Journal of Physiology (1990), 431, pp. 543-556 543With 5 figures

Printed in Great Britain

IS INCREASED METABOLISM IN RATS IN THE COLD MEDIATED BYTHE THYROID?

BY E. M. WHITAKER*, S. H. HUSSAINt, G. R. HERVEY, G. TOBINAND K. M. RAYFIELD

From the Department of Physiology, University of Leeds, Leeds LS2 9NQ

(Received 24 October 1989)

SUMMARY

1. In the rat variation of metabolic heat production is the principal effector ofthermoregulation. There is a continuous relationship between ambient temperatureand metabolic rate over the whole range of tolerable environmental temperature.The mechanism that controls metabolic rate is unknown; this paper reports anattempt to test whether thyroid hormones provide the controlling pathway.

2. First, the changes in metabolic rate and in the plasma concentrations of thyroidstimulating hormone (TSH), triiodothyronine (T3) and thyroxine (T4) were measuredin rats living in a controlled environment, first at 23 °C and then at 6 'C. Metabolicrate increased from approximately 290 to 470 kJ day-' when the temperature waslowered, a factor of ca 1-6, and the diurnal rhythm disappeared. The concentrationof TSH increased from approximately 320 to 450 ng ml-' (with loss of diurnalrhythm) and of T3 from ca 0-7 to 1-0 nmol 1-1, a factor of ca 1-4 in each case. T4concentration did not change.

3. Next, a dose schedule of T3 was found that, when injected i.v. via indwellingjugular cannulae in the same rats in an environment at 23 °C, maintained an increasein T3 concentration rather greater than had been found at 6 'C.

4. This dose of T3, given to the same rats at 23 °C, did not affect metabolic rate(or its diurnal pattern).

5. It is therefore unlikely that the increase in T3 concentration evoked the increasein metabolic rate when ambient temperature was changed from 23 to 6 °C; andtherefore that the thyroid controls variation of metabolic rate in 'everyday'thermoregulation in the rat.

INTRODUCTION

The animal generally taught by physiologists is a chimaera: its physiologicalsystems are an assembly from the various species in which the different systems havebeen principally studied. Its thermoregulatory system is that of man. Thethermoregulatory system, however, shows greater variations among species than any

* To whom reprint requests should be sent.t Present address: School of Pharmaceutical Sciences, University of Malaysia, Minden, Penang

11800, Malaysia.MS 8667

544 E. M. IVH[TAKER AND OTHERS

other. In rodents the major effector of thermoregulation over the whole range oftolerable ambient temperatures is variation of heat production. It produces adramatic effect, changing resting metabolism by a factor of around three, yet itsmechanism in unknown. This paper reports a first step toward identifying itsmechanism.

A BShivering

Thermoneutral zone 0

0~~~~~~~~~~~~~~~~~~~0CD L \Sweating

Cold Hot Cold Hot

Fig. 1. Relationship between environmental temperature and heat production in man (A)and the rat (B), shown diagrammatically. A also shows heat loss by sweating.

To appreciate the experiment it is necessary to understand the differences betweenhuman and rodent thermoregulation. Figure IA shows diagrammatically the patternof thermoregulation in adult man. When ambient temperature falls below a certainlevel - ca 26 °C for an unclothed adult at rest in still air - heat production increases.The source of the additional heat is shivering, a specialized pattern of contraction ofskeletal muscle evoked via well-known nervous pathways. At ambient temperaturesabove ca 26 °C heat production is essentially independent of ambient temperature.Above 30 °C sweating comes into play and provides a powerful means of increasingheat loss. The interval of ca 4 °C, over which neither shivering nor sweating is used,is the 'thermoneutral' zone. Man remains in this zone for most of the time by usinghis uniquely effective behavioural thermoregulation, together with a minorcontribution from vasomotor control (for a fuller account see Hervey, 1988).

In the rat the relationship between heat production (i.e. resting metabolic rate)and ambient temperature is as shown in Fig. 1 B; it is a continuous, slightlycurvilinear relationship that extends over the full range of tolerable ambienttemperatures. This relationship was demonstrated by Benedict & MacLeod (1929),Herrington (1940) and Armitage, Harris, Hervey & Tobin (1984); it has never beensuggested on the basis of any experimental data that the relationship between heatproduction and temperature in the rat is anything other than this. The rat has onlylimited ability to thermoregulate by sweating, vasomotor control or behaviour,and the variation of metabolic rate the relationship describes is the major effector ofthermoregulation. If ambient temperature is increased sufficiently a point is reached- somewhat above 30 °C - at which metabolic rate cannot be reduced further; ratsthen wet their bodies by profuse salivation and if the situation continues are likelyto die. The environmental temperature at which this occurs is clearly 'upper critical'and is in no way comparable to the thermoneutral zone of man.The experiments to be reported were designed to test the proposition that thyroid

METABOLISM AND THYROID HORMONES IN THE COLD

hormones control the change in metabolism with ambient temperature in the rat. Ithas long been known that administration of thyroid hormones leads to increase inmetabolic rate, and that thyroid function increases in rats exposed to cold: thyroidhormones are therefore clearly a candidate for the function of controlling metabolismin relation to temperature. It must be observed, however, that the evidence relatingthyroid function to temperature is complex and there are particular difficulties inregard to the time factor.We wish to emphasize that the intention of the experiments was to investigate

whether the thyroid mediates the response shown diagrammatically in Fig. IB: thatis, the rat's normal, 'everyday' thermoregulation. In this the adjustments ofmetabolic rate take place rapidly, within hours. Although the experimentalprocedure chosen involved changing environmental temperature from 23 to 6 °C, itis important to appreciate that the metabolic response this evoked is part of acontinuous relationship that extends over all environments: it should not be thoughtof as 'a response to cold'.The experimental design was as follows. As a first step oxygen consumption and

the plasma concentrations of the hormones thyroid stimulating hormone (TSH),triiodothyronine (T3) and thyroxine (T4) were measured in rats in a controlledenvironment, first at 23 °C and then at 6 'C. The methods used provided a reasonablycontinuous record and caused minimum disturbance to the rats. The amount ofincrease in the plasma concentration of T3 was noted. Next, intravenous injectionsof T3 were given, at 23 °C, to find a dose schedule that would produce an increase inplasma T3 concentration rather more than had occurred when the ambienttemperature was lowered. Finally, this dose of T3 was administered, at 23 °C, and theincrease in oxygen consumption compared with that found when ambienttemperature was lowered. The T3 concentration produced was also checked.Comparing the increases in metabolism that occurred after the temperature change,and, in the same rats, after artificially increasing T3 concentration to the extentfound when the temperature was changed, should indicate whether the increase inoxygen consumption in response to lowering environmental temperature could- havebeen evoked by T3.

METHODS

The experiments used male Wistar rats 5 months old and weighing 300-350 g at the start. Theyhad previously been kept in a well-ventilated room maintained at 21 +1 °C and 55 + 5% relativehumidity. During the experiments they were housed two per cage in metabolism cages, which were37 x 37 x 15 cm rectangular Perspex chambers with grid floors. The initial temperature inexperiments was 23 'C. For the cold phase of the experiments the temperature was lowered to 6 'C.These temperatures were maintained within + 0 5 °C; the change in temperature was accomplishedin less than an hour. Humidity remained at 55 %. Lighting was on from 07.00 to 19.00 h. The ratshad free access to water and a standard laboratory diet (Oxoid Breeding Diet, Herbert C. Styles,Bewdley, Worcs).

Energy expenditureEnergy expenditure was measured by means of oxygen consumption using a highly accurate,

flow-over, multichannel indirect calorimeter. Details of this and of the methods of calibration anduse have been given by Armitage, Hervey, Rolls, Rowe & Tobin (1983). Four cages contained tworats each; a fifth cage was empty. Each cage was connected to the analysis line in turn for 10 min;

18 PHY 431

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the cycle of operations also included a 10 min period during which the analysers were calibrated,and so lasted 1 h. The empty cage provided blank and recovery checks in alternate cycles. Withcages of this size a period of 10 min on each cage is necessary to allow adequate wash-out and thetime resolution of the system is correspondingly limited. In terms of accuracy our systemconsistently gives agreement within 1-3% between energy expenditure and the sum of energyintake and change in carcass energy over balance periods of 5 days - a performance that we do notthink has been surpassed.

CannulationBlood samples were taken and injections given via chronic indwelling cannulae. A polyethylene

cannula (PP 25, BDH, internal diameter 0 4 mm, external diameter 0-8 mm) was implanted into ajugular vein under 2% halothane in oxygen anaesthesia. The cannula was brought out at the backof the neck and its external portion passed through an anchoring disc sutured to neck muscles. Thecannula then passed through a protective spring coil 16 cm long. Outside the cage it passed to acounterweighted loop passing over a pulley and ultimately to a syringe connection. Thisarrangement kept the cannula above the rat and protected it, while allowing the rat to move freelyaround the cage. The cannula was flushed twice daily with saline containing 100 i.u. heparin ml-'and left filled with saline containing 200 i.u. heparin ml-'. This generally assured patency of thecannula, and so continuance of the experiment, for 10 days. It was, however, still desirable todesign experiments to be completed in as short a time as possible.

Blood collectionBlood samples of 0 7 ml were taken for assays of all three hormones in the first stage of the study,

and of 0-3 ml for T3 only in the second and third stages. The samples were centrifuged immediatelyand the plasma stored at -80 'C. To avoid the taking of samples changing the concentrations ofhormones or other blood constituents the blood removed was replaced with a similar volume ofblood obtained from donor rats kept in the same environment. Assays of donor blood confirmedthat hormone concentrations in it were similar to those in experimental rats. In the first stage ofthe study samples were taken every 4 h omitting 01.00 h, and more frequently during the 24 hfollowing lowering the temperature. In the second and third stages samples were taken as describedbelow.

Hormone administration

T. was given as freeze-dried sterile laevo-iodothyronine sodium BP, Glaxo, dissolved in 0-1 mlsterile saline. This was injected into the cannula, after collection of a sample, and washed in withrat red cells suspended in heparinized saline containing Haemaccel. Doses were determined in thesecond stage of the study as described. In the final experiment 200 ng T3 per day was given as 50 ngat 07.00, 13.00, 19.00 and 01.00 h.

RadioimmunoasmaysTSH was determined in 01 ml samples of plasma with reagents and protocol supplied by

NIADDK, Baltimore, MD, USA. The reference preparation was NIAMDD-rat TSH-RP-1 and theprimary antibody was NIADDK-anti-rat TSH-serum-5. lodinated TSH was prepared frompurified rat TSH (NIADDK-rat TSH-1-6) using the Iodogen method (Fraker & Speck, 1978). Thesensitivity of the assay was 25 ng ml-' and intra- and interassay coefficients of variation were 6 and9% respectively (n = 7).

Total T3 and T4 were determined in 50 #1 samples of plasma with reagents and protocol suppliedby the Department of Nuclear Medicine, Leeds General Infirmary. The sensitivities of T, and T4assays were 0-25 and 9 nmol 1-1 respectively. Intra- and interassay coefficients of variation were forT3 6% and 8% respectively, and for T4 3% and 5% respectively. 'Bound' and 'free' componentswere not separated.

StatisticsThe significance of differences between values for hormone concentrations and for energy

expenditure at the two temperatures was tested by pair t tests. Since the number of days for whichmeasurements were available varied, values for the same rat at the same time of day at the same

546

METABOLISM AND THYROID HORMONES IN THE COLD

temperature were averaged; the resulting pair of means, for one rat at one time of day at 23 andat 6 °C, provided one pair of data for the t test.

Experimental protocolThe effects of cold exposure on thyroid hormones and energy expenditure. Rats from stock were

weighed daily for 10 days, after which four matched groups of two rats each were selected for eachexperiment, by means of a computer program that found groups optimally matched, with equalweighting, for body weight and rate of weight gain. The rats were kept in the metabolism cages fora 7 day adaptation period to allow them to become accustomed to the metabolism cages. Cannulaewere then implanted. Energy expenditure was measured during the last 3 days of the adaptationperiod to establish the normal diurnal pattern of energy expenditure. This pattern was disruptedfor 1-2 days after implanting cannulae; measurements of energy expenditure to be used as datawere therefore started 3 days after surgery. They were continued for 3 days at 23 TC, and for afurther 3 days at 6 'C.

Preliminary studies showed a diurnal rhythm in TSH concentrations, but not in T3 and T4concentrations, at room temperature. Measurements ofTSH were made for 2 days before loweringthe environmental temperature and measurements of T3 and T4 for 1 day; all continued for 3 daysat 6 'C provided the cannulae remained patent.

Establishing the dose of T3 needed to reproduce the increase in concentrationfound on exposure to 6 'C.In a first experiment the effects of single intravenous injections of widely differing doses, 100, 1000and 10000 ng, were studied. Nine rats were cannulated, allowed 2 days to recover and allocated tothree groups. Initial blood samples were taken before injections were started. Each rat was thengiven an injection of T3 at 09.00 h. Blood samples of 0 3 ml were taken 1, 5, 9, 24 and 30 h later.

In a second experiment several days later, repeated injections were given to the same rats, toidentify a dose schedule that would maintain a reasonably steady plasma T3 concentration. Fourrats were given 25 ng and five rats 50 ng T3 every 6 h (100 or 200 ng day-') for 36 h, beginning at07.00 h.

The effects of administered T3 on energy expenditure. The third and crucial stage of the studyrequired measurement of the metabolic response to injection of T3. The dose used was 50 ng every6 h beginning at 07.00 h (200 ng day-'). Eight rats were housed two per cage in metabolism cagesfor an initial 7 days; they were then cannulated; 3 days were allowed for recovery. Measurementof energy expenditure then began, and continued for 3 days. Injections of T3 were then started;measurement of energy expenditure continued for a further 3 days.

RESULTS

Responses to changing ambient temperatureEnergy expenditure

Figure 2 shows the mean hourly pattern of energy expenditure of 23 °C, beforecannulation (panels 1 and 2), after recovery from the effects of the cannulationprocedure (panels 3 and 4) and at 6 'C (panels 5-7). At 23 'C energy expenditureshowed a diurnal cycle; this was not visibly affected by blood sampling andreplacement. Mean energy expenditure over the three days at 23 'C was10-8+0-1 kJ h-' (mean+s.E.M.) during the light phase and 13-5+041 kJ h-' duringthe dark phase.When the room temperature was changed to 6 'C there was a rapid initial increase

in energy expenditure. This took around 4 h and accounted for about 70% of thetotal increase (panel 5); energy expenditure then continued to rise more slowly butappeared to have reached a plateau by the end of the 3 days at 6 'C (panel 7). Meanenergy expenditure over the 3 days at 6 'C was 19-2 +02 kJ h-' during the lightphase and 196 + 02 kJ h-' during the dark phase. The increases in energy

18-2

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E. M. WHITAKER AND OTHERS

expenditure when the temperature was changed from 23 to 6 °C, 6-1 kJ h-1 in thedark and 8-5 kJ h-1 in the light, were highly significant: 95% confidence limits wererespectively 5-5-67 and 8-0-90 kJ h-1. Careful observation of the rats throughoutthe experiment showed piloerection; they were not seen to shiver, nor to increasegeneral locomotor activity.

23 OC 6 oC

30

~2520

c 150.x

10

c 5

007.00 07.00 07.00 07.00 07.00 07.00 07.00 07.00 07.00

Time (h)

Fig. 2. Energy expenditure. Mean of four metabolism cages each holding two rats; all ratstreated similarly; each cage read for 10 min in each hour. Panels 1 and 2, pre-experiment;3 and 4, post-cannulation, starting three days after surgery; 5-7, after lowering ambienttemperature. (For clarity, standard errors are not shown; for n = 4 cages the S.E.M. forone reading was typically ± 0 I kJ h-1, range + 0 01 to+ 0 3).

Hormone concentrationsFigure 3 shows the mean concentrations of plasma TSH, T3 and T4 before and after

lowering ambient temperature. At 23 °C TSH showed a diurnal pattern with amorning peak and a night trough. After the first 24 h at 6 °C the mean concentrationof TSH remained high, and the diurnal pattern was lost. Mean plasma TSH at 23 °Cwas 316+21 ng ml-' and at 6 °C 446+ 17 ng ml-'. This difference was highlysignificant (P < 0-001 in paired t test performed as described). T3 and T4concentrations showed no obvious diurnal rhythms. The mean plasma T3concentration at 23 "C was 0 7 + 0 1nmol 1-1, and at 6 °C 1.0 + 01 nmol 1-(P < 0001). Plasma T4 concentration did not appear to change: the meanconcentration was 31 + 3 nmol 1-1 at 23 °C and 31 + 2 nmol 1-1 at 6 "C.

Replicating T3 concentration found at 6 "C by exogenously administered T3Single intravenous injectionsThe mean concentrations of T3 before injection were 0-20 + 002, 0-22 + 0-03 and

0-30 + 001 nmol 1-1 in the groups that were to receive 10000, 1000 and 100 ngT3 respectively. A dose of 10000 ng resulted in plasma concentrations greater than7 nmol I-', the upper limit of the assay, in all rats at 1 and 5 h post-injection and inone rat at 9 h, and of around 4 nmol 1-1 in the other two rats at 9 h. A dose of 1000 ng

548

METABOLISM AND THYROID HORMONES IN THE COLD

also exceeded 7 nmol 1-1 at 1 h post-injection and substantially exceeded theconcentrations found at 6 °C at 5 h. These doses were therefore considered to be toohigh. A dose of 100 ng exceeded 7 nmol 1-1 at 1 h in one rat and exceeded thephysiological concentrations for 6 °C in the other two, but at 5 h the three rats

A 4 23 °C * 0 - 6 °C -

E

I

CL

BE

0-

B

L.EC

ECl

1.5 r

1 .0-

0.5k

0c

40

w-.5EC

I-*

EClCu

30 -

20 -

10

01 -- -07.00 07.00 07.00 07.00 07.00

19.00 19.00 19.00 19.00 19.0007.00

Time (h)Fig. 3. Plasma concentrations of TSH (A), T3 (B) and T4 (C). The period covered is that

of panels 3-7 in Fig. 2. Means at 4 h intervals, + S.E.M. (n = 6-8).

showed concentrations in the range 0-7-1-25 nmol 1-1, which is comparable to theconcentrations seen at 6 'C. At 9 h T3 concentrations had returned to approximatelypre-treatment level.

Injections at six-hourly intervalsFigure 4 shows the plasma T3 concentrations achieved by injecting 100 and 200 ng

T3 daily in divided doses of 25 and 50 ng every 6 h. The horizontal lines a and b showthe lower and upper concentrations of T3 found at 6 'C. A dose of 100 ng day-'

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550 E. M. WHITAKER AND OTHERS

increased mean plasma T3 concentration from 03 + 0-1 to 12 + 01 nmol 1` over theinjection period. A dose of 200 ng day-' increased it to 1-5 + 0-2 nmol 1-1. Since themean concentration found at 6 °C was I 0+ 0 I nmol 1-1, the dose that gave the closestapproximation would appear to be 100 ng day-'. The increases in plasma T3

3v-002.0E

bE

0t t I t I

07.00 19.00 07.00 19.00 07.00Time (h)

Fig. 4. Plasma T3 concentration in rats given 25 ng (U) and 50 ng (@)T3 i.v. every 6 hat the times indicated by the arrows: mean and 1 S.E.M. a and b, lower and upper limitsof concentration found at 6 'C.

following injections, however, varied widely among individual rats (see standarderror bars in Fig. 4). To ensure that exogenous administration of T3 would at leastequal the concentration at 6 'C in every rat, it was decided that a dose of200 ng day-'(50 ng every 6 h) should be used for the final experiment. In the event the meanplasma concentration this achieved was 3-7 +0 3 nmol 1-1.

Effect on energy expenditure of replicating the T3 concentration found at 6 'C byexogenouws T3

Figure 5 compares the effect on energy expenditure of 200 ng day-' of T3 given at23 'C with that of changing ambient temperature from 23 to 6 'C. Whereas, asalready noted, exposure to 6 'C markedly increased energy expenditure and alsoabolished the diurnal rhythm, injection of 200 ng day-' of T3 had no discernibleeffect. Mean energy expenditure during the day was 9-4 kJ h-1 before treatment withT3 and 9-8 kJ h-1 over the three days of treatment; for the night the correspondingvalues were 12-5 and 12-2 kJ h-1. These changes were not statistically significant: theprecision of measurement of energy expenditure was such that, in a one-tailed test,the minimum increase that would achieve borderline significance at the P < 0-05level would be approximately +0 5 kJ h-1. Even if they had been significant thechanges with T3 treatment, +04 and -03 kJ h-1 for the day and night respectively,

METABOLISM AND THYROID HORMONES IN THE COLD

would clearly be physiologically unimportant, compared with the increases, of + 8-5and + 6-1 kJ h-1 for day and night, that occurred when the ambient temperature waschanged to 6 'C.

25 -2 3 'C - - 6 0C PI

20-

.115

10

___o_____. _ -

X 19.00 19.00 19.00 07.00 07.00 07.00 07.00xx) 07.00 07.00 07.00 19.00 19.00 19.00> 20 Time (h)

4 T3-C

15 -

10

0 - -----19.00 19.00 19.00 07.00 07.00 07.00 07.00

07.00 07.00 07.00 19.00 19.00 19.00Time (h)

Fig. 5. Energy expenditure. Above, effect of changing ambient temperature from 23 to6 °C; below, effect of T3, 200 ng day-' I.v. (s.E.M.s not shown; range (n = 4), ±0-01 to+0-4 kJ h-). The gap in the record omits three days allowed for recovery from thecannulation operation.

DISCUSSION

Although not all physiologists are familiar with the relationship between ambienttemperature and metabolic rate in rodents, it is well defined and in no doubt(Benedict & MacLeod, 1929; Herrington, 1940; Armitage et al. 1984). It provides therat's main means of thermoregulation. However, neither the tissues in whichmetabolic rate varies nor the controlling pathway have been identified.The change in daily energy expenditure found in the first phase of the present

study when the ambient temperature was changed from 23 to 6 'C is consistent withexisting knowledge of rat thermoregulation. Armitage et al. (1984) found that adultmale non-obese Zucker rats increased their metabolic rate from 190 to290 kJ rat-' d-' when the cage temperature was changed from 23 to 6 °C, i.e. anincrease by a factor of 1-5. In the present experiment the larger Wistar rats increasedtheir 24 h metabolic rate from 290 to 470 kJ rat-' d-', a factor of 1-6. Armitage et al.(1984) did not report the exact time taken for metabolic rate to change but theresponse was complete within a day; ambient temperature was, however, changed insmaller steps than in the present experiment.The diurnal cycle in energy expenditure seen at 23 'C is also consistent with

existing knowledge (Morrison, 1968; Armitage et al. 1983).

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The disappearance of the diurnal cycle at 6 °C has not been previously reported,though Armitage et al. (1984) observed that the diurnal cycle was less evident at lowtemperatures. They suggested that the increased energy expenditure would lead toincreased food intake, which might cause feeding to 'spill over' from the night to theday, although the effect of feeding upon energy expenditure they found is not in factlarge enough to account for diurnal variation of energy expenditure. The cycle inlocomotor activity may also be relevant.There is an extensive literature on the responses of the thyroid to cold, but it is

extremely confused and does not give a clear picture. It has recently beencomprehensively reviewed and somewhat clarified by Fregly (1989). Fregly's reviewclassifies studies according to time scale, which makes it possible to identify thosethat might be relevant to 'everyday' thermoregulatory variation of metabolic ratein the rat. There are some reports that plasma TSH increases within hours or evenminutes of exposure to low ambient temperature, but less evidence concerning T3. Astudy by Hefco, Krulich, Illner & Larsen (1975), however, reported increase inplasma TSH and T3 within 2 h of changing the environment of rats from 24 to 1-6 'C.Mori, Kobayashi & Wakabayashi (1978; incidentally to a study of thyrotropin-releasing hormone function) reported increased TSH and T3 concentrations in rats1 h after changing the environment from 22 to 4 'C. Where early increases have beenfound, they may be followed by oscillation between normal and raised values (Itoh,Hiroshige, Koseki & Nakatsugawa, 1966; Hefco et al. 1975). There is evidence butnot unanimity that T4 also increases. Exposure to cold for long enough for the ratsto become acclimated, i.e. for weeks, has been more extensively studied. There isclear evidence, amounting to consensus, that in rats (but not necessarily otherspecies) in this situation TSH and T3 concentrations increase; T4and protein-boundiodine do not change.

It must be expected that the concentrations of thyroid hormones, and the amountby which they increase when, for example, environmental temperature is loweredfrom 23 to 6 °C, will depend upon such factors as strain, sex, age and previousexperience of the animals. In the study of Hefco et al. (1975) the increases in plasmaTSH and T3 concentrations varied widely, from 50 to 300%. Mori et al. (1978) foundthat both approximately doubled at 1 h. The smaller increases seen in the presentstudy could be at least in part attributable to the less severe exposure to cold.The objective of the second phase of the present experiment was successfully

achieved. Measurement of hormone concentrations in the initial phase had shownthat, in the rats used and under the conditions of the experiment, in order to replicateand somewhat exceed the effect of lowering ambient temperature on T3 concentrationit was necessary to maintain a plasma T3 concentration of 1-2 nmol 1-1. In the secondphase 200 ng day-' T3 given as 50 ng every 6 h produced a reasonable steady meanplasma concentration around 1-5 nmol 1-1, but with variation among individual rats.

In the next phase of the experiment this dose produced a mean T3 concentrationof ca 3-7 nmol I-'. The difference reflects the variability in this area ofphysiology evenwhen all steps are taken to achieve uniformity. In previous studies in which T3 hasbeen infused (i.v. or s.c., into thyroidectomized rats) the relationship between thedose and elevation of plasma concentration would appear broadly similar (Silva &Larsen 1977; Connors & Hedge 1981). The fact remains that the requirement was

552

METABOLISM AND THYROID HORMONES IN THE COLD

satisfied that the exogenously produced increase in T3 concentration should be atleast as high as the physiological response at 6 °C without being grossly excessive.Throughout the experiment the use of jugular cannulae ensured that collection ofblood and administration of hormone did not stress the rats and replacement of bloodby a red cell suspension minimized body fluid disturbance.The experimental design assumes that rise in plasma concentration is the form in

which the 'message' reaches the responding tissue; for the purpose of identifying thecontrolling pathway, therefore, it seemed appropriate to measure plasma con-centration rather than, say, hormone secretion rate or clearance. There is no evidenceas to whether binding of T3 in plasma changes during the thyroid response to cold.This could possibly be important to a design in which administration of T3 was usedto mimic secretion by the thyroid in the cold and the dose was judged by totalconcentration in plasma. It would have been difficult to separate free and boundcomponents. The experimental design would only be vitiated if binding did notchange in the cold, i.e. endogenous secretion did raise the concentration of freehormone, whereas for some reason binding capacity increased during exogenousadministration, so that the effect of cold was not really simulated. This seems veryunlikely. There is similarly no reason to suppose that artificially administered T3would dramatically reduce T3 receptor concentrations where endogenous T3, atabout the same concentration, had not done so.The result of the final phase was absolutely clear. Lowering ambient temperature

to 6 °C had led within 24 h to a substantial increase in energy expenditure and lossof the diurnal cycle. Raising plasma T3 concentration artificially, to a higher levelthan had been found at 6 TC, had negligible effect upon energy expenditure and didnot alter the diurnal rhythm. This result leads to the conclusion that the increase inplasma T3 concentration when ambient temperature was lowered was not thecontrolling pathway for the increase in energy expenditure.

This may seem surprising at first sight. That cold increases thyroid function andthat thyroid hormones increase metabolic rate are familiar 'facts': together theyraise an obvious possibility that the thyroid provides the controlling pathway forvariation of metabolism with ambient temperature in the rat. The limited andconfused evidence as to rapid thyroid responses to cold has, however, already beenreferred to. The data on the relationship between thyroid hormones and metabolicrate are equally unsatisfactory.Although increase in metabolic rate in human subjects given thyroid extract by

mouth was reported by Magnus-Levy in 1895, we have been unable to find anyreview dealing with the action of T3 or T4 on metabolic rate in whole animals, andfew papers. None of the latter reports measurements, with controls, of the oxygenconsumption of intact rats given T3or T4 i.v.; doses were s.c. or i.P. and often high,and observations not begun until a day or many days after treatment had begun.Himms-Hagen's (1983) review of 'thermogenic' actions of thyroid hormones reportsonly measurements in isolated tissues (and suffers from the confusion as to what isbeing studied so often associated with the term 'thermogenesis'). Myant & Witney(1967) gave rats 25 /tg T3 i.P. every 12 h; they found an increase by 20% in oxygenconsumption 18 h after the first injection. Rothwell, Saville & Stock (1983) gave'lean' Zucker rats single injections of ca 35 jtg T3 s.c.; they found an increase in

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oxygen consumption of11 % 12 h after the injection (see Table 2 of their paper; not41 % as stated in the text). These doses are of the order of twenty times greater thanwe found reproduced the physiological response to an ambient temperature of 6°C;this suggests that the doses of T3 required to elevate metabolic rate significantly inthe short term may be supraphysiological so far as thermoregulation is concerned.The time factor must be recalled here. There is more evidence concerning long-

term than short-term responses by the thyroid to temperature change, but this isirrelevant to 'everyday' thermoregulation in the rat, which depends uponadjustments of metabolic heat production effected within at most a few hours. Theliterature that describes responses by the thyroid over weeks of exposure to cold mayprovide evidence for a thyroid-mediated mechanism that plays an ancillary role inrodents exposed to long-term cold; this would be quite distinct from 'everyday'thermoregulation, which is at issue here.The continuous nature of the relationship between ambient temperature and

metabolic rate in the rat (Fig. LB; Armitage et al. 1984, Fig.5B should also be bornein mind. It seems rather unlikely that different mechanisms are responsible fordifferent parts of such a relationship. It has, however, never been suggested thatreduction of thyroid secretion is responsible for reduction of metabolic rate in ratsmoved from temperate to hot environments.The question as to what tissues are responsible for the variation in metabolic rate

is clearly of great interest, but we can contribute little to it. Skeletal muscle shouldperhaps be considered first, if only because of the large amount of it in the body andits capacity to vary energy turnover. Our impression is that contraction is notinvolved but this has not been rigorously tested. In the human pathologicalcondition malignant hyperpyrexia, the combination of certain anaesthetics with acongenital biochemical abnormality leads to increased heat production in muscle,without contraction, that, judged by the rate of increase of body temperature, mustbe severalfold. Although pathological in man, this might be a clue to the existenceof a mechanism normal in other species.Brown adipose tissue will undoubtedly be suggested by some. The studies that led

to the discovery of its function in thermoregulation showed that brown adiposetissue is controlled by,-noradrenergic sympathetic nerves: this suggestion wouldtherefore also identify the controlling pathway. A major difficulty with it is that ratsraised to adulthood at conventional animal house temperatures possess very littlefunctioning brown adipose tissue (see review by Hervey & Tobin, 1983). Furthermorepropranolol, which the early studies showed completely blocks the response of brownadipose tissue to cold (Heim & Hull, 1966), does not affect rats' metabolic rate at23°C (Hervey & Tobin, 1982); this suggests that brown adipose tissue does notcontribute to determining whole-body metabolic rate at this temperature, which isin the middle of the range over which thermoregulatory control operates. Ratschronically exposed to cold develop brown adipose tissue, but, as in the case of thethyroid response, both the time factor and the argument from continuity of thetemperature-metabolic rate relationship suggest that this must provide an ancillarymechanism, distinct from 'everyday' thermoregulation.The term 'thermogenesis' could literally mean simply 'heat production', or more

usefully the function of brown adipose tissue in thermoregulation in animals that

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possess it. It has been used in both these senses (Himms-Hagen, 1983) and,unfortunately, in many other contexts also. The tissues responsible have often notbeen identified, and there has not always been reliable evidence that increase inmetabolism actually occurred. Some of the literature on 'non-shivering ther-mogenesis' is reviewed by Hervey & Tobin (1983); although it is often claimed orimplied that administration of noradrenaline provokes this, the increase inmetabolism is actually small and may be non-specific. The 'thermogenesis' area,therefore, is a morass in which it is difficult to build solid physiological theories.On present views, however, catecholamines are probably the next candidate to

consider for the function of controlling pathway in the rat's thermoregulation. Somesort of synergistic action of catecholamines and thyroid hormones may also be apossibility. Further consideration, however, must await further evidence.

We wish to thank the National Institute for Arthritis, Diabetes, Digestive Diseases and Kidney,Bethesda, MD, USA for donating reagents for TSH assay; and Mrs G. Coates for excellent care ofthe animals.

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