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Short communication
Changes in leptin production/secretion induced in response to septic doses of lipopolysaccharides in gonadally intact and ovariectomized female rats Takeshi Iwasa ∗ , Toshiya Matsuzaki, Munkhsaikhan Munkhzaya, Altankhuu Tungalagsuvd, Takako Kawami, Masahiro Murakami, Takeshi Kato, Akira Kuwahara, Toshiyuki Yasui, Minoru Irahara Department of Obstetrics and Gynecology, The University of Tokushima Graduate School, Institute of Health Biosciences, 3-18-15 Kuramoto-Cho, Tokushima 770-8503, Japan
a r t i c l e
i n f o
Article history: Received 1 February 2014 Received in revised form 8 March 2014 Accepted 14 March 2014
Keywords: Ovariectomy Leptin ObRb LPS
a b s t r a c t In addition to its role as a regulator of energy homeostasis, leptin plays a pivotal role in certain immune/inflammatory responses. It has been reported that the synthesis and secretion of leptin are increased during immune stress in male experimental animals, whereas the changes in leptin synthesis and secretion induced by immune stress in females have not been fully elucidated. In this study, we found that during lipopolysaccharide (LPS)-induced immune stress the synthesis and secretion of leptin were decreased in gonadally intact female rats, but increased in ovariectomized rats. However, the LPS-induced increase in the serum leptin level observed in the ovariectomized rats was partially attenuated by estradiol supplementation. These results suggest that the changes in leptin synthesis and secretion induced in response to immune stress in females are affected by the gonadal steroid milieu and that estradiol and other factors are involved in these alterations. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Leptin plays a crucial role as a regulator of appetite and energy expenditure. However, in addition to its role in energy homeostasis it also plays a pivotal role in immune/inflammatory responses (La Cava and Matarese, 2004). In male experimental animals, it was found that the administration of high doses of lipopolysaccharide (LPS) or proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), resulted in the increased synthesis and secretion of leptin (Mastronardi
∗ Corresponding author. Tel.: +81 88 633 7177. E-mail addresses: [email protected], [email protected] (T. Iwasa).
et al., 2001; Grunfeld et al., 1996). Increased serum leptin levels promote febrile and anorectic responses and exert a pro-survival effect under septic conditions (La Cava and Matarese, 2004). Although the immunological roles played by leptin under immune stress conditions have been established in males, they have not been fully elucidated in females. We have already reported that the serum leptin level is increased by LPS injection in pre-pubertal female rats (Iwasa et al., 2011). In addition, it has been reported that reduced leptin levels during starvation attenuate the sustained fever response to LPS in female rats (Inoue and Luheshi, 2010). These results indicate that leptin may be involved in immune/inflammatory responses in females, as well as in males. However, changes in leptin and its receptors under immune stress conditions at adulthood have not been evaluated in females. In this study, the effects
http://dx.doi.org/10.1016/j.jri.2014.03.003 0165-0378/© 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Iwasa, T., et al., Changes in leptin production/secretion induced in response to septic doses of lipopolysaccharides in gonadally intact and ovariectomized female rats. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.03.003
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of the administration of a high dose of LPS on the serum leptin level and the mRNA expression levels of the leptin (ob) and hypothalamic leptin receptor (ObRb) genes were examined in female rats. As the synthesis and secretion of leptin are affected by the gonadal steroid milieu (Meli et al., 2004), gonadally intact, ovariectomized, and estradiol-supplemented ovariectomized female rats were used for the present experiments. The serum IL-1 and TNF-␣ levels of the rats were also measured because these cytokines are involved in the upregulation of leptin expression.
TTG-3 ; R: 5 -GGT TCC CTG GGT GCT CTG A-3 ; 18s rRNA: F: 5 -GAC GGA CCA GAG CGA AAG C-3 ; R: 5 -AAC CTC CGA CTT TCG TTC TTG A-3 . The PCR conditions were as follows: initial denaturation and enzyme activation at 95 ◦ C for 20 s, followed by 45 cycles of denaturation at 95 ◦ C for 3 s and annealing at 63 ◦ C for 30 s (ObRb), 64 ◦ C for 30 s (18s rRNA), or 66 ◦ C for 30 s (ob), before a final extension step at 72 ◦ C for 1 min. Data analysis was performed using one-way (for intra-group comparisons) or two-way (for inter-group comparisons) analysis of variance (ANOVA), followed by Dunnett’s test or the Student’s t test (comparisons between time points).
2. Materials and methods 3. Results and discussion Sixty-one female Sprague–Dawley rats (Charles River Japan, Tokyo, Japan) were used in this study. They were housed in a room under controlled light (12 h light:12 h darkness; lights on at 0800 and off at 2000) and temperature (24 ◦ C) conditions and were allowed free access to commercial laboratory chow (standard diet; 3.6 kcal/g, 13.3% energy provided as fat; Oriental MF, Oriental Yeast Co, Ltd, Osaka, Japan). All animal experiments were conducted in accordance with the ethical standards of the institutional Animal Care and Use Committee of the University of Tokushima. All surgical procedures were carried out under sodium pentobarbital- (60–80 mg/kg; intraperitoneal, i.p.) or sevoflurane-induced anesthesia. In the first experiment, 24 rats were ovariectomized (OVX group) and 23 rats underwent sham surgery (sham group) at 10 weeks of age. After a 6-week recovery period, the rats in the sham and OVX groups were sub-divided into saline-injected and LPS-injected groups (0111:B4; Sigma, St. Louis, MO, USA; 5 mg/kg, i.p.). Blood samples, the whole brain, and visceral (parametrial) fat samples were collected at 6 h (in the saline and LPS-injected groups) or 24 h (only in the LPS-injected groups) post-injection. In the second experiment, 16 rats were ovariectomized at 10 weeks of age. After a 6-week recovery period, each rat was subcutaneously implanted with a Silastic tube (length, 20 mm; inner diameter, 1.5 mm; outer diameter, 2.0 mm) containing 20 g/ml of 17-estradiol (Sigma). The next day, they were subdivided into saline-injected and LPS-injected groups. Blood and tissue samples were collected at 24 h post-injection. In both experiments, hypothalamic explants were dissected from the rats’ whole brains as described previously (Iwasa et al., 2012), and the changes in the rats’ body weight and the total volume of fat were also measured. In addition, the rats’ serum levels of leptin, IL-1, and TNF-␣ were measured in duplicate using radioimmunoassays (rat leptin RIA lit or multi-species leptin RIA kit, Linco Research, St. Charles, MO, USA) or enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN, USA). Total RNA was isolated from the hypothalamus and parametrial fat samples and used to synthesize cDNA. Then, the expression levels of hypothalamic leptin ObRb mRNA and adipose ob mRNA were measured using real-time PCR analysis. The expression level of each gene was normalized to that of 18s rRNA. The forward and reverse primers used were as follows: ob (leptin): F: 5 -GGT CAC CGG TTT GGA CTT CAT-3 , R: 5 -CTG GTC CAT CTT GGA CAA ACT CA-3 ; ObRb (leptin receptor type b): F: 5 -GCA GCT ATG GTC TCA CTT CTT
In the first experiment, it was found that the OVX rats were heavier and displayed lower uterine weights than the sham rats (data not shown). In addition, the OVX rats possessed significantly more (by volume) visceral and subcutaneous fat than the sham rats (Fig. 1). The body weight changes induced by the injection of LPS did not differ between the OVX and sham groups. The serum IL-1 and TNF-␣ levels of saline-injected rats were undetectable with the ELISA kit. Similarly, the serum TNF-␣ levels in rats 24 h after LPS injection were undetectable. 24 h after the injection of LPS, the serum IL-1 levels of the sham rats were significantly higher than those of the OVX rats. Similarly, the serum TNF-␣ levels of the sham rats were significantly higher than those of the OVX rats 6 h after the injection of LPS. Conversely, the serum leptin levels of the LPS-injected sham rats were significantly lower than those of the saline-injected sham rats. In addition, after the injection of LPS the serum leptin levels of the sham rats were significantly lower than those of the OVX rats. Among the OVX rats, the visceral fat ob mRNA levels of the LPS-injected group were significantly higher than those of the saline-injected group. Also, the visceral fat ob mRNA levels of the OVX rats were significantly higher than those of the sham rats. In both groups, the hypothalamic ObRb mRNA levels of the LPS-injected rats were significantly lower than those of the saline-injected rats. In the second experiment (in which estradiol-supplemented rats were used), the serum leptin levels of the LPS-injected rats tended to be higher than those of the saline-injected rats (P = 0.08; Fig. 2), although the body weight of the LPS-injected rats was markedly reduced. On the other hand, ob mRNA expression was not affected by the administration of LPS in these rats. Serum leptin levels in the second experiment were lower than those in the first experiment. Differences in leptin measurement kits (rat-specific kit for the first experiment and the kit for multi-species for the second experiment) may induce these discrepancies. In the present study, we found that the serum leptin level and ob mRNA expression were decreased in gonadally intact female rats during LPS-induced immune stress, whereas LPS increased expression levels in OVX rats. The injection of LPS reduced hypothalamic ObRb mRNA expression in both the OVX and sham rats. We speculate that such changes in ObRb expression are an adaptive response aimed at attenuating leptin-induced anorexia
Please cite this article in press as: Iwasa, T., et al., Changes in leptin production/secretion induced in response to septic doses of lipopolysaccharides in gonadally intact and ovariectomized female rats. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.03.003
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Fig. 1. Fat volume (A), body weight (BW) change (B), the serum levels of cytokines and leptin (C–E), visceral fat ob mRNA levels (F), and hypothalamic ObRb mRNA levels (G) in the sham () and OVX () rats after the injection of saline or LPS. The serum IL-1 and TNF-␣ levels of saline-injected rats were undetectable. The serum TNF-␣ levels in rats 24 h after LPS injection were undetectable. The mRNA expression levels of ObRb and ob were normalized by dividing them by the expression level of 18s rRNA. Data are presented as mean + SEM values. *P < 0.05, **P < 0.01; Student’s t test. † P < 0.05, †† P < 0.01 vs. the saline-injected group; Dunnett’s test. UD: undetectable.
Fig. 2. Body weight (BW) change (A), serum leptin levels (B), visceral fat ob mRNA levels (C), and hypothalamic ObRb mRNA levels (D) in estradiolsupplemented ovariectomized rats at 24 h post-injection of saline or LPS. The ObRb and ob mRNA expression levels were normalized by dividing them by the expression level of 18s rRNA. Data are presented as mean + SEM values.
Please cite this article in press as: Iwasa, T., et al., Changes in leptin production/secretion induced in response to septic doses of lipopolysaccharides in gonadally intact and ovariectomized female rats. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.03.003
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under immune stress conditions. The serum levels of IL-1 and TNF-␣, which positively regulate leptin expression (Mastronardi et al., 2001; Grunfeld et al., 1996), were increased by LPS treatment in both the OVX and sham rats. However, IL-1 at 24 h after LPS injection and TNF-␣ at 6 h after LPS injection in OVX rats were significantly lower than those in sham rats. Therefore, such differences in the responses of cytokines may be responsible for the discrepancies between these groups with regard to the changes in the serum leptin level and ob mRNA expression induced by the LPS injection. Interestingly, the changes in the serum leptin level and the expression level of the ob gene observed in the OVX rats after the injection of LPS were similar to those reported for male rodents, while those observed in the sham rats were opposite to those detected in male rodents (Mastronardi et al., 2001; Grunfeld et al., 1996). As it has been reported that gonadal steroids are involved in regulating the leptin system under basal conditions (Inoue and Luheshi, 2010), we speculated that gonadal steroids might also affect the leptin system during immune stress conditions. Therefore, in the present study we examined the responses of the leptin system to LPS in estradiol-supplemented ovariectomized rats. As a result, we found that the LPS-induced increase in the serum leptin level was partially attenuated in these rats. We speculate that not only estradiol, but also other factors, for example, progesterone, testosterone, and obesity, are involved in the ovariectomy-induced changes in the response of the leptin system to immune stress. Actually, it has been reported that diet-induced obesity increases the leptin response to LPS in adult male rats (Pohl et al., 2009). Further experiments, focused on the ovariectomy-induced hormonal and physiological changes, are needed to clarify the mechanism by which leptin response is altered according to the gonadal steroid milieu in females. To the best of our knowledge, this is the first report to show the relationship between gonadal steroid milieu and leptin response to immune stress in females. The responses of the leptin system have been reported to play important pathological and physiological roles in immune stress conditions. It has been reported that leptin induces the anorectic and febrile responses via the upregulation of hypothalamic pro-inflammatory cytokines. For instance, co-administration of leptin antiserum attenuates the LPS-induced febrile responses in rats (Sachot et al., 2004). It has also been reported that rats with a low serum leptin level show high mortality under septic conditions and that the supplementation of leptin protects against
lethality in these individuals (Faggioni et al., 2000). If this is true, then the sham rats, whose serum leptin levels fell after the injection of LPS, should be more susceptible to immune stress and display higher mortality than the OVX rats under life-threatening septic conditions. Actually, it is reported that, the high, but within the physiological range, estrogen milieu, i.e., the estrous phase and E2-administered condition (Geary et al., 2004), increases the anorectic response to immune stress in female rats. It is also reported that low-dose E2 enhances the production of pro-inflammatory cytokines (IL-1, IL-6, and TNF-␣) (Straub, 2007). Further studies are needed to clarify the pathophysiological roles of the discrepancies in the response of the leptin system to immune stress between gonadally intact female rats and OVX rats. References Faggioni, R., Moser, A., Feingold, K.R., Grunfeld, C., 2000. Reduced leptin levels in starvation increase susceptibility to endotoxin shock. Am. J. Pathol. 156, 1781–1787. Geary, N., Asarian, L., Sheahan, J., Langhans, W., 2004. Estradiol-mediated increases in the anorexia induced by intraperitoneal injection of bacterial lipopolysaccharide in female rats. Physiol. Behav. 82, 251–261. Grunfeld, C., Zhao, C., Fuller, J., Pollock, A., Moser, A., Friedman, J., Feingold, K.R., 1996. Endotoxin and cytokines induce expression of leptin, the ob gene product in hamsters. J. Clin. Invest. 97, 2152–2157. Inoue, W., Luheshi, G.N., 2010. Acute starvation alters lipopolysaccharideinduced fever in leptin-dependent and -independent mechanisms in rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299, 1709–1719. Iwasa, T., Matsuzaki, T., Murakami, M., Kinouchi, R., Gereltsetseg, G., Nakazawa, H., Yamamoto, S., Kuwahara, A., Yasui, T., Irahara, M., 2011. Changes in responsiveness of appetite, leptin and hypothalamic IL-1 and TNF-␣ to lipopolysaccharide in developing rats. J. Neuroimmunol. 236, 10–16. Iwasa, T., Matsuzaki, T., Murakami, M., Kinouchi, R., Gereltsetseg, G., Nakazawa, H., Yamamoto, S., Kuwahara, A., Yasui, T., Irahara, M., 2012. Effects of lipopolysaccharide exposure at different postnatal time points on the response of LH to homotypic stress in adulthood. J. Reprod. Immunol. 94, 155–160. La Cava, A., Matarese, G., 2004. The weight of leptin in immunity. Nat. Rev. Immunol. 4, 371–379. Mastronardi, C.A., Yu, W.H., Srivastava, V.K., Dees, W.L., McCann, S.M., 1472. Lipopolysaccharide-induced leptin release is neurally controlled. Proc. Natl. Acad. Sci. U.S.A. 98, 14270–14725. Meli, R., Pacilio, M., Raso, G.M., Esposito, E., Coppola, A., Nasti, A., Di Carlo, C., Nappi, C., Di Carlo, R., 2004. Estrogen and raloxifene modulate leptin and its receptor in hypothalamus and adipose tissue from ovariectomized rats. Endocrinology 145, 3115–3121. Pohl, J., Woodside, B., Luheshi, G.N., 2009. Changes in hypothalamically mediated acute-phase inflammatory response to lipopolysaccharide in diet-induced obese rats. Endocrinology 150, 4901–4910. Sachot, C., Poole, S., Luhashi, G.N., 2004. Circulation leptin mediates lipopolysaccharide-induced anorexia and fever in rats. J. Physiol. 561, 263–272. Straub, R.H., 2007. The complex role of estrogens in inflammation. Endocr. Rev. 28, 521–574.
Please cite this article in press as: Iwasa, T., et al., Changes in leptin production/secretion induced in response to septic doses of lipopolysaccharides in gonadally intact and ovariectomized female rats. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.03.003
ARTICLE IN PRESS
JRI-2248; No. of Pages 4
Journal of Reproductive Immunology xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Journal of Reproductive Immunology journal homepage: www.elsevier.com/locate/jreprimm
Short communication
Changes in leptin production/secretion induced in response to septic doses of lipopolysaccharides in gonadally intact and ovariectomized female rats Takeshi Iwasa ∗ , Toshiya Matsuzaki, Munkhsaikhan Munkhzaya, Altankhuu Tungalagsuvd, Takako Kawami, Masahiro Murakami, Takeshi Kato, Akira Kuwahara, Toshiyuki Yasui, Minoru Irahara Department of Obstetrics and Gynecology, The University of Tokushima Graduate School, Institute of Health Biosciences, 3-18-15 Kuramoto-Cho, Tokushima 770-8503, Japan
a r t i c l e
i n f o
Article history: Received 1 February 2014 Received in revised form 8 March 2014 Accepted 14 March 2014
Keywords: Ovariectomy Leptin ObRb LPS
a b s t r a c t In addition to its role as a regulator of energy homeostasis, leptin plays a pivotal role in certain immune/inflammatory responses. It has been reported that the synthesis and secretion of leptin are increased during immune stress in male experimental animals, whereas the changes in leptin synthesis and secretion induced by immune stress in females have not been fully elucidated. In this study, we found that during lipopolysaccharide (LPS)-induced immune stress the synthesis and secretion of leptin were decreased in gonadally intact female rats, but increased in ovariectomized rats. However, the LPS-induced increase in the serum leptin level observed in the ovariectomized rats was partially attenuated by estradiol supplementation. These results suggest that the changes in leptin synthesis and secretion induced in response to immune stress in females are affected by the gonadal steroid milieu and that estradiol and other factors are involved in these alterations. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Leptin plays a crucial role as a regulator of appetite and energy expenditure. However, in addition to its role in energy homeostasis it also plays a pivotal role in immune/inflammatory responses (La Cava and Matarese, 2004). In male experimental animals, it was found that the administration of high doses of lipopolysaccharide (LPS) or proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), resulted in the increased synthesis and secretion of leptin (Mastronardi
∗ Corresponding author. Tel.: +81 88 633 7177. E-mail addresses: [email protected], [email protected] (T. Iwasa).
et al., 2001; Grunfeld et al., 1996). Increased serum leptin levels promote febrile and anorectic responses and exert a pro-survival effect under septic conditions (La Cava and Matarese, 2004). Although the immunological roles played by leptin under immune stress conditions have been established in males, they have not been fully elucidated in females. We have already reported that the serum leptin level is increased by LPS injection in pre-pubertal female rats (Iwasa et al., 2011). In addition, it has been reported that reduced leptin levels during starvation attenuate the sustained fever response to LPS in female rats (Inoue and Luheshi, 2010). These results indicate that leptin may be involved in immune/inflammatory responses in females, as well as in males. However, changes in leptin and its receptors under immune stress conditions at adulthood have not been evaluated in females. In this study, the effects
http://dx.doi.org/10.1016/j.jri.2014.03.003 0165-0378/© 2014 Elsevier Ireland Ltd. All rights reserved.
Please cite this article in press as: Iwasa, T., et al., Changes in leptin production/secretion induced in response to septic doses of lipopolysaccharides in gonadally intact and ovariectomized female rats. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.03.003
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of the administration of a high dose of LPS on the serum leptin level and the mRNA expression levels of the leptin (ob) and hypothalamic leptin receptor (ObRb) genes were examined in female rats. As the synthesis and secretion of leptin are affected by the gonadal steroid milieu (Meli et al., 2004), gonadally intact, ovariectomized, and estradiol-supplemented ovariectomized female rats were used for the present experiments. The serum IL-1 and TNF-␣ levels of the rats were also measured because these cytokines are involved in the upregulation of leptin expression.
TTG-3 ; R: 5 -GGT TCC CTG GGT GCT CTG A-3 ; 18s rRNA: F: 5 -GAC GGA CCA GAG CGA AAG C-3 ; R: 5 -AAC CTC CGA CTT TCG TTC TTG A-3 . The PCR conditions were as follows: initial denaturation and enzyme activation at 95 ◦ C for 20 s, followed by 45 cycles of denaturation at 95 ◦ C for 3 s and annealing at 63 ◦ C for 30 s (ObRb), 64 ◦ C for 30 s (18s rRNA), or 66 ◦ C for 30 s (ob), before a final extension step at 72 ◦ C for 1 min. Data analysis was performed using one-way (for intra-group comparisons) or two-way (for inter-group comparisons) analysis of variance (ANOVA), followed by Dunnett’s test or the Student’s t test (comparisons between time points).
2. Materials and methods 3. Results and discussion Sixty-one female Sprague–Dawley rats (Charles River Japan, Tokyo, Japan) were used in this study. They were housed in a room under controlled light (12 h light:12 h darkness; lights on at 0800 and off at 2000) and temperature (24 ◦ C) conditions and were allowed free access to commercial laboratory chow (standard diet; 3.6 kcal/g, 13.3% energy provided as fat; Oriental MF, Oriental Yeast Co, Ltd, Osaka, Japan). All animal experiments were conducted in accordance with the ethical standards of the institutional Animal Care and Use Committee of the University of Tokushima. All surgical procedures were carried out under sodium pentobarbital- (60–80 mg/kg; intraperitoneal, i.p.) or sevoflurane-induced anesthesia. In the first experiment, 24 rats were ovariectomized (OVX group) and 23 rats underwent sham surgery (sham group) at 10 weeks of age. After a 6-week recovery period, the rats in the sham and OVX groups were sub-divided into saline-injected and LPS-injected groups (0111:B4; Sigma, St. Louis, MO, USA; 5 mg/kg, i.p.). Blood samples, the whole brain, and visceral (parametrial) fat samples were collected at 6 h (in the saline and LPS-injected groups) or 24 h (only in the LPS-injected groups) post-injection. In the second experiment, 16 rats were ovariectomized at 10 weeks of age. After a 6-week recovery period, each rat was subcutaneously implanted with a Silastic tube (length, 20 mm; inner diameter, 1.5 mm; outer diameter, 2.0 mm) containing 20 g/ml of 17-estradiol (Sigma). The next day, they were subdivided into saline-injected and LPS-injected groups. Blood and tissue samples were collected at 24 h post-injection. In both experiments, hypothalamic explants were dissected from the rats’ whole brains as described previously (Iwasa et al., 2012), and the changes in the rats’ body weight and the total volume of fat were also measured. In addition, the rats’ serum levels of leptin, IL-1, and TNF-␣ were measured in duplicate using radioimmunoassays (rat leptin RIA lit or multi-species leptin RIA kit, Linco Research, St. Charles, MO, USA) or enzyme-linked immunosorbent assays (R&D Systems, Minneapolis, MN, USA). Total RNA was isolated from the hypothalamus and parametrial fat samples and used to synthesize cDNA. Then, the expression levels of hypothalamic leptin ObRb mRNA and adipose ob mRNA were measured using real-time PCR analysis. The expression level of each gene was normalized to that of 18s rRNA. The forward and reverse primers used were as follows: ob (leptin): F: 5 -GGT CAC CGG TTT GGA CTT CAT-3 , R: 5 -CTG GTC CAT CTT GGA CAA ACT CA-3 ; ObRb (leptin receptor type b): F: 5 -GCA GCT ATG GTC TCA CTT CTT
In the first experiment, it was found that the OVX rats were heavier and displayed lower uterine weights than the sham rats (data not shown). In addition, the OVX rats possessed significantly more (by volume) visceral and subcutaneous fat than the sham rats (Fig. 1). The body weight changes induced by the injection of LPS did not differ between the OVX and sham groups. The serum IL-1 and TNF-␣ levels of saline-injected rats were undetectable with the ELISA kit. Similarly, the serum TNF-␣ levels in rats 24 h after LPS injection were undetectable. 24 h after the injection of LPS, the serum IL-1 levels of the sham rats were significantly higher than those of the OVX rats. Similarly, the serum TNF-␣ levels of the sham rats were significantly higher than those of the OVX rats 6 h after the injection of LPS. Conversely, the serum leptin levels of the LPS-injected sham rats were significantly lower than those of the saline-injected sham rats. In addition, after the injection of LPS the serum leptin levels of the sham rats were significantly lower than those of the OVX rats. Among the OVX rats, the visceral fat ob mRNA levels of the LPS-injected group were significantly higher than those of the saline-injected group. Also, the visceral fat ob mRNA levels of the OVX rats were significantly higher than those of the sham rats. In both groups, the hypothalamic ObRb mRNA levels of the LPS-injected rats were significantly lower than those of the saline-injected rats. In the second experiment (in which estradiol-supplemented rats were used), the serum leptin levels of the LPS-injected rats tended to be higher than those of the saline-injected rats (P = 0.08; Fig. 2), although the body weight of the LPS-injected rats was markedly reduced. On the other hand, ob mRNA expression was not affected by the administration of LPS in these rats. Serum leptin levels in the second experiment were lower than those in the first experiment. Differences in leptin measurement kits (rat-specific kit for the first experiment and the kit for multi-species for the second experiment) may induce these discrepancies. In the present study, we found that the serum leptin level and ob mRNA expression were decreased in gonadally intact female rats during LPS-induced immune stress, whereas LPS increased expression levels in OVX rats. The injection of LPS reduced hypothalamic ObRb mRNA expression in both the OVX and sham rats. We speculate that such changes in ObRb expression are an adaptive response aimed at attenuating leptin-induced anorexia
Please cite this article in press as: Iwasa, T., et al., Changes in leptin production/secretion induced in response to septic doses of lipopolysaccharides in gonadally intact and ovariectomized female rats. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.03.003
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Fig. 1. Fat volume (A), body weight (BW) change (B), the serum levels of cytokines and leptin (C–E), visceral fat ob mRNA levels (F), and hypothalamic ObRb mRNA levels (G) in the sham () and OVX () rats after the injection of saline or LPS. The serum IL-1 and TNF-␣ levels of saline-injected rats were undetectable. The serum TNF-␣ levels in rats 24 h after LPS injection were undetectable. The mRNA expression levels of ObRb and ob were normalized by dividing them by the expression level of 18s rRNA. Data are presented as mean + SEM values. *P < 0.05, **P < 0.01; Student’s t test. † P < 0.05, †† P < 0.01 vs. the saline-injected group; Dunnett’s test. UD: undetectable.
Fig. 2. Body weight (BW) change (A), serum leptin levels (B), visceral fat ob mRNA levels (C), and hypothalamic ObRb mRNA levels (D) in estradiolsupplemented ovariectomized rats at 24 h post-injection of saline or LPS. The ObRb and ob mRNA expression levels were normalized by dividing them by the expression level of 18s rRNA. Data are presented as mean + SEM values.
Please cite this article in press as: Iwasa, T., et al., Changes in leptin production/secretion induced in response to septic doses of lipopolysaccharides in gonadally intact and ovariectomized female rats. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.03.003
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ARTICLE IN PRESS T. Iwasa et al. / Journal of Reproductive Immunology xxx (2014) xxx–xxx
under immune stress conditions. The serum levels of IL-1 and TNF-␣, which positively regulate leptin expression (Mastronardi et al., 2001; Grunfeld et al., 1996), were increased by LPS treatment in both the OVX and sham rats. However, IL-1 at 24 h after LPS injection and TNF-␣ at 6 h after LPS injection in OVX rats were significantly lower than those in sham rats. Therefore, such differences in the responses of cytokines may be responsible for the discrepancies between these groups with regard to the changes in the serum leptin level and ob mRNA expression induced by the LPS injection. Interestingly, the changes in the serum leptin level and the expression level of the ob gene observed in the OVX rats after the injection of LPS were similar to those reported for male rodents, while those observed in the sham rats were opposite to those detected in male rodents (Mastronardi et al., 2001; Grunfeld et al., 1996). As it has been reported that gonadal steroids are involved in regulating the leptin system under basal conditions (Inoue and Luheshi, 2010), we speculated that gonadal steroids might also affect the leptin system during immune stress conditions. Therefore, in the present study we examined the responses of the leptin system to LPS in estradiol-supplemented ovariectomized rats. As a result, we found that the LPS-induced increase in the serum leptin level was partially attenuated in these rats. We speculate that not only estradiol, but also other factors, for example, progesterone, testosterone, and obesity, are involved in the ovariectomy-induced changes in the response of the leptin system to immune stress. Actually, it has been reported that diet-induced obesity increases the leptin response to LPS in adult male rats (Pohl et al., 2009). Further experiments, focused on the ovariectomy-induced hormonal and physiological changes, are needed to clarify the mechanism by which leptin response is altered according to the gonadal steroid milieu in females. To the best of our knowledge, this is the first report to show the relationship between gonadal steroid milieu and leptin response to immune stress in females. The responses of the leptin system have been reported to play important pathological and physiological roles in immune stress conditions. It has been reported that leptin induces the anorectic and febrile responses via the upregulation of hypothalamic pro-inflammatory cytokines. For instance, co-administration of leptin antiserum attenuates the LPS-induced febrile responses in rats (Sachot et al., 2004). It has also been reported that rats with a low serum leptin level show high mortality under septic conditions and that the supplementation of leptin protects against
lethality in these individuals (Faggioni et al., 2000). If this is true, then the sham rats, whose serum leptin levels fell after the injection of LPS, should be more susceptible to immune stress and display higher mortality than the OVX rats under life-threatening septic conditions. Actually, it is reported that, the high, but within the physiological range, estrogen milieu, i.e., the estrous phase and E2-administered condition (Geary et al., 2004), increases the anorectic response to immune stress in female rats. It is also reported that low-dose E2 enhances the production of pro-inflammatory cytokines (IL-1, IL-6, and TNF-␣) (Straub, 2007). Further studies are needed to clarify the pathophysiological roles of the discrepancies in the response of the leptin system to immune stress between gonadally intact female rats and OVX rats. References Faggioni, R., Moser, A., Feingold, K.R., Grunfeld, C., 2000. Reduced leptin levels in starvation increase susceptibility to endotoxin shock. Am. J. Pathol. 156, 1781–1787. Geary, N., Asarian, L., Sheahan, J., Langhans, W., 2004. Estradiol-mediated increases in the anorexia induced by intraperitoneal injection of bacterial lipopolysaccharide in female rats. Physiol. Behav. 82, 251–261. Grunfeld, C., Zhao, C., Fuller, J., Pollock, A., Moser, A., Friedman, J., Feingold, K.R., 1996. Endotoxin and cytokines induce expression of leptin, the ob gene product in hamsters. J. Clin. Invest. 97, 2152–2157. Inoue, W., Luheshi, G.N., 2010. Acute starvation alters lipopolysaccharideinduced fever in leptin-dependent and -independent mechanisms in rats. Am. J. Physiol. Regul. Integr. Comp. Physiol. 299, 1709–1719. Iwasa, T., Matsuzaki, T., Murakami, M., Kinouchi, R., Gereltsetseg, G., Nakazawa, H., Yamamoto, S., Kuwahara, A., Yasui, T., Irahara, M., 2011. Changes in responsiveness of appetite, leptin and hypothalamic IL-1 and TNF-␣ to lipopolysaccharide in developing rats. J. Neuroimmunol. 236, 10–16. Iwasa, T., Matsuzaki, T., Murakami, M., Kinouchi, R., Gereltsetseg, G., Nakazawa, H., Yamamoto, S., Kuwahara, A., Yasui, T., Irahara, M., 2012. Effects of lipopolysaccharide exposure at different postnatal time points on the response of LH to homotypic stress in adulthood. J. Reprod. Immunol. 94, 155–160. La Cava, A., Matarese, G., 2004. The weight of leptin in immunity. Nat. Rev. Immunol. 4, 371–379. Mastronardi, C.A., Yu, W.H., Srivastava, V.K., Dees, W.L., McCann, S.M., 1472. Lipopolysaccharide-induced leptin release is neurally controlled. Proc. Natl. Acad. Sci. U.S.A. 98, 14270–14725. Meli, R., Pacilio, M., Raso, G.M., Esposito, E., Coppola, A., Nasti, A., Di Carlo, C., Nappi, C., Di Carlo, R., 2004. Estrogen and raloxifene modulate leptin and its receptor in hypothalamus and adipose tissue from ovariectomized rats. Endocrinology 145, 3115–3121. Pohl, J., Woodside, B., Luheshi, G.N., 2009. Changes in hypothalamically mediated acute-phase inflammatory response to lipopolysaccharide in diet-induced obese rats. Endocrinology 150, 4901–4910. Sachot, C., Poole, S., Luhashi, G.N., 2004. Circulation leptin mediates lipopolysaccharide-induced anorexia and fever in rats. J. Physiol. 561, 263–272. Straub, R.H., 2007. The complex role of estrogens in inflammation. Endocr. Rev. 28, 521–574.
Please cite this article in press as: Iwasa, T., et al., Changes in leptin production/secretion induced in response to septic doses of lipopolysaccharides in gonadally intact and ovariectomized female rats. J. Reprod. Immunol. (2014), http://dx.doi.org/10.1016/j.jri.2014.03.003
