- Email: [email protected]
Malnutrition during lactation in rats is associated with higher expression of leptin receptor in the pituitary of adult offspring
BASIC NUTRITIONAL INVESTIGATION
Malnutrition During Lactation in Rats Is Associated With Higher Expression of Leptin Receptor in the Pituitary of Adult Offspring Luciana Lea˜o Vicente, MSc, Egberto Gaspar de Moura, PhD, Patricia Cristina Lisboa, PhD, Andrea Monte Alto Costa, PhD, Thaı´s Amadeu, MSc, C. A. Mandarim-de-Lacerda, PhD, and Magna Cottini F. Passos, PhD From the Departamentos de Cieˆncias Fisiolo´gicas, Histologia e Embriologia, and Laborato´rio de Morfometria e Morfologia Cardiovascular, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil; and the Departamento de Nutric¸a˜o Aplicada, Instituto de Nutric¸a˜o, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil OBJECTIVE: Recent studies have shown that leptin receptor is expressed in human and rat pituitary glands. The expression of leptin receptor in rats whose dams were malnourished during lactation has not been previously reported. METHODS: We examined leptin receptor expression in the pituitary gland of adult rats whose dams were assigned to one of the following groups during lactation: control diet, protein-restricted diet (8% protein), or energy-restricted diet (the control diet fed in restricted quantities that were calculated according to the mean ingestion of the protein-restricted group). After weaning, all pups had free access to the control diet until they reached adult age, at which time leptin receptor expression in the pituitary was analyzed by immunohistochemistry. RESULTS: Adult animals from protein- and energy-restricted dams had a higher expression of leptin receptor in pituitary tissue, normal serum leptin concentrations, higher serum tri-iodothyronine concentrations, and lower thyroid-stimulating hormone concentrations than did the control rats. CONCLUSIONS: In the fed state, leptin has a stimulatory effect on release of thyroid-stimulating hormone. The higher expression of leptin receptor in the pituitary of animals from protein- and energy-restricted dams may suggest a postreceptor failure in leptin action. This higher receptor expression may have allowed a greater inhibition of release of thyroid-stimulating hormone. Nutrition 2004;20:924 –928. ©Elsevier Inc. 2004 KEY WORDS: protein malnutrition, energy malnutrition, leptin receptor, pituitary, lactation
INTRODUCTION Leptin acts to regulate the energy available in the peripheral adipose tissue through specific hypothalamic signals and affects body weight, food intake, body temperature, and metabolic rate1–3. Leptin is also involved in the neuroendocrine regulation of pituitary function4. Studies in normal rodents have shown the expression of leptin receptor in the pituitary by reverse transcriptase–polymerase chain reaction5–7, immunoblotting7, and immunohistochemical analysis7. Leptin regulates the function of growth hormone–secreting cells7 and has an acute stimulatory effect on the release of thyroid-stimulating hormone (TSH) in vivo by acting at the hypothalamic level8 –10. In addition, Ortiga-Carvalho et al.10 showed a direct inhibitory effect of leptin on TSH release in pituitary explants, which
This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico and the Fundac¸a˜o de Amparo a Pesquisa do Rio de Janeiro. Correspondence to: Magna Cottini F. Passos, PhD, Departamento de Cieˆncias Fisiolo´gicas, 5o andar, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro, 87, Rio de Janeiro, RJ 20551-030, Brazil. E-mail: [email protected] Nutrition 20:924 –928, 2004 ©Elsevier Inc., 2004. Printed in the United States. All rights reserved.
suggests that leptin may act as an autocrine or paracrine factor at the pituitary level. Despite the close relation between leptin and pituitary hormones, the regulation of the expression of leptin receptor in the pituitary gland has not been extensively studied. We previously showed that protein and energy malnutrition in lactating rats is associated with thyroid dysfunction and body weight alterations in the offspring at 180 d of age11,12. In the present study, we evaluated whether protein or energy restriction during lactation affects the expression of leptin receptor in the pituitary gland of the offspring in adulthood.
MATERIALS AND METHODS Wistar rats were kept in a temperature-controlled (25 ⫾ 1°C) room with an artificial dark–light cycle (lights on from 7:00 AM to 7:00 PM). Three-month-old, virgin female rats were caged with one male rat at a proportion of 2:1. After mating, each female was placed in an individual cage with free access to water and food until delivery. The use of the animals according our experimental design was approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro, which based its analysis on the principles described in the Guide for the Care and Use of Laboratory Animals13. 0899-9007/04/$30.00 doi:10.1016/j.nut.2004.06.014
Nutrition Volume 20, Number 10, 2004
Malnutrition and Pituitary Leptin Receptor
TABLE I.
925
for immunohistochemistry. The animals had free access to water and a normal diet until immediately before death.
COMPOSITION OF THE CONTROL AND LOW-PROTEIN DIETS Ingredient
Control diet*
Soybean ⫹ wheat (g/kg) Cornstarch (g/kg) Soybean oil (g/kg) Vitamin mix (g/kg)‡ Mineral mix (g/kg)‡ Macronutrient composition (% energy) Protein (g/kg) Carbohydrate (g/kg) Fat (g/kg) Total energy (kJ/kg)
Low-protein diet†
230.0 676.0 50.0 4.0 40.0
80.0 826.0 50.0 4.0 40.0
23.0 66.0 11.0 17038.7
8.0 81.0 11.0 17038.7
* Standard rat diet (Nuvilab-NUVITAL Nutrientes LTDA, Parana´ , Brazil). † The low-protein diet was prepared in our laboratory by replacing part of the protein in the control diet with cornstarch. The amount of cornstarch was calculated to make up for the decrease in energy content resulting from the reduction in protein. ‡ Vitamin and mineral mixtures were formulated to meet the American Institute of Nutrition AIN-93G recommendation for rodent diets and contained the recommended amount of iodine14.
The dams were randomly assigned to one of the following groups: a control group, which had free access to a standard laboratory diet containing 23% protein; a protein-restricted (PR) group, which had free access to an isoenergetic, protein-restricted diet containing 8% protein; and an energy-restricted (ER) group, which received a standard laboratory diet in restricted quantities that were calculated according to the mean ingestion of the PR group. Thus, the amount of food consumed by the ER and PR groups was about the same. The composition of the diets, which followed recommended standards, is shown in Table I14. The PR diet was prepared in our laboratory by replacing part of the protein in the control diet with cornstarch. The amount of starch was calculated to make up for the decrease in energy content resulting from the protein reduction. Within 24 h of birth, excess pups were removed, and only six male pups were kept per dam in the control and treatment groups, because it has been shown that this procedure maximizes lactation performance15. The experimental diets were started at birth, which was defined as day 0 of lactation, and were ended at weaning (day 21). After weaning, two animals from each litter were chosen at random and were placed together in a cage with free access to water and a normal diet until they were 150 d old. The remaining four animals from each litter were killed on day 21 (see the paragraph on blood sampling, below). In the dams, food intake and body weight were monitored during the lactation period. In the pups, body weight and food intake were monitored every 4 d, from birth until 150 d of age. The amount of the diet ingested was calculated as the difference between the weight of the food remaining in the food bin (Da) and the amount placed there 4 d before (Di). This was measured in the pups from weaning until 150 d of age. These data were then used to calculate daily food intake according to the following formula: food intake (g)⫽([Di ⫺ Da]/3)/4, where 3 is the number of animals in each cage and 4 is the number of days. Blood samplings were performed at 21 and 150 d of age. At these ages, the animals were killed at 10:00 AM with a lethal dose of pentobarbital, and blood was obtained by cardiac puncture. The pituitary glands from animals 150 d old were excised and prepared
Immunohistochemistry The pituitary glands from the 150-d-old animals were placed in fixative for 12 h at room temperature (freshly prepared 4% [w/v] formaldehyde in 0.1 M phosphate buffer, pH 7.2), embedded in Paraplast plus (Sigma, St. Louis, MO, USA), and sectioned in 3-m thicknesses. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in 0.01 M phosphate buffered saline, and the sections were rinsed successively with distilled water and phosphate buffered saline. The primary antibody used was the polyclonal anti–leptin-R antibody (1:100; antihuman leptin receptor — Long Form, Linco Research, St. Charles, MO, USA). Immunostaining was performed by using the avidin-biotin-peroxidase method (DAKO Corporation, Carpinteria, CA, USA). Stereologic Analysis Our analysis included the use of a video-microscope system composed of a Leica DMRBE microscope (Leica, Wetzlar, Germany) with an oil-immersion objective (100 ⫻, numerical aperture, 1.25), a Kappa video camera (Kappa, Gleichen, Germany), and a Sony Trinitron monitor (Sony, Pencoed, UK). The numerical density per area of the cells staining for leptin receptor in the pituitary was estimated by taking 10 random microscopic fields per animal, with five animals per group. The counts were performed over a 6400m2 frame, and all immunostained cell profiles in the frame that were not intersecting with the forbidden line or its extensions were sampled. The pituitary is an isotropic tissue; thus, the random microscopic fields were observed in coded slices by moving the microscope stage in a blinded manner16,17. After the counts were completed, the group to which each coded slice belonged was recorded. Serum Hormone Concentrations Blood samples were centrifuged to obtain serum, which was stored at ⫺20°C until analyzed. Serum TSH was measured by specific radioimmunoassay with a kit for rat TSH supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD, USA). The results are expressed in terms of the reference preparation provided (RP-3). Total serum tri-iodothyronine (TT3) and total serum thyroxine (TT4) were measured by radioimmunoassay with commercial kits
TABLE II. MATERNAL BODY WEIGHT AND BODY WEIGHT AND SERUM LEPTIN CONCENTRATION OF THE PUPS ON DAY 21 OF LACTATION* C Maternal body weight (g) Pup body weight (g) Pup serum leptin (ng/mL)
225.1 ⫾ 4.6 30.5 ⫾ 1.6 0.8 ⫾ 0.13
PR
ER
197.9 ⫾ 5.8† 176 ⫾ 5.8† 17.1 ⫾ 0.5† 16.5 ⫾ 0.6† 1.5 ⫾ 0.19† 1.4 ⫾ 0.14†
* Values are the mean ⫾ standard error of the mean for five animals per group. † Significantly different from the control group (multiple comparison of means test, P ⬍ 0.05). C, control diet group; ER, energy-restricted diet group; PR, proteinrestricted diet group
926
Vicente et al.
Nutrition Volume 20, Number 10, 2004 TABLE III.
BODY WEIGHT AND SERUM LEPTIN, TT3, TT4, AND TSH CONCENTRATIONS IN ANIMALS AT AGE 150 D*
Body weight (g) Leptin (ng/mL) TT3 (nmol/L) TT4 (nmol/L) TSH (ng/ml)
C
PR
ER
316.5 ⫾ 18.8 1.8 ⫾ 0.17 21.08 ⫾ 1.85 11.77 ⫾ 0.56 3.04 ⫾ 0.42
294.9 ⫾ 8.33† 1.6 ⫾ 0.22 59.28 ⫾ 3.79† 13.38 ⫾ 0.42† 2.21 ⫾ 0.14†
362.5 ⫾ 7.59† 1.8 ⫾ 0.20 64.98 ⫾ 3.83† 11.83 ⫾ 0.09 2.07 ⫾ 0.14†
* Values are the mean ⫾ standard error of the mean for five animals per group. † Significantly different from the control group (multiple comparison of means test, P ⬍ 0.05). C, control diet group; ER, energy-restricted diet group; PR, proteinrestricted diet group; TSH, thyroid-stimulating hormone; TT3, total triiodothyronine; TT4, total thyroxine
(Coat-A-Coat, DPC, Los Angeles, CA, USA). We used control standard curves diluted in iodothyronine-free rat serum (charcoal treated). Leptin concentrations were measured with a commercial kit (Murine Leptin Elisa-DSL-10-24100; Diagnostic Systems Laboratories, Webster, TX, USA). The interassay and intra-assay coefficients of variation were 3.1% and 4.2%, respectively, and the limit of detection was 0.04 ng/100 mL.
II. All these variables were significantly affected by the dietary treatment. The body weight of the pups whose dams were fed the PR or ER diet during lactation was significantly lower (P ⬍ 0.001) than that of the controls at the end of lactation. Serum leptin concentrations were higher in pups from PR and ER dams than in the control group (P ⬍ 0.05). The body weight and serum leptin, TT3, TT4, and TSH concentrations of the animals at 150 d of age are presented in Table III. The animals whose dams were fed the PR diet weighed significantly less than did the control animals (P ⬍ 0.05); in contrast, the animals whose dams were fed the ER diet weighed about 10% more than the controls (P ⬍ 0.01). No significant differences in serum leptin concentrations were observed at this age. The offspring of PR dams had significantly higher serum TT3 (149%, P ⬍ 0.001) and TT4 (12%, P ⬍ 0.05) concentrations than did the controls. In the offspring of ER dams, only the serum TT3 concentration was significantly higher than that in the controls (178%, P ⬍ 0.05). Serum TSH was significantly lower in the offspring of PR and ER dams than in those of the controls (P ⬍ 0.05). Immunohistochemistry showed the presence of leptin receptor immunoreactivity in the pituitary of adult animals (Figure 1). Leptin expression in the pituitary was higher in the rats whose dams were fed the PR and ER diets than in the controls (Figure 2). The quantity of immunostaining cells, analyzed by the stereologic method, is shown in Figure 3. The percentage of immunostaining cells marked by leptin receptor antibody in the pituitary was higher in the offspring of PR and ER dams than in those of the controls (by 22% and 24%, respectively).
Statistical Analysis
DISCUSSION
The data are reported as means ⫾ standard errors of the means. One-way analysis of variance and then the Neuman-Keuls test were used to assess serum hormone concentrations, except for serum TSH, which was analyzed by use of the Kruskal-Wallis multiple-comparison test followed by Dunn’s multiple-comparison test. Differences in numerical density per area across groups were assessed by use of Kruskal-Wallis non-parametric analysis of variance and the Kolmogorov-Smirnov test, with a significance level of 0.0518.
The present data on body weight during lactation reinforce previous findings from our laboratory in which malnourished dams and their pups had significantly lower body weights than did control animals11. Despite the lower body weight of the offspring, serum leptin concentrations were higher at weaning in the pups of malnourished dams than in the control animals. Because leptin is present in breast milk19, leptin may be transferred to the pups through the milk, which could lead to further decreases in food intake and body weight. The higher serum concentration of leptin at weaning could affect the regulation of several factors that contribute to the long-term control of the endocrine system. In the present study, the expression of leptin receptor in the pituitary was higher in the offspring of dams who were malnourished during lactation than in the offspring of the control group. The findings of higher serum concentrations of T4 and T3 and
RESULTS The body weight and serum leptin concentration of the pups and maternal body weight on day 21 of lactation are presented in Table
FIG. 1. Immunohistochemical demonstration of long-form leptin receptor (Ob-Rb) in the pituitary of adult rats whose dams were fed a control diet during lactation. (A) Ob-Rb–like immunoreactivity is seen in the pituitary. (B) No reaction product was observed when the primary antibody was omitted.
Nutrition Volume 20, Number 10, 2004
Malnutrition and Pituitary Leptin Receptor
927
FIG. 2. Immunohistochemical staining for leptin receptor in the pituitary of adult rats whose dams were fed a control (A), protein-restricted (B), and energy-restricted (C) diet during lactation. Magnification 20⫻.
lower serum concentrations of TSH in these animals reinforce the results of our previous studies12,20. The lower serum TSH concentration is easily explained by the negative feedback of thyroid hormones on the hypothalamic-pituitary axis. However, the higher expression of leptin receptor in the pituitary gland could also lead to a greater inhibitory effect of leptin on TSH secretion, because it has been shown that leptin suppresses TSH release in vitro10. Leptin increases thyroid hormone secretion through a direct mechanism that involves its binding to thyroid leptin receptors21. If these receptors are also highly expressed at the thyroid level, we could explain the higher thyroid hormone concentration observed in the present study. However, leptin also has an acute stimulatory effect on TSH secretion in vivo in fed rats10 and in fasting rodents4,22. Therefore,
FIG. 3. Quantity of immunostaining cells in the pituitary of adult rats whose dams were fed a control (solid bars), protein-restricted (open bars), or energy restricted (hatched bars) diet during lactation. Values are mean ⫾ standard error of the mean for five animals. *Significantly different from the control group (Kruskal-Wallis non-parametric analysis of variance and Kolmogorov-Smirnov test, P ⬍ 0.05).
this higher pituitary leptin receptor expression could also be interpreted as a resistance to leptin action on TSH secretion. We postulate that this resistance to leptin could be the result of a greater exposure to leptin at weaning, which could program pituitary thyrotrophs for lower leptin sensitivity later in life. This kind of programming was reported by other investigators who showed that higher leptin concentrations during the neonatal period program for greater body weight in later life in humans23 and in rats24. It has been reported that hypothalamic leptin receptors are upregulated when there is a deficiency of leptin action25. Greater hypothalamic leptin receptor expression was also reported in ER adult ewes with low serum leptin concentrations26. In the present study, however, we found no significant difference in serum leptin concentrations in the adult offspring, which leads us to conclude that it is the serum leptin concentrations at weaning that program the changes in pituitary leptin receptor. In conclusion, we showed that protein or energy restriction during lactation is associated with greater expression of leptin receptor in the pituitary in adult offspring. We suggest that higher leptin action or leptin resistance may explain this finding. Both explanations are plausible according the type of malnutrition. In the offspring of PR dams, higher leptin action is a more appropriate explanation, because those animals were leaner, had higher thyroid function, and had lower concentrations of TSH, all of which are dependent on higher leptin action. In the offspring of ER dams, however, the resistance hypothesis is more likely, because those animals were heavier and, despite having higher T3 concentrations, did not have other indications of thyroid hyperfunction. These data provide new insights into the physiologic importance of nutritional status and consequent changes in leptin concentrations during the neonatal period and suggest that leptin may have an important role in thyroid–pituitary axis regulation in adults. Further studies, such as studies of the expression of leptin receptor in the thyroid and mechanisms of intracellular leptin signaling, are needed to explore the mechanism that leads to the higher pituitary expression of leptin receptor.
928
Vicente et al.
REFERENCES 1. Pelleymounter MA, Cullen MJ, Baker MB, et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995;269:540 2. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 1995;269:546 3. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998;395:763 4. Ahima RS, Saper CB, Flyer JS, Elmquist JK. Leptin regulation of neuroendocrine systems. Front Neuroendocrinol 2000;20:317 5. Zamorano PL, Mahesh VB, De Sevilla LM, et al. Expression and localization of the leptin receptor in endocrine and neuroendocrine tissues of the rat. Neuroendocrinology 1997;65:223 6. Jin L, Zhang S, Burguera BG, et al. Leptin and leptin receptor expression in rat and mouse pituitary cells. Endocrinology 2000;141:333 7. Sone M, Nagata H, Takekoshi S, Osamura RY. Expression and localization of leptin receptor in the normal rat pituitary gland. Cell Tissue Res 2001;305:351 8. Legradi G, Emerson CH, Ahima RS, Flier JS, Lechan RM. Leptin prevents fasting-induced suppresion of prothyrotropin-releasing hormone messenger ribonucleic acid in neurons of the hypothalamic paraventricular nucleus. Endocrinology 1997;138:2569 9. Seoane LM, Carro E, Tovar S, Casanueva FF, Dieguez C. Regulation in vivo TSH secretion by leptin. Regul Pept 2000;92:25 10. Ortiga-Carvalho TM, Oliveira KJ, Soares BA, Pazos-Moura CC. Leptin role in the regulation of thyrotropin secretion in fed state—in vivo and in vitro studies. J Endocrinol 2002;174:121 11. Passos MCF, Ramos CF, Moura EG. Short and long term effects of malnutrition in rats during lactation on the body weight of offspring. Nutr Res 2000;20:1603 12. Passos MCF, Ramos CF, Dutra SCP, Mouc¸ o T, Moura EG. Long-term effects of malnutrition during lactation on the thyroid function of offspring. Horm Metab Res 2002;34:40 13. Bayne K. Revised guide for the care and use of laboratory animals available. Physiologist 1996;39:208
Nutrition Volume 20, Number 10, 2004 14. Reeves PG, Nielsen FH, Fahey GC. AIN-93 Purified diets for laboratory rodents: final report of the American Institute of Nutrition Ad Hoc Writing Committee on the reformulation of the AIN-76 rodent diet. J Nutr 1993;123:1939 15. Fishbeck KL, Rasmussen KM. Effect of repeated cycles on maternal nutritional status, lactational performance and litter growth in ad libitum-fed and chronically food-restricted rats. J Nutr 1987;117:1967 16. Gundersen HJ, Bendtsen TF, Korbo L, et al. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 1988;96:379 17. Mandarim-de-Lacerda CA. Stereological tools in biomedical research. An Acad Bras Cienc 2003;75:469 18. Zar JH. Biostatistical analysis. 4th ed. Upper Saddle River, NJ: Prentice-Hall; 1999 19. Casabiell X, Pin˜ eiro V, Tome´ MA, et al. Presence of leptin in colostrum and/or breast milk from lactating mothers: a potential role in the regulation of neonatal food intake. J Clin Endocrinol Metab 1997;82:4270 20. Dutra SCP, Passos MCF, Lisboa PC, et al. Liver deiodinase activity is increased in adult rats whose mothers were submitted to malnutrition during lactation. Horm Metab Res 2003;35:268 21. Nowak KM, Kaczmarek P, Mackowiak P, et al. Rat thyroid gland expresses the long form of leptin receptors, and leptin stimulates the function of the gland in euthyroid non-fasted animals. Int J Mol Med 2002;9:31 22. Ahima RS, Prabakaran D, Mantzoros C, et al. Role of leptin in the neuroendocrine response to fasting. Nature 1996;382:250 23. Shingal A, Farooqi SI, O’Rahilly, et al. Early nutrition and leptin concentrations in early life. Am J Clin Nutr 2002;75:993. 24. Cravo CO, Teixeira CV, Passos MC, et al. Leptin treatment during the neonatal period is associated with higher food intake and adult body weight in rats. Horm Metab Res 2002;34:400 25. Huang XF, Zhang R. Upregulation of leptin receptor mRNA expression in obese mouse brain. Neuroreport 1997;8:1035 26. Dyer CJ, Simmons JM, Matteri RL, Keisler DH. Leptin receptor mRNA is expressed in ewe anterior pituitary and adipose tissues and is differentially expressed in hypothalamic regions of well-fed and feed-restricted ewes. Domest Anim Endocrinol 1997;14:119
Malnutrition During Lactation in Rats Is Associated With Higher Expression of Leptin Receptor in the Pituitary of Adult Offspring Luciana Lea˜o Vicente, MSc, Egberto Gaspar de Moura, PhD, Patricia Cristina Lisboa, PhD, Andrea Monte Alto Costa, PhD, Thaı´s Amadeu, MSc, C. A. Mandarim-de-Lacerda, PhD, and Magna Cottini F. Passos, PhD From the Departamentos de Cieˆncias Fisiolo´gicas, Histologia e Embriologia, and Laborato´rio de Morfometria e Morfologia Cardiovascular, Instituto de Biologia Roberto Alcantara Gomes, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil; and the Departamento de Nutric¸a˜o Aplicada, Instituto de Nutric¸a˜o, Universidade do Estado do Rio de Janeiro, Rio de Janeiro, Brazil OBJECTIVE: Recent studies have shown that leptin receptor is expressed in human and rat pituitary glands. The expression of leptin receptor in rats whose dams were malnourished during lactation has not been previously reported. METHODS: We examined leptin receptor expression in the pituitary gland of adult rats whose dams were assigned to one of the following groups during lactation: control diet, protein-restricted diet (8% protein), or energy-restricted diet (the control diet fed in restricted quantities that were calculated according to the mean ingestion of the protein-restricted group). After weaning, all pups had free access to the control diet until they reached adult age, at which time leptin receptor expression in the pituitary was analyzed by immunohistochemistry. RESULTS: Adult animals from protein- and energy-restricted dams had a higher expression of leptin receptor in pituitary tissue, normal serum leptin concentrations, higher serum tri-iodothyronine concentrations, and lower thyroid-stimulating hormone concentrations than did the control rats. CONCLUSIONS: In the fed state, leptin has a stimulatory effect on release of thyroid-stimulating hormone. The higher expression of leptin receptor in the pituitary of animals from protein- and energy-restricted dams may suggest a postreceptor failure in leptin action. This higher receptor expression may have allowed a greater inhibition of release of thyroid-stimulating hormone. Nutrition 2004;20:924 –928. ©Elsevier Inc. 2004 KEY WORDS: protein malnutrition, energy malnutrition, leptin receptor, pituitary, lactation
INTRODUCTION Leptin acts to regulate the energy available in the peripheral adipose tissue through specific hypothalamic signals and affects body weight, food intake, body temperature, and metabolic rate1–3. Leptin is also involved in the neuroendocrine regulation of pituitary function4. Studies in normal rodents have shown the expression of leptin receptor in the pituitary by reverse transcriptase–polymerase chain reaction5–7, immunoblotting7, and immunohistochemical analysis7. Leptin regulates the function of growth hormone–secreting cells7 and has an acute stimulatory effect on the release of thyroid-stimulating hormone (TSH) in vivo by acting at the hypothalamic level8 –10. In addition, Ortiga-Carvalho et al.10 showed a direct inhibitory effect of leptin on TSH release in pituitary explants, which
This work was supported by grants from the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico and the Fundac¸a˜o de Amparo a Pesquisa do Rio de Janeiro. Correspondence to: Magna Cottini F. Passos, PhD, Departamento de Cieˆncias Fisiolo´gicas, 5o andar, Instituto de Biologia, Universidade do Estado do Rio de Janeiro, Av. 28 de setembro, 87, Rio de Janeiro, RJ 20551-030, Brazil. E-mail: [email protected] Nutrition 20:924 –928, 2004 ©Elsevier Inc., 2004. Printed in the United States. All rights reserved.
suggests that leptin may act as an autocrine or paracrine factor at the pituitary level. Despite the close relation between leptin and pituitary hormones, the regulation of the expression of leptin receptor in the pituitary gland has not been extensively studied. We previously showed that protein and energy malnutrition in lactating rats is associated with thyroid dysfunction and body weight alterations in the offspring at 180 d of age11,12. In the present study, we evaluated whether protein or energy restriction during lactation affects the expression of leptin receptor in the pituitary gland of the offspring in adulthood.
MATERIALS AND METHODS Wistar rats were kept in a temperature-controlled (25 ⫾ 1°C) room with an artificial dark–light cycle (lights on from 7:00 AM to 7:00 PM). Three-month-old, virgin female rats were caged with one male rat at a proportion of 2:1. After mating, each female was placed in an individual cage with free access to water and food until delivery. The use of the animals according our experimental design was approved by the Animal Care and Use Committee of the Biology Institute of the State University of Rio de Janeiro, which based its analysis on the principles described in the Guide for the Care and Use of Laboratory Animals13. 0899-9007/04/$30.00 doi:10.1016/j.nut.2004.06.014
Nutrition Volume 20, Number 10, 2004
Malnutrition and Pituitary Leptin Receptor
TABLE I.
925
for immunohistochemistry. The animals had free access to water and a normal diet until immediately before death.
COMPOSITION OF THE CONTROL AND LOW-PROTEIN DIETS Ingredient
Control diet*
Soybean ⫹ wheat (g/kg) Cornstarch (g/kg) Soybean oil (g/kg) Vitamin mix (g/kg)‡ Mineral mix (g/kg)‡ Macronutrient composition (% energy) Protein (g/kg) Carbohydrate (g/kg) Fat (g/kg) Total energy (kJ/kg)
Low-protein diet†
230.0 676.0 50.0 4.0 40.0
80.0 826.0 50.0 4.0 40.0
23.0 66.0 11.0 17038.7
8.0 81.0 11.0 17038.7
* Standard rat diet (Nuvilab-NUVITAL Nutrientes LTDA, Parana´ , Brazil). † The low-protein diet was prepared in our laboratory by replacing part of the protein in the control diet with cornstarch. The amount of cornstarch was calculated to make up for the decrease in energy content resulting from the reduction in protein. ‡ Vitamin and mineral mixtures were formulated to meet the American Institute of Nutrition AIN-93G recommendation for rodent diets and contained the recommended amount of iodine14.
The dams were randomly assigned to one of the following groups: a control group, which had free access to a standard laboratory diet containing 23% protein; a protein-restricted (PR) group, which had free access to an isoenergetic, protein-restricted diet containing 8% protein; and an energy-restricted (ER) group, which received a standard laboratory diet in restricted quantities that were calculated according to the mean ingestion of the PR group. Thus, the amount of food consumed by the ER and PR groups was about the same. The composition of the diets, which followed recommended standards, is shown in Table I14. The PR diet was prepared in our laboratory by replacing part of the protein in the control diet with cornstarch. The amount of starch was calculated to make up for the decrease in energy content resulting from the protein reduction. Within 24 h of birth, excess pups were removed, and only six male pups were kept per dam in the control and treatment groups, because it has been shown that this procedure maximizes lactation performance15. The experimental diets were started at birth, which was defined as day 0 of lactation, and were ended at weaning (day 21). After weaning, two animals from each litter were chosen at random and were placed together in a cage with free access to water and a normal diet until they were 150 d old. The remaining four animals from each litter were killed on day 21 (see the paragraph on blood sampling, below). In the dams, food intake and body weight were monitored during the lactation period. In the pups, body weight and food intake were monitored every 4 d, from birth until 150 d of age. The amount of the diet ingested was calculated as the difference between the weight of the food remaining in the food bin (Da) and the amount placed there 4 d before (Di). This was measured in the pups from weaning until 150 d of age. These data were then used to calculate daily food intake according to the following formula: food intake (g)⫽([Di ⫺ Da]/3)/4, where 3 is the number of animals in each cage and 4 is the number of days. Blood samplings were performed at 21 and 150 d of age. At these ages, the animals were killed at 10:00 AM with a lethal dose of pentobarbital, and blood was obtained by cardiac puncture. The pituitary glands from animals 150 d old were excised and prepared
Immunohistochemistry The pituitary glands from the 150-d-old animals were placed in fixative for 12 h at room temperature (freshly prepared 4% [w/v] formaldehyde in 0.1 M phosphate buffer, pH 7.2), embedded in Paraplast plus (Sigma, St. Louis, MO, USA), and sectioned in 3-m thicknesses. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in 0.01 M phosphate buffered saline, and the sections were rinsed successively with distilled water and phosphate buffered saline. The primary antibody used was the polyclonal anti–leptin-R antibody (1:100; antihuman leptin receptor — Long Form, Linco Research, St. Charles, MO, USA). Immunostaining was performed by using the avidin-biotin-peroxidase method (DAKO Corporation, Carpinteria, CA, USA). Stereologic Analysis Our analysis included the use of a video-microscope system composed of a Leica DMRBE microscope (Leica, Wetzlar, Germany) with an oil-immersion objective (100 ⫻, numerical aperture, 1.25), a Kappa video camera (Kappa, Gleichen, Germany), and a Sony Trinitron monitor (Sony, Pencoed, UK). The numerical density per area of the cells staining for leptin receptor in the pituitary was estimated by taking 10 random microscopic fields per animal, with five animals per group. The counts were performed over a 6400m2 frame, and all immunostained cell profiles in the frame that were not intersecting with the forbidden line or its extensions were sampled. The pituitary is an isotropic tissue; thus, the random microscopic fields were observed in coded slices by moving the microscope stage in a blinded manner16,17. After the counts were completed, the group to which each coded slice belonged was recorded. Serum Hormone Concentrations Blood samples were centrifuged to obtain serum, which was stored at ⫺20°C until analyzed. Serum TSH was measured by specific radioimmunoassay with a kit for rat TSH supplied by the National Institute of Diabetes and Digestive and Kidney Diseases (Bethesda, MD, USA). The results are expressed in terms of the reference preparation provided (RP-3). Total serum tri-iodothyronine (TT3) and total serum thyroxine (TT4) were measured by radioimmunoassay with commercial kits
TABLE II. MATERNAL BODY WEIGHT AND BODY WEIGHT AND SERUM LEPTIN CONCENTRATION OF THE PUPS ON DAY 21 OF LACTATION* C Maternal body weight (g) Pup body weight (g) Pup serum leptin (ng/mL)
225.1 ⫾ 4.6 30.5 ⫾ 1.6 0.8 ⫾ 0.13
PR
ER
197.9 ⫾ 5.8† 176 ⫾ 5.8† 17.1 ⫾ 0.5† 16.5 ⫾ 0.6† 1.5 ⫾ 0.19† 1.4 ⫾ 0.14†
* Values are the mean ⫾ standard error of the mean for five animals per group. † Significantly different from the control group (multiple comparison of means test, P ⬍ 0.05). C, control diet group; ER, energy-restricted diet group; PR, proteinrestricted diet group
926
Vicente et al.
Nutrition Volume 20, Number 10, 2004 TABLE III.
BODY WEIGHT AND SERUM LEPTIN, TT3, TT4, AND TSH CONCENTRATIONS IN ANIMALS AT AGE 150 D*
Body weight (g) Leptin (ng/mL) TT3 (nmol/L) TT4 (nmol/L) TSH (ng/ml)
C
PR
ER
316.5 ⫾ 18.8 1.8 ⫾ 0.17 21.08 ⫾ 1.85 11.77 ⫾ 0.56 3.04 ⫾ 0.42
294.9 ⫾ 8.33† 1.6 ⫾ 0.22 59.28 ⫾ 3.79† 13.38 ⫾ 0.42† 2.21 ⫾ 0.14†
362.5 ⫾ 7.59† 1.8 ⫾ 0.20 64.98 ⫾ 3.83† 11.83 ⫾ 0.09 2.07 ⫾ 0.14†
* Values are the mean ⫾ standard error of the mean for five animals per group. † Significantly different from the control group (multiple comparison of means test, P ⬍ 0.05). C, control diet group; ER, energy-restricted diet group; PR, proteinrestricted diet group; TSH, thyroid-stimulating hormone; TT3, total triiodothyronine; TT4, total thyroxine
(Coat-A-Coat, DPC, Los Angeles, CA, USA). We used control standard curves diluted in iodothyronine-free rat serum (charcoal treated). Leptin concentrations were measured with a commercial kit (Murine Leptin Elisa-DSL-10-24100; Diagnostic Systems Laboratories, Webster, TX, USA). The interassay and intra-assay coefficients of variation were 3.1% and 4.2%, respectively, and the limit of detection was 0.04 ng/100 mL.
II. All these variables were significantly affected by the dietary treatment. The body weight of the pups whose dams were fed the PR or ER diet during lactation was significantly lower (P ⬍ 0.001) than that of the controls at the end of lactation. Serum leptin concentrations were higher in pups from PR and ER dams than in the control group (P ⬍ 0.05). The body weight and serum leptin, TT3, TT4, and TSH concentrations of the animals at 150 d of age are presented in Table III. The animals whose dams were fed the PR diet weighed significantly less than did the control animals (P ⬍ 0.05); in contrast, the animals whose dams were fed the ER diet weighed about 10% more than the controls (P ⬍ 0.01). No significant differences in serum leptin concentrations were observed at this age. The offspring of PR dams had significantly higher serum TT3 (149%, P ⬍ 0.001) and TT4 (12%, P ⬍ 0.05) concentrations than did the controls. In the offspring of ER dams, only the serum TT3 concentration was significantly higher than that in the controls (178%, P ⬍ 0.05). Serum TSH was significantly lower in the offspring of PR and ER dams than in those of the controls (P ⬍ 0.05). Immunohistochemistry showed the presence of leptin receptor immunoreactivity in the pituitary of adult animals (Figure 1). Leptin expression in the pituitary was higher in the rats whose dams were fed the PR and ER diets than in the controls (Figure 2). The quantity of immunostaining cells, analyzed by the stereologic method, is shown in Figure 3. The percentage of immunostaining cells marked by leptin receptor antibody in the pituitary was higher in the offspring of PR and ER dams than in those of the controls (by 22% and 24%, respectively).
Statistical Analysis
DISCUSSION
The data are reported as means ⫾ standard errors of the means. One-way analysis of variance and then the Neuman-Keuls test were used to assess serum hormone concentrations, except for serum TSH, which was analyzed by use of the Kruskal-Wallis multiple-comparison test followed by Dunn’s multiple-comparison test. Differences in numerical density per area across groups were assessed by use of Kruskal-Wallis non-parametric analysis of variance and the Kolmogorov-Smirnov test, with a significance level of 0.0518.
The present data on body weight during lactation reinforce previous findings from our laboratory in which malnourished dams and their pups had significantly lower body weights than did control animals11. Despite the lower body weight of the offspring, serum leptin concentrations were higher at weaning in the pups of malnourished dams than in the control animals. Because leptin is present in breast milk19, leptin may be transferred to the pups through the milk, which could lead to further decreases in food intake and body weight. The higher serum concentration of leptin at weaning could affect the regulation of several factors that contribute to the long-term control of the endocrine system. In the present study, the expression of leptin receptor in the pituitary was higher in the offspring of dams who were malnourished during lactation than in the offspring of the control group. The findings of higher serum concentrations of T4 and T3 and
RESULTS The body weight and serum leptin concentration of the pups and maternal body weight on day 21 of lactation are presented in Table
FIG. 1. Immunohistochemical demonstration of long-form leptin receptor (Ob-Rb) in the pituitary of adult rats whose dams were fed a control diet during lactation. (A) Ob-Rb–like immunoreactivity is seen in the pituitary. (B) No reaction product was observed when the primary antibody was omitted.
Nutrition Volume 20, Number 10, 2004
Malnutrition and Pituitary Leptin Receptor
927
FIG. 2. Immunohistochemical staining for leptin receptor in the pituitary of adult rats whose dams were fed a control (A), protein-restricted (B), and energy-restricted (C) diet during lactation. Magnification 20⫻.
lower serum concentrations of TSH in these animals reinforce the results of our previous studies12,20. The lower serum TSH concentration is easily explained by the negative feedback of thyroid hormones on the hypothalamic-pituitary axis. However, the higher expression of leptin receptor in the pituitary gland could also lead to a greater inhibitory effect of leptin on TSH secretion, because it has been shown that leptin suppresses TSH release in vitro10. Leptin increases thyroid hormone secretion through a direct mechanism that involves its binding to thyroid leptin receptors21. If these receptors are also highly expressed at the thyroid level, we could explain the higher thyroid hormone concentration observed in the present study. However, leptin also has an acute stimulatory effect on TSH secretion in vivo in fed rats10 and in fasting rodents4,22. Therefore,
FIG. 3. Quantity of immunostaining cells in the pituitary of adult rats whose dams were fed a control (solid bars), protein-restricted (open bars), or energy restricted (hatched bars) diet during lactation. Values are mean ⫾ standard error of the mean for five animals. *Significantly different from the control group (Kruskal-Wallis non-parametric analysis of variance and Kolmogorov-Smirnov test, P ⬍ 0.05).
this higher pituitary leptin receptor expression could also be interpreted as a resistance to leptin action on TSH secretion. We postulate that this resistance to leptin could be the result of a greater exposure to leptin at weaning, which could program pituitary thyrotrophs for lower leptin sensitivity later in life. This kind of programming was reported by other investigators who showed that higher leptin concentrations during the neonatal period program for greater body weight in later life in humans23 and in rats24. It has been reported that hypothalamic leptin receptors are upregulated when there is a deficiency of leptin action25. Greater hypothalamic leptin receptor expression was also reported in ER adult ewes with low serum leptin concentrations26. In the present study, however, we found no significant difference in serum leptin concentrations in the adult offspring, which leads us to conclude that it is the serum leptin concentrations at weaning that program the changes in pituitary leptin receptor. In conclusion, we showed that protein or energy restriction during lactation is associated with greater expression of leptin receptor in the pituitary in adult offspring. We suggest that higher leptin action or leptin resistance may explain this finding. Both explanations are plausible according the type of malnutrition. In the offspring of PR dams, higher leptin action is a more appropriate explanation, because those animals were leaner, had higher thyroid function, and had lower concentrations of TSH, all of which are dependent on higher leptin action. In the offspring of ER dams, however, the resistance hypothesis is more likely, because those animals were heavier and, despite having higher T3 concentrations, did not have other indications of thyroid hyperfunction. These data provide new insights into the physiologic importance of nutritional status and consequent changes in leptin concentrations during the neonatal period and suggest that leptin may have an important role in thyroid–pituitary axis regulation in adults. Further studies, such as studies of the expression of leptin receptor in the thyroid and mechanisms of intracellular leptin signaling, are needed to explore the mechanism that leads to the higher pituitary expression of leptin receptor.
928
Vicente et al.
REFERENCES 1. Pelleymounter MA, Cullen MJ, Baker MB, et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995;269:540 2. Campfield LA, Smith FJ, Guisez Y, Devos R, Burn P. Recombinant mouse OB protein: evidence for a peripheral signal linking adiposity and central neural networks. Science 1995;269:546 3. Friedman JM, Halaas JL. Leptin and the regulation of body weight in mammals. Nature 1998;395:763 4. Ahima RS, Saper CB, Flyer JS, Elmquist JK. Leptin regulation of neuroendocrine systems. Front Neuroendocrinol 2000;20:317 5. Zamorano PL, Mahesh VB, De Sevilla LM, et al. Expression and localization of the leptin receptor in endocrine and neuroendocrine tissues of the rat. Neuroendocrinology 1997;65:223 6. Jin L, Zhang S, Burguera BG, et al. Leptin and leptin receptor expression in rat and mouse pituitary cells. Endocrinology 2000;141:333 7. Sone M, Nagata H, Takekoshi S, Osamura RY. Expression and localization of leptin receptor in the normal rat pituitary gland. Cell Tissue Res 2001;305:351 8. Legradi G, Emerson CH, Ahima RS, Flier JS, Lechan RM. Leptin prevents fasting-induced suppresion of prothyrotropin-releasing hormone messenger ribonucleic acid in neurons of the hypothalamic paraventricular nucleus. Endocrinology 1997;138:2569 9. Seoane LM, Carro E, Tovar S, Casanueva FF, Dieguez C. Regulation in vivo TSH secretion by leptin. Regul Pept 2000;92:25 10. Ortiga-Carvalho TM, Oliveira KJ, Soares BA, Pazos-Moura CC. Leptin role in the regulation of thyrotropin secretion in fed state—in vivo and in vitro studies. J Endocrinol 2002;174:121 11. Passos MCF, Ramos CF, Moura EG. Short and long term effects of malnutrition in rats during lactation on the body weight of offspring. Nutr Res 2000;20:1603 12. Passos MCF, Ramos CF, Dutra SCP, Mouc¸ o T, Moura EG. Long-term effects of malnutrition during lactation on the thyroid function of offspring. Horm Metab Res 2002;34:40 13. Bayne K. Revised guide for the care and use of laboratory animals available. Physiologist 1996;39:208
Nutrition Volume 20, Number 10, 2004 14. Reeves PG, Nielsen FH, Fahey GC. AIN-93 Purified diets for laboratory rodents: final report of the American Institute of Nutrition Ad Hoc Writing Committee on the reformulation of the AIN-76 rodent diet. J Nutr 1993;123:1939 15. Fishbeck KL, Rasmussen KM. Effect of repeated cycles on maternal nutritional status, lactational performance and litter growth in ad libitum-fed and chronically food-restricted rats. J Nutr 1987;117:1967 16. Gundersen HJ, Bendtsen TF, Korbo L, et al. Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 1988;96:379 17. Mandarim-de-Lacerda CA. Stereological tools in biomedical research. An Acad Bras Cienc 2003;75:469 18. Zar JH. Biostatistical analysis. 4th ed. Upper Saddle River, NJ: Prentice-Hall; 1999 19. Casabiell X, Pin˜ eiro V, Tome´ MA, et al. Presence of leptin in colostrum and/or breast milk from lactating mothers: a potential role in the regulation of neonatal food intake. J Clin Endocrinol Metab 1997;82:4270 20. Dutra SCP, Passos MCF, Lisboa PC, et al. Liver deiodinase activity is increased in adult rats whose mothers were submitted to malnutrition during lactation. Horm Metab Res 2003;35:268 21. Nowak KM, Kaczmarek P, Mackowiak P, et al. Rat thyroid gland expresses the long form of leptin receptors, and leptin stimulates the function of the gland in euthyroid non-fasted animals. Int J Mol Med 2002;9:31 22. Ahima RS, Prabakaran D, Mantzoros C, et al. Role of leptin in the neuroendocrine response to fasting. Nature 1996;382:250 23. Shingal A, Farooqi SI, O’Rahilly, et al. Early nutrition and leptin concentrations in early life. Am J Clin Nutr 2002;75:993. 24. Cravo CO, Teixeira CV, Passos MC, et al. Leptin treatment during the neonatal period is associated with higher food intake and adult body weight in rats. Horm Metab Res 2002;34:400 25. Huang XF, Zhang R. Upregulation of leptin receptor mRNA expression in obese mouse brain. Neuroreport 1997;8:1035 26. Dyer CJ, Simmons JM, Matteri RL, Keisler DH. Leptin receptor mRNA is expressed in ewe anterior pituitary and adipose tissues and is differentially expressed in hypothalamic regions of well-fed and feed-restricted ewes. Domest Anim Endocrinol 1997;14:119