Estradiol-induced hypophagia is associated with the differential mRNA expression of hypothalamic neuropeptides

Braz J Med Biol Res, August 2010, Volume 43(8) 759-766

Estradiol-induced hypophagia is associated with the differential mRNA expression of hypothalamic neuropeptides

L.E.C.M. Silva¹, M. Castro², F.C. Amaral², J. Antunes-Rodrigues¹ and L.L.K. Elias¹

¹Departamento de Fisiologia, ²Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, SP, Brasil

^{Correspondence and Footnotes}

Abstract

Estradiol participates in the control of energy homeostasis, as demonstrated by an increase in food intake and in body weight gain after ovariectomy in rats. In the present study, female Wistar rats (200-230 g, N = 5-15 per group), with free access to chow, were individually housed in metabolic cages. We investigated food intake, body weight, plasma leptin levels, measured by specific radioimmunoassay, and the hypothalamic mRNA expression of orexigenic and anorexigenic neuropeptides, determined by real-time PCR, in ovariectomized rats with (OVX+E) and without (OVX) estradiol cypionate treatment (10 µg/kg body weight, sc, for 8 days). Hormonal and mRNA expression were determined at pre-feeding and 4 h after food intake. OVX+E rats showed lower food intake, less body weight gain and lower plasma leptin levels. In the OVX+E group, we also observed a reduction of neuropeptide Y (NPY), agouti-related protein (AgRP) and cocaine- and amphetamine-regulated transcript (CART) mRNA expression in the arcuate nucleus and a decrease in orexin A in the lateral hypothalamic area (LHA). There was an increase in leptin receptor (LepRb), melanocortin-4 receptor (MC4-R), CART, and mainly corticotropin-releasing hormone (CRH) mRNA in the paraventricular nucleus and LepRb and CART mRNA in the LHA. These data show that hypophagia induced by estradiol treatment is associated with reduced hypothalamic expression of orexigenic peptides such as NPY, AgRP and orexin A, and increased expression of the anorexigenic mediators MC4-R, LepRb and CRH. In conclusion, estradiol decreases food intake, and this effect seems to be mediated by peripheral factors such as leptin and the differential mRNA expression of neuropeptides in the hypothalamus.

Key words: Estradiol; Leptin; Orexigenic and anorexigenic neuropeptides; Hypothalamus

Introduction

Gonadal hormones influence food intake and body weight in many mammalian species, including mice, rats, and women (1,2). In female rats, ovariectomy increases food intake and body weight, and these changes can be reversed by treatment with estrogen alone (2,3). Additionally, the energy balance of female rats oscillates as a function of the estrous cycle (3). In humans, abdominal fat increases in postmenopausal women, and estrogen replacement therapy prevents increases in body weight and fat accumulation (4).

The hypothalamus and peripheral signals of the energy store play an important role in the control of energy homeostasis. Several neuropeptides expressed in the hypothalamus possess orexigenic or anorexigenic effects and also affect energy expenditure (5). The paraventricular (PVN) and arcuate (ARC) nuclei and lateral hypothalamic area (LHA) are hypothalamic regions involved in the neuronal circuitry that controls food intake. Neurons in these regions are responsive to leptin, a hormone whose actions are mediated by the long form of the leptin receptor (LepRb) (5).

The ARC contains two distinct neuronal populations that express the cocaine- and amphetamine-regulated transcript (CART) and proopiomelanocortin (POMC), and activation of these neurons is associated with a reduction in food intake (5). The cleavage of POMC in the ARC derives the α-melanocyte-stimulating hormone (α-MSH), which inhibits food intake through hypothalamic melanocortin-3 (MC3-R) and melanocortin-4 (MC4-R) receptors. The other neuronal population co-expresses orexigenic peptides, neuropeptide Y (NPY) and agouti-related protein (AgRP) (5). Both neuronal populations project to second-order neurons located in the PVN, LHA and perifornical area. In the PVN, there is high expression of corticotropin-releasing hormone (CRH) (5), known to affect the energy balance through its effects on food intake and energy expenditure (6). The LHA contains neurons expressing melanin-concentrating hormone (MCH) (7) and orexin A (8), two neuropeptides with potent effects on feeding, as demonstrated by the increase in food intake after intracerebroventricular (icv) administration of these peptides (9,10).

Neurons of the PVN and LHA also express MC4-R, which has a significant role in the regulation of appetite. The icv injection of an MC4-R-specific antagonist increases food intake (11), and targeted deletion of the MC4-R in mice leads to hyperphagia, maturity onset obesity and hyperinsulinemia (12).

Leptin, the product of the lep gene (13), is mainly produced and secreted by white adipocytes in proportion to fat mass, signaling the repletion of body energy stores to the hypothalamus (5). In the ARC, leptin inhibits NPY/AgRP neurons and activates POMC/CART neurons (5). In fact, exogenous administration of leptin results in body weight loss, reduced adiposity, and appetite suppression (14).

Although the effect of estradiol on feeding behavior and body weight is well established, the mechanisms underlying these effects of estradiol on the central nervous system are unknown. To evaluate whether estradiol modulates responses to orexigenic or anorexigenic signals in the hypothalamus, the present study determined food intake, body weight changes and the hypothalamic expression of mediators involved in the control of energy homeostasis before and after food intake in ovariectomized rats with and without estradiol treatment.

Material and Methods

Animals

Female Wistar rats (200-230 g) were housed individually in metabolic cages and maintained in a room with constant temperature (23 ± 2°C) and under a 12/12-h light/dark cycle (dark onset: 18:00 h). Rats were given free access to rat chow and tap water. The estrous cycle was assessed with daily vaginal smears, and only those animals having three consecutive estrous cycles were included in this study.

All procedures performed were approved by Ethics Committee for Animal Use of the School of Medicine of Ribeirão Preto (Protocol #004/2006).

Effect of estradiol on food intake, body weight gain, plasma leptin levels, and hypothalamic mRNA expression

After three consecutive estrous cycles, the animals were subjected to bilateral ovariectomy under anesthesia with 2.5% 2.2.2-tribromoethanol (1 mL/100 g body weight, ip; Aldrich, USA). On the next day, rats were treated with estradiol cypionate (OVX+E, 10 µg/kg body weight, sc; Pfizer, Brazil) or vehicle (corn oil: 0.1 mL/rat, sc, OVX) between 9:00 and 10:00 h for 8 days. Food intake and body weight were determined daily during this period. On the 8th day, food was removed from the metabolic cage at 16:00 h and was returned to the cage at dark onset (18:00 h). One set of OVX and OVX+E rats was decapitated at 17:00 h (pre-feeding) and another subgroup of rats (OVX and OVX+E) was decapitated at 22:00 h (4 h after feeding). Trunk blood and brain tissues from these animals were collected under RNase-free conditions for the determination of plasma leptin levels and mRNA expression, respectively.

Measurement of plasma leptin levels

Plasma leptin concentration was measured by radioimmunoassay using the commercial kit Murine Leptin (Linco Research, USA) according to the manufacturer protocol. The assay sensitivity and intra-assay coefficient of variation were 0.5 ng/mL and 1.6%, respectively.

Microdissection

Coronal sections of 600 (PVN) and 1500 µm (ARC and LHA) thickness were cut from frozen brain in a cryostat. Punches were then immediately obtained based on the rat brain atlas coordinates of Paxinos and Watson (15). With a stainless steel punch needle 1.5 mm in diameter, bilateral PVN punches were taken from sections between -1.7 and -2.4 mm relative to the bregma, and bilateral ARC and LHA punches were taken from sections between -2.4 and 3.8 mm relative to the bregma. Punches containing the selected hypothalamic regions were stored at 4°C in RNAlater reagent (Ambion, USA) for a maximum of 48 h until RNA isolation. All procedures were performed under standard RNase-free conditions to avoid exogenous RNase contamination.

RNA isolation and cDNA synthesis

Total RNA was extracted using Trizol reagent (Invitrogen, USA) according to manufacturer instructions. Total RNA was quantified by spectrophotometric absorption at 260 nm (Eppendorf Biophotometer, USA). The quality and integrity of the isolated RNA were demonstrated using agarose gel (1.2%) stained with ethidium bromide. cDNA was synthesized from 250 ηg of total RNA using a high-capacity cDNA reverse transcription kit (Applied Biosystems, USA).

Quantitative real-time PCR

Quantitative real-time PCR was performed using Applied Biosystems 7500 real-time PCR system under the following conditions: initial denaturation at 95°C for 10 min during the first stage and a denaturation (95°C for 15 s) and annealing/extension (60°C for 1 min) steps for 40 amplification cycles in the second stage. The following Taqman^® Gene Expression Assays (Applied Biosystems, USA) were used in this study: Rn 01431703_g1 (AgRP), Rn 00567382_m1 (CART), Rn 01462137 (CRH), Rn 01491866_s1 (MC4-R), Rn 01410145_m1 (NPY), Rn 00561465_m1 (LepRb), Rn 00565995_m1 (orexin A), Rn 00595020_m1 (POMC), Rn 00561766_g1 (pro-MCH). Each PCR assay was performed in triplicate. Water instead of cDNA was used as a negative control. β-actin, glyceraldehyde-3-phosphate dehydrogenase and 18S rRNA were used as housekeeping genes to normalize the reactions. The relative quantitation was determined by the DDCT method. For each sample, the threshold cycle (Ct: the cycle at which emission intensity rises above baseline values) was determined and normalized to the average of housekeeping genes (DCT = Ct_unknown - Ct_{housekeeping genes}). The fold change of mRNA in the unknown sample (OVX+E at 17:00 or 22:00 h) relative to the respective control group (OVX at 17:00 or 22:00 h) was determined by 2^-DDCT, where DDCT = DCT_unknown - DCT_{control group}). Data are reported as relative mRNA expression to that of the control group.

Data analysis

Data are reported as means ± SEM. Statistical analysis was performed using the SigmaStat 3.1 software. Data concerning 24-h food intake, body weight gain and mRNA expression were analyzed by the unpaired t-test. Plasma leptin levels were analyzed by two-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls post hoc test. Differences between means were considered to be significant when P < 0.05.

Results

Food intake, body weight and plasma leptin levels

Estradiol treatment (N = 15) induced lower food intake (P < 0.01; Figure 1) and body weight gain compared to the untreated OVX group (N = 13; P < 0.01).

The OVX+E group showed higher plasma leptin levels compared to the OVX group at 17:00 h (pre-feeding: 2.4 ± 0.3 vs 1.6 ± 0.3 ηg/mL) and at 22:00 h (post-feeding: 3.7 ± 0.3 vs 2.5 ± 0.3 ηg/mL; Figure 1). Plasma leptin levels were higher (P < 0.05) at 22:00 h than at 17:00 h in both OVX and OVX+E groups.

Hypothalamic mRNA expression

Figure 2 shows a significant reduction (P < 0.05) in NPY and AgRP mRNA expression in the ARC at 17:00 h in estradiol-treated animals. In addition, estradiol treatment decreased CART mRNA expression in this nucleus both at 17:00 and 22:00 h. There was no difference in POMC or LepRb mRNA expression in the ARC at 17:00 and 22:00 h in either the OVX or OVX+E group.

LepRb, MC4-R, CRH, and CART mRNA expression in the PVN is shown in Figure 3. At 17:00 h, LepRb, MC4-R, CART, and mainly CRH mRNA expression levels were significantly higher in the OVX+E group compared to the OVX group. However, no significant difference in mRNA levels of these neuropeptides between the OVX and OVX+E groups was found at 22:00 h. Figure 4 shows LepRb, MC4-R, orexin A, pro-MCH, and CART mRNA expression in the LHA of OVX and OVX+E groups. There was no difference in the MC4-R and pro-MCH mRNA expression between the OVX and OVX+E groups, either at 17:00 or 22:00 h. It was also observed that LepRb and CART mRNA expression at 22:00 h were higher in the OVX+E group compared to the OVX group, with no difference at 17:00 h. Orexin A mRNA expression was lower at 17:00 h in the OVX+E rats, with no difference between groups at 22:00 h.

Figure 1.
Mean daily food intake (Panel A), total body weight gain (Panel B) after 8 days of treatment with oil (OVX, N = 13) or estradiol (OVX+E, N = 15) in ovariectomized rats. Plasma leptin levels (Panel C) in the pre-feeding (17:00 h) and post-feeding (22:00 h) periods of OVX (N = 7-8) and OVX+E (N = 7-8) groups. Data are reported as means ± SEM. *P < 0.05 (unpaired t-test or two-way ANOVA followed by the Student-Newman-Keuls post hoc test).

Figure 2.
Relative NPY, AgRP, CART, LepRb, and POMC mRNA expression in the arcuate nucleus at 17:00 h (pre-feeding) and 22:00 h (post-feeding) of ovariectomized rats with (OVX+E) and without estradiol treatment (OVX), N = 4-7 per group. Values are reported as means ± SEM. LepRb = leptin receptor (Panel A); NPY = neuropeptide Y (Panel B); AgRP = agouti-related protein (Panel C); CART = cocaine- and amphetamine-regulated transcript (Panel D); POMC = proopiomelanocortin (Panel E). *P < 0.05 (unpaired t-test).

Figure 3.
Relative LepRb, MC4-R, CART, and CRH mRNA expression in the paraventricular nucleus at 17:00 h (pre-feeding) and 22:00 h (post-feeding) of ovariectomized rats with (OVX+E) and without estradiol treatment (OVX), N = 4-8 per group. Data are reported as means ± SEM. LepRb = leptin receptor (Panel A); MC4-R = melanocortin-4 receptor (Panel B); CART = cocaine- and amphetamine-regulated transcript (Panel C); CRH = corticotropin-releasing hormone (Panel D). *P < 0.05 (unpaired t-test).

Figure 4.
Relative LepRb, MC4-R, orexin A, pro-MCH, and CART mRNA expression in the lateral hypothalamic area at 17:00 h (pre-feeding) and 22:00 h (post-feeding) of ovariectomized rats with (OVX+E) and without estradiol treatment (OVX), N = 5-8 per group. Data are reported as means ± SEM. LepRb = leptin receptor (Panel A); MC4-R = melanocortin-4 receptor (Panel B); orexin A (Panel C); pro-MCH = pro-melanin-concentrating hormone (Panel D); CART = cocaine- and amphetamine-regulated transcript (Panel E). *P < 0.05 (unpaired t-test).

Discussion

The present study demonstrated that estradiol treatment of OVX rats induced lower food intake, less body weight gain and lower plasma leptin levels both before and after feeding. Furthermore, the mRNA expression of several neuropetides in specific hypothalamic nuclei showed that estradiol can modify the mRNA expression of neuropeptides and receptors localized in the ARC, PVN and LHA.

The lower food intake and reduced body weight gain of the OVX+E group is in agreement with the well-established role of estradiol in modulating energy homeostasis. Indeed, variation of food intake and body weight of adult female rats can be correlated with the estrous cycle, with lower food intake during the peri-ovulatory phase (2).

We observed increased plasma leptin levels at 17:00 and 22:00 h in estradiol-treated animals. These data agree with a previous report (16), but differ from data presented in another study, which reports that estradiol treatment does not modify plasma leptin levels (17). The reason for this discrepancy may be related to the experimental protocols, duration of estradiol treatment and feeding status. To reinforce the notion that estrogens affect leptin secretion, it has been shown that estradiol has a direct effect on white adipocytes, increasing the synthesis and secretion of leptin (16), as well as its mRNA expression (18). The presence of estrogen receptors in adipocytes (19) provides a possible mechanism by which estrogen may modulate food intake, increasing leptin release.

We also observed increased plasma leptin levels in the post-feeding period (22:00 h), when compared to the pre-feeding period (17:00 h) in both groups (OVX and OVX+E). Leptin secretion is related to adiposity; however, leptin gene expression in white adipose tissue and circulating leptin are also regulated by acute or short-term stimulation (20,21). These data suggest that food intake may modulate leptin secretion and reinforce the notion that it may play a role in the process of satiety, modulating meal size (22). The increase in plasma leptin levels after feeding may be mediated, in part, by insulin, which directly stimulates the synthesis and secretion of leptin by adipocytes (23).

It has been demonstrated that estrogen-deficient rats show insensitivity to central leptin administration, which may contribute to their increased body weight (24). We observed an increase of LepRb mRNA expression in the PVN in the pre-feeding period (17:00 h) and in the LHA in the post-feeding period (22:00 h) in the OVX+E group. Using the whole hypothalamus, Rocha et al. (17) demonstrated an increase in LepRb mRNA expression in intact rats treated with estradiol. The abundance of leptin receptors is likely to be an important determinant of leptin sensitivity (25). Therefore, though no change of LepRb mRNA expression was observed in the ARC in the present study, increased expression of this receptor in the PVN and LHA might contribute to the lower food intake by OVX+E rats.

Our data showed that estradiol treatment reduced NPY and AgRP mRNA expression in the ARC that could be due to higher plasma leptin levels in the OVX+E group. This result is consistent with a report from Pelletier et al. (26), which showed that estradiol administration to OVX female mice reduced NPY mRNA expression in the ARC. The present results reinforce the idea that the interaction of leptin and estrogen signaling in hypothalamic regions is related to energy homeostasis (27,28). It is also important to point out that estradiol may also interact with the action of ghrelin on NPY neurons by attenuating the orexigenic effects of this peptide produced by the stomach (2).

In the ARC, we found a decrease of CART expression in OVX+E rats. CART neurons in the ARC have been shown to project to areas containing neurons that show immunoreactivity to gonadotropin-releasing hormone (GnRH) (29). Therefore, CART neurons from the ARC, under the influence of estradiol, might be involved in the control of the hypothalamic-pituitary-ovarian axis.

It is interesting to note that, in spite of the co-localization of CART and POMC in the ARC, CART was reduced in OVX+E rats, but there was no change in POMC mRNA expression in these rats. This result agrees with previous reports using different experimental models (30,31) that also showed a differential expression of CART and POMC mRNAs, suggesting a distinct intracellular regulation of these neuropeptides.

We observed a marked increase of CRH mRNA in the PVN of OVX+E rats during the pre-feeding period. CRH has also been shown to mediate the anorexigenic effects of leptin (32). Besides having anorexigenic effects, CRH has been shown to increase sympathetic nerve activity in brown adipose tissue involved in the control of thermogenesis (6) and also to mediate leptin actions on the expression of uncoupling protein mRNA in this tissue (33). Parvocellular PVN neurons sensitive to estrogen have been shown to possess reciprocal projections with neurons in the nucleus of the solitary tract, which participate in the PVN-hindbrain pathway involved in the control of energy homeostasis (2). Thus, our result suggests that estradiol, by increasing CRH mRNA expression in the PVN, may contribute to diminished food intake and to enhanced energy expenditure.

We also observed that estradiol increased CART gene expression in the PVN during the pre-feeding period. Wang et al. (34) showed that the injection of this neuropeptide into the PVN decreased NPY-induced feeding and body weight. Thus, our data indicate that the increased CART in the PVN could mediate the inhibitory effect of estradiol on food intake. In addition, estradiol treatment induced an increase in MC4-R mRNA expression in the PVN during the pre-feeding period. The activation of this receptor decreases food intake and also stimulates sympathetic outflow to brown adipose tissue and, consequently, thermogenesis (35). A decrease in MC4-R expression after food intake was also observed in the group treated with estradiol. This result is consistent with previous studies that demonstrated a down-regulation of this receptor after feeding (36,37). On the basis of the present study, we suggest that increased downstream MC4-R signaling may also contribute to the inhibition of food intake and the enhancement of energy expenditure induced by estradiol.

The hypophagic effect of estradiol was also associated with a reduction of orexin A mRNA expression in the LHA at 17:00 h, indicating that change in this neuropeptide expression also contributes to the decrease in food intake observed in OVX+E rats. There is substantial evidence demonstrating that orexin has other physiological effects, being involved in the control of sexual behavior and GnRH and LH release (38). The significance of the differential expression of orexin mRNA found in the present study and its participation in the hypothalamic-pituitary-ovarian axis remains to be established. We observed that in the LHA there was no difference in MCH mRNA expression, but there was higher CART expression at 22:00 h in the OVX+ E group. It was shown that CART colocalizes extensively with MCH in the LHA (39). Though the functions of CART in this area are not well established, it could counteract the orexigenic effect of MCH (39).

The results of the present study provide evidence that estradiol affects energy homeostasis through its effects on food intake, which are mediated by peripheral factors, such as leptin released by white adipose tissue and by differential expression of hypothalamic neuropeptides.

References

1. Wade GN. Gonadal hormones and behavioral regulation of body weight. Physiol Behav 1972; 8: 523-534.

2. Butera PC. Estradiol and the control of food intake. Physiol Behav 2010; 99: 175-180.

3. Asarian L, Geary N. Modulation of appetite by gonadal steroid hormones. Philos Trans R Soc Lond B Biol Sci 2006; 361: 1251-1263.

4. Tchernof A, Poehlman ET, Despres JP. Body fat distribution, the menopause transition, and hormone replacement therapy. Diabetes Metab 2000; 26: 12-20.

5. Woods SC, D’Alessio DA. Central control of body weight and appetite. J Clin Endocrinol Metab 2008; 93: S37-S50.

6. LeFeuvre RA, Rothwell NJ, Stock MJ. Activation of brown fat thermogenesis in response to central injection of corticotropin releasing hormone in the rat. Neuropharmacology 1987; 26: 1217-1221.

7. Marsh DJ, Weingarth DT, Novi DE, Chen HY, Trumbauer ME, Chen AS, et al. Melanin-concentrating hormone 1 receptor-deficient mice are lean, hyperactive, and hyperphagic and have altered metabolism. Proc Natl Acad Sci U S A 2002; 99: 3240-3245.

8. Sakurai T, Amemiya A, Ishii M, Matsuzaki I, Chemelli RM, Tanaka H, et al. Orexins and orexin receptors: a family of hypothalamic neuropeptides and G protein-coupled receptors that regulate feeding behavior. Cell 1998; 92: 573-585.

9. Korczynski W, Ceregrzyn M, Matyjek R, Kato I, Kuwahara A, Wolinski J, et al. Central and local (enteric) action of orexins. J Physiol Pharmacol 2006; 57 (Suppl 6): 17-42.

10. Qu D, Ludwig DS, Gammeltoft S, Piper M, Pelleymounter MA, Cullen MJ, et al. A role for melanin-concentrating hormone in the central regulation of feeding behaviour. Nature 1996; 380: 243-247.

11. Fan W, Boston BA, Kesterson RA, Hruby VJ, Cone RD. Role of melanocortinergic neurons in feeding and the agouti obesity syndrome. Nature 1997; 385: 165-168.

12. Huszar D, Lynch CA, Fairchild-Huntress V, Dunmore JH, Fang Q, Berkemeier LR, et al. Targeted disruption of the melanocortin-4 receptor results in obesity in mice. Cell 1997; 88: 131-141.

13. Zhang Y, Proenca R, Maffei M, Barone M, Leopold L, Friedman JM. Positional cloning of the mouse obese gene and its human homologue. Nature 1994; 372: 425-432.

14. Pelleymounter MA, Cullen MJ, Baker MB, Hecht R, Winters D, Boone T, et al. Effects of the obese gene product on body weight regulation in ob/ob mice. Science 1995; 269: 540-543.

15. Paxinos G, Watson C. The rat brain in stereotaxic coordinates. San Diego: Academic Press; 1997.

16. Brann DW, De Sevilla L, Zamorano PL, Mahesh VB. Regulation of leptin gene expression and secretion by steroid hormones. Steroids 1999; 64: 659-663.

17. Rocha M, Bing C, Williams G, Puerta M. Physiologic estradiol levels enhance hypothalamic expression of the long form of the leptin receptor in intact rats. J Nutr Biochem 2004; 15: 328-334.

18. Machinal F, Dieudonne MN, Leneveu MC, Pecquery R, Giudicelli Y. In vivo and in vitro ob gene expression and leptin secretion in rat adipocytes: evidence for a regional specific regulation by sex steroid hormones. Endocrinology 1999; 140: 1567-1574.

19. Wade GN, Gray JM. Cytoplasmic 17 beta-[³H]estradiol binding in rat adipose tissues. Endocrinology 1978; 103: 1695-1701.

20. Grill HJ. Leptin and the systems neuroscience of meal size control. Front Neuroendocrinol 2010; 31: 61-78.

21. Germano CM, de Castro M, Rorato R, Laguna MT, Antunes-Rodrigues J, Elias CF, et al. Time course effects of adrenalectomy and food intake on cocaine- and amphetamine-regulated transcript expression in the hypothalamus. Brain Res 2007; 1166: 55-64.

22. Saladin R, De Vos P, Guerre-Millo M, Leturque A, Girard J, Staels B, et al. Transient increase in obese gene expression after food intake or insulin administration. Nature 1995; 377: 527-529.

23. Barr VA, Malide D, Zarnowski MJ, Taylor SI, Cushman SW. Insulin stimulates both leptin secretion and production by rat white adipose tissue. Endocrinology 1997; 138: 4463-4472.

24. Ainslie DA, Morris MJ, Wittert G, Turnbull H, Proietto J, Thorburn AW. Estrogen deficiency causes central leptin insensitivity and increased hypothalamic neuropeptide Y. Int J Obes Relat Metab Disord 2001; 25: 1680-1688.

25. Elmquist JK, Maratos-Flier E, Saper CB, Flier JS. Unraveling the central nervous system pathways underlying responses to leptin. Nat Neurosci 1998; 1: 445-450.

26. Pelletier G, Li S, Luu-The V, Labrie F. Oestrogenic regulation of pro-opiomelanocortin, neuropeptide Y and corticotrophin-releasing hormone mRNAs in mouse hypothalamus. J Neuroendocrinol 2007; 19: 426-431.

27. Shughrue PJ, Lane MV, Merchenthaler I. Comparative distribution of estrogen receptor-alpha and -beta mRNA in the rat central nervous system. J Comp Neurol 1997; 388: 507-525.

28. Gao Q, Horvath TL. Cross-talk between estrogen and leptin signaling in the hypothalamus. Am J Physiol Endocrinol Metab 2008; 294: E817-E826.

29. Rondini TA, Baddini SP, Sousa LF, Bittencourt JC, Elias CF. Hypothalamic cocaine- and amphetamine-regulated transcript neurons project to areas expressing gonadotropin releasing hormone immunoreactivity and to the anteroventral periventricular nucleus in male and female rats. Neuroscience 2004; 125: 735-748.

30. Borges BC, Antunes-Rodrigues J, Castro M, Bittencourt JC, Elias CF, Elias LL. Expression of hypothalamic neuropeptides and the desensitization of pituitary-adrenal axis and hypophagia in the endotoxin tolerance. Horm Behav 2007; 52: 508-519.

31. Vrang N, Larsen PJ, Tang-Christensen M, Larsen LK, Kristensen P. Hypothalamic cocaine-amphetamine regulated transcript (CART) is regulated by glucocorticoids. Brain Res 2003; 965: 45-50.

32. Uehara Y, Shimizu H, Ohtani K, Sato N, Mori M. Hypothalamic corticotropin-releasing hormone is a mediator of the anorexigenic effect of leptin. Diabetes 1998; 47: 890-893.

33. Masaki T, Yoshimichi G, Chiba S, Yasuda T, Noguchi H, Kakuma T, et al. Corticotropin-releasing hormone-mediated pathway of leptin to regulate feeding, adiposity, and uncoupling protein expression in mice. Endocrinology 2003; 144: 3547-3554.

34. Wang C, Billington CJ, Levine AS, Kotz CM. Effect of CART in the hypothalamic paraventricular nucleus on feeding and uncoupling protein gene expression. Neuroreport 2000; 11: 3251-3255.

35. Haynes WG, Morgan DA, Djalali A, Sivitz WI, Mark AL. Interactions between the melanocortin system and leptin in control of sympathetic nerve traffic. Hypertension 1999; 33: 542-547.

36. Shinyama H, Masuzaki H, Fang H, Flier JS. Regulation of melanocortin-4 receptor signaling: agonist-mediated desensitization and internalization. Endocrinology 2003; 144: 1301-1314.

37. Germano CM, Castro M, Rorato R, Costa DB, Antunes-Rodrigues J, Elias CF, et al. Downregulation of melanocortin-4 receptor during refeeding and its modulation by adrenalectomy in rats. Horm Metab Res 2008; 40: 842-847.

38. Russell SH, Small CJ, Kennedy AR, Stanley SA, Seth A, Murphy KG, et al. Orexin A interactions in the hypothalamo-pituitary gonadal axis. Endocrinology 2001; 142: 5294-5302.

39. Vrang N. Anatomy of hypothalamic CART neurons. Peptides 2006; 27: 1970-1980.

Acknowledgments

The authors thank Maria Valci Aparecida dos Santos Silva, Marina Holanda, and Rubens Fernando de Melo for excellent technical assistance. Research supported by FAPESP, CAPES and CNPq.

Address for correspondence: L.L.K. Elias, Departamento de Fisiologia, FMRP, USP, Avenida Bandeirantes, 3900, 14049-900 Ribeirão Preto, SP, Brasil. Fax: [phone]. E-mail: [e-mail]

Received December 7, 2009. Accepted May 25, 2010. Available online July 2, 2010. Published August 13, 2010.

The Brazilian Journal of Medical and Biological Research is partially financed by

Correspondence and Footnotes

Publication Dates

• Publication in this collection
  17 Aug 2010
• Date of issue
  Aug 2010

History

• Accepted
  25 May 2010
• Received
  07 Dec 2009