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J Appl Physiol 91: 603-612, 2001;
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Vol. 91, Issue 2, 603-612, August 2001

Hypoxic metabolic response of the golden-mantled ground squirrel

Renata C. H. Barros1, Mary E. Zimmer2, Luiz G. S. Branco3, and William K. Milsom2

1 Departamento de Fisiologia, Faculdade de Medicina de Ribeirão Preto, and 3 Departamento Morfologia, Estomatologia, and Fisiologia, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, 14040-904 Ribeirão Preto, São Paulo, Brazil; and 2 Department of Zoology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the magnitude of the hypoxic metabolic response in golden-mantled ground squirrels to determine whether the shift in thermoregulatory set point (Tset) and subsequent fall in body temperature (Tb) and metabolic rate observed in small mammals were greater in a species that routinely experiences hypoxic burrows and hibernates. We measured the effects of changing ambient temperature (Ta; 6-29°C) on metabolism (O2 consumption and CO2 production), Tb, ventilation, and heart rate in normoxia and hypoxia (7% O2). The magnitude of the hypoxia-induced falls in Tb and metabolism of the squirrels was larger than that of other rodents. Metabolic rate was not simply suppressed but was regulated to assist the initial fall in Tb and then acted to slow this fall and stabilize Tb at a new, lower level. When Ta was reduced during 7% O2, animals were able to maintain or elevate their metabolic rates, suggesting that O2 was not limiting. The slope of the relationship between temperature-corrected O2 consumption and Ta extrapolated to a Tset in hypoxia equals the actual Tb. The data suggest that Tset was proportionately related to Ta in hypoxia and that there was a shift from increasing ventilation to increasing O2 extraction as the primary strategy employed to meet increasing metabolic demands under hypoxia. The animals were neither hypothermic nor hypometabolic, as Tb and metabolic rate appeared to be tightly regulated at new but lower levels as a result of a coordinated hypoxic metabolic response.

body temperature set point; hypometabolism; ventilation; hypothermia


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MUCH EMPHASIS HAS BEEN PLACED on studies of the cardiorespiratory responses of mammals that serve to attenuate changes in O2 tissue supply during hypoxia. Thermoregulatory mechanisms that decrease O2 demand, however, may be just as important for homeostasis under these circumstances (9, 15). Hypoxia leads to a drop in body temperature (Tb) in newborns and adults of many species, including humans (10), and leads animals to select ambient temperatures (Ta) that favor Tb values below those seen in normoxia, indirectly suggesting that there has been a drop in the set point for thermoregulation (Tset) (19, 42). The simplest possibility of an inability to acquire sufficient O2 to defend a normal Tb seems extremely unlikely in view of the available literature, which contradicts this idea (14, 15, 35).

Maintenance of normal physiological function in mammals, however, usually requires a constant Tb of 37°C. Lowering Tb 10-15°C for an extended period of time can lead to hypothermic dysfunction of several organ systems, which can result in death (cf. Ref. 2). Thus reductions in Tb that serve to reduce O2 demands under hypoxic conditions must normally be restricted by the capacity of the system to tolerate such decreases in Tb and rates of energy production. An exception is seen in hibernating mammals. These animals can decrease their Tb to 2-7°C, reduce their heart rate (HR) from 300 to 2-10 beats/min, reduce their O2 consumption (VO2) as much as 50-fold, and start breathing episodically during hibernation (cf. Refs. 2, 25). These amazing alterations of whole animal physiology are completely reversible and represent an adaptation to conserve energy reserves during extended periods of severe changes in climate and food availability.

As with most hibernating animals, the golden-mantled ground squirrel Spermophilus lateralis is also fossorial, living much of its time in a hypoxic, hypercarbic environment (26). The fractional concentration of O2 of burrow gas is typically ~16% but may be as low as 10% (22). Considering that this animal has the ability to reduce its Tb drastically under some conditions and that it faces hypoxia in its natural environment, one might hypothesize that this species should produce a well-regulated decrease in Tb during hypoxia as a physiological adaptation serving to attenuate such energetically costly responses to hypoxia as increases in cardiac output and ventilation.

The goal of this study was to test this hypothesis by examining the relationship between metabolism and Ta during normoxia and hypoxia. We wished to test the hypothesis that hypoxia in squirrels decreases the Tset and, in doing so, reduces thermal conductance and the lower critical limit of the thermoneutral zone. We also wished to assess the influence of such a regulated drop in Tb during hypoxia on cardiorespiratory control.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals. Experiments were performed on adult golden-mantled ground squirrels Spermophilus lateralis (Rodentia: Sciuridae) during the nonhibernating season (April-August). Animals were purchased from a collector in Redding, CA, housed in a controlled-environment chamber at an Ta of 21 ± 1°C under a 12:12-h light-dark cycle photoperiod, and fed laboratory chow supplemented with sunflower seeds ad libitum.

The study consisted of measuring VO2 and Tb in animals at different Ta, first during normoxia (breathing 21% inspired O2) and a week later during hypoxia (breathing 7% inspired O2). Measurements of pulmonary ventilation (VE), HR, and CO2 production (VCO2), as well as electrical activity from the cortex [electroencephalogram (EEG)] and a postural muscle [electromyogram (EMG)], were also obtained simultaneously.

Surgical procedures. Animals (241 ± 25.0 g) were anesthetized with pentobarbital sodium (Somnotol, 6.5 mg/100 g) administered intraperitoneally. Incision sites were shaved, treated with a depilatory cream, thoroughly cleaned with distilled water, and sterilized with ethanol. Each animal was submitted to a paramedian laparotomy, and a catheter (PE-50 tubing) with a blind end was chronically implanted in the peritoneal cavity. The external (open) portion of the catheter was passed under the skin and emerged at the interscapular area of the animal. The wound was then closed, and the implanted catheter was used for the insertion of a thermocouple to measure Tb during experiments. Subsequently, animals were placed in a Kopf stereotaxic device. A longitudinal incision was performed from the top of the head to the interscapular area for insertion of several electrodes. Four stainless steel EEG electrodes were implanted in the surface of the skull, one above each frontal and each occipital lobe, to monitor cerebral activity. Two EMG electrodes were sewn into the dorsal neck musculature (anterior trapezius) to monitor shivering thermogenesis. Two electrocardiogram (ECG) electrodes were sutured to the muscle wall on either side of the rib cage to measure HR. The wire leads from all eight electrodes were connected to amphenol pin strips that were affixed upright on the skull surface with dental acrylic. The wound was then closed around the headpiece and treated with a topical antibiotic (Flamazine, 2% silver sulfadiazene). Experiments were initiated 1 wk after surgery.

VE measurements. VE was measured by using the whole body plethysmographic (barometric) technique in an open-flow system (21, 25, 29). The plethysmograph consisted of two identical Plexiglas 1-liter chambers, with one being the animal chamber and the other the reference chamber. The flow of the gas passing through both chambers was controlled by a gas-mixing flowmeter (model GF-3/MP, Cameron Instrument) and set at a rate of 1,000 ml/min. Excurrent flow was also controlled by a flowmeter to ensure an equal flow of 500 ml/min in each chamber. The pressure signal was detected by a differential pressure transducer (model DP103-18, Validyne), amplified (model 7P122E, low-level direct-current amplifier, Grass Instruments), and recorded on a chart recorder. The system was calibrated dynamically as described by McArthur and Milsom (25). Two respiratory variables were measured, breathing frequency (f) and tidal volume (VT), from which pulmonary VE was calculated. Respiratory variables are reported at BTPS.

VO2 and VCO2 measurements. Concentrations of O2 (%O2) and of CO2 (%CO2) were measured in the outflow gas from the open-flow system using a polarographic O2 analyzer (model OM11, Beckman) and an infrared CO2 analyzer (model LB2, Beckman), respectively. These analyzers were frequently calibrated with gases that had been prepared by a precision gas-mixing flowmeter (model GF-3/MP, Cameron Instrument). The respirometer consisted of a Plexiglas 1-liter chamber (animal chamber). The flow of gas passing through the chamber was monitored by a flowmeter and set at a rate of 500 ml/min. Excurrent flow was passed through a drying column (Drierite) before entering the cells of the CO2 analyzer and then passed through a CO2 scavenger before entering the O2 analyzer. VO2 was calculated by subtracting the fractional concentration of O2 in the outflow gas from that in the inflow gas and multiplying by the constant gas flow through the chamber. The VCO2 was calculated by subtracting the fractional concentration of CO2 in the inflow gas from that in the outflow gas and multiplying by the constant gas flow through the chamber. Barometric pressure and chamber temperature were used to convert measures to STPD.

From the VE and metabolic data, O2 delivery, O2 extraction, and the air convection requirement (ACR; or ventilatory equivalent) were calculated. O2 delivery was calculated as the alveolar ventilation [VE - dead space ventilation (VDS)] multiplied by the fractional concentration of O2 in inspired air. This was calculated from VE, f, VT, and VDS [the latter taken from Milsom and Reid (27)]. Knowing the amount of O2 delivered to the gas exchange surface and the metabolic rate or amount consumed, we calculated the percentage of O2 that must have been extracted from the respired gas by dividing VO2 by the O2 delivery and multiplying by 100. For these calculations, all values had to be converted into STPD.

Protocol. Each squirrel was placed in the animal chamber flushed with normoxic air at an Ta of 25°C. The animal chamber was set inside an environmental chamber to allow measurements at different Ta. The contact pins in the animal's headpiece were connected to long wire leads, and a thermocouple was inserted into the peritoneal catheter. All leads were then fed out of the chamber. The EEG, EMG, and ECG signals were amplified (model 7P511K, Grass Instruments), recorded on a polygraph (model 79E, Grass Instruments), and recorded along with VE and the metabolic variables on a computer data-acquisition system (WYNDAQ, DATAQ Instruments) sampling at 100 Hz per channel. The thermocouple was connected to a digital monitor (Sensortek). The animals were then left in the animal chamber for a stabilization period of at least 30 min. After the animal had settled down, control measurements at 25°C were initiated. During the next 3 h, Ta was gradually increased to 29°C and then decreased to 6°C, in such a way that the animal stayed at least 10 min at an Ta of 29, 27, 25, 23, 21, 19, 17, 15, 13, 10, 8, and 6°C. The identical protocol was followed during hypoxia, except that each animal was exposed to hypoxic poikilocapnic air (7% inspired O2) for at least 3 h before the beginning of the gradual changes in Ta and that Ta was only decreased from the starting temperature of 25°C. This period of time was chosen because it was the time necessary to achieve a new and stable value of VO2 and Tb during hypoxia. Thus the total period of hypoxic exposure was ~6 h. Normoxic and hypoxic experiments were performed on the same animal within a 1-wk interval. Sleep states were identified from the EEG and EMG recordings, and only data obtained during periods of quiet wakefulness were analyzed. Changes in state between wake and sleep can alter metabolism by as much as 50% in this species (based on unpublished results and as we observed during our experiments), and we wished to control for such confounding influences. Recording sessions lasted between 4 and 7 h and were conducted during the same period each day to control for possible circadian changes.

Statistical analysis. Values in this study are given as means ± SE (unless otherwise stated). The effects of hypoxia over time on VO2, Tb, VCO2, VT, f, VE, and HR were evaluated using a one-way ANOVA. The Tukey-Kramer multiple-comparisons test was applied as a post hoc test. Comparison of the effects of different levels of Ta vs. 25°C and of hypoxia vs. air on VO2, Tb, VCO2, VT, f, VE, and HR was assessed using a two-way ANOVA. P values < 0.05 were assumed to be significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Hypoxia at constant Ta. Figure 1 illustrates the metabolic changes that occurred during 180-200 min of hypoxia in squirrels. This amount of time was necessary to achieve new and stable values of VO2, Tb, and VCO2. Whereas VO2 and VCO2 had stabilized at significantly reduced values after 1 h of 7% O2 exposure (Fig. 1, A and C), the fall in Tb only began to stabilize after 150 min (Fig. 1B). Note that, during the first 10 min, both VO2 and VCO2 had a tendency to increase, whereas Tb began to fall immediately.


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  		Fig. 1.   Effect of 180-200 min of hypoxia on O2 consumption (VO2; A), body temperature (Tb; B), and CO2 production (VCO2; C) of golden-mantled ground squirrels. Values are expressed as means ± SE (n = 7, 5, and 7, respectively). * Significant effect of hypoxia compared with normoxia, P < 0.05 (1-way ANOVA).

Chronic hypoxia caused cardiorespiratory changes in squirrels, as illustrated in Fig. 2. The change in f in response to hypoxia was biphasic (Fig. 2B); there was an initial increase followed by a decline back to resting levels. Whereas hypoxia did not elicit significant alterations in HR (Fig. 2D), HR tended to change in parallel with f. Whereas the changes in VT during 200 min of hypoxia were also not significant, VT tended to increase initially, then fall, and subsequently increase slowly after 1 h of 7% O2 exposure (Fig. 2A). The net result was that VE increased briefly and then immediately declined toward baseline values (Fig. 2C). Between the first and fourth hour of hypoxic exposure, the rise in VT and fall in f were roughly offsetting, and thus VE remained relatively constant.


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  		Fig. 2.   Effect of 180-200 min of hypoxia on tidal volume (VT; A), respiratory frequency (f, in breaths/min; B), ventilation (VE; C), and heart rate (HR, in beats/min; D). Values are expressed as means ± SE (n = 7, 7, 7, and 5, respectively). * Significant effect of hypoxia compared with control, P < 0.05 (1-way ANOVA).

These changes in metabolism and cardiorespiratory variables occurred concurrently with the fall in Tb. If the values for metabolic rate are corrected for the fall in Tb, assuming a constant ratio of change in VO2 by increasing 10°C in Tb (Q10) of either 2 or 3 (Fig. 3A), one sees that metabolism is elevated initially, then falls to or below starting levels, and then rises again. Thus metabolism appears to be suppressed only transiently after the first 10 min. After the first hour, metabolism falls only as fast (Q10 of 2) or slower (Q10 of 3) than would be predicted by the changes in Tb alone.


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  		Fig. 3.   Effect of 180-200 min of hypoxia on VO2 (A) and VE (B) with data presented as actual values () and as values corrected to a constant 38°C with a Q10 of 2 (triangle ) or 3 (black-triangle). Also shown are effects of 180-200 min of hypoxia on the air convection requirement [ratio of VE to VO2 (VE/VO2); C], as well as O2 extraction from lung gas (D).

The changes in the ACR [VE-to-VO2 ratio (VE/VO2)] (Fig. 3C) reveal that, at the onset of hypoxia, relative to metabolic rate, VE increases as would be expected during hypoxic exposure. This increase is not sustained, however, but falls again during the first hour. As a consequence, to sustain metabolism under these hypoxic conditions, O2 extraction from lung gas must increase (Fig. 3D) during the first hour. After the first hour, VE rises again relative to metabolism, and O2 extraction returns to near normal levels.

Hypoxia with changing Ta. Figure 4 illustrates the effects of changing Ta with and without hypoxia on the metabolism and Tb of squirrels. During air breathing, VO2 started to increase as Ta fell <23°C, and this increase became significant as Ta fell <15°C. The increase in VCO2 also became significant when Ta fell <13°C. Tb remained unchanged throughout.


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  		Fig. 4.   Effect of changing ambient temperature (Ta) during normoxia or hypoxia on VO2 (A), Tb (B), and VCO2 (C). Values are expressed as means ± SE (n = 7, 5, and 7, respectively). + Significant effect of hypoxia compared with normoxia, P < 0.05 (2-way ANOVA). * Significant effect of different Ta values compared with 25°C, P < 0.05 (2-way ANOVA). The dashed lines in B are linear regressions for the data, and the dotted line indicates the Ta at which these regressions intersect.

After ~3 h of 7% O2 exposure at 25°C, VO2, VCO2, and Tb had decreased to new steady-state values (Fig. 1). Then, as Ta was reduced during continued hypoxia, VO2 and VCO2 increased slowly. The increase in VO2 became significant only at 6°C, whereas the increase in VCO2 did not reach significance. Tb fell along with Ta, and this fall was significant <15°C Ta (Fig. 4).

The relationships of VO2 and VCO2 with Ta below the thermoneutral zone are roughly linear. The straight lines that describe these relationships in mammals should intersect the abscissa when Tb equals Ta, and, for animals breathing air, there is a good correlation between the values of these intercepts (37 and 40.5°C for VO2 and VCO2, respectively) and the measured Tb (38°C) (Fig. 5). Because Tb fell with decreasing Ta in the hypoxic animals, we again temperature corrected the data to analyze what the relationship between metabolism and Ta would have been had the Tb remained constant. This is shown in Fig. 5, C and D, for values of VO2 and VCO2 corrected to both 38 and 33°C, the Tb values of normoxic and hypoxic animals, respectively, at the starting Ta of 25°C. In both cases, the regression lines for the relationships between VO2 and VCO2 with Ta intersect the abscissa at 33°C, the Tb of the animals in hypoxia at the starting Ta, suggesting that this is their new, "effective" set point (at 25°C on 7% O2) for Tb regulation.


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  		Fig. 5.   Effect of changing Ta during normoxia on VO2 (A) and VCO2 (B). Also shown is effect of changing Ta during hypoxia on VO2 (C) and VCO2 (D) for actual values () and values corrected with a Q10 of 3 to a constant 33°C () or 38°C (black-triangle). Lines are linear regressions through all data, and intersects with the abscissa indicate thermoregulatory set point.

The effects of decreasing Ta on the cardiorespiratory responses of squirrels during normoxia and hypoxia are shown in Fig. 6. During air breathing, VE tended to increase in parallel with metabolism with decreasing Ta, but this was not significant. There was a significant increase in VT at 15 and 13°C; however, f did not change with decreasing Ta (Fig. 6, A, B, and C). After 3 h of chronic hypoxia, all respiratory variables had returned to near normoxic levels (Fig. 2), and subsequent changes in Ta had no significant effect on VE, VT, or f. HR was not significantly altered by changing Ta during normoxia or hypoxia (Fig. 6D). Figure 7 depicts the changes in O2 delivery {FIO2 * [f * (VT - VDS)], where FIO2 is the inspired fraction of O2} that accompany the changes in VE and ambient O2 levels during decreasing Ta in both normoxia and hypoxia, as well as the changes in O2 demand (i.e., VO2). In general, the changes in O2 supply do not parallel the changes in O2 demand, and this is also illustrated by the changes in ACR (VE/VO2) (Fig. 7C). The net result is that O2 extraction must rise as Ta falls, more so in hypoxia than normoxia (Fig. 7D).


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  		Fig. 6.   Effect of changing Ta during normoxia or hypoxia on VT (A), f (B; in breaths/min), VE (C), and HR (D; in beats/min). Values are expressed as means ± SE (n = 7, 7, 7, and 5, respectively). * Significant effect of different Ta values compared with 25°C, P < 0.05 (2-way ANOVA).



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  		Fig. 7.   Effect of changing Ta during normoxia and hypoxia on O2 delivery to the lungs (inspired O2 fraction * VE; A), VO2 (B), the air convection requirement (VE/VO2; C), as well as O2 extraction from the lung gas (D).

Figure 8 shows the EMG recording of one animal to illustrate the effects of Ta and hypoxia on shivering thermogenesis. Note that there was no increase in EMG activity in hypoxia, despite the reduction in Tb to 33°C. Exposure to decreasing Ta caused an increase in the muscular electrical activity (muscle tone) during air breathing but not during hypoxia, even at 6°C. However, at the end of the hypoxic exposure, with the onset of air breathing the animal showed a sharp increase in muscle tone, and shivering thermogenesis could be observed behaviorally. The time course of this increased shivering response was ~30 min.


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  		Fig. 8.   Electromyogram (EMG) recordings from individual squirrels illustrating the effects of cold (6°C) on muscular electrical activity during normoxia (top), hypoxia (bottom), as well as after the return to air during cold exposure (bottom).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

One major question that was addressed in this study was whether the hypoxia-induced decrease in metabolism and Tb seen in small mammals results from a regulated decrease in Tb set point or from substrate (O2) limitation to thermoregulatory effectors. Although many previous studies (4, 8, 9, 15, 19, 28, 35) have addressed this issue, and most indirectly suggest that there is a drop in Tb set point, and exclude the alternate hypothesis, the novelty of our study is in 1) choice of species, 2) experimental design, 3) completeness of the data set, and 4) the use of Q10-corrected data for interpreting the results. Considering this, the present data also strongly (but indirectly) support the suggestion that there is a drop in Tb set point based on several findings. 1) Metabolic rate is not simply suppressed but appears to be regulated to assist the initial fall in Tb but then act to slow this fall and stabilize Tb at a new, lower level. 2) If Ta is reduced during hypoxia, animals are able to maintain or elevate their metabolic rates, suggesting that O2 is not limiting. 3) The slope of the relationship between temperature-corrected VO2 and Ta extrapolates to an "effective" Tb set point in hypoxia equal to the actual Tb.

These data, along with those of the previous studies mentioned above, imply that these animals are neither hypothermic nor hypometabolic. Such terms imply that animals have Tb values and metabolic rates below their physiological set points, and this does not appear to be the case (i.e., "reduced" does not necessarily infer "hypo"). If the set points have indeed changed, then they are not below desired levels. Both appear to be tightly regulated at new but lower levels in what could best be called "the hypoxic metabolic response." A regulated reduction in Tb during chronic hypoxia will both reduce resting O2 demands and reduce the energetically costly increases in cardiac output and ventilation induced by acute hypoxia.

Effects of hypoxia on metabolic responses at constant Ta. Values obtained for metabolic and cardiorespiratory variables in control squirrels in the present study are generally consistent with those obtained in previous studies on this species and on other fossorial rodents (17, 25, 41). As expected, sustained hypoxia caused a drop in VO2, VCO2, and Tb. However, the magnitude of the hypoxia-induced falls in Tb and metabolic rate of adult golden-mantled ground squirrels was larger than that of adult Sprague-Dawley rats after steady-state levels were reached (~3 h) and considering similar experimental conditions such as level of hypoxia, exposure period, and Ta (31). This could reflect an increased exposure to hypoxic burrow conditions and may, at the appropriate time, by initiating a fall in metabolism and Tb, contribute significantly to the entrance into hibernation.

The kinetics of the initial changes reveal several things. First, Tb begins to fall immediately, whereas VO2 and VCO2 tend to rise for the first 15 min. The rising trend in metabolic rate most likely reflects the classic physiological response to acute hypoxia, characterized by cardiorespiratory responses (hyperventilation and increased cardiac output) that serve to attenuate changes in O2 supply (30, 33). The rise in VCO2 also reflects the hyperpnea associated with the hypoxic stimulus. For Tb to fall while this trend is occurring, however, the animals must have transferred enough heat to the environment to loose not only the heat generated by this elevated metabolism (~7% increase) but also some of the stored body heat. This is strongly suggestive that the animals were recruiting heat loss mechanisms to actively reduce Tb, an indication of a reduction in Tb set point. It is suggestive only, however, given that these changes, individually, are not significant and given the slow time constant for measures of changes in metabolism.

After the first 15 min, metabolic rate began to fall. Because VO2 fell rapidly during the remainder of the first hour whereas Tb continued to fall slowly, this change in metabolism could not result primarily from a Q10 effect of the change in Tb. It must represent an active metabolic reduction.

Both VO2 and VCO2 reached new steady-state levels by the end of the first hour. This drop in VO2 was at least 1 ml · min-1 · kg-1 larger compared with Sprague-Dawley rats (31). Tb continued to fall throughout the hypoxic exposure, likely due to the long time constant for passive heat exchange (30). Again, the magnitude of the drop in Tb in squirrels, once a new steady-state level was reached, was at least 2°C larger than that of nonfossorial rodents (31). However, it would be reasonable to expect metabolic rate to fall further during this period because of the fall in Tb. Maintenance of a stable VO2 after the first hour, while Tb continued to fall slowly, requires a relative increase in metabolism, and this must represent a reduction or removal of the active metabolic suppression. Recent studies (6, 31) suggest that more prolonged hypoxic exposure not only leads to removal of the metabolic suppression but may even lead to an increase in metabolism (6), but this would appear to be over a different time course and involving different mechanisms than those indicated here.

Both the initial metabolic suppression and subsequent relative increase in metabolism become clear when the data for metabolic rate are temperature corrected (Fig. 3A). Because Tb must have Q10 effects on metabolic processes, these complicate data interpretation. VO2 will be changing during this period for two sets of reasons: because of active processes suppressing metabolism, and because of passive temperature effects. Temperature correcting the data removes the latter effects, but to do so properly requires knowledge of the Q10 for these passive processes. It is well established that the Q10 for metabolic rate of the golden-mantled ground squirrel, even during stages of torpor or hibernation, is also between 2 and 3, the normal range for most chemical and physiological processes (16). When this correction was done, it would appear that, after the first hour, to regulate Tb, metabolism must not fall as fast as would be predicted from the changes in Tb alone. This suggests that metabolic rate is not simply suppressed by hypoxia but is regulated in an intricate fashion to assist the initial fall in Tb and then slow the rate at which Tb falls to its new level and maintain it there.

It has been suggested that the hypoxic reduction in metabolism may correspond to a drop in a "facultative" component of the normoxic VO2 (30). This is supported by studies demonstrating a drop in energy utilization, with no compensatory increase in glycolysis and lactate production during hypoxia (12, 32), and maintenance of biosynthetic reactions that do not utilize oxidative phosphorylation (34). Thus, on return to normoxia, there is no aerobic debt to repay (30). Further evidence that a reduction in Tb is a beneficial response to hypoxia in mammals includes observations that 1) a hypoxic reduction in Tb increases survival in rats (43), and that 2) a 1°C drop in brain temperatures in rats is adequate to prevent a fall in brain ATP levels at low O2 partial pressures (20 Torr) (5). Such benefits should be even more important to fossorial animals such as ground squirrels, which are more likely to encounter hypoxic environments. This regulated reduction in Tb during chronic hypoxia will reduce both resting O2 demands and the energetically costly increases in cardiac output and ventilation induced by acute hypoxia. Furthermore, by initiating a fall in metabolism and Tb at the appropriate time, this process may also contribute significantly to the entrance into hibernation. All such speculation, however, must be tempered by the recent observations noted above, suggesting that the hypoxic metabolic response is not sustained during more prolonged hypoxic exposure and may even be replaced by an increase in metabolism (6, 31). It will be interesting to see whether this is also true for small hibernating and fossorial species of mammals.

Effects of hypoxia on cardiorespiratory responses. The squirrels in the present study showed a biphasic ventilatory response to sustained hypoxia characterized by an immediate but brief increase in ventilation followed by a progressive decline toward baseline values. This response has been described especially in newborn and anesthetized mammals, although the time course in the present study is relatively slow (33, 38). It is interesting that a fossorial species that exhibits a decreased sensitivity to hypoxia and hypercarbia (3, 39) also demonstrates a biphasic ventilatory response (Fig. 2), suggesting that it may be adaptive in fossorial animals too. The initial rise in ventilation was due primarily to alterations in f, because VT fell initially, as already reported in a previous study on this species (41). It is not uncommon to see a fall in VT in association with the powerful increase in f during hypoxia. This is largely due to the removal of CO2 by the hyperpnea. After the first hour, VT slowly increases again, as the f falls, and CO2 levels presumably return to normal. Among the factors that are bound to have contributed to the shape of the f response in the present study are the classic short-term ventilatory depression (33), the fall in arterial PCO2 (PaCO2) due to the relative hyperventilation, and the falls in Tb and metabolic rate.

Interestingly, the changes in HR seemed to parallel the changes in f, although the changes were not significant. HR has been reported to be maintained or elevated during hypoxic exposure in other mammals also (cf. Ref. 7).

The changes in breathing pattern along with changes in the ratio of ventilation to metabolism (the ACR or ventilatory equivalent; VE/VO2) have significant consequences for O2 extraction. The ACR increased initially, reflecting the hyperpnea typical of the hypoxic ventilatory response and accounting for the increase in VCO2 relative to VO2. This ratio then fell as f and VT both fell faster than VO2, reflecting the biphasic ventilatory response. To meet the changing metabolic demands with these changes in breathing pattern, O2 extraction must have risen (due to both the hypoxia and the biphasic ventilatory response). The data indicate that, at its peak, O2 extraction from lung gas must have reached almost 50% (Fig. 3D). The ACR then rose again to a relatively high and somewhat stable value after 2 h, during which O2 extraction fell back close to starting values. At the end of the 3-h period, ventilation, VO2, and O2 extraction all seemed to have stabilized. Given the high level of VE/VO2 and VCO2, CO2 conduction must have decreased, suggesting the animals were maintaining a new but lower PaCO2 and alveolar-arterial CO2 difference [see also Rohlicek et al. (35)]. These data suggest that, just as during entrance into hibernation, the hypoxic metabolic response is associated with a regulated shift in PaCO2.

Effects of different Ta and hypoxia on metabolic responses. The relationship between Ta and metabolic rate (VO2 or VCO2) in the present study for animals breathing air (Fig. 5) suggests that the lower critical temperature for the thermoneutral zone of the golden-mantled ground squirrel is ~23°C. This is low for a small rodent and is in accordance with expectations for animals living in cold environments (37). Below this Ta, passive adjustments of heat loss through alterations in thermal conductance via vasomotor responses, postural changes, and regulation of effective insulation no longer appeared sufficient to maintain Tb. Maintenance of a constant Tb was now associated with a linear increase in metabolic rate. The slope of the relationship between Ta and VO2 below the thermoneutral zone is reflective of the thermal conductance of the animal (36). Down to the lowest Ta used in this study (6°C), normoxic animals were able to maintain Tb by roughly doubling VO2. This is somewhat greater than expected and may reflect the fact that these experiments were conducted in the summer period when the fat deposits and thermal insulation of these animals were not fully developed. It is widely maintained that the negative correlations describing the relationships between Ta and VO2 and VCO2 in mammals should intersect the abscissa where Ta equals Tb (36), and, for golden-mantled ground squirrels breathing air, there was a good correlation between the values of these intercepts (37 and 40.5°C for VO2 and VCO2, respectively) and the measured Tb (38°C) (Fig. 5). Others have not always found such tight correlations in rodents (17, 18).

The relationships between Ta and VO2 and VCO2 for hypoxic animals should also intersect the abscissa where Tb equals Ta. This assumes, however, that all data are collected at a constant Tb. For the golden-mantled ground squirrels in the present study, such analysis was confounded by the fact that, whereas Tb had stabilized after 3 h of hypoxia at constant Ta, it subsequently fell as Ta was reduced. Thus at each level of Ta, there was a new, unique relationship between Tb and VO2. To circumvent this problem, we temperature corrected the data to analyze what the changes in metabolic rate would be if temperature had remained constant (we corrected the data to the Tb of both animals breathing air and 7% O2 at 25°C) (Fig. 5, C and D). Such a correction is essential for this regression analysis to work, and when the data were temperature corrected to either constant Tb, the relationships did indeed intersect the abscissa at a value equal to the Tb (33°C) of animals at that Ta (i.e., at 25°C).

These observations raise several important points. First, VO2 increased as Ta fell, even when it was not temperature corrected, indicating that O2 was not limiting as a substrate as has also been suggested by others (1, 11, 12, 15, 35). Second, the animals appeared to be regulating their Tb at a new, effective set point, but a Tset that was proportionately related to Ta; the lower the Ta, the greater the reduction in Tset brought about by the hypoxia, as has also been shown by others (14, 35). Third, the slopes of the temperature-corrected data suggest that thermal conductance was not affected by hypoxia: there was simply a downward shift in the relationship between Ta and VO2. This is a theoretical abstraction, however, because Tb did fall with Ta and thus effective conductance was lowered by this dynamic process. Whereas the uncorrected data suggest that the metabolic threshold for thermogenesis was reduced to a lower Ta (i.e., the lower critical temperature of the thermoneutral zone was reduced), this is not so clear from the temperature-corrected data. In either case, however, the data do suggest that the thermoregulatory system was still functioning normally. Kuhnen et al. (23) have observed a shift in the thermogenic threshold in hypoxic hamsters. Finally, our data show that, whereas shivering thermogenesis was not present in hypoxic animals at the lowest levels of Ta used in this study, once the hypoxia was reversed, and presumably Tset was restored to normoxic levels, shivering thermogenesis was immediately instituted for rewarming, as previously reported in cats by Gautier et al. (15). The time course of the return of the shivering response from hypoxia to normoxia was so fast that ~30 min were sufficient to restore Tb to normal levels. Wistar rats need almost 1 h to reach basal Tb, even when exposed to only a 30-min period of hypoxia (7% O2) (4). This ability of the squirrels to produce heat rapidly is consistent with their ability to arouse from hibernation and elevate Tb rapidly from much lower levels. All of these observations are consistent with there having been a regulated change in Tset with no impairment of thermoregulatory capacity.

Effects of different Ta and hypoxia on cardiorespiratory responses. For animals breathing air, initially with the fall in Ta, ventilation and O2 delivery increased along with metabolism, primarily because of increases in VT. Similar results have been reported in other rodents (13, 20). The ACR was constant between 25 and 15°C. Neither respiratory rate nor HR changed significantly. Below 15°C, when the metabolic rate was increasing significantly, ventilation and O2 delivery were maintained constant, and thus VE/VO2 fell and the rise in VO2 arose from an increase in O2 extraction.

During hypoxia, there were no significant changes in ventilation (VT or f, and hence O2 delivery) or HR with the fall in Ta. These data are in accordance with the results reported by Maskrey (24) in rats, showing that, when body core cooling and hypoxia are delivered together, they combine to depress respiration. Because metabolic rate did increase as Ta fell, however, VE/VO2 fell and O2 extraction must have risen again. This was particularly significant for the hypoxic animals in which O2 extraction is estimated to have risen to >40%.

Interpretation of these data is difficult. It is not clear why increases in O2 extraction rather than ventilation appear to have been employed to match increases in O2 uptake and increases in metabolic rate. It is also not clear how the increase in O2 extraction arose because HR did not increase. Whereas such factors as increases in hematocrit, stroke volume, or Hb O2 affinity could contribute to such an increase, these possibilities remain to be explored. It is clear, however, that there has been a shift in the primary strategy employed to meet increasing metabolic demands under hypoxic conditions: from increasing ventilation to increasing O2 extraction.

Conclusions. The present study strongly suggests that hypoxia produces a decrease in the effective Tb set point but little change in thermogenic capacity in the golden-mantled ground squirrel. Metabolic rate was not simply suppressed but appeared to be regulated to assist the initial fall in Tb and then acted to slow this fall and stabilize Tb at a new, lower level. When Ta was reduced during hypoxia, animals were able to maintain or elevate their metabolic rates, suggesting that O2 was not limiting. The slope of the relationship between temperature-corrected VO2 and Ta extrapolated to an effective Tb set point in hypoxia equal to the actual Tb. Finally, the squirrels showed a biphasic ventilatory response to sustained hypoxia, leading to decreased demands for O2. The animals were neither hypothermic nor hypometabolic, as both Tb and metabolic rate appeared to be tightly regulated at new but lower levels as a result of a coordinated hypoxic metabolic response. This regulated reduction in Tb during hypoxia will reduce both resting O2 demands and the energetically costly increases in cardiac output and ventilation induced by acute hypoxia. The magnitude of the hypoxia-induced falls in Tb and metabolic rate of adult golden-mantled ground squirrels were larger than those expected for adult rodents. This perhaps reflects an increased exposure to hypoxic burrow conditions (over generations or during development) and may, at the appropriate time, by initiating a fall in metabolism and Tb, contribute significantly to the entrance into hibernation. Finally, there has been a shift from increasing ventilation to increasing O2 extraction as the primary strategy employed to meet increasing metabolic demands under hypoxic conditions for reasons that are not yet apparent.


    ACKNOWLEDGEMENTS

This work was supported by the National Sciences and Engineering Research Council of Canada, the Fundação de Amparo a Pesquisa do Estado de São Paolo (FAPESP), and the Conselho Nacional de Desenvolvimento Científico e Tecnológico. R. C. H. Barros was the recipient of a FAPESP (98/02993-5) postgraduate scholarship.


    FOOTNOTES

Address for reprint requests and other correspondence: R. C. H. Barros, Departamento de Morfologia, Estomatologia e Fisiologia, Faculdade de Odontologia de Ribeirão Preto, Universidade de São Paulo, Avenida do Café s/n°, 14040-904, Ribeirão Preto, SP, Brazil (E-mail: habenchu{at}rfi.fmrp.usp.br).

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.

Received 1 August 2000; accepted in final form 12 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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