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Am J Physiol Regul Integr Comp Physiol 294: R1968-R1979, 2008. First published April 16, 2008; doi:10.1152/ajpregu.00778.2007
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SLEEP AND TEMPERATURE REGULATION

Loss of prokineticin receptor 2 signaling predisposes mice to torpor

Preeti H. Jethwa,1 Helen I'Anson,2 Amy Warner,1 Hayden M. Prosser,3 Michael H. Hastings,4 Elizabeth S. Maywood,4 and Francis J. P. Ebling1

1School of Biomedical Sciences, University of Nottingham Medical School, Nottingham; 2Department of Biology, Washington and Lee University, Lexington, Virginia; 3The Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge; and 4Medical Research Council Laboratory of Molecular Biology, Neurobiology Division, Cambridge, United Kingdom

Submitted 25 October 2007 ; accepted in final form 10 April 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The genes encoding prokineticin 2 polypeptide (Prok2) and its cognate receptor (Prokr2/Gpcr73l1) are widely expressed in both the suprachiasmatic nucleus and its hypothalamic targets, and this signaling pathway has been implicated in the circadian regulation of behavior and physiology. We have previously observed that the targeted null mutation of Prokr2 disrupts circadian coordination of cycles of locomotor activity and thermoregulation. We have now observed spontaneous but sporadic bouts of torpor in the majority of these transgenic mice lacking Prokr2 signaling. During these torpor bouts, which lasted for up to 8 h, body temperature and locomotor activity decreased markedly. Oxygen consumption and carbon dioxide production also decreased, and there was a decrease in respiratory quotient. These spontaneous torpor bouts generally began toward the end of the dark phase or in the early light phase when the mice were maintained on a 12:12-h light-dark cycle and persisted when mice were exposed to continuous darkness. Periods of food deprivation (16–24 h) induced a substantial decrease in body temperature in all mice, but the duration and depth of hypothermia was significantly greater in mice lacking Prokr2 signaling compared with heterozygous and wild-type littermates. Likewise, when tested in metabolic cages, food deprivation produced greater decreases in oxygen consumption and carbon dioxide production in the transgenic mice than controls. We conclude that Prokr2 signaling plays a role in hypothalamic regulation of energy balance, and loss of this pathway results in physiological and behavioral responses normally only detected when mice are in negative energy balance.

metabolic rate; thermoregulation; CLAMS; radiotelemetry

PROKINETICINS ARE SMALL, SECRETED proteins (Prok1 and Prok2) that share ~44% amino acid sequence identity with a highly conserved NH2 terminus (15). These prokineticins are thought to influence a wide variety of physiological processes both in the central nervous system and in peripheral tissues, including intestinal contraction, hyperalgesia, spermatogenesis, neuronal survival, circadian rhythmicity, angiogensis, ingestive behavior, and hematopoesis (for reviews, see Refs. 17 and 31). These proteins are the cognate ligands for two closely related G protein-coupled receptors (Prokr1 and Prokr2) which share 87% amino acid sequence identity and exhibit the greatest differences in the NH2-terminal sequences (16, 25). There are differences in the distribution of these receptors: Prokr1 is mainly located in peripheral tissues, while Prokr2 is predominantly expressed in the brain (16), particularly in the suprachiasmatic nucleus (SCN) (19) but also in many other hypothalamic regions (6). The expression pattern of Prok2 in the SCN is highly circadian, and intracerebroventricular treatment of rats with Prok2 inhibits nocturnal locomotor activity (4, 5). It has, therefore, been proposed that Prok2 functions as a humoral output signal communicating circadian information from the SCN to regions of the brain controlling motor output (4–6).

In support of this hypothesized role for Prok2 as a circadian output signal from the SCN, we and others have generated mice in which the genes encoding either Prok2 (14) or Prokr2 (20, 23) are mutated and rendered dysfunctional. The common phenotypic feature of targeted ablation of either the ligand or receptor was that the amplitude of circadian rhythms of body temperature and locomotor activity was decreased (14, 23), and there were also changes in the profile of the rhythm of body temperature such that the duration of the nocturnal elevation in body temperature was shorter in mice lacking Prok2 signaling compared with control mice (23). In mice expressing the mutated Prokr2 gene, we also occasionally observed very low light phase body temperatures that appeared to be bouts of torpor. Torpor bouts are not routinely observed in laboratory strains of mice maintained on ad libitum feed at constant temperature, so this phenotypic trait is indicative of altered energy metabolism. The purpose of the present study was, therefore, to carry out observations for an extended period of time to characterize further these torpor bouts, and to determine whether changes in energy intake or expenditure might contribute to the occurrence of the periods of hypothermia.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. All in vivo experimental procedures were approved by the University of Nottingham Local Ethical Review Committee and were carried out in accordance with the Animals Scientific Procedures Act (United Kingdom) 1986. Male and female transgenic mice were generated as previously described (23), thus carried functionally null alleles for the Prokr2 gene (prokr2Brdm1), henceforth abbreviated as m. Homozygous mutant mice (Prokr2m/m) and their littermates (Prokr2+/+ and Prokr2+/m) were obtained as adults from The Wellcome Trust Sanger Institute (Hinxton, UK), and studies were carried out in two separate batches starting 8 mo apart. All studies were carried out on both batches of mice using the same protocols. The first batch consisted of four males (2 Prokr2m/m mice and 2 littermates) and four females (also 2 Prokr2m/m and 2 littermates). Studies 1–4 (see below) were carried out sequentially in these mice. The second batch consisted of five males (2 Prokr2m/m mice and 3 littermates) and three females (2 Prokr2m/m and 1 littermate), but one Prokr2m/m female died before the various studies could be completed so data from this mouse have been excluded from analysis. Studies 3 and 4 were carried out initially in the second batch of mice, and then studies 1 and 2 where conducted. The mice were housed in individual cages under controlled temperature (21 ± 1°C) and on a photocycle of 12:12-h light-dark cycle (12L:12D); lights off at 17:00 h with ad libitum access to food intake and water unless stated otherwise. A dim red light (<10 lux) was present continuously during the dark phase (DD). Studies did not commence until 2 wk after transport from Hinxton to allow a habituation period, at which point the mice were aged between ~4 and 8 mo (Table 1).


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  		Table 1. Age, body weight, and tissues weight of the study population

 
Radiotelemetry. Mice were anesthetized with a mixture of ketamine (Vetalar 100 mg/kg ip; Fort Dodge Animal Health, Southampton, UK) and medetomidine (Dormitor 1 mg/kg ip; Pfizer, Kent, UK) in a ratio of 1:4. Analgesia was maintained via subcutaneous injection of carprofen (50 mg/kg Rimadyl; Pfizer, Kent, UK) and administered before surgery. Telemetry devices (TA10TA-F20; Data Sciences International, St Paul, MN) were placed in the peritoneal cavity of the mice. Following surgery, the animals were given the anesthetic reversal atipamezole (1 mg/kg ip Antisedan; Pfizer, Kent, UK) and also 0.9% saline (10 µl/g body wt sc). Signals from the radiotelemetry implants were detected using RPC-1 receivers and processed using ART version 3.1 silver software (Data Sciences International).

Study 1: effects of Prokr2 gene mutation on body temperature rhythms in entrained and free-running conditions. Body temperature was monitored in all ad libitum-fed Prokr2+/+, Prokr2+/m, and Prokr2m/m mice for 10 s in every 2 min over a period of ~4 wk in a 12:12-h light-dark cycle (12L:12D). The photoperiod was then switched to DD, i.e., continual dim red light at the same intensity as during the dark phase of the 12L:12D photocycle, and mice were monitored for a further 3 wk.

Study 2: effects of food deprivation on body temperature in Prokr2m/m mice. After the evaluations of body temperature rhythms in 12L:12D and in DD, the mice bearing radiotelemetry implants were returned to a 12L:12D photocycle for 14 days and maintained on ad libitum feed prior to starting this study. Access to food but not water was then completely removed from mice just before lights out and replaced 16 h later. Body temperature was monitored throughout this study as described in study 1. After a further 7 days on ad libitum feed, access to food but not water was completely removed from mice 6 h before lights out, and replaced 24 h later, and body temperature was monitored for a further 7 days.

Study 3: feeding behavior and metabolic rate in Prokr2m/m mice. VO2, VCO2, locomotor activity, and various parameters of eating behavior (frequency and duration of feeding bouts, food consumption per bout, and total food intake) were measured using a Columbus Instruments Comprehensive Lab Animal Monitoring System (CLAMS; Linton Instrumentation, Linton, UK/Columbus Instruments, Columbus, OH) as previously described (11). This is a modified open-circuit calorimeter, and the configuration used consisted of eight chambers in which mice were studied individually. The chambers had feeders positioned in the middle of the cages, and the weight of each removal of food was recorded along with the timing and duration of this feeding bout. For subsequent analysis a "meal" was defined as a bout of food intake of >0.02 g. We operated the system with an air intake of 0.6 l/min/chamber and an extracted outflow of 0.4 l/min. The chambers have a volume of ~2.7 liters and thus get ~14 air changes/h. A sample of air was extracted from each chamber every 9 min for sequential analysis of CO2 and then O2; the room air (i.e., input air to the chambers) was similarly analyzed every 9 min. Water was provided by dropper bottles, but intake was not recorded. Activity was recorded when two or more consecutive infrared beams positioned ~2 cm apart were broken. All measurements were taken at an ambient temperature of 21–22°C. Metabolic gas values and feeding behaviors were monitored over a 96-h period in ad libitum-fed mice. For the purposes of data analysis and presentation, the first 12 h of data were discarded since the high activity levels and high VO2 indicated that the mice were incompletely habituated to the metabolic cages.

Study 4: effects of food deprivation on the metabolic rate of Prokr2m/m mice. Mice were placed in the CLAMS apparatus (as above, in study 3), but after a habituation period of at least 24 h beginning an hour before lights out food was withdrawn for 16 h. Drinking water was available at all times. Metabolic gases and activity were monitored over this period and for a further 24 h. Food intake was also assessed before and after the period of deprivation so that the effects of the loss of Prokr2 signaling on compensatory hyperphagia could be determined.

Autopsy. After the completion of studies, all mice were autopsied. For the first batch of animals this was 7 days after the last period of food deprivation, and for the mice in the second batch this was 28 days after the last period of food deprivation. Following euthanasia with pentobarbitoal sodium (Euthatal; Rhone Merieux, Harlow, UK), the reproductive tissues and surrounding fat depots were dissected, and their wet weight was recorded.

Data analysis. In all analyses of body temperature, a moving average was calculated in 1-h epochs based upon the values for 10-s intervals sampled every 2 min using the ART version 3.1 silver software (Data Sciences International). These data are generally presented in figures as representative individuals or as group mean ± SE. The analysis of mean nocturnal (i.e., mean for the 12-h dark phase) and mean diurnal (i.e., mean for the 12-h light phase) body temperatures was based on 28 days of data while mice were on ad libitum feed and entrained to 12L:12D; data for the first week after surgery were disregarded. Values were calculated for individual mice, and these were compared using a two-way ANOVA (effect of genotype vs. effect of gender; Prism 4.0; San Diego, CA). The nocturnal maximum and diurnal minimum values for each mouse over the 28-day period were compared using the same ANOVA model. The minimum values for the hourly temperature epochs during the periods of 16- or 24-h food deprivation were also compared using two-way ANOVA.

For analysis of data obtained from the CLAMS apparatus, mean values for VO2, VCO2, respiratory quotient (RQ), locomotor activity, and parameters of food intake were calculated for the 12-h light phase and the 12-h dark phase, and then analyzed using two-way ANOVA with genotype and gender as the main effects. For the analysis of the effects of food deprivation, the physiological parameters were calculated for 1-h time bins, and two-factor ANOVAs carried out with genotype and time (repeated measures) as the main factors because data from one female were lost due to technical failure, leaving data from just two female Prokr2m/m mice. Where a significant time x genotype interaction was identified, the effects of genotype at specific time points were revealed by post hoc Bonferroni tests (version 4.0; Prism, San Diego, CA).

A torpor bout was defined as such if the hourly mean body temperature fell below 33.0°C for two or more consecutive hours. The rationale for this is that the group mean ± SD minimum diurnal body temperature in both genders in wild-type mice was 34.8 ± 0.4°C (Table 2), thus 33.0°C represents a cut-off 4 SD below the minimum temperature that would be expected in an ad libitum-fed wild-type mouse. The criterion of 33.0°C for the onset of a torpor bout is more stringent than that which would be predicted by the model recently developed by Willis (29). The incidence of torpor bouts was compared between genotypes by a Fisher's exact probability test (version 4.0; Prism). For all analyses, data from Prokr2+/+and Prokr2+/m mice were considered as a single control group because preliminary analyses revealed no differences in any parameters between these genotypes. P < 0.05 was considered statistically significant in all analyses.


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  		Table 2. Analysis of core body temperature and incidence of torpor bouts

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Study 1: effects of Prokr2 gene mutation on body temperature rhythms in entrained and free-running conditions. The control mice (Prokr2+/+and Prokr2+/m) all displayed robust daily rhythms in body temperature while entrained to the 12L:12D photocycle, and clear circadian rhythms, which free ran when the mice were exposed to DD (Figs. 1, top and Fig. 2, top). The range in hourly mean body temperature varied by over 2°C (Figs. 1 and 2), and in females there was evidence of raised nocturnal temperatures at regular 5- or 6-day intervals, presumably reflecting cyclical estrus activity (Fig. 2, top 2 panels). Maximum and mean nocturnal body temperatures were significantly lower in Prokr2m/m mice of both sexes (Table 2), and correspondingly minimum and mean diurnal body temperatures were also significantly lower in Prokr2m/m mice (Table 2). The circadian organization of the temperature rhythms in Prokr2m/m mice was less defined. In general, a nocturnal increase in temperature occurred in such mice on 12L:12D, but the rise was truncated, i.e., returned to diurnal values long before the onset of the light phase. Strikingly, five of seven Prokr2m/m mice displayed bouts of torpor, i.e., decreases in body temperature below 33°C lasting 2 or more hours (Figs. 1 and 2, bottom 2 panels). Such torpor bouts were never observed in control mice (P < 0.005). These torpor bouts were sporadic in appearance, varied greatly in frequency between individual Prokr2m/m mice, and were not synchronized between individuals (Figs. 1 and 2). Three of four Prokr2m/m males displayed torpor bouts (e.g., see Fig. 1), the bouts occurring in these on average 6–10 days apart (Table 2), and two of three Prokr2m/m females displayed torpor bouts (Fig. 2), one at a very high frequency (Fig. 2, bottom), the other very infrequently (Fig. 2, middle). The propensity to display torpor did not correlate with the body weight of the individual mice, nor did it correlate with weight gain over the experimental period (data not shown). In general, the timing of the onset of individual torpor bouts was restricted to the late dark phase/early light phase (Fig. 3), though in some individuals the timing was far more precise (Fig. 3A) than in others (Fig. 3B). The duration of individual torpor bouts also varied within individuals (e.g., Fig. 3C) and between individuals (e.g., Fig. 3A vs. Fig. 3B), but in no individuals did the bouts last longer than 7 h (Fig. 3).


Figure 1
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  		Fig. 1. Representative body temperature (Tb) traces in male mice for 5 days at the end of a period of entrainment to a 12:12-h light-dark (12L:12D) photocycle and then for 15 days on continual dim red light (DD). Traces are shown for a Prokr2+/+ (top) and a Prokr2+/m mouse (2nd panel), and for two Prokr2m/m littermates (bottom 2 panels). Note the sporadic bouts of torpor in the Prokr2m/m males.

 

Figure 2
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  		Fig. 2. Representative body temperature traces in female mice for 5 days at the end of a period of entrainment to a 12L:12D photocycle and then for 15 days on DD. Traces are shown for a Prokr2+/+ (top) and a Prokr2+/m mouse (2nd panel) and for two Prokr2m/m littermates (bottom 2 panels). *Likely proestrous days. Note the single bout of torpor but disorganized circadian temperature profile in one Prokr2m/m female (3rd panel), and the almost daily occurrence of torpor in the other Prokr2m/m female (bottom).

 

Figure 3
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  		Fig. 3. Examples of torpor bouts in individual Prokr2m/m mice on a 12L:12D photocycle. Note the similarity of profile and timing of the initiation of the torpor bouts in the late dark phase (black bar) in mouse A (top) compared with the more variable timing in mouse B (middle). In mouse C the onset is restricted to the late dark phase/early light phase (white bar), but the duration and depth of the torpor bout varies considerably.

 
Study 2: effects of food deprivation on body temperature in Prokr2m/m mice. Removal of food for 16 h (Fig. 4) or 24 h (Fig. 5) produced a marked decrease in diurnal body temperature in both the control and Prokr2m/m mice, but the magnitude of the hypothermia was significantly greater in the latter group (Table 2). In the study where 16 h of food deprivation was initiated shortly before lights off, mean body temperature in the control mice during the period of food deprivation descended to 31.9°C (males) and 29.5°C (females) compared with a mean of 34.8°C during the light phase while on ad libitum feed (Fig. 4, Table 2). In contrast, all Prokr2m/m mice entered torpor during the light phase when food deprived; mean body temperature fell to 22.4°C and 23.0°C for males and females, respectively (Fig. 4, Table 2). A similar outcome was observed in the study where 24-h food deprivation was initiated midway through the light phase (Fig. 5). Although the magnitude of hypothermia was somewhat less than in the 16-h study in both the control and Prokr2m/m mice (Table 2), there was an extended effect of the 24-h food deprivation such that despite being returned to ad libitum feed, the majority of both the male and female Prokr2m/m mice showed a second bout of hypothermia 24 h after the initial response (Fig. 5).


Figure 4
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  		Fig. 4. Body temperature in control (Prokr2+/+/Prokr2+/m) and Prokr2m/m mice on a 12L:12D photocycle subject to a 16-h period of food deprivation (FR) beginning at hour 35 (hatched bar). Values are group mean ± SE, n = 5 control and n = 4 Prokr2m/m males (top), and n = 3 for both control and Prokr2m/m females (bottom). Note the lower body temperature in the Prokr2m/m mice compared with the controls both before and after the food deprivation, and the far greater hypothermic response to food deprivation.

 

Figure 5
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  		Fig. 5. Body temperature in control (Prokr2+/+/Prokr2+/m) and Prokr2m/m mice on a 12L:12D photocycle subject to a 24-h period of FR beginning at hour 30 (hatched bar). Values are group mean ± SE, n = 5 control and n = 4 Prokr2m/m males (top), and n = 3 for both control and Prokr2m/m females (bottom). Note the greater hypothermic response to food deprivation in the Prokr2m/m mice and their second bout of hypothermia on the day that they are returned to ad libitum diet.

 
Study 3: feeding behavior and metabolic rate in Prokr2m/m mice. Control male (Fig. 6, left) and female (Fig. 7, left) mice displayed robust 24-h variations in VO2, VCO2, RQ, and feeding behavior while entrained to 12L:12D photocycle. The Prokr2m/m mice generally had less clearly defined circadian patterns of metabolic gas exchange (e.g., Fig. 7, right), and in four of seven Prokr2m/m mice we observed one or more bouts of torpor during housing in the CLAMS cages (examples shown in Figs. 6, right and 7, right). During these bouts of torpor, VO2 dropped to below 200 ml·h–1·kg0.75, and RQ decreased to ~0.7 (Figs. 6 and 7). RQ values <0.7 occurred in some individual samples during torpor bouts (Fig. 6), but we consider these to be artefacts, because when VO2 and VCO2 are extremely low, the ratio of these is much more sensitive to the variance (error) and limited resolution of the oxygen and carbon dioxide detectors. For subsequent statistical analyses of the impact of the Prokr2m/m mutation, data from 24-h periods in which torpor bouts occurred were excluded from comparison. During these nontorpor cycles, the Prokr2m/m mice had a significantly lower VO2 and VCO2 than their littermate controls during both the light and dark phases of the 12L:12D photocycle (Table 3), but RQ did not differ between the genotypes (Table 3). The lower VO2 and VCO2 occurred in both male and female Prokr2m/m mice (Table 3). There was a high degree of variability in the amount of locomotor activity within groups (Table 3), but during the dark phase there were significantly greater levels of activity in female mice (Table 3), and in both sexes, significantly lower levels of activity in the Prokr2m/m mice (Table 3). During the light phase no significant effects of either genotype or gender on activity levels were detected (Table 3). Overall food intake was significantly lower in the Prokr2m/m mice in both the dark and light phases (Table 3). In the light phase there was a significant genotype x gender interaction suggesting that the decrease in food intake in mice only occurred in the females (Table 3). There were no significant differences in individual meal sizes between the genotypes, but there was a reduced frequency of meals during the light phase in the Prokr2m/m mice (Table 3).


Figure 6
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  		Fig. 6. Representative examples of VO2, VCO2, respiratory quotient, locomotor activity, and feeding bouts in a male wild-type mouse (left) and a male Prokr2m/m mouse (right). Mice were entrained to a 12L:12D photocycle. Values for metabolic gases and activity were collected in 9-min epochs; bouts of food intake were recorded in real time. Note the two bouts of torpor and the reduced amplitude of the rhythm of O2 uptake and CO2 production in the Prokr2m/m mouse.

 

Figure 7
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  		Fig. 7. Representative examples of VO2, VCO2, RQ, locomotor activity, and feeding bouts in a female Prokr2+/+ mouse (left) and a female Prokr2m/m mouse (right). Mice were entrained to a 12L:12D photocycle. Values for metabolic gases and activity were collected in 9-min epochs; bouts of food intake were recorded in real time. Note the 2 bouts of torpor and absence of clear rhythmicity of O2 uptake and CO2 production in the Prokr2m/m mouse.

 

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  		Table 3. Analysis of gas exchange and feeding behavior

 
Study 4: effects of food deprivation on metabolic rate in Prokr2m/m mice. Food deprivation beginning shortly before the onset of the dark phase caused a rapid decrease in VO2 and VCO2 in both groups of mice (Fig. 8; effect of time VO2: F = 11.9, P < 0.001 VCO2: F = 22.0, P < 0.001). The decreases in these parameters were significantly greater in the Prokr2m/m mice compared with their littermates (time x genotype interaction VO2: F = 8.0, P < 0.05, VCO2: F = 10.1, P < 0.05). Post hoc Bonferroni tests revealed that the significantly lower decreases in VO2 and VCO2 in the Prokr2m/m mice occurred after 11-h food deprivation during the last 3 h of the dark phase (Fig. 8). There was also a significant decrease in RQ in both groups (Fig. 8, effect of time F = 45.2, P < 0.0001), but this did not differ between the genotypes. There was no significant difference in the degree of locomotor activity between the two groups during food deprivation. Both groups showed a similar degree of compensatory hyperphagia when returned to the ad libitum food supply, the 24-h food intake in the Prokr2m/m mice increased by 41 ± 9% compared with the 24 h prior to food deprivation, and in the control littermates food intake correspondingly increased by 54 ± 7%.


Figure 8
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  		Fig. 8. Metabolic parameters in control mice (Prokr2+/+/Prokr2+/m, bullet) and Prokr2m/m mice ({circ}) entrained to a 12L:12D photocycle and subject a 16-h period of FR (hatched bar) beginning at just before onset of the dark phase; depiction of VO2, VCO2, RQ, and locomotor activity. Values are group mean ± SE, n = 8 control and n = 7 Prokr2m/m mice (both sexes combined). *P < 0.05 vs. control mice. Note that both VO2 and VCO2 are lower in the Prokr2m/m mice compared with controls and that all mice rapidly decrease RQ during food deprivation.

 
Body and organ weights. At the start of the study (mice aged ~5 mo) there was no overall effect of genotype on body weight, but there was a significant gender x genotype interaction, thus male Prokr2m/m mice weighed less than their littermate controls, whereas female Prokr2m/m mice weighed more (Table 1). At the end of the study these significant differences in body weight had been lost (Table 1), and there were no significant differences in intra-abdominal fat depot weights (Table 1). There were clear differences (P < 0.001) in the weight of the reproductive organs. Three of the four Prokr2m/m males had undeveloped testes (<10 mg paired weight), the other had testes of intermediate size (paired weight 98 mg vs. range 166–249 mg in wild-type males). Likewise, all the Prokr2m/m females had an undeveloped uterus (<20 mg, range for wild-types 88–326 mg).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The major finding of the present study is that targeted genetic disruption of Prokr2-mediated signaling predisposes mice to torpor, a transient hypometabolic state. The majority of Prokr2m/m mice displayed spontaneous bouts of torpor despite being maintained at room temperature (21–22°C) on ad libitum food, and collectively the Prokr2m/m mice displayed significantly greater hypothermic and hypometabolic responses when challenged with acute food deprivation. Although standard laboratory housing conditions of 21–22°C might be considered a mild thermal stress, as this temperature is below the thermoneutral zone for mice (22), none of the heterozygote or wild-type littermates (controls) housed in these same conditions showed spontaneous torpor bouts. Similarly, as expected, while all control mice showed a depression of body temperature and VO2 during acute food deprivation, these physiological responses were significantly less than those seen in the Prokr2m/m mice. Torpor is a physiological tactic that has evolved in small mammals as a means to reduce energy expenditure during metabolic stress, the main situations being exposure to cold ambient temperatures or severe food shortage. Many small seasonal rodents have evolved the capacity to enter torpor spontaneously in anticipation of metabolically challenging conditions, for example short winter photoperiods will induce spontaneous bouts of torpor in Siberian hamsters, Phodopus sungorus (26, 28) and in several species of wild mice (7, 17). Moreover, within the 24-h light-dark cycle, circadian mechanisms play a key role in timing each individual torpor bout; ablation of the hypothalamic SCN results in random timing of torpor bouts in food-restricted hamsters (24). However, in laboratory strains of mice, torpor has only been observed as a response to cold exposure (22), food deprivation (10), or to hormonal or pharmacological manipulations (8). The occurrence of spontaneous torpor bouts in Prokr2m/m mice therefore strongly implicates Prokr2-mediated signaling in the control of energy metabolism and thermoregulation, although the level at which this occurs is not yet known. It was noticeable that the timing of the onset of these torpor bouts generally occurred toward the end of the dark phase or in the early light phase, consistent with previous observations that the underlying circadian system functions normally in Prokr2m/m mice, despite some deficits in circadian output mechanisms (4, 23).

We can exclude the possibility that the occurrence of spontaneous torpor reflects an abnormality in sensing of ambient temperature in the Prokr2m/m mice because we saw no other signs of cold stress, for example shivering or piloerection. We also consider it unlikely that the impaired reproductive development and function clearly evident in the mice used in the present studies [and evident in other mutant mice with impaired Prokr2 signaling (20)] would predispose these mice to torpor. While it is well established that high levels of circulating androgens can block the occurrence of torpor in hamsters exposed to short photoperiods (1), the opposite is not the case. Removal of testosterone from hamsters by means of surgical castration did not induce torpor bouts (28). Moreover, we have conducted studies in another strain of mutant mice that are hypogonadal (3) and never observed spontaneous torpor bouts in such mice (Ebling FJP, unpublished data), so loss of bioactive gonadotropin-releasing hormone per se is unlikely to contribute to the prevalence of torpor.

As torpor in mice occurs as a compensatory response to negative energy balance it is possible that Prokr2m/m mice have a deficiency in nutrient absorption from the gastrointestinal tract, particularly since prokineticins are expressed in the gut and act as potent stimulators of smooth muscle in the ileum (15). However, there are several lines of evidence that would argue against this. First, there is very limited expression of Prokr2 in the gastrointestinal tract, its expression being restricted just to the ilium-cecum junction, whereas Prokr1 is expressed widely in the stomach and small and large intestine (16). Second, one would predict that mice with impaired nutrient absorption would show a compensatory increase in food intake; however, we observed that food intake was reduced in the Prokr2m/m mice rather than increased. Third, one might expect some degree of growth impairment and reduced body weight if nutrient absorption were impaired, but we observed at the end of this study that body weight and abdominal fat depots were similar in Prokr2m/m mice and their intact littermates. In other cohorts of transgenic and littermate control mice we also noted similar levels of adiposity and body weight (Ebling FJP and Prosser HM, unpublished observation). Any early developmental effects of the mutation on growth could result from impaired olfaction and failure to locate and compete for maternal milk or may reflect the abnormally low sex steroids concentrations in the mutant mice (20, 21). Finally, if gut absorption were impaired, one might expect a lower respiratory quotient in Prokr2m/m mice reflecting greater reliance on catabolism of fat than carbohydrate. We did not observe this, despite observing an overall reduction in metabolic rate (i.e., lower VO2 and VCO2).

We consider it likely that the propensity of Prokr2m/m mice to enter torpor reflects an alteration in energy sensing and/or control of energy metabolism in hypothalamic and brain stem structures. Recent studies in the mouse indicate that Prokr2 gene expression is abundant in several structures known to be important in sensing of nutrients and peripheral endocrine signals (arcuate nucleus, area postrema, nucleus tractus solitarius) and in regulation of food intake, including the paraventricular nucleus and dorsomedial nucleus of the hypothalamus (6). One interpretation of the present data is that the loss of Prokr2 signaling primarily causes a decrease in food intake, and compensatory strategies are therefore engaged to conserve energy expenditure. In support of this, we observed significantly lower food intake in Prokr2m/m mice, and infer that energy expenditure is decreased because even in cycles where torpor bouts did not occur, Prokr2m/m mice displayed a lower core body temperature and lower oxygen consumption across the circadian cycle compared with their littermates. Moreover, Prokr2m/m mice exhibited a greater response to food deprivation, and in the study where such mice were food deprived for 24 h we even observed a second bout of hypothermia 24 h after the initial response. Although we cannot rule out the possibility that this was simply a coincidental bout of spontaneous torpor, it seems more probable that the initial period of food deprivation produced a more extended period of negative energy balance such that the Prokr2m/m mice were more predisposed to hypothermia and thus displayed a second bout of torpor when the circadian phase was appropriate. We also observed in the study in the metabolic cages that the RQ tended to take longer to increase after refeeding in the Prokr2m/m mice compared with their littermates, despite the fact that they showed a comparable degree of hyperphagia after the food deprivation as did their control littermates, indicating that the period of food deprivation had induced a greater dependence upon catabolism of fat reserves. Previous studies in mice have demonstrated that the depth and duration of torpor bouts induced by food deprivation can be enhanced by additional treatment with ghrelin (8), so it will be important to determine whether Prokr2m/m mice might have increased circulating concentrations of ghrelin or an increased responsiveness to ghrelin, as this may be a key factor in their propensity to show torpor.

One interesting though speculative possibility is that whatever the precise nature of the underlying metabolic disturbance in Prokr2m/m mice, it arises from the disruption of circadian output in these mutants. There is recent evidence that Prok2 functions as a signal from the SCN communicating circadian rhythmicity to Prokr2-expressing hypothalamic regions; loss of this signaling pathway results in less precise timing of patterns of locomotor activity and in a reduced overall degree of activity, resulting in attenuated amplitude of rhythmicity (4, 23). A relationship between circadian dysfunction and metabolic abnormalities has been proposed recently (for a review, see Ref. 13). One line of evidence for this link comprises observations of altered insulin production, and glucose and lipid metabolism in humans (9) and laboratory animals (2) subjected to enforced changes in environmental light-dark cycles. In addition, metabolic dysfunction has been observed in mice where the molecular basis of circadian rhythm generation has been perturbed (12, 27). Interestingly, disruption of rhythm generation in such clock mutants was associated with hyperphagia and increased energy storage leading to obesity (27), whereas disruption of circadian communication and the consequent dampened amplitude of locomotor and body temperature rhythms in the present study appears to be associated with mild hypophagia and decreased energy expenditure. This is reminiscent of a recent study by Zhang et al. (30) where exposure of mice to constant darkness induced hypophagia and weight loss, decreases in blood glucose, and increases in circulating fatty acids; phenomena indicative of increased catabolism of fat reserves (30). However, torpor was not observed during the studies in DD (30), whereas in the present study, the propensity for torpor in the Prokr2m/m mice appears to have conserved energy expenditure such that catabolism of fat depots did not occur to an extent that would cause weight loss.

Perspectives and Significance

To date, the principal role of signaling via the Prok2 receptor in the hypothalamus has been considered to be the communication of circadian information. Our observations of hypothermia and spontaneous bouts of torpor in mice with disrupted Prokr2 signaling suggest an additional important role in sensing and control of energy balance. The loss of Prokr2 signaling predisposes the mice to a strategy to conserve energy; it is an intriguing hypothesis that this may be a consequence of an underlying attenuation of circadian rhythmicity. Our findings also raise the possibility that pharmacological enhancement of Prokr2 signaling would promote greater energy expenditure and be a potential therapeutic strategy for promotion and maintenance of body weight loss.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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REFERENCES
 
This research was supported by the Biotechnology and Biological Sciences Research Council, UK Project Grants BBS/B/10765 and BB/D525064/1 and an ISIS Fellowship (to H. I'Anson), by the Wellcome Trust (to H. M. Prosser), and by the Medical Research Council, Cambridge, UK (to M. H. Hastings, E. S. Maywood).


   ACKNOWLEDGMENTS
 
We thank the staff at Biomedical Sciences Support Unit, University of Nottingham, Nottingham, UK, for assistance with animal care. We thank Professor Allan Bradley for support with these studies.


   FOOTNOTES
 

Address for reprint requests and other correspondence: F. J. P. Ebling, School of Biomedical Sciences, Univ. of Nottingham Medical School, Queen's Medical Centre, Nottingham, NG7 2UH, UK (e-mail: fran.ebling{at}nottingham.ac.uk)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 

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