Effects of high-tryptophan diet on pre- and postnatal development in rats: a morphological study
- First Online:
- Cite this article as:
- Castrogiovanni, P., Musumeci, G., Trovato, F.M. et al. Eur J Nutr (2014) 53: 297. doi:10.1007/s00394-013-0528-4
- 343 Downloads
Tryptophan is an essential amino acid, precursor of serotonin. Serotonin (5HT) regulates the secretion of pituitary growth hormone (GH), which in turn stimulates the liver to produce insulin-like growth factor-I (IGF-I) that is necessary for development and growth. The aim of our study was to investigate the effects of an excess of tryptophan in the diet of pregnant rats on the differentiation of skeletal muscle tissue.
We conducted an immunohistochemical study on the IGF-I expression in hepatic and muscle tissues in offspring, and then, we associated this molecular data with morphological effects on the structure of the muscle fibers and hepatic tissue at different postnatal weeks, from birth to sexual maturity. Measurements of 5HT, GH in blood, and of tryptophan hydroxylase (Tph) activity in gastrointestinal tracts tissue were also taken.
Hyperserotonemia and higher values of Tph activity were detected in both pregnant rats and pups. Very low levels of GH were detected in experimental pups. Morphological alterations of the muscle fibers and lower IGF-I expression in hepatic and muscle tissue in pups were found.
Our data suggest that an excess of tryptophan in the diet causes hyperserotonemia in fetus. Hyperserotonemia results in an excess of serotonin in the brain where it has an adverse effect on the development of serotonergic neurons. The affected neurons do not regulate optimally the secretion of pituitary GH that consequently decreases. This limits stimulation in the liver to produce IGF-I, crucial for development and growth of pups.
KeywordsTryptophan Serotonin Growth hormone Insulin-like growth factor Development Diet
Tryptophan (Trp) is an essential amino acid assumed only through diet, because human organism is unable to synthesize it. Trp is involved in highly relevant biochemical and physiological processes. l-Trp is the enantiomer used in nature as a precursor of functional molecules such as niacin, serotonin, and epiphyseal melatonin [1, 2, 3]. Serotonin (5HT) is one of the most important molecules derived from l-Trp. It is a tryptamine monoamine neurotransmitter synthesized in serotonergic neurons in the central nervous system (CNS), as well as in the enterochromaffin cells of the gastrointestinal tract . In the brain, 5HT regulates serotonergic growth and maturation of some cerebral regions in the developing brain , while in the adult brain, it has the role of a neurotransmitter regulating function and plasticity [6, 7]. A fundamental activity of 5HT is its influence in the secretion of pituitary growth hormone (GH). Actually, data on the exact role of 5HT on the release of GH are controversial [8, 9, 10, 11, 12], and this is probably due to the complexity of the serotonergic system and the many receptor subtypes involved [13, 14, 15, 16, 17]. Importance of GH in pre- and postnatal development is well known [5, 18, 19, 20, 21]. Particularly, in our study, we have considered the production of insulin-like growth factors (IGFs) through pituitary GH stimulation . IGFs are a group of peptide hormones with anabolic functions, mainly produced in the liver after GH stimulation. IGFs especially promote differentiation of myoblasts and osteoblastic tissue , fundamental for development and growth in all vertebrates studied to date [18, 19, 24, 25]. IGFs are detectable in many fetal tissues since the first trimester of pregnancy, and the concentrations of IGFs in the fetal circulation increase during the whole pregnancy . Though IGFs are not necessary for the survival of the fetus, a lack of these growth factors has an effect on the development of many tissues, as shown in many studies [21, 26]. The nutritional status of the mother is crucial in determining the future pattern of growth and metabolism in the child; thus, the fetus is affected by the mother’s nutritional deficiency, and it permanently slows the growth rate . In addition, also in the first period of life, diet proteins will influence GH secretion , so an excess intake of Trp in maternal diet may result in dysregulation of the peripheral 5HT homeostasis leading to hyperserotonemia. The mechanism of hyperserotonemia and its relation to central 5HT dysfunction are not yet fully understood. Possible alterations in the expression of one of the 5HT elements could lead to the dysregulation of 5HT transmission in the brain, and this could lead to a lower production of pituitary GH and hepatic IGF-I with related consequences on development and growth [28, 29]. In the present study, we have tested, in a rat model, effects of a diet with an excess of Trp on hyperserotonemia, liver production of IGF-I, and the latter’s expression on skeletal muscle tissue and possible morphological alteration of this one, to emphasize the importance of diet during pregnancy and in the early postnatal period.
Materials and methods
Breeding and housing of animals
In our study, we used 10 healthy female Sprague Dawley rats (Charles River Laboratories, Wilmington, MA, USA), with an average body weight of 200 ± 40 g. Rats were individually housed in polycarbonate cages during the entire stabling period and were permitted free cage activity without joint immobilization. Rats were housed at steady temperature (20–23 °C) and humidity, with free access to water and food and photoperiod of 12 h light/dark. Female rats were mated with males of the same strain and age in 2:1 ratio, respectively. Pregnancy was confirmed in all females by the presence of sperm in vaginal plug, and the male rats were removed from the cage. Females remained singly housed until weaning of the pups. After weaning, pups were housed 3–4 per cage, in polycarbonate cages at steady temperature (20–23 °C) and humidity, with free access to water and food and photoperiod of 12 h light/dark. All efforts were made to reduce the number of animals used and to minimize animal suffering. Experiment was performed in accordance with the European Communities Council Directive (86/609/EEC) and Italian Animal Protection Law (116/1992).
Control five pregnant rats fed from gestional day 1, ad libitum, with control rat chow containing all essential amino acids. The protein content was about 30 %, and maize and casein were the main sources of amino acids. Normally in the control chow, Trp was present in an amount of 0.22 g in 100 g of pellets.
Experimental five pregnant rats fed from gestional day 1, ad libitum, with experimental rat chow containing all essential amino acids. The protein content was about 30 %, and maize and casein were the main sources of amino acids. In the experimental chow, Trp (Sigma, USA) was added during the preparation of pellets up to an amount of 1 g in 100 g of pellets.
Both diets were prepared by Laboratorio Dottori Piccioni, Gessate (Milano), Italy.
After weaning, pups were housed 3–4 per cage and they continued to feed on with the same diet as their mothers during the postnatal development.
first postnatal week, n = 10 pups (5 controls and 5 experimental).
second postnatal week, n = 10 pups (5 controls and 5 experimental).
third/fourth postnatal weeks, n = 10 pups (5 controls and 5 experimental).
fifth/tenth postnatal weeks, n = 10 pups (5 controls and 5 experimental).
nineteenth/twentieth postnatal weeks, n = 10 pups (5 controls and 5 experimental).
The pups at different postnatal weeks were killed by an intravenous lethal injection of Tanax (Hoechst Roussel VetGmbH, Wiesbaden, Germany) under Furane®-narcosis. Gastrointestinal tract, liver, and muscle samples were collected from 10 rats of every age group for a total of 50 samples. Gastrointestinal tract samples were used to perform Tph activity measurement; liver and muscle samples were used to perform IGF-1 immunohistochemical evaluation, and simultaneously, tissue specimens from several organs were collected for further investigations. We also collected the blood from both control and experimental pregnant rats (5 days before the parturition) and pups (before they were killed). The whole blood was used to perform serotonin (5HT) and growth hormone (GH) quantification in pups; platelet-free plasma (PFP) and platelet-rich plasma (PRP) were used to perform serotonin (5HT) quantification in pregnant rats. Under light ether narcosis, 800 μl of pup blood or 1.5 ml of adult blood was withdrawn from the jugular vein into syringes preloaded with 200 or 500 μl of 3.13 % trisodium citrate anticoagulant, as previously described .
Measurement of the peripheral 5HT and GH concentrations
After thorough mixing, samples were transferred from syringes into microtubes. 5HT and GH concentrations were determined using a commercially available ELISA kit (DRG Instruments GmbH, Germany), according to the manufacturer’s instructions. 5HT concentrations were determined in whole blood for pups and in platelet-free plasma and platelet-rich plasma for pregnant rats. Results of 5HT concentration were expressed in μM of WB, PFP, and PRP. Growth hormone (GH) concentrations were determined in whole blood of pups. Results of GH concentration were expressed in μg/l of WB.
Measurement of tryptophan hydroxylase (Tph) activity
Frozen gastrointestinal tract samples were homogenized in 5 vols of 50 mM Tris-acetate buffer (pH 7.5), 1 mM dithiothreitol (Sigma, USA), 8 mM l-tryptophan (Sigma, USA), and 0.5 mM 6,7-dimethyl-5,6,7,8-tetra-hydropteridine (Sigma, USA). The homogenate was incubated for 30 min at 37 °C. 6 M perchloric acid was added to stop the reaction. 5HT produced by reaction was determined with HPLC. The oxidation potential of the electrochemical detector was set at +700 mV. Protein assays were performed by the method of Lowry et al.  with bovine serum albumin as standard. Tph activity was expressed in Katal.
Samples were fixed in 10 % buffered formalin, following overnight wash; specimens were treated as previously described . Sections 4–5 μm in thickness were obtained according to routine procedures, mounted on sialane-coated slides and stored at room temperature. Slides were dewaxed in xylene, hydrated using graded ethanol, and stained for routine histologic evaluation by Haematoxylin and Eosin (H&E) for the morphological structure. Paraffin sections (~5 μm) were deparaffinized in xylene for 15 min, rehydrated and incubated for 30 min in 0.3 % H2O2/methanol to quench endogenous peroxidase activity then rinsed for 20 min with phosphate-buffered saline (PBS; Bio-Optica, Milano, Italy). Sections were irradiated (5 min × 3) in capped polypropylene slide-holders with citrate buffer (10 mM citric acid, 0.05 % Tween 20, pH 6.0, Bio-Optica, Milan, Italy), using a microwave oven (750 W) to unmask antigenic sites. Sections were incubated with diluted goat polyclonal anti-IGF-I antibody (Santa Cruz Biotechnology Inc, California, USA), (diluted 1:50 in PBS) overnight at 4 °C. The secondary antibody, Dako LSAB+ system-HRP, was applied according to the manufacturer’s instructions (Dako, Denmark). The immunoreaction was visualized by incubating the sections for 4 min in a 0.1 % 3.3′-diaminobenzidine and 0.02 % hydrogen peroxide solution (DAB substrate kit, Vector Laboratories, CA, USA). Sections were lightly counterstained with Mayer’s haematoxylin (Histolab Products AB, Goteborg, Sweden) mounted in GVA mount (Zymed, Laboratories Inc., San Francisco, CA, USA) observed with an Olympus BX41 light microscope (Japan) and photographed with a digital camera (Nikon, Japan).
Evaluation of immunohistochemistry
The IGF-I staining status was identified as either negative or positive. Immunohistochemistry positive staining was defined as cytoplasmic/membranous and nuclear detection of brown chromogen in muscle fibers and in hepatic tissue via evaluation by light microscopy as previously described [33, 34]. Stain intensity and the proportion of immunopositive muscle fibers and hepatocytes were also assessed by light microscopy. Intensity of staining was graded as follows: no detectable staining, weak, moderate, strong, and very strong staining. The percentage of IGF-I immunopositive muscle fibers and hepatocytes was independently evaluated by 4 investigators (2 anatomical morphologists and 2 histologists) and scored as a percentage of the final number of 100 muscle fibers/hepatocytes. Counting was performed at 200× magnification. Positive and negative controls were performed to test the specific reaction of primary antibodies used in this study at a protein level. Positive staining was defined as the presence of brown chromogen detection distributed within the sarcoplasm (between the myofibrils of the muscle fibers) and in hepatic tissue. Sections of colon tissue also were used for anti-IGF-I positive control. The positive immunolabeling was both perinuclear and cytoplasmic. Sections of rat muscle and liver were randomly drawn from experimental sample for the negative control. These were then treated with normal rabbit serum instead of the specific antibodies.
Computerized morphometry measurements and image analysis
Fifteen fields randomly selected from each section were analyzed, and the percent area stained with IGF-I antibody was calculated using an image analyzer (Image-Pro Plus 4.5.1, Immagini and Computer, Milan, Italy), which quantifies the level of positive immunolabelling in each field, as described previously . Digital pictures were taken using the Axioplan Zeiss light microscope and photographed using the Canon digital camera. Evaluations were made by four blinded investigators, whose evaluations were assumed to be correct if values were not significantly different. In case of disputes concerning the interpretation, the case has been revised to reach a unanimous agreement.
Statistical analysis was performed using SPSS software (SPSS® release 19.0, Chicago, IL, USA). The immunohistochemistry Cohen’s kappa was applied as previously described [36, 37]. The chi-square test and Fisher’s Exact test were used to examine the association between IGF-I expressions in experimental rats versus control ones at different postnatal weeks. Comparisons between more than two groups were tested using analysis of variance (ANOVA) and Bonferroni’s test. Unvaried analyses were performed using the Kaplan–Meier method. Multivariate analysis was performed using Cox proportional hazards model. In all statistical analyses, a p value <0.05 was considered significant.
Morphological and immunohistochemical evaluations
Interobserver agreement, determined using the Kappa coefficient, was 0.94 (almost perfect).
Measurement of the peripheral 5HT and GH concentrations
The levels of GH in the WB are significantly lower in experimental pups (0.55 ± 0.16 μg/l) in comparison with control groups (1.9 ± 0.85 μg/l; p < 0.01; Fig. 7b). Whereas in the control rats, there was a physiologic gradual increase of GH levels as age increases followed by a subsequent decrease, significant differences in GH concentration were not detected between different groups of age in experimental rats.
Measurement of Tph activity in gastrointestinal tract samples
In previous studies , we investigated the lack of Trp in diet of pregnant rats to analyze the effects on offspring. We showed impaired growth and development in pups of experimental rats and also alterations in the sexual development in growing rats. Based on the most recent studies [30, 39], in our research, we investigated how high-Trp diet of pregnant rats, since the first day of pregnancy, affected normal differentiation of skeletal muscle tissue. Particularly, we conducted an immunohistochemical study on the IGF-I expression in hepatic and muscle tissues, and then, we associated this molecular data to morphological effects on the structure of the muscle fibers and liver in rats at different postnatal weeks, from birth to sexual maturity.
Literature widely reports the link between Trp in the diet and production of 5HT, influence of 5HT on the GH production, and hence the GH-dependence on hepatic IGF-I production [1, 5, 8, 18, 23, 24, 30]. Furthermore, the most recent literature shows the possible negative influence of the excess of 5HT on the differentiation of serotonergic neurons of the raphe nuclei in the brainstem with consequent reduction in the GH production [30, 39]. It has a direct effect on hepatic production of IGF-I, particularly in the postnatal period in which IGF-I influences tissue differentiation and global growth [40, 41].
Its well known that in CNS the function of 5HT is also to induce pituitary GH production with a complex hormonal mechanism [13, 14, 15, 16, 17, 42]. An alteration in some elements of this process could result in a dysfunction in the GH production with consequences for development and growth. The most recent literature shows that the hyperserotonemia induced in experimental conditions in pregnant rats causes disorders in the offspring, such as a lower body mass and a lower survival rate [30, 39]. In addition, data show that high 5HT levels could inhibit the development of 5HT neurons and lead to anatomical and functional alterations of the brain .
The hypothesis of our study is that a high intake of Trp in the diet of both pregnant rats and pups results in increased production of 5HT in enterochromaffin cells, thereby resulting in hyperserotonemia in both pregnant rats and pups, as we report in our results. During fetal and early postnatal development, the blood brain barrier is not yet formed [30, 43], so 5HT can freely move to the central compartment. In the CNS, the excess of 5HT prevents the normal differentiation of serotonergic neurons of the raphe nuclei with consequences for the possibility that their serotonergic processes could reach the hypothalamus . Therefore, the regulating function of 5HT is blocked and this determines a lower production of GH by the pituitary gland and IGF-I by the liver. Finally, low levels of IGF-I have negative consequences for muscular and osteoblastic tissue differentiation and for body growth.
In the present study, we investigated this hypothesis by IGF-I immunohistochemical staining of muscle fibers and hepatic tissue in rats at different postnatal weeks, from birth to sexual maturity, and the results were encouraging and in accordance with data from other authors. Our results showed that at birth and in the early postnatal days, IGF-I expression was similar in both experimental and control rats (p > 0.05). In this period, the hyperserotonemia induced by the experimental conditions, and the blood brain barrier not yet formed, resulted in presence of 5HT in the pituitary tissue where, through a local paracrine mechanism, GH production is induced [13, 14, 15, 16, 17]. In subsequent phases of postnatal development, the IGF-I expression was significantly reduced in experimental rats compared with control ones (p < 0.05). This relevant datum could be explained by the negative effect that the excess of 5HT would have on the development of some regions of the CNS, especially on serotonergic neurons of the raphe nuclei of the brainstem . Impaired serotonergic neurons were not able to send their axons to the hypothalamus; thus, the decreased pituitary GH production resulted in a decreased hepatic production of IGF-I in postnatal period. After reaching sexual maturity, about three months of age, the IGF-I expression decreased both in experimental and control rats and in adult IGF-I expression, it was similar in experimental and control rats (p > 0.05). This datum was explained by the physiological reduction in the IGF-I production, as occurs on completion of its function in body growth [44, 45].
In conclusion, we found that Trp affects development before and after birth. Through a concatenation of effects mediated by 5HT and GH, Trp loading during pregnancy decreased IGF-I expression in muscle and this had an effect on the normal development of muscle tissue as shown in the morphological study of sample. This is an example of how diet modification during pregnancy may have long-time effects upon postnatal development. Further studies are needed to better understand the interactions between endocrine, neurocrine systems, and diet on development and growth.
The study was funded by the Department of Bio-Medical Sciences, University of Catania. The authors would like to thank Prof. Iain Halliday for commenting and making corrections to the paper and Mr. Pietro Asero for technical support in the laboratory.
Conflict of interest
The authors declare that they have no conflict of interest.
Experiment was performed in accordance with the European Communities Council Directive (86/609/EEC) and Italian Animal Protection Law (116/1992).