PRACE ORYGINALNE/ORIGINAL PAPERS
Effect of tributyltin on the food intake and brain neuropeptide expression in rats
Wpływ tributylocyny na przyjmowanie pokarmu i ekspresję neuropeptydów w mózgu szczurów
1Henan Open Laboratory of key subjects of Environmental and Animal Products Safety, College of Animal Science and Technology, Henan University of Science and Technology, Henan, China
2Laboratory Animal Center, No. 150 Central Hospital of PLA, Henan, China
Jiliang Zhang M.D., Henan Open Laboratory of key subjects of Environmental and Animal Products Safety, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, Henan, 471003, China, e-mail: firstname.lastname@example.org
Introduction: Tributyltin (TBT) is a largely diffused environmental pollutant. Several studies have demonstrated that TBT is involved in the development of obesity. However, few studies addressing the effects of TBT on the brain neuropeptides involved in appetite and body weight homeostasis have been published.
Material and methods: Experiments were carried out on female and male Sprague-Dawley rats. Animals were exposed to TBT (0.5 μg/kg body weight) for 54 days. The hepatic triglyceride and total cholesterol were determined using commercial enzyme kits. The NPY, AgRP POMC and CART mRNA expression in brains were quantified by real-time PCR.
Results: TBT exposure resulted in significant increases in the hepatic total cholesterol and triglyceride concentration of both male and female rats. Interestingly, increases in body weight and fat mass were only found in the TBT-treated male rats. TBT exposure also led to a significant increase in food intake by the female rats, while no change was observed in the male rats. Moreover, the neuropeptides expression was different between males and females after TBT exposure. TBT induced brain NPY expression in the female rats, and depressed brain POMC, AgRP and CART expression in the males.
Conclusions: TBT can increase food intake in female rats, which is associated with the disturbance of NPY in brains. TBT had sex-different effects on brain NPY, AgRP POMC and CART mRNA expression, which indicates a complex neuroendocrine mechanism of TBT.
(Endokrynol Pol 2014; 65 (6): 485–490)
Key words: tributyltin; body weight; food intake; neuropeptides
Wstęp: Tributylocyna (TBT) jest powszechnie występującym w środowisku zanieczyszczeniem. Prowadzone dotychczas badania wykazały, że obecność TBT może mieć związek z rozwojem otyłości. Niewiele jest jednak doniesień na temat wpływu TBT na układ neuropeptydów w mózgowiu regulujących łaknienie i utrzymanie masy ciała.
Materiał i metody: Doświadczenia przeprowadzono na szczurach obu płci szczepu Sprague-Dawley. Zwierzętom podawano przez 54 dni TBT w dawce 0,5 μg/kg masy ciała. Stężenie triglicerydów i całkowite stężenie cholesterolu w wątrobie oznaczano przy użyciu komercyjnych zestawów analitycznych. Obecność mRNA NPY, AgRP POMC i CART w mózgach szczurów oznaczano metodą PCR w czasie rzeczywistym (real time-PCR).
Wyniki: Ekspozycja na TBT powodowała istotne zwiększenie całkowitego stężenia cholesterolu i trójglicerydów w wątrobie zarówno samców, jak i samic szczura. Co ciekawe, zwiększenie masy ciała i masy tkanki tłuszczowej odnotowano jedynie u samców, którym podawano TBT Stwierdzono także istotne zwiększenie ilości pokarmu przyjmowanego przez samice, natomiast nie obserwowano takich zmian u samców. Ponadto, odnotowano różnice w ekspresji neuropeptydów w mózgowiu samic i samców szczura, którym podawano TBT Ekspozycja na TBT nasilała ekspresję NPY w mózgach samic, ale równocześnie zmniejszała ekspresję POMC, AgRP i CART w mózgach samców szczura.
Wnioski: Ekspozycja na TBT może zwiększać ilość pokarmu spożywanego przez samice szczura, co wiąże się z zaburzeniem układu NPY w mózgowiu. Trybutylocyna wywiera odmienny wpływ na ekspresję mRNA NPY, AgRP POMC i CART w mózgach samców i samic szczura, co wskazuje na istnienie złożonego mechanizmu działania tej substancji na układ neuroendokrynny.
(Endokrynol Pol 2014; 65 (6): 485–490)
Słowa kluczowe: tributylocyna; masa ciała; przyjmowanie pokarmu; neuropeptydy
Organotin compounds, such as tributyltin (TBT), are used in a wide variety of consumer products, for example in agriculture and industry as biocide, heat stabiliser and chemical catalyst . Although TBT has been banned from paints in the European Union since 2003 (EC Regulation 782/2003), it is still found at high levels in marine and freshwater ecosystems exceeding toxicity levels . The contamination level of organotin compounds in food, particularly in fish and shellfish, remains high . Recently, it was reported that TBT was also found in dust collected from houses . High levels of butyltins were detected in human liver tissue and blood samples. For example, total butyltin concentrations in human livers collected from Poland are in the range 2.4–11 ng/g wet weight , and butyltin concentrations in the livers of Japanese are in the range 59–96 ng/g wet weight . Kannan et al.  reported that TBT levels in human blood collected from the United States were up to 8.18 ng/mL.
In mammals, TBT is toxic to a number of organs [8–10]. Recently, TBT was shown to affect adipogenic differentiation by activating peroxisome proliferator-activated receptor (PPAR) γ and retinoid X receptor (RXR) in vitro . Prenatal exposure to environmental TBT predisposes multipotent stem cells to become adipocytes in mice . When pregnant mice are injected daily from gestational days 12–18 with TBT (0.05 or 0.5 mg/kg body weight intraperitoneally), an increase in lipid accumulation or an increase in mature adipocytes is observed in the pups . Recent reports indicate that TBT increases body weight in male mice . In our previous study, we have found that chronic and repeated exposure to low doses of TBT disturbs levels of key hormones linked to energy homeostasis . These findings suggest a peripheral role of TBT on obesity. However, few studies on the effects of TBT on the central nervous system have been reported. Hypothalamus acts as the control centre for hunger and satiety. Part of the hypothalamus, the arcuate nucleus, includes neurons that coexpress peptides that stimulate food intake and weight gain, especially neuropeptide Y (NPY) and agouti related peptide (AgRP), as well as those expressing proopiomelanocortin (POMC) and cocaine- and amphetamine regulated transcript (CART) which inhibit feeding and promote weight loss. Together, these neurons and peptides control the sensations of hunger and satiety and thereby regulate appetite and energy balance .
Therefore, this study was designed to investigate the effects of TBT on body weight gain and determine alterations of hypothalamic expression of the neuropeptides involved in food intake regulation in rats.
Material and methods
TBT chloride was obtained from Fluka AG, Switzerland, with a purity of greater than 97%. All other chemicals were of analytical grade and were obtained from commercial sources. The TBT was dissolved in 100% ethanol and diluted with 0.85% (g/g) sodium chloride. The final TBT concentration was 0.1 μg/mL, and the final ethanol concentration was 0.1 ml/mL volume.
Experimental species and treatment
All animal experiments were conducted according to the research protocols approved by the Institutional Animal Care and Use Committee, Henan University of Science and Technology. Sprague-Dawley rats were purchased from Zhengzhou University (China), housed in individual wire-mesh cages, in the same room at 24 ± 1°C under a 12-h light-dark cycle. Before the treatment, the average body weight of the males and females was 218.58 ± 18.49 and 187.85 ± 8.56 g respectively. After a quarantine period, 16 male or female rats with adequate weight gain and without clinical signs were divided randomly into two experimental groups. One group was orally administered by gavage once every three days with 0.5 μg/kg TBT, and the other group received an equal volume of vehicle (5 mL/kg). Body weight was recorded on the day of oral administration of TBT or vehicle, and actual dosing volumes were adjusted according to the body weights recorded. Food intake was determined every two days throughout the duration of the experiment. The amount of food remaining at the end of the two day period was individually weighed and subtracted from the original quantity provided. Bedding was searched thoroughly to ensure complete removal of all remaining food. All rats had ad libitum access to water.
The rats were sacrificed 54 days after the exposure began and fasted for 12 h before necropsy. The fat mass (the epididymal and retroperitoneal fat deposits) was weighed. The adiposity index was calculated as the quotient of the fat mass (g) and the final body weight of the animal (g). The isolated brains were flash frozen in liquid nitrogen for analysis of NPY, AgRP POMC and CART mRNA expression. The isolated livers were stored at –80 °C for biochemical analysis.
Extraction of total lipids from the livers was performed as described by Folch et al.  in the presence of butylated hydroxytoluene as an antioxidant. Triglyceride and total cholesterol were determined using commercial enzyme kits purchased from Nanjing Jiancheng Bioengineering Institute (China) according to the manufacturer’s instructions.
Reverse-transcriptional real-time PCR (RT-PCR)
Levels of NPY, AgRP, POMC and CART mRNA were determined by quantitative real-time PCR using SYBR Green chemistry on a Rotor-Gene 3000 (Applied Biosystems, USA) using the housekeeping gene P-actin as internal control according to the methods of our laboratory . The Relative Expression Software Tool(REST 2008©-version 2) was used to calculate the relative expression. The real time quantitative PCR primers and the PCR efficiencies are shown in Table I.
Table I. Sequences of forward and reverse primers used for real-time RT-PCR
Tabela I. Sekwencje starterów (lewy starter i prawy starter) stosowanych do analizy PCR w czasie rzeczywistym (RT-PCR)
|Target||Sequence||Accession number||PCR efficiency (%)|
|NPY||(F)5’ CGCTCTATCCCTGCTCGTGT 3’
(R)5’ GGTCTTCAAGCCTTGTTCTGG 3’
|AGRP||(F)5’ AAGAAGAACCGGAACAAATGC 3’
(R)5’ GCAGGACTCGTGCAGCCTTA 3’
|POMC||(F)5’ AACGGAGATGAACAGCCCTTGAC 3’
(R)5’ CGACTCGTTCTCGGCGACATT 3’
|CART||(F)5’ GCCAAGTCCCCATGTGTGAC 3’
(R)5’ CACCCCTTCACAAGCACTTCA 3’
|P-actin||(F)5’ CCGTAAAGACCTCTATGCCAACA 3’
(R)5’ CGGACTCATCGTACTCCTGCT 3’
Results were reported as means ± standard error of measurement (S.E.M.). Significant differences between means were analysed with one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc using statistical software, SPSS Version 11.0 (SPSS Inc., Chicago, IL, USA) for Windows, except mRNA expression, which was performed using the Pair Wise Fixed Reallocation Randomization Test© . In all cases, a value of p < 0.05 was used to indicate significant differences.
The average body weights of rats after TBT exposure for 54 days are shown in Figure 1. Significant increases of body weights at days 6, 12, 18, 24 and 30 were found in the male rats exposed to TBT compared to the control (Fig. 1A). However, in the female rats, no significant change of body weight was observed in the TBT exposure group compared to the control (Fig. 1B).
In the male rats, TBT exposure resulted in a significant increase (by 1.56-fold) in fat mass compared to the control (Table II). However, in the female rats, there was no significant difference of fat mass between the exposure group and the control (Table II). Although increases of adiposity index were found in both male and female rats after TBT exposure, no significant changes were found (Table II).
Table II. Effects of TBT on the fat mass and adiposity index in male and female rats
Tabela II. Wpływ TBT na masę tkanki tłuszczowej i wskaźnik otłuszczenia u samców i samic
|Fat mass (g)||Adiposity index|
|Control||7.98 ± 0.96||9.09 ± 1.47||2.06 ± 0.51||3.09 ± 0.40|
|TBT||12.3 ± 1.94*||7.69 ± 1.08||3.56 ± 0.72||3.58 ± 0.34|
In both male and female rats, TBT exposure resulted in significant increases (by 1.44- and 1.31-fold, respectively) of the hepatic total cholesterol (Table III). The hepatic triglyceride also increased significantly (by 1.86- and 1.24-fold, respectively) after exposure to TBT in both male and female rats (Table III).
Table III. Effects of TBT on the hepatic total cholesterol and triglyceride in male and female rats
Tabela III. Wpływ TBT na całkowite stężenie cholesterolu i triglicerydów u samców i samic
|Total cholesterol [mmol/L]||Triglyceride [mmol/L]|
|Control||0.84 ± 0.24||1.18 ± 0.15||0.23 ± 0.11||0.17 ± 0.11|
|tbt||1.21 ± 0.13*||1.55 ± 0.32*||0.43 ± 0.08*||0.21 ± 0.08*|
In the male rats, no significant changes of food intake and the ratio of food intake and body weight were found between the exposure group and the control (Table IV). However, in the female rats, TBT exposure resulted in a significant increase (by 1.50- and 1.12-fold) in food intake and the ratio of food intake and body weight compared to the control (Table IV).
Table IV Effects of TBT on the food intake (g/2 day/rat) in male and female rats
Tabela IV Wpływ TBT na ilość przyjmowanego pokarmu (g/2 × dziennie/zwierzę) u samców i samic
|Food intake/body weight||Food intake (g)|
|Control||0.094 ± 0.002||0.094 ± 0.001||29.15 ± 1.68||20.17 ± 0.30|
|TBT||0.096 ± 0.003||0.104 ± 0.004*||28.25 ± 0.43||22.52 ± 0.87*|
No significant change in the brain expression of NPY was observed in the male rats (Fig. 2A). However, in female rats, the brain expression of NPY was significantly increased (by 1.93-fold) in the TBT exposure group compared to the control (Fig. 2A).
TBT exposure significantly decreased (by 0.42-fold) the brain expression of AgRP in male rats compared to the control (Fig. 2B). No significant change of AgRP expression in the brain was observed in the female rats (Fig. 2B).
Although a decrease of brain POMC expression was found in the male rats after TBT exposure, no significant changes were found (Fig. 2C). No significant change of brain POMC expression was also observed in the female rats (Fig. 2C).
TBT exposure significantly decreased (by 0.38-fold) the brain expression of CART in male rats compared to the control (Fig. 2D). No significant change of CART expression in the brain was observed in the female rats (Fig. 2D).
TBT is a largely diffused environmental pollutant, and the level in food, particularly in fish and shellfish, remains high. The average intake of TBT by humans from market-bought seafood has been estimated to vary worldwide between 0.18 and 2.6 μg per day per person . It is reported that mean concentrations of TBT in fish muscle collected from Taiwanese harbours is 308.7 ng/g wet weight . Using the average seafood consumption data of 0.067 kg/day provided by Lee et al. , the dose of TBT used in the present study is below those doses to which people may be exposed. Recently, a human epidemiological study showed that placenta TBT is associated with increased weight gain during the first three months of life , which indicated a role for TBT pollution in the development of obesity. In the present study, 0.5 μg/kg TBT treatment already produced a prominent obesity-related effect. However, the Tolerable Daily Intake of organotins established by the European Food Safety Authority is 0.250 μg/kg body weight . Therefore, the health impact of TBT on human might be underestimated.
TBT is considered as a kind of obesogen, chiefly for its action on fat tissue inducing the differentiation of preadipocytes from adipocytes [11, 13, 24]. In our previous study, hepatic dysfunction, as well as a rise in plasmatic levels of insulin, leptin, and resistin, was observed . In the present study, TBT exposure resulted in significant increases of the hepatic total cholesterol and triglyceride in both male and female rats. The liver plays a central role in coordinating whole body metabolism. Changes in hepatic lipid metabolism might contribute to the obesity effect of TBT exposure. Interestingly, the increases of fat mass and body weight were only found in the male rats. It has been reported that male mice are more susceptible to obesity than female mice, and ovarian hormones might provide protection against weight gain . In addition, the ovary is one of the most dynamic endocrine organs in females. A substantial energy source is necessary for its activity in puberty. In our previous study, TBT exposure induced an increase of interstitial ectopic lipid accumulation and total lipids in the ovaries of fish . Thus, no change of fat mass and body weight in females might be also associated with the high-energy demand of ovaries.
Obesity is not a purely metabolic disease and its genesis is also due to an imbalance of neuroendocrine mechanisms acting under the control of specific neural circuits located in the hypothalamus . TBT can be transported to the brains of fish by axonal transport . In rats, it has been reported that TBT disrupted blood-brain barrier and increased Sn accumulation in the brain regions . Therefore, the brain might be a potential target organ of TBT Neurotoxicological studies have demonstrated that the levels of brain dopamine, norephinephrine and serotonin decrease in a dose-dependent manner after ingestion of high doses of TBT in mice . It is also reported that acute exposure to TBT induces c-fos activation in the hypothalamic arcuate nucleus of adult male mice .
It is well known that hypothalamic orexigenic (i.e. NPY and AgRP) and anorexigenic (i.e. POMC and CART) neuropeptides play an important role in the regulation of appetite and body weight homeostasis . In the present study, we observed that TBT exposure leads to a significant increase of NPY expression in the female rats, which could explain the observed increase of food intake in the females. However, no change of food intake was observed in the male rats, which might be associated with the opposite effects on appetite of decrease of AgRP and CART. The increase in body weight in the male rats may be caused by complex factors, not only by food intake. In addition, except NPY, the brain expression of AgRP POMC and CART was depressed by TBT exposure in male rats. It is reported that TBT induces oxidative damage, inflammation and apoptosis via disturbance in the blood-brain barrier and metal homeostasis in rat brains . In our previous study, TBT induced brain astrocyte apoptosis in rockfish . Thus, the no change or depression of neuropeptides expression in male rats would be due to the cytotoxicity of TBT. There is some evidence showing that NPY-expressing neurons in the hypothalamus concentrate 17β-oestradiol and the sex steroid oestrogen may play a role in the regulation of NPY synthesis [34, 35]. Therefore, we suspect that the sex-different effects of TBT on brain neuropeptides expression might be due to the neuro-protection of 17β-oestradiol in the brain, where females have higher 17β-oestradiol levels compared to males .
TBT can increase the food intake in female rats, which is associated with the disturbance of NPY in brains. TBT had sex-different effects on brain NPY, AgRP POMC and CART mRNA expression, which indicates a complex neuroendocrine mechanism of TBT. The obesity induced by TBT might be caused by complex factors, not only by food intake. Elucidation of this mechanism requires further study.
The present study was supported by the National Natural Science Foundation of China (41301562), the National Training Programmes of Innovation and Entrepreneurship for Undergraduates of China (201310464054), and the Youth Fund Projects of Henan University of Science and Technology (13000943).
- Zuo Z., Chen S., Wu T. et al. Tributyltin causes obesity and hepatic steatosis in male mice. Environ Toxicol 2011; 26: 79–85.
- Antizar-Ladislao B. Environmental levels, toxicity and human exposure to tributyltin (TIN)-contaminated marine environment. A review. Environ Int 2008; 34: 292–308.
- Sousa A., Laranjeiro F., Takahashi S. et al. Imposex and organotin prevalence in a European post-legislative scenario: temporal trends from 2003 to 2008. Chemosphere 2009; 77: 566–573.
- Kannan K., Takahashi S., Fujiwara N. et al. Organotin compounds, including butyltins and octyltins, in house dust from Albany, New York, USA. Arch Environ Contam Toxicol 2010; 58: 901–907.
- Kannan K., Falandysz J. Butyltin residues in sediment, fish, fish-eating birds, harbour porpoise and human tissues from the Polish coast of the Baltic Sea. Mar Pollut Bull 1997; 34: 203–207.
- Takahashi S., Mukai H., Tanabe S. et al. Butyltin residues in livers of humans and wild terrestrial mammals and in plastic products. Environ Pollut 1999; 106: 213–218.
- Kannan K., Senthilkumar K., Giesy J.P. Occurrence of butyltin compounds in human blood. Environ Sci Technol 1999; 33: 1776–1779.
- Whalen M.M., Loganathan B.G., Kannan K. Immunotoxicity of environmentally relevant concentrations of butyltins on human natural killer cells in vitro. Environ Res 1999; 81: 108–116.
- da Silva de Assis H.C., Sanchez-Chardi A., Dos Reis R.C. et al. Subchronic toxic effects of tributyltin (TBT) and inorganic lead (PbII) in rats. Environ Toxicol Pharmacol 2005; 19: 113–120.
- Mitra S., Gera R., Singh V. et al. Comparative toxicity of low dose tributyltin chloride on serum, liver, lung and kidney following subchronic exposure. Food Chem Toxicol 2014; 64: 335–343.
- le Maire A., Grimaldi M., Roecklin D. et al. Activation of RXR-PPAR heterodimers by organotin environmental endocrine disruptors. EMBO Rep 2009; 10: 367–373.
- Kirchner S., Kieu T., Chow C. et al. Prenatal exposure to the environmental obesogen tributyltin predisposes multipotent stem cells to become adipocytes. Mol Endocrinol 2010; 24: 526–539.
- Grün F., Blumberg B. Environmental obesogens: organotins and endocrine disruption via nuclear receptor signaling. Endocrinology 2006; 147: 50–55.
- Si J., Wu X., Wan C. et al. Peripubertal exposure to low doses of tributyltin chloride affects the homeostasis of serum T, E2, LH, and body weight of male mice. Environ Toxicol 2011; 26: 307–314.
- Schwartz M.W., Woods S.C., Porte Jr D. et al. Central nervous system control of food intake. Nature 2000; 404: 661–671.
- Folch J., Lees M., Sloane Stanley G.H. A simple method for the isolation and purification of total lipids from animal tissues. J Biol Chem 1957; 226: 497–509.
- Zhang J., Zuo Z., He C. et al. Inhibition of thyroidal status related to depression of testicular development in Sebastiscus marmoratus exposed to tributyltin. Aquat Toxicol 2009; 94: 62–67.
- Pfaffl M.W., Graham W.H., Dempfle L. Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 2002; 30: 1–10.
- Tsuda T., Inoue T., Kojima M. et al. Daily intakes of tributyltin and triphenyltin compounds from meals. J AOAC Int 1995; 78: 941–943.
- Lee C.C., Wang T., Hsieh C.Y. et al. Organotin contamination in fishes with different living patterns and its implications for human health risk in Taiwan. Environ Pollut 2005; 137: 198–208.
- Lee C.C., Hsieh C.Y., Tien C.J. Factors influencing organotin distribution in different marine environmental compartments, and their potential health risk. Chemosphere 2006; 65: 547–559.
- Rantakokko P., Main K.M., Wohlfart-Veje C. et al. Association of placenta organotin concentrations with growth and ponderal index in 110 newborn boys from Finland during the first 18 months of life: a cohort study. Environ Health 2014; 13: 45.
- EFSA. Opinion of the scientific panel on contaminants in the food chain on a request from the commission to assess the health risks to consumers associated with exposure to organotins in foodstuffs. EFSA J 2004; 102: 1–119.
- Penza M., Jeremic M., Marrazzo E. et al. The environmental chemical tributyltin chloride (TBT) shows both estrogenic and adipogenic activities in mice which might depend on the exposure dose. Toxicol Appl Pharmacol 2011; 255: 65–75.
- Hong J., Stubbins R.E., Smith R.R. et al. Differential susceptibility to obesity between male, female and ovariectomized female mice. Nutr J 2009; 8: 11.
- Zhang J., Zuo Z., Xiong J. et al. Tributyltin exposure causes lipotoxicity responses in the ovaries of rockfish, Sebastiscus marmoratus. Chemosphere 2013; 90: 1294–1299.
- King P.J. The hypothalamus and obesity. Curr Drug Targets 2005; 6: 225–240
- Rouleau C., Xiong Z., Pacepavicius G. et al. Uptake of waterborne tributyltin in the brain of fish: axonal transport as a proposed mechanism. Environ Sci Technol 2003; 37: 3298–3302.
- Mitra S., Siddiqui W.A., Khandelwal S. Differential susceptibility of brain regions to tributyltin chloride toxicity. Environ Toxicol 2014; DOI: 10.1002/tox.22009.
- Elsabbagh H.S., Moussa S.Z., El-tawil O.S. Neurotoxicologic sequelae of tributyltin intoxication in rats. Pharmacol Res 2002; 45: 201–206.
- Bo E., Viglietti-Panzica C., Panzica G.C. Acute exposure to tributyltin induces c-fos activation in the hypothalamic arcuate nucleus of adult male mice. Neurotoxicology 2011; 32: 277–280.
- Mitra S., Gera R., Siddiqui W.A. et al. Tributyltin induces oxidative damage, inflammation and apoptosis via disturbance in blood-brain barrier and metal homeostasis in cerebral cortex of rat brain: An in vivo and in vitro study. Toxicology 2013; 310: 39–52.
- Zhang J., Zuo Z., Chen R. et al. Tributyltin exposure causes brain damage in Sebastiscus marmoratus. Chemosphere 2008; 73: 337–343.
- Sar M., Sahu A., Crowley W.R. et al. Localization of neuropeptide-Y immunoreactivity in estradiol-concentrating cells in the hypothalamus. Endocrinology 1990; 127: 2752–2756.
- Sinchak K., Wagner E.J. Estradiol signaling in the regulation of reproduction and energy balance. Front Neuroendocrin 2012; 33: 342–363.
- Zhang Q.G., Wang R., Tang H. et al. Brain-derived estrogen exerts anti-inflammatory and neuroprotective actions in the rat hippocampus. Mol Cell Endocrinol 2014; 389: 84–91.