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Prokineticin 2 is involved in the thermoregulation and energy expenditure
Regulatory Peptides 179 (2012) 84–90
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Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
Prokineticin 2 is involved in the thermoregulation and energy expenditure Wenbai Zhou a, b, Jia-Da Li a, c,⁎, Wang-Ping Hu a, d, Michelle Y. Cheng a, Qun-Yong Zhou a,⁎⁎ a
Department of Pharmacology, University of California, Irvine, CA 92697, USA Department of Endocrinology and Metabolism, Huashan Hospital, Shanghai, China c The State Key Laboratory of Medical Genetics, Central South University of China, Changsha, Hunan, China d Department of Pharmacology, Xianning College, Xianning, Hubei, China b
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Article history: Received 21 March 2012 Received in revised form 28 June 2012 Accepted 27 August 2012 Available online 4 September 2012 Keywords: Prokineticin 2 Torpor Fasting Arousal
a b s t r a c t Animals have developed adaptive strategies to survive tough situations such as food shortage. However, the underlying molecular mechanism is not fully understood. Here, we provided evidence that the regulatory peptide prokineticin 2 (PK2) played an important role in such an adaptation. The PK2 expression was rapidly induced in the hypothalamic paraventricular nucleus (PVN) after fasting, which can be mimicked by 2-deoxy-D-glucose (2-DG) injection. The fasting-induced arousal was absent in the PK2-deficient (PK2 −/−) mice. Furthermore, PK2 −/− mice showed less energy expenditure and body weight loss than wild-type (WT) controls upon fasting. As a result, PK2 −/− mice entered torpor after fasting. Supply of limited food (equal to 5% of body weight) daily during fasting rescued the body weight loss and hypothermal phenotype in WT mice, but not in PK2 −/− mice. Our study thus demonstrated PK2 as a regulator in the thermoregulation and energy expenditure. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Prokineticins, including prokineticin 1 (PK1) and prokineticin 2 (PK2), are a pair of regulatory peptides with a similar molecular weight of ~10 kDa [1]. PK1 and PK2 are the cognate ligands for two closely related G-protein-coupled receptors (PKR1 and PKR2) [2–4]. In the central nervous system, PK2 is expressed in the suprachiasmatic nucleus (SCN), islands of Calleja, medial preoptic area (MPA), olfactory bulb, nucleus accumbens shell, hypothalamic arcuate nucleus and amygdale, while PKR2 is widely expressed throughout the brain [5]. PK2 is reported to be an output molecule for the SCN clock. In the SCN, the circadian pacemaker, PK2 expression is highly circadian and regulated by the central clock machinery [6]. Lacking of PK2 genes in the mice results in reduced circadian rhythmicity in a variety of behavior and physiology, including locomotion, sleep/wake cycle, body temperature, food intake, hormone level, emotional conditions, and energy metabolism
Abbreviations: PK2, prokineticin 2; PVN, hypothalamic paraventricular nucleus; 2-DG, 2-deoxy-D-glucose; SCN, suprachiasmatic nucleus; SNS, sympathetic nervous system; RQ , respiratory quotient; PK2 −/−, PK2-deficient; NREM, non-rapid eye movement; REM, rapid eye movement; DMH, dorsomedial hypothalamus; VLPO, ventrolateral preoptic nucleus; CNS, central nervous system; MPA, medial preoptic area; Arc, arcuate nucleus. ⁎ Correspondence to: J.-D. Li, The State Key Laboratory of Medical Genetics, Central South University of China, Changsha, 410078, Hunan, China. Fax: +86 731 84805339. ⁎⁎ Correspondence to: Q.-Y. Zhou, Dept of Pharmacology, Univ. of California, Irvine, CA92697, USA. Fax: + 1 9498244855. E-mail addresses: [email protected] (J.-D. Li), [email protected] (Q.-Y. Zhou). 0167-0115/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.regpep.2012.08.003
[7,8]. The circadian system may affect thermoregulation depending on the time of day and feeding condition [9,10]. During the food deprivation, the SCN is activated, associating with activation of other hypothalamic areas like the hypothalamic paraventricular nucleus (PVN), which plays important roles in the regulation of the sympathetic nervous system (SNS). SNS regulates the heat production through general activity, shivering, and brown fat thermogenesis [11]. Thus, both of the SCN and PVN are involved in energy expenditure. Recently, Jethwa et al. [12] demonstrated that prokineticin receptor 2 (PKR2) signaling was crucial in the thermoregulation. They observed that null mutation of PKR2 (PKR2m/m) mice showed sporadic bouts of torpor under ad libitum feeding at constant temperature. During the torpor, PKR2 m/m mice showed behavioral hyporesponsive, as decreasing locomotor activities, oxygen consumption and respiratory quotient (RQ). As they removed food for a period, PKR2m/m mice and their littermates all displayed low body temperature, in which PKR2 m/m mice had a deeper and longer hypothermia associated with greater decreased oxygen consumption and RQ compared to their littermates. They suggested that PKR2 signaling pathway is involved in the regulation of energy balance and thermoregulation. As PK2 and PKR2 are likely a pair in the central nervous system, we investigated the role of PK2 in the thermoregulation using PK2-deficient (PK2−/−) mice in the study. 2. Materials and methods 2.1. Animals PK2−/− mice were generated by homologous recombination as previously described [13]. PK2−/− mice and their littermate wild-type
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(WT) mice in a C57BL/6 × 129/Ola hybrid background were used in all experiments. We have monitored the body weight change of WT and PK2−/− mice under regular chow feeding. There is dramatic body weight difference between male WT and PK2−/− mice, whereas the body weight in female mice is quite similar (data not shown). To avoid potential confound caused by the basal body weight, we only used females of 3–5 months of age in this study. We did not observe difference in the food intake of WT and PK2 −/− mice under basal conditions [8]. Before the experiments, the mice were group-housed (3–5 animals per cage) under controlled conditions [temperature, 20± 2 °C; relative humidity, 50–60%; 12:12-h light–dark (LD) cycle, lights on at 7:00 AM and lights off at 7:00 PM] and had free access to food and water [14]. During experiments, mice were individually housed. All procedures regarding the care and use of animals were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. 2.2. In situ hybridization Mice were food-deprived starting from the lights off and killed at 0, 4, 8, 16 and 24 h after food deprivation. For re-feeding experiments, an independent group of mice was supplied with food pellets after 24-h food deprivation, and mice were killed at 15, 30, 60, 120 and 240 min after re-feeding. For 2-deoxy-D-glucose (2-DG) experiment, mice were injected with 2-DG (250 mg/kg) or saline at 9 am, and returned to their home cages without food. These mice were killed at 0, 2, 4 and 8 h after injection. Brains were sectioned and processed for in situ hybridization as described previously [15]. This DNA sequence used for PK2 probe is nucleotides 1–528 of mouse Prok2 (GenBank accession no. AF487280). We have used this probe in a variety of studies [6,14,16–18]. We verified this probe by hybridizing with brain slices with a sense cRNA, which make no signal. Antisense and sense cRNA probes were generated by in vitro transcription in the presence of 35 S-labeled UTP (1200 Ci/mmol). The PK2 mRNA distributions were analyzed in autoradiograms. Specific hybridization signals were quantitatively analyzed using a video-based computer image analysis system (MCID, Imaging Research, St. Catharine's, Ontario, Canada). A calibration curve of optical density versus radioactivity (dpm/mg tissue wet weight) was constructed using 14C-standards. Specific hybridization signals in PVN were obtained by subtracting background values obtained from adjacent brain areas that have no hybridization signal.
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personal computer every 5 min. The mice were allowed to recover for at least 2 weeks before the experiments. The ambient temperature was constant at 21 °C unless otherwise indicated. 2.5. Energy expenditure and oxygen consumption A comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH) was used to monitor oxygen (O2) and carbon dioxide (CO2) gas fractions at both the inlet and outlet ports to each of 4 test chambers, which were supplied with corn cob bedding. The airflow was 0.65 L/min, and air from each chamber was sampled and analyzed every 10 s. Experimental mice were either fasted or had free access to food. The mice were allowed to familiar the cages for 2 days, and then were recorded. 2.6. Statistical analyses A repeated-measures ANOVA followed by an unpaired t test was used to analyze the data for differences between genotypes or treatments. All statistical analysis was performed using Prism 4.4 (GraphPad Software, San Diego, CA). 3. Results 3.1. Fasting induced PK2 expression in the PVN We previously showed that light induced PK2 expression in the SCN [16]. To study the effect of food deprivation on PK2 induction, we subjected wild-type (WT) mice to fasting and detected the PK2 mRNA expression throughout the brain using in situ hybridization. Under ad lib feeding conditions, PK2 was barely detectable in the PVN (Fig. 1A); however, the expression of PK2 was significantly induced in the PVN after fasting (Fig. 1B). The induction occurred as early as 4 h after fasting (Fig. 1C). The fasting-induced PK2 expression was restricted to the PVN as no significant induction was found in the other brain areas (data not shown). When food pellets were supplied to the fasted mice (e.g. re-feeding), the PK2 level in the PVN rapidly fell to the basal undetectable level within 4 h (Fig. 1C). Moreover, 2-deoxy-D-glucose (2-DG), an inhibitor of glucose utilization, also rapidly induced PK2 expression in the PVN (Fig. 1D), implying that the induction was a response to the dwindling energy reserves.
2.3. Recording and analysis of sleep/wake 3.2. PK2 −/− mice displayed torpor upon fasting The electroencephalogram (EEG) and electromyogram (EMG) signals from mice were recorded and analyzed as described [7]. The mice were connected to a wire tether system (Plastics One, Roanoke, VA) for the collection of EEG and EMG signals. This swivel system allowed the animal unrestricted movement throughout the recording cage. After at least 5 days of adaptation to the recording environment, a 48-h baseline EEG/EMG recording was collected on a LD cycle with lights on at 7:00 AM and off at 7:00 PM. Mice were recorded concurrently in matched littermate pairs of PK2 −/− and WT mice. EEG/EMG signals were amplified using a Grass Telefactor Model 15LT with 15A94 amplifier (Grass Instruments, West Warwick, RI) and filtered (EEG: 0.3–100 Hz, EMG: 30–300 Hz) before being digitized at a sampling rate of 128 Hz and stored on a computer. 2.4. Core body temperature monitoring A radio transmitter device (G2 E-mitter; Mini-Mitter, Sunriver, OR) used to measure body temperature and locomotion activity simultaneously was implanted in the abdominal cavity by sterile technique under general anesthesia [19]. Body temperature and locomotion activity were recorded by a receiver board (ER-4000 energizer receiver; Mini-Mitter) underneath the cage and were stored in a
Torpor is generally defined as reduction in body temperature below 31 °C [20]. Previously, Jethaw et al. [12] reported a portion of PKR2 m/m mice showed spontaneous torpor under normal feeding conditions. However, no spontaneous torpor was observed in PK2 −/− mice under ad lib feeding and constant ambient temperature conditions (21 °C). We did not even observe torpor in ad lib-fed PK2 −/− mice at 4 °C (data not shown). Jethaw et al. [12] also reported that a severe torpor occurred in PKR2 m/m mice during fasting. Thus, we subjected PK2 −/− mice to fasting and monitored their body temperature using telemetries. Both WT and PK2 −/− mice showed a decreased body temperature during fasting; however, the body temperature of PK2 −/− mice was much lower (genotype: F (1) = 19.13, P b 0.0001, two-way ANOVA). PK2 −/− mice entered torpor after fasting, i.e. the core body temperature dropped below 30 °C, whereas WT mice were still able to keep the core body temperature above 33 °C (Fig. 2A). After prolonged fasting (> 36 h), WT mice displayed a transitional body temperature dipping close to 30 °C, whereas PK2 −/− mice showed a period of ~ 4 h of torpor, when the body temperature dropped to as low as 25 °C (Fig. 2A). Interestingly, most of the torpors occurred at the transition from the active phase to the inactive phase (night to day).
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Fig. 1. Induction of PK2 mRNA in PVN during fasting. (A, B) Autoradiograms depicted PK2 mRNA expression in PVN under normal feeding (A) or after 24 hour fasting (B). (C) Time course of PK2 mRNA expression in PVN during the 24 hour fasting or re-feeding after 24 hour fasting (n = 3–4 animals). (D) PK2 mRNA expression in PVN in mice treated with saline or 2-DG. Mice were returned to their home cages without food after injections. PK2 mRNA in PVN was significantly higher at 2 and 4 h after 2-DG injection (n = 3–4 animals, *P b 0.05, unpaired t test).
To see if the genotypic difference in core body temperature is due to the difference in activity, we also analyzed the locomotor activity during fasting simultaneously. Both WT and PK2 −/− mice showed increased locomotion upon fasting (especially at the first day of fasting), although PK2 −/− mice moved less than WT mice during the dark phase under fasting or normal feeding conditions (genotype: F (1) = 26.37, P b 0.0001, two-way ANOVA). Nevertheless, during the
period when PK2 −/− mice showed torpor (ZT20 to ZT4, ZT0 is defined as the time when light is turned on), no difference in activity was seen between WT and PK2 −/− mice (Fig. 2B). 3.3. Altered energy expenditure in PK2 −/− mice during fasting We also monitored the energy expenditure of WT and PK2 −/− mice during fasting with an indirect metabolic system. Consistent with the body temperature recording, the energy expenditure dropped in both WT and PK2 −/− mice during fasting as compared with ad lib feeding. However, during certain periods, especially at the end of dark period and beginning of light phase, the oxygen consumption in PK2 −/− mice was significantly lower than WT mice (genotype: F (1) = 4.548, P b 0.01, two-way ANOVA; Fig. 3A), suggesting lower energy expenditure in PK2 −/− mice. Mammals were able to mobilize lipid during fasting to keep the core body temperature relatively stable. Consistent with the reduced energy expenditure in PK2−/− mice during fasting, the body weight loss was significantly less in PK2−/− mice than WT controls (P b 0.01, unpaired t test) (Fig. 3B). 3.4. Absence of fasting-induced arousal in PK2 −/− mice
Fig. 2. Body temperature and locomotor activity during the 48 h of fasting. (A) Decrease in body temperature during fasting in both genotypes. Note the greater hypothermic response to fasting in the PK2−/− mice (n = 5 mice/genotype). (B) Induced locomotor activity in both genotypes during the 48 h of fasting. Note locomotor activity was lower in PK2−/− mice compared to the WT mice (n = 5 mice/genotype).
Animals respond to reduced food availability by becoming more wakeful and active, a phenomenon called fasting-induced arousal. To examine whether PK2 signaling is required for the adaptive arousal response to fasting, we recorded states of wakefulness, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep during fasting. After baseline (fed ad lib) recording, animals were fasted for 36 h beginning with the onset of the dark phase. Analysis of sleep/wake states revealed significant increases of arousal in WT mice during fasting (wakefulness, F (1) = 17.28, P b 0.0001; NREM, F (1) = 42.52, P b 0.0001; two-way ANOVA treatment effect; Fig. 4A and B). In particular, there was a robust increase in arousal after 18 h of fasting. In contrast, PK2 −/− mice exhibited no appreciable increase in arousal during the entire fasting period (wakefulness, F
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Fig. 3. Energy expenditure in the both genotypes during the 48 h of fasting. (A) Oxygen consumption during fasting in both genotypes. Note that oxygen consumption was lower in PK2−/− mice (n = 6 mice/genotype). (B) Marked decrease in body weight in WT mice during the 24 h or 48 h of starvation compared to that of PK2−/− mice. n = 6 mice/genotype, **P b 0.05, unpaired t test.
(1) = 0.75, P = 0.3869; NREM, F (1) = 2.049, P = 0.1534; two-way ANOVA treatment effect; Fig. 4C and D). 3.5. Restricted food supply rescued the fasting-induced phenotypes in the WT but not in PK2 −/− mice To investigate whether restricted food supply can rescue the hypothermal phenotype in fasted animals, we provided a food pellet equal to 5% of their body weight everyday to the fasted WT and PK2 −/− mice. As shown in Fig. 5A, both WT and PK2 −/− mice showed a decreased body temperature during restricted food supply; however, the body temperature of PK2 −/− mice was much lower (Genotype: F (1) = 5.10, P b 0.0001, two-way ANOVA). The hypothermal phenotype was not likely a consequence of difference in locomotion, either (Fig. 5B). This amount of food significantly increased the core body temperature in WT mice compared to the WT mice under the fasting (treatment: F (1) = 8.213, P b 0.01, two-way ANOVA; Fig. 5C). However, the body temperature of PK2 −/− mice was almost identical to that of fasting (treatment: F (1) = 0.5366, P > 0.05, two-way ANOVA; Fig. 5D). Furthermore, restricted food supply resulted in significantly less body weight loss in WT mice as compared with fasting (P b 0.01); however, the body weight loss during the first day of restricted feeding was similar to that during fasting in PK2 −/− mice (Fig. 5E). The fact that restricted food pellet rescued the hypothermal and body weight drop in WT mice but not in PK2 −/− mice implied a deficit of food utilization in PK2 −/− mice during re-feeding period. 4. Discussion In the real world, animals always face food shortage and thus evolve adaptive strategies to survive these tough situations. During fasting, small laboratory animals such as mice become aroused and active so as to maintain body temperature in a normal range and be ready for food supply [21]. Once food is applied, mice ingested the food and utilized the food to compensate the body weight loss. In these processes, the regulatory peptide PK2 seems to play important roles. First, fasting significantly induced PK2 expression in the PVN, and this induction disappeared after re-feeding. Second, PK2 −/− mice failed to display the fasting-induced arousal. Third, PK2 −/−
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mice were ready to enter torpor after fasting. Last, food applied after fasting rescued the hypothermal phenotypes in WT mice but not in PK2 −/− mice, the possible explanation is that PK2 −/− mice were not as efficient as WT mice to utilize food. Metabolic adaptations to fasting include changes in fuel usage, increased metabolic efficiency, and minimization of energy expenditure [22]. Energy storage is important for the adaptation and the mice deficient in the adipose tissue readily enter torpor during fasting [23]. However, these mice usually exhaust all energy storage during short-time fasting. It is unlikely that the torpor in PK2 −/− mice was caused by alteration in adipose tissue as PK2 −/− mice displayed a slightly more fat than WT controls (data not shown). In contrast to the rapid body weight loss observed in adipose-deficient mice, PK2 −/− mice lost less body weight than WT controls. PK2 and PKR2 are a pair of ligand–receptor in the central nervous system (CNS). As both PK2−/− and PKR2−/− mice entered torpor during fasting, we favored the idea that the defect of PK2/PKR2 signaling in the CNS contributed to the torpor phenotypes. PKR2 is highly expressed in the forebrain involved in the regulation of body temperature, arousal as well as sensing of nutrients, such as the PVN and the dorsomedial hypothalamus (DMH) [6]. The PVN is a very crucial site within the forebrain that involved in the hormonal endocrine, neural control, sympathetic nerve activity, and body temperature regulation. The PVN could detect energy homeostasis and play roles in energy intake and energy expenditure [24,25]. There are thermosensitive neurons locating in the PVN, which can be activated when the core body temperature is altered. Thus, the PVN could sense the body temperature change through these neurons and make response by activating the sympathetic nerves system [26]. The PK2 receptor, PKR2, is expressed in the PVN and previous studies have reported that PK2 could influence the excitability of different subpopulations of PVN neurons, as magnocellular, parvocellular preautonomic, and parvocellular neuroendocrine neurons within the PVN [27,28]. A possible explanation was that PK2 facilitated the PVN to sense the hypothermia, activated the sympathetic nerve system which in turn led to increasing activity, expanding the wake periods, and using body energy storage. PK2 was induced in the PVN by fasting, implying a possible intra-PVN projection of PK2 within the PVN. Gardiner et al. [29] found a decrease in hypothalamic PK2 mRNA expression following fasting in rats. In our in situ hybridization experiment, we only see significant increase of PK2 mRNA in the PVN. As PK2 is also highly expressed in other hypothalamic areas, such as the SCN, the arcuate nucleus (Arc), and the medical preoptic area (MPA) [5,17], it is possible that there is change in these areas during fasting which we did not detect. The PKR2 is also highly expressed in the DMH, an important brain area involved in the body temperature and arousal regulation [30]. The DMH is known to heavily innervate the ventrolateral preoptic nucleus (VLPO) and has been shown to be a critical nucleus for arousal regulation in response to restricted feeding [31]. Therefore, PK2 might participate in fasting-induced arousal through the DMH– VLPO pathway. PK2 might be originated from the SCN, MPA or PVN and project to the DMH to participate arousal and/or body temperature regulation [30]. PK2 is reported to be an important output factor that transmits the circadian information from the circadian pacemaker SCN. In support of this, we and others have observed a reduced circadian rhythmicity in the mice deficient in either PK2 or PKR2 [6,16]. Interestingly, the circadian rhythms of body temperature, locomotion and metabolism and sleep–wake cycle are essentially intact during fasting (at least in the first 2-day fasting). Activity may be a major force to maintain the core body temperature during the active phase, while another switch to catabolize fat depots is turned on during the inactive phase so as to keep the core body temperature in a relatively normal range during the inactive phase. If the switch is disabled or did not respond correctly, energy expenditure does not occur to an extent, which causes the animals entering torpor. It is worthy to note that
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Fig. 4. Sleep–wake patterns in WT and PK2−/− mice. (A, C) Distribution of wake, non-rapid eye movement (NREM) sleep in 2-h intervals under a 12 h: 12 h light: dark (LD) cycle for WT (A) and PK2−/− (C) mice. (B, D) Amounts of sleep–wake states (wake, NREM, REM) in WT (B) and PK2−/− (D) mice. (n = 6 mice/genotype, **P b 0.01, unpaired t test).
the fasted PK2 −/− mice displayed torpor during the transition from active phase to inactive phase, when the PK2 in the SCN is expressed at the peak levels. It is likely that PK2 from the SCN may serve as the switch signal to turn on energy expenditure during the inactive phase. Our observations of hypothermia and torpor in PK2 −/− mice during fasting suggested that PK2 might play an important role in energy intake and expenditure. There was an intriguing hypothesis that
enhancement of PK2 signaling in PVN during fasting might help mice to endure the tough situation via alternation to fat catabolism and maintaining body temperature. Acknowledgments This work was supported by the Projects in the Major State Basic Research Development Program of China (973 Program) (2012CB517904),
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Fig. 5. Different responses to fasting (F) and to food restriction (FR) in PK2−/− mice and WT mice. (A) Decrease in body temperature during the food restriction in WT mice and PK2−/− mice (n = 5 mice/genotype). (B) Locomotor activity during the food restriction in WT mice and PK2−/− mice (n = 5 mice/genotype). (C, D) Body temperature during fasting and food restriction in WT mice or PK2−/− mice (n = 5 mice/genotype). (E) Decrease of body weight during the 48 h of fasting or 48 h of food restriction in both genotypes (n = 6 mice/genotype, *P b 0.05, **P b 0.01, unpaired t test).
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Contents lists available at SciVerse ScienceDirect
Regulatory Peptides journal homepage: www.elsevier.com/locate/regpep
Prokineticin 2 is involved in the thermoregulation and energy expenditure Wenbai Zhou a, b, Jia-Da Li a, c,⁎, Wang-Ping Hu a, d, Michelle Y. Cheng a, Qun-Yong Zhou a,⁎⁎ a
Department of Pharmacology, University of California, Irvine, CA 92697, USA Department of Endocrinology and Metabolism, Huashan Hospital, Shanghai, China c The State Key Laboratory of Medical Genetics, Central South University of China, Changsha, Hunan, China d Department of Pharmacology, Xianning College, Xianning, Hubei, China b
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Article history: Received 21 March 2012 Received in revised form 28 June 2012 Accepted 27 August 2012 Available online 4 September 2012 Keywords: Prokineticin 2 Torpor Fasting Arousal
a b s t r a c t Animals have developed adaptive strategies to survive tough situations such as food shortage. However, the underlying molecular mechanism is not fully understood. Here, we provided evidence that the regulatory peptide prokineticin 2 (PK2) played an important role in such an adaptation. The PK2 expression was rapidly induced in the hypothalamic paraventricular nucleus (PVN) after fasting, which can be mimicked by 2-deoxy-D-glucose (2-DG) injection. The fasting-induced arousal was absent in the PK2-deficient (PK2 −/−) mice. Furthermore, PK2 −/− mice showed less energy expenditure and body weight loss than wild-type (WT) controls upon fasting. As a result, PK2 −/− mice entered torpor after fasting. Supply of limited food (equal to 5% of body weight) daily during fasting rescued the body weight loss and hypothermal phenotype in WT mice, but not in PK2 −/− mice. Our study thus demonstrated PK2 as a regulator in the thermoregulation and energy expenditure. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Prokineticins, including prokineticin 1 (PK1) and prokineticin 2 (PK2), are a pair of regulatory peptides with a similar molecular weight of ~10 kDa [1]. PK1 and PK2 are the cognate ligands for two closely related G-protein-coupled receptors (PKR1 and PKR2) [2–4]. In the central nervous system, PK2 is expressed in the suprachiasmatic nucleus (SCN), islands of Calleja, medial preoptic area (MPA), olfactory bulb, nucleus accumbens shell, hypothalamic arcuate nucleus and amygdale, while PKR2 is widely expressed throughout the brain [5]. PK2 is reported to be an output molecule for the SCN clock. In the SCN, the circadian pacemaker, PK2 expression is highly circadian and regulated by the central clock machinery [6]. Lacking of PK2 genes in the mice results in reduced circadian rhythmicity in a variety of behavior and physiology, including locomotion, sleep/wake cycle, body temperature, food intake, hormone level, emotional conditions, and energy metabolism
Abbreviations: PK2, prokineticin 2; PVN, hypothalamic paraventricular nucleus; 2-DG, 2-deoxy-D-glucose; SCN, suprachiasmatic nucleus; SNS, sympathetic nervous system; RQ , respiratory quotient; PK2 −/−, PK2-deficient; NREM, non-rapid eye movement; REM, rapid eye movement; DMH, dorsomedial hypothalamus; VLPO, ventrolateral preoptic nucleus; CNS, central nervous system; MPA, medial preoptic area; Arc, arcuate nucleus. ⁎ Correspondence to: J.-D. Li, The State Key Laboratory of Medical Genetics, Central South University of China, Changsha, 410078, Hunan, China. Fax: +86 731 84805339. ⁎⁎ Correspondence to: Q.-Y. Zhou, Dept of Pharmacology, Univ. of California, Irvine, CA92697, USA. Fax: + 1 9498244855. E-mail addresses: [email protected] (J.-D. Li), [email protected] (Q.-Y. Zhou). 0167-0115/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.regpep.2012.08.003
[7,8]. The circadian system may affect thermoregulation depending on the time of day and feeding condition [9,10]. During the food deprivation, the SCN is activated, associating with activation of other hypothalamic areas like the hypothalamic paraventricular nucleus (PVN), which plays important roles in the regulation of the sympathetic nervous system (SNS). SNS regulates the heat production through general activity, shivering, and brown fat thermogenesis [11]. Thus, both of the SCN and PVN are involved in energy expenditure. Recently, Jethwa et al. [12] demonstrated that prokineticin receptor 2 (PKR2) signaling was crucial in the thermoregulation. They observed that null mutation of PKR2 (PKR2m/m) mice showed sporadic bouts of torpor under ad libitum feeding at constant temperature. During the torpor, PKR2 m/m mice showed behavioral hyporesponsive, as decreasing locomotor activities, oxygen consumption and respiratory quotient (RQ). As they removed food for a period, PKR2m/m mice and their littermates all displayed low body temperature, in which PKR2 m/m mice had a deeper and longer hypothermia associated with greater decreased oxygen consumption and RQ compared to their littermates. They suggested that PKR2 signaling pathway is involved in the regulation of energy balance and thermoregulation. As PK2 and PKR2 are likely a pair in the central nervous system, we investigated the role of PK2 in the thermoregulation using PK2-deficient (PK2−/−) mice in the study. 2. Materials and methods 2.1. Animals PK2−/− mice were generated by homologous recombination as previously described [13]. PK2−/− mice and their littermate wild-type
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(WT) mice in a C57BL/6 × 129/Ola hybrid background were used in all experiments. We have monitored the body weight change of WT and PK2−/− mice under regular chow feeding. There is dramatic body weight difference between male WT and PK2−/− mice, whereas the body weight in female mice is quite similar (data not shown). To avoid potential confound caused by the basal body weight, we only used females of 3–5 months of age in this study. We did not observe difference in the food intake of WT and PK2 −/− mice under basal conditions [8]. Before the experiments, the mice were group-housed (3–5 animals per cage) under controlled conditions [temperature, 20± 2 °C; relative humidity, 50–60%; 12:12-h light–dark (LD) cycle, lights on at 7:00 AM and lights off at 7:00 PM] and had free access to food and water [14]. During experiments, mice were individually housed. All procedures regarding the care and use of animals were approved by the Institutional Animal Care and Use Committee of the University of California, Irvine. 2.2. In situ hybridization Mice were food-deprived starting from the lights off and killed at 0, 4, 8, 16 and 24 h after food deprivation. For re-feeding experiments, an independent group of mice was supplied with food pellets after 24-h food deprivation, and mice were killed at 15, 30, 60, 120 and 240 min after re-feeding. For 2-deoxy-D-glucose (2-DG) experiment, mice were injected with 2-DG (250 mg/kg) or saline at 9 am, and returned to their home cages without food. These mice were killed at 0, 2, 4 and 8 h after injection. Brains were sectioned and processed for in situ hybridization as described previously [15]. This DNA sequence used for PK2 probe is nucleotides 1–528 of mouse Prok2 (GenBank accession no. AF487280). We have used this probe in a variety of studies [6,14,16–18]. We verified this probe by hybridizing with brain slices with a sense cRNA, which make no signal. Antisense and sense cRNA probes were generated by in vitro transcription in the presence of 35 S-labeled UTP (1200 Ci/mmol). The PK2 mRNA distributions were analyzed in autoradiograms. Specific hybridization signals were quantitatively analyzed using a video-based computer image analysis system (MCID, Imaging Research, St. Catharine's, Ontario, Canada). A calibration curve of optical density versus radioactivity (dpm/mg tissue wet weight) was constructed using 14C-standards. Specific hybridization signals in PVN were obtained by subtracting background values obtained from adjacent brain areas that have no hybridization signal.
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personal computer every 5 min. The mice were allowed to recover for at least 2 weeks before the experiments. The ambient temperature was constant at 21 °C unless otherwise indicated. 2.5. Energy expenditure and oxygen consumption A comprehensive Lab Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH) was used to monitor oxygen (O2) and carbon dioxide (CO2) gas fractions at both the inlet and outlet ports to each of 4 test chambers, which were supplied with corn cob bedding. The airflow was 0.65 L/min, and air from each chamber was sampled and analyzed every 10 s. Experimental mice were either fasted or had free access to food. The mice were allowed to familiar the cages for 2 days, and then were recorded. 2.6. Statistical analyses A repeated-measures ANOVA followed by an unpaired t test was used to analyze the data for differences between genotypes or treatments. All statistical analysis was performed using Prism 4.4 (GraphPad Software, San Diego, CA). 3. Results 3.1. Fasting induced PK2 expression in the PVN We previously showed that light induced PK2 expression in the SCN [16]. To study the effect of food deprivation on PK2 induction, we subjected wild-type (WT) mice to fasting and detected the PK2 mRNA expression throughout the brain using in situ hybridization. Under ad lib feeding conditions, PK2 was barely detectable in the PVN (Fig. 1A); however, the expression of PK2 was significantly induced in the PVN after fasting (Fig. 1B). The induction occurred as early as 4 h after fasting (Fig. 1C). The fasting-induced PK2 expression was restricted to the PVN as no significant induction was found in the other brain areas (data not shown). When food pellets were supplied to the fasted mice (e.g. re-feeding), the PK2 level in the PVN rapidly fell to the basal undetectable level within 4 h (Fig. 1C). Moreover, 2-deoxy-D-glucose (2-DG), an inhibitor of glucose utilization, also rapidly induced PK2 expression in the PVN (Fig. 1D), implying that the induction was a response to the dwindling energy reserves.
2.3. Recording and analysis of sleep/wake 3.2. PK2 −/− mice displayed torpor upon fasting The electroencephalogram (EEG) and electromyogram (EMG) signals from mice were recorded and analyzed as described [7]. The mice were connected to a wire tether system (Plastics One, Roanoke, VA) for the collection of EEG and EMG signals. This swivel system allowed the animal unrestricted movement throughout the recording cage. After at least 5 days of adaptation to the recording environment, a 48-h baseline EEG/EMG recording was collected on a LD cycle with lights on at 7:00 AM and off at 7:00 PM. Mice were recorded concurrently in matched littermate pairs of PK2 −/− and WT mice. EEG/EMG signals were amplified using a Grass Telefactor Model 15LT with 15A94 amplifier (Grass Instruments, West Warwick, RI) and filtered (EEG: 0.3–100 Hz, EMG: 30–300 Hz) before being digitized at a sampling rate of 128 Hz and stored on a computer. 2.4. Core body temperature monitoring A radio transmitter device (G2 E-mitter; Mini-Mitter, Sunriver, OR) used to measure body temperature and locomotion activity simultaneously was implanted in the abdominal cavity by sterile technique under general anesthesia [19]. Body temperature and locomotion activity were recorded by a receiver board (ER-4000 energizer receiver; Mini-Mitter) underneath the cage and were stored in a
Torpor is generally defined as reduction in body temperature below 31 °C [20]. Previously, Jethaw et al. [12] reported a portion of PKR2 m/m mice showed spontaneous torpor under normal feeding conditions. However, no spontaneous torpor was observed in PK2 −/− mice under ad lib feeding and constant ambient temperature conditions (21 °C). We did not even observe torpor in ad lib-fed PK2 −/− mice at 4 °C (data not shown). Jethaw et al. [12] also reported that a severe torpor occurred in PKR2 m/m mice during fasting. Thus, we subjected PK2 −/− mice to fasting and monitored their body temperature using telemetries. Both WT and PK2 −/− mice showed a decreased body temperature during fasting; however, the body temperature of PK2 −/− mice was much lower (genotype: F (1) = 19.13, P b 0.0001, two-way ANOVA). PK2 −/− mice entered torpor after fasting, i.e. the core body temperature dropped below 30 °C, whereas WT mice were still able to keep the core body temperature above 33 °C (Fig. 2A). After prolonged fasting (> 36 h), WT mice displayed a transitional body temperature dipping close to 30 °C, whereas PK2 −/− mice showed a period of ~ 4 h of torpor, when the body temperature dropped to as low as 25 °C (Fig. 2A). Interestingly, most of the torpors occurred at the transition from the active phase to the inactive phase (night to day).
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Fig. 1. Induction of PK2 mRNA in PVN during fasting. (A, B) Autoradiograms depicted PK2 mRNA expression in PVN under normal feeding (A) or after 24 hour fasting (B). (C) Time course of PK2 mRNA expression in PVN during the 24 hour fasting or re-feeding after 24 hour fasting (n = 3–4 animals). (D) PK2 mRNA expression in PVN in mice treated with saline or 2-DG. Mice were returned to their home cages without food after injections. PK2 mRNA in PVN was significantly higher at 2 and 4 h after 2-DG injection (n = 3–4 animals, *P b 0.05, unpaired t test).
To see if the genotypic difference in core body temperature is due to the difference in activity, we also analyzed the locomotor activity during fasting simultaneously. Both WT and PK2 −/− mice showed increased locomotion upon fasting (especially at the first day of fasting), although PK2 −/− mice moved less than WT mice during the dark phase under fasting or normal feeding conditions (genotype: F (1) = 26.37, P b 0.0001, two-way ANOVA). Nevertheless, during the
period when PK2 −/− mice showed torpor (ZT20 to ZT4, ZT0 is defined as the time when light is turned on), no difference in activity was seen between WT and PK2 −/− mice (Fig. 2B). 3.3. Altered energy expenditure in PK2 −/− mice during fasting We also monitored the energy expenditure of WT and PK2 −/− mice during fasting with an indirect metabolic system. Consistent with the body temperature recording, the energy expenditure dropped in both WT and PK2 −/− mice during fasting as compared with ad lib feeding. However, during certain periods, especially at the end of dark period and beginning of light phase, the oxygen consumption in PK2 −/− mice was significantly lower than WT mice (genotype: F (1) = 4.548, P b 0.01, two-way ANOVA; Fig. 3A), suggesting lower energy expenditure in PK2 −/− mice. Mammals were able to mobilize lipid during fasting to keep the core body temperature relatively stable. Consistent with the reduced energy expenditure in PK2−/− mice during fasting, the body weight loss was significantly less in PK2−/− mice than WT controls (P b 0.01, unpaired t test) (Fig. 3B). 3.4. Absence of fasting-induced arousal in PK2 −/− mice
Fig. 2. Body temperature and locomotor activity during the 48 h of fasting. (A) Decrease in body temperature during fasting in both genotypes. Note the greater hypothermic response to fasting in the PK2−/− mice (n = 5 mice/genotype). (B) Induced locomotor activity in both genotypes during the 48 h of fasting. Note locomotor activity was lower in PK2−/− mice compared to the WT mice (n = 5 mice/genotype).
Animals respond to reduced food availability by becoming more wakeful and active, a phenomenon called fasting-induced arousal. To examine whether PK2 signaling is required for the adaptive arousal response to fasting, we recorded states of wakefulness, non-rapid eye movement (NREM) sleep, and rapid eye movement (REM) sleep during fasting. After baseline (fed ad lib) recording, animals were fasted for 36 h beginning with the onset of the dark phase. Analysis of sleep/wake states revealed significant increases of arousal in WT mice during fasting (wakefulness, F (1) = 17.28, P b 0.0001; NREM, F (1) = 42.52, P b 0.0001; two-way ANOVA treatment effect; Fig. 4A and B). In particular, there was a robust increase in arousal after 18 h of fasting. In contrast, PK2 −/− mice exhibited no appreciable increase in arousal during the entire fasting period (wakefulness, F
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Fig. 3. Energy expenditure in the both genotypes during the 48 h of fasting. (A) Oxygen consumption during fasting in both genotypes. Note that oxygen consumption was lower in PK2−/− mice (n = 6 mice/genotype). (B) Marked decrease in body weight in WT mice during the 24 h or 48 h of starvation compared to that of PK2−/− mice. n = 6 mice/genotype, **P b 0.05, unpaired t test.
(1) = 0.75, P = 0.3869; NREM, F (1) = 2.049, P = 0.1534; two-way ANOVA treatment effect; Fig. 4C and D). 3.5. Restricted food supply rescued the fasting-induced phenotypes in the WT but not in PK2 −/− mice To investigate whether restricted food supply can rescue the hypothermal phenotype in fasted animals, we provided a food pellet equal to 5% of their body weight everyday to the fasted WT and PK2 −/− mice. As shown in Fig. 5A, both WT and PK2 −/− mice showed a decreased body temperature during restricted food supply; however, the body temperature of PK2 −/− mice was much lower (Genotype: F (1) = 5.10, P b 0.0001, two-way ANOVA). The hypothermal phenotype was not likely a consequence of difference in locomotion, either (Fig. 5B). This amount of food significantly increased the core body temperature in WT mice compared to the WT mice under the fasting (treatment: F (1) = 8.213, P b 0.01, two-way ANOVA; Fig. 5C). However, the body temperature of PK2 −/− mice was almost identical to that of fasting (treatment: F (1) = 0.5366, P > 0.05, two-way ANOVA; Fig. 5D). Furthermore, restricted food supply resulted in significantly less body weight loss in WT mice as compared with fasting (P b 0.01); however, the body weight loss during the first day of restricted feeding was similar to that during fasting in PK2 −/− mice (Fig. 5E). The fact that restricted food pellet rescued the hypothermal and body weight drop in WT mice but not in PK2 −/− mice implied a deficit of food utilization in PK2 −/− mice during re-feeding period. 4. Discussion In the real world, animals always face food shortage and thus evolve adaptive strategies to survive these tough situations. During fasting, small laboratory animals such as mice become aroused and active so as to maintain body temperature in a normal range and be ready for food supply [21]. Once food is applied, mice ingested the food and utilized the food to compensate the body weight loss. In these processes, the regulatory peptide PK2 seems to play important roles. First, fasting significantly induced PK2 expression in the PVN, and this induction disappeared after re-feeding. Second, PK2 −/− mice failed to display the fasting-induced arousal. Third, PK2 −/−
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mice were ready to enter torpor after fasting. Last, food applied after fasting rescued the hypothermal phenotypes in WT mice but not in PK2 −/− mice, the possible explanation is that PK2 −/− mice were not as efficient as WT mice to utilize food. Metabolic adaptations to fasting include changes in fuel usage, increased metabolic efficiency, and minimization of energy expenditure [22]. Energy storage is important for the adaptation and the mice deficient in the adipose tissue readily enter torpor during fasting [23]. However, these mice usually exhaust all energy storage during short-time fasting. It is unlikely that the torpor in PK2 −/− mice was caused by alteration in adipose tissue as PK2 −/− mice displayed a slightly more fat than WT controls (data not shown). In contrast to the rapid body weight loss observed in adipose-deficient mice, PK2 −/− mice lost less body weight than WT controls. PK2 and PKR2 are a pair of ligand–receptor in the central nervous system (CNS). As both PK2−/− and PKR2−/− mice entered torpor during fasting, we favored the idea that the defect of PK2/PKR2 signaling in the CNS contributed to the torpor phenotypes. PKR2 is highly expressed in the forebrain involved in the regulation of body temperature, arousal as well as sensing of nutrients, such as the PVN and the dorsomedial hypothalamus (DMH) [6]. The PVN is a very crucial site within the forebrain that involved in the hormonal endocrine, neural control, sympathetic nerve activity, and body temperature regulation. The PVN could detect energy homeostasis and play roles in energy intake and energy expenditure [24,25]. There are thermosensitive neurons locating in the PVN, which can be activated when the core body temperature is altered. Thus, the PVN could sense the body temperature change through these neurons and make response by activating the sympathetic nerves system [26]. The PK2 receptor, PKR2, is expressed in the PVN and previous studies have reported that PK2 could influence the excitability of different subpopulations of PVN neurons, as magnocellular, parvocellular preautonomic, and parvocellular neuroendocrine neurons within the PVN [27,28]. A possible explanation was that PK2 facilitated the PVN to sense the hypothermia, activated the sympathetic nerve system which in turn led to increasing activity, expanding the wake periods, and using body energy storage. PK2 was induced in the PVN by fasting, implying a possible intra-PVN projection of PK2 within the PVN. Gardiner et al. [29] found a decrease in hypothalamic PK2 mRNA expression following fasting in rats. In our in situ hybridization experiment, we only see significant increase of PK2 mRNA in the PVN. As PK2 is also highly expressed in other hypothalamic areas, such as the SCN, the arcuate nucleus (Arc), and the medical preoptic area (MPA) [5,17], it is possible that there is change in these areas during fasting which we did not detect. The PKR2 is also highly expressed in the DMH, an important brain area involved in the body temperature and arousal regulation [30]. The DMH is known to heavily innervate the ventrolateral preoptic nucleus (VLPO) and has been shown to be a critical nucleus for arousal regulation in response to restricted feeding [31]. Therefore, PK2 might participate in fasting-induced arousal through the DMH– VLPO pathway. PK2 might be originated from the SCN, MPA or PVN and project to the DMH to participate arousal and/or body temperature regulation [30]. PK2 is reported to be an important output factor that transmits the circadian information from the circadian pacemaker SCN. In support of this, we and others have observed a reduced circadian rhythmicity in the mice deficient in either PK2 or PKR2 [6,16]. Interestingly, the circadian rhythms of body temperature, locomotion and metabolism and sleep–wake cycle are essentially intact during fasting (at least in the first 2-day fasting). Activity may be a major force to maintain the core body temperature during the active phase, while another switch to catabolize fat depots is turned on during the inactive phase so as to keep the core body temperature in a relatively normal range during the inactive phase. If the switch is disabled or did not respond correctly, energy expenditure does not occur to an extent, which causes the animals entering torpor. It is worthy to note that
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Fig. 4. Sleep–wake patterns in WT and PK2−/− mice. (A, C) Distribution of wake, non-rapid eye movement (NREM) sleep in 2-h intervals under a 12 h: 12 h light: dark (LD) cycle for WT (A) and PK2−/− (C) mice. (B, D) Amounts of sleep–wake states (wake, NREM, REM) in WT (B) and PK2−/− (D) mice. (n = 6 mice/genotype, **P b 0.01, unpaired t test).
the fasted PK2 −/− mice displayed torpor during the transition from active phase to inactive phase, when the PK2 in the SCN is expressed at the peak levels. It is likely that PK2 from the SCN may serve as the switch signal to turn on energy expenditure during the inactive phase. Our observations of hypothermia and torpor in PK2 −/− mice during fasting suggested that PK2 might play an important role in energy intake and expenditure. There was an intriguing hypothesis that
enhancement of PK2 signaling in PVN during fasting might help mice to endure the tough situation via alternation to fat catabolism and maintaining body temperature. Acknowledgments This work was supported by the Projects in the Major State Basic Research Development Program of China (973 Program) (2012CB517904),
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Fig. 5. Different responses to fasting (F) and to food restriction (FR) in PK2−/− mice and WT mice. (A) Decrease in body temperature during the food restriction in WT mice and PK2−/− mice (n = 5 mice/genotype). (B) Locomotor activity during the food restriction in WT mice and PK2−/− mice (n = 5 mice/genotype). (C, D) Body temperature during fasting and food restriction in WT mice or PK2−/− mice (n = 5 mice/genotype). (E) Decrease of body weight during the 48 h of fasting or 48 h of food restriction in both genotypes (n = 6 mice/genotype, *P b 0.05, **P b 0.01, unpaired t test).
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