Lateral hypothalamic serotonin is not stimulated during central leptin hypophagia
Mônica Marques Telles
a,b, Thaís Girão da Silva
a, Regina Lúcia Harumi Watanabe
a,
Iracema Senna de Andrade
a, Debora Estadella
a, Cláudia Maria Oller Nascimento
a,
Lila Missae Oyama
a, Eliane Beraldi Ribeiro
a,⁎
aDepartamento de Fisiologia, Disciplina de Fisiologia da Nutrição, Universidade Federal de São Paulo (UNIFESP), São Paulo/SP, Brazil bDepartamento de Ciências Biológicas, Universidade Federal de São Paulo (UNIFESP), Diadema/SP, Brazil
a b s t r a c t
a r t i c l e
i n f o
Article history:
Received 2 February 2012
Received in revised form 12 September 2012 Accepted 3 March 2013
Available online 19 March 2013
Keywords: Adipokines Food intake In vivo microdialysis Leptin
Serotonin
Whether leptin targets the hypothalamic serotonergic system to inhibit food intake is not established. We examined the effect of a short-term i.c.v. leptin treatment on serotonin microdialysate levels in rat lateral hypothalamus. Adipose tissue gene expression was also evaluated.
Male rats received four daily injections of leptin (5μg) or vehicle (with pair-feeding to leptin-induced intake) and afifth injection during collection of LH microdialysates. We found that serotonin and 5-HIAA levels were not affected by the leptin pre-treatment, as basal levels were similar between the leptin and the pair-fed group. These levels remained unaltered after the acute leptin injection.
For gene expression studies, rats were pre-treated withfive daily injections of either leptin (5μg) or vehicle (with either pair-feeding or ad libitum intake). mRNA levels of resistin, adiponectin, lipoprotein lipase, and PPAR-gamma were unaltered by either leptin or pair-feeding. Leptin gene expression was significantly re-duced by leptin but not by pair-feeding, in both the retroperitoneal (−74%) and the epididymal (−99%) de-pots while no differences were observed in the subcutaneous depot.
The observations confirmed the absence of an acute stimulatory effect of central leptin on serotonin release in the lateral hypothalamus and showed that the pre-treatment with leptin failed to modify this pattern. This indicates that components of the serotonergic system are probably not directly affected by leptin. Addition-ally, the central effect of leptin was able to downregulate its own adipose tissue gene expression in a depot-specific manner while other adipokine genes were not affected.
© 2013 Elsevier B.V.
1. Introduction
It is well recognized that the adipocyte hormone leptin plays a pivotal role in signaling to the central nervous system (CNS) infor-mation about the amount of energy stored in white adipose tissue
(WAT), thus influencing the central mechanisms involved in the
maintenance of energy balance. This effect results, at least in part, from the stimulatory effect of leptin on central anorexigenic factors such as proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART), and neurotensin, with a concomitant inhibitory effect on central orexigenic factors such as neuropeptide Y (NPY), agouti related peptide (AgRP), melanin-concentrating
hor-mone (MCH) and orexins[1,2].
The neurotransmitter serotonin has catabolic effects on energy homeostasis, reducing food intake and body weight and stimulating energy expenditure[3–6]. Several lines of studies have examined a
putative role of serotonin as one catabolic system targeted by leptin, thus contributing to leptin hypophagia. The results are so far highly inconclusive.
Serotonergic raphe neurons have been shown to lack the long iso-form of leptin receptors (Ob-Rb)[7]and serotonin turnover was un-affected in several brain areas of normal rats after 3 days of intraperitoneal leptin infusion[8]. Basal and potassium-stimulated serotonin overflow were unaltered by the addition of leptin to
syn-aptosomes or superfusion preparations of rat hypothalami[9,10].
These findings indicated that leptin did not induce serotonergic
stimulation.
However, opposite results have been reported. Serotonin raphe neurons have been found to express Ob-Rb[11–14], peripheral leptin has been shown to activate Ob-Rb signal transduction in brainstem and lateral hypothalamic sites[15,16], and leptin has been shown to stimulate hypothalamic serotonin turnover[17,18]. These data favor a stimulatory role of leptin on the serotonergic system.
Functional studies have also led to contradictoryfindings, as leptin hypophagia has been found to involve activation of 5-HT2B and
5-HT2C receptors [19,20]while it reportedly failed to be affected
after serotonergic depletion or 5-HT2C receptor knockout[21]. ⁎ Corresponding author at: Universidade Federal de São Paulo, UNIFESP, Departamento de
Fisiologia, Rua Botucatu, 862, 2° andar, Vila Clementino, São Paulo, SP 04023-062, Brazil. Tel./Fax: +55 11 5573 9525.
E-mail address:[email protected](E.B. Ribeiro).
0167-0115 © 2013 Elsevier B.V.
http://dx.doi.org/10.1016/j.regpep.2013.03.027
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Regulatory Peptides
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Open access under the Elsevier OA license.
We have previously demonstrated that one single central leptin in-jection failed to“per se”alter serotonin extracellular levels in the lateral hypothalamus (LH) while it exacerbated the feeding-induced serotonin levels. Thesefindings suggested that, rather than interacting directly with components of the serotonergic system, leptin probably targeted the mechanisms involved in the serotonergic-stimulating effect of food[22].
In view of the controversial findings mentioned above, we
performed the present study with the objective of investigating further the ability of leptin to interact with the serotonergic system. To specifi -cally evaluate whether a series of repeated leptin central injections would evidence an effect independent of food intake, we measured ex-tracellular levels of 5-HT and its metabolite 5-hidroxyindolacetic acid, in the lateral hypothalamus of rats pre-treated with leptin or vehicle for 4 days. We chose the in vivo brain microdialysis technique, which allows a direct measurement of 5-HT extracellular levels in the awake animal.
Besides the central effect of leptin on neurotransmitters and neuro-peptides involved in energy balance, it has been postulated that central leptin also modulates WAT activity. A recent study demonstrated that a 7-day central leptin infusion up-regulated WAT mRNA and protein levels of resistin but failed to modify resistin plasma levels[23]. In addi-tion, it has been demonstrated that leptin inhibited its own expression in adipocytes via beta 3 adrenoceptor-mediated sympathetic activity [24]. Thesefindings agree with other studies showing that the central effect of leptin on WAT activity or metabolism occurred via the sympa-thetic nervous system[25–27]. It is possible that central leptin modu-lates WAT gene expression of other adipokines involved in energy metabolism.
Additional experiments were then performed to evaluate the ef-fect of the leptin treatment on leptin, adiponectin, resistin, PPARγ and lipoprotein lipase gene expressions, in epididymal, retroperitone-al and subcutaneous depots of white adipose tissue.
2. Materials and methods
2.1. Animals and surgery
The Committee on Animal Research Ethics of the Federal University of São Paulo approved the procedures used in the present experiments.
Since weaning, male Wistar rats were housed five per cage and
maintained in controlled conditions of lighting (12 h light: 12 h dark) and temperature (24 ± 1 °C), with free access to standard balanced rat chow (4% fat, 22% protein, Nuvital Nutrients, Columbo, PR, Brazil) and water.
At 12 to 14 weeks of age the animals used for microdialysis experi-ments were anesthetized with ketamine/xylaxine (67/13 mg/kg) and
stereotaxically [28]implanted with both a 21-gauge guide cannula
aimed at the right lateral hypothalamus (A−2.5 mm, L−1.6 mm, V
−7.7 mm from bregma) and a 23-gauge cannula aimed at the left
later-al ventricle (A−0.9 mm, L +1.6 mm, V−2.6 mm from bregma). For
the animals used for northern blotting experiments, a single brain can-nula was implanted, aimed at the left lateral ventricle. The cancan-nulas were secured to the skull with screws and dental cement and the ani-mals were individually caged thereafter.
2.2. Microdialysis experiments
Four days after surgery, the animals were divided in 2 groups. The leptin group received 4 daily i.c.v. injections (5μL) of 5.0μg of leptin and was fed ad libitum. The pair-fed control group consisted of ani-mals receiving 4 daily i.c.v. injections of vehicle (CSF, artificial
cere-brospinal fluid) and being pair-fed to the leptin animals. Food
intake was recorded 24 h after each injection and body weight was recorded 24 h after thefirst 3 injections. The injections were performed around noon.
At 18:00 h, after the fourth injection, a concentric custom-constructed microdialysis probe (1.5 mm of effective membrane length) was inserted through the hypothalamic cannula andfixed to it with a small drop of dental cement. The details of probe construction have been previously de-scribed[29,30]. The animals were then connected to a swivel system, which allowed continuous probe perfusion with CSF by a microperfusion pump (Carnegie Medicin, Solna, Sweden). CSF composition was: 145 mM NaCl, 2.7 mM KCl, 1.0 mM MgCl2, 1.2 mM CaCl2, 2.0 mM Na2HPO4,
pH 7.4. Overnight perfusion was performed at 1.0μL/min.
At 6:00 h, probeflow rate was adjusted to 2.5μL/min and food was withdrawn from the cage, so that the animals were fasted for 6 h before the microdialysis experiments. Collection of 20-min dialysate samples was started around noon. Samples were collected into 10μL of 0.5 M perchloric acid and immediately injected into an HPLC system. Baseline samples were collected until 5-HT levels were stable, the last three sam-ples being averaged to yield the mean baseline level (100% value). 5μL of CSF vehicle was then injected through the i.c.v. cannula and three ad-ditional samples were collected (samples V1–V3). An i.c.v. injection of 5μg of leptin was then administered to all animals. Nine additional
20-min dialysate samples were collected (samples L1–L9). No food
was provided during microdialysate sampling.
2.3. HPLC analysis
Dialysate levels of 5-HT and 5-HIAA were measured by high per-formance liquid chromatography with electrochemical detection. The system (ESA Inc., Chelmsford, Mass.) consisted of a model 580
pump with two PEEK pulse dampers in series, a 50μL Rheodyne
PEEK sample loop, a 3μm MD150 C column, a model 5020 guard
cell set at 300 mV, a model 5014B analytical cell set at−175 and
150 mV, and a model 5200A detector. The mobile phase consisted
of 75 mM sodium phosphate, 1.5 mM octanesulfonic acid, 50μM
EDTA, 100μL/L triethylamine, and 10% v/v acetonitrile at pH 3.0.
The flow rate was 0.6 mL/min. The detection limit for 5-HT was
1.5 pg/50μL at a signal to noise ratio of 3:1.
2.4. Histological analysis
For verification of microdialysis membrane and i.c.v. cannula posi-tioning, at the termination of the experiments all animals were deeply anesthetized and perfused with 0.9% saline followed by 10% formalin and 40μm brain sections were examined under a microscope, following staining with Cresyl Violet. Only data from rats with correct probe and cannula placements were included in the analysis.
2.5. Northern blotting analysis
Three groups were performed for northern blot experiments. One group received 6 daily i.c.v. injections (5μL) of 5.0μg of leptin and was fed ad libitum (leptin group). Two control groups were performed, consisting of animals receiving 6 daily i.c.v. injections of vehicle (CSF, ar-tificial cerebrospinalfluid) and either fed ad libitum (Ad libitum group) or pair-fed to the leptin rats (pair-fed group). The injections were performed around noon. After thefifth injection, at 6:00 a.m., food was withdrawn and, at noon, the sixth injection was administered. One hour later, the animals were anesthetized, killed by decapitation, and samples from epididymal, retroperitoneal and subcutaneous white adipose tissue depots were quickly removed and inserted into vials containing RNA later® (Ambion) and frozen at−70 °C. RNA
ex-traction was performed according to the method described by[31].
Adipokines mRNA levels were measured by Northern blotting using an-tisense oligonucleotide probes in conjunction with chemiluminescence detection[32]. Signals were collected by exposure of the membrane to
X-ray film and autoradiographs were quantitated by densitometry
with image-analysis software (MCID Basic Software—Image Research
Probes (Oswell DNA Services, UK) sequences were as follows:
– leptin: 5′-GGTCTGCGGCAGGGAGCAGCTCTTGGAGAAGGC-3′
– adiponectin: 5′-GTTGCAGTGGAATTTGCCAGTGCTGCCGTCA-3′
– resistin: 5′-CGGACGTCCCATGAGCCACAGCCAGAGCCA-3′
– lipoprotein lipase: 5′-GCCAGCAGCATGGGCTCCAAGGCTGTACCC-3′
– PPARγ: 5′-CACGTTCAGCAAGCCTGGGCGGTCTCCACT-3′
– 18S rRNA: 5′-CGCCTGCTGCCTTCCTTGGATGTGGTAGCCG.
2.6. Statistical analysis
The results are given as Means ± S.E.M. 5-HT and 5-HIAA data were expressed as percentage of the mean baseline microdialysate level of each animal. The data were submitted to ANOVA for repeated measures followed by Tukey's post hoc test, for comparisons among the samples collected throughout the experimental period, in the same group. For comparison between pair-fed and leptin groups, in the same sample, the Student'st-test was used. Northern Blotting data were expressed as densitometric arbitrary units of target mRNA/18S rRNA signal ratio. The data were submitted to Kruskal Wallis analysis of variance followed by Dunn's post hoc test. Significance was set at pb0.05.
3. Results
3.1. Microdialysis experiments
Brain histological analysis showed that the microdialysis membranes were correctly positioned in the LH, ranging from−2.3 to−2.8 mm from bregma, in the 11 rats analyzed in the present study (Fig. 1).
Fig. 2shows that daily food intake was similar between the pair-fed and the leptin groups throughout the treatment period. After 4 daily leptin injections, the mean food intake (12.2 ± 3.3 g) tended to be lower than that after thefirst injection (20.4 ± 2.1 g), but no statistical
significance was reached (p = 0.246). Body weight gain was also
similar between the pair-fed and the leptin rats, both showing weight loss during treatments.
Baseline levels of 5-HT and 5-HIAA in LH microdialysates were similar between leptin and pair-fed groups (Table 1).Fig. 3shows 5-HT and 5-HIAA levels in 20-min. LH microdialysate samples collected at baseline and after the injection of CSF-vehicle or leptin. It can be seen that 5-HT levels failed to be significantly modified by either injection in leptin-treated (F(12,75)= 0.029) or vehicle-treated pair-fed rats (F(12,62)=
0.273). 5-HIAA levels were not modified after leptin injection in either group, i.e., the pre-treated with leptin (F(12,77)= 0.156) or the pair-fed
group (F(12,64)= 0.104).
3.2. Gene expression experiments
Fig. 4shows that the daily food intakes were similar between the pair-fed and the leptin groups, except at day 4. Leptin inhibited food intake since the second injection day, with a maximal 54% reduction occurring at day 5. Body mass gain was similar among the groups at days 1, 2, and 3. At day 4, the pair-fed and leptin animals had signif-icantly lower body mass gain that of the ad libitum group.
Fig. 5shows that the short-term leptin i.c.v. treatment led to re-duction of leptin gene expression in both epididymal (99%) and retro-peritoneal (74%) adipose tissue depots in comparison to either ad libitum or pair-fed groups, but failed to do so in the subcutaneous depot (χ2= 0.961; p = 0.634). No significant differences were ob-served between ad libitum and pair-fed rats. Gene expression of adiponectin, resistin, LPL, and PPARγwere similar among the groups in all tissues (data not shown).
4. Discussion
Thefirst aim of the present study was to evaluate the effect of a short pre-treatment with leptin on the LH serotonin extracellular levels induced by an acute leptin injection. During the pre-treatment
Bregma -2.56 mm
with leptin, food intake was inhibited and the intake of the pair-fed group was thus adjusted to match this effect.
Basal levels of both serotonin and 5-HIAA in LH microdialysates were similar between the leptin pre-treated and the pair-fed animals. Data about the effect of feeding status on the serotonergic system are controversial. We have previously reported that the ingestion of food acutely stimulated the serotonergic system in the LH of normal rats, in relation to fasting levels [22,29,33]. Serotonin release from rat raphe tissue in vitro has been shown to be unchanged after 48 h of fasting but enhanced after 72 h[34]while a 72-hour fast failed to change rat brain serotonin turnover[8]. Some studies have associated partial food restriction with lowering of central serotonergic activity, such as decreased hypothalamic 5-HT turnover in rats and decreased brainL-tryptophan availability in dieting human females[35,36]. In
light of these observations, it is possible that the pair-feeding regimen used in the present study caused a reduction in baseline serotonergic activity and that leptin failed to further modify this effect.
After the collection of basal microdialysates, both the leptin pre-treated and the vehicle pre-treated animals received one i.c.v. in-jection of vehicle. Serotonin levels remained stable in the three microdialysate samples collected after vehicle injection. This present observation in previously food-restricted (pair-fed) rats agrees with that from our early work, in which 5-HT also remained stable in ad libitum fed rats[22].
We next evaluated whether one leptin i.c.v. injection would acute-ly affect the LH serotonergic activity. We failed to observe alterations
in either serotonin or 5-HIAA levels, regardless that the animals had been pre-treated with leptin or vehicle for 4 days. The results
ob-served in the pair-fed group confirm our previous results in ad
libitum rats in which one single i.c.v injection of leptin failed to affect hypothalamic 5-HT release[22]. Since we have previously been able to detect shifts in 5-HT extracellular levels in response to different treatments, using the same methodological settings as used in the present experiments[4,22,29,33,37], it is assured that the present ob-servation is of physiological significance.
Some lines of evidence indicated that serotonin could be one cat-abolic system directly targeted by leptin while other data pointed to a lack of such an interaction. For example, serotonin neurons of the mesencephalic raphe nuclei have been found to express leptin recep-tors[11–13]but Ob-Rb reportedly did not co-localize with serotonin in dorsal raphe neurons[21]. Additionally, serotonin release by rat synaptosomes or superfused hypothalami were unaltered by the ad-dition of leptin[9,10]and serotonin turnover was unaffected in sever-al brain areas of normsever-al rats after 3 days of constant intraperitonesever-al leptin infusion[8]. On the other hand, chronic i.c.v. leptin decreased serotonin transporter binding sites in the frontal cortex, although
not in the raphe nuclei[38]. Moreover, down regulation of dorsal
raphe serotonin transporter expression has been associated with lep-tin deficiency[39].
We have previously observed that, while not able to per se modify serotonin extracellular levels in the rat LH, one leptin central injection caused an exacerbation of the feeding-induced serotonin release, probably by interacting with other mechanisms linking feeding to Leptin
Pair-fed 0
5 10 15 20 25 30 35
1 2 3 4 Days
Food intake (g/day)
1 2 3
-15 -10 -5 0 5 10 15
Days
Body weight gain (g/day)
a
b
Fig. 2.Food intake (a) and body mass gain (b) measured after 1, 2, 3, or 4 daily i.c.v. injections of either 5μg of leptin followed by ad libitum feeding (n = 6) or vehicle followed by pair-feeding to leptin rats (n = 5). Values are means ± S.E.M.
Table 1
Mean basal dialysate levels of 5-HT (pg/50μL) and 5-HIAA (ng/50μL) of leptin-treated (n = 6) and pair-fed animals (n = 5). Values are means ± S.E.M.
5-HT (pg/50μL) 5-HIAA (ng/50μL)
Leptin 2.55 ± 0.75 3.70 ± 1.03
Pair-fed 3.39 ± 0.58 4.14 ± 1.03
5-HIAA (% baseline) CSF Lep
0 40 80 120 160 200
B V1 V2 V3 L1 L2 L3 L4 L5 L6 L7 L8 L9
5-HT (% baseline)
Lep CSF
0 40 80 120 160 200
B V1 V2 V3 L1 L2 L3 L4 L5 L6 L7 L8 L9 Pairfed Leptin
Pairfed Leptin
b
a
serotonergic activation. We concluded that serotonin contributed to leptin hypophagia, although leptin affected the serotonin system probably via indirect mechanisms[22]. In the present study, after a series of repeated injections, we failed tofind evidence supporting the existence of leptin's effects on serotonergic function independent of food intake, at least reflected in LH extracellular levels.
Central leptin actions have been shown to include a modulation of white adipose tissue function. In the present study, we evaluated whether the short term i.c.v. leptin treatment was able to modify white adipose tissue expression of genes involved in the maintenance of energy balance and/or regulation of metabolism.
We found leptin gene expression to be reduced by central leptin in both the retroperitoneal and the epididymal adipose tissue depots. Thesefindings agree with previous data[25,27]and support the sug-gestion that, at least in these depots, a homeostatic mechanism may be operative to control central leptin activity by negative feedback.
That this was a depot-specific effect was indicated by the lack of an effect on leptin gene expression in the subcutaneous depot, simi-larly to previous work[40]. It is interesting that several studies have found the subcutaneous depot to present a different response in rela-tion to the other depots analyzed, with respect to gene expression profile in obese individuals, depot size in growth-restricted newborn infants and fasting effect in rats[40–42].
The effects of central leptin on adipose tissue metabolism have been proposed to be mediated by the sympathetic nervous system, based on several lines of evidence, including the demonstrations
that fat pad denervation orβ3-adrenergic receptors gene knockout
or dopamineβ-hydroxilase gene knockout abolished central leptin
effects on lipogenic enzymes and on ob gene expression in WAT [25–27]. Moreover, the lack of a rapid effect of intraperitoneal leptin has been taken as to suggest an indirect mechanism for the downregulation by leptin of its own gene expression in WAT[43].
Alternatively, central leptin-induced downregulation of ob gene expression in white adipose tissue could have resulted from a direct effect on the tissue, after leakage of leptin to the systemic circulation during the six daily intracerebroventricular injections performed, of
5μg/rat/day. Although we did not determine serum leptin levels,
other authors have reported that i.c.v. leptin failed to modify these levels after dosages higher than the one used in the present study. In-deed, it has been shown that leptin infusion in the lateral cerebral ventricle of rats, at the dosage of 12μg/day per 4 days, failed to in-crease serum leptin levels, indicating no leakage of the hormone into the circulating blood[44]. Thesefindings corroborate the notion that, rather than a direct stimulation of WAT leptin receptors, the re-sults of the present experiments are likely to reflect an indirect action of leptin.
Our results have also shown that central leptin failed to modify adiponectin, LPL, resistin and PPARγgene expression. Available data about leptin effects on these adipose tissue genes are limited and conflicting. Epididymal fat expression of resistin and adiponectin
0 0.5 1 1.5 2
AU
Ad libitum Pair-fed Leptin
0 0.75 1.5 2.25
AU
*
#
0 0.5 1 1.5
AU
*
#
a
b
c
Ad libitum Pair-fed Leptin
Ad libitum Pair-fed Leptin
Fig. 5.Leptin gene expression, expressed as arbitrary units (AU), in (a) epididymal (n = 6), (b) subcutaneous (n = 4–6) and (c) retroperitoneal (n = 3–5) white adi-pose tissue of leptin, pair-fed and ad libitum groups. *pb0.05 vs. ad libitum; # pb0.05 vs. pair-fed.
Ad Libitum Pair-fed Leptin
a
b
-15 -10 -5 0 5 10 15
Days
Body weight gain (g/day)
1 2 3 4
*
*
0 5 10 15 20 25 30 35
1 2 3 4 5 Days
Food intake (g/day)
*
*
*
*
*
*
*
*
# §
§
was unaffected while subcutaneous fat resistin but not adiponectin expression was diminished after 4 daily peripheral leptin injections [40]. Epididymal but not retroperitoneal fat expression of resistin was increased after 7 daily central leptin injections[23]. Further stud-ies are warranted in order to clarify the role of central leptin on these white adipose tissue genes.
In summary, the present data confirmed previous data showing
that leptin per se did not have an acute stimulatory effect on seroto-nin release in the lateral hypothalamus and extended thefindings to demonstrate that the pre-treatment with the protein did not modify this pattern. Additionally, the central effect of leptin was able to mod-ulate its own adipose tissue gene expression in a depot-specific man-ner while other adipokine genes were not affected.
Acknowledgments
The gene expression experiments were performed at the Obesity Biology Research Unit, University of Liverpool, UK, in the laboratory of Professor Paul Trayhurn, to whom the authors are indebted.
The authors are grateful for the worthy support given by Leif Hunter, Dr Bohan Wang and Dr Muhammad R. Peeraully.
This study was supported by grants from the State of Sao Paulo Re-search Foundation (FAPESP, Brazil), the National Council for Scientific and Technological Development (CNPq, Brazil), and the Coordination for the Improvement of Higher Education Personnel (Capes, Brazil).
The authors declare no conflicts of interest.
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