~OUKNALOF
Vol.
38, No.
APPLIED
3,
March
PHYsIoLocy 1975. Printed
in U.S.A*
Nonshivering
thermogenesis
in rats under
severe cold
and cold conditions
0. HfiROUX, E. PAGk, J. LEBLANC, J. LEDUC, R. GILBERT, A. VILLEMAIRE, AND P, RIVEST Station Biologique de St, Hi&~~/yte, Unimx’te de Montreal, Montreal,
H~ROUX, 0, E. PAGI~, J. LEBLANC, J. LEDUC, R. GILBERT, A. VILLEMAIRE, AND P. RXVEST. Nonshivering thermogenesis and co/d resistance irz rats under se~cre cold conditions. J. Appl. Physiol. 38(3) : 436-442. 1975.-Following either chronic exposure to 6”C, or outdoor winter exposure, or chronic treatment with tyramine rats were exposed to - 40°C and their oxygen consumption and colonic temperature monitored. Fall in body temperature with time of exposure followed a sigmoid curve which had an inflection point around 32.9”C. Both the time required for body temperature to reach this point and hypothermic resistance defined as the total 02 consumed up to the inflection time were useful indices of resistance to severe cold. Three days before the cold tests, capacity for norepinephrine-induced nonshivering thermogenesis was measured in all animals by examination of their metabolic response to tyramine. The magnitude of response to tyramine correlated well with hypothermic resistance only for those rats chronically treated with tyramine. It is concluded that it is impossible to predict with any reasonable degree of confidence the cold resistance of a rat from its tyramine response. In cold-acclimated rats, factors in addition to norepinephrine sensitivity are significantly involved in cold resistance and deserve further studies. cold
acclimation;
cold
resistance
resistance
at - 40°C
THE FIRST OBSERVATIONS that chronic exposure of rats to moderately cold temperature was associated with the development of increased resistance to cold were made by Ogle and Mills (13) in 1933 and Gelineo (3) in 1934It then became apparent that this conditioning or acclimation to cold extended the lower limits of environmental temperatures at which peak metabolic rate was reached or at which body temperature or body weight could be maintained. Subsequent research revealed that apparently the most significant metabolic alteration which occurs during acclimation to cold is the development of a potential to produce heat by a new thermogenic mechanism called nonshivering thermogenesis (NST). This potential is expressed through the action of norepinephrine (NE) liberated by the sympathetic nervous system (8) and it has been shown that the capacity for NST can be measured by the metabolic response of the animal to exogenous NE
creased resistance of cold-acclimated rats to severe cold: a) during acclimation to cold, the gradual development of NST as indicated by both increased calorigenic response to NE and decreased shivering paralleling an increase in cold resistance (2); b) at very cold temperatures (e.g., -40°C) the metabolic rate of the cold-acclimated rat is greater than that of the warm-acclimated rat and the difference has been shown to be due to the ability of the former to produce heat by NST in addition to shivering (7). It has been stated, furthermore, that calorigenic response to NE may be used to measure the degree to which a rat has become cold-acclimated (9). Because increased cold resistance is associated with acclimation to cold it might then be assumed that the calorigenic response of a rat to NE could provide a good index of its cold resistance. But the validity of this assumption is questioned by the obscrvation that during deacclimation of the cold-acclimated rat, resistance to severe cold decreases faster than NE sensitivity (4, 5, 12). Furthermore, among cold-acclimated rats (6) the variability in cold resistance is much greater than the variability in NE sensitivity (8). This seems to suggest that the degree of cold resistance exhibited by an animal is a function not only of its capacity for NE-induced NST, but also of other factors. However, since cold resistance and NE-induced calorigenesis were not measured in the same group of animals, the relative significance of factors other than NST is not apparent. If NE-induced NST is primarily responsible for the increased cold resistance of cold-acclimated rats, one would expect the cold resistance and the capacity for NE-induced calorigenesis, each measured in the same animals, to be highly correlated. Verification of this would permit measurement of cold resistance by measurement of calorigenic response to NE. The latter test is preferable to and more practical than exposure of the animal to severe cold to measure its cold resistance in terms either of survival time or of rate of fall in body temperature. This communication presents results of various experiments designed to determine whether the most cold resistant rats are those that have the greatest NE-induced calorigenesis and therefore the greatest capacity for NST. EXPERIMENTAL
m
Quebec, Canada
CONDITIONS
(9)
Certain observations that NE-induced NST This paper
have led is primarily
has been designated
NRCC
investigators responsible 14502.
to believe for the in-
Animals,
Caging, Environmental
Conditions
Group I: Sprague-Dawley rats, 7 wk at 6°C (October). Twelve adult male Sprague-Dawley rats of an average body
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NONSHIVERING
THERMQGENESIS
AND
COLD
437
RESISTANCI%
weight of 200 g were transferred to 6°C and kept in individual wire-mesh cages for 7 wk, at which time they had reached a body weight of 380 g. They were fed Master laboratory chow and received tap water ad libitum. Lights were on from 800 to 2000 h. Metabolic response to tyramine (see below) was measured twice on these animals at an interval of 3 days and 3 days after the second test, they were exposed to -40°C to measure their cold resistance. Group II: Sprague-Hawley rats, kebt at 6°C for dzxerent lengths of dinze (July-August). Eighty adult male SpragueDawley rats, of an average body weight of 186 g were distributed at random among the following eight subgroups subgroup &0 day at 6°C (kept at 23OC since weaning) 112-1 4 days at 6°C &-lo days at 6°C &-15 days at 6°C I&,-22 days at 6°C I&--28 days at 6°C UT-37 days at 6°C I&-37 days at 6°C After the indicated time at 6”C, the metabolic response of each rat to tyramine was measured as described below and the rats were returned to their respective acclimation rooms. Three days later each rat was exposed to -40°C for a cold-resistance test. Twenty-four hours after the tyramine test, the eight rats of grouf I& were surgically adrenalectomized at room temperature and returned to 6°C the next day. Two days later cold resistance at -40°C was measured. For these animals, tap water was replaced by 0.9 % NaCl solution after adrenalectomy. While this study was being pursued using SpragueDawley rats, the opportunity arose to test with our procedures other varieties of rats, which had been made cold resistant by difierent treatments. These were a group of hooded rats exposed to outdoor winter conditions and a group of Wistar rats chronically treated with tyramine. t&up III: outdoor hooded rats. Ten adult male hooded rats were moved outdoors in August to 20 x 24 x 9 in wire-mesh cages supplied with shavings. They were kept 10 per cage, fed Purina laboratory chow in cubes and were given tepid tap water twice a day in a bowl. (See (4) for details concerning weather conditions prevailing outdoors where these animals were kept.) In May of the following year they were brought into the laboratory for measurement of response to tyramine and then returned outdoors. Three days later cold resistance at -40°C was measured. When tested with tyramine, their average body weight was 371 g. Group IV: tyramine-treated Wisfar rats. Twelve male Wistar rats of an average weight of 200 g were kept in individual cages at 28°C and given Purina laboratory chow and tap water ad libitum. For 1.5 mo beginning in November they received daily subcutaneous injections of tyramine (10 mg/kg body wt) dispersed in oil. Two days after the end of this treatment, tyramine response was measured on these animals whose average body weight was 398 g. Three days later cold resistance at -40°C was measured.
M&dr a) Metabolic response to tyramine was measured at 27-28°C in the closed-circuit apparatus previously described (14). This test was chosen in preference to a NE test for the following reasons: I) a maximum response is always obtained with a dose of 20 mg/kg ip without any apparent signs of toxicity; 2) the response to tyramine may be a more physiological measurement of capacity for NST since tyramine liberates endogenous noradrenaline; 3) there is a linear relationship between metabolic response to tyramine and metabolic response to NE. After allowing 30 min for thermal equilibration of the chamber, the resting rate of 02 consumption of the rat was measured for 30 min. The rat was then injected intraperitoneally with tyramine hydrochloride (20 mg/kg) in 0.9 c/o NaCl and 0~ consumption was recorded for 60 min. The maximal metabolic response occurred between 10 and 20 min after the injection. The response was expressed as the mean percentage increase in 0 2 consumption above the resting level during this 1%min period. b) Measurement of cold resistance. the animal was placed in a chamber immersed in an antifreeze bath kept at -45°C (11). The temperature in the chamber varied between -40 and - 43°C and increased at a rate of about l”C/h+ Oxygen consumption (corrected to STP) was measured in a closed-circuit system similar to the one used for the tyramine test. Ten minutes were allowed for equilibrium to be attained in the system before commencement of measurements of oxygen consumption. For groups II and III, in addition to measuring oxygen consumption, colonic temperature was continuously monitored by a telemetric system employing a small (28 x 7 mm) radiotransmitter inserted into the rat’s rectum (10). Colonic temperatures were not monitored for groups I and IV because the radiotransmitters were not available at the time these groups were studied.
I
32
40
28
38
3
34
it 3
32
i
-36
24
- 20 .-‘c
-$ I6
VE 0” 12 >
30
:
28
F
26 24
0 5
22
5
20
s
8 4 1
TIME
(min)
FIG. 1. Typical changes in the rate of oxygen consumption and in the body temperature of rats exposed to -40°C. Oxygen consumption, o and broken line; coIonic temperature, l and fufl line. F, = failure point, I, = inflection point, It = inflection time, and Ft = failure time.
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438
Hl&OUX
When rats were exposed to -40°C in the metabolic chamber, rectal temperature immediately began to decline. The fall in rectal temperature with increasing time of exposure to -40°C followed a sigmoid curve which had an inflection point (IP) at about 32.9”C. This point was located at the intersection of the two lines traced over the initial concave portion and the subsequent convex position of the curve. The time required to reach the inflection point was called the inflection time (It) (Fig. 1). During exposure of a rat to -440°C its oxygen consumption either gradually declined or it fluctuated around a certain level for some time until it abruptly dropped. The point on the oxygen consumption curve, at which this abrupt drop occurred, was called the failure point (Fp) and the time taken to reach the failure point was called the failure time (FJ. Ft varied considerably from one rat to another. The total oxygen consumed in the interval between 10 min (the time at which measurement of 02 consumption was begun) and I, or F, can be construed as a measure of the metabolic effort made by an animal to combat hypothermia. This value, expressed in milliliters of oxygen, defines what we call hypothermic resistance (HR). HRi and HRf distinguish hypothermic resistance computed from inflection point and failure point, respectively. Since temperature was much less variable from one reading to another than oxygen consumption, HRi could be more accurately determined than HRf. The calculation of HRf for those groups of rats for which body temperature was not measured is justified, however, because of a very high degree of correlation (r = 0.95 with n = 78) between HRi and HRf. RESULTS
The possibility stores sufficiently
that the tyramine test might reduce NE to adversely affect cold resistance meas-
TABLE 1. Metabolic res;bonse to tyramine in rats (grouj acclimated to 6°C fur 28 days and tested twice at an interval of 3 duys
-_~-~-
-. -. ~.-Rat
So.
Body
4 5 8 9 10 11 13 14 15 18 20 23 Avg
&
Weights
380
+
Results are expressed above the resting level tyramine hydrochloride
5.0
~--
---
1st Tyramine Test Tyramine Response
2nd Tyramine test Tyramine Response, %
144 117 121 125 142 120 138 108 101 122 135 169
109 102 138 139 144 113 133 114 107 145 123 163
392 400 382 378 380 360 378 380 336 390 394 385 SE
I>
125.5
=I= 5.28
as percent increase between 10th and 20th at a dose of 20 mg/kg
127 in min body
zt
5.46
02 consumption after injection wt.
of
ET
-* I-I - -0 -.-. - .-.1* ---r----~
AL.
15 18 g 13
/ O. “d . .,:\ ,, \
\
PIG. 2. Change oxygen consumption 30 days). Number
with time of exposure of twelve rats (group for each line represents
to -40°C I) acchmated rat number.
'r
in
2. Failure time (FJ, hypothermia resistance (H&j, and metabolic res;bonse to tyramine in rats (group I> acclimated to 6°C for 30 days --, -
the rate of to 6°C (for
TABLE
Rat Xo.
r 1;’ 20 15 4 9 10 13 18 23 8 14 Means =t SE (excluding the 1st 4 rats)
Body
Wt, g
Ft, min
HRf
I
ml
HRf/(Fk - lo>,* ml *min-1
versus tyramine response far rats acclimuted to 6°C fur dl@rent lengths of time (group II> ---.----.. . _~~~--. ,~---_~--_No. of Days
Subgroup 111 JII2
113 114
115 116 117 11s
at 6°C --
No, of Rats
0 4 10 15 22 28 37 37 adrenalectolnized
Correlation
11 12 11 12 12 12 8” r
-o-739* -0.409 0.511 -0.139 0.321 -0.249 0.245 -0.034 -.-
.” * Statistically
significant
Coef r
at 5% level.
not maintain a relatively constant rate of oxygen uptake for any appreciable length of time after the first 10 minutes; in panel (b) are five oxygen consumption curves for rats that maintained a relatively constant level of oxygen uptake for about 30 to 50 minutes; in panel (c) are the curves for four rats that maintained an essentially constant level of 02 consumption for at least 90 minutes. The test for one rat was interrupted at 130 minutes, at which time F, had not been reached. Table 2 summarizes the results of all the measurements (tyramine response, Ft and HRf) made on the rats of group I: The very considerable variation among the rats in their Ft and HRf is readily apparent. Division of HRf by Ft-10 min gives the average rate of 02 consumption (ml 0 29min-l) during the resistance period. It is apparent from these calculations that there is little variation among the rats in rate of 02 consumption before Ft is reached. The great variability of HRf therefore results from variation in Ft which is a measure of the length of time the animals can maintain a high rate of 02 consumption. It is worth noting that when the average rate of oxygen consumption at -40°C between 10 min and Ft (i-e., 24.7 mlvmin-l) is expressed as calm h-l* body wt”b5, the value obtained (viz., 365) is in close agreement with the values reported by Hart and Jansky (7) and Depocas (2). 1Most pertinent to the present study, however, is the lack of correlation (r = 0.0003) between HRf and tyramine response. Group II: Exposure to 6°C
fur Di$erentLengths
439 to 6*C, both the average tyramine response and the average cold resistance as evaluated by HRi increased (Table 3). Neither tyramine response nor HRi could be corrected for differences in body weight, because the relatively small number of animals in each group did not allow accurate calculation of the regression coefficients between these responses and body weights. Rather than assuming some coefficient such as 0.5, 0.66, or 0.75, it appeared preferable to report the absolute values. Nevertheless, the increase in HRi and tyramine response with time of cold acclimation can hardly be totally attributable to increase in body weight since weight increased by only 25 %, whereas HRj increased three- to fourfold and tyramine response more than doubled. When the degree of hypothermic resistance at -40°C was compared with the tyramine response of individual rats which were in the process of becoming acclimated to 6°C (groups 112 to 11,) no significant correlation between these two parameters was found at any time of acclimation (Table 4). For the control rats (group 1,) kept at 23°C a significant negative correlation was observed (Table 4 and Fig. 3). When all the data for subgroups 11,117 were pooled, a significant positive correlation (r = 0.297, n = 72, I’ < 0.02) was found between the tyramine response (TR) and the HRi according to the equation HR; = 242 + 7.32 TR. It is noteworthy that there was no effect of length of acclimation time on the rectal temperature at the inflection point, The average rectal temperature at the inflection point for the rats of the six cold-exposed groups (112-11~) was 32.98 =t 0.26”C. With increasing period of acclimation to 6°C there was, however, a gradual increase in It which reached a maximum around the 22nd day. The relationship between inflection time (It) and number of days (x) at 6°C could be represented by the following quadratic equation : It = a + bx + CX? The values of the constants were a = 20.8 XII 7.9 b = 3.0 & 1.1 c = 0.05 It 0.03
ii!-
HRi = -5.811 TR + 394.9 r = -0.739 kl=IO, paal)
0
of Time FIG.
a) Hypothermic resistance (HI&) vs. tyramine general, with increasing period of acclimation
response. In of the rats
response relation
3. Regression of hypothermia resistance (FIR;) in 23°C-acclirnated rats (group 111) (July-August). coefficient.
on tyramine r = Cor-
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HfiKOUX
5 4
0’
4
I5
20
VQ, mox (-4Q”Chll-
?Gr’
25
1
FXG. 4. Regression Of maximum VOZ after tyramine injection on maximum i02 at -440°C of rats acclilnated to 6°C for different lengths of time. Individual rats are represented by the arabic numbers corresponding to the different subgroups: group III = 0 days at 6°C; group 112 = 4 days at 6°C; group 113 = 10 days at 6°C; group I14 = 15 days at 6°C; group 115 = 22 days at 6°C; group I16 = 28 days at 6°C; group I& = 37 days at 6’C. TABLE 5. Comparison of tyramine response nnd cold resistance in intact and adrenalectomised co/d-acclimated rats (37 days at 6’C) ~ ------v-P__, _ ----__._-^.,Group
IIT
Intact
wt
response, 70
Tyramine HR
i/I
t-10,
Inflection
ml/mint time,
min
241 *11 78.4 43.0 18.2 zto.97
are
means =t SE, with no. different at the 5% between 10 min of exposure
Group IV:
Tyramine- Treated
Wistar Rats
Since changes in body temperature during exposure to -40°C were not recorded for the rats of this group, hypothermic resistance was calculated from Ft. When tyramine response and HRf were related, a significant correlation (r = 0.899) was found (Fig. 6). DISCUSSION
(6) (6) (5) (5)
258 +9 60.9 +4.1 16.1 dzO.77 24.6 +8.0
17 AI9 -17.5 zt6.4* -2.10 ztl.24 -31.2
(9)
(8)
l 12.9* ~__
~_._ Values * Significantly at -40°C
Hypothermic resistunce US. tyramine response. After spending the whole winter outdoors, rats were tested in May. No significant correlation was observed between hypothermic resistance and tyramine response (r = 0.335, n = 10; see Fig. 5). The average colonic temperature at the inflection point was 32.5 =t 024°C.
-__
118
Adrenalectomized .-__
55.8 l 4.3
Outdoor Hooded Rats
Diff
_--~---~-~.----
Body
Group
AT,.
c> Efect of bilateral adrenalectomy on tyramine response and hypothermic resistance of cold-acclimated rats (group 118). Intact and adrenalectomized cold-acclimated rats did not differ significantly in body weight (Table 5). Tyramine response, average oxygen uptake between 10 min and the inflection point and inflection time (taken as an index of cold resistance) were not significantly related to body weight. Both tyramine response and inflection time, but not average oxygen uptake, were significantly reduced by adrenalectomy. Group III:
10
ET
of animals given in parentheses. level. t Average 02 uptake and inflection time.
b) Com@rison of the maximum rates of 02 consumption induced by tyrclmine and by exposure to - 40°C. In both the tyramine and cold resistance tests, the oxygen consumption during each 5-min period was measured and the maximum rate of 02 consumption (~OZ & was calculated from the 5-min period during which total oxygen consumption was the highest. Within none of the subgroups, Ul-117, was there a significant correlation between ii02 max (-40°C) and Tj02 max (tyramine). When, however, all the data for subgroups U&IT were pooled a significant positive correlation was found between the i/o2 lllaX at -40°C (X) and (Y) according to the equation the VOZ nlax (tyramine) Y = 3.55 + 0.359X (Fig. 4). This regression equation reveals that to a vo 2 lllax (-40°C) of 20 ml responds a ‘ii02 max (tyramine) of lo.83 ml min-I. If the maximum response to tyramine is a true measure of the maximum nonshivering thermogenic capacity of these animals, the data indicate that about 54 % of the ii02 max at -40°C was due to NST and about 46 % to other thermogenie mechanisms, such as shivering. l
min-l
An enhanced metabolic response to tyramine (i.e., to endogenously released norepinephrine (I 2)) has been confirmed in the present study for rats made more cold resistant by three different treatments, namely, chronic cold exposure (groups 1 and U), outdoor winter exposure (group IU), and chronic treatment of tyramine (group IV). For the Sprague-Dawley and hooded rats whose cold resistance was increased by exposure to cold, the degree of cold resistance of an individual rat could not be evaluated from its tyramine response, since there was for any given group no significant correlation between tyramine response and hypothermic resistance at -40°C. When the data for all 72 rats of group IL were pooled, a HRi’924TR I
q
0.335
+62cl73 (n
q
0
IO, p-Go.3 1
0
z k
1600
cor-
l
90
0
FIG. 5. Regression response of hooded Carrel ati on coefficient
20
rats
40
7
f
100 IlO 140 $0 8’0 TYRAMINE RESPONSE (TR)
of hypothermia (group 111) .
kept
resistance outside
1
I60
(IIRi) on tyramine over the winter. r =
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NOMHIVERING
THERMOGENESIS
HRf= 18 02
TR-953.3+
AND
COLD
RESISTANCE
402.7
ml 0, /“r =0.903
-
1600
w I
800
%
600
(n=8,p
kept at 28°C. r = Corcoefficient.
statistically significant correlation between tvramine response and HRi was found, but the low correlation coefficient (0.297) shows that it would not be possible to make more than a qualitative prediction of cold resistance from the tyramine response. For Wistar rats chronically treated with tyramine there was, however, a strong and significant correlation between metabolic response to tyramine and cold resistance. The high correlation coefficient (0399) indicates that a fairly accurate prediction of the degree of cold resistance of any individual rjat in that particular group could be made from its metabol -c response to tyramine. It seems unlikely that the difference between the coldrats with respect acclimated rats and tyramine-treated to presence or absence of correlation between tyramine response and cold resistance is due to the different strain of animals. It is more probable that in the chronically coldexposed rats, in addition to the increase in NE sensitivity, which also occurred in the rats chronicallv treated with tyramine, were other physiological adjustments there which greatly contributed to their greater hypothermic would preresistance. Since resistance to hypothermia sumably be a function of all the adjustments which occur during chronic cold exposure, it is not surprising that no correlation was found between hypothermic resistance and only one of these adjustments, namely, nonshivering thermogenesis as measured by increased metabolic sensitivi ty to NE. Rats treated with tyramine, but not chronitally exposed to cold, develop a greater norepinephrine sensitivity but apparently do not undergo the other physiological adjustments which further enhance cold resistance. The significant correlation between tyramine response and hvpothermic resistance in the case of the tyramine-treated ,
441 rats suggests that the increase in hypothermic resistance in these animals is primarily due to an increased capacity for NST. In cold-acclimated rats, NE-induced NST can, according to the equation given in Fig. 4, account for slightly more than 50 % of the heat produced at -440°C. Hypothermia resistance, as measured in this study, however, is a function not only of the rate of heat production, but also of the length of time a high rate of heat production can be maintained. The latter appears to be at least partially dependent on the presence of the adrenals, since rats without their adrenals reach a level of heat production which is essentially normal, but they fail to maintain it. This failure to maintain a high rate of heat production could then be attributed to the absence of one or more of the hormones originating from the adrenal. Further experiments in which only the medulla will be removed should help in identifying the hormonal factors responsible for the maintenance of a high rate of heat production under conditions of severe cold. In 1951, Sellers et al. (15) found that in a cold environment, the metabolic rate of a non-cold-acclimated rat increased for a brief period to as high or almost as high a level as that of the cold-acclimated animal but was not maintained. The difference between the acclimated and nonacclimated animal appeared to lie in an ability, acquired during acclimation, to continue producing heat at the increased rate. The present results demonstrate further that even within a group of similarly cold-acclimated rats, the individuals may have essentially identical initial increases in heat production, while their survival at -40°C may not be the same because of diflerences in the length of time this extra heat can be produced. Whereas, the inflection time varies from one rat to the other, the body temperature at the inflection point is invariably around 329°C. The significance of this apparently critical temperature remains to be elucidated. The factors that account for individual difierences in hypothermic resistance may be quite numerous and they need not be exclusively of metabolic origin. Peripheral vascular control of heat conservation, for example, may be an important factor. This aspect of cold acclimation is more diff-icult to study and for that reason has been somewhat neglected in studies on laboratory animals. It is concluded from this study that I) because there is no correlation between tyramine response and hypothermic resistance for a group of rats acclimated to cold, resistance to severe cold must be dependent, to a very significant degree, upon factors other than capacity for NST. 2) variation in the ability of cold-acclimated rats to resist lethal hypothermia is primarily due to variations in the length of time a high metabolic rate can be maintained, rather than to variation in maximum metabolic capacity. From a rat’s maximum $%2 in response to tyramine it is possible to predict with reasonable accuracy its voz Illax at -4O”C, but it is not possible to predict the length of time the animal can maintain the high rate of oxygen consumption, or how long it will resist lethal hypothermia. Thus, tyramine response can be used to predict maximum metabolic capacity, but not metabolic endurance. 3) The adrenals appear to be essential for rats to maintain a high metabolic rate in a severely cold environment.
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442
HfiROUX
Further studies on cold resistance towards the limiting factors responsible tain a high rate of heat production exposure. The authors acknowledge bonneau from the National Oakson from Lava1 University The the body temperature.
should be directed for failure to mainduring severe cold
the technical collaboration of R. CharResearch Council of Ottawa and G. who developed the transmitters for collaboration of Mr. Jean-Guy PiIon,
Director
ET
AL
of the
‘
Vol.
38, No.
APPLIED
3,
March
PHYsIoLocy 1975. Printed
in U.S.A*
Nonshivering
thermogenesis
in rats under
severe cold
and cold conditions
0. HfiROUX, E. PAGk, J. LEBLANC, J. LEDUC, R. GILBERT, A. VILLEMAIRE, AND P, RIVEST Station Biologique de St, Hi&~~/yte, Unimx’te de Montreal, Montreal,
H~ROUX, 0, E. PAGI~, J. LEBLANC, J. LEDUC, R. GILBERT, A. VILLEMAIRE, AND P. RXVEST. Nonshivering thermogenesis and co/d resistance irz rats under se~cre cold conditions. J. Appl. Physiol. 38(3) : 436-442. 1975.-Following either chronic exposure to 6”C, or outdoor winter exposure, or chronic treatment with tyramine rats were exposed to - 40°C and their oxygen consumption and colonic temperature monitored. Fall in body temperature with time of exposure followed a sigmoid curve which had an inflection point around 32.9”C. Both the time required for body temperature to reach this point and hypothermic resistance defined as the total 02 consumed up to the inflection time were useful indices of resistance to severe cold. Three days before the cold tests, capacity for norepinephrine-induced nonshivering thermogenesis was measured in all animals by examination of their metabolic response to tyramine. The magnitude of response to tyramine correlated well with hypothermic resistance only for those rats chronically treated with tyramine. It is concluded that it is impossible to predict with any reasonable degree of confidence the cold resistance of a rat from its tyramine response. In cold-acclimated rats, factors in addition to norepinephrine sensitivity are significantly involved in cold resistance and deserve further studies. cold
acclimation;
cold
resistance
resistance
at - 40°C
THE FIRST OBSERVATIONS that chronic exposure of rats to moderately cold temperature was associated with the development of increased resistance to cold were made by Ogle and Mills (13) in 1933 and Gelineo (3) in 1934It then became apparent that this conditioning or acclimation to cold extended the lower limits of environmental temperatures at which peak metabolic rate was reached or at which body temperature or body weight could be maintained. Subsequent research revealed that apparently the most significant metabolic alteration which occurs during acclimation to cold is the development of a potential to produce heat by a new thermogenic mechanism called nonshivering thermogenesis (NST). This potential is expressed through the action of norepinephrine (NE) liberated by the sympathetic nervous system (8) and it has been shown that the capacity for NST can be measured by the metabolic response of the animal to exogenous NE
creased resistance of cold-acclimated rats to severe cold: a) during acclimation to cold, the gradual development of NST as indicated by both increased calorigenic response to NE and decreased shivering paralleling an increase in cold resistance (2); b) at very cold temperatures (e.g., -40°C) the metabolic rate of the cold-acclimated rat is greater than that of the warm-acclimated rat and the difference has been shown to be due to the ability of the former to produce heat by NST in addition to shivering (7). It has been stated, furthermore, that calorigenic response to NE may be used to measure the degree to which a rat has become cold-acclimated (9). Because increased cold resistance is associated with acclimation to cold it might then be assumed that the calorigenic response of a rat to NE could provide a good index of its cold resistance. But the validity of this assumption is questioned by the obscrvation that during deacclimation of the cold-acclimated rat, resistance to severe cold decreases faster than NE sensitivity (4, 5, 12). Furthermore, among cold-acclimated rats (6) the variability in cold resistance is much greater than the variability in NE sensitivity (8). This seems to suggest that the degree of cold resistance exhibited by an animal is a function not only of its capacity for NE-induced NST, but also of other factors. However, since cold resistance and NE-induced calorigenesis were not measured in the same group of animals, the relative significance of factors other than NST is not apparent. If NE-induced NST is primarily responsible for the increased cold resistance of cold-acclimated rats, one would expect the cold resistance and the capacity for NE-induced calorigenesis, each measured in the same animals, to be highly correlated. Verification of this would permit measurement of cold resistance by measurement of calorigenic response to NE. The latter test is preferable to and more practical than exposure of the animal to severe cold to measure its cold resistance in terms either of survival time or of rate of fall in body temperature. This communication presents results of various experiments designed to determine whether the most cold resistant rats are those that have the greatest NE-induced calorigenesis and therefore the greatest capacity for NST. EXPERIMENTAL
m
Quebec, Canada
CONDITIONS
(9)
Certain observations that NE-induced NST This paper
have led is primarily
has been designated
NRCC
investigators responsible 14502.
to believe for the in-
Animals,
Caging, Environmental
Conditions
Group I: Sprague-Dawley rats, 7 wk at 6°C (October). Twelve adult male Sprague-Dawley rats of an average body
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NONSHIVERING
THERMQGENESIS
AND
COLD
437
RESISTANCI%
weight of 200 g were transferred to 6°C and kept in individual wire-mesh cages for 7 wk, at which time they had reached a body weight of 380 g. They were fed Master laboratory chow and received tap water ad libitum. Lights were on from 800 to 2000 h. Metabolic response to tyramine (see below) was measured twice on these animals at an interval of 3 days and 3 days after the second test, they were exposed to -40°C to measure their cold resistance. Group II: Sprague-Hawley rats, kebt at 6°C for dzxerent lengths of dinze (July-August). Eighty adult male SpragueDawley rats, of an average body weight of 186 g were distributed at random among the following eight subgroups subgroup &0 day at 6°C (kept at 23OC since weaning) 112-1 4 days at 6°C &-lo days at 6°C &-15 days at 6°C I&,-22 days at 6°C I&--28 days at 6°C UT-37 days at 6°C I&-37 days at 6°C After the indicated time at 6”C, the metabolic response of each rat to tyramine was measured as described below and the rats were returned to their respective acclimation rooms. Three days later each rat was exposed to -40°C for a cold-resistance test. Twenty-four hours after the tyramine test, the eight rats of grouf I& were surgically adrenalectomized at room temperature and returned to 6°C the next day. Two days later cold resistance at -40°C was measured. For these animals, tap water was replaced by 0.9 % NaCl solution after adrenalectomy. While this study was being pursued using SpragueDawley rats, the opportunity arose to test with our procedures other varieties of rats, which had been made cold resistant by difierent treatments. These were a group of hooded rats exposed to outdoor winter conditions and a group of Wistar rats chronically treated with tyramine. t&up III: outdoor hooded rats. Ten adult male hooded rats were moved outdoors in August to 20 x 24 x 9 in wire-mesh cages supplied with shavings. They were kept 10 per cage, fed Purina laboratory chow in cubes and were given tepid tap water twice a day in a bowl. (See (4) for details concerning weather conditions prevailing outdoors where these animals were kept.) In May of the following year they were brought into the laboratory for measurement of response to tyramine and then returned outdoors. Three days later cold resistance at -40°C was measured. When tested with tyramine, their average body weight was 371 g. Group IV: tyramine-treated Wisfar rats. Twelve male Wistar rats of an average weight of 200 g were kept in individual cages at 28°C and given Purina laboratory chow and tap water ad libitum. For 1.5 mo beginning in November they received daily subcutaneous injections of tyramine (10 mg/kg body wt) dispersed in oil. Two days after the end of this treatment, tyramine response was measured on these animals whose average body weight was 398 g. Three days later cold resistance at -40°C was measured.
M&dr a) Metabolic response to tyramine was measured at 27-28°C in the closed-circuit apparatus previously described (14). This test was chosen in preference to a NE test for the following reasons: I) a maximum response is always obtained with a dose of 20 mg/kg ip without any apparent signs of toxicity; 2) the response to tyramine may be a more physiological measurement of capacity for NST since tyramine liberates endogenous noradrenaline; 3) there is a linear relationship between metabolic response to tyramine and metabolic response to NE. After allowing 30 min for thermal equilibration of the chamber, the resting rate of 02 consumption of the rat was measured for 30 min. The rat was then injected intraperitoneally with tyramine hydrochloride (20 mg/kg) in 0.9 c/o NaCl and 0~ consumption was recorded for 60 min. The maximal metabolic response occurred between 10 and 20 min after the injection. The response was expressed as the mean percentage increase in 0 2 consumption above the resting level during this 1%min period. b) Measurement of cold resistance. the animal was placed in a chamber immersed in an antifreeze bath kept at -45°C (11). The temperature in the chamber varied between -40 and - 43°C and increased at a rate of about l”C/h+ Oxygen consumption (corrected to STP) was measured in a closed-circuit system similar to the one used for the tyramine test. Ten minutes were allowed for equilibrium to be attained in the system before commencement of measurements of oxygen consumption. For groups II and III, in addition to measuring oxygen consumption, colonic temperature was continuously monitored by a telemetric system employing a small (28 x 7 mm) radiotransmitter inserted into the rat’s rectum (10). Colonic temperatures were not monitored for groups I and IV because the radiotransmitters were not available at the time these groups were studied.
I
32
40
28
38
3
34
it 3
32
i
-36
24
- 20 .-‘c
-$ I6
VE 0” 12 >
30
:
28
F
26 24
0 5
22
5
20
s
8 4 1
TIME
(min)
FIG. 1. Typical changes in the rate of oxygen consumption and in the body temperature of rats exposed to -40°C. Oxygen consumption, o and broken line; coIonic temperature, l and fufl line. F, = failure point, I, = inflection point, It = inflection time, and Ft = failure time.
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438
Hl&OUX
When rats were exposed to -40°C in the metabolic chamber, rectal temperature immediately began to decline. The fall in rectal temperature with increasing time of exposure to -40°C followed a sigmoid curve which had an inflection point (IP) at about 32.9”C. This point was located at the intersection of the two lines traced over the initial concave portion and the subsequent convex position of the curve. The time required to reach the inflection point was called the inflection time (It) (Fig. 1). During exposure of a rat to -440°C its oxygen consumption either gradually declined or it fluctuated around a certain level for some time until it abruptly dropped. The point on the oxygen consumption curve, at which this abrupt drop occurred, was called the failure point (Fp) and the time taken to reach the failure point was called the failure time (FJ. Ft varied considerably from one rat to another. The total oxygen consumed in the interval between 10 min (the time at which measurement of 02 consumption was begun) and I, or F, can be construed as a measure of the metabolic effort made by an animal to combat hypothermia. This value, expressed in milliliters of oxygen, defines what we call hypothermic resistance (HR). HRi and HRf distinguish hypothermic resistance computed from inflection point and failure point, respectively. Since temperature was much less variable from one reading to another than oxygen consumption, HRi could be more accurately determined than HRf. The calculation of HRf for those groups of rats for which body temperature was not measured is justified, however, because of a very high degree of correlation (r = 0.95 with n = 78) between HRi and HRf. RESULTS
The possibility stores sufficiently
that the tyramine test might reduce NE to adversely affect cold resistance meas-
TABLE 1. Metabolic res;bonse to tyramine in rats (grouj acclimated to 6°C fur 28 days and tested twice at an interval of 3 duys
-_~-~-
-. -. ~.-Rat
So.
Body
4 5 8 9 10 11 13 14 15 18 20 23 Avg
&
Weights
380
+
Results are expressed above the resting level tyramine hydrochloride
5.0
~--
---
1st Tyramine Test Tyramine Response
2nd Tyramine test Tyramine Response, %
144 117 121 125 142 120 138 108 101 122 135 169
109 102 138 139 144 113 133 114 107 145 123 163
392 400 382 378 380 360 378 380 336 390 394 385 SE
I>
125.5
=I= 5.28
as percent increase between 10th and 20th at a dose of 20 mg/kg
127 in min body
zt
5.46
02 consumption after injection wt.
of
ET
-* I-I - -0 -.-. - .-.1* ---r----~
AL.
15 18 g 13
/ O. “d . .,:\ ,, \
\
PIG. 2. Change oxygen consumption 30 days). Number
with time of exposure of twelve rats (group for each line represents
to -40°C I) acchmated rat number.
'r
in
2. Failure time (FJ, hypothermia resistance (H&j, and metabolic res;bonse to tyramine in rats (group I> acclimated to 6°C for 30 days --, -
the rate of to 6°C (for
TABLE
Rat Xo.
r 1;’ 20 15 4 9 10 13 18 23 8 14 Means =t SE (excluding the 1st 4 rats)
Body
Wt, g
Ft, min
HRf
I
ml
HRf/(Fk - lo>,* ml *min-1
versus tyramine response far rats acclimuted to 6°C fur dl@rent lengths of time (group II> ---.----.. . _~~~--. ,~---_~--_No. of Days
Subgroup 111 JII2
113 114
115 116 117 11s
at 6°C --
No, of Rats
0 4 10 15 22 28 37 37 adrenalectolnized
Correlation
11 12 11 12 12 12 8” r
-o-739* -0.409 0.511 -0.139 0.321 -0.249 0.245 -0.034 -.-
.” * Statistically
significant
Coef r
at 5% level.
not maintain a relatively constant rate of oxygen uptake for any appreciable length of time after the first 10 minutes; in panel (b) are five oxygen consumption curves for rats that maintained a relatively constant level of oxygen uptake for about 30 to 50 minutes; in panel (c) are the curves for four rats that maintained an essentially constant level of 02 consumption for at least 90 minutes. The test for one rat was interrupted at 130 minutes, at which time F, had not been reached. Table 2 summarizes the results of all the measurements (tyramine response, Ft and HRf) made on the rats of group I: The very considerable variation among the rats in their Ft and HRf is readily apparent. Division of HRf by Ft-10 min gives the average rate of 02 consumption (ml 0 29min-l) during the resistance period. It is apparent from these calculations that there is little variation among the rats in rate of 02 consumption before Ft is reached. The great variability of HRf therefore results from variation in Ft which is a measure of the length of time the animals can maintain a high rate of 02 consumption. It is worth noting that when the average rate of oxygen consumption at -40°C between 10 min and Ft (i-e., 24.7 mlvmin-l) is expressed as calm h-l* body wt”b5, the value obtained (viz., 365) is in close agreement with the values reported by Hart and Jansky (7) and Depocas (2). 1Most pertinent to the present study, however, is the lack of correlation (r = 0.0003) between HRf and tyramine response. Group II: Exposure to 6°C
fur Di$erentLengths
439 to 6*C, both the average tyramine response and the average cold resistance as evaluated by HRi increased (Table 3). Neither tyramine response nor HRi could be corrected for differences in body weight, because the relatively small number of animals in each group did not allow accurate calculation of the regression coefficients between these responses and body weights. Rather than assuming some coefficient such as 0.5, 0.66, or 0.75, it appeared preferable to report the absolute values. Nevertheless, the increase in HRi and tyramine response with time of cold acclimation can hardly be totally attributable to increase in body weight since weight increased by only 25 %, whereas HRj increased three- to fourfold and tyramine response more than doubled. When the degree of hypothermic resistance at -40°C was compared with the tyramine response of individual rats which were in the process of becoming acclimated to 6°C (groups 112 to 11,) no significant correlation between these two parameters was found at any time of acclimation (Table 4). For the control rats (group 1,) kept at 23°C a significant negative correlation was observed (Table 4 and Fig. 3). When all the data for subgroups 11,117 were pooled, a significant positive correlation (r = 0.297, n = 72, I’ < 0.02) was found between the tyramine response (TR) and the HRi according to the equation HR; = 242 + 7.32 TR. It is noteworthy that there was no effect of length of acclimation time on the rectal temperature at the inflection point, The average rectal temperature at the inflection point for the rats of the six cold-exposed groups (112-11~) was 32.98 =t 0.26”C. With increasing period of acclimation to 6°C there was, however, a gradual increase in It which reached a maximum around the 22nd day. The relationship between inflection time (It) and number of days (x) at 6°C could be represented by the following quadratic equation : It = a + bx + CX? The values of the constants were a = 20.8 XII 7.9 b = 3.0 & 1.1 c = 0.05 It 0.03
ii!-
HRi = -5.811 TR + 394.9 r = -0.739 kl=IO, paal)
0
of Time FIG.
a) Hypothermic resistance (HI&) vs. tyramine general, with increasing period of acclimation
response. In of the rats
response relation
3. Regression of hypothermia resistance (FIR;) in 23°C-acclirnated rats (group 111) (July-August). coefficient.
on tyramine r = Cor-
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HfiKOUX
5 4
0’
4
I5
20
VQ, mox (-4Q”Chll-
?Gr’
25
1
FXG. 4. Regression Of maximum VOZ after tyramine injection on maximum i02 at -440°C of rats acclilnated to 6°C for different lengths of time. Individual rats are represented by the arabic numbers corresponding to the different subgroups: group III = 0 days at 6°C; group 112 = 4 days at 6°C; group 113 = 10 days at 6°C; group I14 = 15 days at 6°C; group 115 = 22 days at 6°C; group I16 = 28 days at 6°C; group I& = 37 days at 6’C. TABLE 5. Comparison of tyramine response nnd cold resistance in intact and adrenalectomised co/d-acclimated rats (37 days at 6’C) ~ ------v-P__, _ ----__._-^.,Group
IIT
Intact
wt
response, 70
Tyramine HR
i/I
t-10,
Inflection
ml/mint time,
min
241 *11 78.4 43.0 18.2 zto.97
are
means =t SE, with no. different at the 5% between 10 min of exposure
Group IV:
Tyramine- Treated
Wistar Rats
Since changes in body temperature during exposure to -40°C were not recorded for the rats of this group, hypothermic resistance was calculated from Ft. When tyramine response and HRf were related, a significant correlation (r = 0.899) was found (Fig. 6). DISCUSSION
(6) (6) (5) (5)
258 +9 60.9 +4.1 16.1 dzO.77 24.6 +8.0
17 AI9 -17.5 zt6.4* -2.10 ztl.24 -31.2
(9)
(8)
l 12.9* ~__
~_._ Values * Significantly at -40°C
Hypothermic resistunce US. tyramine response. After spending the whole winter outdoors, rats were tested in May. No significant correlation was observed between hypothermic resistance and tyramine response (r = 0.335, n = 10; see Fig. 5). The average colonic temperature at the inflection point was 32.5 =t 024°C.
-__
118
Adrenalectomized .-__
55.8 l 4.3
Outdoor Hooded Rats
Diff
_--~---~-~.----
Body
Group
AT,.
c> Efect of bilateral adrenalectomy on tyramine response and hypothermic resistance of cold-acclimated rats (group 118). Intact and adrenalectomized cold-acclimated rats did not differ significantly in body weight (Table 5). Tyramine response, average oxygen uptake between 10 min and the inflection point and inflection time (taken as an index of cold resistance) were not significantly related to body weight. Both tyramine response and inflection time, but not average oxygen uptake, were significantly reduced by adrenalectomy. Group III:
10
ET
of animals given in parentheses. level. t Average 02 uptake and inflection time.
b) Com@rison of the maximum rates of 02 consumption induced by tyrclmine and by exposure to - 40°C. In both the tyramine and cold resistance tests, the oxygen consumption during each 5-min period was measured and the maximum rate of 02 consumption (~OZ & was calculated from the 5-min period during which total oxygen consumption was the highest. Within none of the subgroups, Ul-117, was there a significant correlation between ii02 max (-40°C) and Tj02 max (tyramine). When, however, all the data for subgroups U&IT were pooled a significant positive correlation was found between the i/o2 lllaX at -40°C (X) and (Y) according to the equation the VOZ nlax (tyramine) Y = 3.55 + 0.359X (Fig. 4). This regression equation reveals that to a vo 2 lllax (-40°C) of 20 ml responds a ‘ii02 max (tyramine) of lo.83 ml min-I. If the maximum response to tyramine is a true measure of the maximum nonshivering thermogenic capacity of these animals, the data indicate that about 54 % of the ii02 max at -40°C was due to NST and about 46 % to other thermogenie mechanisms, such as shivering. l
min-l
An enhanced metabolic response to tyramine (i.e., to endogenously released norepinephrine (I 2)) has been confirmed in the present study for rats made more cold resistant by three different treatments, namely, chronic cold exposure (groups 1 and U), outdoor winter exposure (group IU), and chronic treatment of tyramine (group IV). For the Sprague-Dawley and hooded rats whose cold resistance was increased by exposure to cold, the degree of cold resistance of an individual rat could not be evaluated from its tyramine response, since there was for any given group no significant correlation between tyramine response and hypothermic resistance at -40°C. When the data for all 72 rats of group IL were pooled, a HRi’924TR I
q
0.335
+62cl73 (n
q
0
IO, p-Go.3 1
0
z k
1600
cor-
l
90
0
FIG. 5. Regression response of hooded Carrel ati on coefficient
20
rats
40
7
f
100 IlO 140 $0 8’0 TYRAMINE RESPONSE (TR)
of hypothermia (group 111) .
kept
resistance outside
1
I60
(IIRi) on tyramine over the winter. r =
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NOMHIVERING
THERMOGENESIS
HRf= 18 02
TR-953.3+
AND
COLD
RESISTANCE
402.7
ml 0, /“r =0.903
-
1600
w I
800
%
600
(n=8,p
kept at 28°C. r = Corcoefficient.
statistically significant correlation between tvramine response and HRi was found, but the low correlation coefficient (0.297) shows that it would not be possible to make more than a qualitative prediction of cold resistance from the tyramine response. For Wistar rats chronically treated with tyramine there was, however, a strong and significant correlation between metabolic response to tyramine and cold resistance. The high correlation coefficient (0399) indicates that a fairly accurate prediction of the degree of cold resistance of any individual rjat in that particular group could be made from its metabol -c response to tyramine. It seems unlikely that the difference between the coldrats with respect acclimated rats and tyramine-treated to presence or absence of correlation between tyramine response and cold resistance is due to the different strain of animals. It is more probable that in the chronically coldexposed rats, in addition to the increase in NE sensitivity, which also occurred in the rats chronicallv treated with tyramine, were other physiological adjustments there which greatly contributed to their greater hypothermic would preresistance. Since resistance to hypothermia sumably be a function of all the adjustments which occur during chronic cold exposure, it is not surprising that no correlation was found between hypothermic resistance and only one of these adjustments, namely, nonshivering thermogenesis as measured by increased metabolic sensitivi ty to NE. Rats treated with tyramine, but not chronitally exposed to cold, develop a greater norepinephrine sensitivity but apparently do not undergo the other physiological adjustments which further enhance cold resistance. The significant correlation between tyramine response and hvpothermic resistance in the case of the tyramine-treated ,
441 rats suggests that the increase in hypothermic resistance in these animals is primarily due to an increased capacity for NST. In cold-acclimated rats, NE-induced NST can, according to the equation given in Fig. 4, account for slightly more than 50 % of the heat produced at -440°C. Hypothermia resistance, as measured in this study, however, is a function not only of the rate of heat production, but also of the length of time a high rate of heat production can be maintained. The latter appears to be at least partially dependent on the presence of the adrenals, since rats without their adrenals reach a level of heat production which is essentially normal, but they fail to maintain it. This failure to maintain a high rate of heat production could then be attributed to the absence of one or more of the hormones originating from the adrenal. Further experiments in which only the medulla will be removed should help in identifying the hormonal factors responsible for the maintenance of a high rate of heat production under conditions of severe cold. In 1951, Sellers et al. (15) found that in a cold environment, the metabolic rate of a non-cold-acclimated rat increased for a brief period to as high or almost as high a level as that of the cold-acclimated animal but was not maintained. The difference between the acclimated and nonacclimated animal appeared to lie in an ability, acquired during acclimation, to continue producing heat at the increased rate. The present results demonstrate further that even within a group of similarly cold-acclimated rats, the individuals may have essentially identical initial increases in heat production, while their survival at -40°C may not be the same because of diflerences in the length of time this extra heat can be produced. Whereas, the inflection time varies from one rat to the other, the body temperature at the inflection point is invariably around 329°C. The significance of this apparently critical temperature remains to be elucidated. The factors that account for individual difierences in hypothermic resistance may be quite numerous and they need not be exclusively of metabolic origin. Peripheral vascular control of heat conservation, for example, may be an important factor. This aspect of cold acclimation is more diff-icult to study and for that reason has been somewhat neglected in studies on laboratory animals. It is concluded from this study that I) because there is no correlation between tyramine response and hypothermic resistance for a group of rats acclimated to cold, resistance to severe cold must be dependent, to a very significant degree, upon factors other than capacity for NST. 2) variation in the ability of cold-acclimated rats to resist lethal hypothermia is primarily due to variations in the length of time a high metabolic rate can be maintained, rather than to variation in maximum metabolic capacity. From a rat’s maximum $%2 in response to tyramine it is possible to predict with reasonable accuracy its voz Illax at -4O”C, but it is not possible to predict the length of time the animal can maintain the high rate of oxygen consumption, or how long it will resist lethal hypothermia. Thus, tyramine response can be used to predict maximum metabolic capacity, but not metabolic endurance. 3) The adrenals appear to be essential for rats to maintain a high metabolic rate in a severely cold environment.
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442
HfiROUX
Further studies on cold resistance towards the limiting factors responsible tain a high rate of heat production exposure. The authors acknowledge bonneau from the National Oakson from Lava1 University The the body temperature.
should be directed for failure to mainduring severe cold
the technical collaboration of R. CharResearch Council of Ottawa and G. who developed the transmitters for collaboration of Mr. Jean-Guy PiIon,
Director
ET
AL
of the
‘
