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Increase of T3 secreted through the milk in protein restricted lactating rats
Nutrition Research 21 (2001) 917–924 www.elsevier.com/locate/nutres
Increase of T3 secreted through the milk in protein restricted lactating rats M.C.F. Passosa, C.F. Ramosb, T. Mouc¸oc, E.G. Mourad,* a
Department of Applied Nutrition, State University of Rio de Janeiro, 20550-030, Rio de Janeiro, RJ, Brazil b Department of Anatomy, State University of Rio de Janeiro, 20550-030, Rio de Janeiro, RJ, Brazil c Laboratory Miguelote Viana, State University of Rio de Janeiro, 20550-030, Rio de Janeiro, RJ, Brazil d Department of Physiological Science, State University of Rio de Janeiro, 20550-030, Rio de Janeiro, RJ, Brazil Received 14 July 2000; received in revised form 3 January 2001; accepted 31 January 2001
Abstract Lactating rats were fed an 8% protein-restricted diet (PR), a 23% protein diet (C), and an energy-restricted pair-fed to PR group (PF). Thyroxine (T4) and triiodothyronine (T3) concentrations were analyzed in milk and in the serum of mothers and pups on different days of lactation. Serum T3 was always higher (p ⬍ 0.01) in PR mothers, while T4 was lower (p ⬍ 0.05) on days 4 and 21. PR group showed higher T3 (p ⬍ 0.001) on days 4, 8, and 12, while PF showed higher T3 in milk (p ⬍ 0.05) on days 12 and 16. In PR pups serum T3 was higher (p ⬍ 0.05), while T4 was lower on day 4 and both were lower (p ⬍ 0.05) on day 12. In PF pups T3 was lower (p ⬍ 0.05) on day 21, and T4 was lower (p ⬍ 0.01) on day 12. These data suggest a higher transfer of T3 for the PR pups that could be important for their nervous system development. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Triiodothyronine; Protein malnutrition; Milk; Rats; Lactation
1. Introduction Energy restriction is associated with low serum T3 concentration and normal or low serum T4 in adult animals [1–3] and humans [4,5] studies. Thyroid function is also affected in adult animals submitted to protein malnutrition [6 – 8]. However, there are scanty data about the effects of protein malnutrition during lactation on thyroid function of mothers and their offspring. Most authors studied protein malnutrition over the gestational period or both the * Corresponding author. Tel.: ⫹021-5876134; fax: ⫹021-5876129. E-mail address: [email protected] (E.G. Moura). 0271-5317/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 2 9 4 - 9
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gestational and lactational periods [9 –11]. We have showed recently, that in post-weaned rats whose dams were fed a low-protein diet during lactation period, the total serum T3 concentration was lower than that of the control group [12]. There is a considerable doubt about the extent to which nutritional status influences lactation performance and its implications for infant growth and nutritional status, since changes in iodine metabolism could have consequences for the thyroid function of the neonate, with possible impairment in central nervous system development. Although the presence of thyroid hormones in milk of rats [13,14] and humans [14 –17] is well documented, there are conflicting findings about their concentrations. Varma et al [15] found easily measurable levels of T3, low levels of reverse triiodothyronine (rT3) and tracer amounts of T4. Low, but detectable, concentrations of T4 in human milk were reported by Sack et al. [16]. In contrast, T3 was frequently reported in easily measurable concentrations, but the quoted levels differ among authors. These contradictory findings can be due to differences in analytical methods used, which, were developed for thyroid hormones determination in the serum and not in the milk [17]. Compared with thyroid hormones concentrations in blood, T4 in milk represents only a small fraction (about 1/30), whereas T3 approximates 1/3 of that in serum [18]. No study to our knowledge has been specifically designed to evaluated the concentration of T3 in the milk of lactating rats submitted a protein or energy-restricted diet. Previously we showed that the lactating rats submitted to a protein-restricted diet during lactation, when compared to control animals, presented at the end of lactation lower 131I uptake by the thyroid, higher serum TSH and T3 concentration and higher 131I uptake by the mammary gland, suggesting an adaptive change that could provide more iodine and thyroid hormone to their offspring [19]. The aim of the present study was evaluated the T3 and T4 concentrations in the milk of protein or energyrestricted lactating rats at different stages of lactation, comparing with T3 and T4 serum concentrations in mothers and their offspring submitted to similar conditions. 2. Materials and methods Wistar rats were kept in a room with controlled temperature (25 ⫾ 1°C) and with artificial dark-light cycle (lights on from 7:00 a.m. to 7:00 p.m.). 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 parturition. The use and handling of experimental animals followed the principles described in the Guide for the Care and Use of Laboratory Animals [20]. Dams were randomly assigned to one of the following groups: control group (C), with free access to a standard laboratory diet containing 23% protein, protein-restricted group (PR), with free access to an isoenergy and protein-restricted diet containing 8% protein; and an energyrestricted group, (pair fed, PF) receiving a standard laboratory diet in restricted quantities, which were calculated according to the mean ingestion of the PR group. In this way, the amounts of food consumed to both PF and PR groups were about the same. Table 1 shows the composition of the diets. Within 24 hours of birth, excess pups were removed, so that only 6 male pups were kept per dam, because it has been shown that this procedure maximizes lactation performance [22]. Malnutrition was started at birth, which was defined as day 0 (d0) of lactation, and was ended at weaning (d21).
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Table 1 Composition of the control and low-protein diets Control* Ingredients (g/Kg) Soybean ⫹ wheat Corn starch Soybean oil Vitamin mix** Mineral mix** Macronutrient composition (%) Protein Carbohydrate Fat Total energy (KJ/Kg)
Low-protein#
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 diet for rats (Nuvilab-NUVITAL Nutrientes LTDA, Parana´, Brazil). The low-protein diet was prepared in our laboratory using the control diet and replacing part of its protein with corn starch. The amount of the latter was calculated so as to make up for the decrease in energy content due to protein reduction. **Vitamin and mineral mixtures were formulated to meet the American Institute of Nutrition AIN-93G recommendation for rodent diets [21]. #
During lactation milk samples were obtained from all females on days 4, 8, 12, 16 and 21. Before milking females were separated from their litters for 2 hours. While separated, dams were allowed access to the appropriate diets, after which they were lightly anesthetized and injected subcutaneously with 5 IU oxytocin (Eurofarma, SP, Brazil) [23]. Milk samples were then obtained by gently squeezing the left thoracic and abdominal teats and stored at ⫺20°C until analysis. As blood withdraw could affect lactational performance we decide to collect blood from other similar experimental groups, on days 4, 12, and 21, in both dams and their pups. The fat was extracted from the milk with ether and ethanol, according Jansson et al [17]. The total T3 and T4 concentrations in the milk and the total T3 and free T4 in the serum were measured by chemiluminescent enzyme immunoassay (Access Immunoassay System, Sanofi Diagnostic Pasteur, INC, USA). The sensitivity of T3 and T4 assays were 0.1 nmol/L and 0.0015 nmol/L, respectively. The intra-assay and inter-assay errors for T3 were 7.95%, for both, and for T4 were 5.6% and 7%, respectively. The data are reported as means ⫾ SEM and analyzed by the Two-way analysis of variance followed by Newman Keuls test to determine which groups were divergent. The level of significance was set at p ⬍ 0.05. 3. Results The effects of protein or energy-restricted diet on serum T3 concentration are shown in Fig. 1A. Serum T3 concentration was higher in PR mothers during all stages of lactation compared to the control group (46%, 75% and 46%, days 4, 12 and 21, respectively, p ⬍ 0.01) and PF group (28%, 57% and 50%, days 4, 12 and 21, respectively, p ⬍ 0.01). By contrary, serum T4 concentration was lower in PR mothers in the beginning (day 4, 19%, p ⬍ 0.05) and at the end (day 21, 34%, p ⬍ 0.05) of lactation (Fig. 1B). No alteration was found on PF mothers. T3 concentration in the milk increased (p ⬍ 0.01) during lactation in the control group.
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Fig. 1. Serum T3 (A) and T4 (B) concentrations in the mothers of control (black bars), protein-restriction (white bars), and energy-restriction (hatched bars) diets groups during lactation. Values are given as the mean ⫾ SEM. Significant differences between either the diet-restricted groups and controls (*), or between the two dietrestricted groups (#), were determined by a multiple comparison of means test with the level of significance set at p ⬍ 0.05 (see Material and Methods). The numbers of animals studied are shown in parentheses.
Higher concentration (p ⬍ 0.001) of this hormone was found in PR mothers on days 4 (1975%), 8 (165%), and 12 (156%) of lactation, compared to the controls. T3 concentration was also higher (p ⬍ 0.05) in the milk of PF mothers, but only at days 12 (126%) and 16 (128%) of lactation (Fig. 2). T4 concentration was undetectable in the milk of controls and malnourished lactating rats. Fig. 3 depicts the serum T3 and T4 concentrations in the pups during lactation. Serum T3 concentration in the pups of PR mothers was higher on day 4 (46%, p ⬍ 0.05) and lower (43%, p ⬍ 0.05) on day 12 of lactation compared to control group. This hormone was not altered in the pups of PF mothers on days 4 and 12, but at the end of lactation it was found lower (27%, p ⬍ 0.05) compared to the control group (Fig. 3A). Serum T4 concentration in the pups of PR mothers was lower (p ⬍ 0.05) on days 4 (20%) and 12 (52%) of lactation compared to control group, while in the pups of PF mothers it was lower only at 12th (32%, p ⬍ 0.01) day of lactation (Fig. 3B).
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Fig. 2. Milk T3 concentration in the control (black bars), protein-restriction (white bars), and energy-restriction (hatched bars) diets groups during lactation. Values are given as the mean ⫾ SEM. Significant differences between either the diet-restricted groups and controls (*), or between the two diet-restricted groups (#), were determined by a multiple comparison of means test with the level of significance set at p ⬍ 0.05 (see Material and Methods). The numbers of animals studied are shown in parentheses.
4. Discussion Our experimental design included the pair fed group with the aim to identify protein-restriction specific changes. The alterations observed in the pair fed group are different from those observed in the PR group and reinforce our previous studies where other parameters, such as, thyroid iodine uptake [19], milk composition and body weight of adult offspring [24] changes differently according to the kind of malnutrition. In these previous papers we also demonstrated that both protein or energy restricted-diets were associated with a similar decrease in food intake and body weight of dams and in the body weight of pups from day 6 until weaning [19,24]. We showed here that protein restriction during lactation is associated with higher serum T3 concentrations in lactating rats. These findings of reduced T4 with increased T3 in the serum of protein-restricted lactating rats suggests increased T3 production by the thyroid or by peripheral T4 to T3 conversion. These data are similar to the findings of other reports, which demonstrated that the increase in serum T3 also occurs in non-lactating proteinrestricted rats [6 – 8] leading to specific metabolic changes [7,25]. The metabolic importance of a higher serum T3 concentration associated to protein deficiency is unknown. In contrast, none alteration of these hormones was found in the serum of lactating rats with energy restriction in our study. This data is in disagreement with previous studies, where rats were submitted to an energy restriction during gestation and lactation [1] or when adults [2,3] or in humans [4,5]. In these studies they found low serum T3 concentration with normal or low serum T4. In the energy restriction group we could expect a reduction of T3 serum concentration on the mother. However, this effect is related to degree and duration of energy restriction and the stage of life that the energy restriction is started. In our experimental model, the energy restriction could not be enough to produce significant changes in T3 serum concentration. Anyway, the point here is that the changes are different according to the kind
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Fig. 3. Serum T3 (A) and T4 (B) concentrations in the pups of control (black bars), protein-restriction (white bars), and energy-restriction (hatched bars) diets groups during lactation. Values are given as the mean ⫾ SEM. Significant differences between either the diet-restricted groups and controls (*), or between the two dietrestricted groups (#), were determined by a multiple comparison of means test with the level of significance set at p ⬍ 0.05 (see Material and Methods). The numbers of animals studied are shown in parentheses.
of malnutrition. In the two malnourished groups of mothers there was a significant reduction in body weight [19,24]. So, the differences in T3 serum concentration seems to be mainly due to specific protein reduction in the diet. We observed that T3 concentration in the milk increased through the lactation in control animals, which is in agreement with previous studies [26]. This could be related to a maturation of postulated carrier T3 systems in mammary gland. However, despite an organic anion transporter be postulated as a thyroid hormone carrier in central nervous system and liver [27], the mechanism by which thyroid hormones are transported across the bloodmammary gland barrier is unknown. Here we measured, for the first time, thyroid hormones in the milk of malnourished rats. We showed that T3 is secreted in a different pattern according the nutritional status of the mother. In protein restriction, it is secreted in a higher concentration since the first days of lactation. The higher serum T3 concentration of protein restricted mothers could explain, in part, this higher T3 secreted into the milk, but we cannot discard other additional effects, since in the PF group, T3 also increase in the milk, despite no increase in serum T3 concentration of mothers. Variations in hormone uptake and in hormone concentrations among tissue are explained by
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differences in cellular specific binding power, by differences in the capillary circulation and the plasma membrane carrier system for the thyroid hormones, which may facilitate diffusion (bloodbrain barrier and muscle), active transport (hepatocytes) or internalization (fibroblasts) [28]. The increase in T3 secreted into the milk could be, also, due to a higher 5⬘-deiodinase activity in the mammary gland and milk. Some authors [29,30] relate that type I 5⬘deiodinase is decreased in the liver and increased in the mammary gland of lactating rats, suggesting that 5⬘DI activity is regulated in a different way in mammary gland. If this is true, this activity could change differently in tissues according to different nutritional conditions. Besides, Slebodzinski [31] showed that the whole milk or its cellular components (namely macrophages, lymphocytes and granulocytes) possess deiodinating enzyme system converting T4 into T3, adding an additional step of regulation, since milk composition does change in malnourished rats and could regulate this deiodinase activity. The higher T3 concentration in the milk of protein restricted lactating rats, in the beginning of lactation, suggests a higher transfer of T3 for the pups, since the serum T3 concentration in these pups are significantly increased, despite of lower serum T4 concentration. However, the lower protein concentration of this milk [24] could increase T3 production in those pups in the same way that occurs with their mothers. So, this T3 transferred to milk could be very important for the thyroid function regulation of pups until the first 4 days of life, which is more critical for the thyroid hormone action on central nervous system development. As malnutrition continues during lactation, this adaptive mechanism does not persist through the 12th day. However, this phase does not seems to be so critical for the neural development of the animal. At the end of lactation, all hormonal concentrations are similar to the controls, suggesting that other adaptive mechanisms could play some role, such as iodine metabolism. Comparing the effects of the two kinds of malnutrition on the serum thyroid hormone concentration in the pups, it seems that protein restriction is associated with a more efficient adaptive mechanism that assures more T3 in the beginning and normalization at the end of lactation. Acknowledgments This work was supported by a grant from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and by funds from Po´s-Graduac¸a˜o em Biologia (PGBN-UERJ). References [1] Oberkotter LV, Rasmussen KM. Changes in plasma thyroid hormone concentration in chronically foodrestricted female rats and their offspring during suckling. J Nutr 1992;122:435– 41. [2] Hugues JN, Enjalbert A, Burger AG, Voirol MJ, Sebaoun J, Epelbaum J. Sensitivity of thyrotropin (TSH) secretion to 3,5,3⬘-triiodothyronine and TSH-releasing hormone in rat during starvation. Endocrinology 1986;119:253–60. [3] Rodriguez F, Mellado M, Montoya E, Jolin T. Sensitivity of thyrotropin secretion to TSH-releasing hormone in food-restricted rats. Acta Endocrinol (Copenh) 1991;124:194 –202. [4] Spencer CA, Lum SMC, Wilber JF, et al. Dynamics of serum thyrotropin and thyroid hormone changes in fasting. J Clin Endocrinol Metab 1983;56:883–7. [5] LoPresti JS, Gray D, Nicoloff JT. Influence of fasting and refeeding on 3,3,5⬘-triiodothyronine metabolism in man. J Clin Endocrinol Metab 1991;72:130 – 4. [6] Tulp OL, Krupp PP, Danforth EJ, Horton ES. Characteristics of thyroid function in experimental protein malnutrition. J Nutr 1979;109:1321–32. [7] Sawaya AL, Lunn PG. Evidence suggesting that the elevated plasma triiodothyronine concentration of rats fed on protein deficient diets is physiologically active. Br J Nutr 1985;53:175– 81.
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[8] Rouaze-Romet M, Savu L, Vranckx R, Bleiberg-Daniel F, Le Moullac B, Gouache P, Nunez EA. Re-expression of thyroxine-binding globulin in post-weaning rats during protein or energy malnutrition. Acta Endocrinologica 1992;127:441– 8. [9] Coleoni AH, Munaro N, Recu´pero AR, Cherubini O. Nuclear triiodothyronine receptors and metabolic response in perinatally protein-deprived rats. Acta Endocrinol 1983;104:450 –5. [10] Shrader RE, Hasting-Roberts MM, Ferlatte MI, Zeman FJ. Effect of prenatal protein restriction on fetal and neonatal thyroid morphology in the rat. J Nutr 1977;107:213–20. [11] Friedman DJ, Zeman F. Thyroid, pituitary, and hypothalamic hormone levels in neonatal rat pups following maternal protein deficiency. Proc Soc Exp Biol Med 1979;16:275–9. [12] Ramos CF, Lima APS, Teixeira CV, Brito PD, Moura EG. Thyroid function in post-weaning rats whose dams were fed a low-protein diet during suckling. Braz J Med Biol Res 1997;30:133–7. [13] Strbak V, Macho L, Knopp J, Struha´rova´ E. Thyroxine content in mother milk and regulation of thyroid function of suckling rats. Endocrinologia Experimentalis 1974;8:59 – 69. [14] Oberkotter LV, Tenore A. Separation and radioimmunoassay of T3 and T4 in human breast milk. Hormone Res 1983;17:11– 8. [15] Varma SK, Collins M, Row A, Haller WS, Varma K. Thyroxine, triiodothyronine, and reverse triiodothyronine concentrations in human milk. J Pediatr 1978;93(5):803– 6. [16] Sack J, Amado O, Lunenfeld B. Thyroxine content in human milk. J Clin Endocrinol Metab 1977;45:171–3. [17] Jansson L, Ivarsson S, Larsson I, Ekman R. Tri-iodothyronine, and thyroxyne in human milk. Acta Pædiatr Scand 1983;72:703–5. [18] Slebodzinski AB, Nowak J, Gawecka H, Sechman A. Thyroid hormones and insulin in milk: a comparative study. Endocr Exper 1986;20:247–55. [19] Ramos CF, Teixeira CV, Passos MCF, Pazos-Moura CC, Lisboa PC, Curty FH, Moura EG. Low-protein diet changes thyroid function in lactating rats. Proc Soc Exp Biol Med 2000;224(4):256 – 63. [20] Bayne K. Revised guide for the care and use of laboratory animals available. Am Phys Soc Physiol 1996;39(4):199, 208 –11. [21] 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 –51. [22] Fischbeck KL, Rasmussen KM. Effect of repeated reproductive cycles on maternal nutritional status, lactational performance and litter growth in ad libitum fed and chronically food-restricted rats. J Nutr 1987;117:1967–75. [23] Pine AP, Jessop NS, Oldham JD. Maternal protein reserves and their influence on lactational performance in rats: the effects of dietary protein restriction and stage of lactation on milk composition. J Nutr 1994;72:815–30. [24] Passos MCF, Ramos CF, Moura EG. Short and long term effects of malnutrition in rats during lactation on the body weight of offspring. Nutrition Research 2000;20(11):1605–14. [25] Claeyssens S, Lavoinne A, Ragot-Fresel M, Bois-Joyeux, Chanez M, Peret J. Metabolic changes in rats fed a low protein diet during post-weaning growth. Metabolism 1990;39:676 – 68. [26] Kodolvsky O. Hormones in milk: minireview. Life Sci 1980;26:1833– 6. [27] Abe T, Kakyo M, Sakagami H, Tokui T, Nishio T, Tanemoto M, Nomura H, Hebert SC, Matsuno S, Kondo H, Yawo H. Molecular characterization and tissue distribution of a new organic anion transporter subtype (oatp3) that transportes thyroid hormones and taurocholate and comparison with oatp2. J Biol Chem 1998;273(35): 22395– 401. [28] Green WL. The thyroid. New York: Elsevier, 1987. [29] Valverde RC, Aceves C. Circulating thyronines and peripheral monodeiodination in lactating rats. Endocrinology 1989;124:1340 – 4. [30] Jack LJW, Kahl S, St Germain DL, Capuco AV. Tissue distribution, and regulation of 5⬘deiodinase process in lactating rats. J Endocrinol 1994;142:205–15. [31] Slebodzinski AB, Brzezinska-Slebodzinski E. Local generation of triiodothyronine by the mammary gland as a source of measurable quantities of the hormone in milk. Endocrine Regulations 1991;25:83–9.
Increase of T3 secreted through the milk in protein restricted lactating rats M.C.F. Passosa, C.F. Ramosb, T. Mouc¸oc, E.G. Mourad,* a
Department of Applied Nutrition, State University of Rio de Janeiro, 20550-030, Rio de Janeiro, RJ, Brazil b Department of Anatomy, State University of Rio de Janeiro, 20550-030, Rio de Janeiro, RJ, Brazil c Laboratory Miguelote Viana, State University of Rio de Janeiro, 20550-030, Rio de Janeiro, RJ, Brazil d Department of Physiological Science, State University of Rio de Janeiro, 20550-030, Rio de Janeiro, RJ, Brazil Received 14 July 2000; received in revised form 3 January 2001; accepted 31 January 2001
Abstract Lactating rats were fed an 8% protein-restricted diet (PR), a 23% protein diet (C), and an energy-restricted pair-fed to PR group (PF). Thyroxine (T4) and triiodothyronine (T3) concentrations were analyzed in milk and in the serum of mothers and pups on different days of lactation. Serum T3 was always higher (p ⬍ 0.01) in PR mothers, while T4 was lower (p ⬍ 0.05) on days 4 and 21. PR group showed higher T3 (p ⬍ 0.001) on days 4, 8, and 12, while PF showed higher T3 in milk (p ⬍ 0.05) on days 12 and 16. In PR pups serum T3 was higher (p ⬍ 0.05), while T4 was lower on day 4 and both were lower (p ⬍ 0.05) on day 12. In PF pups T3 was lower (p ⬍ 0.05) on day 21, and T4 was lower (p ⬍ 0.01) on day 12. These data suggest a higher transfer of T3 for the PR pups that could be important for their nervous system development. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Triiodothyronine; Protein malnutrition; Milk; Rats; Lactation
1. Introduction Energy restriction is associated with low serum T3 concentration and normal or low serum T4 in adult animals [1–3] and humans [4,5] studies. Thyroid function is also affected in adult animals submitted to protein malnutrition [6 – 8]. However, there are scanty data about the effects of protein malnutrition during lactation on thyroid function of mothers and their offspring. Most authors studied protein malnutrition over the gestational period or both the * Corresponding author. Tel.: ⫹021-5876134; fax: ⫹021-5876129. E-mail address: [email protected] (E.G. Moura). 0271-5317/01/$ – see front matter © 2001 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 1 ) 0 0 2 9 4 - 9
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gestational and lactational periods [9 –11]. We have showed recently, that in post-weaned rats whose dams were fed a low-protein diet during lactation period, the total serum T3 concentration was lower than that of the control group [12]. There is a considerable doubt about the extent to which nutritional status influences lactation performance and its implications for infant growth and nutritional status, since changes in iodine metabolism could have consequences for the thyroid function of the neonate, with possible impairment in central nervous system development. Although the presence of thyroid hormones in milk of rats [13,14] and humans [14 –17] is well documented, there are conflicting findings about their concentrations. Varma et al [15] found easily measurable levels of T3, low levels of reverse triiodothyronine (rT3) and tracer amounts of T4. Low, but detectable, concentrations of T4 in human milk were reported by Sack et al. [16]. In contrast, T3 was frequently reported in easily measurable concentrations, but the quoted levels differ among authors. These contradictory findings can be due to differences in analytical methods used, which, were developed for thyroid hormones determination in the serum and not in the milk [17]. Compared with thyroid hormones concentrations in blood, T4 in milk represents only a small fraction (about 1/30), whereas T3 approximates 1/3 of that in serum [18]. No study to our knowledge has been specifically designed to evaluated the concentration of T3 in the milk of lactating rats submitted a protein or energy-restricted diet. Previously we showed that the lactating rats submitted to a protein-restricted diet during lactation, when compared to control animals, presented at the end of lactation lower 131I uptake by the thyroid, higher serum TSH and T3 concentration and higher 131I uptake by the mammary gland, suggesting an adaptive change that could provide more iodine and thyroid hormone to their offspring [19]. The aim of the present study was evaluated the T3 and T4 concentrations in the milk of protein or energyrestricted lactating rats at different stages of lactation, comparing with T3 and T4 serum concentrations in mothers and their offspring submitted to similar conditions. 2. Materials and methods Wistar rats were kept in a room with controlled temperature (25 ⫾ 1°C) and with artificial dark-light cycle (lights on from 7:00 a.m. to 7:00 p.m.). 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 parturition. The use and handling of experimental animals followed the principles described in the Guide for the Care and Use of Laboratory Animals [20]. Dams were randomly assigned to one of the following groups: control group (C), with free access to a standard laboratory diet containing 23% protein, protein-restricted group (PR), with free access to an isoenergy and protein-restricted diet containing 8% protein; and an energyrestricted group, (pair fed, PF) receiving a standard laboratory diet in restricted quantities, which were calculated according to the mean ingestion of the PR group. In this way, the amounts of food consumed to both PF and PR groups were about the same. Table 1 shows the composition of the diets. Within 24 hours of birth, excess pups were removed, so that only 6 male pups were kept per dam, because it has been shown that this procedure maximizes lactation performance [22]. Malnutrition was started at birth, which was defined as day 0 (d0) of lactation, and was ended at weaning (d21).
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Table 1 Composition of the control and low-protein diets Control* Ingredients (g/Kg) Soybean ⫹ wheat Corn starch Soybean oil Vitamin mix** Mineral mix** Macronutrient composition (%) Protein Carbohydrate Fat Total energy (KJ/Kg)
Low-protein#
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 diet for rats (Nuvilab-NUVITAL Nutrientes LTDA, Parana´, Brazil). The low-protein diet was prepared in our laboratory using the control diet and replacing part of its protein with corn starch. The amount of the latter was calculated so as to make up for the decrease in energy content due to protein reduction. **Vitamin and mineral mixtures were formulated to meet the American Institute of Nutrition AIN-93G recommendation for rodent diets [21]. #
During lactation milk samples were obtained from all females on days 4, 8, 12, 16 and 21. Before milking females were separated from their litters for 2 hours. While separated, dams were allowed access to the appropriate diets, after which they were lightly anesthetized and injected subcutaneously with 5 IU oxytocin (Eurofarma, SP, Brazil) [23]. Milk samples were then obtained by gently squeezing the left thoracic and abdominal teats and stored at ⫺20°C until analysis. As blood withdraw could affect lactational performance we decide to collect blood from other similar experimental groups, on days 4, 12, and 21, in both dams and their pups. The fat was extracted from the milk with ether and ethanol, according Jansson et al [17]. The total T3 and T4 concentrations in the milk and the total T3 and free T4 in the serum were measured by chemiluminescent enzyme immunoassay (Access Immunoassay System, Sanofi Diagnostic Pasteur, INC, USA). The sensitivity of T3 and T4 assays were 0.1 nmol/L and 0.0015 nmol/L, respectively. The intra-assay and inter-assay errors for T3 were 7.95%, for both, and for T4 were 5.6% and 7%, respectively. The data are reported as means ⫾ SEM and analyzed by the Two-way analysis of variance followed by Newman Keuls test to determine which groups were divergent. The level of significance was set at p ⬍ 0.05. 3. Results The effects of protein or energy-restricted diet on serum T3 concentration are shown in Fig. 1A. Serum T3 concentration was higher in PR mothers during all stages of lactation compared to the control group (46%, 75% and 46%, days 4, 12 and 21, respectively, p ⬍ 0.01) and PF group (28%, 57% and 50%, days 4, 12 and 21, respectively, p ⬍ 0.01). By contrary, serum T4 concentration was lower in PR mothers in the beginning (day 4, 19%, p ⬍ 0.05) and at the end (day 21, 34%, p ⬍ 0.05) of lactation (Fig. 1B). No alteration was found on PF mothers. T3 concentration in the milk increased (p ⬍ 0.01) during lactation in the control group.
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Fig. 1. Serum T3 (A) and T4 (B) concentrations in the mothers of control (black bars), protein-restriction (white bars), and energy-restriction (hatched bars) diets groups during lactation. Values are given as the mean ⫾ SEM. Significant differences between either the diet-restricted groups and controls (*), or between the two dietrestricted groups (#), were determined by a multiple comparison of means test with the level of significance set at p ⬍ 0.05 (see Material and Methods). The numbers of animals studied are shown in parentheses.
Higher concentration (p ⬍ 0.001) of this hormone was found in PR mothers on days 4 (1975%), 8 (165%), and 12 (156%) of lactation, compared to the controls. T3 concentration was also higher (p ⬍ 0.05) in the milk of PF mothers, but only at days 12 (126%) and 16 (128%) of lactation (Fig. 2). T4 concentration was undetectable in the milk of controls and malnourished lactating rats. Fig. 3 depicts the serum T3 and T4 concentrations in the pups during lactation. Serum T3 concentration in the pups of PR mothers was higher on day 4 (46%, p ⬍ 0.05) and lower (43%, p ⬍ 0.05) on day 12 of lactation compared to control group. This hormone was not altered in the pups of PF mothers on days 4 and 12, but at the end of lactation it was found lower (27%, p ⬍ 0.05) compared to the control group (Fig. 3A). Serum T4 concentration in the pups of PR mothers was lower (p ⬍ 0.05) on days 4 (20%) and 12 (52%) of lactation compared to control group, while in the pups of PF mothers it was lower only at 12th (32%, p ⬍ 0.01) day of lactation (Fig. 3B).
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Fig. 2. Milk T3 concentration in the control (black bars), protein-restriction (white bars), and energy-restriction (hatched bars) diets groups during lactation. Values are given as the mean ⫾ SEM. Significant differences between either the diet-restricted groups and controls (*), or between the two diet-restricted groups (#), were determined by a multiple comparison of means test with the level of significance set at p ⬍ 0.05 (see Material and Methods). The numbers of animals studied are shown in parentheses.
4. Discussion Our experimental design included the pair fed group with the aim to identify protein-restriction specific changes. The alterations observed in the pair fed group are different from those observed in the PR group and reinforce our previous studies where other parameters, such as, thyroid iodine uptake [19], milk composition and body weight of adult offspring [24] changes differently according to the kind of malnutrition. In these previous papers we also demonstrated that both protein or energy restricted-diets were associated with a similar decrease in food intake and body weight of dams and in the body weight of pups from day 6 until weaning [19,24]. We showed here that protein restriction during lactation is associated with higher serum T3 concentrations in lactating rats. These findings of reduced T4 with increased T3 in the serum of protein-restricted lactating rats suggests increased T3 production by the thyroid or by peripheral T4 to T3 conversion. These data are similar to the findings of other reports, which demonstrated that the increase in serum T3 also occurs in non-lactating proteinrestricted rats [6 – 8] leading to specific metabolic changes [7,25]. The metabolic importance of a higher serum T3 concentration associated to protein deficiency is unknown. In contrast, none alteration of these hormones was found in the serum of lactating rats with energy restriction in our study. This data is in disagreement with previous studies, where rats were submitted to an energy restriction during gestation and lactation [1] or when adults [2,3] or in humans [4,5]. In these studies they found low serum T3 concentration with normal or low serum T4. In the energy restriction group we could expect a reduction of T3 serum concentration on the mother. However, this effect is related to degree and duration of energy restriction and the stage of life that the energy restriction is started. In our experimental model, the energy restriction could not be enough to produce significant changes in T3 serum concentration. Anyway, the point here is that the changes are different according to the kind
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Fig. 3. Serum T3 (A) and T4 (B) concentrations in the pups of control (black bars), protein-restriction (white bars), and energy-restriction (hatched bars) diets groups during lactation. Values are given as the mean ⫾ SEM. Significant differences between either the diet-restricted groups and controls (*), or between the two dietrestricted groups (#), were determined by a multiple comparison of means test with the level of significance set at p ⬍ 0.05 (see Material and Methods). The numbers of animals studied are shown in parentheses.
of malnutrition. In the two malnourished groups of mothers there was a significant reduction in body weight [19,24]. So, the differences in T3 serum concentration seems to be mainly due to specific protein reduction in the diet. We observed that T3 concentration in the milk increased through the lactation in control animals, which is in agreement with previous studies [26]. This could be related to a maturation of postulated carrier T3 systems in mammary gland. However, despite an organic anion transporter be postulated as a thyroid hormone carrier in central nervous system and liver [27], the mechanism by which thyroid hormones are transported across the bloodmammary gland barrier is unknown. Here we measured, for the first time, thyroid hormones in the milk of malnourished rats. We showed that T3 is secreted in a different pattern according the nutritional status of the mother. In protein restriction, it is secreted in a higher concentration since the first days of lactation. The higher serum T3 concentration of protein restricted mothers could explain, in part, this higher T3 secreted into the milk, but we cannot discard other additional effects, since in the PF group, T3 also increase in the milk, despite no increase in serum T3 concentration of mothers. Variations in hormone uptake and in hormone concentrations among tissue are explained by
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differences in cellular specific binding power, by differences in the capillary circulation and the plasma membrane carrier system for the thyroid hormones, which may facilitate diffusion (bloodbrain barrier and muscle), active transport (hepatocytes) or internalization (fibroblasts) [28]. The increase in T3 secreted into the milk could be, also, due to a higher 5⬘-deiodinase activity in the mammary gland and milk. Some authors [29,30] relate that type I 5⬘deiodinase is decreased in the liver and increased in the mammary gland of lactating rats, suggesting that 5⬘DI activity is regulated in a different way in mammary gland. If this is true, this activity could change differently in tissues according to different nutritional conditions. Besides, Slebodzinski [31] showed that the whole milk or its cellular components (namely macrophages, lymphocytes and granulocytes) possess deiodinating enzyme system converting T4 into T3, adding an additional step of regulation, since milk composition does change in malnourished rats and could regulate this deiodinase activity. The higher T3 concentration in the milk of protein restricted lactating rats, in the beginning of lactation, suggests a higher transfer of T3 for the pups, since the serum T3 concentration in these pups are significantly increased, despite of lower serum T4 concentration. However, the lower protein concentration of this milk [24] could increase T3 production in those pups in the same way that occurs with their mothers. So, this T3 transferred to milk could be very important for the thyroid function regulation of pups until the first 4 days of life, which is more critical for the thyroid hormone action on central nervous system development. As malnutrition continues during lactation, this adaptive mechanism does not persist through the 12th day. However, this phase does not seems to be so critical for the neural development of the animal. At the end of lactation, all hormonal concentrations are similar to the controls, suggesting that other adaptive mechanisms could play some role, such as iodine metabolism. Comparing the effects of the two kinds of malnutrition on the serum thyroid hormone concentration in the pups, it seems that protein restriction is associated with a more efficient adaptive mechanism that assures more T3 in the beginning and normalization at the end of lactation. Acknowledgments This work was supported by a grant from Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and by funds from Po´s-Graduac¸a˜o em Biologia (PGBN-UERJ). References [1] Oberkotter LV, Rasmussen KM. Changes in plasma thyroid hormone concentration in chronically foodrestricted female rats and their offspring during suckling. J Nutr 1992;122:435– 41. [2] Hugues JN, Enjalbert A, Burger AG, Voirol MJ, Sebaoun J, Epelbaum J. Sensitivity of thyrotropin (TSH) secretion to 3,5,3⬘-triiodothyronine and TSH-releasing hormone in rat during starvation. Endocrinology 1986;119:253–60. [3] Rodriguez F, Mellado M, Montoya E, Jolin T. Sensitivity of thyrotropin secretion to TSH-releasing hormone in food-restricted rats. Acta Endocrinol (Copenh) 1991;124:194 –202. [4] Spencer CA, Lum SMC, Wilber JF, et al. Dynamics of serum thyrotropin and thyroid hormone changes in fasting. J Clin Endocrinol Metab 1983;56:883–7. [5] LoPresti JS, Gray D, Nicoloff JT. Influence of fasting and refeeding on 3,3,5⬘-triiodothyronine metabolism in man. J Clin Endocrinol Metab 1991;72:130 – 4. [6] Tulp OL, Krupp PP, Danforth EJ, Horton ES. Characteristics of thyroid function in experimental protein malnutrition. J Nutr 1979;109:1321–32. [7] Sawaya AL, Lunn PG. Evidence suggesting that the elevated plasma triiodothyronine concentration of rats fed on protein deficient diets is physiologically active. Br J Nutr 1985;53:175– 81.
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