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Study on the expression of c-Fos protein in the brain of rats after ingestion of food rich in lycopene
Neuroscience Letters 536 (2013) 1–5
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Study on the expression of c-Fos protein in the brain of rats after ingestion of food rich in lycopene Kiyoshige Takayama a,∗ , Eri Nishiko a , Gaku Matsumoto b , Takahiro Inakuma b a b
Department of Laboratory Sciences, Gunma University School of Health Science, 3-39-15 Showa, Maebashi, Gunma 371-8514, Japan Research Institute, Kagome Co. Ltd., 17 Nishitomiyama, Nasushiobara, Tochigi 329-2762, Japan
h i g h l i g h t s
Lycopene contained in tomato belongs to carotenoid such as beta-carotene. Continuous intake of lycopene-rich food induced accumulation of lycopene in the plasma and liver. Continuous intake of lycopene-rich food induced the stimulation of neurons in the hypothalamus. Continuous intake of lycopene-rich food induced expression of c-Fos in the PVH, VMH and SO.
a r t i c l e
i n f o
Article history: Received 14 July 2012 Received in revised form 31 October 2012 Accepted 20 December 2012 Keywords: Lycopene c-Fos Brain
a b s t r a c t Lycopene, a reddish pigment contained in tomato, belongs to the carotenoid family along with betacarotene and rutein. This study examined whether administration of lycopene to rats would induce excitation of neurons in the central nervous system. Continuous intake of lycopene-rich food was found to induce accumulation of lycopene in the plasma and liver, and stimulated central neurons in the paraventricular nucleus, ventromedial nucleus and supraoptic nucleus of the hypothalamus, which are known to be involved in the functions of athrocytosis and water drinking. These findings suggest that lycopene may have some influence on feeding and water-drinking behaviors. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lycopene, a reddish pigment contained in tomato, is a carotenoid pigment similar to beta-carotene and rutein [1]. Lycopene is known to have strong scavenging activity against singlet oxygen [2], and acts as an anti-oxidant inhibiting cell-damage caused by free radicals [3,4]. A recent study revealed that ingestion of tomato or lycopene may be correlated with decreased morbidity from certain cancers: lycopene intake reportedly has preventive effects against carcinoma of the prostate [5], bladder cancer [6] and large bowel cancer [7]. Furthermore, lycopene may decrease levels of low-density lipoprotein and total cholesterol in plasma, and thus may help prevent arteriosclerosis and subsequent cardiac infarction or stroke [8]. The brain is known to consume a larger amount of oxygen than other tissues by producing active oxygen, and is very sensitive to oxidative stress [9]. This oxidative stress has been considered to contribute to diseases such as Alzheimer disease or Parkinson disease [10], and attention has focused on substances showing
∗ Corresponding author. Tel.: +81 27 220 8943; fax: +81 27 220 8943. E-mail address: [email protected] (K. Takayama). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.12.035
anti-oxidant activity in the brain. Hsiao et al. reported that injection of lycopene exerted anti-oxidant effects resulting in reduced infarct volume in ischemia/reperfusion brain injury [11]. The proto-oncogene c-fos is well known to induce c-Fos protein transiently within neurons in response to various kinds of stimulations, and the induction of c-Fos protein in neurons starts about 20 min after stimulation [12]. Our laboratory has examined neural expression of c-Fos protein after peripheral administration of a number of peptides, including gastrin, galanin, ghrelin, leptin and apelin [13–17]. All of these peptides were found to induce expression of c-Fos protein in various nuclei in the brain. Our first experiment in the present investigation examined whether lycopene administered peripherally would induce c-Fos protein in the brain, while the second experiment surveyed the neuronal expression of c-Fos protein throughout the central nervous system (CNS) after ingestion of lycopene-rich food. 2. Materials and methods All of the steps to the experimental procedures were conducted in compliance with the Guiding Principles for the Care and Use of Experimental Animals approved by the Gunma University Institute of Experimental Animal Research.
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K. Takayama et al. / Neuroscience Letters 536 (2013) 1–5
Experiments were performed using male SPF Wistar rats weighing 240–270 g (9–10 weeks old; SLC Japan, Shizuoka, Japan). To avoid any restlessness of a single rat, 6 rats were housed in a cage in a quiet room under conditions of regulated illumination (12 h light, 12 h dark; lights on at 08:00 h) and constant temperature (22 ± 2 ◦ C) with ad libitum access to water and rat chow (MF diet; Oriental Yeast, Tokyo, Japan). Initially, rats were intraperitoneally injected with lycopene (10 mg/rat in 0.6 ml dimethyl sulfoxide (DMSO)) in the test experiments, while 0.6 ml of DMSO without lycopene was injected in a similar manner in control experiments. At 90 min after the injection, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (80 mg/kg; Abbott Park, IL, USA), and perfused via the left ventricle with about 20 ml of saline solution to flush out the blood. This was immediately followed by 100 ml of 0.5% glutaraldehyde and 4% paraformaldehyde (PFA) (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer (PB) (pH 7.4) under pressure of 100–120 mmHg, and 400 ml of 4% PFA in PB under hydrostatic pressure. The brains were removed and cut into two blocks at the level between superior and inferior colliculi, fixed in 4% PFA in PB for a further 1.5 h at 4 ◦ C, and then soaked in a stepwise manner in 10%, 20%, and 25% sucrose in PB at 4 ◦ C. The brains were frozen and cut into serial transverse sections at 40 m thickness. Brain sections were collected in plates containing PB chilled by ice water. One group of every fourth section was rinsed in 0.1 M Tris–saline (TS) (pH 7.4) three times, incubated with 0.5% bovine serum albumin (BSA) in TS for 20 min and incubated with sheep anti-c-Fos polyclonal antiserum (1:100, sc52; Santa Cruz Biotech, Santa Cruz, CA, USA) in 0.5% BSA at 4 ◦ C for 16 h. All the following procedures were conducted at room temperature. The sections were rinsed three times in TS, then incubated in avidin and biotin solution, respectively, for 15 min each. Thereafter, sections were incubated for 20 min with 2% normal rabbit serum in TS to block nonspecific binding, and incubated for 60 min with anti-sheep immunoglobulin (Ig)G in TS. After rinsing three times in TS, sections were incubated for 60 min in Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA), and then treated with diaminobenzidine (DAB)/nickel solution containing 0.003% H2 O2 . Then, sections were rinsed twice in TS and rinsed further in PB, mounted on glass slides and dried. Sections on slides were dehydrated in a graded ethanol series (50%, 70%, 95%, 99%), infiltrated with xylene and cover-slipped in Permount (Fisher, Fair Lawn, NJ, USA). In the control experiments, rats (n = 4) were sham-operated and injected with 200 l of 0.9% NaCl solution. Brain sections were processed as described above. Localization of c-Fos protein in neuronal nuclei was visualized as black precipitates of nickel-intensified DAB reaction products. The c-Fos-immunoreactive (c-Fos-ir) neurons were surveyed under bright-field microscopy. Brain histology was checked against the rat brain atlas of Swanson [18]. In the second experiment, rats ingested normal food placed in a metal food box for 11 days before starting experiments. Afterwards, rats were arbitrarily divided into 2 groups, with 2 rats placed in each cage. Twelve rats in 6 cages were used for test experiments, and those in the other 6 cages for control experiments. In the test experiment, rats ingested lycopene-rich food (0.2% lycopene-containing MF diet) freely for 24 h, while control rats were similarly provided with normal food (MF diet). Body weights were measured once a week during the experiments. After starting the ingestion of each food in the test and control experiments, rats were processed for immunohistochemical staining of c-Fos protein using the methods described above, and for weighing amounts of lycopene contained in various organs every week. One rat from each group was chosen for c-Fos immunohistochemical staining and the other for measuring the lycopene content of various tissues. Experiments lasted as long as 6 weeks.
2.1. Extraction of lycopene contained in internal organs 2.1.1. Extraction from tissues Tissue (0.1 g) was homogenized with a 9-fold volume of PB in a centrifuge tube (10 ml), and stirred for 1 min with a 10-l internal standard (IS) (trans--Apo-8 -carotenal; Sigma–Aldrich, St. Louis, MO, USA), 50 l of 2 mM ␣-tocopherol and 3 ml of MeOH/CH2 Cl2 (2:1, v/v). Two milliliters of hexane was added and stirred. After centrifuging 3000 rpm for 5 min, the hexane layer was removed. Afterwards, the solution was evaporated to dry, and the residual lipid was saponified using 2 ml of 60% KOH at 60 ◦ C for 45 min. To the solution was added 2 ml of distilled water, 2 ml of ethanol and 8 ml of hexane before centrifuging at 3000 rpm to remove the hexane layer. The hexane solution was then evaporated under centrifugation. The residue was dissolved in 50 l of methanol/CH2 Cl2 (1:4) and filtered, and analyzed with HPLC as described below. 2.1.2. Extraction from blood serum To 500 l of serum sample was added IS, 1 ml of 0.01% BHT containing ethanol, and 4 ml of hexane/CH2 Cl2 (4:1). This mixture was centrifuged at 2000 rpm for 10 min. The supernatant was recovered and 1 ml of hexane/CH2 Cl2 (4:1) was added, stirred and centrifuged. The supernatant was again removed, the solvent evaporated, and then 100 l of ethanol was added to dissolve the extracts. After filtration, the filtrate was analyzed with HPLC. 2.2. HPLC analysis A previously described method [19] was used for quantifying lycopene in tissues and serum. HPLC analysis was performed using a photodiode detector (SPD-M10; Shimadzu, Kyoto, Japan) with a wavelength of 460 nm and a C30 carotenoid column (250 mm × 4.6 mm, 5 m; YMC, Wilmington, NC, USA). Lycopene content was quantified using an internal standard. 2.3. Statistical analysis Results are expressed are mean ± standard error of the mean, and the Mann–Whitney U-test was performed to compare results between test and control groups. Values of P < 0.05 were taken as statistically significant. 3. Results 3.1. Survey of c-Fos-ir neurons after intraperitoneal injection of lycopene The c-Fos protein was found in several nuclei in the brain, including the ventromedial nucleus hypothalamus (VMH), paraventricular nucleus hypothalamus (PVH), supraoptic nucleus (SO), central nucleus amygdala (CEA), paraventricular nucleus thalamus (PVT), periaqueductal gray matter (PAG), lateral parabrachial nucleus (PBL), and the complex of the solitary tract nucleus and dorsal motor nucleus of the vagus nerve (NTS/DMX) (Table 1). The sites of these nuclei were identified according to the atlas by Paxinos and Watson [18]. 3.2. Measurement of body weight of rat and of amount of lycopene in internal organs Body weights of rats in the test and control groups increased during experimental periods by 39 ± 2 g and 37 ± 6 g, respectively, showing no significant differences between groups (Table 2). Similarly, no significant differences were observed in any tissue weights between the test and control groups (Table 3). On the other hand, great increases in lycopene concentration were seen in blood
K. Takayama et al. / Neuroscience Letters 536 (2013) 1–5
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Table 1 Expression of c-Fos-immunoreactive neurons in the brain after intraperitoneal injection of lycopene in DMSO (test experiment, n = 3). In the control experiment, no lycopene was contained in the DMSO (n = 3). Number of c-Fos-immunoreactive neurons in one experiment: −, no; +, 2–10; ++, 11–50; +++, >50. Nucleus
Test experiment
Control experiment
SFO SO PAH VMH LH CEA PVT PAG LC Pbl VLM NTS/DMX AP
+ +++ +++ ++ + +++ ++ ++ + +++ ++ +++ +
− + + + − + + + − + + + −
Abbreviations: AP, area postrema; CEA, central nucleus amygdale; LC, locus, coeruleus; LH, lateral hypothalamic nucleus; NTS/DMX, complex of solitary tract nucleus and dorsal motor nucleus of vagus nerve; PAG, periaqueductal gray matter; PAH, paraventricular nucleus hypothalamus; Pbl, lateral parabrachial nucleus; PVT, paraventricular nucleus thalamus; SFO, subfornical organ; SO, supraoptic nucleus; VLM, ventrolateral medulla; VMH, ventromedial hypothalamic area.
Fig. 1. Number of c-Fos-immunoreactive neurons in control (n = 4) and test groups (n = 4). Mean ± standard error of the mean. PVH, paraventricular nucleus hypothalamus; SO, supraoptic nucleus; VMH, ventromedial nucleus hypothalamus.
4. Discussion In the present study, continuous intake of lycopene was not found to influence the weights of internal organs, although lycopene accumulation in the liver and serum was confirmed. These results support those obtained in human investigations [20,21]. Measurements of the lycopene content in blood serum and tissues in humans under daily intake of foods have revealed that lycopene distributes to many tissues, not only in the blood serum and liver, but also in the adrenal glands, testes and ovaries [22]. As in humans, orally administered lycopene was absorbed into the rat body and accumulated in organs such as blood serum and liver. In the ingestion experiment, continuous intake of lycopenerich foods induced expression of c-Fos-positive neurons in the VMH, PVH and SO. This result was in accordance with findings from the experiment after intraperitoneal injection of lycopene. In addition, some nuclei only showed c-Fos-positive neurons after intraperitoneal injection of lycopene, not after continuous intake of lycopene-containing foods. The concentration of lycopene in blood serum after intake of lycopene-containing food may be much lower than that after intraperitoneal injection, because the lycopene has to be absorbed through the alimentary canal into the blood. The difference in the distribution of c-Fos-positive neurons might be due to the different concentrations of lycopene, as well as to the duration of stimulation by lycopene. Neurons in the PVH, SO and VMH might be stimulated continuously regardless of the concentration of lycopene in the blood, while neurons in other nuclei might be stimulated only transiently and acutely by higher concentrations of lycopene in the blood. Neurons in the VMH, in which c-Fos was found to be induced after administration of lycopene, are known to be involved in the
Table 2 Changes in body weight and body weight gains in test (n = 12) and control rats (n = 12) during experiments (mean ± standard error of the mean). Group
Control Test
Body weight (g)
Body weight gain
Beginning (3 day)
After 25 or 26 days
285 ± 2 284 ± 2
324 ± 3 321 ± 6
39 ± 2 37 ± 6
serum (14.46 ± 5.5 ng/ml) and liver (6.89 ± 1.52 g/g-wet) in the test group, but not in the control group. However, almost no lycopene was detected in the brain in the test group.
3.3. Survey of c-Fos-ir neurons after ingestion of lycopene-rich food A survey of c-Fos-ir neurons was conducted using whole-brain sections from the medulla oblongata to the cerebral cortex. These c-Fos-ir neurons were particularly prominent in areas of the hypothalamus such as the PVH, SO and VMH. Numbers of c-Fos-ir neurons in these nuclei were counted. Statistical analysis was performed and significant differences were found in numbers of cFos-ir neurons in the PVH and VMH between test and control rats, although no significant differences were seen for the SO (Fig. 1). Representative photomicrographs of c-Fos-ir neurons in the PVH and VMH are shown in Fig. 2.
Table 3 Weights of various tissues in rats in the test and control groups (n = 12 each) and ratio of tissue weights to body weight (n = 6). n.d.: not detected. Tissue organ
Weight (g) Control
Brain Liver Kidney Testis Spleen Lung Stomach Heart Blood plasma
1.9 11.1 2.1 3.3 0.7 1.4 2.2 0.8
± ± ± ± ± ± ± ±
Test 0 0.4 0.2 0.2 0 0.1 0.1 0
1.9 11.1 2.2 3.2 0.7 1.4 2.2 0.9
± ± ± ± ± ± ± ±
0 0.2 0.1 0.1 0 0 0.2 0.1
Ratio of organ weight to body weight (%)
Concentration in lycopene
Control
Control
Test
n.d. n.d.
n.d. 6.89 ± 1.52 (g/g-wet)
n.d.
14.46 ± 5.5 (ng/ml)
0.6 3.5 0.6 1.0 0.2 0.4 0.7 0.3
± ± ± ± ± ± ± ±
0 0.1 0 0.1 0 0 0 0
Test 0.6 3.4 0.6 1.0 0.2 0.4 0.7 0.3
± ± ± ± ± ± ± ±
0 0.1 0 0 0 0 0.1 0
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Fig. 2. Representative microphotographs of c-Fos-immunoreactive neurons in the paraventricular nucleus hypothalamus (PVH) and ventromedial nucleus hypothalamus (VMH) in test rat (A) and control rat (B).
beginning of the fighting action by PAG neurons projecting from the medial nucleus of the amygdala, and in the endocrine functions of the pancreas and adrenal medulla through neurons in the PAG, medulla oblongata and spinal cord [23]. VMH is also known as the satiety center, and damage to this area can induce hyperphagia and obesity, while stimulation inhibits food intake [24]. The regulation of food intake by VMH neurons has been known to be entirely through glucose or free fatty acids. Experiments by Oomura et al. showed that the injection of glucose into the carotid artery resulted in the excitation of VMH neurons [25], and that free fatty acids inhibited glucoreceptive neurons (GRNs) in the VMH [26]. GRNs are reportedly reactive to various satiety-inducing substances that are increased in plasma and cerebrospinal fluid after intake of food, including glucose, insulin, glucagon, carcitonin, corticotropin-releasing hormone (CRH), interleukin-1, acidic fibroblast growth factor, leptin, ␣-melanocyte-stimulating hormone, and various hunger-inducing substances which are increased under hunger states (such as free fatty acids, orexin and ghrelin) [26]. The present results that lycopene induced expression of c-Fos in the VMH neurons thus suggest that lycopene might stimulate GRNs, as satiety-inducing neurons and hunger-inducing neurons in the VMH. The PVH is located in the rostral half of the periventricular area of the hypothalamus, and is subdivided into gigantocellular and parvocellular areas. The neurons of the gigantocellular parts are known to play roles in the synthesis of vasopressin and oxytocin, which accelerate reabsorption of water into the nephron and stimulate smooth muscle of the uterus and lactiferous glands, respectively. The neurons in the parvocellular parts project to the median eminence to regulate the function of the frontal lobe of the pituitary gland and behavioral regulation of motives and various appetites. Neurons in the PVH also play roles in regulation of athrocytosis
and water intake, cardiovascular control and regulation of synthesis of hormones controlling the pituitary frontal lobe hormones such as adrenocorticotropic hormone. The gigantocellular neurons in the SO play a role in the regulation of posterior pituitary hormones by synthesizing vasopressin and oxytocin. These neuronal hormones are transported through the axons to the posterior lobes for secretion into the blood [23]. Neurons in the PVH are particularly deeply involved in the behaviors of food and water intake and are known to be strongly stimulated by food intake among the neurons in the hypothalamus. Stimulation of PVH neurons by norepinephrine reportedly leads to increased food intake behavior and subsequent hyperglycemia and hyperinsulinemia [27]. The PVH is known to contain vasopressin-secreting neurons and to receive projections from angiotensin-sensitive areas of the central preoptic area [28]. Damage to periventricular regions including the PVH has also been reported to induce hyperphagia and adiposis, representing similar responses to the satiety center of the VMH [29]. While the neurons in the PVH and VMH involved in the regulation of food intake and water drinking were found to be excited, neurons in the lateral hypothalamic area (LH) or the DMH acting as regulatory mediators between the LH and VMH displayed c-Fos-ir neurons after the intake of lycopene-rich food. This indicates that cFos-ir neurons in the VMH and PVH may be involved in the function of the satiety center itself. In the present study, the concentration of lycopene in the serum and liver increased in test groups, but the concentration of lycopene in brain tissues was below the limit of detection. In a recent study, lycopene was reportedly detected in the brain [30]. Whether lycopene can pass through the blood brain barrier (BBB) has not been reported. Considering that c-Fos-ir neurons were found in neither the circumventricular organs such as the area postrema,
K. Takayama et al. / Neuroscience Letters 536 (2013) 1–5
organum vasculosum of the lamina terminalis or subfornical organ, which are known to lack a BBB, nor in the neurons of the medulla oblongata projecting from peripheral afferent sensory fibers, it might be suggested that lycopene could pass through the BBB to stimulate central neurons directly. In summary, continuous intake of lycopene-rich food was found to induce accumulation of lycopene in the serum and liver, and also to stimulate central neurons involved in athrocytosis and water drinking. Such findings suggest that lycopene may have some influence on feeding and water-drinking behaviors. The differences in results between experiments after intraperitoneal injection of lycopene and after 4 weeks of continuous intake of lycopene-rich food as shown in the present study remain to be clarified. References [1] G. Britton, Carotenoids 1: structure and properties of carotenoids in relation to function, FASEB Journal 9 (1995) 1551–1558. [2] P.D. Masco, S. Kaiser, H. Sies, Lycopene as the most efficient biological carotenoid singlet oxygen quencher, Archives of Biochemistry and Biophysics 274 (1989) 532–538. [3] N.I. Krinsky, Mechanisms of action of biological antioxidants, Proceedings of the Society for Experimental Biology and Medicine 200 (1992) 248–254. [4] S.K. Clinton, Lycopene: chemistry, biology, and implications for human health and disease, Nutrition Reviews 56 (1998) 35–41. [5] R.S. Gunasekera, K. Sewgobind, S. Desai, L. Dunn, H.S. Black, W.L. McKeehan, B. Patil, Lycopene and lutein inhibit proliferation in rat prostate carcinoma cells, Nutrition and Cancer 58 (2007) 171–177. [6] E. Okajima, M. Tsutsumi, S. Ozono, H. Akai, A. Denda, H. Nishino, S. Oshima, H. Sakamoto, Y. Konishi, Inhibitory effect of tomato juice on Rrat urinary bladder carcinogenesis after N-Butyl-N-(4-hydroxybutyl)nitrosamine initiation, Japanese Journal of Cancer Research 89 (1998) 22–26. [7] T. Narisawa, Y. Fukaura, M. Hasebe, S. Nomura, S. Oshima, H. Sakamoto, T. Inakuma, Y. Ishiguro, J. Takayasu, H. Nishino, Prevention of N-methylnitrosourea-induced colon carcinogenesis in F344 rats by lycopene and tomato juice rich in lycopene, Japanese Journal of Cancer Research 89 (1998) 1003–1008. [8] Y.-M. Hsu, C.-H. Lai, C.-Y. Chang, C.-T. Fan, C.-T. Chen, C.-H. Wu, Characterizing the lipid-lowering effects and antioxidant mechanisms of tomato paste, Bioscience, Biotechnology, and Biochemistry 72 (2008) 677–685. [9] B.H. Juurlink, P.G. Paterson, Review of oxidative stress in brain and spinal cord injury, suggestions for pharmacological and nutritional management strategies, Journal of Spinal Cord Medicine 21 (1998) 309–334. [10] H. Suganuma, T. Hirano, Y.Y. Arimoto, T. Inakuma, Effect of tomato intake on striatal monoamine level in a mouse model of experimental Parkinson’s disease, Journal of Nutritional Science and Vitaminology 48 (2002) 251–254. [11] G. Hsiao, T.H. Fong, N.H. Tzu, K.H. Lin, D.S. Chou, J.R. Sheu, A potent antioxidant, lycopene, affords neuroprotection against microglia activation and focal cerebral ischemia in rats, In Vivo 18 (2004) 351–356. [12] M. Dragunow, R. Faull, The use of c-fos as metabolic marker in neuronal pathway tracing, Journal of Neuroscience Methods 29 (1989) 261–265.
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[13] K. Yakabi, H. Iwabuchi, T. Nakamura, K. Endo, Y. Fukunaga, I. Kumaki, K. Takayama, Neuronal expression of c-Fos protein in the brain after intravenous injection of gastrin in rats, Neuroscience Letters 317 (2002) 57–60. [14] K. Hayashi, K. Takayama, Neuronal expression of c-Fos protein in the brain after intraperitoneal injection of galanin in Wistar rats, Annals of Gunma University School of Health Sciences 27 (2006) 27–31. [15] K. Takayama, Y. Johno, K. Hayashi, K. Yakabi, T. Tanaka, S. Ro, Expression of c-Fos protein in the brain after intravenous injection of ghrelin in rats, Neuroscience Letters 417 (2007) 292–296. [16] K. Takayama, H. Kawata, R. Takahara, K. Tomita, Neuronal expression of cFos protein in the brain after intraperitoneal injection of leptin in Wistar rats, Annals of Gunma University School of Health Sciences 28 (2007) 1–8. [17] K. Takayama, H. Iwazaki, M. Hirabayashi, K. Yakabi, S. Ro, Distribution of c-Fos immunoreactive neurons in the brain after intraperitoneal injection of apelin12 in Wistar rats, Neuroscience Letters 431 (2008) 247–250. [18] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1997. [19] K. Yoshida, H. Yokoyama, T. Oteki, G. Matsumoto, K. Aizawa, T. Inakuma, Evaluation of the effect of dietary lycopene, the main carotenoid in tomato (Lycopersicon esculentum), on the in vivo renal reducing ability by a radiofrequency electron paramagnetic resonance method, Journal of Agricultural and Food Chemistry 59 (2011) 2966–2971. [20] H. Sakamoto, S. Oshima, F. Ojima, Y. Ishiguro, M. Ogawa, H. Fukuba, Changes of serum carotenoids and retinol levels after continual ingestion of carrot or tomato juice in human subjects, Japan Society of Nutrition and Food Science 50 (1997) 21–24. [21] H. Sakamoto, H. Mori, F. Ojima, Y. Ishiguro, S. Arimoto, Y. Imae, T. Nanba, M. Ogawa, H. Fukuba, Elevation of serum carotenoids after continual ingestion of tomato juice, Japan Society of Nutrition and Food Science 47 (1994) 93–99. [22] W. Stahl, W. Schwarz, A.R. Sundquist, H. Sies, Cis-trans isomers of lycopene and beta-carotene in human serum and tissues, Archives of Biochemistry and Biophysics 294 (1992) 173–177. [23] R. Nieuwenhuys, J. Voogd, C. van Huijzen, The Human Central Nervous System. A Synopsis and Atlas, Igakusyoin Pub. Comp., Tokyo, 1991. [24] S. Ishi, T. Shibasaki, Molecular biology of feeding behavior, Bunshi Seishin Igaku 3 (2003) 185–190. [25] Y. Oomura, K. Kimura, H. Ooyama, M. Iki, M. Kuniyoshi, Reciprocal activities of the ventromedial and lateral hypothalamic areas of cats, Science 143 (1964) 484–485. [26] Y. Oomura, Neural mechanism of feeding behavior, Clinical Neuroscience 24 (2006) 887–891. [27] P.G.M. Luiten, G.J. Ter Horst, A.B.S. Steffens, The hypothalamus, intrinsic connections and outflow pathways to the emdocrine system in relation to the control of feeding and metabolism, Progress in Neurobiology 28 (1987) 1–54. [28] L.W. Swanson, G.J. Mogenson, Neural mechanisms for the functional coupling of autonomic, endocrine and somatomotor responses in adaptive behavior, Brain Research Reviews 3 (1981) 1–34. [29] A. Honma, H. Kawano, S. Honma, Y. Kawano, Mechanism of destruction of hypothalamus and induction of satiety by the administration of gold thioglucose, Journal of Japan Society for the Study of Obesity 9 (2003) 108–113. [30] N.E. Craft, T.B. Haitema, K.M. Garnett, K.A. Fitch, C.K. Dorey, Carotenoid, tocopherol, and retinol concentrations in elderly human brain, Journal of Nutrition, Health & Aging 8 (2004) 156–162.
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Study on the expression of c-Fos protein in the brain of rats after ingestion of food rich in lycopene Kiyoshige Takayama a,∗ , Eri Nishiko a , Gaku Matsumoto b , Takahiro Inakuma b a b
Department of Laboratory Sciences, Gunma University School of Health Science, 3-39-15 Showa, Maebashi, Gunma 371-8514, Japan Research Institute, Kagome Co. Ltd., 17 Nishitomiyama, Nasushiobara, Tochigi 329-2762, Japan
h i g h l i g h t s
Lycopene contained in tomato belongs to carotenoid such as beta-carotene. Continuous intake of lycopene-rich food induced accumulation of lycopene in the plasma and liver. Continuous intake of lycopene-rich food induced the stimulation of neurons in the hypothalamus. Continuous intake of lycopene-rich food induced expression of c-Fos in the PVH, VMH and SO.
a r t i c l e
i n f o
Article history: Received 14 July 2012 Received in revised form 31 October 2012 Accepted 20 December 2012 Keywords: Lycopene c-Fos Brain
a b s t r a c t Lycopene, a reddish pigment contained in tomato, belongs to the carotenoid family along with betacarotene and rutein. This study examined whether administration of lycopene to rats would induce excitation of neurons in the central nervous system. Continuous intake of lycopene-rich food was found to induce accumulation of lycopene in the plasma and liver, and stimulated central neurons in the paraventricular nucleus, ventromedial nucleus and supraoptic nucleus of the hypothalamus, which are known to be involved in the functions of athrocytosis and water drinking. These findings suggest that lycopene may have some influence on feeding and water-drinking behaviors. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Lycopene, a reddish pigment contained in tomato, is a carotenoid pigment similar to beta-carotene and rutein [1]. Lycopene is known to have strong scavenging activity against singlet oxygen [2], and acts as an anti-oxidant inhibiting cell-damage caused by free radicals [3,4]. A recent study revealed that ingestion of tomato or lycopene may be correlated with decreased morbidity from certain cancers: lycopene intake reportedly has preventive effects against carcinoma of the prostate [5], bladder cancer [6] and large bowel cancer [7]. Furthermore, lycopene may decrease levels of low-density lipoprotein and total cholesterol in plasma, and thus may help prevent arteriosclerosis and subsequent cardiac infarction or stroke [8]. The brain is known to consume a larger amount of oxygen than other tissues by producing active oxygen, and is very sensitive to oxidative stress [9]. This oxidative stress has been considered to contribute to diseases such as Alzheimer disease or Parkinson disease [10], and attention has focused on substances showing
∗ Corresponding author. Tel.: +81 27 220 8943; fax: +81 27 220 8943. E-mail address: [email protected] (K. Takayama). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.12.035
anti-oxidant activity in the brain. Hsiao et al. reported that injection of lycopene exerted anti-oxidant effects resulting in reduced infarct volume in ischemia/reperfusion brain injury [11]. The proto-oncogene c-fos is well known to induce c-Fos protein transiently within neurons in response to various kinds of stimulations, and the induction of c-Fos protein in neurons starts about 20 min after stimulation [12]. Our laboratory has examined neural expression of c-Fos protein after peripheral administration of a number of peptides, including gastrin, galanin, ghrelin, leptin and apelin [13–17]. All of these peptides were found to induce expression of c-Fos protein in various nuclei in the brain. Our first experiment in the present investigation examined whether lycopene administered peripherally would induce c-Fos protein in the brain, while the second experiment surveyed the neuronal expression of c-Fos protein throughout the central nervous system (CNS) after ingestion of lycopene-rich food. 2. Materials and methods All of the steps to the experimental procedures were conducted in compliance with the Guiding Principles for the Care and Use of Experimental Animals approved by the Gunma University Institute of Experimental Animal Research.
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Experiments were performed using male SPF Wistar rats weighing 240–270 g (9–10 weeks old; SLC Japan, Shizuoka, Japan). To avoid any restlessness of a single rat, 6 rats were housed in a cage in a quiet room under conditions of regulated illumination (12 h light, 12 h dark; lights on at 08:00 h) and constant temperature (22 ± 2 ◦ C) with ad libitum access to water and rat chow (MF diet; Oriental Yeast, Tokyo, Japan). Initially, rats were intraperitoneally injected with lycopene (10 mg/rat in 0.6 ml dimethyl sulfoxide (DMSO)) in the test experiments, while 0.6 ml of DMSO without lycopene was injected in a similar manner in control experiments. At 90 min after the injection, rats were anesthetized by intraperitoneal injection of pentobarbital sodium (80 mg/kg; Abbott Park, IL, USA), and perfused via the left ventricle with about 20 ml of saline solution to flush out the blood. This was immediately followed by 100 ml of 0.5% glutaraldehyde and 4% paraformaldehyde (PFA) (Merck, Darmstadt, Germany) in 0.1 M phosphate buffer (PB) (pH 7.4) under pressure of 100–120 mmHg, and 400 ml of 4% PFA in PB under hydrostatic pressure. The brains were removed and cut into two blocks at the level between superior and inferior colliculi, fixed in 4% PFA in PB for a further 1.5 h at 4 ◦ C, and then soaked in a stepwise manner in 10%, 20%, and 25% sucrose in PB at 4 ◦ C. The brains were frozen and cut into serial transverse sections at 40 m thickness. Brain sections were collected in plates containing PB chilled by ice water. One group of every fourth section was rinsed in 0.1 M Tris–saline (TS) (pH 7.4) three times, incubated with 0.5% bovine serum albumin (BSA) in TS for 20 min and incubated with sheep anti-c-Fos polyclonal antiserum (1:100, sc52; Santa Cruz Biotech, Santa Cruz, CA, USA) in 0.5% BSA at 4 ◦ C for 16 h. All the following procedures were conducted at room temperature. The sections were rinsed three times in TS, then incubated in avidin and biotin solution, respectively, for 15 min each. Thereafter, sections were incubated for 20 min with 2% normal rabbit serum in TS to block nonspecific binding, and incubated for 60 min with anti-sheep immunoglobulin (Ig)G in TS. After rinsing three times in TS, sections were incubated for 60 min in Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA), and then treated with diaminobenzidine (DAB)/nickel solution containing 0.003% H2 O2 . Then, sections were rinsed twice in TS and rinsed further in PB, mounted on glass slides and dried. Sections on slides were dehydrated in a graded ethanol series (50%, 70%, 95%, 99%), infiltrated with xylene and cover-slipped in Permount (Fisher, Fair Lawn, NJ, USA). In the control experiments, rats (n = 4) were sham-operated and injected with 200 l of 0.9% NaCl solution. Brain sections were processed as described above. Localization of c-Fos protein in neuronal nuclei was visualized as black precipitates of nickel-intensified DAB reaction products. The c-Fos-immunoreactive (c-Fos-ir) neurons were surveyed under bright-field microscopy. Brain histology was checked against the rat brain atlas of Swanson [18]. In the second experiment, rats ingested normal food placed in a metal food box for 11 days before starting experiments. Afterwards, rats were arbitrarily divided into 2 groups, with 2 rats placed in each cage. Twelve rats in 6 cages were used for test experiments, and those in the other 6 cages for control experiments. In the test experiment, rats ingested lycopene-rich food (0.2% lycopene-containing MF diet) freely for 24 h, while control rats were similarly provided with normal food (MF diet). Body weights were measured once a week during the experiments. After starting the ingestion of each food in the test and control experiments, rats were processed for immunohistochemical staining of c-Fos protein using the methods described above, and for weighing amounts of lycopene contained in various organs every week. One rat from each group was chosen for c-Fos immunohistochemical staining and the other for measuring the lycopene content of various tissues. Experiments lasted as long as 6 weeks.
2.1. Extraction of lycopene contained in internal organs 2.1.1. Extraction from tissues Tissue (0.1 g) was homogenized with a 9-fold volume of PB in a centrifuge tube (10 ml), and stirred for 1 min with a 10-l internal standard (IS) (trans--Apo-8 -carotenal; Sigma–Aldrich, St. Louis, MO, USA), 50 l of 2 mM ␣-tocopherol and 3 ml of MeOH/CH2 Cl2 (2:1, v/v). Two milliliters of hexane was added and stirred. After centrifuging 3000 rpm for 5 min, the hexane layer was removed. Afterwards, the solution was evaporated to dry, and the residual lipid was saponified using 2 ml of 60% KOH at 60 ◦ C for 45 min. To the solution was added 2 ml of distilled water, 2 ml of ethanol and 8 ml of hexane before centrifuging at 3000 rpm to remove the hexane layer. The hexane solution was then evaporated under centrifugation. The residue was dissolved in 50 l of methanol/CH2 Cl2 (1:4) and filtered, and analyzed with HPLC as described below. 2.1.2. Extraction from blood serum To 500 l of serum sample was added IS, 1 ml of 0.01% BHT containing ethanol, and 4 ml of hexane/CH2 Cl2 (4:1). This mixture was centrifuged at 2000 rpm for 10 min. The supernatant was recovered and 1 ml of hexane/CH2 Cl2 (4:1) was added, stirred and centrifuged. The supernatant was again removed, the solvent evaporated, and then 100 l of ethanol was added to dissolve the extracts. After filtration, the filtrate was analyzed with HPLC. 2.2. HPLC analysis A previously described method [19] was used for quantifying lycopene in tissues and serum. HPLC analysis was performed using a photodiode detector (SPD-M10; Shimadzu, Kyoto, Japan) with a wavelength of 460 nm and a C30 carotenoid column (250 mm × 4.6 mm, 5 m; YMC, Wilmington, NC, USA). Lycopene content was quantified using an internal standard. 2.3. Statistical analysis Results are expressed are mean ± standard error of the mean, and the Mann–Whitney U-test was performed to compare results between test and control groups. Values of P < 0.05 were taken as statistically significant. 3. Results 3.1. Survey of c-Fos-ir neurons after intraperitoneal injection of lycopene The c-Fos protein was found in several nuclei in the brain, including the ventromedial nucleus hypothalamus (VMH), paraventricular nucleus hypothalamus (PVH), supraoptic nucleus (SO), central nucleus amygdala (CEA), paraventricular nucleus thalamus (PVT), periaqueductal gray matter (PAG), lateral parabrachial nucleus (PBL), and the complex of the solitary tract nucleus and dorsal motor nucleus of the vagus nerve (NTS/DMX) (Table 1). The sites of these nuclei were identified according to the atlas by Paxinos and Watson [18]. 3.2. Measurement of body weight of rat and of amount of lycopene in internal organs Body weights of rats in the test and control groups increased during experimental periods by 39 ± 2 g and 37 ± 6 g, respectively, showing no significant differences between groups (Table 2). Similarly, no significant differences were observed in any tissue weights between the test and control groups (Table 3). On the other hand, great increases in lycopene concentration were seen in blood
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Table 1 Expression of c-Fos-immunoreactive neurons in the brain after intraperitoneal injection of lycopene in DMSO (test experiment, n = 3). In the control experiment, no lycopene was contained in the DMSO (n = 3). Number of c-Fos-immunoreactive neurons in one experiment: −, no; +, 2–10; ++, 11–50; +++, >50. Nucleus
Test experiment
Control experiment
SFO SO PAH VMH LH CEA PVT PAG LC Pbl VLM NTS/DMX AP
+ +++ +++ ++ + +++ ++ ++ + +++ ++ +++ +
− + + + − + + + − + + + −
Abbreviations: AP, area postrema; CEA, central nucleus amygdale; LC, locus, coeruleus; LH, lateral hypothalamic nucleus; NTS/DMX, complex of solitary tract nucleus and dorsal motor nucleus of vagus nerve; PAG, periaqueductal gray matter; PAH, paraventricular nucleus hypothalamus; Pbl, lateral parabrachial nucleus; PVT, paraventricular nucleus thalamus; SFO, subfornical organ; SO, supraoptic nucleus; VLM, ventrolateral medulla; VMH, ventromedial hypothalamic area.
Fig. 1. Number of c-Fos-immunoreactive neurons in control (n = 4) and test groups (n = 4). Mean ± standard error of the mean. PVH, paraventricular nucleus hypothalamus; SO, supraoptic nucleus; VMH, ventromedial nucleus hypothalamus.
4. Discussion In the present study, continuous intake of lycopene was not found to influence the weights of internal organs, although lycopene accumulation in the liver and serum was confirmed. These results support those obtained in human investigations [20,21]. Measurements of the lycopene content in blood serum and tissues in humans under daily intake of foods have revealed that lycopene distributes to many tissues, not only in the blood serum and liver, but also in the adrenal glands, testes and ovaries [22]. As in humans, orally administered lycopene was absorbed into the rat body and accumulated in organs such as blood serum and liver. In the ingestion experiment, continuous intake of lycopenerich foods induced expression of c-Fos-positive neurons in the VMH, PVH and SO. This result was in accordance with findings from the experiment after intraperitoneal injection of lycopene. In addition, some nuclei only showed c-Fos-positive neurons after intraperitoneal injection of lycopene, not after continuous intake of lycopene-containing foods. The concentration of lycopene in blood serum after intake of lycopene-containing food may be much lower than that after intraperitoneal injection, because the lycopene has to be absorbed through the alimentary canal into the blood. The difference in the distribution of c-Fos-positive neurons might be due to the different concentrations of lycopene, as well as to the duration of stimulation by lycopene. Neurons in the PVH, SO and VMH might be stimulated continuously regardless of the concentration of lycopene in the blood, while neurons in other nuclei might be stimulated only transiently and acutely by higher concentrations of lycopene in the blood. Neurons in the VMH, in which c-Fos was found to be induced after administration of lycopene, are known to be involved in the
Table 2 Changes in body weight and body weight gains in test (n = 12) and control rats (n = 12) during experiments (mean ± standard error of the mean). Group
Control Test
Body weight (g)
Body weight gain
Beginning (3 day)
After 25 or 26 days
285 ± 2 284 ± 2
324 ± 3 321 ± 6
39 ± 2 37 ± 6
serum (14.46 ± 5.5 ng/ml) and liver (6.89 ± 1.52 g/g-wet) in the test group, but not in the control group. However, almost no lycopene was detected in the brain in the test group.
3.3. Survey of c-Fos-ir neurons after ingestion of lycopene-rich food A survey of c-Fos-ir neurons was conducted using whole-brain sections from the medulla oblongata to the cerebral cortex. These c-Fos-ir neurons were particularly prominent in areas of the hypothalamus such as the PVH, SO and VMH. Numbers of c-Fos-ir neurons in these nuclei were counted. Statistical analysis was performed and significant differences were found in numbers of cFos-ir neurons in the PVH and VMH between test and control rats, although no significant differences were seen for the SO (Fig. 1). Representative photomicrographs of c-Fos-ir neurons in the PVH and VMH are shown in Fig. 2.
Table 3 Weights of various tissues in rats in the test and control groups (n = 12 each) and ratio of tissue weights to body weight (n = 6). n.d.: not detected. Tissue organ
Weight (g) Control
Brain Liver Kidney Testis Spleen Lung Stomach Heart Blood plasma
1.9 11.1 2.1 3.3 0.7 1.4 2.2 0.8
± ± ± ± ± ± ± ±
Test 0 0.4 0.2 0.2 0 0.1 0.1 0
1.9 11.1 2.2 3.2 0.7 1.4 2.2 0.9
± ± ± ± ± ± ± ±
0 0.2 0.1 0.1 0 0 0.2 0.1
Ratio of organ weight to body weight (%)
Concentration in lycopene
Control
Control
Test
n.d. n.d.
n.d. 6.89 ± 1.52 (g/g-wet)
n.d.
14.46 ± 5.5 (ng/ml)
0.6 3.5 0.6 1.0 0.2 0.4 0.7 0.3
± ± ± ± ± ± ± ±
0 0.1 0 0.1 0 0 0 0
Test 0.6 3.4 0.6 1.0 0.2 0.4 0.7 0.3
± ± ± ± ± ± ± ±
0 0.1 0 0 0 0 0.1 0
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Fig. 2. Representative microphotographs of c-Fos-immunoreactive neurons in the paraventricular nucleus hypothalamus (PVH) and ventromedial nucleus hypothalamus (VMH) in test rat (A) and control rat (B).
beginning of the fighting action by PAG neurons projecting from the medial nucleus of the amygdala, and in the endocrine functions of the pancreas and adrenal medulla through neurons in the PAG, medulla oblongata and spinal cord [23]. VMH is also known as the satiety center, and damage to this area can induce hyperphagia and obesity, while stimulation inhibits food intake [24]. The regulation of food intake by VMH neurons has been known to be entirely through glucose or free fatty acids. Experiments by Oomura et al. showed that the injection of glucose into the carotid artery resulted in the excitation of VMH neurons [25], and that free fatty acids inhibited glucoreceptive neurons (GRNs) in the VMH [26]. GRNs are reportedly reactive to various satiety-inducing substances that are increased in plasma and cerebrospinal fluid after intake of food, including glucose, insulin, glucagon, carcitonin, corticotropin-releasing hormone (CRH), interleukin-1, acidic fibroblast growth factor, leptin, ␣-melanocyte-stimulating hormone, and various hunger-inducing substances which are increased under hunger states (such as free fatty acids, orexin and ghrelin) [26]. The present results that lycopene induced expression of c-Fos in the VMH neurons thus suggest that lycopene might stimulate GRNs, as satiety-inducing neurons and hunger-inducing neurons in the VMH. The PVH is located in the rostral half of the periventricular area of the hypothalamus, and is subdivided into gigantocellular and parvocellular areas. The neurons of the gigantocellular parts are known to play roles in the synthesis of vasopressin and oxytocin, which accelerate reabsorption of water into the nephron and stimulate smooth muscle of the uterus and lactiferous glands, respectively. The neurons in the parvocellular parts project to the median eminence to regulate the function of the frontal lobe of the pituitary gland and behavioral regulation of motives and various appetites. Neurons in the PVH also play roles in regulation of athrocytosis
and water intake, cardiovascular control and regulation of synthesis of hormones controlling the pituitary frontal lobe hormones such as adrenocorticotropic hormone. The gigantocellular neurons in the SO play a role in the regulation of posterior pituitary hormones by synthesizing vasopressin and oxytocin. These neuronal hormones are transported through the axons to the posterior lobes for secretion into the blood [23]. Neurons in the PVH are particularly deeply involved in the behaviors of food and water intake and are known to be strongly stimulated by food intake among the neurons in the hypothalamus. Stimulation of PVH neurons by norepinephrine reportedly leads to increased food intake behavior and subsequent hyperglycemia and hyperinsulinemia [27]. The PVH is known to contain vasopressin-secreting neurons and to receive projections from angiotensin-sensitive areas of the central preoptic area [28]. Damage to periventricular regions including the PVH has also been reported to induce hyperphagia and adiposis, representing similar responses to the satiety center of the VMH [29]. While the neurons in the PVH and VMH involved in the regulation of food intake and water drinking were found to be excited, neurons in the lateral hypothalamic area (LH) or the DMH acting as regulatory mediators between the LH and VMH displayed c-Fos-ir neurons after the intake of lycopene-rich food. This indicates that cFos-ir neurons in the VMH and PVH may be involved in the function of the satiety center itself. In the present study, the concentration of lycopene in the serum and liver increased in test groups, but the concentration of lycopene in brain tissues was below the limit of detection. In a recent study, lycopene was reportedly detected in the brain [30]. Whether lycopene can pass through the blood brain barrier (BBB) has not been reported. Considering that c-Fos-ir neurons were found in neither the circumventricular organs such as the area postrema,
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organum vasculosum of the lamina terminalis or subfornical organ, which are known to lack a BBB, nor in the neurons of the medulla oblongata projecting from peripheral afferent sensory fibers, it might be suggested that lycopene could pass through the BBB to stimulate central neurons directly. In summary, continuous intake of lycopene-rich food was found to induce accumulation of lycopene in the serum and liver, and also to stimulate central neurons involved in athrocytosis and water drinking. Such findings suggest that lycopene may have some influence on feeding and water-drinking behaviors. The differences in results between experiments after intraperitoneal injection of lycopene and after 4 weeks of continuous intake of lycopene-rich food as shown in the present study remain to be clarified. References [1] G. Britton, Carotenoids 1: structure and properties of carotenoids in relation to function, FASEB Journal 9 (1995) 1551–1558. [2] P.D. Masco, S. Kaiser, H. Sies, Lycopene as the most efficient biological carotenoid singlet oxygen quencher, Archives of Biochemistry and Biophysics 274 (1989) 532–538. [3] N.I. Krinsky, Mechanisms of action of biological antioxidants, Proceedings of the Society for Experimental Biology and Medicine 200 (1992) 248–254. [4] S.K. Clinton, Lycopene: chemistry, biology, and implications for human health and disease, Nutrition Reviews 56 (1998) 35–41. [5] R.S. Gunasekera, K. Sewgobind, S. Desai, L. Dunn, H.S. Black, W.L. McKeehan, B. Patil, Lycopene and lutein inhibit proliferation in rat prostate carcinoma cells, Nutrition and Cancer 58 (2007) 171–177. [6] E. Okajima, M. Tsutsumi, S. Ozono, H. Akai, A. Denda, H. Nishino, S. Oshima, H. Sakamoto, Y. Konishi, Inhibitory effect of tomato juice on Rrat urinary bladder carcinogenesis after N-Butyl-N-(4-hydroxybutyl)nitrosamine initiation, Japanese Journal of Cancer Research 89 (1998) 22–26. [7] T. Narisawa, Y. Fukaura, M. Hasebe, S. Nomura, S. Oshima, H. Sakamoto, T. Inakuma, Y. Ishiguro, J. Takayasu, H. Nishino, Prevention of N-methylnitrosourea-induced colon carcinogenesis in F344 rats by lycopene and tomato juice rich in lycopene, Japanese Journal of Cancer Research 89 (1998) 1003–1008. [8] Y.-M. Hsu, C.-H. Lai, C.-Y. Chang, C.-T. Fan, C.-T. Chen, C.-H. Wu, Characterizing the lipid-lowering effects and antioxidant mechanisms of tomato paste, Bioscience, Biotechnology, and Biochemistry 72 (2008) 677–685. [9] B.H. Juurlink, P.G. Paterson, Review of oxidative stress in brain and spinal cord injury, suggestions for pharmacological and nutritional management strategies, Journal of Spinal Cord Medicine 21 (1998) 309–334. [10] H. Suganuma, T. Hirano, Y.Y. Arimoto, T. Inakuma, Effect of tomato intake on striatal monoamine level in a mouse model of experimental Parkinson’s disease, Journal of Nutritional Science and Vitaminology 48 (2002) 251–254. [11] G. Hsiao, T.H. Fong, N.H. Tzu, K.H. Lin, D.S. Chou, J.R. Sheu, A potent antioxidant, lycopene, affords neuroprotection against microglia activation and focal cerebral ischemia in rats, In Vivo 18 (2004) 351–356. [12] M. Dragunow, R. Faull, The use of c-fos as metabolic marker in neuronal pathway tracing, Journal of Neuroscience Methods 29 (1989) 261–265.
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[13] K. Yakabi, H. Iwabuchi, T. Nakamura, K. Endo, Y. Fukunaga, I. Kumaki, K. Takayama, Neuronal expression of c-Fos protein in the brain after intravenous injection of gastrin in rats, Neuroscience Letters 317 (2002) 57–60. [14] K. Hayashi, K. Takayama, Neuronal expression of c-Fos protein in the brain after intraperitoneal injection of galanin in Wistar rats, Annals of Gunma University School of Health Sciences 27 (2006) 27–31. [15] K. Takayama, Y. Johno, K. Hayashi, K. Yakabi, T. Tanaka, S. Ro, Expression of c-Fos protein in the brain after intravenous injection of ghrelin in rats, Neuroscience Letters 417 (2007) 292–296. [16] K. Takayama, H. Kawata, R. Takahara, K. Tomita, Neuronal expression of cFos protein in the brain after intraperitoneal injection of leptin in Wistar rats, Annals of Gunma University School of Health Sciences 28 (2007) 1–8. [17] K. Takayama, H. Iwazaki, M. Hirabayashi, K. Yakabi, S. Ro, Distribution of c-Fos immunoreactive neurons in the brain after intraperitoneal injection of apelin12 in Wistar rats, Neuroscience Letters 431 (2008) 247–250. [18] G. Paxinos, C. Watson, The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1997. [19] K. Yoshida, H. Yokoyama, T. Oteki, G. Matsumoto, K. Aizawa, T. Inakuma, Evaluation of the effect of dietary lycopene, the main carotenoid in tomato (Lycopersicon esculentum), on the in vivo renal reducing ability by a radiofrequency electron paramagnetic resonance method, Journal of Agricultural and Food Chemistry 59 (2011) 2966–2971. [20] H. Sakamoto, S. Oshima, F. Ojima, Y. Ishiguro, M. Ogawa, H. Fukuba, Changes of serum carotenoids and retinol levels after continual ingestion of carrot or tomato juice in human subjects, Japan Society of Nutrition and Food Science 50 (1997) 21–24. [21] H. Sakamoto, H. Mori, F. Ojima, Y. Ishiguro, S. Arimoto, Y. Imae, T. Nanba, M. Ogawa, H. Fukuba, Elevation of serum carotenoids after continual ingestion of tomato juice, Japan Society of Nutrition and Food Science 47 (1994) 93–99. [22] W. Stahl, W. Schwarz, A.R. Sundquist, H. Sies, Cis-trans isomers of lycopene and beta-carotene in human serum and tissues, Archives of Biochemistry and Biophysics 294 (1992) 173–177. [23] R. Nieuwenhuys, J. Voogd, C. van Huijzen, The Human Central Nervous System. A Synopsis and Atlas, Igakusyoin Pub. Comp., Tokyo, 1991. [24] S. Ishi, T. Shibasaki, Molecular biology of feeding behavior, Bunshi Seishin Igaku 3 (2003) 185–190. [25] Y. Oomura, K. Kimura, H. Ooyama, M. Iki, M. Kuniyoshi, Reciprocal activities of the ventromedial and lateral hypothalamic areas of cats, Science 143 (1964) 484–485. [26] Y. Oomura, Neural mechanism of feeding behavior, Clinical Neuroscience 24 (2006) 887–891. [27] P.G.M. Luiten, G.J. Ter Horst, A.B.S. Steffens, The hypothalamus, intrinsic connections and outflow pathways to the emdocrine system in relation to the control of feeding and metabolism, Progress in Neurobiology 28 (1987) 1–54. [28] L.W. Swanson, G.J. Mogenson, Neural mechanisms for the functional coupling of autonomic, endocrine and somatomotor responses in adaptive behavior, Brain Research Reviews 3 (1981) 1–34. [29] A. Honma, H. Kawano, S. Honma, Y. Kawano, Mechanism of destruction of hypothalamus and induction of satiety by the administration of gold thioglucose, Journal of Japan Society for the Study of Obesity 9 (2003) 108–113. [30] N.E. Craft, T.B. Haitema, K.M. Garnett, K.A. Fitch, C.K. Dorey, Carotenoid, tocopherol, and retinol concentrations in elderly human brain, Journal of Nutrition, Health & Aging 8 (2004) 156–162.