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Arcuate NPY neurons sense and integrate peripheral metabolic signals to control feeding
Neuropeptides 46 (2012) 315–319
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Arcuate NPY neurons sense and integrate peripheral metabolic signals to control feeding Daisuke Kohno, Toshihiko Yada ⇑ Division of Integrative Physiology, Department of Physiology, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan
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Article history: Received 30 July 2012 Accepted 12 September 2012 Available online 26 October 2012 Keywords: Neuropeptide Y (NPY) Arcuate nucleus (ARC) Ghrelin Leptin Insulin Glucose Feeding
a b s t r a c t NPY neuron in the hypothalamic arcuate nucleus is a key feeding center. Studies have shown that NPY neuron in the arcuate nucleus has a role to induce food intake. The arcuate nucleus is structurally unique with lacking blood brain barrier. Peripheral energy signals including hormones and nutrition can reach the arcuate nucleus. In this review, we discuss sensing and integrating peripheral signals in NPY neurons. In the arcuate nucleus, ghrelin mainly activates NPY neurons. Leptin and insulin suppress the ghrelininduced activation in 30–40% of the ghrelin-activated NPY neurons. Lowering glucose concentration activates 40% of NPY neurons. These results indicate that NPY neuron in the arcuate nucleus is a feeding center in which major peripheral energy signals are directly sensed and integrated. Furthermore, there are subpopulations of NPY neurons in regard to their responsiveness to peripheral signals. These findings suggest that NPY neuron in the arcuate nucleus is an essential feeding center to induce food intake in response to peripheral metabolic state. Ó 2012 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7.
NPY neurons in the arcuate nucleus are important orexigenic neurons. . . . . . . . . . . . . . . . Structural characteristics of the arcuate nucleus and inputs of peripheral energy signals . Ghrelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. NPY neurons in the arcuate nucleus are important orexigenic neurons Neuropeptide Y (NPY) is a 36 amino acid peptide found by Tatemoto K. and Mutt V. from the porcine brain in 1982 (Tatemoto et al., 1982). NPY is widely expressed in the neurons of the central and peripheral nervous systems (Gray and Morley, 1986). The arcuate nucleus (ARC) in the hypothalamus is a region where ⇑ Corresponding author. Address: Division of Integrative Physiology, Department of Physiology, Jichi Medical University School of Medicine, Shimotsuke, Tochigi 329-0498, Japan. Tel.: +81 285 58 7320; fax: +81 285 44 9962. E-mail address: [email protected] (T. Yada). 0143-4179/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.npep.2012.09.004
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NPY neurons are abundantly distributed. NPY neurons in the ARC play an important role in feeding regulation. NPY secretion in the hypothalamus is increased during fasting (Kalra et al., 1991), and the injection of NPY in the hypothalamus strongly increases food intake (Stanley et al., 1986). Moreover, recently developed genetic technologies, optogenetics and DREADD, have shown that the cellular activation of AgRP neurons, which are largely overlapped with NPY neurons in the ARC (Hahn et al., 1998), induces acute and robust food intake (Aponte et al., 2011; Krashes et al., 2011), suggesting NPY/AgRP neurons in the ARC are the principal inducer of feeding. Inversely, the selective ablation of NPY/AgRP neurons from adult mice causes marked reduction of feeding and body weight (Gropp et al., 2005; Luquet et al., 2005), suggesting NPY/
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AgRP neurons are indispensable for the induction of feeding. Therefore, NPY neurons in the ARC are essential for feeding control and thereby the population of NPY neurons in the ARC is a feeding center. NPY neurons are also expressed in some other brain regions including dorsomedial hypothalamus (DMH), nucleus accumbens, hippocampus and brain stem. In regard to feeding, the expression level of NPY in the DMH is transiently increased in some limited periods of life when energy balance is negatively regulated including early postnatal and lactating periods, suggesting that NPY neurons in the DMH contribute to the induction of feeding in specific life stages (Mercer et al., 2011).
rons in response to ghrelin (Kohno et al., 2003, 2007, 2008a,b). Our studies showed that ghrelin at 10 12–10 8 M directly increases [Ca2+]i in 21–41% of ARC neurons (Fig. 1A and B) (Kohno et al., 2003, 2007). Immunocytochemistry following calcium imaging proved that more than 80% of ghrelin-responding neurons in ARC are NPY neurons (Fig. 1C and D) (Kohno et al., 2003, 2007). These results indicate that NPY neurons are the major target of ghrelin in the ARC. Since both ghrelin and NPY have strong orexigenic effects, the axis from the stomach ghrelin-producing cells to the ARC NPY neurons and subsequent NPY secretion may be an essential pathway for the feeding induction.
2. Structural characteristics of the arcuate nucleus and inputs of peripheral energy signals
4. Leptin
The ventromedial ARC, where NPY neurons are abundantly localized, has unique characteristics in structure. The ventromedial ARC, as well as adjacent median eminence, is considered a circumventricular organ, which lacks blood–brain barrier (Ciofi, 2011; Rodriguez et al., 2010; Shaver et al., 1992). The endothelia of capillaries in this area are fenestrated and thereby the permeability of molecules from blood to brain is much higher in this area compared to most of other hypothalamic regions. Therefore, peripheral molecules should be able to reach NPY neurons directly from blood flow. However it is known that some metabolic effects of peripheral signals are mediated by brain, neuron populations for these effects are not fully understood. Because of the characteristic as a circumventricular organ, it is reasonable to hypothesize that ARC neurons including NPY neurons receive peripheral signals. We have been investigating the control of NPY neurons by peripheral molecules, including ghrelin, leptin, insulin, and glucose. To examine the direct effect of peripheral molecules on NPY neurons, we employed the single-cell calcium imaging followed by cell-type identification with immunocytochemistry (Muroya et al., 1999; Yada et al., 1994). Cells were prepared acutely from adult rats or mice, and then, dispersed on coverslips. Fura-2 AM, a calcium concentration indicator, was loaded into cells, and intracellular calcium concentration ([Ca2+]i) of each cell was measured under a fluorescence microscope every 10–30 s for 1–2 h. We monitored the alteration of [Ca2+]i, which represents cellular activity, over time. To identify the cell property, immunocytochemistry using antiNPY antibody was performed. With this method, we could examine the direct action of peripheral molecules. We review our recent findings regarding NPY populations that respond to peripheral signals. 3. Ghrelin Ghrelin is a potent orexigenic peptide produced mainly from stomach (Kojima et al., 1999). Many studies have suggested close link between ghrelin and NPY neurons in the ARC. A ghrelin receptor, GHSR, is highly expressed in the ARC (Zigman et al., 2006). In the rat ARC, GHSR is expressed in 94% of NPY, 30% of somatostatin, 20% of the growth hormone releasing hormone, and 8% of proopiomelanocortin (POMC) neurons (Willesen et al., 1999). Thus, GHSR is expressed in almost all NPY neurons in the ARC, whereas the expression rates of GHSR in other neurons in the ARC are not so high. Consistently, c-Fos is abundantly and selectively expressed in NPY neurons in the ARC after the administration of ghrelin (Wang et al., 2002). Moreover, the blocking of NPY-mediated pathway with the central administrations of anti-NPY IgG, anti-AgRP IgG, or NPY Y1 or Y5 receptor antagonist inhibits ghrelin-induced feeding (Asakawa et al., 2001; Nakazato et al., 2001; Shintani et al., 2001), suggesting that NPY neurons mediate ghrelin-induced food intake. To investigate the direct effect of ghrelin on NPY neurons in the ARC, we observed the [Ca2+]i alteration in ARC NPY neu-
Leptin, a hormone released from white adipose tissue, is one of a few hormones that negatively regulate feeding and energy homeostasis (Williams et al., 2009; Zhang et al., 1994). Leptin works in the brain especially in the ARC. The central injection of leptin in ob/ob mouse reduces food intake and body weight (Campfield et al., 1995), and the neuron-selective deletion of leptin receptor leads to obesity (Cohen et al., 2001). Moreover, the selective restoration of leptin receptor in the ARC of the leptin receptor knockout mouse recovers satiety function and decreases body weight (Coppari et al., 2005), suggesting that the ARC is the major region for the leptin reception to reduce food intake. Plasma levels of ghrelin and leptin are inversely correlated (Cummings and Foster, 2003), and also leptin antagonizes food intake induced by ghrelin (Nakazato et al., 2001). Therefore, the balance between ghrelin and leptin could be essential for the regulation of feeding. We investigated the effect of leptin on NPY neurons. Leptin dosedependently inhibited ghrelin-induced increases in [Ca2+]i in ARC neurons (Fig. 2A–C). This inhibition was observed in about 45% of ghrelin-responding ARC neurons, and the 80% of leptin inhibition was observed in NPY neurons (Fig. 2C and D). Therefore, ghrelin and leptin are interacting in one cell in about one half of NPY neurons in the ARC. This reciprocal regulation of NPY neurons by ghrelin and leptin may be an essential mechanism controlling feeding. 5. Insulin Plasma Insulin level deceases during fasting and increases after food intake. In other words, the pattern of plasma insulin level is inversely correlated to the plasma ghrelin pattern (Cummings et al., 2001). The major role of insulin is to decrease blood glucose level, but the contribution of insulin to the control of feeding is also suggested. Insulin receptor is widely distributed in the brain including NPY neurons (Havrankova et al., 1978; Maejima et al., 2011). The central infusion of insulin decreases food intake and body weight (Woods et al., 1979). The brain selective deletion of insulin receptor causes obesity on HFD (Bruning et al., 2000). We investigated the effect of insulin on NPY neurons. Insulin at 10 12 M suppressed about 60% of ghrelin responding neurons in the ARC, and more than 70% of the ghrelin-activated insulin-inhibited ARC neurons were NPY neurons (Fig. 3A) (Maejima et al., 2011). These things indicate that ghrelin and insulin reciprocally regulate NPY neurons in the ARC. The suppression of NPY neurons might be the mechanism underlying the insulin’s anorexigenic effect. 6. Glucose Glucose is a fundamental energy nutrient. Glucose concentration in the blood reflects the energy status of the body, and the glucose concentration rises after eating and falls during fasting. The most characterized glucose-sensing cell in a whole body is the pancreatic b-cells, which increase their cellular activity in response to
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Ghrelin 10-10 M
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Fig. 1. Ghrelin increases cytosolic calcium concentration ([Ca2+]i) in NPY neurons. A. Ghrelin increased [Ca2+]i in an arcuate nucleus (ARC) neuron. B. Percentage of ARC neurons with [Ca2+]i increases in response to ghrelin. C. An ARC neuron responded to ghrelin at 10 10 M and was stained positively by immunocytochemistry with anti-NPY antibody. Scale bar, 10 lm. D. Percentage of NPY-immunoreactive neurons among ghrelin-responding ARC neurons. (The figures were modified from Kohno et al., 2003, 2007).
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-log[Leptin] (M) Fig. 2. Leptin suppresses ghrelin-induced [Ca2+]i increases in NPY neurons. A. Ghrelin at 10 10 M consistently increased [Ca2+]i in ARC neurons. B. Leptin at 10 12 M suppressed ghrelin-induced [Ca2+]i increase (left panel) in a neuron that was proven to be NPY-immunoreactive (right panel). Scale bar, 10 lm. C. Percentage of leptin-suppressed neurons among ARC neurons responded to ghrelin with [Ca2+]i increases. D. Percentage of NPY-immunoreactive neurons among ARC neurons in which ghrelin-induced [Ca2+]i increases were suppressed by leptin. (The figures were modified from Kohno et al., 2007).
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Fig. 3. Responses to insulin and lowering glucose concentration in ARC neurons. A. Insulin suppressed ghrelin-induced [Ca2+]i increase in an ARC neuron. B. Lowering glucose concentration from 10 to 2.8 mM increased [Ca2+]i in a ghrelin-responding ARC neuron. (Figure B was modified from Kohno et al., 2003).
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Fig. 4. Subpopulations of ARC NPY neurons with regard to their responsiveness to peripheral energy signals.
hyperglycemia. Some hypothalamic neurons have ability to sense glucose concentration (Anand et al., 1964; Oomura et al., 1969, 1974). Two types of glucose-sensing neurons exist in the hypothalamus; glucose-inhibited (or glucose-sensitive) neuron, which increases its activity when the extracellular glucose concentration decreases, and the glucose-responsive (or glucose-excited) neuron, which activates its cellular activity when the extracellular glucose concentration increases. Our studies have shown that about 40% of NPY neurons are glucose-inhibited neurons. Intriguingly, a large majority of glucose-inhibited neurons in ARC are NPY neurons (Muroya et al., 1999). Also, more than half of ghrelin-responding ARC neurons are glucose-inhibited neurons (Fig. 3B) (Kohno et al., 2003). These results suggest that glucose is the major regulator of NPY neurons. 7. Summary Many studies have shown clearly that NPY/AgRP neurons in the ARC play pivotal role for the induction of feeding. Our studies have shown that many of NPY neurons in the ARC are activated by ghrelin and low glucose while leptin and insulin negatively regulate
NPY neurons (Fig. 4). These results indicate NPY neurons in the ARC are the major site to receive and integrate multiple peripheral energy signals. These inputs on NPY neurons may be essential to control energy homeostasis, similarly to POMC neurons that also play an essential role by receiving peripheral signals (Balthasar et al., 2004; Hill et al., 2010). Clarifying overlapping and/or segregation between NPY subpopulations would be the important subject for the future studies. Acknowledgements This work was supported by Grant-in-Aid for Scientific Research (B) (18390065, 20390061, 23390044), for Challenging Exploratory Research (22659044, 24659101) and for Scientific Research on Innovative Areas (23115715) from Japan Society for the Promotion of Science (JSPS), Strategic Research Program for Brain Sciences (10036069) by the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), MEXT-Supported Programs for Strategic Research Foundation at Private Universities (2008–2012, 2011–2015) Grant from Japan Diabetes Foundation, and Basic Science Research Award from Sumitomo Foundation to
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T.Y. This study was subsidized by JKA through its promotion funds from KEIRIN RACE to T.Y.
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Arcuate NPY neurons sense and integrate peripheral metabolic signals to control feeding Daisuke Kohno, Toshihiko Yada ⇑ Division of Integrative Physiology, Department of Physiology, Jichi Medical University School of Medicine, Tochigi 329-0498, Japan
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Article history: Received 30 July 2012 Accepted 12 September 2012 Available online 26 October 2012 Keywords: Neuropeptide Y (NPY) Arcuate nucleus (ARC) Ghrelin Leptin Insulin Glucose Feeding
a b s t r a c t NPY neuron in the hypothalamic arcuate nucleus is a key feeding center. Studies have shown that NPY neuron in the arcuate nucleus has a role to induce food intake. The arcuate nucleus is structurally unique with lacking blood brain barrier. Peripheral energy signals including hormones and nutrition can reach the arcuate nucleus. In this review, we discuss sensing and integrating peripheral signals in NPY neurons. In the arcuate nucleus, ghrelin mainly activates NPY neurons. Leptin and insulin suppress the ghrelininduced activation in 30–40% of the ghrelin-activated NPY neurons. Lowering glucose concentration activates 40% of NPY neurons. These results indicate that NPY neuron in the arcuate nucleus is a feeding center in which major peripheral energy signals are directly sensed and integrated. Furthermore, there are subpopulations of NPY neurons in regard to their responsiveness to peripheral signals. These findings suggest that NPY neuron in the arcuate nucleus is an essential feeding center to induce food intake in response to peripheral metabolic state. Ó 2012 Elsevier Ltd. All rights reserved.
Contents 1. 2. 3. 4. 5. 6. 7.
NPY neurons in the arcuate nucleus are important orexigenic neurons. . . . . . . . . . . . . . . . Structural characteristics of the arcuate nucleus and inputs of peripheral energy signals . Ghrelin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Leptin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Insulin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Glucose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. NPY neurons in the arcuate nucleus are important orexigenic neurons Neuropeptide Y (NPY) is a 36 amino acid peptide found by Tatemoto K. and Mutt V. from the porcine brain in 1982 (Tatemoto et al., 1982). NPY is widely expressed in the neurons of the central and peripheral nervous systems (Gray and Morley, 1986). The arcuate nucleus (ARC) in the hypothalamus is a region where ⇑ Corresponding author. Address: Division of Integrative Physiology, Department of Physiology, Jichi Medical University School of Medicine, Shimotsuke, Tochigi 329-0498, Japan. Tel.: +81 285 58 7320; fax: +81 285 44 9962. E-mail address: [email protected] (T. Yada). 0143-4179/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.npep.2012.09.004
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NPY neurons are abundantly distributed. NPY neurons in the ARC play an important role in feeding regulation. NPY secretion in the hypothalamus is increased during fasting (Kalra et al., 1991), and the injection of NPY in the hypothalamus strongly increases food intake (Stanley et al., 1986). Moreover, recently developed genetic technologies, optogenetics and DREADD, have shown that the cellular activation of AgRP neurons, which are largely overlapped with NPY neurons in the ARC (Hahn et al., 1998), induces acute and robust food intake (Aponte et al., 2011; Krashes et al., 2011), suggesting NPY/AgRP neurons in the ARC are the principal inducer of feeding. Inversely, the selective ablation of NPY/AgRP neurons from adult mice causes marked reduction of feeding and body weight (Gropp et al., 2005; Luquet et al., 2005), suggesting NPY/
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AgRP neurons are indispensable for the induction of feeding. Therefore, NPY neurons in the ARC are essential for feeding control and thereby the population of NPY neurons in the ARC is a feeding center. NPY neurons are also expressed in some other brain regions including dorsomedial hypothalamus (DMH), nucleus accumbens, hippocampus and brain stem. In regard to feeding, the expression level of NPY in the DMH is transiently increased in some limited periods of life when energy balance is negatively regulated including early postnatal and lactating periods, suggesting that NPY neurons in the DMH contribute to the induction of feeding in specific life stages (Mercer et al., 2011).
rons in response to ghrelin (Kohno et al., 2003, 2007, 2008a,b). Our studies showed that ghrelin at 10 12–10 8 M directly increases [Ca2+]i in 21–41% of ARC neurons (Fig. 1A and B) (Kohno et al., 2003, 2007). Immunocytochemistry following calcium imaging proved that more than 80% of ghrelin-responding neurons in ARC are NPY neurons (Fig. 1C and D) (Kohno et al., 2003, 2007). These results indicate that NPY neurons are the major target of ghrelin in the ARC. Since both ghrelin and NPY have strong orexigenic effects, the axis from the stomach ghrelin-producing cells to the ARC NPY neurons and subsequent NPY secretion may be an essential pathway for the feeding induction.
2. Structural characteristics of the arcuate nucleus and inputs of peripheral energy signals
4. Leptin
The ventromedial ARC, where NPY neurons are abundantly localized, has unique characteristics in structure. The ventromedial ARC, as well as adjacent median eminence, is considered a circumventricular organ, which lacks blood–brain barrier (Ciofi, 2011; Rodriguez et al., 2010; Shaver et al., 1992). The endothelia of capillaries in this area are fenestrated and thereby the permeability of molecules from blood to brain is much higher in this area compared to most of other hypothalamic regions. Therefore, peripheral molecules should be able to reach NPY neurons directly from blood flow. However it is known that some metabolic effects of peripheral signals are mediated by brain, neuron populations for these effects are not fully understood. Because of the characteristic as a circumventricular organ, it is reasonable to hypothesize that ARC neurons including NPY neurons receive peripheral signals. We have been investigating the control of NPY neurons by peripheral molecules, including ghrelin, leptin, insulin, and glucose. To examine the direct effect of peripheral molecules on NPY neurons, we employed the single-cell calcium imaging followed by cell-type identification with immunocytochemistry (Muroya et al., 1999; Yada et al., 1994). Cells were prepared acutely from adult rats or mice, and then, dispersed on coverslips. Fura-2 AM, a calcium concentration indicator, was loaded into cells, and intracellular calcium concentration ([Ca2+]i) of each cell was measured under a fluorescence microscope every 10–30 s for 1–2 h. We monitored the alteration of [Ca2+]i, which represents cellular activity, over time. To identify the cell property, immunocytochemistry using antiNPY antibody was performed. With this method, we could examine the direct action of peripheral molecules. We review our recent findings regarding NPY populations that respond to peripheral signals. 3. Ghrelin Ghrelin is a potent orexigenic peptide produced mainly from stomach (Kojima et al., 1999). Many studies have suggested close link between ghrelin and NPY neurons in the ARC. A ghrelin receptor, GHSR, is highly expressed in the ARC (Zigman et al., 2006). In the rat ARC, GHSR is expressed in 94% of NPY, 30% of somatostatin, 20% of the growth hormone releasing hormone, and 8% of proopiomelanocortin (POMC) neurons (Willesen et al., 1999). Thus, GHSR is expressed in almost all NPY neurons in the ARC, whereas the expression rates of GHSR in other neurons in the ARC are not so high. Consistently, c-Fos is abundantly and selectively expressed in NPY neurons in the ARC after the administration of ghrelin (Wang et al., 2002). Moreover, the blocking of NPY-mediated pathway with the central administrations of anti-NPY IgG, anti-AgRP IgG, or NPY Y1 or Y5 receptor antagonist inhibits ghrelin-induced feeding (Asakawa et al., 2001; Nakazato et al., 2001; Shintani et al., 2001), suggesting that NPY neurons mediate ghrelin-induced food intake. To investigate the direct effect of ghrelin on NPY neurons in the ARC, we observed the [Ca2+]i alteration in ARC NPY neu-
Leptin, a hormone released from white adipose tissue, is one of a few hormones that negatively regulate feeding and energy homeostasis (Williams et al., 2009; Zhang et al., 1994). Leptin works in the brain especially in the ARC. The central injection of leptin in ob/ob mouse reduces food intake and body weight (Campfield et al., 1995), and the neuron-selective deletion of leptin receptor leads to obesity (Cohen et al., 2001). Moreover, the selective restoration of leptin receptor in the ARC of the leptin receptor knockout mouse recovers satiety function and decreases body weight (Coppari et al., 2005), suggesting that the ARC is the major region for the leptin reception to reduce food intake. Plasma levels of ghrelin and leptin are inversely correlated (Cummings and Foster, 2003), and also leptin antagonizes food intake induced by ghrelin (Nakazato et al., 2001). Therefore, the balance between ghrelin and leptin could be essential for the regulation of feeding. We investigated the effect of leptin on NPY neurons. Leptin dosedependently inhibited ghrelin-induced increases in [Ca2+]i in ARC neurons (Fig. 2A–C). This inhibition was observed in about 45% of ghrelin-responding ARC neurons, and the 80% of leptin inhibition was observed in NPY neurons (Fig. 2C and D). Therefore, ghrelin and leptin are interacting in one cell in about one half of NPY neurons in the ARC. This reciprocal regulation of NPY neurons by ghrelin and leptin may be an essential mechanism controlling feeding. 5. Insulin Plasma Insulin level deceases during fasting and increases after food intake. In other words, the pattern of plasma insulin level is inversely correlated to the plasma ghrelin pattern (Cummings et al., 2001). The major role of insulin is to decrease blood glucose level, but the contribution of insulin to the control of feeding is also suggested. Insulin receptor is widely distributed in the brain including NPY neurons (Havrankova et al., 1978; Maejima et al., 2011). The central infusion of insulin decreases food intake and body weight (Woods et al., 1979). The brain selective deletion of insulin receptor causes obesity on HFD (Bruning et al., 2000). We investigated the effect of insulin on NPY neurons. Insulin at 10 12 M suppressed about 60% of ghrelin responding neurons in the ARC, and more than 70% of the ghrelin-activated insulin-inhibited ARC neurons were NPY neurons (Fig. 3A) (Maejima et al., 2011). These things indicate that ghrelin and insulin reciprocally regulate NPY neurons in the ARC. The suppression of NPY neurons might be the mechanism underlying the insulin’s anorexigenic effect. 6. Glucose Glucose is a fundamental energy nutrient. Glucose concentration in the blood reflects the energy status of the body, and the glucose concentration rises after eating and falls during fasting. The most characterized glucose-sensing cell in a whole body is the pancreatic b-cells, which increase their cellular activity in response to
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Fig. 1. Ghrelin increases cytosolic calcium concentration ([Ca2+]i) in NPY neurons. A. Ghrelin increased [Ca2+]i in an arcuate nucleus (ARC) neuron. B. Percentage of ARC neurons with [Ca2+]i increases in response to ghrelin. C. An ARC neuron responded to ghrelin at 10 10 M and was stained positively by immunocytochemistry with anti-NPY antibody. Scale bar, 10 lm. D. Percentage of NPY-immunoreactive neurons among ghrelin-responding ARC neurons. (The figures were modified from Kohno et al., 2003, 2007).
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-log[Leptin] (M) Fig. 2. Leptin suppresses ghrelin-induced [Ca2+]i increases in NPY neurons. A. Ghrelin at 10 10 M consistently increased [Ca2+]i in ARC neurons. B. Leptin at 10 12 M suppressed ghrelin-induced [Ca2+]i increase (left panel) in a neuron that was proven to be NPY-immunoreactive (right panel). Scale bar, 10 lm. C. Percentage of leptin-suppressed neurons among ARC neurons responded to ghrelin with [Ca2+]i increases. D. Percentage of NPY-immunoreactive neurons among ARC neurons in which ghrelin-induced [Ca2+]i increases were suppressed by leptin. (The figures were modified from Kohno et al., 2007).
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Fig. 3. Responses to insulin and lowering glucose concentration in ARC neurons. A. Insulin suppressed ghrelin-induced [Ca2+]i increase in an ARC neuron. B. Lowering glucose concentration from 10 to 2.8 mM increased [Ca2+]i in a ghrelin-responding ARC neuron. (Figure B was modified from Kohno et al., 2003).
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Fig. 4. Subpopulations of ARC NPY neurons with regard to their responsiveness to peripheral energy signals.
hyperglycemia. Some hypothalamic neurons have ability to sense glucose concentration (Anand et al., 1964; Oomura et al., 1969, 1974). Two types of glucose-sensing neurons exist in the hypothalamus; glucose-inhibited (or glucose-sensitive) neuron, which increases its activity when the extracellular glucose concentration decreases, and the glucose-responsive (or glucose-excited) neuron, which activates its cellular activity when the extracellular glucose concentration increases. Our studies have shown that about 40% of NPY neurons are glucose-inhibited neurons. Intriguingly, a large majority of glucose-inhibited neurons in ARC are NPY neurons (Muroya et al., 1999). Also, more than half of ghrelin-responding ARC neurons are glucose-inhibited neurons (Fig. 3B) (Kohno et al., 2003). These results suggest that glucose is the major regulator of NPY neurons. 7. Summary Many studies have shown clearly that NPY/AgRP neurons in the ARC play pivotal role for the induction of feeding. Our studies have shown that many of NPY neurons in the ARC are activated by ghrelin and low glucose while leptin and insulin negatively regulate
NPY neurons (Fig. 4). These results indicate NPY neurons in the ARC are the major site to receive and integrate multiple peripheral energy signals. These inputs on NPY neurons may be essential to control energy homeostasis, similarly to POMC neurons that also play an essential role by receiving peripheral signals (Balthasar et al., 2004; Hill et al., 2010). Clarifying overlapping and/or segregation between NPY subpopulations would be the important subject for the future studies. Acknowledgements This work was supported by Grant-in-Aid for Scientific Research (B) (18390065, 20390061, 23390044), for Challenging Exploratory Research (22659044, 24659101) and for Scientific Research on Innovative Areas (23115715) from Japan Society for the Promotion of Science (JSPS), Strategic Research Program for Brain Sciences (10036069) by the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT), MEXT-Supported Programs for Strategic Research Foundation at Private Universities (2008–2012, 2011–2015) Grant from Japan Diabetes Foundation, and Basic Science Research Award from Sumitomo Foundation to
D. Kohno, T. Yada / Neuropeptides 46 (2012) 315–319
T.Y. This study was subsidized by JKA through its promotion funds from KEIRIN RACE to T.Y.
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