biochemical aspects of acclimation to cold

d. therm. Biol. Vol. 8,. pp. 85 to 90, 1983 0306-4565 83 010085-06S05.00 0 Printed in Great Britain. All rights reserved Copyright © 1983 Pergamon Press Ltd

REVIEW

BIOCHEMICAL ASPECTS OF ACCLIMATION TO COLD

BARBARA CANNON and JAN NEDERGAARD The Wenner-Gren Institute, University of Stockholm, Norrtullsgatan 16, S-113 45 Stockholm, Sweden

Abstract--Acclimation to cold in mammals is manifested by an increase in non-shivering thermogenesis which can be measured experimentally as an increased thermogenic response to injected norepinephrine. The functional parallel to this adaptation is an "'activation" of brown adipose tissue. The biochemical parameter most informative of the degree-of-activation is probably the "'content" of thermogenin in the brown adipose tissue of the cold-acclimated animal.

INTRODUCTION

IT IS EVIDENCE for the force of evolution that it has been able to adapt life to a variety of surroundings and to spread life on an earth where the temperature passes through all possibilities from a glittering cold with creaking snow to a radiant heat with a shadow- less sun.

In the biochemical acclimation to cold--which is one of the manifestations of this adapta t ion-- the problem has been solved in two principally distinct ways: one for the poikilotherms, another for the homeotherms.

In poikilotherms the problem is to make life efficient and functional at a range of different temperatures within the organism itself. Here evolution may have selected isoenzymes which, e.g. by increased affinity, compensate for the decrease in activity which is a thermodynamic consequence of a lowered working temperature. It may also be necessary to change membrane composition so that a reasonable degree of fluidity is maintained at lower temperatures, and changes in homeostatic levels of, e.g., electrolytes or acid-base balance may be necessary to allow the processes of life to be maintained in an altered physical environment.

In homeotherms the situation is, in principle, quite different. Changes in isoenzyme composition, membrane fluidity or electrolytes do not serve any obvious purpose, as it is exactly the point of homeothermia that the reaction temperature for chemical and physical processes is kept constant. Although several studies have found changes in such parameters as a consequence of acclimation to cold, their physiological significance is not immediately evident (Cannon & Polnaszek, 1976), and some of the described phenomena may indeed be secondary effects of, e.g., an increased food intake in the cold.

Rather. the solution to the problem for birds and mammals must be to strengthen the effector mechanisms for temperature control, i.e. to decrease heat loss or to increase heat production.

A decrease in heat loss can be obtained by mechanisms such as a decreased blood flow to the periphery, an increased insulation of the body, behavioural mechanisms etc. Here we shall not discuss these acclimation possibilities further.

In the question of both birds and mammals it is clear that these animals have means of increasing the heat production of the body, and these means are augmented during acclimation to cold. In the question of birds it must be admitted that we know very little of the physiological and biochemical mechanisms behind this phenomenon.

However, in the question of mammals we now have some understanding of the mechanism.

DELINEATION OF THE PROBLEM: THE MANIFESTATION OF ACCLIMATION

TO COLD

In order to delineate the problem, we have described in Fig. I the principal changes in metabolism caused by acclimation to cold.

As seen, an animal living at thermoneutral temperatures (here stated as 28°C) has a certain basal metabolism. This is naturally in principle a "non-shivering thermogenesis" (NST) as this is a heat production which does not involve muscle contraction, but this is not a thermoreyulatory NST (cf. Bligh & Johnson, 1973).

When such an animal is placed in a cold environment (here 5°C), it must - - in order to remain in heat balance--increase its heat production, and it does this through muscle contractions: shivering.

If, instead, such an animal is injected with norepinephrine (NE), an increase in metabolism can often be observed. This is relatively small, and the cause of it is not fully known. Here we call it "metabolic". It may principally be divided into two parts:

One part which may represent "the effector organ for NST (i.e. brown adipose tissue (BAT), see below) being slightly activated even at thermoneutral temperatures, e.g. as a consequence of diet-induced thermogenesis (Rothwell & Stock, 1979) or simply representing a minimal level of brown fat being present.

The other part may stem from organs not involved in, and not being effector organs for, "true" (i.e. thermoregulatory) NST, e.g. the muscles and the liver. It does seem that both muscles (Mejsnar & Jansky,

85

86 BARBARA CANNON and JAN NEDERGAARD

BMR,

shivering

I

Control

28"C 5"C 28"C+NE

bolic

Cold-acclimated

| NST,

i t cold- i n d uce d

28"C 5"C 2B*C+NE

Fig. 1. Delineation of the problem. This principal sketch indicates the differences in response to cold stress and NE injection between control and cold-acclimated animals. The size of the bars (which indicate heat production or Oz consumption) is arbitrary although the relative responses indicated are of the order seen in rats. 28°C indicates the thermoneutral zone for the animal; 28°C + NE indicates the response to NE injection at this temperature. The effect of acclimation to cold is marked as "cold-

induced". For further discussion see text.

1971; Grubb & Folk, 1976, 1977) and liver can respond to NE addition with a slightly increased metabolism. For liver this is seen both when NE is perfused through the liver (Sugano et al., 1980), in liver slices from catecholamine-treated animals (Bern- stein et al., 1975) and even with liver cells in vitro as a direct consequence of NE addition (Dehaye et al., 1981). There is reason to think that these effects are due to c(-adrenergic stimulation. These responses may be related to the "non-NE-thermogenesis" discussed by Jansky (1978), especially as they-- i f they are c(-adrenergic in na ture - -may be more sensitive to epinephrine than to NE. It is important to realize that these effects of NE on liver or muscles have never been demonstrated to be augmented by acclimation to cold; thus this again is not a thermoregulatory NST. It should be noted that as a consequence of the existence of these phenomena, there is no simple re- lation between the response to NE injection in a non- cold-acclimated animal and the potential of "the effector organ for NST (brown fat) in that animal. The greater part of NE-stimulated metabolism in such animals may result from a stimulation of other organs.

In the cold-accl imated mammal the picture is different. As seen in Fig. 1, if a cold-acclimated mammal is placed at thermoneutral temperatures (28°C) it does not display an elevated metabolism; the basal metabolic rate is virtually unchanged.

However, if this cold-acclimated animal is observed at the temperature to which it is acclimated (here 5°C), it displays an elevated metabol ism--but not increased shivering. Thus the capacity for NST has increased. This is the ' true' (i.e. thermoregulatory) N S T , developed as an alternative to shivering.

Parallel to this, the response to NE injection is increased so that this level and the level of heat production in the cold is almost the same.

It is this increased response to NE injection, induced by acclimation to cold, to which we shall restrict ourselves here.

THE EFFECTOR ORGAN FOR NST

There is today no doubt that the main effector organ for cold-induced (thermoregulatory) NST is BAT. Although Heim & Hull (1966) had already rea- lized that BAT was the effector organ for NST in the newborn rabbit (i.e. most of the increased 02 consumption takes place there), it took 12 years before Foster & Frydman (1978) demonstrated that this was also the case in the traditional experimental animal: the adult rat. In a cold-acclimated rat injected with NE. more than a third of the total blood flow is di- rected to the brown fat, and this blood is virtually depleted of O : during the passage through the tissue. This means that BAT has an immense capacity for 02 consumption, and the development of this capacity is the biochemical mechanism behind acclimation to cold.

BIOCHEMICAL PARAMETERS FOR AN

INCREASED "'DEG R EE-OF-ACTI VATION" OF BAT

Thus, BAT must possess a biochemical mechanism which allows for its potent ability to consume O2 and thus to produce heat. Several parameters have been utilized in order to quantify and characterize the development of the biochemical acclimation in BAT. Here, we shall briefly discuss some of these parameters and their advantages and disadvantages.

( I ) T issue wet wei.qht

The first observation of a connection between acclimation to cold and BAT was that of Pag6 & Babi- neau (1950) that the wet weight of the tissue was twice as high in cold-acclimated rats as in control rats. Although this observation did point in the correct direction, this simple parameter is the one which gives the greatest difficulty in interpretation. This is because an increase in wet weight may indicate an activation of the tissue; it may however also reflect a lowered

Biochemical aspects of

degree-of-activation of the tissue, resulting from a de- position instead of a combustion of lipids in BAT and through this to an increase in weight. This fact has, e.g., blurred the discussion of the effect of thyroid hormone on the tissue. Hyperthyroidism leads to an increase in wet weight (Heick et al., 1973; Sundin, 1981), but (as discussed below) this is actually due to a deactivation of the tissue.

(2) D N A

Activation of brown fat is generally accompanied by hyperplasia (e.g. Cameron & Smith, 1964; Thom- son et al., 1969), and this increase in cell number may be taken as a measure of an activation. However, there are examples of activation of the tissue which are not followed by an increase in D N A , notably this seems to be the case during cold acclimation of the Djungarian hamster (Rafael et al., 1981).

(3) Protein content

An increase in the lipid-free dry weight (i.e. protein content) of the tissue is seen as an effect of acclimation to cold (Pag6 & Babineau, 1950), but due to the unspecificity of such changes, rate-limiting enzymes should be searched for.

200

I00

nmol 0 mi n. mg

cold 02

contr¢

Polm, corn

\

cold I - - NS

control ~ ' I

~ P

FCCP I rain

Fig. 2. Demonstration of the rate-limiting step for 02 consumption of BAT mitochondria in vitro. Mitochondria were isolated from control and cold-acclimated hamsters as described by Sundin et at. (1982). After addition of substrate (Palmcarn: palmitoyl-carnitine, 50/~M in presence of 3 mM malate) a spontaneous rate of respiration occurs. This can be totally inhibited by the addition of GDP (2.5 mMk indicating that this rate is fully dependent on the activity of thermogenin. After addition of the artificial uncoupler FCCP ( 10/~M), the respiration is only limited by the oxidative enzymes. Adapted from Sundin et al. (1982).

acclimation to cold 87

(4) CytOchrome-c-oxidase ( C O X )

Following the work by Jansky (1963; Jansky et al.. 1969), a series of investigators have measured this activity because it may be taken as a measure of the maximal O2-consuming ability of a tissue.

This may be criticized because other O2-consuming reactions could occur. However, the only such activity reported, the peroxisomal acyl-CoA-oxidase, has even in brown fat of cold-acclimated rats a negligible activity in comparison with COX (Nedergaard et al.. 1980).

Thus, although it is correct that the activity of COX determines the upper level of the oxidation rate in the tissue, experiments in vitro and in vivo do not seem to indicate that COX activity is normally the rate-limiting factor.

With experiments in vitro (Fig. 2) it can be seen that isolated brown fat mitochondria presented with a substrate (here palmitoyl-carnitine) do not spon- taneously utilize their full potential for oxidation of that substrate. Only when an artificial uncoupler (here FCCP) is added does the oxidation sequence (the de- hydrogenases of the fl-oxidation, the citric-acid cycle and the COX) work at top speed. It will be noted from Fig. 2, that this oxidation rate (per mg mitochondrial protein) is unchanged by acclimation to cold.

Similarly, there are situations in vivo where the COX capacity is unchanged but the thermogenic capacity is changed. One is the obese (ob/ob) mouse compared to control ("lean") mice. These mice have a reduced thermogenic response, which occurs before the onset of obesity (Trayhurn et al., 1977). The wet weight of brown fat of the mice is increased (cf. (1)), but the protein and the COX activity are unchanged (Himms-Hagen & Desauteis, 1978). Thus the differ- ence between normal mice and ob/ob mice cannot be explained by such parameters.

(5) Thermogenin concentration (nmol/mq mitochondrial protein)

From a long series of studies it is now evident that the ability of BAT to allow respiration to proceed without it being coupled to ATP-synthesis is due to a probably brown-fat-specific protein: thermogenin (for reviews see Nicholis, 1979; Cannon et al., 1981 ; Lind- berg et al., 1981 ; Nedergaard & Lindberg, 1982).

Thermogenin (cf. Fig. 3) is a polypeptide of mol. wt 32,000, which is able to bind purine nucleotides, e.g. GDP, and which is at least a part of the "natural protonophore" of the inner mitochondrial membrane of BAT. The amount of this protein (in its active form) can easily be estimated by measuring the ability of GDP to bind to isolated mitochondria. Due to the very high affinity of thermogenin for GDP (Kin below 1/JM (Sundin & Cannon, 1980)), the binding of GDP is specific to thermogenin in the range below l0/aM, i.e. there is only one binding site for GDP (Nichoils, 1976; Sundin & Cannon, 1980). Thus, competition studies, performed with a 100-fold excess (as is normally the case for receptor studies (cf. Svoboda et al., 1979)) of GDP, totally abolish this binding (H'~rdefelt & Sundim unpublished): i.e. there is no unspecific binding.


T. Momentary GDP] GT P L, - - acy I - Co A A D P | ~ / ATPJ ~ /

Y o ° . - " o . -

11. "Short- term' ? ]2I. 'Lon(~- term"

Fig. 3. Regulation of thermogenin activity. The role of thermogenin is to allow OH- to pass through the mitochondrial membrane, thus uncoupling the mitochondria. Three different modes of regulation are

indicated on the figure, i-thermogenin indicates the hypothetical inactive form.

It is the concentration of thermogenin in the mitochondria which--at least in vitro---determines the maximal rate of thermogenesis observed. One example of this is seen in Fig. 2: the "spontaneous" rate of oxidation of added substrate is increased as an effect of acclimation to cold. That all of this respiration can be inhibited by the addition of GDP makes it evident that the respiration is fully dependent upon thermogenin.

The concentration of thermogenin in the mitochondria increases dramatically during the 3 weeks when adaptation to cold occurs in, e.g., the rat (Desautels et al., 1978; Sundin & Cannon, 1980), up to a level of about 0.7 nmol per mg mitochondrial protein (Sundin & Cannon, 1980).

However, after this phase, the specific concentration starts to decrease, and after some weeks more of acclimation, the specific concentration is close to starting values (Desautels et al., 1978; Sundin & Can- non. 1980). During this phase, the capacity of the rat for NST remains elevated (Depocas, 1960). Therefore the simple correlation between thermogenin concentration and NST does not at first glance seem to hold.

(6) Total content of thermoyenin

However, not only are there changes in thermogenin concentration (per mg mitochondrial protein) during acclimation; the total amount of mitochondrial protein also increases. As the increase in other mitochondrial enzymes seems to be slower than the increase in thermogenin, this obviously leads to a decrease in the relative content of thermogenin. When the thermogenin concentration and the total amount of mitochondrial protein are multiplied together, a measure of total thermogenin content of BAT is obtained, and this value remains elevated and constant during prolonged acclimation to cold (Sundin & Cannon, 1980). This thus seems to be the best biochemical parameter for acclimation to cold.

CONSEQUENCES OF THE USE OF THERMOGENIN "'CONTENT" AS A

BIOCHEMICAL CRITERION FOR ACCLIMATION

The identification of thermogenin "'content" as the rate-limiting step for thermogenesis makes several observations more comprehensible.

First, the increase in thermogenin content during acclimation to cold is vast (nearly 50-fold increase). This is naturally much more than the increase in NE- stimulated respiratory rate observed after acclimation to cold. but--as discussed above (Fig. I)--in the non- cold-acclimated animal, the major part of the response to a NE injection may take place in non-BAT. Only when an animal is acclimated to cold is BAT thermogenesis dominating, and then there is probably a simple relationship between thermogenin content and the capacity for thermoregulatory NST.

Secondly, hyperthyroidism leads to a decrease in thermogenin content (Sundin, 1981). Thus--as discussed by Sundin (1981)---the increased basal metabolism caused by hyperthyroidism decreases the need for brown-fat heat production, and brown fat is deactivated {and brown-fat wet weight increased due to lipid accumulation).

Thirdly, the obese (ob/ob) mouse has, despite un- altered protein and COX content in the tissue, a lowered thermogenin content (Desautels & Himms- Hagen, 1978). As this occurs before the onset of obesity {at least in the db/db obese mouse (Goodbody & Trayhurn, 1981)) the lack of thermogenin has been suggested to be both the cause of obesity and the cause of the diminished NST.

THE REGULATION OF THERMOGENIN

If it is the amount of thermogenin which is decisive for the thermogenic capacity of BAT, great interest

Biochemical aspects of acclimation to cold St;

must be placed on the control of the activity of this enzyme.

We suggest that it is possible {as sketched in Fig. 3) to discuss three different types of regulation of thermogenin activity: the momentary regulation, the short-term regulation and the long-term regulation.

I. The momentary reyulation

The momentary regulation is the background for the rapid (time-course of minutes) switch-on and switch-off of thermogenesis in the tissue as an effect of changes in acute thermoregulatory demand. The negative modulators are in this case probably cytoso- lic purine nucleotides; the positive modulators (acti- vators) may- -as we suggested earl ier--be acyl-CoAs (Cannon et al., 1977).

I1. Short-term reyulation

A series of disparate observations may perhaps be understood if an inactive form of thermogenin (with approximately the same molecular weight) is postu- lated. This "i-thermogenin" could then be activated and deactivated (e.g. by phosphorylation) as a short- term effect of constant stimulation. Such a putative i-form would fit with the fact that hibernators (e.g. hamsters) do not show any change in the amount of protein of tool. wt of 32,000, despite the fact that GDP-binding (Himms-Hagen & Gwilliam, 1980) and degree-of-uncoupling is increased during acclimation to cold (Sundin et al., 1982). Also some effects of diet- induced thermogenesis could be fitted to such a model (Himms-Hagen et al., 1981), as well as the ability of injected NE to elevate thermogenin content in the absence of protein synthesis (Desauteis & Himms-Hagen, 1979). We consider this a short-term effect, because it may also be the mechanism behind the very rapid (hours to a day) changes in thermogenin content (determined as E D P binding) seen immediately at the onset or cessation of a cold stress (Desautels & Himms-Hagen, 1979). These rapid changes also appear not to require protein synthesis.

III . Loncl-term re qulation

This would be the type of regulation involving the synthesis of protein and would require days to weeks.

THE INDUCERS OF REGULATION OF THERMOGENIN

The hormonal and neuronal factors behind the suggested short-term and long-term changes in thermogenin content are as yet largely unknown. Through pharmacological studies on brown fat in situ, we and others have initiated the search for the agents respon- sible for these changes. As yet, the results are incon- clusive, although some pharmacological treatments definitely lead to changes in thermogenin content.

Probably the resolution of the regulatory mechanisms behind acclimation to cold will result from a combination of such results obtained in vivo with results obtained in vitro, i.e. from studies on differen- tiating brown fat cells grown in cell culture under controlled condi t ions--a promising system for future studies.

Acknowledgement--Our group is supported by the Swedish Natural Science Research Council.

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Key Word lndex--Thermogenesis: cold acclimation: norepinephrine: brown fat: mitochondria: thermogenin.

biochemical aspects of acclimation to cold

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