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Central administration of insulin suppresses food intake in chicks
Neuroscience Letters 423 (2007) 153–157
Central administration of insulin suppresses food intake in chicks Kazuhisa Honda a , Hiroshi Kamisoyama a , Takaoki Saneyasu a , Kunio Sugahara b , Shin Hasegawa a,∗ b
a Department of Animal Science, Faculty of Agriculture, Kobe University, Kobe 657-8501, Japan Department of Animal Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan
Received 16 March 2007; received in revised form 1 June 2007; accepted 14 July 2007
Abstract Although the orexigenic action of peptide hormones such as ghrelin and growth hormone releasing peptide is different between chickens and mammals, the anorexigenic action of peptide hormones is similar in both species. For example, central administration of peptide hormones such as leptin, cholecystokinin or glucagon has been shown to suppress food intake behavior in chickens and mammals. Central administration of insulin suppresses food intake in mammals. However, the anorexigenic action of insulin in chickens has not yet been identified. In the present study, we investigated the effects of central administration of insulin on food intake in chicks. Intracerebroventricular administration of insulin in chicks significantly suppressed food intake. Central administration of insulin significantly upregulated mRNA levels of proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART) and corticotropin-releasing factor (CRF), but did not influence mRNA levels of neuropeptide Y and agouti-related protein in the hypothalamus. These results suggest that alpha-melanocyte stimulating hormone (␣-MSH, an anorexigenic peptide from the post-translational cleavage of POMC), CART and CRF are involved in the anorexigenic action of insulin in chicks. Furthermore, central administration of ␣-MSH or CART significantly suppressed food intake. In addition, ␣-MSH significantly upregulated CRF mRNA expression, suggesting that the anorexigenic action of ␣-MSH is mediated by CRF. Our findings demonstrate that insulin functions in chicks as an appetite-suppressive peptide in the central nervous system and suggest that this anorexigenic action is mediated by CART, ␣-MSH and CRF. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Food intake; Chicken; Insulin; Proopiomelanocortin; Cocaine- and amphetamine-regulated transcript; Corticotropin-releasing factor
In mammals, the central regulatory mechanisms of food intake are known to be closely related to energy metabolism [15,19]. For example, insulin and leptin are sensed by neurons in the hypothalamic arcuate nucleus (ARC); they act as important peripheral hormones in energy metabolism, and such hormones influence the regulation of food intake [15,19]. The ARC contains two functionally different neurons: (a) neurons that suppress food intake by releasing alpha-melanocyte stimulating hormone (␣-MSH), cocaine- and amphetamine-regulated transcript (CART), or both; and (b) neurons that stimulate food intake by releasing neuropeptide Y (NPY), agouti-related protein (AgRP), or both. Intracerebroventriular administration of insulin in rats increases proopiomelanocortin (POMC, a precursor of ␣-MSH and adrenocorticotropic hormone) mRNA levels [1]. Li et al. suggest that CART-producing neurons in the ARC
∗
Corresponding author. Tel.: +81 78 803 5808; fax: +81 78 803 5808. E-mail address: [email protected] (S. Hasegawa).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.07.004
are sensitive to changes in plasma insulin levels [14]. Central and peripheral administration of insulin in rats suppresses food intake and decreases NPY mRNA [16,21]. Subcutaneous administration of insulin in rats decreases AgRP mRNA levels [16]. These observations suggest that ␣-MSH, CART, NPY and AgRP act as downstream molecules of insulin-induced appetite suppressive pathways in mammals. In addition, peripheral administration of insulin increases corticotropin-releasing factor (CRF) mRNA levels in rat hypothalamus [2,23], and there is evidence that the anorexigenic action of ␣-MSH [7] or CART [27] is mediated by CRF. It is therefore likely that CRF is also involved in the anorexigenic action of insulin and acts as a downstream molecule in the ␣-MSH- or CART-induced appetite suppressive pathway. The regulatory mechanisms of food intake in poultry have been extensively studied in recent decades [12,17]. Growing evidence indicates that, although the orexigenic action of peptide hormones such as ghrelin and growth hormone releasing peptide is different between chickens and mammals, the
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K. Honda et al. / Neuroscience Letters 423 (2007) 153–157
anorexigenic action of peptide hormones is similar in both species [17]. For example, central administration of anorexigenic peptide hormones such as leptin [4], cholecystokinin [5] or glucagon [9] has been shown to have the same effects on food intake in chickens as in mammals [10,15,19]. Insulin receptors exist in chicken brain [20], and peripheral administration of insulin can suppress food intake in chickens [22]. However, the effects of central administration of insulin on food intake have not yet been identified. In this study, we focused on the functional roles of insulin in the central regulation of food intake in chicks. The results show that insulin suppresses food intake via hypothalamic anorexigenic neuropeptides. Day-old male chicks (White Leghorn) were purchased from a local hatchery (Ghen Corporation, Gifu, Japan). They were given free access to water and a commercial chick starter diet (Nippon Formula Feed Mfg. Co., Ltd., Kanagawa, Japan). Room temperature was maintained at 32 ± 2 ◦ C. All experimental procedures followed the guidelines for the care and use of experimental animals at the Rokkodai Campus of Kobe University in Japan. Insulin was purchased from Wako Pure Chemical Industries, Ltd. (bovine insulin, Osaka, Japan). All primers were purchased from Hokkaido System Science Co., Ltd. (Hokkaido, Japan). In Experiment 1, 8-day-old chicks were divided into four groups. Insulin was dissolved in a 0.85% (w/v) saline solution containing 0.1% (w/v) Evans Blue. Either insulin (0.1, 0.4, 2, or 10 g) or saline (as a control) was intracerebroventricularly administered according to the method of Davis et al. [3] at a volume of 10 l after 3 h of fasting. Food intake was measured at 30, 60 and 120 min after administration. At the end of the experiment, the chicks were sacrificed by decapitation. Verification of injection was made by observation of the presence of Evans Blue dye in the lateral ventricle. In Experiment 2, 8-day-old chicks were divided into two groups, and either 10 g insulin or saline (as a control) was intracerebroventricularly administered as described in Experiment 1. The significant difference in food intake between control and insulin group was found at 60 and 120 min but not at 30 min after administration of 10 g insulin in Experiment 1. According to this evidence, we measured POMC, CART, NPY, AgRP and CRF mRNA levels at 60 min after central administration of insulin. At that point, the chicks were sacrificed. Their brains were removed within 1 min of decapitation, frozen on powdered dry ice, weighed and stored at −80 ◦ C for further analysis. The hypothalamus was dissected from the frozen brain by referring to a stereotaxic atlas drawn by Kuenzel and Masson [13] and weighed. Total RNA was extracted from the hypothalamus using the Sepazol-RNA I (Nacalai Tesque, Inc., Kyoto, Japan). Firststrand cDNA was synthesized from 5 g of total RNA treated with DNase I (Invitrogen, Carlsbad, California, USA) using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, California, USA) with random primers. PCR was performed using TaKaRa Ex Taq (Takara Bio, Inc., Siga, Japan). Complementary DNA of POMC (GenBank accession no. AB019555), CART (GenBank accession no. BI394769), NPY (GenBank accession no. M87294), and AgRP (GenBank accession no. AB029443) were amplified with the following
primers: POMC sense, 5 -AGA TGG AGA AGG GTT GGA A-3 ; POMC antisense, 5 -CGT TGG GGT ACA CCT TGA-3 ; CART sense, 5 -CCG CAC TAC GAG AAG AAG-3 ; CART antisense, 5 -AGG CAC TTG AGA AGA AAG G-3 ; NPY sense, 5 -CTT GTC GCT GCT GAT CTG-3 ; NPY antisense, 5 -GCC TCA GAG CCG AGT AGT-3 ; AgRP sense, 5 -GCA GGA AGG TGA TGG TAA C-3 ; AgRP antisense, 5 -GTC ACA GCA GGG GAT CTG-3 . Complementary DNA of CRF was amplified with specific primers as described previously [9]. As an internal standard, chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank accession no. NM 204305) was also amplified, using the following primers: GAPDH sense, 5 -GGT GCT AAG CGT GTT ATC ATC TCA-3 ; GAPDH antisense, 5 -CAT GGT TGA CAC CCA TCA CAA-3 . Initially, we performed amplification experiments in each gene and determined the cycle number that the amplification products in each gene were between 20 and 40 cycle number and also within the linear portion of the amplification curve. Then, PCR reactions were performed as follows: 94 ◦ C for 5 min; 30 cycles (NPY and AgRP), 33 cycles (POMC and CART), 35 cycles (CRF) or 24 cycles (GAPDH) of 94 ◦ C for 30 s, 55 ◦ C for 1 min and 72 ◦ C for 1 min; and final extension at 72 ◦ C for 4 min. The PCR products were analyzed by ethidium bromide (Nacalai Tesque, Inc., Kyoto, Japan) staining on 1% agarose gel. The fluorescence from PCR products was detected and quantified with Typhoon 9400 (Amersham Biosciences Corp., Piscataway, New Jersey, USA). In Experiment 3, 8-day-old chicks were divided into three groups. Central administration of 1 g ␣-MSH [11] or 5 g CART [26] suppresses food intake in chicks. We followed these studies and either 1 g of ␣-MSH, 5 g CART or saline (as a control) was intracerebroventricularly administered as described in Experiment 1. Food intake was measured at 30, 60 and 120 min after administration. We found that central administration of ␣-MSH or CART in chicks significantly suppressed food intake (p < 0.05) throughout the experimental period. Therefore, we measured CRF mRNA levels at 30 min after central administration of ␣-MSH or CART. 8-day-old chicks were divided into three groups and either 1 g of ␣-MSH, 5 g CART or saline (as a control) was intracerebroventricularly administered as described in Experiment 1. Hypothalamic mRNA of CRF and GAPDH were determined as described in Experiment 2. Data from Experiments 1 and 3 were analyzed by the Tukey–Kramer test. Data from Experiment 2 were analyzed by Student’s t-test. All statistics was performed using the commercial package StatView version 5 (SAS Institute, Cary, North Carolina, USA, 1998). Central administration of insulin in chicks significantly suppressed food intake (p < 0.05) in a dose-dependent manner (Experiment 1, Fig. 1). It is known that insulin upregulates mRNA of POMC, whereas this peptide downregulates mRNA of NPY and AgRP in the hypothalamus [15,19]. CART-producing neurons in the ARC are thought to be sensitive to plasma insulin levels [14]. Therefore, we next examined whether ␣-MSH, CART, NPY and AgRP, act as downstream molecules in the insulin-induced anorexigenic pathway in chicks. Insulin administration significantly upregulated mRNA expressions of POMC
K. Honda et al. / Neuroscience Letters 423 (2007) 153–157
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Fig. 1. Effect of central administration of insulin on cumulative food intake in chicks. Data are means ± S.E.M. The number of chicks used is shown in parentheses. Groups with different letters are significantly different (p < 0.05) at each time point.
and CART (p < 0.05, Experiment 2, Fig. 2), but did not influence NPY and AgRP mRNA levels (Experiment 2, Fig. 3). Thus, these results suggest that insulin functions as an appetite-suppressive peptide and that insulin selectively modulates mRNA levels of
Fig. 3. Effect of central administration of insulin on hypothalamic mRNA levels of NPY and AgRP in chicks. Data are means ± S.E.M. of six chicks in each group.
Fig. 2. Effect of central administration of insulin on hypothalamic mRNA levels of POMC and CART in chicks. Data are means ± S.E.M. of six chicks in each group. *Significant with respect to saline (p < 0.05).
POMC and CART to suppress food intake. Insulin receptors exist in avian brains [20]. However, up to now, there is no report demonstrating the distribution of insulin receptor in the chicken brain. Our results showed that central administration of insulin did not influence NPY and AgRP mRNA levels in hypothalamus. Thus, it seems that insulin receptor might not be expressed in hypothalamic NPY and AgRP neurons in chickens. Chan et al. [2] and Suda et al. [23] have reported that peripheral administration of insulin increases CRF mRNA levels in rat hypothalamus. Central administration of CRF suppresses food intake in chicks [6,25]. Therefore, we next examined the effects of insulin on CRF mRNA level in chicken hypothalamus. Central administration of insulin significantly upregulated mRNA expression of CRF (p < 0.05, Experiment 2, Fig. 4). These findings and our results suggest that CRF is also involved in anorexigenic action of insulin in chicks. In mammalian hypothalamus, POMC-producing neurons project from ARC to the paraventricular nucleus (PVN) in hypothalamus, and PVN contains CRF-producing neurons [15,19]. Central administration of CART in rats suppresses food intake with inducing Fos-like immunoreactivity in CRFcontaining neurons in the PVN [27]. These findings suggest that anorexigenic signals from POMC- or CART-producing neu-
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Fig. 4. Effect of central administration of insulin on hypothalamic CRF mRNA levels in chicks. Data are means ± S.E.M. of six chicks in each group. *Significant with respect to saline (p < 0.05).
Fig. 5. Effect of central administration of ␣-MSH or CART on hypothalamic CRF mRNA levels in chicks. Data are means ± S.E.M. of six chicks in each group. Groups with different letters are significantly different (p < 0.05).
rons in the ARC are integrated to CRF neuron in the PVN. Immunohistochemical studies have shown that hypothalamic POMC- or CRF-producing neurons in chickens localize in the same nuclei as those in mammals [8,18]. For example, POMC-immunoreactive perikarya are present in infundibular nuclei (equivalent of the mammalian ARC) [8], and CRFimmunoreactive perikarya are present in the PVN [18]. The CRF receptor antagonist astressin significantly reversed the anorexigenic action of ␣-MSH in chicks [24]. These observations raise the question whether anorexigenic action of ␣-MSH or CART is mediated by CRF in chickens as well as in mammals. Therefore, we finally examined whether hypothalamic CRF acts as a downstream molecule in the ␣-MSH- or CART-induced appetite suppressive pathway in chicks. Central administration of ␣-MSH or CART in chicks significantly suppressed food intake (p < 0.05) throughout the experimental period (from 30 to 120 min after administration). At 30 min after administration, food intake was 0.91 ± 0.19 g in control (saline alone), and 0.09 ± 0.03 g and 0.33 ± 0.14 g in ␣-MSH and CART injections, respectively; at 60 min, 1.54 ± 0.32 g in control, and 0.10 ± 0.03 g and 0.36 ± 0.13 g in ␣-MSH and CART, respectively; at 120 min, 2.21 ± 0.24 g in control, and 0.10 ± 0.03 g and 0.36 ± 0.13 g in ␣-MSH and CART, respectively (means ± S.E.M. [n = 5 in each group]). Central administration of ␣-MSH significantly upregulated CRF mRNA but central administration of CART did not influence CRF mRNA (Experiment 3, Fig. 5). Thus, these results suggest the involvement of CRF in ␣-MSH-induced but not CART-induced anorexigenic action in chicks. In summary, we studied the functional roles of insulin in the central regulation of food intake in chicks. Our data clearly showed that central administration of insulin significantly suppressed food intake in chicks, and this anorexigenic action was suggested to be mediated by CART, ␣-MSH and CRF. Our results suggest that, unlike its action in mammals, insulin selectively modulates mRNA levels of hypothalamic anorexigenic neuropeptides, which are likely one of the regulatory mechanisms of food intake in chickens.
Acknowledgements We thank Dr. Takao Sakai (Department of Biomedical Engineering, The Cleveland Clinic, Cleveland, Ohio, USA) for helpful discussions and Ms. Christine Kassuba for editorial assistance. This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture of Japan. References [1] S.C. Benoit, E.L. Air, L.M. Coolen, R. Strauss, A. Jackman, D.J. Clegg, R.J. Seeley, S.C. Woods, The catabolic action of insulin in the brain is mediated by melanocortins, J. Neurosci. 22 (2002) 9048–9052. [2] O. Chan, K. Inouye, E. Akirav, E. Park, M.C. Riddell, M. Vranic, S.G. Matthews, Insulin alone increases hypothalamo–pituitary–adrenal activity, and diabetes lowers peak stress responses, Endocrinology 146 (2005) 1382–1390. [3] J.L. Davis, D.T. Masuoka, L.K. Gerbrandt, A. Cherkin, Autoradiographic distribution of l-proline in chicks after intracerebral injection, Physiol. Behav. 22 (1979) 693–695. [4] D.M. Denbow, S. Meade, A. Robertson, J.P. McMurtry, M. Richards, C. Ashwell, Leptin-induced decrease in food intake in chickens, Physiol. Behav. 69 (2000) 359–362. [5] D.M. Denbow, R.D. Myers, Eating, drinking and temperature responses to intracerebroventricular cholecystokinin in the chick, Peptides 3 (1982) 739–743. [6] D.M. Denbow, N. Snapir, M. Furuse, Inhibition of food intake by CRF in chickens, Physiol. Behav. 66 (1999) 645–649. [7] C. Fekete, G. Legradi, E. Mihaly, J.B. Tatro, W.M. Rand, R.M. Lechan, ␣Melanocyte stimulating hormone prevents fasting-induced suppression of corticotropin-releasing hormone gene expression in the rat hypothalamic paraventricular nucleus, Neurosci. Lett. 289 (2000) 152–156. [8] H.H. Gerets, K. Peeters, L. Arckens, F. Vandesande, L.R. Berghman, Sequence and distribution of pro-opiomelanocortin in the pituitary and the brain of the chicken (Gallus gallus), J. Comp. Neurol. 417 (2000) 250–262. [9] K. Honda, H. Kamisoyama, N. Saito, Y. Kurose, K. Sugahara, S. Hasegawa, Central administration of glucagon suppresses food intake in chicks, Neurosci. Lett. 416 (2007) 198–201. [10] A. Inokuchi, Y. Oomura, H. Nishimura, Effect of intracerebroventricularly infused glucagon on feeding behavior, Physiol. Behav. 33 (1984) 397–400. [11] S. Kawakami, T. Bungo, R. Ando, A. Ohgushi, M. Shimojo, Y. Masuda, M. Furuse, Central administration of a-melanocyte stimulating hormone
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Central administration of insulin suppresses food intake in chicks Kazuhisa Honda a , Hiroshi Kamisoyama a , Takaoki Saneyasu a , Kunio Sugahara b , Shin Hasegawa a,∗ b
a Department of Animal Science, Faculty of Agriculture, Kobe University, Kobe 657-8501, Japan Department of Animal Science, Faculty of Agriculture, Utsunomiya University, Utsunomiya 321-8505, Japan
Received 16 March 2007; received in revised form 1 June 2007; accepted 14 July 2007
Abstract Although the orexigenic action of peptide hormones such as ghrelin and growth hormone releasing peptide is different between chickens and mammals, the anorexigenic action of peptide hormones is similar in both species. For example, central administration of peptide hormones such as leptin, cholecystokinin or glucagon has been shown to suppress food intake behavior in chickens and mammals. Central administration of insulin suppresses food intake in mammals. However, the anorexigenic action of insulin in chickens has not yet been identified. In the present study, we investigated the effects of central administration of insulin on food intake in chicks. Intracerebroventricular administration of insulin in chicks significantly suppressed food intake. Central administration of insulin significantly upregulated mRNA levels of proopiomelanocortin (POMC), cocaine- and amphetamine-regulated transcript (CART) and corticotropin-releasing factor (CRF), but did not influence mRNA levels of neuropeptide Y and agouti-related protein in the hypothalamus. These results suggest that alpha-melanocyte stimulating hormone (␣-MSH, an anorexigenic peptide from the post-translational cleavage of POMC), CART and CRF are involved in the anorexigenic action of insulin in chicks. Furthermore, central administration of ␣-MSH or CART significantly suppressed food intake. In addition, ␣-MSH significantly upregulated CRF mRNA expression, suggesting that the anorexigenic action of ␣-MSH is mediated by CRF. Our findings demonstrate that insulin functions in chicks as an appetite-suppressive peptide in the central nervous system and suggest that this anorexigenic action is mediated by CART, ␣-MSH and CRF. © 2007 Elsevier Ireland Ltd. All rights reserved. Keywords: Food intake; Chicken; Insulin; Proopiomelanocortin; Cocaine- and amphetamine-regulated transcript; Corticotropin-releasing factor
In mammals, the central regulatory mechanisms of food intake are known to be closely related to energy metabolism [15,19]. For example, insulin and leptin are sensed by neurons in the hypothalamic arcuate nucleus (ARC); they act as important peripheral hormones in energy metabolism, and such hormones influence the regulation of food intake [15,19]. The ARC contains two functionally different neurons: (a) neurons that suppress food intake by releasing alpha-melanocyte stimulating hormone (␣-MSH), cocaine- and amphetamine-regulated transcript (CART), or both; and (b) neurons that stimulate food intake by releasing neuropeptide Y (NPY), agouti-related protein (AgRP), or both. Intracerebroventriular administration of insulin in rats increases proopiomelanocortin (POMC, a precursor of ␣-MSH and adrenocorticotropic hormone) mRNA levels [1]. Li et al. suggest that CART-producing neurons in the ARC
∗
Corresponding author. Tel.: +81 78 803 5808; fax: +81 78 803 5808. E-mail address: [email protected] (S. Hasegawa).
0304-3940/$ – see front matter © 2007 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2007.07.004
are sensitive to changes in plasma insulin levels [14]. Central and peripheral administration of insulin in rats suppresses food intake and decreases NPY mRNA [16,21]. Subcutaneous administration of insulin in rats decreases AgRP mRNA levels [16]. These observations suggest that ␣-MSH, CART, NPY and AgRP act as downstream molecules of insulin-induced appetite suppressive pathways in mammals. In addition, peripheral administration of insulin increases corticotropin-releasing factor (CRF) mRNA levels in rat hypothalamus [2,23], and there is evidence that the anorexigenic action of ␣-MSH [7] or CART [27] is mediated by CRF. It is therefore likely that CRF is also involved in the anorexigenic action of insulin and acts as a downstream molecule in the ␣-MSH- or CART-induced appetite suppressive pathway. The regulatory mechanisms of food intake in poultry have been extensively studied in recent decades [12,17]. Growing evidence indicates that, although the orexigenic action of peptide hormones such as ghrelin and growth hormone releasing peptide is different between chickens and mammals, the
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anorexigenic action of peptide hormones is similar in both species [17]. For example, central administration of anorexigenic peptide hormones such as leptin [4], cholecystokinin [5] or glucagon [9] has been shown to have the same effects on food intake in chickens as in mammals [10,15,19]. Insulin receptors exist in chicken brain [20], and peripheral administration of insulin can suppress food intake in chickens [22]. However, the effects of central administration of insulin on food intake have not yet been identified. In this study, we focused on the functional roles of insulin in the central regulation of food intake in chicks. The results show that insulin suppresses food intake via hypothalamic anorexigenic neuropeptides. Day-old male chicks (White Leghorn) were purchased from a local hatchery (Ghen Corporation, Gifu, Japan). They were given free access to water and a commercial chick starter diet (Nippon Formula Feed Mfg. Co., Ltd., Kanagawa, Japan). Room temperature was maintained at 32 ± 2 ◦ C. All experimental procedures followed the guidelines for the care and use of experimental animals at the Rokkodai Campus of Kobe University in Japan. Insulin was purchased from Wako Pure Chemical Industries, Ltd. (bovine insulin, Osaka, Japan). All primers were purchased from Hokkaido System Science Co., Ltd. (Hokkaido, Japan). In Experiment 1, 8-day-old chicks were divided into four groups. Insulin was dissolved in a 0.85% (w/v) saline solution containing 0.1% (w/v) Evans Blue. Either insulin (0.1, 0.4, 2, or 10 g) or saline (as a control) was intracerebroventricularly administered according to the method of Davis et al. [3] at a volume of 10 l after 3 h of fasting. Food intake was measured at 30, 60 and 120 min after administration. At the end of the experiment, the chicks were sacrificed by decapitation. Verification of injection was made by observation of the presence of Evans Blue dye in the lateral ventricle. In Experiment 2, 8-day-old chicks were divided into two groups, and either 10 g insulin or saline (as a control) was intracerebroventricularly administered as described in Experiment 1. The significant difference in food intake between control and insulin group was found at 60 and 120 min but not at 30 min after administration of 10 g insulin in Experiment 1. According to this evidence, we measured POMC, CART, NPY, AgRP and CRF mRNA levels at 60 min after central administration of insulin. At that point, the chicks were sacrificed. Their brains were removed within 1 min of decapitation, frozen on powdered dry ice, weighed and stored at −80 ◦ C for further analysis. The hypothalamus was dissected from the frozen brain by referring to a stereotaxic atlas drawn by Kuenzel and Masson [13] and weighed. Total RNA was extracted from the hypothalamus using the Sepazol-RNA I (Nacalai Tesque, Inc., Kyoto, Japan). Firststrand cDNA was synthesized from 5 g of total RNA treated with DNase I (Invitrogen, Carlsbad, California, USA) using the SuperScript III First Strand Synthesis System for RT-PCR (Invitrogen, Carlsbad, California, USA) with random primers. PCR was performed using TaKaRa Ex Taq (Takara Bio, Inc., Siga, Japan). Complementary DNA of POMC (GenBank accession no. AB019555), CART (GenBank accession no. BI394769), NPY (GenBank accession no. M87294), and AgRP (GenBank accession no. AB029443) were amplified with the following
primers: POMC sense, 5 -AGA TGG AGA AGG GTT GGA A-3 ; POMC antisense, 5 -CGT TGG GGT ACA CCT TGA-3 ; CART sense, 5 -CCG CAC TAC GAG AAG AAG-3 ; CART antisense, 5 -AGG CAC TTG AGA AGA AAG G-3 ; NPY sense, 5 -CTT GTC GCT GCT GAT CTG-3 ; NPY antisense, 5 -GCC TCA GAG CCG AGT AGT-3 ; AgRP sense, 5 -GCA GGA AGG TGA TGG TAA C-3 ; AgRP antisense, 5 -GTC ACA GCA GGG GAT CTG-3 . Complementary DNA of CRF was amplified with specific primers as described previously [9]. As an internal standard, chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH, GenBank accession no. NM 204305) was also amplified, using the following primers: GAPDH sense, 5 -GGT GCT AAG CGT GTT ATC ATC TCA-3 ; GAPDH antisense, 5 -CAT GGT TGA CAC CCA TCA CAA-3 . Initially, we performed amplification experiments in each gene and determined the cycle number that the amplification products in each gene were between 20 and 40 cycle number and also within the linear portion of the amplification curve. Then, PCR reactions were performed as follows: 94 ◦ C for 5 min; 30 cycles (NPY and AgRP), 33 cycles (POMC and CART), 35 cycles (CRF) or 24 cycles (GAPDH) of 94 ◦ C for 30 s, 55 ◦ C for 1 min and 72 ◦ C for 1 min; and final extension at 72 ◦ C for 4 min. The PCR products were analyzed by ethidium bromide (Nacalai Tesque, Inc., Kyoto, Japan) staining on 1% agarose gel. The fluorescence from PCR products was detected and quantified with Typhoon 9400 (Amersham Biosciences Corp., Piscataway, New Jersey, USA). In Experiment 3, 8-day-old chicks were divided into three groups. Central administration of 1 g ␣-MSH [11] or 5 g CART [26] suppresses food intake in chicks. We followed these studies and either 1 g of ␣-MSH, 5 g CART or saline (as a control) was intracerebroventricularly administered as described in Experiment 1. Food intake was measured at 30, 60 and 120 min after administration. We found that central administration of ␣-MSH or CART in chicks significantly suppressed food intake (p < 0.05) throughout the experimental period. Therefore, we measured CRF mRNA levels at 30 min after central administration of ␣-MSH or CART. 8-day-old chicks were divided into three groups and either 1 g of ␣-MSH, 5 g CART or saline (as a control) was intracerebroventricularly administered as described in Experiment 1. Hypothalamic mRNA of CRF and GAPDH were determined as described in Experiment 2. Data from Experiments 1 and 3 were analyzed by the Tukey–Kramer test. Data from Experiment 2 were analyzed by Student’s t-test. All statistics was performed using the commercial package StatView version 5 (SAS Institute, Cary, North Carolina, USA, 1998). Central administration of insulin in chicks significantly suppressed food intake (p < 0.05) in a dose-dependent manner (Experiment 1, Fig. 1). It is known that insulin upregulates mRNA of POMC, whereas this peptide downregulates mRNA of NPY and AgRP in the hypothalamus [15,19]. CART-producing neurons in the ARC are thought to be sensitive to plasma insulin levels [14]. Therefore, we next examined whether ␣-MSH, CART, NPY and AgRP, act as downstream molecules in the insulin-induced anorexigenic pathway in chicks. Insulin administration significantly upregulated mRNA expressions of POMC
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Fig. 1. Effect of central administration of insulin on cumulative food intake in chicks. Data are means ± S.E.M. The number of chicks used is shown in parentheses. Groups with different letters are significantly different (p < 0.05) at each time point.
and CART (p < 0.05, Experiment 2, Fig. 2), but did not influence NPY and AgRP mRNA levels (Experiment 2, Fig. 3). Thus, these results suggest that insulin functions as an appetite-suppressive peptide and that insulin selectively modulates mRNA levels of
Fig. 3. Effect of central administration of insulin on hypothalamic mRNA levels of NPY and AgRP in chicks. Data are means ± S.E.M. of six chicks in each group.
Fig. 2. Effect of central administration of insulin on hypothalamic mRNA levels of POMC and CART in chicks. Data are means ± S.E.M. of six chicks in each group. *Significant with respect to saline (p < 0.05).
POMC and CART to suppress food intake. Insulin receptors exist in avian brains [20]. However, up to now, there is no report demonstrating the distribution of insulin receptor in the chicken brain. Our results showed that central administration of insulin did not influence NPY and AgRP mRNA levels in hypothalamus. Thus, it seems that insulin receptor might not be expressed in hypothalamic NPY and AgRP neurons in chickens. Chan et al. [2] and Suda et al. [23] have reported that peripheral administration of insulin increases CRF mRNA levels in rat hypothalamus. Central administration of CRF suppresses food intake in chicks [6,25]. Therefore, we next examined the effects of insulin on CRF mRNA level in chicken hypothalamus. Central administration of insulin significantly upregulated mRNA expression of CRF (p < 0.05, Experiment 2, Fig. 4). These findings and our results suggest that CRF is also involved in anorexigenic action of insulin in chicks. In mammalian hypothalamus, POMC-producing neurons project from ARC to the paraventricular nucleus (PVN) in hypothalamus, and PVN contains CRF-producing neurons [15,19]. Central administration of CART in rats suppresses food intake with inducing Fos-like immunoreactivity in CRFcontaining neurons in the PVN [27]. These findings suggest that anorexigenic signals from POMC- or CART-producing neu-
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Fig. 4. Effect of central administration of insulin on hypothalamic CRF mRNA levels in chicks. Data are means ± S.E.M. of six chicks in each group. *Significant with respect to saline (p < 0.05).
Fig. 5. Effect of central administration of ␣-MSH or CART on hypothalamic CRF mRNA levels in chicks. Data are means ± S.E.M. of six chicks in each group. Groups with different letters are significantly different (p < 0.05).
rons in the ARC are integrated to CRF neuron in the PVN. Immunohistochemical studies have shown that hypothalamic POMC- or CRF-producing neurons in chickens localize in the same nuclei as those in mammals [8,18]. For example, POMC-immunoreactive perikarya are present in infundibular nuclei (equivalent of the mammalian ARC) [8], and CRFimmunoreactive perikarya are present in the PVN [18]. The CRF receptor antagonist astressin significantly reversed the anorexigenic action of ␣-MSH in chicks [24]. These observations raise the question whether anorexigenic action of ␣-MSH or CART is mediated by CRF in chickens as well as in mammals. Therefore, we finally examined whether hypothalamic CRF acts as a downstream molecule in the ␣-MSH- or CART-induced appetite suppressive pathway in chicks. Central administration of ␣-MSH or CART in chicks significantly suppressed food intake (p < 0.05) throughout the experimental period (from 30 to 120 min after administration). At 30 min after administration, food intake was 0.91 ± 0.19 g in control (saline alone), and 0.09 ± 0.03 g and 0.33 ± 0.14 g in ␣-MSH and CART injections, respectively; at 60 min, 1.54 ± 0.32 g in control, and 0.10 ± 0.03 g and 0.36 ± 0.13 g in ␣-MSH and CART, respectively; at 120 min, 2.21 ± 0.24 g in control, and 0.10 ± 0.03 g and 0.36 ± 0.13 g in ␣-MSH and CART, respectively (means ± S.E.M. [n = 5 in each group]). Central administration of ␣-MSH significantly upregulated CRF mRNA but central administration of CART did not influence CRF mRNA (Experiment 3, Fig. 5). Thus, these results suggest the involvement of CRF in ␣-MSH-induced but not CART-induced anorexigenic action in chicks. In summary, we studied the functional roles of insulin in the central regulation of food intake in chicks. Our data clearly showed that central administration of insulin significantly suppressed food intake in chicks, and this anorexigenic action was suggested to be mediated by CART, ␣-MSH and CRF. Our results suggest that, unlike its action in mammals, insulin selectively modulates mRNA levels of hypothalamic anorexigenic neuropeptides, which are likely one of the regulatory mechanisms of food intake in chickens.
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