Nutr Dev 2006;46:49–56.
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Nutrition journal homepage: www.nutritionjrnl.com
Basic nutritional investigation
Protein restriction inhibits gastric cell proliferation during rat postnatal growth in parallel to ghrelin changes Ariane Kasai M.S., Patrıcia Gama Ph.D. *, Eliana Parisi Alvares Ph.D. ~o Paulo, Sa ~o Paulo, Brazil Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sa
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 April 2011 Accepted 11 October 2011
Objective: Gastric development depends directly on the proliferation and differentiation of epithelial cells, and these processes are controlled by multiple elements, such as diet, hormones, and growth factors. Protein restriction affects gastrointestinal functions, but its effects on gastric growth are not fully understood. Methods: The present study evaluated cell proliferation in the gastric epithelia of rats subjected to protein restriction since gestation. Because ghrelin is increasingly expressed from the fetal to the weaning stages and might be part of growth regulation, its distribution in the stomach of rats was investigated at 14, 30, and 50 d old. Results: Although the protein restriction at 8% increased the intake of food and body weight, the body mass was lower (P < 0.05). The stomach and intestine were also smaller but increased proportionately throughout treatment. Cell proliferation was estimated through DNA synthesis and metaphase indices, and lower rates (P < 0.05) were detected at the different ages. The inhibition was concomitant with a larger number of ghrelin-immunolabeled cells at 30 and 50 d postnatally. Conclusion: Protein restriction impairs cell proliferation in the gastric epithelium, and a ghrelin upsurge under this condition is parallel to lower gastric and body growth rates. Ó 2012 Elsevier Inc. All rights reserved.
Keywords: Protein restriction Stomach Cell proliferation Ghrelin Rat
Introduction Gastric epithelial cells begin to differentiate before birth but are not fully mature until the end of the third postnatal week [1, 2]. During this period, changes in the dietary pattern and feeding conditions can disturb epithelial cell proliferation, differentiation, and death, which can affect the growth of gastric glands [3–5]. Accordingly, protein restriction during pregnancy and lactation decreases body and stomach weights and decreases the small intestinal length in weanling rats [6]. However, few studies have addressed the effects of undernutrition on gastric histophysiology. Majumdar [7] reported that deficient feeding during the first 2 wk postnatally retards the growth concomitant with lower gastrin levels. In addition, greater mucous and prostaglandin secretions [8] and even lower pH [9] have been observed in adult rats subjected to protein restriction during development.
This study was supported by FAPESP 2004/10807-5. Ariane Kasai, M.S., was the recipient of a CNPq fellowship. * Corresponding author: Tel.: þ55-11-3091-7303; fax: þ55-11-3091-7402. E-mail address: [email protected] (P. Gama). 0899-9007/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.nut.2011.10.003
The ontogenesis of the gastrointestinal tract is coordinated by a complex interaction of hormones, growth factors, milk-borne molecules, luminal microbes, and genetic programming [10–13]. Among these elements, ghrelin, a peptide first described as a natural ligand for the growth hormone (GH) secretagogue receptor [14], increases from birth to the fourth postnatal week in rats [15]. This orexigenic hormone is synthesized mainly in the gastric mucosa, in which endocrine cells express and modify the molecules through the action of ghrelin O-acyl-transferase [16]. Although this intracellular event is required for the production of acylated peptide, the des-acylated form is dominant in the blood, and the mechanism that regulates the differential release remains unknown [17]. Ghrelin levels can be influenced by age, dietary pattern, and feeding condition. In adult rats, plasma ghrelin was found to increase after the intake of a low-protein diet [18], calorie restriction [19], and fasting [20]. In pups, although similar observations were made, the content of ghrelin in the stomach decreased in fasted neonatal rats, indicating depletion from the gastric mucosa into the blood [21]. Functionally, ghrelin triggers GH release [14], induces food intake [17], and, for epithelial kinetics, reverses the apoptotic
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response induced by lipids in pancreatic b-cells [22] or by fasting in the intestinal mucosa [23]. Different studies have suggested that ghrelin acts through the paracrine pathways and the endocrine route [24]. Considering the importance of the interaction between feeding and hormones in the control of gastrointestinal postnatal development, we evaluated the effects of protein restriction on gastric cell proliferation and ghrelin distribution through the suckling, postweaning, and early adulthood stages. Material and methods Animals and protein restriction Wistar rats from the animal colony of the Department of Cell and Develop~o Paulo (Sa ~o mental Biology, Institute of Biomedical Sciences, University of Sa Paulo, SP, Brazil) were used according to the protocol reviewed and approved by the ethical committee on animal experimentation (CEEA no. 82/2005). Animals were maintained at 22 C under 12-h light/12-h dark schedules. Rats (200–250 g of body weight) were mated, and pregnancy was confirmed by checking the presence of spermatozoa in vaginal smears. Pregnant rats were randomly separated into two groups: control rats were fed an AIN-93G [25] normal-protein diet stria e Come rcio Ltda., Sa ~o Paulo, SP, Brazil) containing 20% casein (Rhoster Indu and protein-restricted (PR) rats were fed an AIN-93G isocaloric PR diet containing stria e Come rcio Ltda.; Table 1). All animals were fed an 8% casein (Rhoster Indu amount of chow larger than the daily average consumption previously calculated in the laboratory. The same diet offered throughout gestation was used after delivery and weaning, which was set at 21 d. Litters were culled to eight pups around the third day. During the gestational and lactation periods for dams and after the onset of weaning for pups, the food intake was calculated daily by subtracting the amount of chow left on the top cover and bed from the amount offered. The body weight was registered throughout the experimentation period. Water was offered ad libitum. Because previous studies have shown that fasting alters the kinetics of the gastric epithelium and the expression of different growth factors [3,10,12], the animals used in the present protocols were not prevented from eating.
cells were counted inside the proliferative compartment of the gland in randomly chosen fields using an ocular grid (Zeiss Integration Eyepiece I Kpl 8, Zeiss, Hamburg, Germany) at 800 magnification. At 14 d, the proliferative compartment in the corpus region of the stomach contains the entire gland, whereas at 30 d it covers the isthmus and neck glandular areas [3]. Only longitudinally sectioned areas of the gastric gland were used. The MI was reported as the number of metaphase cells per total number of cells counted multiplied by 100. In addition to the evaluation of the MI, we studied the DNA synthesis index (SI). Animals were injected intraperitoneally with bromodeoxyuridine (BrDU; Sigma, St. Louis, MO, USA; 100 mg/kg of body weight) 1 h before the sacrifice. Briefly, after being cleared of paraffin and rehydrated, the gastric sections were immersed in phosphate buffered saline (PBS; pH 7.4) 5 mmol/L, and the endogenous peroxidase was blocked with 0.5% H2O2 in methanol for 10 min. After being washed with water and PBS, the sections were incubated with 0.1% pepsin (Merck, Darmstadt, Germany) in 0.1 N HCl for 20 min in a water bath at 37 C with subsequent rinsing in PBS. The monoclonal anti-BrDU antibody (GE Healthcare, Buckinghamshire, UK) and PBS were applied to the sections at 1:100 (overnight at 4 C). Slides were washed with PBS and then incubated with the secondary antibody conjugated with peroxidase (GE Healthcare) for 1 h at room temperature (RT). BrDU immunolabeling was detected by covering sections with 0.05% 3,30 -diaminobenzidine tetrahydrochloride in PBS containing H2O2. All slides were counterstained with Mayer hematoxylin. Negative controls were performed through substitution of the primary antibody by PBS. The SI was determined according to the same criteria described for MI. The SI was reported as the number of BrDU-labeled nuclei per total number of epithelial nuclei counted multiplied by 100. Ghrelin detection in gastric mucosa
Rats 14, 30, and 50 d old were anesthetized intraperitoneally with a 1:1 (v/v) mixture of ketamine and xylazine (Anasedan and Dopalen, Vetbrands, S~ ao Paulo, SP, Brazil) at 0.5 mL/100 g of body weight. The stomach was opened and rinsed with saline, and serosal side was dried and weighed before fixation, which was performed in 10 % formalin for immunohistochemistry or in Bouin liquid for morphologic and metaphase index (MI) studies. Samples were embedded in paraffin wax. The small intestine was collected in all experiments and the length was measured.
Sections were cleared of paraffin, rehydrated with PBS containing 0.1% (v/v) Triton X-100 (10 min at RT), washed with PBS, and incubated with 0.5% (v/v) H2O2 in methanol for 10 min to block the endogenous peroxidase activity. Then the slides were washed in water and PBS and incubated with 0.05% pepsin (Merck) in 0.1 N HCl for 20 min in a water bath at 37 C for antigen retrieval. Subsequently, sections were washed in PBS containing 0.1% (w/v) bovine serum albumin (10 min) and non-specific binding was blocked with 20% (v/v) goat serum (1 h at RT). Tissue sections were incubated overnight at 4 C with polyclonal chicken anti-ghrelin antibody at 1:100 (Abcam, Cambridge, MA, USA). After washing with PBS and incubating with a biotinylated secondary antibody (2 h at RT; Jackson ImmunoResearch Laboratories, West Grove, PA, USA), the sections were treated with a streptavidin–peroxidase complex (Jackson ImmunoResearch Laboratories) for 1 h at RT. Peroxidase activity was developed by 3,30 diaminobenzidine tetrahydrochloride (Sigma) as described earlier. Negative control samples were incubated with goat serum. Sections were analyzed under a light microscope (Nikon) at 800 magnification. The presence and localization of ghrelin-labeled cells in the corpus of the stomach were observed and the number of immunostained cells was estimated in the microscopic fields. Approximately 30 fields per animal were studied. Results were obtained as the number of labeled cells per field.
Cell proliferation determination
Plasma ghrelin detection
To estimate the MI in the gastric epithelium, each animal received vincristine ao Paulo, SP, Brazil) 0.5 mg/kg of body sulfate (Zodiac Produtos Farmaceuticos, S~ weight intraperitoneally 2 h before euthanasia. After the collection and histologic procedures, non-serial 5-mm sections from the stomach wall were obtained and stained with hematoxylin and eosin. The MI was assessed by counting the number of cells arrested at metaphase under light microscopy (Nikon, Tokyo, Japan). Approximately 2500 epithelial
Blood was collected by puncture from the abdominal aorta, immediately transferred to polypropylene microtubes containing ethylenediaminetetraacetic acid (1 mg/mL) and aprotinin (0.6 TIU/mL), and centrifuged (14 000 rpm at 4 C for 15 min). Plasma samples were stored at 80 C until assayed. Ghrelin was measured using a commercial enzymatic immunoassay kit (Phoenix Pharmaceuticals, Burlingame, CA, USA) according to the manufacturer’s instructions. The sensitivity of the assay was 0.1 ng/mL. The intra- and interassay coefficients of variation reported were lower than 5% and lower than 14%, respectively.
Tissue collection
ˇ
Table 1 Composition of isocaloric (control) and protein-restricted diets* Ingredients (%)
Control
Protein restrictiony
Casein (protein) Cornstarch Dextrinized cornstarch Sucrose Soybean oil Cellulose–fiber AIN-93G mineral mix AIN-93 vitamin mix L-Cystine Choline bitartrate tert-Butyl-hydroquinone
20.00 39.75 13.20 10.00 7.00 5.00 3.50 1.00 0.30 0.25 0.0014
8.00 51.75 13.20 10.00 7.00 5.00 3.50 1.00 0.30 0.25 0.0014
* y
Prepared according the AIN-93G [25]. Isocaloric diet (3.8 kcal/g).
Statistical analysis Results were reported as mean standard deviation, and GraphPad Prism 5.1 (GraphPad Prism Software, Inc., San Diego, CA, USA) was used for statistical purposes. The data were analyzed by analysis of variance followed by the Tukey test, and statistical significance level was considered at P < 0.05.
Results Food intake did not differ between dams fed the normal control diet and those receiving the PR chow. Similarly, protein restriction did not influence feeding after the onset of weaning (data not shown). However, when the intake was adjusted for body weight (grams of chow per gram body of weight), the diet consumption was significantly higher in the PR rats (P < 0.05;
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Fig. 1B). It should be noted that this ratio was not considered before weaning, because throughout the suckling period, milk was the only source of nutrients and the parameter was not accessible. Also, when the different ages were compared, the intake did not change in the control or PR group. Body weight was registered for the dams during pregnancy and lactation. After starting treatment, the control and PR dams showed 29.5% and 15.2% weight gains, respectively. When pups and developing animals were considered, a lower body weight was observed in the PR group compared with the control group feeding (P < 0.05; Fig. 1A). Of note, we registered a difference on the fifth postnatal day; although the control pups progressively gained mass (P < 0.05), PR rats grew at a much lower rate. Stomach weight and intestinal length were recorded and these organs were significantly smaller in the PR group (P < 0.05; Fig. 1C, D), except for the animals at 14 d, when treatment did not exert a significant response. Also, the stomach and intestines grew proportionately through the period evaluated in the control and PR rats. We should mention that we did not record gross morphologic changes in the histologic organization of these organs. Cell proliferation was estimated in the gastric mucosa of pups and developing rats under protein restriction to determine whether the decrease of the organ might be a consequence of a lower cell division and was correlated to ghrelin production. We found that protein restriction changed the extension of the proliferative compartment in the gastric epithelium. It is known that proliferating cells are distributed in the entire gland until weaning, after which they are restricted to the isthmus/neck region [3]. Protein restriction disturbed the compartment by spreading BrDU-labeled cells along the gland (Fig. 2A–E). Moreover, protein restriction decreased the DNA SI (P < 0.05) compared with the control group during the suckling, postweaning, and early adulthood stages (Fig. 2F). In addition, a major IS decrease was recorded at 30 d, which might be caused
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by the high index detected in control rats and by the low and unchanged indices obtained for the PR group. These results were corroborated by the MI determined for 14- and 30-d-old rats (Table 2). To investigate the distribution of ghrelin-producing cells during protein restriction, we used immunohistochemistry to label the gastric epithelium and the enzymatic immunoassay to measure plasma ghrelin. First, ghrelin immunolabeling was more restricted to the basal region of the gland, and we confirmed that the hormone was confined to the cytoplasm of the endocrine cells (Fig. 3A–E). Second, protein restriction triggered different effects on the number of ghrelin-labeled cells depending on the age studied. Accordingly, although we did not record changes at 14 d, the population of ghrelin-positive cells significantly increased at 30 and 50 d in the gastric epithelium of PR rats (P < 0.05; Fig. 3F). Moreover, the effect was stronger at 30 d compared with 50 d (P < 0.05). The concentration of plasma ghrelin was lower at 14 d that at 30 d. Similarly to what was described earlier, protein restriction did not influence the levels of ghrelin at 14 d, but it significantly increased the hormonal concentration at 30 d (Table 2).
Discussion Different studies have reported the effects of protein restriction on body growth and metabolism, although only a few have aimed to describe to consequences of a low-protein diet on gastric development. In the present study, we compared the effects of regular feeding with those of protein restriction on the gastric mucosa from pregnancy to early adulthood in rats. Interestingly, the present results indicated for the first time that protein restriction significantly inhibited epithelial cell proliferation in the gastric mucosa throughout the developmental
Fig. 1. Growth and food intake evaluation in rats subjected to protein restriction (black bars) or fed normal protein levels (white bars) throughout gestation and postnatal development. (A) Body weight (grams) at 14, 30, and 50 d postnatally. (B) Food intake/body weight registered after the onset of weaning. (C) Stomach weight and (D) intestinal length throughout development. Values are presented as mean SD (n ¼ 5 animals/group). Experiments were repeated in duplicate. * P < 0.05 compared with controls; # P < 0.05 compared to the younger group on the same treatment.
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Fig. 2. Cell proliferation in the gastric epithelium of rats subjected to protein restriction throughout gestation and postnatal development. (A–E) Distribution of bromodeoxyuridine-labeled cells in the gastric gland of control animals at 14 d (A) and 50 d (D) and protein-restricted rats at 14 d (B) and 50 d (E). (C) Negative control. Original magnifications 40 in A–C and 20 in D and E. (F) The IS obtained for the control (white bars) and protein-restricted (black bars) groups. Values are presented as mean SD (n ¼ 4/group). * P < 0.05 versus control; # P < 0.05 versus the younger group on the same treatment. b, base region in the mucosa; f, foveola; g, gland; i, isthmus region in the mucosa; n, neck region in the mucosa; SI, DNA synthesis index.
period in parallel with an increasing density of ghrelinproducing cells in the stomach. Consistent with previous observations, we found that protein restriction markedly decreased body weight, despite the increased food intake. Weaver et al. [6] showed that protein restriction during the gestation and lactation stages induces
Table 2 Metaphase index and plasma ghrelin levels after protein restriction 14 d old Control
30 d old PR
Control
PR
Metaphase index (%) 1.76 0.16 1 0.04* 2.2 0.37 0.73 0.1* Plasma ghrelin (ng/mL) 0.84 0.22 0.85 0.34 1.97 0.06 2.68 0.3* PR, protein restriction * P < 0.05 compared with control at the same age.
a growth deficit throughout development and the consequences can be recorded in adults, even when protein levels are recovered after weaning. Using different feeding models, Young et al. [26] and Desai et al. [27] also described a long-lasting effect on growth retardation in malnourished pups, which includes the development of digestive organs. In the present study, we recorded decreased stomach mass and intestinal length in animals subjected to protein restriction, but these grew proportionately to aging, which corroborates a recent study that showed that long-term dietary restriction can induce the increase of gut size [19]. Interestingly, we also observed that the effect of protein restriction was more severe to the stomach than to the intestine, and such a difference might represent an adaptive mechanism to guarantee the absorption of nutrients from the diet. Because this function is essential to maintaining metabolism and growth,
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Fig. 3. Ghrelin distribution in the gastric epithelium of rats subjected to protein restriction throughout gestation and postnatal development. (A–E) Distribution of ghrelinlabeled cells in the gastric gland of control animals at 14 d (A) and 30 d (D) and protein-restricted rats at 14 d (B) and 30 d (E). (C) Negative control. Original magnifications 40 in A–C, 20 in D and E; 100 in inset in B. (F) Number of ghrelin-producing cells per field obtained for the control (white bars) and protein-restricted (black bars) groups. Values are presented as mean SD (n ¼ 4). * P < 0.05 versus control; # P < 0.05 versus younger group on the same treatment.
most studies have focused on the response of the small intestinal epithelium to dietary changes and reported that protein restriction inhibits cell proliferation in the mucosa [28–30]. We focused on the stomach, and our results demonstrated that epithelial renewal was decreased in rats subjected to protein restriction in the suckling, postweaning, and early adulthood stages. The decrease of cell proliferation in the gastric epithelium has been detected under other different feeding conditions, such as fasting [1,3,4], indicating that the presence and type of food might interfere with epithelial kinetics. Curiously, when ages were compared, a proliferative peak at 30 d in regularly fed rats and a constant rate of DNA synthesis during protein restriction were noted, which indicated a more profound response in 30-d-old animals. In addition, we observed that protein restriction altered the proliferative compartment, which is defined as the isthmus/neck region of the gland [31], by spreading the dividing cells outside this area. The molecular events behind
such changes should be investigated, but other studies have reported the influence of dietary pattern on the distribution of proliferative cells [4,5]. The epithelial cell proliferation in the gastrointestinal tract is regulated by the interaction of multiple agents, which include special feeding conditions, hormones, and growth factors [5,10, 12]. As mentioned earlier, the gastric epithelium was responsive to long-term protein restriction, which permanently affected stomach growth. Because ghrelin is a hormone involved in the control of food intake and its effects on cellular turnover have been recently uncovered in digestive organs [22–24], we evaluated the density of ghrelin-producing cells in the gastric mucosa. We found that protein restriction promoted different effects on this endocrine cell population depending on the developmental stage. Accordingly, at suckling, we did not detect changes, whereas in weaned and young adult rats we found an increase of ghrelin-immunoreactive cells. It is important to note
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that that a peak was observed at 30 d. Previous reports have consistently shown that food and calorie restrictions induce higher expressions of ghrelin O-acyl-transferase and ghrelin mRNA levels after short-term treatment [32,33], and that such a spike is followed by a decrease after months of dietary deprivation [19]. The effects of increased ghrelin can be diverse on the organs, as recently reviewed [34]. Our results indicated that gastric cell proliferation was inhibited by protein restriction in parallel with a high production of ghrelin, especially in weaned rats. Although few studies have evaluated the action of this molecule as a hormone or paracrine factor on cell kinetics, there is a consensus about its role in apoptosis suppression in the intestinal mucosa [23] and pancreatic cells [22]. In the stomach, however, a dual age-dependent effect has been described, and Warzecha et al. [35] demonstrated that ghrelin can inhibit or stimulate cell proliferation when administered to suckling or peripubertal rats, respectively. In another study, Ceranowicz et al. [24] showed that ghrelin accelerated the healing of gastric ulcers by increasing DNA synthesis in adult animals, and they postulated that this response should be indirect and mediated by GH and insulin-like growth factor-1. Comparatively, we agree with the idea that ghrelin might be differentially correlated to cell proliferation in the gastric epithelium, and that variation could reflect a change in the sensibility or functionality during development. However, because we observed constant low proliferative indices in the gastric epithelium of PR rats, other molecules could interact with ghrelin or alter its action, and the possible candidates for these functions are GH secretagogue receptor, leptin, corticosterone, and growth factors [5,12,36,37]. Altogether, our results indicated a correlation among protein restriction, inhibition of gastric cell proliferation, and high ghrelin levels, suggesting that the permanent situation of malnutrition might interfere not only with metabolism but also with cellular kinetics in the gastric mucosa, which ultimately would affect stomach growth. Acknowledgments The authors thank Cruz Alberto Mendoza Rigonati for technical assistance. References [1] Alvares EP. Extensive networks of TMPase positive basal lysosomes are present in fetal rat gastric epithelium before overt differentiation. J Submicrosc Cytol Pathol 1994;26:515–23. [2] Zhu L, Hatakeyama J, Zhang B, Makdisi J, Ender C, Forte JG. Novel insights of the gastric gland organization revealed by chief cell specific expression of moesin. Am J Physiol Gastrointest Liver Physiol 2009;296:G185–95. [3] Alvares EP, Gama P. Fasting enhances cell proliferation of gastric epithelium during the suckling period in rats. Braz J Med Biol Res 1993;26:869–73. [4] Gama P, Alvares EP. Early weaning and prolonged nursing induce changes in cell proliferation in the gastric epithelium of developing rats. J Nutr 2000;130:2594–8. [5] Osaki L, Figueiredo PM, Alvares EP, Gama P. EGFR is involved in the control of gastric cell proliferation through the activation of MAPK and Src signalling pathways in early-weaned rats. Cell Prolif 2011;44:174–82. [6] Weaver LT, Desai M, Austin S, Arthur HM, Lucas A, Hales CN. Effects of protein restriction in early life on growth and function of the gastrointestinal tract of the rat. J Pediatr Gastroenterol Nutr 1998;27:553–9. [7] Majumdar AP. Postnatal undernutrition: effect of epidermal growth factor on growth and function of the gastrointestinal tract in rats. J Pediatr Gastroenterol Nutr 1984;3:618–25. [8] Paula AC, Gracioso JS, Toma W, Bezerra R, Saad MA, De Lucca IM, et al. Is gastric ulceration different in normal and malnourished rats? Br J Nutr 2005;93:47–52. [9] Montoya CA, Leterme P, Lalles JP. A protein-free diet alters small intestinal architecture and digestive enzyme activities in rats. Reprod Nutr Dev 2006;46:49–56.
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