HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: 15/11/2048 00-0536fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by GOLOZOUBOVA, V. Articles by NEDERGAARD, J. Search for Related Content PubMed PubMed Citation Articles by GOLOZOUBOVA, V. Articles by NEDERGAARD, J.

Only UCP1 can mediate adaptive nonshivering thermogenesis in the cold ¹

The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-106 91, Stockholm, Sweden; and
^* Department of Biology, University of Oulu, FI-90014 Oulu, Finland

³Correspondence: The Wenner-Gren Institute, The Arrhenius Laboratories F3, Stockholm University, SE-106 91, Stockholm, Sweden. E-mail: jan{at}metabol.su.se

SPECIFIC AIM

The question of the existence of mechanisms for adaptive nonshivering thermogenesis other than that mediated by the brown fat-specific uncoupling protein 1 (UCP-1) was explored in mice, in which this protein had been genetically ablated. The physiological demand for recruitment of such putative alternative mechanisms was enhanced by exposing the animals to severe chronic cold, necessitating a large amount of extra heat production; any potential alternative molecular mechanism for adaptive nonshivering thermogenesis should then become manifest.

PRINCIPAL FINDINGS

1. UCP1-ablated mice can develop cold tolerance
The body temperature of 30°C^- acclimated wild-type mice acutely exposed to 4°C decreased rapidly (Fig. 1A ). However, 24°C^- and 18°C^- acclimated wild-type mice demonstrated no problem in maintaining normal body temperature when exposed to 4°C (Fig. 1A ); these mice thus showed the expected effect of acclimation to temperatures below thermoneutrality.

View larger version (27K): [in this window] [in a new window]   Figure 1. Body temperature and oxygen consumption (thermogenesis) of wild-type (WT) (filled symbols) and UCP1-ablated (open symbols) mice. A–C) Data presented from experiments in which mice acclimated to 30°C, 24°C, or 18°C were acutely exposed to 4°C. The body temperature of WT (A) and UCP1-ablated (B) mice was measured upon acute exposure to 4°C. C) WT and UCP1-ablated mice acclimated to 30°C or to 18°C were placed in a metabolic chamber at 4°C at the time indicated by the arrow, and the rate of oxygen consumption monitored for 1 h. Means of four animals (SE omitted). Resting metabolic rate (RMR) was not markedly different between the groups. (Animals acclimated to 30°C failed to maintain body temperature.) D–F) Effects of prolonged acclimation to 4°C. D) Body temperature of WT and UCP1-ablated mice monitored for 1 wk after transfer from 18°C to 4°C. E) Oxygen consumption of cold-acclimated (more than 3 wk at 4°C) WT and UCP1-ablated mice in the cold. F) Norepinephrine (NE, 1 mg/kg BW) was injected 30°C. Means ± SE of four mice.

Under identical conditions, the body temperature of 30°C^- acclimated UCP1-ablated mice decreased even faster than that of the wild-type mice (Fig. 1B ). UCP1-ablated mice acclimated to 24°C tolerated cold somewhat better than 30°C^- acclimated mice, but still succumbed to cold after 2 h of cold exposure. Unexpectedly, UCP1-ablated mice acclimated to 18°C could sustain their normal body temperature at 4°C (Fig. 1A , B ).

2. In the cold, UCP1-ablated mice produce as much heat as do wild-type mice
The surprising ability of the UCP1-ablated mice, acclimated to 18°C, to tolerate acute severe cold exposure could be due to decreased heat dissipation (improved insulation, etc.). However, in acute cold (Fig. 1C ), the 18°C^- acclimated, UCP1-ablated mice demonstrated exactly the same fourfold increase in oxygen consumption as wild-type mice (Fig. 1C ). Therefore, reduced heat dissipation was not the reason for the improved cold tolerance. Acclimation of these mice to 18°C had instead resulted in an impressive ability to sustain the requisite increase in oxygen consumption under conditions of acute severe cold stress, despite the total absence of UCP1 in these mice.

3. Even UCP1-ablated mice can survive in the cold for an extended time
Remarkably, the UCP1-ablated mice could sustain their body temperature at 4°C not only for hours (Fig. 1B ) but for days (Fig. 1D ), even weeks (not shown). At 4°C, their metabolic rate remained at the same high level (Fig. 1E ) as that observed during the initial acute cold exposure (Fig. 1C ) and was not different from that in the wild-type mice (Fig. 1E ). Thus, despite UCP1 ablation, these mice had developed a mechanism for heat production efficient enough to support prolonged survival in the cold. In these mice, any alternative mechanisms for adaptive nonshivering thermogenesis could therefore be optimally examined.

4. The mechanism for heat production evolved in UCP1-ablated mice is not adrenergic
In UCP1-ablated mice we have as yet been unable to observe any recruitment of adrenergically induce thermogenesis. To test the possibility that a novel, adrenergically induced but UCP1-independent nonshivering thermogenesis could have evolved in these mice exposed to a more severe cold stress than UCP1-ablated mice earlier studied, norepinephrine was injected at thermoneutrality into mice acclimated to 4°C. As expected, wild-type animals responded with a massive increase in oxygen consumption (Fig. 1F ) which was as high as that observed in the cold (Fig. 1E ). In contrast, the effect of norepinephrine injection was minor in the cold-acclimated UCP1-ablated mice and did not differ significantly from the effect of saline (not shown). Thus, the heat production that had developed in these mice was not an adrenergically stimulated thermogenesis. Rather, an alternative mechanism of sustained heat production was used by the cold-acclimated UCP1-ablated mice.

5. No induction of any alternative mechanism for adaptive nonshivering thermogenesis in UCP1-ablated mice
Two types of thermoregulatory heat production are recognized: shivering and nonshivering thermogenesis. To test whether an alternative nonshivering thermogenesis had developed in the UCP1-ablated mice, we examined the effect of cold acclimation on shivering intensity (Fig. 2 ).

View larger version (28K): [in this window] [in a new window]   Figure 2. Shivering in wild-type and UCP1-ablated mice. EMG recordings on wild-type and UCP1-ablated mice acclimated to 30 or to 4°C for more than 3 wk. Safety pin-type electrodes (2 measuring, 1 ground) were inserted under the skin over the back muscles of the mice. The EMG was recorded as rectified, integrated, and noise-filtered data. Examples of 4 min segments of recordings are shown in panels A, B, D, and E. Each set of recordings at 4°C (broken line) and at 30°C (solid line) was made on the same animal on the same experimental occasion. Mean minimal muscle electrical activity of wild-type (C) and UCP1-ablated (F) mice acclimated to 30°C (WA) and to 4°C (CA) and examined at 30°C and at 4°C is shown. Data are means ± SE of 5 animals in each group. ***Significant (P0.001, unpaired t test) effect of acclimation.

The muscular electrical activity of wild-type mice acclimated to 30°C and examined at 30°C was low and stable (Fig. 2A ). When acutely exposed to 4°C, these mice shivered intensely, and muscular electrical activity increased markedly (Fig. 2A , 2C ). The oxygen consumption was correspondingly increased (cf. Fig. 1C ). Cold acclimation had a clear effect on their muscle electrical activity at 4°C: these mice no longer shivered (Fig. 2B, C ). Their oxygen consumption remained very high (cf. Fig. 1E ): fourfold higher than that at 30°C. Thus, as expected, cold acclimation of wild-type mice resulted in the development of ‘classical’ adaptive nonshivering thermogenesis: oxygen consumption remained elevated but shivering was substituted by adaptive nonshivering thermogenesis.

In the 30°C^- acclimated UCP1-ablated mice, basal muscle electrical activity was not different from that of the corresponding wild-type animals (Fig. 2D , F ). Upon acute exposure to 4°C, these mice shivered as intensely as the corresponding wild-type mice (Fig. 2D , F ). However, cold acclimation did not result in any cessation of shivering. Rather, the UCP1-ablated mice continued to shiver unabatedly in the cold (Fig. 2E , 2F ); the muscle activity of the cold-acclimated UCP1-ablated mice was as high as that of the warm-acclimated mice (Fig. 2F ). Thus, in the UCP1-ablated mice, shivering remained the only mechanism of heat production even after cold acclimation.

6. Decreased long-term survival in the cold in mice without UCP1
All wild-type mice survived at 4°C for more than 6 months. However, although UCP1-ablated mice could survive for weeks, between wk 10 and 20 most UCP1-ablated mice died. Thus, even the ‘improved’ shivering that had developed in the UCP1-ablated mice during cold acclimation was not sufficient for long-term survival in the cold. We could not identify cause of death.

CONCLUSIONS

UCP1-ablated mice are unable to produce heat in their brown adipose tissue, and their survival in the cold is thus fully dependent on heat from other sources. One of the innate limitations of gene ablation, the possible induction of compensatory mechanisms, was an advantage in this case, as it was exactly the search for alternative mechanisms for adaptive thermogenesis that was the purpose of the present study. Remarkably, the UCP1-ablated mice survived for prolonged periods (many weeks) in surroundings (4°C) where they were obligated to constantly produce heat at fourfold resting levels. However, despite these extreme requirements for adaptive thermogenesis, no substitution of shivering by any adaptive nonshivering thermogenic process occurred. Thus, the following conclusions may be drawn.

All adaptive nonshivering thermogenesis in the cold results from the activity of UCP1
Although it was originally suspected that adaptive nonshivering thermogenesis was located in muscle, the recognition of the heat-producing ability of brown adipose tissue made this tissue an alternative candidate. However, that this minor tissue should be solely responsible for the dramatic augmentation in metabolism seen after cold acclimation has remained in dispute, and the idea that an alternative and additional mechanism to that in brown adipose tissue could exist has persisted. It is, however, the outcome of the present investigation that no adaptive nonshivering thermogenesis exists in the absence of UCP1 (Fig. 2) .

No muscular adaptive nonshivering thermogenesis
Muscle is the tissue that has most consistently been invoked as being responsible for non-brown fat-derived nonshivering thermogenesis, primarily due to its large mass and high potential metabolic capacity. However, evidence for the existence of muscle-derived thermogenesis is only circumstantial; it is limited to theoretical considerations of possible mechanisms and to observations of alterations in muscle morphology, enzyme complement or gene expression. Our experiments indicate that muscle adaptive nonshivering thermogenesis cannot be the reason for the alterations in the muscles. Instead, shivering may be considered a muscle training process. Even in wild-type mice, muscle training occurs at least during the initial period of acclimation and some alterations observed in muscles of these animals are very similar to observations on the effect of endurance training with respect to e.g., muscle capillary density and mitochondrial oxidative capacity. In the UCP1-ablated mice the initial acclimation to 18°C apparently gave sufficient muscular training to allow for the subsequent survival at 4°C.

In addition to muscle, other organs have been suggested to be sites of adaptive nonshivering thermogenesis, for example. However, our studies do not support the existence of adaptive thermogenesis localized to visceral organs.

No adaptive nonshivering thermogenesis can be induced by cold through other members of the uncoupling protein family
From cDNA libraries, sequences corresponding to proteins more closely related to the original uncoupling protein (UCP, thermogenin, UCP1) than to any other known protein have been identified; the corresponding proteins have been named UCP2 and UCP3. The function of these UCP1-like proteins remains unclear, but it has when ectopically expressed, they can uncouple mitochondria. If UCP2 and UCP3 were to function as mitochondrial uncouplers, as does UCP1, they could be responsible for any UCP1-independent nonshivering thermogenesis. The present results can therefore also be seen as an analysis of the ability of UCP2/UCP3, etc., to function as mediators of ‘alternative’ cold acclimation-recruited adaptive thermogenesis. The results clearly demonstrate that in the absence of UCP1, the organism has no ability to develop any cold-recruited adaptive nonshivering thermogenesis induced by any endogenous hormone or neurotransmitter in any organ in the body (Fig. 3 ). Thus, UCP2, UCP3, or any other protein except UCP1, related or not, cannot be involved in cold acclimation-recruited nonshivering thermogenesis. Mediation of cold-recruited nonshivering thermogenesis remains, therefore, the function exclusively of UCP1.

View larger version (34K): [in this window] [in a new window]   Figure 3. No adaptive nonshivering thermogenesis can be recruited by cold exposure in animals without UCP1. When acutely exposed to cold, both UCP1-ablated (left diagram) and wild-type mice increase their metabolism through shivering. In UCP1-ablated mice, shivering intensity does not decrease with time, even after many weeks in cold. In contrast, in animals with UCP1, the capacity for nonshivering thermogenesis is successively increased (i.e., adaptive thermogenesis is induced) and shivering therefore declines. The ability to perform adaptive nonshivering thermogenesis therefore resides in UCP1 alone. Thus, no UCP1-independent ‘thermogenic’ processes in different cells of the body (including substrate cycles, mitochondrial proton leak, Na+/K+-ATPase, ‘new’ uncoupling proteins UCP2/UCP3, etc.) show an adaptive response (which would have led to decreased shivering). All adaptive nonshivering thermogenesis recruited by cold acclimation emanates from UCP1.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0536fje ; to cite this article, use FASEB J. (July 9, 2001) 10.1096/fj.00-0536fje

² Present address: Department of Medicine, Royal Brisbane Hospital, Herston, Queensland 4029, Australia.

This Article Full Text (PDF) All Versions of this Article: 15/11/2048 00-0536fjev1    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by GOLOZOUBOVA, V. Articles by NEDERGAARD, J. Search for Related Content PubMed PubMed Citation Articles by GOLOZOUBOVA, V. Articles by NEDERGAARD, J.