Dual Regulatory Effects of Orexins on Sympathetic Nerve Activity Innervating Brown Adipose Tissue in Rats
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内分泌学杂志 2005年第6期
Department of Internal Medicine I, Faculty of Medicine, Oita University, Hasama, Oita 879-5593, Japan
Address all correspondence and requests for reprints to: Professor Hironobu Yoshimatsu, M.D., Ph.D., Department of Internal Medicine I, Faculty of Medicine, Oita University, 1–1 Idaigaoka, Hasama, Oita 879-5593, Japan. E-mail: hiroy@oita-med.ac.jp.
Abstract
This study examined how orexin regulates the activity of the sympathetic nerves that innervate brown adipose tissue (BAT) in rats. Infusion of orexin A at a dose of 0.3 nmol into the third cerebral ventricle decreased BAT sympathetic nerve activity, compared with the effect of PBS (P < 0.05), whereas infusion of orexin B at the same dose caused a significant increase (P < 0.05). Pretreatment with a third cerebral ventricle injection of 2.24 μmol/kg -fluoromethylhistidine, an irreversible inhibitor of the histamine-synthesizing enzyme histidine decarboxylase, attenuated the orexin B-induced response of BAT sympathetic nerve activity, but not that induced by orexin A. These results indicate that orexins may regulate both BAT energy expenditure and thermogenesis through their dual effects on sympathetic nerve activity. In particular, orexin B regulates BAT sympathetic nerve activity via neuronal histamine in the hypothalamus.
Introduction
OREXIN A AND OREXIN B are hypothalamic neuropeptides that are derived from a 130-amino-acid precursor, prepro-orexin and are potent agonists at both the orexin-1 (OX1) and orexin-2 (OX2) receptors. Orexin neurons are located in the lateral hypothalamic area (LHA), from which they innervate almost all regions of the brain except the cerebellum (1, 2). Especially dense projections are observed in specific regions of the thalamus, limbic system, and aminergic nuclei, such as the noradrenergic locus coeruleus (LC), serotonergic raphe nucleus, and histaminergic tuberomammillary nucleus (TMN) (3, 4). Concomitantly, the presence of orexin receptors has been identified in these nuclei, namely OX1R in the LC and OX2R in the TMN (5). Hypothalamic histamine neurons, with cell bodies in the TMN, have been shown to be involved in the regulation of a variety of physiological functions, such as feeding behavior, drinking behavior, and thermoregulation, as well as the autonomic nervous and neuroendocrine systems (6). The orexin-histamine signaling pathway plays an important role in the regulation of the sleep-wakefulness cycle, although its involvement in other hypothalamic functions remains unclear (7).
Brown adipose tissue (BAT) plays a major role in energy expenditure and thermogenesis via the function of uncoupling protein 1. The regulation of energy expenditure in BAT is under the control of the sympathetic nervous system and higher brain structures. Discrete hypothalamic nuclei and various orexigenic or anorexigenic neuropeptides regulate BAT metabolism by affecting efferent sympathetic nerve activity (8, 9, 10, 11). Hypothalamic neuronal histamine, an anorexigenic substance, increases BAT sympathetic nerve activity (12). Orexins affect energy balance and thermoregulation. Orexin A has been shown to induce hypothermia and food intake, and both orexins increase renal sympathetic nerve activity (13, 14, 15, 16). Therefore, it is highly probable that orexins regulate BAT energy expenditure and thermogenesis by influencing sympathetic nerve activity. We thus hypothesized that hypothalamic neuronal histamine may mediate orexin signals to the sympathetic nerves that innervate BAT, because the close relationship between these two systems has been recognized both anatomically and functionally (17, 18). This study therefore examined the effects of central administration of orexins on electrophysiological sympathetic nerve activity in BAT and modulation of these responses by neuronal histamine.
Materials and Methods
Animals
Mature male Sprague-Dawley rats (8–10 wk old) (Seac Yoshitomi, Fukuoka, Japan) were maintained on a 12-h light, 12-h dark photoperiod (lights on at 0700 h) in a room with controlled temperature (21 ± 1 C) and humidity (55 ± 5%). The rats were allowed free access to food (pelleted rodent chow no. CE-2; Clea Japan, Tokyo, Japan) and water. All studies were conducted in accordance with the Oita Medical University Guidelines, which are based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Reagents
Orexin A, orexin B, and -fluoromethylhistidine (FMH) (all purchased from Sigma Chemical Co., St. Louis, MO) were each dissolved in PBS to final concentrations of 30 μM, 30 μM, and 67 mM, respectively. Each solution was freshly prepared on the day of use. The pH of each solution was adjusted to 6.4–7.2.
Cerebroventricular cannula implantation
Each rat was fixed in a stereotaxic apparatus (Narishige, Tokyo, Japan) under sodium pentobarbital anesthesia (45 mg/kg, ip), and a stainless steel guide cannula (23 gauge) was surgically implanted into the third cerebral ventricle (i3vt). A stainless steel wire stylet (29 gauge) was left in place in the guide cannula to maintain patency and prevent leakage of cerebrospinal fluid. Surgery was carried out at least 1 wk before infusion of the test solutions; the details of the surgical procedure have been described elsewhere (19).
Measurement of sympathetic nerve activity
Electrophysiological recordings were made under anesthesia (urethane, 0.8 g/kg; -chloralose, 80 mg/kg) using 20 cannulated rats. After dissection of the fine branches of the sympathetic nerves that innervate the interscapular BAT, the nerves were transected where they entered the BAT. Nerve activity was measured using a pair of silver-wire electrodes, which were immersed in a mixture of liquid paraffin and white petroleum jelly to prevent dehydration of the nerve tissue. The action potentials were amplified and filtered with low- and high-frequency cutoffs. The nerve signal was distinguished from background noise using a window discriminator. All nerve activity was analyzed based on values obtained after conversion of the raw data to standard pulses using an analog-to-digital converter. Impulses were integrated by a rate meter with a reset time of 5 sec and recorded using a chart recorder. Details of this nerve recording technique have been described elsewhere (8, 20). After determination of the background firing rate of the sympathetic nerves, changes in nerve activity were measured for up to 60 min after each bolus i3vt infusion with orexin A (0–3 nmol/rat, 1.0 μl/min, 10 min), orexin B (0–3 nmol/rat, 1.0 μl/min, 10 min), FMH (0–3 μmol/kg, 1.0 μl/min, 10 min), or PBS (n = 4 for each treatment). The doses of orexins and FMH were determined by previous studies (21, 22) and in our preliminary study. Infusions of orexins were performed 2 h after pretreatment with FMH at 1300 h. Nerve activity was measured at 10-min intervals, beginning 20 min before and continuing for 60 min after administration of the test solution. Nerve activity values recorded after the administration of each test solution were expressed as the percentage difference from the baseline value (at 0 min). The baseline 100% on figures signifies the 60-min averages score of BAT sympathetic nerve activity before the orexin treatment. For the analysis of dose dependence, orexins intracerebroventricular (icv) or PBS icv infusion was performed, and sympathetic nerve activity in BAT was recorded at 20 and 40 min after the infusion.
Statistical analysis
Differences among the groups were assessed using two-way ANOVA and post hoc Fisher’s protected least-significant difference test. A two-sided P value of less than 0.05 was considered statistically significant. The statistical analysis of dose dependence was assessed by the Spearman’s correlation coefficient by rank.
Results
Dose-dependent effects of orexins on BAT sympathetic nerve activity
We observed the dose-dependent effects of orexin A and orexin B. The BAT sympathetic nerve activity changes were dependent on the doses of orexins when given icv (orexin A: 0 nmol, 100.0 ± 4.5%; 0.03 nmol, 87.4 ± 6.1%; 0.3 nmol, 63.2 ± 6.4%; and 3 nmol, 47.4 ± 6.9%; P < 0.01) (orexin B: 0 nmol, 100.0 ± 5.3%; 0.03 nmol, 135.2 ± 7.3%; 0.3 nmol, 240.8 ± 11.2%; and 3 nmol, 322.0 ± 21.0%; P < 0.01) (Table 1).
TABLE 1. Dose-dependent effects of orexin A and orexin B on BAT sympathetic nerve activity
The effect of i3vt infusion of orexin A on BAT sympathetic nerve activity
Figure 1A shows the typical response of BAT sympathetic nerve activity after infusion of orexin A into the i3vt. Sympathetic nerve activity rapidly decreased after central administration of orexin A. The changes in sympathetic nerve activity in response to orexin A treatment differed significantly from those induced by PBS (P < 0.05; Fig. 1B).
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FIG. 1. A, Rate meter plots of BAT sympathetic nerve activity after infusion of orexin A (0.3 nmol) into the i3vt. Vertical axis shows nerve impulses per 5 sec. Horizontal bars, 10-min time scale; bold horizontal bar, orexin A infusion. B, Percentage differences in sympathetic nerve activity from baseline (100%) after i3vt infusion of orexin A (0.3 nmol) or PBS (control group). The baseline 100% signifies the 60-min averages score of BAT sympathetic nerve activity before the orexin treatment. All data are means ± SEM (n = 4 per group). , Orexin A-treated animals; , control. *, P < 0.05; **, P < 0.01 vs. control.
The effect of pretreatment with FMH on changes in BAT sympathetic nerve activity induced by orexin A
Figure 2A shows the typical responses of BAT sympathetic nerve activity to infusion of orexin A in rats pretreated with an infusion of FMH into the i3vt. The mean changes in sympathetic nerve activity induced by orexin A after pretreatment with FMH are shown in Fig. 2B. Sympathetic nerve activity decreased rapidly after central administration of orexin A in pretreated rats; these changes differed significantly from those induced by PBS (P < 0.05) but did not differ significantly from those induced by orexin A in rats pretreated only with PBS (P > 0.05).
FIG. 2. A, Rate meter plots of BAT sympathetic nerve activity after i3vt injection of orexin A (0.3 nmol) 2 h after pretreatment with FMH (0.67 μmol). Vertical axis shows nerve impulses per 5 sec. Horizontal bars, 10-min time scale; bold bar, orexin A infusion. B, Percentage differences in sympathetic nerve activity from baseline after i3vt injection of orexin A (0.3 nmol), orexin A (0.3 nmol) with FMH (0.67 μmol) pretreatment, or PBS (control). The baseline 100% signifies the 60-min averages score of BAT sympathetic nerve activity before the orexin treatment. All data are means ± SEM (n = 4 per group). , Orexin A-treated animals; , orexin A-treated animals with FMH pretreatment; , control. *, P < 0.05;, **, P < 0.01 vs. control.
The effect of i3vt infusion of orexin B on BAT sympathetic nerve activity
A typical response of BAT sympathetic nerve activity to i3vt infusion of orexin B is shown in Fig. 3A. Sympathetic nerve activity gradually increased after central administration of orexin B. The mean change in sympathetic nerve activity in response to orexin B treatment was statistically significant compared with that induced by PBS (P < 0.05; Fig. 3B).
FIG. 3. A, Rate meter plots of BAT sympathetic nerve activity after infusion of orexin B (0.3 nmol) into the i3vt. Vertical axis shows nerve impulses per 5 sec. Horizontal bars, 10-min time scale; bold horizontal bar, orexin B infusion. B, Percentage differences in sympathetic nerve activity from baseline after i3vt infusion of orexin B (0.3 nmol) or PBS. The baseline 100% signifies the 60-min averages score of BAT sympathetic nerve activity before the orexin treatment. All data are means ± SEM (n = 4 per group). , Orexin B-treated animals; , control. *, P < 0.05; **, P < 0.01 vs. control.
The effect of pretreatment with FMH on changes in BAT sympathetic nerve activity induced by orexin B
Figure 4A shows the typical responses of BAT sympathetic nerve activity to infusion of orexin B in rats pretreated with an infusion of FMH into the i3vt. Sympathetic nerve activity gradually increased after central administration of orexin B in rats pretreated with FMH. The changes in sympathetic nerve activity in response to orexin B were significantly attenuated in pretreated rats compared with those induced by orexin B in rats pretreated with only PBS (P < 0.05; Fig. 4B).
FIG. 4. A, Rate meter plots of BAT sympathetic nerve activity after i3vt injection of orexin B (0.3 nmol) 2 h after pretreatment with FMH (0.67 μmol). Vertical axis shows nerve impulses per 5 sec. Horizontal bars, 10-min time scale; bold bar, orexin B infusion. B, Percentage differences in sympathetic nerve activity from baseline (100%) after i3vt injection of orexin B (0.3 nmol), orexin B (0.3 nmol) with FMH (0.67 μmol) pretreatment, or PBS (control). All data are means ± SEM (n = 4 per group). , Orexin B-treated animals; , orexin B-treated animals with FMH pretreatment; , control. *, P < 0.05; **, P < 0.01 vs. control; +, P < 0.05; ++, P < 0.01 vs. FMH pretreatment.
Discussion
This study demonstrated that i3vt infusion of orexin A or orexin B dose-dependently decreased or increased BAT sympathetic nerve activity in anesthetized rats, respectively. Depletion of neuronal histamine by pretreatment with FMH attenuated the sympathetic nerve responses induced by orexin B but not those induced by orexin A, indicating that the action of orexin B is mediated by neuronal histamine. Thus, it is likely that the dual effects of orexin on BAT sympathetic nerve may depend on differences in peptide subtype and/or its target neuronal substance. We present here a discussion of how orexin differentiates its actions on sympathetic nerves, as well as the effects on neuronal histamine.
The actions of orexins are mediated via two G protein-coupled receptors, OX1R and OX2R (1, 2). The receptor binding affinity of orexin A is 10–100 times greater than orexin B for OX1R, whereas both orexins are equipotent for OX2R (2, 23, 24, 25). Distinct expression patterns of the two receptor subtypes have been identified throughout the brain (5). Considering the i3vt infusion site used in this study, the target site for the binding of orexin to receptors may be either the hypothalamus or the brain stem. From this standpoint, the ventromedial hypothalamic nucleus, the A5 cell group, and the LC, all of which express high levels of OX1R mRNA, may be candidates as targets of orexin A, because these nuclei have been identified as central origins of efferent pathways to the BAT and to each other (26).
On the other hand, high-density expression of OX2R has been observed in the arcuate nucleus (ARC), the LHA, the paraventricular nucleus (PVN), the ventral premammillary nucleus, and the TMN, and moderate expression levels have been found in the medial preoptic area and the dorsal motor nucleus of the vagus (5). The PVN and medial preoptic area were identified as major hypothalamic origins of efferent pathways to BAT in a neuroanatomical study (26). In fact, chemical stimulation of these nuclei increased BAT sympathetic nerve activity (8). From the ARC, the LHA (especially orexin neurons themselves), and the PVN, direct neuronal projections to the intermediolateral cell column of the spinal cord, a location of sympathetic preganglionic neurons, have been identified (27, 28). Among these nuclei, cumulative evidence indicates the importance of the LHA and the ARC in orexin-A-OX2R signaling to suppress BAT sympathetic nerve activity. Electrophysiological studies have demonstrated an excitatory effect of orexin A on LHA neuronal activity and an inhibitory influence of this area on BAT sympathetic nerve activity (8), indicating that the LHA is a target site of orexin A for regulation of the sympathetic innervation of BAT.
In contrast to the suppressive effects of orexin A, central administration of orexin B in the present study increased BAT sympathetic nerve activity. In addition, this orexin B-induced response was attenuated by histamine depletion caused by pretreatment with FMH. As described above, the presence of orexin neurons, as well as the orexin receptor OXR2, has been identified in the TMN, a site of origin for histamine neurons (29). It has been demonstrated that neuronal histamine mediates orexin signaling in the regulation of the sleep-wakefulness cycle (30). Finally, central administration of histamine has been shown to increase BAT sympathetic nerve activity (12). Taken together, the evidence suggests that it is highly probable that orexin B regulates BAT sympathetic nerve activity in an excitatory manner through OX2R in the TMN. Our unpublished observation demonstrated that FMH treatment, but not neuropeptide Y antagonist, influenced orexin B-induced feeding and/or sympathetic nerve activity. The results suggested that neuronal histamine, but not neuropeptide Y, might be closely involved in the orexin B-induced feeding behavior and sympathetic nerve activity. Further study is necessary to clarify the details of this mechanism.
Considering both the present and previous studies, the results suggest that OXR in hypothalamus or the brain stem may mediate the orexin-induced effect on BAT sympathetic nerve activity. In addition, modulation of OXR in the outside of the hypothalamus, such as cerebral cortex, spinal cord, and the cisterna magna, is also possible. Previous studies demonstrated that orexin could have direct effects at the level of the sympathetic preganglionic neurons (31). Beyond BAT sympathetic nerve activity, further studies that examine the sympathetic nerve activity of renal and lumber nerves may be merited.
Functional studies have recently demonstrated that central administration of orexin A or orexin B induces hypothermia or hyperthermia, respectively (14, 15). These results are consistent with the present study, because the time course of BAT thermogenesis, as assessed by local BAT temperature, was similar to that observed for BAT sympathetic nerve activity (32). On the other hand, previous studies demonstrated an orexin A-induced hyperthermic response in unanesthetized animals (33, 34). It is well known that arousal state and muscle movement affect body temperature; in the unanesthetized condition, administration of orexin A induced an increase in arousal level and consequent arbitrary movements, both of which may elevate body temperature. There was the possibility that anesthesia might modulate BAT sympathetic nerve activity and thermogenesis because anesthesia is a factor, but not the sole mechanism, regulating arousal state and body temperature. Because the present study was limited to the acute phase of orexins’ effects in anesthetized rats, it is possible that differences in the state of arousal may determine the effect of orexins on body temperature. In addition, the timing of infusion might influence the orexin-induced sympathetic nerve activity because the effects of orexins are clearly dependent on the sleep-wake cycle (30).
In summary, we demonstrated that central administration of orexins induces dual effects on BAT sympathetic nerve activity: inhibition by orexin A and stimulation by orexin B. Hypothalamic neuronal histamine is involved in the latter response of BAT sympathetic nerve activity.
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Address all correspondence and requests for reprints to: Professor Hironobu Yoshimatsu, M.D., Ph.D., Department of Internal Medicine I, Faculty of Medicine, Oita University, 1–1 Idaigaoka, Hasama, Oita 879-5593, Japan. E-mail: hiroy@oita-med.ac.jp.
Abstract
This study examined how orexin regulates the activity of the sympathetic nerves that innervate brown adipose tissue (BAT) in rats. Infusion of orexin A at a dose of 0.3 nmol into the third cerebral ventricle decreased BAT sympathetic nerve activity, compared with the effect of PBS (P < 0.05), whereas infusion of orexin B at the same dose caused a significant increase (P < 0.05). Pretreatment with a third cerebral ventricle injection of 2.24 μmol/kg -fluoromethylhistidine, an irreversible inhibitor of the histamine-synthesizing enzyme histidine decarboxylase, attenuated the orexin B-induced response of BAT sympathetic nerve activity, but not that induced by orexin A. These results indicate that orexins may regulate both BAT energy expenditure and thermogenesis through their dual effects on sympathetic nerve activity. In particular, orexin B regulates BAT sympathetic nerve activity via neuronal histamine in the hypothalamus.
Introduction
OREXIN A AND OREXIN B are hypothalamic neuropeptides that are derived from a 130-amino-acid precursor, prepro-orexin and are potent agonists at both the orexin-1 (OX1) and orexin-2 (OX2) receptors. Orexin neurons are located in the lateral hypothalamic area (LHA), from which they innervate almost all regions of the brain except the cerebellum (1, 2). Especially dense projections are observed in specific regions of the thalamus, limbic system, and aminergic nuclei, such as the noradrenergic locus coeruleus (LC), serotonergic raphe nucleus, and histaminergic tuberomammillary nucleus (TMN) (3, 4). Concomitantly, the presence of orexin receptors has been identified in these nuclei, namely OX1R in the LC and OX2R in the TMN (5). Hypothalamic histamine neurons, with cell bodies in the TMN, have been shown to be involved in the regulation of a variety of physiological functions, such as feeding behavior, drinking behavior, and thermoregulation, as well as the autonomic nervous and neuroendocrine systems (6). The orexin-histamine signaling pathway plays an important role in the regulation of the sleep-wakefulness cycle, although its involvement in other hypothalamic functions remains unclear (7).
Brown adipose tissue (BAT) plays a major role in energy expenditure and thermogenesis via the function of uncoupling protein 1. The regulation of energy expenditure in BAT is under the control of the sympathetic nervous system and higher brain structures. Discrete hypothalamic nuclei and various orexigenic or anorexigenic neuropeptides regulate BAT metabolism by affecting efferent sympathetic nerve activity (8, 9, 10, 11). Hypothalamic neuronal histamine, an anorexigenic substance, increases BAT sympathetic nerve activity (12). Orexins affect energy balance and thermoregulation. Orexin A has been shown to induce hypothermia and food intake, and both orexins increase renal sympathetic nerve activity (13, 14, 15, 16). Therefore, it is highly probable that orexins regulate BAT energy expenditure and thermogenesis by influencing sympathetic nerve activity. We thus hypothesized that hypothalamic neuronal histamine may mediate orexin signals to the sympathetic nerves that innervate BAT, because the close relationship between these two systems has been recognized both anatomically and functionally (17, 18). This study therefore examined the effects of central administration of orexins on electrophysiological sympathetic nerve activity in BAT and modulation of these responses by neuronal histamine.
Materials and Methods
Animals
Mature male Sprague-Dawley rats (8–10 wk old) (Seac Yoshitomi, Fukuoka, Japan) were maintained on a 12-h light, 12-h dark photoperiod (lights on at 0700 h) in a room with controlled temperature (21 ± 1 C) and humidity (55 ± 5%). The rats were allowed free access to food (pelleted rodent chow no. CE-2; Clea Japan, Tokyo, Japan) and water. All studies were conducted in accordance with the Oita Medical University Guidelines, which are based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
Reagents
Orexin A, orexin B, and -fluoromethylhistidine (FMH) (all purchased from Sigma Chemical Co., St. Louis, MO) were each dissolved in PBS to final concentrations of 30 μM, 30 μM, and 67 mM, respectively. Each solution was freshly prepared on the day of use. The pH of each solution was adjusted to 6.4–7.2.
Cerebroventricular cannula implantation
Each rat was fixed in a stereotaxic apparatus (Narishige, Tokyo, Japan) under sodium pentobarbital anesthesia (45 mg/kg, ip), and a stainless steel guide cannula (23 gauge) was surgically implanted into the third cerebral ventricle (i3vt). A stainless steel wire stylet (29 gauge) was left in place in the guide cannula to maintain patency and prevent leakage of cerebrospinal fluid. Surgery was carried out at least 1 wk before infusion of the test solutions; the details of the surgical procedure have been described elsewhere (19).
Measurement of sympathetic nerve activity
Electrophysiological recordings were made under anesthesia (urethane, 0.8 g/kg; -chloralose, 80 mg/kg) using 20 cannulated rats. After dissection of the fine branches of the sympathetic nerves that innervate the interscapular BAT, the nerves were transected where they entered the BAT. Nerve activity was measured using a pair of silver-wire electrodes, which were immersed in a mixture of liquid paraffin and white petroleum jelly to prevent dehydration of the nerve tissue. The action potentials were amplified and filtered with low- and high-frequency cutoffs. The nerve signal was distinguished from background noise using a window discriminator. All nerve activity was analyzed based on values obtained after conversion of the raw data to standard pulses using an analog-to-digital converter. Impulses were integrated by a rate meter with a reset time of 5 sec and recorded using a chart recorder. Details of this nerve recording technique have been described elsewhere (8, 20). After determination of the background firing rate of the sympathetic nerves, changes in nerve activity were measured for up to 60 min after each bolus i3vt infusion with orexin A (0–3 nmol/rat, 1.0 μl/min, 10 min), orexin B (0–3 nmol/rat, 1.0 μl/min, 10 min), FMH (0–3 μmol/kg, 1.0 μl/min, 10 min), or PBS (n = 4 for each treatment). The doses of orexins and FMH were determined by previous studies (21, 22) and in our preliminary study. Infusions of orexins were performed 2 h after pretreatment with FMH at 1300 h. Nerve activity was measured at 10-min intervals, beginning 20 min before and continuing for 60 min after administration of the test solution. Nerve activity values recorded after the administration of each test solution were expressed as the percentage difference from the baseline value (at 0 min). The baseline 100% on figures signifies the 60-min averages score of BAT sympathetic nerve activity before the orexin treatment. For the analysis of dose dependence, orexins intracerebroventricular (icv) or PBS icv infusion was performed, and sympathetic nerve activity in BAT was recorded at 20 and 40 min after the infusion.
Statistical analysis
Differences among the groups were assessed using two-way ANOVA and post hoc Fisher’s protected least-significant difference test. A two-sided P value of less than 0.05 was considered statistically significant. The statistical analysis of dose dependence was assessed by the Spearman’s correlation coefficient by rank.
Results
Dose-dependent effects of orexins on BAT sympathetic nerve activity
We observed the dose-dependent effects of orexin A and orexin B. The BAT sympathetic nerve activity changes were dependent on the doses of orexins when given icv (orexin A: 0 nmol, 100.0 ± 4.5%; 0.03 nmol, 87.4 ± 6.1%; 0.3 nmol, 63.2 ± 6.4%; and 3 nmol, 47.4 ± 6.9%; P < 0.01) (orexin B: 0 nmol, 100.0 ± 5.3%; 0.03 nmol, 135.2 ± 7.3%; 0.3 nmol, 240.8 ± 11.2%; and 3 nmol, 322.0 ± 21.0%; P < 0.01) (Table 1).
TABLE 1. Dose-dependent effects of orexin A and orexin B on BAT sympathetic nerve activity
The effect of i3vt infusion of orexin A on BAT sympathetic nerve activity
Figure 1A shows the typical response of BAT sympathetic nerve activity after infusion of orexin A into the i3vt. Sympathetic nerve activity rapidly decreased after central administration of orexin A. The changes in sympathetic nerve activity in response to orexin A treatment differed significantly from those induced by PBS (P < 0.05; Fig. 1B).
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FIG. 1. A, Rate meter plots of BAT sympathetic nerve activity after infusion of orexin A (0.3 nmol) into the i3vt. Vertical axis shows nerve impulses per 5 sec. Horizontal bars, 10-min time scale; bold horizontal bar, orexin A infusion. B, Percentage differences in sympathetic nerve activity from baseline (100%) after i3vt infusion of orexin A (0.3 nmol) or PBS (control group). The baseline 100% signifies the 60-min averages score of BAT sympathetic nerve activity before the orexin treatment. All data are means ± SEM (n = 4 per group). , Orexin A-treated animals; , control. *, P < 0.05; **, P < 0.01 vs. control.
The effect of pretreatment with FMH on changes in BAT sympathetic nerve activity induced by orexin A
Figure 2A shows the typical responses of BAT sympathetic nerve activity to infusion of orexin A in rats pretreated with an infusion of FMH into the i3vt. The mean changes in sympathetic nerve activity induced by orexin A after pretreatment with FMH are shown in Fig. 2B. Sympathetic nerve activity decreased rapidly after central administration of orexin A in pretreated rats; these changes differed significantly from those induced by PBS (P < 0.05) but did not differ significantly from those induced by orexin A in rats pretreated only with PBS (P > 0.05).
FIG. 2. A, Rate meter plots of BAT sympathetic nerve activity after i3vt injection of orexin A (0.3 nmol) 2 h after pretreatment with FMH (0.67 μmol). Vertical axis shows nerve impulses per 5 sec. Horizontal bars, 10-min time scale; bold bar, orexin A infusion. B, Percentage differences in sympathetic nerve activity from baseline after i3vt injection of orexin A (0.3 nmol), orexin A (0.3 nmol) with FMH (0.67 μmol) pretreatment, or PBS (control). The baseline 100% signifies the 60-min averages score of BAT sympathetic nerve activity before the orexin treatment. All data are means ± SEM (n = 4 per group). , Orexin A-treated animals; , orexin A-treated animals with FMH pretreatment; , control. *, P < 0.05;, **, P < 0.01 vs. control.
The effect of i3vt infusion of orexin B on BAT sympathetic nerve activity
A typical response of BAT sympathetic nerve activity to i3vt infusion of orexin B is shown in Fig. 3A. Sympathetic nerve activity gradually increased after central administration of orexin B. The mean change in sympathetic nerve activity in response to orexin B treatment was statistically significant compared with that induced by PBS (P < 0.05; Fig. 3B).
FIG. 3. A, Rate meter plots of BAT sympathetic nerve activity after infusion of orexin B (0.3 nmol) into the i3vt. Vertical axis shows nerve impulses per 5 sec. Horizontal bars, 10-min time scale; bold horizontal bar, orexin B infusion. B, Percentage differences in sympathetic nerve activity from baseline after i3vt infusion of orexin B (0.3 nmol) or PBS. The baseline 100% signifies the 60-min averages score of BAT sympathetic nerve activity before the orexin treatment. All data are means ± SEM (n = 4 per group). , Orexin B-treated animals; , control. *, P < 0.05; **, P < 0.01 vs. control.
The effect of pretreatment with FMH on changes in BAT sympathetic nerve activity induced by orexin B
Figure 4A shows the typical responses of BAT sympathetic nerve activity to infusion of orexin B in rats pretreated with an infusion of FMH into the i3vt. Sympathetic nerve activity gradually increased after central administration of orexin B in rats pretreated with FMH. The changes in sympathetic nerve activity in response to orexin B were significantly attenuated in pretreated rats compared with those induced by orexin B in rats pretreated with only PBS (P < 0.05; Fig. 4B).
FIG. 4. A, Rate meter plots of BAT sympathetic nerve activity after i3vt injection of orexin B (0.3 nmol) 2 h after pretreatment with FMH (0.67 μmol). Vertical axis shows nerve impulses per 5 sec. Horizontal bars, 10-min time scale; bold bar, orexin B infusion. B, Percentage differences in sympathetic nerve activity from baseline (100%) after i3vt injection of orexin B (0.3 nmol), orexin B (0.3 nmol) with FMH (0.67 μmol) pretreatment, or PBS (control). All data are means ± SEM (n = 4 per group). , Orexin B-treated animals; , orexin B-treated animals with FMH pretreatment; , control. *, P < 0.05; **, P < 0.01 vs. control; +, P < 0.05; ++, P < 0.01 vs. FMH pretreatment.
Discussion
This study demonstrated that i3vt infusion of orexin A or orexin B dose-dependently decreased or increased BAT sympathetic nerve activity in anesthetized rats, respectively. Depletion of neuronal histamine by pretreatment with FMH attenuated the sympathetic nerve responses induced by orexin B but not those induced by orexin A, indicating that the action of orexin B is mediated by neuronal histamine. Thus, it is likely that the dual effects of orexin on BAT sympathetic nerve may depend on differences in peptide subtype and/or its target neuronal substance. We present here a discussion of how orexin differentiates its actions on sympathetic nerves, as well as the effects on neuronal histamine.
The actions of orexins are mediated via two G protein-coupled receptors, OX1R and OX2R (1, 2). The receptor binding affinity of orexin A is 10–100 times greater than orexin B for OX1R, whereas both orexins are equipotent for OX2R (2, 23, 24, 25). Distinct expression patterns of the two receptor subtypes have been identified throughout the brain (5). Considering the i3vt infusion site used in this study, the target site for the binding of orexin to receptors may be either the hypothalamus or the brain stem. From this standpoint, the ventromedial hypothalamic nucleus, the A5 cell group, and the LC, all of which express high levels of OX1R mRNA, may be candidates as targets of orexin A, because these nuclei have been identified as central origins of efferent pathways to the BAT and to each other (26).
On the other hand, high-density expression of OX2R has been observed in the arcuate nucleus (ARC), the LHA, the paraventricular nucleus (PVN), the ventral premammillary nucleus, and the TMN, and moderate expression levels have been found in the medial preoptic area and the dorsal motor nucleus of the vagus (5). The PVN and medial preoptic area were identified as major hypothalamic origins of efferent pathways to BAT in a neuroanatomical study (26). In fact, chemical stimulation of these nuclei increased BAT sympathetic nerve activity (8). From the ARC, the LHA (especially orexin neurons themselves), and the PVN, direct neuronal projections to the intermediolateral cell column of the spinal cord, a location of sympathetic preganglionic neurons, have been identified (27, 28). Among these nuclei, cumulative evidence indicates the importance of the LHA and the ARC in orexin-A-OX2R signaling to suppress BAT sympathetic nerve activity. Electrophysiological studies have demonstrated an excitatory effect of orexin A on LHA neuronal activity and an inhibitory influence of this area on BAT sympathetic nerve activity (8), indicating that the LHA is a target site of orexin A for regulation of the sympathetic innervation of BAT.
In contrast to the suppressive effects of orexin A, central administration of orexin B in the present study increased BAT sympathetic nerve activity. In addition, this orexin B-induced response was attenuated by histamine depletion caused by pretreatment with FMH. As described above, the presence of orexin neurons, as well as the orexin receptor OXR2, has been identified in the TMN, a site of origin for histamine neurons (29). It has been demonstrated that neuronal histamine mediates orexin signaling in the regulation of the sleep-wakefulness cycle (30). Finally, central administration of histamine has been shown to increase BAT sympathetic nerve activity (12). Taken together, the evidence suggests that it is highly probable that orexin B regulates BAT sympathetic nerve activity in an excitatory manner through OX2R in the TMN. Our unpublished observation demonstrated that FMH treatment, but not neuropeptide Y antagonist, influenced orexin B-induced feeding and/or sympathetic nerve activity. The results suggested that neuronal histamine, but not neuropeptide Y, might be closely involved in the orexin B-induced feeding behavior and sympathetic nerve activity. Further study is necessary to clarify the details of this mechanism.
Considering both the present and previous studies, the results suggest that OXR in hypothalamus or the brain stem may mediate the orexin-induced effect on BAT sympathetic nerve activity. In addition, modulation of OXR in the outside of the hypothalamus, such as cerebral cortex, spinal cord, and the cisterna magna, is also possible. Previous studies demonstrated that orexin could have direct effects at the level of the sympathetic preganglionic neurons (31). Beyond BAT sympathetic nerve activity, further studies that examine the sympathetic nerve activity of renal and lumber nerves may be merited.
Functional studies have recently demonstrated that central administration of orexin A or orexin B induces hypothermia or hyperthermia, respectively (14, 15). These results are consistent with the present study, because the time course of BAT thermogenesis, as assessed by local BAT temperature, was similar to that observed for BAT sympathetic nerve activity (32). On the other hand, previous studies demonstrated an orexin A-induced hyperthermic response in unanesthetized animals (33, 34). It is well known that arousal state and muscle movement affect body temperature; in the unanesthetized condition, administration of orexin A induced an increase in arousal level and consequent arbitrary movements, both of which may elevate body temperature. There was the possibility that anesthesia might modulate BAT sympathetic nerve activity and thermogenesis because anesthesia is a factor, but not the sole mechanism, regulating arousal state and body temperature. Because the present study was limited to the acute phase of orexins’ effects in anesthetized rats, it is possible that differences in the state of arousal may determine the effect of orexins on body temperature. In addition, the timing of infusion might influence the orexin-induced sympathetic nerve activity because the effects of orexins are clearly dependent on the sleep-wake cycle (30).
In summary, we demonstrated that central administration of orexins induces dual effects on BAT sympathetic nerve activity: inhibition by orexin A and stimulation by orexin B. Hypothalamic neuronal histamine is involved in the latter response of BAT sympathetic nerve activity.
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