Pflügers Archiv - European Journal of Physiology

, Volume 465, Issue 12, pp 1687–1699

The central administration of C75, a fatty acid synthase inhibitor, activates sympathetic outflow and thermogenesis in interscapular brown adipose tissue

Authors

  • Priscila Cassolla
    • Department of Physiology, School of Medicine of Ribeirão PretoUniversity of São Paulo
  • Ernane Torres Uchoa
    • Department of Physiology, School of Medicine of Ribeirão PretoUniversity of São Paulo
  • Frederico Sander Mansur Machado
    • Department of Physiology and Biophysics, Institute of Biological SciencesFederal University of Minas Gerais
  • Juliana Bohnen Guimarães
    • Department of Physiology and Biophysics, Institute of Biological SciencesFederal University of Minas Gerais
  • Maria Antonieta Rissato Garófalo
    • Department of Physiology, School of Medicine of Ribeirão PretoUniversity of São Paulo
  • Nilton de Almeida Brito
    • Department of Physiological Sciences, Biological Sciences CenterState University of Maringá
  • Lucila Leico Kagohara Elias
    • Department of Physiology, School of Medicine of Ribeirão PretoUniversity of São Paulo
  • Cândido Celso Coimbra
    • Department of Physiology and Biophysics, Institute of Biological SciencesFederal University of Minas Gerais
  • Isis do Carmo Kettelhut
    • Department of Biochemistry and Immunology, School of Medicine of Ribeirão PretoUniversity of São Paulo
    • Department of Physiology, School of Medicine of Ribeirão PretoUniversity of São Paulo
Integrative physiology

DOI: 10.1007/s00424-013-1301-5

Cite this article as:
Cassolla, P., Uchoa, E.T., Mansur Machado, F.S. et al. Pflugers Arch - Eur J Physiol (2013) 465: 1687. doi:10.1007/s00424-013-1301-5

Abstract

The present work investigated the participation of interscapular brown adipose tissue (IBAT), which is an important site for thermogenesis, in the anti-obesity effects of C75, a synthetic inhibitor of fatty acid synthase (FAS). We report that a single intracerebroventricular (i.c.v.) injection of C75 induced hypophagia and weight loss in fasted male Wistar rats. Furthermore, C75 induced a rapid increase in core body temperature and an increase in heat dissipation. In parallel, C75 stimulated IBAT thermogenesis, which was evidenced by a marked increase in the IBAT temperature that preceded the rise in the core body temperature and an increase in the mRNA levels of uncoupling protein-1. As with C75, an i.c.v. injection of cerulenin, a natural FAS inhibitor, increased the core body and IBAT temperatures. The sympathetic IBAT denervation attenuated all of the thermoregulatory effects of FAS inhibitors as well as the C75 effect on weight loss and hypophagia. C75 induced the expression of Fos in the paraventricular nucleus, preoptic area, dorsomedial nucleus, ventromedial nucleus, and raphé pallidus, all of which support a central role of FAS in regulating IBAT thermogenesis. These data indicate a role for IBAT in the increase in body temperature and hypophagia that is induced by FAS inhibitors and suggest new mechanisms explaining the weight loss induced by these compounds.

Keywords

Brown adipose tissueFatty acid synthase inhibitorsThermogenesisSympathetic activation

Introduction

Brown adipose tissue (BAT) is a specialized tissue that produces heat and controls energy homeostasis [17]. In mammals, BAT is distributed in discrete deposits in many locations in the body, including the interscapular, cervical, periaortic, axillary, intercostal, and perirenal regions [4, 13, 19, 42]. BAT thermogenesis occurs through the uncoupling of oxidative phosphorylation from electron transport by mitochondrial uncoupling protein-1 (UCP1) activity [22]. The activation of this process (i.e., non-shivering thermogenesis) is under the strict control of the sympathetic nervous system (SNS). Interscapular brown adipose tissue (IBAT) is richly innervated by the noradrenergic neurons of the efferent branches of the SNS [4], which triggers the breakdown of triglycerides into fatty acids. The free fatty acids (FFA) serve not only as an energy source for thermogenesis but also act as the stimulators of the BAT thermogenic activity [33]. The sympathetic activity in the BAT is controlled by the brain autonomic centers, including the hypothalamus and the brainstem, which are the regions involved in cold-induced thermogenesis [31] and energy balance regulation [40]. Recent evidence indicates that BAT is functionally present in adult humans and that its amount is inversely correlated with body mass index; this evidence suggests a potential role of BAT as a new target for anti-obesity drugs [11, 52, 53].

C75(trans-4-carboxy-5-octyl-3-methylenebutyrolactone) is a synthetic inhibitor of fatty acid synthase (FAS) and has been proposed as an anti-obesity agent because its administration decreases food intake and body weight in rodents [24]. The central administration of C75 suppresses the expression of orexigenic neuropeptides (e.g., neuropeptide Y and agouti-related peptide (AgRP)) and induces the expression of anorexigenic neuropeptides (e.g., α-melanocyte-stimulating hormone (α-MSH) and cocaine and amphetamine-regulated transcript (CART)) in the hypothalamus, which is known to be involved in the regulation of feeding behavior [46]. Additionally, Thupari et al. have demonstrated that C75 also induces weight loss by increasing overall energy expenditure [49]. Indeed, it has been shown that chronic treatment with C75 alters the expression of genes involved in fatty acid metabolism to favor fatty acid oxidation in the white adipose tissue of diet-induced obese mice [50]. Despite the controversial role of UCP3 as a heat-producing truly uncoupling protein, other studies have shown that the intracerebroventricular (i.c.v.) injection of C75 increases its gene expression in skeletal muscle in both lean and obese (ob/ob) mice [7, 9]. Because these effects occur very rapidly (i.e., ≤ 2 h after the injection of the C75) and increase the muscle expression of the β-adrenergic signaling molecules (e.g., norepinephrine, β3-adrenergic receptor, and cAMP) they appear to be transmitted from the brain to the periphery by the SNS [9]. It is unknown, however, whether the central injection of C75 affects other peripheral tissues, the actions of which could contribute to body weight loss.

The present work was undertaken to investigate the centrally mediated effects of the FAS inhibitor C75 on feeding behavior and the sympathetically mediated thermogenesis in the IBAT, which is the major BAT deposit in the rat [4, 13]. The regions within the hypothalamus and the brainstem that are activated by this compound were also investigated.

Methods

Animals

Male Wistar rats (220 ± 20 g) were obtained from the University of São Paulo’s Animal Breeding Center and were housed in groups of four or five per cage in a room kept under a 12/12-h light/dark cycle (lights on at 06:00 a.m.) at 25 ± 2 °C. The rats were given access to water and standard rodent chow ad libitum (Nuvilab CR1, Nuvital, Brazil; 22 % protein, 55 % carbohydrate, and 4.5 % lipid) at least 2 days before the surgical procedures. All of the experiments were performed beginning at 08:00 a.m. in a quiet air-conditioned (25 ± 1°C) room in individually separated 24-h-fasted animals. For most experiments, the animals were randomly divided into three treatment groups as follows: C75 (single i.c.v. injection of 150 μg in RPMI medium 1640 vehicle), control (single i.c.v. injection of RPMI medium 1640), and denervated C75 (the animals were previously submitted to bilateral IBAT sympathetic denervation and treated with a single i.c.v. injection of C75). In preliminary experiments, we tested the anorectic effect of three doses (20, 100, and 150 μg), and the dose of 150 μg was effective. A separate group of control and IBAT-denervated animals was treated with cerulenin (single i.c.v. injection of 150 μg in RPMI medium 1640 vehicle). At the end of experiments, the animals were euthanized with an overdose of anesthetic or by decapitation. C75, cerulenin, and RPMI medium 1640 were purchased from Sigma-Aldrich (São Paulo, Brazil).

Stereotaxic surgical and drug injection

To implant the guide cannula into the right lateral ventricle, the animals were anesthetized with an intraperitoneal (i.p.) injection of Ketamine/Xylazine cocktail (5:3.5; 1 mL kg−1 body weight) and fixed in a stereotaxic apparatus (David-Kopff). The coordinates were adapted from those of Paxinos and Watson [37] and were as follows: anterior–posterior −1.5 mm (from the bregma), dorsal–ventral−3.4 mm (from the skull surface), and medial–lateral +2.5 mm. After the surgery, a veterinary poly-antibiotic preparation was given via intramuscular injection (0.5 mL kg−1 body weight, Pentabiótico® 48,000 U; Fort Dodge, Campinas, Brazil). After a 6-day recovery period, the animals were submitted to food deprivation for 24 h before the experiments. After approximately 1 h of being housed in the experimental room, the injection of C75/cerulenin or vehicle alone was performed using a calibrated Hamilton syringe. At the end of each experiment, Evans blue dye was injected (5 μL) into the brains, and cannula placement was verified after immersing the brains in 10 % paraformaldehyde for 2 days.

IBAT bilateral sympathetic denervation

IBAT denervation was performed immediately after the stereotaxic surgery. After identification, five branches from each side (right and left) of the intercostal nerve bundles were isolated, and a section of approximately 5 mm was removed from these nerves. The effectiveness of this technique was shown by the drop in the norepinephrine content in the IBAT on post-operative day 7 (Fig. 4c).

Telemetric measurement of body temperature and spontaneous locomotor activity

Core body temperature and spontaneous locomotor activity measurements were obtained every 5 s using an E-Mitter transponder (PDT4000 E-Mitter; Mini Mitter Co., Sunriver, OR) that was implanted i.p. at the time of the stereotaxic surgery. The signal, which emitted from the transponder and was sent to the energizer/receiver plate (ER−4000, Mini Mitter Co.), was decoded by Vital View software (Mini Mitter). During acclimatization, the basal values were recorded for 30 min, and the recording continued for 20, 8 or 6 h after the injections. The means of these parameters were calculated every 30 min or every 15 min.

Telemetric measurement of IBAT temperature

Small transponders (IPPT-3000 model; BMDS Co., DE, USA) were implanted under the IBAT at the time of stereotaxic surgery. The temperature data were recorded every 30 min for 8 h or every 15 min for 6 h using the data acquisition system DAS 7008-9 (BMDS Co.).

Heat dissipation

The skin tail temperature was measured on the lateral surface ∼ 2 cm from the base of the tail [56] every 5 min for 4 h using a thermocouple (AG 2000 model; Braile Biomédica, São Paulo, Brazil).

Metabolites, hormones, and catecholamine measurements

Plasma glucose and FFA were determined using commercial kits from Labtest (Lagoa Santa, Brazil) and Randox FFA (São Paulo, Brazil), respectively. Insulin was measured using ELISA, and corticosterone was measured using radioimmunoassay. Total thyroxine levels were measured with a solid-phase, competitive, chemiluminescent enzyme immunoassay. To determine the catecholamine plasma levels and the IBAT norepinephrine content [25], the tissue and plasma were stored at −80 °C until the assays were conducted. The catecholamines were assayed as previously described [15] using HPLC (LC-7A, Shimadzu Instruments) with a Spherisorb ODS-2 (5 μm) (Sigma-Aldrich) reversed-phase column. The lipid content in the IBAT was determined using gravimetric method.

Quantitative real-time RT-PCR

IBAT from decapitated rats was quickly dissected on ice, weighed, and stored at −80 °C [3]. The total RNA was subsequently isolated from individual IBAT using TRIzol (Invitrogen®, Carlsbad, CA). Reverse transcription into cDNA was performed using 2 μg of total cellular RNA, 20 pmol oligonucleotide (dT) primer (Invitrogen®), and Advantage ImProm-II reverse transcriptase (Promega®, Madison, WI). Real-time PCR was conducted using an ABI7000 sequence detection system (Applied Biosystems®, Foster City, CA), a SuperScript III Platinum SYBR Green One-Step RT-qPCR Kit with ROX (Invitrogen®), and primers for rat UCP1 (forward 5′-CCGGTGGATGTGGTAAAAAC-3′ and reverse 5′-ATCCGAGTCGCAGAAAAGAA-3′) and β-actin (forward 5′-TTGCTGACAGGATGCAGAAG-3′ and reverse 5′-CAGTGAGGCCAGGATAGAGC-3′) genes. The relative quantitation of mRNA levels was plotted as the fold increase compared with the control group values. The transcripts were normalized to β-actin levels. The level of the target transcripts was calculated using the standard curve method [10].

Perfusion, tissue preparation, and immunohistochemistry

The animals were anesthetized with i.p. injections of 2.5 % tribromoethanol (1 mL per 100 g−1 BW). The rats were then transcardially perfused with 200 mL of cold isotonic saline containing heparin (50 IU L−1) followed by 500 mL of cold 4 % paraformaldehyde solution in a 0.1 M phosphate buffer (PB) at pH 7.2. The brain was removed, fixed for 1 h in 4 % paraformaldehyde solution, and stored at 4°C in PB containing 30 % sucrose. Coronal sections of 30 μm were obtained in a cryostat (Microm) and collected in PB. Sections were processed for Fos immunoreactivity, with an overnight incubation at room temperature with an anti-Fos rabbit antibody (Ab-5; Oncogene Science, Manhasset, NY, USA) diluted 1:10,000 in 0.1 M PB containing 2 % normal donkey serum and 0.3 % Triton X-100. Then, free-floating sections were washed with PB and incubated with biotinylated donkey anti-rabbit antibody (1:200 dilution in 1.5 % normal donkey serum-PB; Jackson ImmunoResearch Laboratories Inc., West Grove, PA) followed by the avidin–biotin–peroxidase complex (Vector, 1:200 in PB), which were both conducted for 1 h at room temperature. The blue–black labeling of the cell nucleus was detected using diaminobenzidine hydrochloride (DAB, Sigma Chemical Co., St. Louis, MO, USA), which was intensified with 1 % cobalt chloride and 1 % nickel ammonium sulfate. Finally, the sections were mounted on gelatinized slides, air-dried overnight, dehydrated, cleared in xylene, and placed under a coverslip with Entellan (New Jersey, USA).

The hypothalamic and brainstem nuclei were identified according to the rat atlas of Paxinos and Watson [38]. The preoptic area (POA) was considered at the level −0.80 mm from the bregma, and the medial (PaMP) and posterior (PaPo) parvocellular subdivisions of the paraventricular nucleus (PVN) were considered at −1.80 and −2.12 mm from the bregma, respectively. The retrochiasmatic nucleus of the hypothalamus (RCA) was considered at the level −1.80 mm from the bregma, and the arcuate (ARC), dorsomedial (DMH), and ventromedial (VMH) nuclei of the hypothalamus were considered at the level −2.12 to −3.30 mm from the bregma. The locus coeruleus (LC), raphé pallidus (RPa), and nucleus of solitary tract (NTS) were considered at −9.30 to −10.04, −12.30 to −14.30, and −13.68 to −14.08 mm from the bregma, respectively. The Fos immunoreactive neurons were quantified with the aid of a computerized system that included a Leica microscope equipped with a DC 200 Leica digital camera attached to a contrast enhancement device. The Fos immunoreactive neurons, indicated by black staining, were identified when the nuclear structure demonstrated a clear immunoreactivity compared to the background level. The visual counting of the Fos immunoreactive neurons was performed in sections from six animals in each experimental condition with the participants blinded to the experimental protocols.

Experimental protocols

Experiments 1 and 2: Effects of C75 on food intake and body weight in intact and IBAT-denervated rats

The animals kept in metabolic cages were fasted for 24 h and then injected with either C75 or vehicle into the lateral ventricle at 08:00 a.m. Food was provided after the injection, and food consumption was measured at 1 h (0–1-h interval), 3 h (1–3-h interval), and 12 h (3–12-h interval) from the time of the injection. In another set of control animals, C75, and IBAT-denervated C75 rats, the food intake, body weight, and fluid intake were monitored daily each morning over the 3 days following the injection.

Experiment 3: Effects of C75 on plasma levels of FFA and glucose in awake rats

On the morning of the day preceding the i.c.v. injections, a Silastic catheter was implanted into the right jugular vein in the control and the C75 rats under anesthesia, which were submitted to food deprivation. Blood samples (0.08 mL, replaced with saline) were collected immediately before (0 time) and 15, 30, 60, 120, and 180 min after the i.c.v. injection of C75 or vehicle to determine the plasma glucose and FFA concentration.

Experiments 4 and 5: Effects of C75 or cerulenin on thermoregulatory mechanisms in intact and IBAT-denervated rats

Six days after the stereotaxic surgery and the implantation of the transponders, the intact and IBAT-denervated animals were fasted for 24 h and then injected with vehicle, C75, or cerulenin. Before and soon after the injections, the core body temperature and spontaneous locomotor activity were recorded every 30 min for 20 h, and thermal index was calculated. In a second series of rats, the skin tail temperature (heat dissipation) was measured every 15 min for 4 h. In a third series of rats, the IBAT temperature was recorded every 30 min for 8 h.

Experiment 6: Correlation between core body and IBAT temperature changes induced by C75 in intact rats

In order to estimate the influence of the IBAT temperature on core body temperature, the temperatures in IBAT and core body were simultaneously recorded every 15 min for 6 h in the same animals.

Experiments 7 and 8: Effects of C75 on IBAT thermogenesis, norepinephrine and lipid levels, and hormones in intact and IBAT-denervated rats

At 1 and/or 4 h after the i.c.v. injection, the animals were decapitated, and their trunk blood and IBAT were collected. The IBAT was rapidly weighed, homogenized, or frozen in liquid nitrogen and stored at 80 °C to determine the UCP1 mRNA, the norepinephrine level, and lipid level. Hormone (insulin, corticosterone, and thyroxine) and catecholamine levels were measured in the plasma samples of the intact animals.

Experiment 9: Effects of C75 on neuronal activation in the hypothalamus and brainstem

Six days after the stereotaxic surgery, the animals were fasted for 24 h and injected with vehicle or C75. At 90 min after the i.c.v injection, the animals were transcardially perfused for brain tissue collection and immunohistochemistry studies.

Statistical analyses

The distribution and variance homogeneity were tested, and the appropriate statistical test was employed (as indicated in the figure legends). Multiple comparisons were made using two-way ANOVA and the Bonferroni test or one-way ANOVA and the Newman–Keuls test. Unpaired Student’s t test was used for comparing two groups. The program GraphPad Prism 5.0 (San Diego, CA, USA) was used to conduct the statistical analyses. The data are expressed as the mean ± SEM. The differences were considered significant at P < 0.05.

Results

C75 acutely reduces food intake and body weight, and IBAT sympathetic denervation attenuates such effects

The anorectic effect of C75 was already detectable 3 h after the injection (Fig. 1a), and food intake was significantly inhibited by ≥ 80 % during the first 24 h after the i.c.v. injection (Fig. 1b). Food intake was unchanged versus control on day 2 and 3 after the i.c.v. administration of C75. Interestingly, the decrease in food intake during the first 24 h was attenuated in the IBAT sympathetic denervated rats (Fig. 1b). As shown in Fig. 1c, C75 significantly induced weight loss at both 24 h (8 %) and 48 h (2 %). IBAT denervation attenuated (80 %) the C75-induced weight loss at 24 h and completely prevented the effect at 48 h (Fig. 1c). C75 produced no significant differences in water intake when compared with the controls at either time point (data not shown).
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Fig. 1

Effect of C75 on food intake and body weight in intact and IBAT-denervated rats. Standard chow (40 g) was offered to 24 h-fasted animals (either submitted or not to IBAT denervation) after i.c.v. injections of C75 or vehicle. a Food intake at 0–1, 1–3, and 3–12 h of intervals. b Food intake at each 24-h period for 3 days. c Percent body weight change at each 24-h period for 3 days. The values represent means ± SEM (n = 6–8/group). Two-way ANOVA and Bonferroni test were used for the statistical analyses. *P < 0.05 vs. control group and #P < 0.05 vs. C75 group

Central administration of C75 induces a transitory and slight increase in plasma glucose and plasma FFA concentration

The C75 treatment significantly increased the plasma FFA at 30 min, 1 h, and 3 h and glucose concentration at 1 h after the i.c.v. injection in non-anesthetized, freely moving rats (Fig. 2). However, these effects were not correlated with any detectable change in the plasma levels of insulin, catecholamines, thyroxine, and corticosterone, which remained similar to the control values 1 and 4 h after the C75 injection (Table 1).
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Fig. 2

Temporal profile of C75-induced changes on plasma levels of free fatty acids (a) and glucose (b) in awake rats. Animals fasted for 24 h with a cannula in the jugular vein received i.c.v. injections of C75 or vehicle, and blood was collected in 15-min intervals for 180 min. The values represent means ± SEM (n = 7/group). Two-way ANOVA and Bonferroni test were used for statistical analyses. *P < 0.05 vs. control group

Table 1

Effect of C75 on the plasma concentration of hormones

 

1 h post-i.c.v injection

4 h post-i.c.v. injection

 

Control

C75

Denervated C75

Control

C75

Denervated C75

Insulin (ng. dL−1)

17.6 ± 1.3

19.9 ± 3.5

20.8 ± 2.6

32.5 ± 6.6

29.2 ± 3.0

38.9 ± 6.8

Norepinephrine (ng. dL−1)

388 ± 48

388 ± 31

340 ± 61

302 ± 44

390 ± 51

369 ± 45

Epinephrine (μg. dL−1)

1.5 ± 0.1

1.5 ± 0.2

1.5 ± 0.1

1.0 ± 0.1

1.2 ± 0.1

1.2 ± 0.1

Total thyroxine (μg. dL−1)

2.2 ± 0.4

2.9 ± 0.1

2.2 ± 0.3

2.1 ± 0.2

2.1 ± 0.1

2.2 ± 0.3

Corticosterone (μg. dL−1)

27.8 ± 2.0

27.8 ± 5.7

27.8 ± 3.7

Values are presented as mean ± SEM (n = 6/group)

IBAT denervation reduces hyperthermia and heat loss evoked by C75

As shown in Fig. 3a, b, the C75 injection increased the core body temperature over 20 h of analysis without altering the spontaneous motor activity. Furthermore, IBAT denervation attenuated the increase of core body temperature that was promoted by C75.
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Fig. 3

Temporal profile of C75-induced changes on core body temperature, physical activity, and skin tail temperature in intact and IBAT-denervated awake rats. The temperature and physical activity parameters were recorded after i.c.v. injections of C75 or vehicle in 24-h-fasted animals. a Change of core body temperature for 20 h (abdominal measurements at 30-min intervals). b Spontaneous locomotor activity for 20 h (measured using the same transponder as core body temperature). c Skin tail temperature changes measured for 4 h in 15-min intervals . d–e The AUC calculated from the core body temperature values (thermal index) for 9 h and from the skin tail temperature for 4 h, respectively. The values represent means ± SEM (n = 7/group). Two-way ANOVA and Bonferroni test were used to analyze the core body and the skin tail temperatures, and the spontaneous locomotor activity, and one-way ANOVA and Newman–Keuls test were used to analyze the heat storage rate and the AUC. *P < 0.05 vs. control group; #P < 0.05 vs. C75 group

To estimate the heat exchange between the body and environment, the skin tail temperature was measured (Fig. 3c, e). The C75 injection enhanced the tail heat loss mechanisms by increasing the skin tail temperature over 4 h of analysis. This effect was completely abolished by IBAT denervation, which suggests that the heat loss is triggered as a thermoregulatory mechanism to compensate for the C75-evoked hyperthermia.

C75 stimulates IBAT thermogenesis via sympathetic innervation

To further investigate the involvement of the IBAT thermogenesis in the thermoregulatory responses that were induced by the C75 injection, the IBAT temperature was assessed in intact and IBAT-denervated rats. As shown in Fig. 4a, C75 induced a marked increase in the IBAT temperature from 1 until 8 h after the injection, and this thermal effect was completely abolished by the IBAT sympathetic denervation. To estimate the kinetic of the temperature changes induced by C75, the IBAT and core body temperatures were simultaneously recorded in the same animals. Figure 4b shows that the increase in IBAT temperature started a few minutes before the core body temperature. There was an increase of norepinephrine content in the IBAT 1 h after the C75 injection (Fig. 4c) which was associated with a decrease in the total lipid content in this tissue (Fig. 4d). The UCP1 mRNA expression was upregulated (∼ 2.5 times) in the IBAT at 4 h after the C75 injection (Fig. 4e). IBAT denervation completely abolished the C75-induced decrease of the IBAT lipid content and the increase of IBAT norepinephrine and UCP1 expression.
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Fig. 4

Effect of C75 on IBAT temperature, total lipids, and mRNA UCP1 expression in intact and denervated rats. The BAT thermogenesis markers were analyzed in the IBAT after i.c.v. injections of C75 or vehicle in innervated or IBAT-denervated 24-h-fasted animals. a Temporal profile of C75-induced changes in IBAT temperature in awake rats for 8 h (change of basal values at 30-min intervals). b Simultaneous changes of core body and IBAT temperatures in awake rats for 6 h (change of basal values at 15-min intervals). c Content of norepinephrine in the BAT after 1 h. d Content of total lipids in the IBAT after 1 h. e UCP1 mRNA expression in the IBAT at 1 and 4 h after the injection. The values represent means ± SEM (n = 4–7/group). Two-way ANOVA and Bonferroni test were used to analyze the IBAT temperature change, and one-way ANOVA and Newman–Keuls test were used to analyze norepinephrine, total lipids and mRNA expression. *P < 0.05 vs. control group; #P < 0.05 vs. C75 group

Resembling C75, the central administration of cerulenin induces thermoregulatory effects

As shown in Fig. 5a–c, the i.c.v. administration of cerulenin, a natural FAS inhibitor, increased the core body and IBAT temperatures without altering the spontaneous locomotor activity of the animals. The cerulenin-induced stimulatory effects on core body and IBAT temperatures, which were recorded in different sets of animals, started 3 h after the injection and lasted for 4 and 2 h, respectively. Both effects were abolished by IBAT sympathetic denervation.
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Fig. 5

Temporal profile of cerulenin-induced changes on core body temperature, physical activity, and IBAT temperature in intact and IBAT-denervated awake rats. Cerulenin or vehicle were i.c.v. injected into innervated or IBAT-denervated 24-h-fasted animals. The temporal profile of cerulenin-induced changes on a core body temperature (abdominal measurements at 10-min intervals), b spontaneous locomotor activity (measured using the same transponder as core body temperature), and c IBAT temperature (change of basal values at 30-min intervals) in awake rats for 8 h. The values represent means ± SEM (n = 4–7/group). Two-way ANOVA and Bonferroni test were used for statistical analyses. *P < 0.05 vs. control group; #P < 0.05 vs. cerulenin group

C75 increases neuronal activation in the hypothalamus and brainstem

To evaluate the brain areas that are involved in the hypophagic and thermoregulatory responses induced by C75, the Fos expression was investigated 90 min after injection in the brain nuclei that is known to participate in the regulation of food intake and thermogenesis. Figure 6c shows that C75 increased the number of Fos immunoreactive neurons in the POA (2.1 times), medial (4.7 times) and posterior (4.0 times) parvocellular subdivisions of the PVN, VMH (3.1 times), DMH (2.6 times), LC (9.0 times), and RPa (2.8 times), with no effects on the RCA, ARC, or NTS. Representative photomicrographs of Fos expression in the POA, DMH, LC, PaMP, PaPo, and RPa are shown in Fig. 6a, b.
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Fig. 6

Effects of C75 on neuronal activation in the hypothalamus and brainstem. Fos expression was analyzed after 90 min of i.c.v. injections of C75 or vehicle in 24-h-fasted animals. a Representative photomicrographs (× 20) of coronal sections showing Fos immunoreactivity in the POA, DMH, and LC; in the bottom left, the insert depicts the area where the photomicrograph was taken at × 10 magnification; bar scale = 100 μm. b Representative photomicrographs (× 40) of coronal sections showing Fos immunoreactivity in the PaMP, PaPO, and RPa; at the bottom left, the insert depicts the area where the photomicrograph was taken at × 20 magnification; scale bar = 50 μm. c Fos expression was analyzed in POA, PaMP, PaPo, RCA, ARC, VMH, DMH, LC, RPa, and NTS. The values represent means ± SEM (n = 6/group). Student’s t test was used for statistical analyses. *P < 0.05 vs. control group

Discussion

The present study shows for the first time that in fasted rats, the central administration of a single injection of C75, a potent inhibitor of FAS, increased IBAT thermogenesis, an effect that was associated with activating key hypothalamic and brainstem BAT thermogenesis-related regions and was completely blocked by the sympathetic denervation of IBAT.

The present data show that C75 induces hypophagia and weight loss in fasted rats, which is consistent with previous studies [7, 21, 24, 27, 28, 39]. However, the negative energy balance caused by FAS inhibitors is not entirely the result of suppressed food intake; it is also the result of increased energy expenditure. In fact, indirect calorimetry experiments showed that C75 administration increases whole-body O2 consumption [49], an effect that is generally regulated by the autonomic nervous system. Cha et al. [9] were the first to demonstrate that the SNS is implicated in the transmission of the C75 signal from the brain to the skeletal muscle. In these studies, it was demonstrated that the sympathetic activation induced by C75 increased the fatty acid oxidation and the expression of UCP3 in skeletal muscles. In agreement with this notion, the present study shows that in awake rats, C75 induces a transient increase in plasma FFA and glucose concentrations, suggesting that the mild sympathoexcitation that is evoked by C75 elicits the activation of lipolysis in white adipose tissue and hepatic glucose production.

The innervation of BAT by the SNS is uncontroversial, and its activation by the central nervous system functions as the principal stimulator of BAT thermogenesis [17]. In this context, the IBAT sympathetic denervation is a powerful tool to eliminate the connection between the central signals to the IBAT, reducing the rise of body temperature, which is partially dependent on this mechanism of heat production.

Because IBAT denervation attenuated the increase in the core body temperature induced by both C75 (Fig. 3a) and cerulenin (Fig. 5a), a natural inhibitor of FAS, we conclude that at least part of the FAS inhibitor-evoked hyperthermia can be attributed to IBAT thermogenesis. This conclusion appears consistent with the following findings: (1) the local IBAT temperature is clearly increased by FAS inhibitors (C75 and cerulenin) and is completely abolished in IBAT-denervated rats (Figs. 4a and 5c); (2) a relationship between the core body and IBAT temperature increase induced by C75 is observed during the heating phase, and such increase in IBAT precedes the core body temperature (Fig. 4b); (3) because the physical activity recorded here remained unchanged after these treatments, the observed thermogenic effects of FAS inhibitors cannot be ascribed to motor activity changes (Figs. 3b and 5b); and (4) the enhanced heat loss, estimated by the skin tail temperature (Fig. 3c, e) in C75-treated rats, excludes the possibility that the hyperthermia is caused by a decrease in heat dissipation (peripheral vasoconstriction). The stimulatory effect of C75 on heat loss can be postulated to be the primary autonomic defense against hyperthermia to achieve control values of core body temperature, as observed in the present study 9 h after the C75 injection. Moreover, the increase in the core body temperature in the IBAT-denervated rats after 6 h of C75 injection (Fig. 3a, d) suggests that other sources of heat were stimulated at the same time as IBAT as, for example, in other deposits of BAT or even in the skeletal muscle, as previously suggested [8]. In contrast to the core body temperature (Fig. 3a), the IBAT temperature in the C75-treated denervated rats did not change during this time (Fig. 4a). Given that both these parameters were registered in different set of rats, these results are difficult to explain. However, one possibility is that the suppressive effect of denervation may have been higher on the core body temperature in the rats used for IBAT measurements than in those used in the core body temperature experiments.

It is well established that BAT thermogenesis is triggered by the release of norepinephrine from its sympathetic nerve terminals, stimulating the β-adrenoceptors, which in turn promote lipolysis and a cascade of intracellular events that end in the activation of UCP1 [4], a key thermogenic protein that is expressed exclusively in BAT. UCP1 dissipates the proton gradient across the inner mitochondrial membrane to produce heat, rather than ATP. For this function, an increase in the fuel oxidation is necessary to raise the production of NADH and FADH2, which donate electrons to the electron-transport chain [22]. Consistent with the hypothesis that IBAT thermogenesis is activated by the central administration of C75 via a direct sympathetic innervation, the early rise in central temperature occurred without any change in the plasma levels of insulin, catecholamines, thyroxine, or corticosterone (Table 1), and the C75-induced IBAT norepinephrine increase was closely correlated with a lower total lipid content (indirect evidence of local lipolysis). Furthermore, this increase coincides with the peak response of core body temperature and the onset of heat production by IBAT. Because the stimulating effect of C75 on UCP1 mRNA expression is not sufficiently rapid (i.e., it does not occur within 1 h of i.c.v. C75 treatment; Fig. 4g) to account for the rapid activation of heat production by IBAT, the gene expression of this enzyme appears not to be involved in the short-term mechanism by which thermogenesis is activated by C75. This is due to a direct stimulation of UCP1 activity by the fatty acids that are derived from lipolysis in the tissue, which precede the effect of norepinephrine on mRNA production [4].

The present data strongly suggest that central administration of C75 triggers sympathetically mediated thermogenesis in IBAT. Because during non-shivering thermogenesis, more than half of all oxygen taken up is consumed by IBAT [4]; such a thermogenic response might be expected to contribute to whole-body energy expenditure and result in weight loss after the central injection of FAS inhibitors. Accordingly, the present data show that the weight loss that was induced by C75 was attenuated in IBAT-denervated rats compared to the controls during the first 2 days of treatment (Fig. 1c). However, caution should be taken in asserting that BAT was the responsible for mediating the weight loss induced by C75. It should be noted that surgical sympathetic denervation was conducted only in the IBAT, which is estimated as 30–40 % of the total BAT in the rat. Collectively, it is most likely that the acute decrease in body weight induced by C75 is due to the decrease in food intake. It was previously postulated [18] that satiety is induced by the high level of core body temperature, which is induced by the episode of stimulated BAT thermogenesis, and the meal size depends on the balance between BAT thermogenesis and heat loss. This view is consistent with the finding that IBAT denervation attenuated the reduced food intake that is induced by C75 during the first 24 h after the injection. However, the notion that BAT themogenesis could inhibit feeding is not consistent with the observation that UCP-1 KO mice do not eat more than WT mice [35], and that the anorexigenic effect of a single injection of leptin is not different between the two groups [34].

The present study shows that the hypophagic effect induced by C75 is associated with enhanced neuronal activation, as assessed by c-Fos expression, in the brain nuclei involved in the regulation of food intake, such as PVN and VMH (Fig. 6). Indeed, the presence of FAS in these hypothalamic nuclei was demonstrated by Kim et al. [20] and Sangiao-Alvarellos et al. [44], indicating that C75 may act directly on neurons in the PVN and VMH. The activation of neurons in the PVN and VMH could account for the reduction in food intake that was observed after the C75 injection because these hypothalamic nuclei are involved in the mediation of the anorexigenic responses that are induced by several treatments, including leptin [2], lipopolysaccharide [41], adrenalectomy [51], and cholecystokinin [30]. PVN neurons express different neuropeptides that are involved in the regulation of energy homeostasis, including the anorexigenic neuropeptides oxytocin, corticotrophin-releasing factor, and thyrotropin-releasing factor [45]. In fact, C75 has been demonstrated to increase the expression of the anorexigenic neuropeptides oxytocin, α-MSH, and corticotrophin-releasing factor in the hypothalamus [20, 46], which is in agreement with the higher activation of neurons in the PVN observed in the present study and in other murine experiments [14, 29].

In addition to their direct effect on food intake, the activation of PVN and VMH neurons in response to C75 could also participate in the enhancement of thermogenesis in IBAT. The primary activation of the PVN and the VMH after the C75 treatment might contribute to the activation of other brain areas (observed in the present work) that are involved in activating IBAT (Fig. 6). PVN neurons have been shown to project to RPa and LC [1, 16, 26], which in turn also project to RPa [16, 43]. RPa neurons are known to contain sympathetic premotor neurons that project to the sympathetic preganglionic neurons in the intermediolateral cell column of the thoracic spinal cord, controlling thermogenesis in the IBAT [32]. Projections from the POA, PVN, DMH, and LC to the RPa have been observed in the literature [16, 57]. Accordingly, the injection of retrograde and trans-synaptic tracer in the IBAT have demonstrated that the neurons in these nuclei and in the RPa polysynaptically innervate the IBAT [5, 36, 54, 57]. Additionally, it is known that VMH and PVN project to the POA [6, 23], which has a pivotal role in thermoregulation [47]. The thermogenic responses induced by POA stimulation has been associated with VMH and DMH neuron activation [12, 48]. In fact, POA projects to the RPa and DMH, which in turn also projects to the RPa [55]. Thus, the stimulation of RPa neurons by the projections from the POA, PVN, DMH, and LC could increase body temperature by activating the sympathetic nervous system in the IBAT.

In summary, the data presented here revealed that the central administration of the FAS inhibitor C75 rapidly increases the sympathetic outflow to IBAT, which leads to the activation of thermogenesis and, consequently, the increase of core body temperature, an effect that is rapidly counter-regulated by heat loss mechanisms. These thermal effects associated with hypophagia may contribute to the weight loss induced by this compound. Additionally, the current study demonstrated that C75 stimulates a brain circuits located in the hypothalamus and brainstem involved in SNS-mediated control of BAT thermogenesis and energy homeostasis. The ability of FAS inhibitors to stimulate IBAT, a tissue that is metabolically inactive in most overweight or obese subjects, suggests that modulating this pathway may serve as a potential target for weight control.

Acknowledgments

We thank José Roberto da Silva (Laboratory of Endocrinology; HCFMRP-USP) for the determination of plasma corticosterone, and Dr. Léa Maria Zanini Maciel and Giselle Aparecida Caixe de Carvalho Paixão (Laboratory of Thyroid and Neonatal Screening; HCFMRP-USP) for the determination of plasma total thyroxine. We are also indebted to Elza Aparecida Filippin, Neusa Maria Zanon, Lilian Zorzenon Carmo de Paula, Maria Valci Aparecida dos Santos, and Victor Diaz Galban for their technical assistance. This work was supported by grants from the Fundação de Amparoà Pesquisa do Estado de São Paulo (Fapesp 08/06694–6, 09/07584–2, 10/11083–6, and 10/11015–0) and from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq 140094/07–5, 306101/09–2, 303786/08–6, and 305149/12–1).

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