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Developmental changes in the hypothalamic mRNA expression levels of brain-derived neurotrophic factor and serum leptin levels: Their responses to fasting in male and female rats
Int. J. Devl Neuroscience 54 (2016) 1–5
Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Developmental changes in the hypothalamic mRNA expression levels of brain-derived neurotrophic factor and serum leptin levels: Their responses to fasting in male and female rats Takeshi Iwasa ∗ , Toshiya Matsuzaki, Kiyohito Yano, Munkhsaikhan Munkhzaya, Altankhuu Tungalagsuvd, Maira Yiliyasi, Akira Kuwahara, Minoru Irahara Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-Cho, Tokushima 770-8503, Japan
a r t i c l e
i n f o
Article history: Received 26 May 2016 Received in revised form 4 August 2016 Accepted 8 August 2016 Available online 9 August 2016 Keywords: BDNF Leptin Hypothalamus Development
a b s t r a c t The actions and responses of hypothalamic appetite regulatory factors change markedly during the neonatal to pre-pubertal period in order to maintain appropriate metabolic and nutritional conditions. In this study, we examined the developmental changes in the hypothalamic mRNA levels of brain-derived neurotrophic factor (BDNF), which is a potent anorectic factor and the changes in the sensitivity of the hypothalamic expression of this factor to fasting during the neonatal to pre-pubertal period. Under fed conditions, hypothalamic BDNF mRNA expression decreased during development in both male and female rats. Similarly, the serum levels of leptin, which is a positive regulator of hypothalamic BDNF expression, also tended to fall during the developmental period. The serum leptin level and the hypothalamic BDNF mRNA level were found to be positively correlated in both sexes under the fed conditions. Hypothalamic BDNF mRNA expression was decreased by 24 h fasting (separating the rats from their mothers) in the early neonatal period (postnatal day 10) in both males and females, but no such changes were seen at postnatal day 20. Twenty-four hours’ fasting (food deprivation) did not affect hypothalamic BDNF mRNA expression in the pre-pubertal period (postnatal day 30). On the other hand, the rats’ serum leptin levels were decreased by 24 h fasting (separating the rats from their mothers at postnatal day 10 and 20, and food deprivation at postnatal day 30) throughout the early neonatal to pre-pubertal period. The correlation between serum leptin and hypothalamic BDNF mRNA levels was not significant under the fasted conditions. It can be speculated that leptin partially regulates hypothalamic BDNF mRNA levels, but only in fed conditions. Such changes in hypothalamic BDNF expression might play a role in maintaining appropriate metabolic and nutritional conditions and promoting normal physical development. In addition, because maternal separation induces a negative energy balance and short- and long-term stress responses, it is also possible that reductions in hypothalamic BDNF mRNA levels in the early neonatal period (postnatal day 10) may be partially induced by stress responses of the maternal deprivation. © 2016 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction The actions and responses of hypothalamic appetite regulatory factors change markedly during the neonatal to pre-pubertal period in order to maintain appropriate metabolic and nutritional conditions. We have shown that the basal expression levels of some hypothalamic orexigenic factors and their responses to fasting change during the neonatal to pre-pubertal period (Iwasa et al., 2015, 2016). On the other hand, as far as we know, there are only
∗ Corresponding author. E-mail address: [email protected] (T. Iwasa). http://dx.doi.org/10.1016/j.ijdevneu.2016.08.001 0736-5748/© 2016 ISDN. Published by Elsevier Ltd. All rights reserved.
limited data about the developmental changes in the expression of hypothalamic anorexigenic factors. Brain-derived neurotrophic factor (BDNF), which is a neurotrophin, is widely expressed in the central nervous system (Lewin and Brade, 1996) and is known to be involved in the development and maintenance of the nervous system (Huang and Reichardt, 2001). Recently, it has been shown that hypothalamic BDNF plays important roles in the regulation of appetite and energy metabolism. BDNF mRNA is expressed at high levels in the ventromedial nucleus of the hypothalamus (VMH) and paraventricular nucleus (PVN), areas that are associated with appetite and metabolism (Wang et al., 2007a,b; Xu and Xie, 2016). The chronic intracerebroventricular infusion of BDNF reduced the appetite and
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T. Iwasa et al. / Int. J. Devl Neuroscience 54 (2016) 1–5
body weight of adult rats (Lapchak and Hefti, 1992), and the direct injection of BDNF into the VMH and PVN reduced food intake and increased energy expenditure (Wang et al., 2007a,b; Xu and Xie, 2016). In addition, 24 h food deprivation reduced the VMH BDNF mRNA levels of rats (Liu et al., 2014), and the injection of leptin, which is a potent anorectic factor derived from adipose tissue, increased the VMH BDNF mRNA levels of mice (Komori et al., 2006). These results suggest that hypothalamic BDNF is a potent anorectic factor and that the BDNF mRNA level might be partially regulated by leptin. Human studies have also indicated that the BDNF pathway plays a pivotal role in the energy regulation system (Cordeira and Rios, 2011). We speculate that, as has been found for other factors, the basal hypothalamic expression levels of BDNF and their responses to fasting change during development because the serum leptin level varies markedly during this period (Iwasa et al., 2014). In the present study, the developmental changes in the hypothalamic BDNF mRNA level and the changes in their sensitivity to fasting during the neonatal to pre-pubertal period were examined. In addition, the relationship between hypothalamic BDNF mRNA and serum leptin levels were evaluated under fed and fasted conditions. 2. Materials and methods 2.1. Animals Pregnant Sprague-Dawley rats were purchased (Charles River Japan Inc., Tokyo, Japan) and housed under controlled lighting (14 h light, 10 h dark) and temperature (24 ◦ C) conditions. The day on which the pups were born was defined as postnatal day 1. Twelve pups were randomly assigned to each dam on postnatal day 2 and weaned on postnatal day 21. Rats of both sexes were randomly selected from each dam on postnatal days 10 and 20 and divided into the fed and fasting groups (n = 7–8 per group) (n = 7 for the postnatal day 10 fed males, n = 8 in the other groups). Specifically, 3 or 4 rats of each sex were selected from each dam and randomly assigned to the fed and fasting groups on postnatal days 10 and 20. To control litter size to 10–12 per dam, pups were moved to other dams and were fostered until weaning. After weaning, pups of the same sex were housed at 3–4 per cage and were randomly assigned to the fed and fasting groups on postnatal day 30. The fasting groups were subjected to 24 h maternal (postnatal days 10 and 20) or food deprivation (postnatal day 30). It has been reported that 24 h maternal deprivation in the neonatal period induces body weight reduction and increases stress responses, e.g., it activates the hypothalamic-pituitary-adrenal axis (Levine et al., 1991; Smith et al., 1997; Schmidt et al., 2006). The experimental design is summarized in Fig. 1. All animal experiments were conducted in accordance with the ethical standards of the animal care and use committee of the University of Tokushima. 2.2. Hormone assay and quantitative real-time polymerase chain reaction Serum leptin levels were measured using a 125Iradioimmunoassay (RIA) kit (multi-species leptin RIA kit, Linco Research Inc., MO, USA). Whole hypothalamic explants were dissected from the frozen brains, as described previously (Iwasa et al., 2015, 2016; Munkhzaya et al., 2015). Total RNA was isolated using a TRIzol® reagent kit (Invitrogen Co., Carlsbad, CA, USA) and an RNeasy mini kit® (Qiagen Gmbh, Hilden, Germany). Five g of total RNA were used for the cDNA synthesis. cDNA was synthesized with oligo (deoxythymidine) primers at 50 ◦ C using the SuperScript III first-strand synthesis system for the real-time polymerase chain reaction (PCR; Invitrogen Co.). The PCR analysis
Day10 fed (n = 7) 24h maternal deprivation (n = 8)
Male
Day20 Fed (n = 8) 24h maternal deprivation (n = 8) Day30 Fed (n = 8) 24h food deprivation (n = 8)
Birth Day10 fed (n = 7) 24h maternal deprivation (n = 8)
Female
Day20 Fed (n = 8) 24h maternal deprivation (n = 8) Day30 Fed (n = 8) 24h food deprivation (n = 8)
Fig. 1. Experimental design of this study.
was performed using the StepOnePlusTM real-time PCR system (PE Applied Biosystems, Foster City, CA, USA). Standard curves, which were generated by serially diluting an abundant sample at least 4 times, were used for the relative quantification of each mRNA expression level. The mRNA expression levels of BDNF were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The following forward and reverse primers were used: BDNF: F: 5 - AGC TGA GCG TGT GTG ACA GT −3 , R: 5 - ACC CAT GGG ATT ACA CTT GG −3 ; GAPDH: F: 5 - ATG GCA CAG TCA AGG CTG AGA −3 , R: 5 - CGC TCC TGG AAG ATG GTG AT- 3 . The PCR cycling conditions were as follows: initial denaturation and enzyme activation at 95 ◦ C for 10 min, followed by 45 cycles of denaturation at 95 ◦ C for 15 s, annealing at 63 ◦ C for 30 s (BDNF) or 65 ◦ C for 30 s (GAPDH), and extension at 72 ◦ C for 1 min. GAPDH mRNA expression was not affected by development or fasting in either sex. In addition, its expression did not differ between the sexes at any of the examined ages. 2.3. Statistical analyses All data are expressed as mean ± SEM values. The statistical analyses were performed using one-way or two-way analysis of variance (ANOVA) together with the Tukey-Kramer post-hoc test or the Student’s t test. Correlation analyses were performed using Pearson’s (when the data were normally distributed) or Spearman’s (when the data were not normally distributed) correlation coefficient as appropriate. Statistical significance was defined as P < 0.05. 3. Results Under the fed conditions, the hypothalamic BDNF mRNA level differed significantly among the examined age groups in both the male (one-way ANOVA; F(2,23) = 77.8, P < 0.01) and female (oneway ANOVA; F(2,23) = 7.05, P < 0.01) rats (Fig. 2A). Hypothalamic BDNF mRNA expression fell with age in both sexes. However, under the fed conditions the patterns of change in hypothalamic BDNF mRNA expression seen during development differed between males and females (two-way ANOVA; F(2,46) = 19.4, P < 0.01). The hypothalamic BDNF mRNA levels of the male rats were significantly higher than those of the females on postnatal days 10 and 20, but not on postnatal day 30 (Student’s t test) (Fig. 2A). The patterns of change in hypothalamic BDNF mRNA expression seen during development differed between the fed and 24 h fasted conditions in both males (two-way ANOVA; F(2,46) = 5.93, P < 0.05) and
T. Iwasa et al. / Int. J. Devl Neuroscience 54 (2016) 1–5
** BDNF mRNA/GAPDH (relative fold)
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Fig. 2. Developmental changes in hypothalamic BDNF mRNA expression (A) and the serum leptin level (B) seen in male and female rats under fed and fasted conditions. The mRNA levels of BDNF were normalized to the mRNA levels of GAPDH, and the value observed on day 10 in the male rats under the fed conditions was defined as 1.0. Data are expressed as mean ± SEM values. Different letters (a-c) indicate significant differences (P < 0.05) within the same sex under the fed conditions (Tukey-Kramer post-hoc test). * P < 0.05, ** P < 0.01 between the fed and fasted groups at the same postnatal age (Student’s t test); # P < 0.05 compared with the value for the fed females at the same postnatal age (Student’s t test).
females (two-way ANOVA; F(2,46) = 8.57, P < 0.01). In both sexes, the hypothalamic BDNF mRNA levels of the 24 h fasted groups were significantly lower than those of the fed groups on postnatal day 10 (Fig. 2A). The hypothalamic BDNF mRNA levels did not differ between fed and 24 h fasted groups on postnatal day 30 (P = 0.08 in males and P = 0.05 in females) (Student’s t test). On the other hand, the hypothalamic BDNF mRNA levels of the fed and 24 h fasted rats did not differ on postnatal day 20 in either sex. Under the fed conditions, significant differences in the serum leptin level were seen among the examined age groups in both the male (one-way ANOVA; F(2,23) = 22.4, P < 0.01) and female (oneway ANOVA; F(2,23) = 13.4, P < 0.01) rats (Fig. 2B). The serum leptin level fell with age in both the male and female rats. In both sexes, the serum leptin levels of the 24 h fasted groups were significantly lower than those of the fed groups at all examined ages (Student’s t test) (Fig. 2B). The patterns of change in the serum leptin level did not differ between males and females (two-way ANOVA; F(2,46) = 3.34, P = 0.07). Under the fed conditions, the hypothalamic BDNF mRNA level and the serum leptin level were found to be positively correlated in both the male and female rats (Fig. 3A, B). On the other hand, no such correlation was observed in either sex under the 24 h fasted conditions (Fig. 3C, D). 4. Discussion In this study, we showed that under the fed conditions hypothalamic BDNF mRNA expression decreased during development in both male and female rats. Similarly, the serum leptin level also tended to fall during the developmental period. In addition, significant positive correlations were detected between the hypothalamic BDNF mRNA level and the serum leptin level under the fed conditions. As it has been reported that leptin acts as a positive regulator
of hypothalamic BDNF expression in rodents (Komori et al., 2006), we speculate that the developmental changes in hypothalamic BDNF mRNA levels observed in this study might have been partially induced by alterations in the serum leptin level. The patterns of change in hypothalamic BDNF mRNA expression and the serum leptin level seen under the fed conditions differed somewhat; i.e., the serum leptin level, but not the hypothalamic BDNF mRNA level, was slightly higher at postnatal day 30 than at postnatal day 20. As hypothalamic BDNF mRNA levels are also regulated by other factors, such as glucocorticoids (Jeanneteau et al., 2012), it is possible that these factors also affect the developmental changes in hypothalamic BDNF mRNA expression. We assume that the changes in hypothalamic BDNF levels that occur during the neonatal to prepubertal period contribute to appropriate appetite regulation and nutritional conditions and promote normal physical development during this period. In the present study, the male rats exhibited significantly higher hypothalamic BDNF mRNA levels on postnatal days 10 and 20 than the female rats. As the serum leptin levels of the male rats also tended to be higher than those of the females at these ages, it is possible that the higher serum leptin levels upregulated hypothalamic BDNF expression in the male rats. Another possibility is that differences in the gonadal steroid milieu affect such sex-dependent differences; however, as far as we know, there are no data about the relationships between gonadal steroids and hypothalamic BDNF mRNA expression. Further studies are needed to investigate these hypotheses. As noted above, it has been shown that food deprivation decreases hypothalamic BDNF mRNA expression in adult rats (Liu et al., 2014). In this study, hypothalamic BDNF mRNA expression was significantly reduced or tended to be reduced by 24 h maternal/food deprivation on postnatal days 10 and 30, respectively, in both male and female rats. On the other hand, hypothalamic BDNF mRNA expression was not affected by food deprivation on postna-
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T. Iwasa et al. / Int. J. Devl Neuroscience 54 (2016) 1–5
Fig. 3. Correlations between the hypothalamic BDNF mRNA levels and serum leptin levels of male or female rats under the fed (Spearman’s correlation coefficient) or fasted conditions (Pearson’s correlation coefficient). The mRNA levels of BDNF were normalized to the mRNA levels of GAPDH, and the value observed on day 10 in the male rats under the fed conditions was defined as 1.0. Regression lines are only shown for statistically significant correlations.
tal day 20 in either sex. These results indicate that the sensitivity of hypothalamic BDNF expression to fasting might already be established in the early neonatal period; i.e., postnatal day 10, but that it might be temporarily suppressed around postnatal day 20. However, we could not identify the reason for such suppression in the present study. In addition, no correlation between the serum leptin level and the hypothalamic BDNF mRNA level was detected under the fasted conditions, and the fasting-induced changes in hypothalamic BDNF mRNA expression and the serum leptin level differed somewhat. We assume that there might be a threshold leptin level for regulating hypothalamic BDNF mRNA expression; i.e., above the threshold leptin positively regulates hypothalamic BDNF mRNA levels, whereas below it leptin has no effect on them. Furthermore, other factors might be the major contributors to the fasting-induced changes in hypothalamic BDNF mRNA levels. In summary, we showed that under fed conditions hypothalamic BDNF mRNA expression decreased during development in both male and female rats, and that there were some sex-dependent differences in hypothalamic BDNF mRNA expression. Leptin might contribute to such changes in hypothalamic BDNF mRNA expression. In addition, the sensitivity of hypothalamic BDNF mRNA expression to fasting might already be established in the early neonatal period, but then temporarily suppressed during development. We speculate that such changes in hypothalamic BDNF expression might help to maintain appropriate metabolic and nutritional conditions and promote normal physical development. Another possibility is that, as noted above, because maternal separation induces a negative energy balance as well as short- and
long-term stress responses, such as activation of the HPA axis (Levine et al., 1991; Smith et al., 1997; Schmidt et al., 2006), it is possible that the reductions in hypothalamic BDNF mRNA expression seen in the early neonatal period (postnatal day 10) in this study might also contribute to stress responses.
References Cordeira, J., Rios, M., 2011. Weighing in the role of BDNF in the central control of eating behavior. Mol. Neurobiol. 44, 441–448. Huang, E.J., Reichardt, L.F., 2001. Neurotrophins; roles in neuronal development and function. Annu. Rev. Neurosci. 24, 677–736. Iwasa, T., Matsuzaki, T., Munkhzaya, M., Tungalagsuvd, A., Kawami, T., Murakami, M., Yamasaki, M., Kato, T., Kuwahara, A., 2014. Pre-pubertal serum leptin levels and sensitivity to central leptin injection of prenatally undernourished female rats. Int. J. Dev. Neurosci. 35, 52–54. Iwasa, T., Matsuzaki, T., Munkhzaya, M., Tungalagsuvd, A., Kuwahara, A., Yasui, T., Irahara, M., 2015. Developmental changes in the hypothalamic mRNA levels of prepro-orexin and orexin receptors and their sensitivities to fasting in male and female rats. Int. J. Dev. Neurosci. 46, 51–54. Iwasa, T., Matsuzaki, T., Tungalagsuvd, A., Munkhzaya, M., Yiliyasi, M., Kuwahara, A., Irahara, M., 2016. Developmental changes in the hypothalamic mRNA levels of nucleobindin-2 (NUCB2) and their sensitivity to fasting in male and female rats. Int. J. Dev. Neurosci. 49, 46–49. Jeanneteau, F.D., Lambert, W.M., Ismaili, N., Bath, K.G., Lee, F.S., Garabedian, M.J., Chao, M.V., 2012. BDNF and glucocorticoids regulate corticotrophin-releasing hormone (CRH) homeostasis in the hypothalamus. Proc. Natl. Acad. Sci. U. S. A. 109, 1305–1310. Komori, T., Morikawa, Y., Nanjo, K., Senba, E., 2006. Induction of brain-derived neurotrophic factor by leptin in the ventromedial hypothalamus. Neuroscience 139, 1107–1115. Lapchak, P.A., Hefti, F., 1992. BDNF and NGF treatment in lesioned rats: effects on cholinergic function and weight gain. Neuroreport 3, 405–408.
T. Iwasa et al. / Int. J. Devl Neuroscience 54 (2016) 1–5 Levine, S., Huchton, D.M., Wiener, S.G., Rosenfeld, P., 1991. Time course of the effect of maternal deprivation on the hypothalamic—pituitary—adrenal axis in the infant rat. Dev. Psychobiol. 24, 547–548. Lewin, G.R., Brade, Y.A., 1996. Physiology of the neurotrophins. Annu. Rev. Neurosci. 19, 289–317. Liu, X., Zhu, Z., Kalyani, M., Janik, J.M., Shi, H., 2014. Effects of energy status and diet on Bdnf expression in the ventromedial hypothalamus of male and female rats. Physiol. Behav. 130, 99–107. Munkhzaya, M., Matsuzaki, T., Iwasa, T., Tungalagsuvd, A., Kawami, T., Kato, T., Kuwahara, A., Irahara, M., 2015. The suppressive effect of immune stress on LH secretion is absent in the early neonatal period in rats. Int. J. Dev. Neurosci. 46, 38–43. Schmidt, M.V., Levine, S., Alam, S., Harbich, D., Sterlemann, V., Ganea, K., de Kloet, E.R., Holsboer, F., Muller, M.B., 2006. Metabolic signals modulate
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hypothalamic—pituitary—adrenal axis activation during maternal separation of the neonatal mouse. J. Neuroendocrinol. 18, 865–874. Smith, M.A., Kim, S.Y., van Oers, H.J., Levine, S., 1997. Maternal deprivation and stress induce immediate early genes in the infant rat brain. Endocrinology 138, 4622–4628. Wang, C.F., Bomberg, E., Billington, C., Levine, A., Kotz, C.M., 2007a. Brain-derived neurotrophic factor in the hypothalamic paraventricular nucleus reduces energy intake. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1003–R1012. Wang, C.F., Bomberg, E., Levine, A., Billington, C., Kotz, C.M., 2007b. Brain-derived neurotrophic factor in the ventromedial nucleus of the hypothalamus reduces energy intake. Am. J. Physiol. Regul. Integr. Comp. Physiol. 293, R1037–R1045. Xu, B., Xie, X., 2016. Neurotrophic factor control of satiety and body weight. Nat. Rev. Neurosci. 17, 282–292.
Contents lists available at ScienceDirect
International Journal of Developmental Neuroscience journal homepage: www.elsevier.com/locate/ijdevneu
Developmental changes in the hypothalamic mRNA expression levels of brain-derived neurotrophic factor and serum leptin levels: Their responses to fasting in male and female rats Takeshi Iwasa ∗ , Toshiya Matsuzaki, Kiyohito Yano, Munkhsaikhan Munkhzaya, Altankhuu Tungalagsuvd, Maira Yiliyasi, Akira Kuwahara, Minoru Irahara Department of Obstetrics and Gynecology, Institute of Biomedical Sciences, Tokushima University Graduate School, 3-18-15 Kuramoto-Cho, Tokushima 770-8503, Japan
a r t i c l e
i n f o
Article history: Received 26 May 2016 Received in revised form 4 August 2016 Accepted 8 August 2016 Available online 9 August 2016 Keywords: BDNF Leptin Hypothalamus Development
a b s t r a c t The actions and responses of hypothalamic appetite regulatory factors change markedly during the neonatal to pre-pubertal period in order to maintain appropriate metabolic and nutritional conditions. In this study, we examined the developmental changes in the hypothalamic mRNA levels of brain-derived neurotrophic factor (BDNF), which is a potent anorectic factor and the changes in the sensitivity of the hypothalamic expression of this factor to fasting during the neonatal to pre-pubertal period. Under fed conditions, hypothalamic BDNF mRNA expression decreased during development in both male and female rats. Similarly, the serum levels of leptin, which is a positive regulator of hypothalamic BDNF expression, also tended to fall during the developmental period. The serum leptin level and the hypothalamic BDNF mRNA level were found to be positively correlated in both sexes under the fed conditions. Hypothalamic BDNF mRNA expression was decreased by 24 h fasting (separating the rats from their mothers) in the early neonatal period (postnatal day 10) in both males and females, but no such changes were seen at postnatal day 20. Twenty-four hours’ fasting (food deprivation) did not affect hypothalamic BDNF mRNA expression in the pre-pubertal period (postnatal day 30). On the other hand, the rats’ serum leptin levels were decreased by 24 h fasting (separating the rats from their mothers at postnatal day 10 and 20, and food deprivation at postnatal day 30) throughout the early neonatal to pre-pubertal period. The correlation between serum leptin and hypothalamic BDNF mRNA levels was not significant under the fasted conditions. It can be speculated that leptin partially regulates hypothalamic BDNF mRNA levels, but only in fed conditions. Such changes in hypothalamic BDNF expression might play a role in maintaining appropriate metabolic and nutritional conditions and promoting normal physical development. In addition, because maternal separation induces a negative energy balance and short- and long-term stress responses, it is also possible that reductions in hypothalamic BDNF mRNA levels in the early neonatal period (postnatal day 10) may be partially induced by stress responses of the maternal deprivation. © 2016 ISDN. Published by Elsevier Ltd. All rights reserved.
1. Introduction The actions and responses of hypothalamic appetite regulatory factors change markedly during the neonatal to pre-pubertal period in order to maintain appropriate metabolic and nutritional conditions. We have shown that the basal expression levels of some hypothalamic orexigenic factors and their responses to fasting change during the neonatal to pre-pubertal period (Iwasa et al., 2015, 2016). On the other hand, as far as we know, there are only
∗ Corresponding author. E-mail address: [email protected] (T. Iwasa). http://dx.doi.org/10.1016/j.ijdevneu.2016.08.001 0736-5748/© 2016 ISDN. Published by Elsevier Ltd. All rights reserved.
limited data about the developmental changes in the expression of hypothalamic anorexigenic factors. Brain-derived neurotrophic factor (BDNF), which is a neurotrophin, is widely expressed in the central nervous system (Lewin and Brade, 1996) and is known to be involved in the development and maintenance of the nervous system (Huang and Reichardt, 2001). Recently, it has been shown that hypothalamic BDNF plays important roles in the regulation of appetite and energy metabolism. BDNF mRNA is expressed at high levels in the ventromedial nucleus of the hypothalamus (VMH) and paraventricular nucleus (PVN), areas that are associated with appetite and metabolism (Wang et al., 2007a,b; Xu and Xie, 2016). The chronic intracerebroventricular infusion of BDNF reduced the appetite and
2
T. Iwasa et al. / Int. J. Devl Neuroscience 54 (2016) 1–5
body weight of adult rats (Lapchak and Hefti, 1992), and the direct injection of BDNF into the VMH and PVN reduced food intake and increased energy expenditure (Wang et al., 2007a,b; Xu and Xie, 2016). In addition, 24 h food deprivation reduced the VMH BDNF mRNA levels of rats (Liu et al., 2014), and the injection of leptin, which is a potent anorectic factor derived from adipose tissue, increased the VMH BDNF mRNA levels of mice (Komori et al., 2006). These results suggest that hypothalamic BDNF is a potent anorectic factor and that the BDNF mRNA level might be partially regulated by leptin. Human studies have also indicated that the BDNF pathway plays a pivotal role in the energy regulation system (Cordeira and Rios, 2011). We speculate that, as has been found for other factors, the basal hypothalamic expression levels of BDNF and their responses to fasting change during development because the serum leptin level varies markedly during this period (Iwasa et al., 2014). In the present study, the developmental changes in the hypothalamic BDNF mRNA level and the changes in their sensitivity to fasting during the neonatal to pre-pubertal period were examined. In addition, the relationship between hypothalamic BDNF mRNA and serum leptin levels were evaluated under fed and fasted conditions. 2. Materials and methods 2.1. Animals Pregnant Sprague-Dawley rats were purchased (Charles River Japan Inc., Tokyo, Japan) and housed under controlled lighting (14 h light, 10 h dark) and temperature (24 ◦ C) conditions. The day on which the pups were born was defined as postnatal day 1. Twelve pups were randomly assigned to each dam on postnatal day 2 and weaned on postnatal day 21. Rats of both sexes were randomly selected from each dam on postnatal days 10 and 20 and divided into the fed and fasting groups (n = 7–8 per group) (n = 7 for the postnatal day 10 fed males, n = 8 in the other groups). Specifically, 3 or 4 rats of each sex were selected from each dam and randomly assigned to the fed and fasting groups on postnatal days 10 and 20. To control litter size to 10–12 per dam, pups were moved to other dams and were fostered until weaning. After weaning, pups of the same sex were housed at 3–4 per cage and were randomly assigned to the fed and fasting groups on postnatal day 30. The fasting groups were subjected to 24 h maternal (postnatal days 10 and 20) or food deprivation (postnatal day 30). It has been reported that 24 h maternal deprivation in the neonatal period induces body weight reduction and increases stress responses, e.g., it activates the hypothalamic-pituitary-adrenal axis (Levine et al., 1991; Smith et al., 1997; Schmidt et al., 2006). The experimental design is summarized in Fig. 1. All animal experiments were conducted in accordance with the ethical standards of the animal care and use committee of the University of Tokushima. 2.2. Hormone assay and quantitative real-time polymerase chain reaction Serum leptin levels were measured using a 125Iradioimmunoassay (RIA) kit (multi-species leptin RIA kit, Linco Research Inc., MO, USA). Whole hypothalamic explants were dissected from the frozen brains, as described previously (Iwasa et al., 2015, 2016; Munkhzaya et al., 2015). Total RNA was isolated using a TRIzol® reagent kit (Invitrogen Co., Carlsbad, CA, USA) and an RNeasy mini kit® (Qiagen Gmbh, Hilden, Germany). Five g of total RNA were used for the cDNA synthesis. cDNA was synthesized with oligo (deoxythymidine) primers at 50 ◦ C using the SuperScript III first-strand synthesis system for the real-time polymerase chain reaction (PCR; Invitrogen Co.). The PCR analysis
Day10 fed (n = 7) 24h maternal deprivation (n = 8)
Male
Day20 Fed (n = 8) 24h maternal deprivation (n = 8) Day30 Fed (n = 8) 24h food deprivation (n = 8)
Birth Day10 fed (n = 7) 24h maternal deprivation (n = 8)
Female
Day20 Fed (n = 8) 24h maternal deprivation (n = 8) Day30 Fed (n = 8) 24h food deprivation (n = 8)
Fig. 1. Experimental design of this study.
was performed using the StepOnePlusTM real-time PCR system (PE Applied Biosystems, Foster City, CA, USA). Standard curves, which were generated by serially diluting an abundant sample at least 4 times, were used for the relative quantification of each mRNA expression level. The mRNA expression levels of BDNF were normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The following forward and reverse primers were used: BDNF: F: 5 - AGC TGA GCG TGT GTG ACA GT −3 , R: 5 - ACC CAT GGG ATT ACA CTT GG −3 ; GAPDH: F: 5 - ATG GCA CAG TCA AGG CTG AGA −3 , R: 5 - CGC TCC TGG AAG ATG GTG AT- 3 . The PCR cycling conditions were as follows: initial denaturation and enzyme activation at 95 ◦ C for 10 min, followed by 45 cycles of denaturation at 95 ◦ C for 15 s, annealing at 63 ◦ C for 30 s (BDNF) or 65 ◦ C for 30 s (GAPDH), and extension at 72 ◦ C for 1 min. GAPDH mRNA expression was not affected by development or fasting in either sex. In addition, its expression did not differ between the sexes at any of the examined ages. 2.3. Statistical analyses All data are expressed as mean ± SEM values. The statistical analyses were performed using one-way or two-way analysis of variance (ANOVA) together with the Tukey-Kramer post-hoc test or the Student’s t test. Correlation analyses were performed using Pearson’s (when the data were normally distributed) or Spearman’s (when the data were not normally distributed) correlation coefficient as appropriate. Statistical significance was defined as P < 0.05. 3. Results Under the fed conditions, the hypothalamic BDNF mRNA level differed significantly among the examined age groups in both the male (one-way ANOVA; F(2,23) = 77.8, P < 0.01) and female (oneway ANOVA; F(2,23) = 7.05, P < 0.01) rats (Fig. 2A). Hypothalamic BDNF mRNA expression fell with age in both sexes. However, under the fed conditions the patterns of change in hypothalamic BDNF mRNA expression seen during development differed between males and females (two-way ANOVA; F(2,46) = 19.4, P < 0.01). The hypothalamic BDNF mRNA levels of the male rats were significantly higher than those of the females on postnatal days 10 and 20, but not on postnatal day 30 (Student’s t test) (Fig. 2A). The patterns of change in hypothalamic BDNF mRNA expression seen during development differed between the fed and 24 h fasted conditions in both males (two-way ANOVA; F(2,46) = 5.93, P < 0.05) and
T. Iwasa et al. / Int. J. Devl Neuroscience 54 (2016) 1–5
** BDNF mRNA/GAPDH (relative fold)
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Fig. 2. Developmental changes in hypothalamic BDNF mRNA expression (A) and the serum leptin level (B) seen in male and female rats under fed and fasted conditions. The mRNA levels of BDNF were normalized to the mRNA levels of GAPDH, and the value observed on day 10 in the male rats under the fed conditions was defined as 1.0. Data are expressed as mean ± SEM values. Different letters (a-c) indicate significant differences (P < 0.05) within the same sex under the fed conditions (Tukey-Kramer post-hoc test). * P < 0.05, ** P < 0.01 between the fed and fasted groups at the same postnatal age (Student’s t test); # P < 0.05 compared with the value for the fed females at the same postnatal age (Student’s t test).
females (two-way ANOVA; F(2,46) = 8.57, P < 0.01). In both sexes, the hypothalamic BDNF mRNA levels of the 24 h fasted groups were significantly lower than those of the fed groups on postnatal day 10 (Fig. 2A). The hypothalamic BDNF mRNA levels did not differ between fed and 24 h fasted groups on postnatal day 30 (P = 0.08 in males and P = 0.05 in females) (Student’s t test). On the other hand, the hypothalamic BDNF mRNA levels of the fed and 24 h fasted rats did not differ on postnatal day 20 in either sex. Under the fed conditions, significant differences in the serum leptin level were seen among the examined age groups in both the male (one-way ANOVA; F(2,23) = 22.4, P < 0.01) and female (oneway ANOVA; F(2,23) = 13.4, P < 0.01) rats (Fig. 2B). The serum leptin level fell with age in both the male and female rats. In both sexes, the serum leptin levels of the 24 h fasted groups were significantly lower than those of the fed groups at all examined ages (Student’s t test) (Fig. 2B). The patterns of change in the serum leptin level did not differ between males and females (two-way ANOVA; F(2,46) = 3.34, P = 0.07). Under the fed conditions, the hypothalamic BDNF mRNA level and the serum leptin level were found to be positively correlated in both the male and female rats (Fig. 3A, B). On the other hand, no such correlation was observed in either sex under the 24 h fasted conditions (Fig. 3C, D). 4. Discussion In this study, we showed that under the fed conditions hypothalamic BDNF mRNA expression decreased during development in both male and female rats. Similarly, the serum leptin level also tended to fall during the developmental period. In addition, significant positive correlations were detected between the hypothalamic BDNF mRNA level and the serum leptin level under the fed conditions. As it has been reported that leptin acts as a positive regulator
of hypothalamic BDNF expression in rodents (Komori et al., 2006), we speculate that the developmental changes in hypothalamic BDNF mRNA levels observed in this study might have been partially induced by alterations in the serum leptin level. The patterns of change in hypothalamic BDNF mRNA expression and the serum leptin level seen under the fed conditions differed somewhat; i.e., the serum leptin level, but not the hypothalamic BDNF mRNA level, was slightly higher at postnatal day 30 than at postnatal day 20. As hypothalamic BDNF mRNA levels are also regulated by other factors, such as glucocorticoids (Jeanneteau et al., 2012), it is possible that these factors also affect the developmental changes in hypothalamic BDNF mRNA expression. We assume that the changes in hypothalamic BDNF levels that occur during the neonatal to prepubertal period contribute to appropriate appetite regulation and nutritional conditions and promote normal physical development during this period. In the present study, the male rats exhibited significantly higher hypothalamic BDNF mRNA levels on postnatal days 10 and 20 than the female rats. As the serum leptin levels of the male rats also tended to be higher than those of the females at these ages, it is possible that the higher serum leptin levels upregulated hypothalamic BDNF expression in the male rats. Another possibility is that differences in the gonadal steroid milieu affect such sex-dependent differences; however, as far as we know, there are no data about the relationships between gonadal steroids and hypothalamic BDNF mRNA expression. Further studies are needed to investigate these hypotheses. As noted above, it has been shown that food deprivation decreases hypothalamic BDNF mRNA expression in adult rats (Liu et al., 2014). In this study, hypothalamic BDNF mRNA expression was significantly reduced or tended to be reduced by 24 h maternal/food deprivation on postnatal days 10 and 30, respectively, in both male and female rats. On the other hand, hypothalamic BDNF mRNA expression was not affected by food deprivation on postna-
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Fig. 3. Correlations between the hypothalamic BDNF mRNA levels and serum leptin levels of male or female rats under the fed (Spearman’s correlation coefficient) or fasted conditions (Pearson’s correlation coefficient). The mRNA levels of BDNF were normalized to the mRNA levels of GAPDH, and the value observed on day 10 in the male rats under the fed conditions was defined as 1.0. Regression lines are only shown for statistically significant correlations.
tal day 20 in either sex. These results indicate that the sensitivity of hypothalamic BDNF expression to fasting might already be established in the early neonatal period; i.e., postnatal day 10, but that it might be temporarily suppressed around postnatal day 20. However, we could not identify the reason for such suppression in the present study. In addition, no correlation between the serum leptin level and the hypothalamic BDNF mRNA level was detected under the fasted conditions, and the fasting-induced changes in hypothalamic BDNF mRNA expression and the serum leptin level differed somewhat. We assume that there might be a threshold leptin level for regulating hypothalamic BDNF mRNA expression; i.e., above the threshold leptin positively regulates hypothalamic BDNF mRNA levels, whereas below it leptin has no effect on them. Furthermore, other factors might be the major contributors to the fasting-induced changes in hypothalamic BDNF mRNA levels. In summary, we showed that under fed conditions hypothalamic BDNF mRNA expression decreased during development in both male and female rats, and that there were some sex-dependent differences in hypothalamic BDNF mRNA expression. Leptin might contribute to such changes in hypothalamic BDNF mRNA expression. In addition, the sensitivity of hypothalamic BDNF mRNA expression to fasting might already be established in the early neonatal period, but then temporarily suppressed during development. We speculate that such changes in hypothalamic BDNF expression might help to maintain appropriate metabolic and nutritional conditions and promote normal physical development. Another possibility is that, as noted above, because maternal separation induces a negative energy balance as well as short- and
long-term stress responses, such as activation of the HPA axis (Levine et al., 1991; Smith et al., 1997; Schmidt et al., 2006), it is possible that the reductions in hypothalamic BDNF mRNA expression seen in the early neonatal period (postnatal day 10) in this study might also contribute to stress responses.
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