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Special Review Series Biogenesis and Physiological Adaptation of Mitochondria

Translation and Integration

Cold-induced recruitment of brown adipose tissue


thermogenesis
Martin Klingenspor
Animal Physiology, Department of Biology, Philipps-University, Marburg, Germany

Experimental Physiology :

Non-shivering thermogenesis in brown adipose tissue is the main mechanism for thermoregulatory heat
production in small mammals and newborns. During cold acclimation the sympathetic innervation triggers
the recruitment of brown adipose tissue by hyperplasia, which involves the proliferation and differentiation of
precursor cells, and by hypertrophy of mature brown adipocytes. Mitochondrial biogenesis and increased
synthesis of the uncoupling protein 1 (UCP-1) are hallmarks of the thermogenic recruitment process. The
severalfold increase of mitochondrial protein content during cold acclimation recruits a large capacity for
oxidative phosphorylation. However, UCP-1 increases proton leakage across the inner membrane of brown
adipocyte mitochondria and thereby dissipates proton motive force as heat instead of ATP synthesis. During
recent years considerable progress has been achieved in the analysis of transcriptional mechanisms controlling
Ucp1 gene expression. However, so far only little is known about the molecular basis of cold-induced
mitochondrial biogenesis in brown adipose tissue. Experimental Physiology (2003) 88.1, 141148.

Abbreviations
AR, adrenergic receptor
c-FOS, immediate early gene (transcription factor)
COX, cytochrome c oxidase
CRE, cAMP response element
CREB, cAMP response element binding protein
DIO-2, deiodinase type 2
mtTFA, mitochondrial transcription factor
NF-E2, nuclear factor-erythroid 2
NFE212, member of the NF-E2 transcription factor family
NRF-1, nuclear respiratory factor 1
NRF-2, nuclear respiratory factor 2
NST, non-shivering thermogenesis
PGC-1, peroxisome proliferator-activated receptor g
coactivator
PPARg, peroxisome proliferator-activated receptor g
PPRE, PPAR response element
RAR, retinoic acid receptor
RARE, retinoic acid response element
RXR, retinoid X receptor
TR, thyroid receptor
TRE, thyroid hormone response element
UCP-1, uncoupling protein

Publication of The Physiological Society


2508

In a cold environment endothermic animals dispose of


different thermoregulatory mechanisms to prevent hypothermia. Thermoregulatory heat production may be
increased by either non-shivering thermogenesis (NST),
shivering, or physical activity, and heat loss may be decreased
by improving fur insulation (pilus erection), cutaneous
vasoconstriction, shallow hypothermia or behavioural
changes (change of posture, huddling). In small mammals
and neonates NST in brown adipose tissue is the most
important thermoregulatory mechanism to defend normothermia in the cold (Himms-Hagen, 1984). Prolonged cold
exposure causes the adaptive recruitment of NST capacity
by hyperplasia of brown adipose tissue and by hypertrophy
of mature brown adipocytes which results in a severalfold
increase in the capacity for NST. Early studies demonstrated
that in cold-acclimated animals brown adipose tissue may
dissipate heat at a power of 300400 W kg_1 tissue, and
contributes more than ~6070 % of total NST (Foster &
Frydman, 1979; Heldmaier & Buchberger, 1985; Puchalski
et al. 1987). This emphasized the outstanding importance
of this thermogenic organ for NST, but also suggested the
existence of other unknown mechanisms and sites of NST
in the body.
The morphological features of brown adipose tissue suit
the function of this thermogenic organ (Cinti, 2001). The
dense vascularization ensures a sufficient supply of oxygen
and metabolic substrates, and the direct sympathetic

Email: klingens@mailer.uni-marburg.de

Experimental Physiology :

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142

M. Klingenspor

innervation of brown adipocytes mediates the central


control of NST. In contrast to white adipocytes, brown
adipocytes store triglycerides in multilocular lipid
droplets and are rich in mitochondria, showing multiple
invaginations of the internal membrane (cristae). These
mitochondria are uniquely equipped with a protontranslocating membrane protein, the uncoupling protein 1
(UCP-1), which cold-exposed animals use to dissipate
proton motive force as heat and maintain high rates of
substrate oxidation in the absence of ATP synthesis (Nicholls
& Rial, 1999). As hallmarks of adaptive recruitment of
thermogenic capacity during cold acclimation, expression
of the Ucp1 gene and mitochondrial biogenesis are strongly
increased (Klaus et al. 1991). The physiological importance
of UCP-1 for NST has been underlined by the thermoregulatory disorder observed in UCP-1_/_ mice, which
develop severe hypothermia when acutely exposed to 4 C
(Enerbck et al. 1997). After successive cold acclimation,
instead of acute exposure, these mice are able to maintain
normothermia at 4 C, but they do so solely by shivering
thermogenesis (Golozoubova et al. 2001; Nedergaard et al.
2001). Novel UCPs have been identified which show a
wider tissue distribution than UCP-1, but most likely have
no functional significance for NST (Ricquier & Bouillaud,
2000; see article by Nedergaard & Cannon in this issue).
Sympathetic stimulation of non-shivering
thermogenesis
The acute stimulation of NST and the recruitment of
increased NST capacity in response to cold exposure are
primarily mediated by the sympathetic neuronal connection
between the brain and brown adipose tissue (Seydoux &
Girardier, 1978). In cold-exposed animals, sympathetic
nerve fibres which densely innervate brown adipose tissue
increase noradrenaline release in the direct vicinity of
brown adipocytes. Mapping of the central nervous system
origins of sympathetic nerve fibres using a transneuronal
viral tract tracing technique revealed most viral staining in
the paraventricular nucleus and the medial preoptic area of
the hypothalamus (Bamshad et al. 1999). In the rat, cooling
of the preoptic area with implanted thermodes elicits NST
in brown adipose tissue (Banet et al. 1978). In rats acutely
exposed to cold, c-FOS expression mapping in the brain,
which is regarded as an indirect marker of neuronal activity,
revealed strong activation of the preoptic area (Baffi &
Palkovits, 2000). These findings correspond to the
established view that the preoptic area integrates peripheral
and central thermal information in the rostral hypothalamus (Boulant, 2000).
Noradrenaline released by sympathetic nerve fibres in
brown adipose tissue binds with different affinities to
multiple a and b adrenergic receptor (AR) isoforms
expressed in brown adipocytes. Brown adipose tissue
hyperplasia is stimulated through b1-AR (Himms-Hagen
et al. 1994; Bronnikov et al. 1999; Klaus et al. 2001),
whereas the lipolytic and thermogenic action is primarily
mediated by b3-AR (Zhao et al. 1998; Chaudhry &

Exp Physiol 88.1

Granneman, 1999), but the functional significance of


adrenergic receptor heterogeneity has not been finally
resolved.
b-AR signalling is coupled to the adenylyl cyclasemediated rise of intracellular cAMP level, which, as a
second messenger, stimulates lipolysis and uncoupled
respiration in brown adipocytes. Increased cAMP levels
release the catalytic subunits of protein kinase A which
phosphorylate multiple targets including hormone-sensitive
lipase (Shih & Taberner, 1995) and perilipin (Chaudhry &
Granneman, 1999). The fatty acids released by increased
lipolysis have a dual role (Nicholls & Rial, 1999). Firstly,
fatty acids activate UCP-1, which causes the dissipation of
proton motive force as heat and results in a reduction of
the mitochondrial membrane potential. Secondly, free
fatty acids are metabolized by mitochondrial b-oxidation.
In the uncoupled state the respiratory chain will exhibit
maximal oxygen consumption as there is no feedback
inhibition by proton motive force.
The role of thyroid hormones in non-shivering
thermogenesis
A complex role of thyroid hormones in the control of nonshivering thermogenesis in brown adipose tissue is evident.
Thyroidectomy does not alter the thermogenic properties
of brown adipose tissue in warm-acclimatized rats, but
strongly impairs the ability to activate non-shivering
thermogenesis in the cold (Triandafillou et al. 1982). On
the other hand, thyroidectomy of cold-acclimated rats
does not affect brown adipose tissue thermogenic capacity
(Zaninovich et al. 2002). The function of thyroid hormones
in the control of thermogenic responses in brown adipose
tissue is tightly linked to the noradrenergic signalling
pathways in brown adipocytes (Bianco & Silva, 1987).
Activation of the type 2 deiodinase (DIO-2), which
catalyses the conversion of T4 to T3 in brown adipocytes,
occurs rapidly in response to cold-induced noradrenergic
stimulation of brown adipose tissue and results in the mere
saturation of nuclear thyroid receptors (TR) with T3.
Brown adipocytes express different thyroid hormone
receptors, of which TRb appears to primarily regulate
Ucp1 gene transcription, whereas TRa is probably required
for the maintenance of b-adrenergic responsiveness
(Ribeiro et al. 2001). In Djungarian hamsters surgical
denervation of brown adipose tissue almost completely
abolished the cold-induced rise in DIO-2 activity (Meywirth
et al. 1990) and UCP-1 mRNA level (Klingenspor et al.
1994). Furthermore, the pharmacological inhibition of
DIO-2 activity with iopanoic acid decreases the coldinduced rise in UCP-1 mRNA level (Reiter et al. 1990).
Expectedly, Dio2_/_ mice develop moderate hypothermia
when exposed to cold for 24 h and show a blunted increase
of brown adipose tissue temperature in response to
noradrenaline infusion, which is caused by a reduced
coupling of noradrenergic signalling to adenylyl cyclase in
brown adipocytes (de Jesus et al. 2001). Concurrently, the
cold-induced rise of UCP-1 mRNA levels in brown adipose

Exp Physiol 88.1

Cold-induced mitochondrial biogenesis

Experimental Physiology :

Translation and Integration

tissue is blunted in Dio2_/_ mice as compared to wild-type


mice. The resulting disorder of NST in brown adipose
tissue of Dio2_/_ mice can be normalized by T3 treatment.
Hyperplasia and hypertrophy of brown adipose tissue
In rats and mice hyperplasia and hypertrophy provide two
distinct mechanisms by which noradrenaline triggers the
adaptive recruitment of brown adipose tissue. Hyperplasia
of brown adipose tissue is due to a transient increase in the
proliferation of endothelial cells, interstitial cells and preadipocytes followed by the differentiation of preadipocytes
to brown adipocytes (Bukowiecki et al. 1982; Bronnikov
et al. 1999). The differentiation programme of brown
adipocytes is characterized by the induction of Ucp1 gene
expression and mitochondrial biogenesis and thus is
fundamentally different from adipogenesis of white
adipocytes (Klaus, 1997).
In contrast to white adipose tissue, only little is known
about the molecular mechanisms of adipogenesis in brown
adipose tissue. Different lines of evidence suggest that the
determination towards either the brown or the white
adipocyte lineage occurs at the precursor cell level.
Preadipocytes isolated from brown and white adipose
tissue and grown under identical culture conditions
preferentially differentiate to white and brown adipocytes,
respectively (Klaus et al. 1995). Distinct differences in
electron microscopic features of precursor cells in brown
and white adipose tissue have been observed (Cinti, 2001).
Furthermore expression profiling experiments have
identified several genes which are differentially expressed
in preadipocytes isolated from white and brown adipose
tissue (Boeuf et al. 2001; Moulin et al. 2001). The biological
function of these genes in preadipocyte determination and
differentiation of brown adipocytes remains to be elucidated.
However, the observation of different phenotypes at the
structural and molecular level strongly suggests that
preadipocytes are already predetermined to become either
brown or white.
In parallel to the development of brown adipocytes during
cold acclimation, hypertrophy of terminally differentiated
brown adipocytes occurs. In warm-acclimated animals
these brown adipocytes already express basal levels of UCP-1
and contain a relatively high number of mitochondria as
compared to preadipocytes. In response to noradrenaline
they increase the synthesis of UCP-1 and mitochondrial
proteins. Clearly, the regulation of Ucp1 gene expression
and mitochondrial biogenesis in mature brown adipocytes
must be different as compared to differentiating brown
preadipocytes, but little is currently known about what
distinguishes these two processes at the molecular level. In
the following, the transcriptional mechanisms controlling
Ucp1 gene expression and mitochondrial biogenesis will be
briefly discussed.
Transcriptional control of the Ucp1 gene
Promoter studies have provided insight to the transcriptional
mechanisms controlling the Ucp1 gene (Silva & Rabelo,

143

1997). In the mouse Ucp1 gene, a ~200 bp enhancer


element 2.32.5 kb upstream to the start of transcription
confers differentiation-dependent expression and cAMP
responsiveness of the gene (Cassard-Doulcier et al. 1993;
Kozak et al. 1994; Larose et al. 1996). A similar enhancer
element is also present in the rat and human Ucp1 gene. In
this enhancer region functional cis-acting response elements
have been identified which act as DNA binding sites for
different members of the nuclear receptor family of
transcription factors, including the peroxisome proliferatoractivated receptor g (PPARg), the retinoic X receptor
(RXR), the retinoic acid receptor (RAR) and the thyroid
receptor (TR). Their corresponding ligands, synthetic
thiazoledinediones, retinoids and triiodothyronine,
respectively, promote transactivation of the Ucp1 gene.
Differentiation-dependent cAMP responsiveness of the Ucp1
enhancer can be conveyed by PPARg. The coexpression of
PPARg and RXRa activates the Ucp1 enhancer in brown
preadipocytes treated with dibutyryl-cAMP (Sears et al.
1996). The finding suggests a key role of PPARg in brown
adipocyte differentiation, but this nuclear receptor is also
essential for the differentiation of white adipocytes, which
do not express UCP-1. The search for physical interaction
partners of PPARg in brown adipocytes led to the
identification of the novel PPARg coactivator (PGC-1), a
transcriptional regulator preferentially expressed in
metabolically active organs such as brown adipose tissue,
heart, kidney, brain and skeletal muscle (Puigserver et al.
1998). The coexpression of PPARg and PGC-1 in brown
adipocytes may therefore be involved in cell-specific
expression of UCP-1.
Currently, four mechanisms are known to confer noradrenergic stimulation of transcriptional activity of the
Ucp1 enhancer in differentiated brown adipocytes (Fig. 1).
Firstly, several putative cAMP response elements were
identified in the Ucp1 promoter of which functional
significance was confirmed by mutational analysis (Kozak
et al. 1994). A rapid increase of in the concentration of the
phosphorylated cAMP response element binding protein
(pCREB) occurs in response to noradrenergic stimulation
of cultured brown adipocytes (Thonberg et al. 2002). Rim
& Kozak (2002) reported that pCREB binds as a homodimer to the CRE2 in the Ucp1 enhancer region and activates
transcription. As a second noradrenergic mechanism of
transcriptional activation, a functional nuclear factorerythroid 2 (NF-E2) response element was found which
upon binding of NFE212, a member of the NF-E2 family,
transactivates Ucp1 gene expression in a cAMP-dependent
manner (Rim & Kozak, 2002).
Two other mechanisms of noradrenergic stimulation of
Ucp1 gene expression exist, which involve cAMP-mediated
upregulation of DIO-2 and PGC-1 synthesis. Cold exposure
causes noradrenergic stimulation of DIO-2 activity and
increase of T3 synthesis which stimulates the transcriptional
activity of TRb at the Ucp1 enhancer (Silva & Rabelo,
1997). Furthermore, in brown adipose tissue of mice

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M. Klingenspor

exposed to cold, and in differentiated brown adipocytes


treated with a b-adrenergic agonist, PGC-1 mRNA levels
are increased (Puigserver et al. 1998). Coexpression studies
in brown adipocytes have demonstrated that PGC-1
strongly coactivates PPARg/RXRa and TRb/RXRa in a
ligand- (troglitazone, T3 and 9-cis retinoic acid, respectively)
and cAMP-dependent manner.
Transcriptional control of mitochondrial biogenesis
A broader role of PGC-1 has been suggested in the
coordination of mitochondrial biogenesis. In myocytes
PGC-1 enforces the gene expression of nuclear respiration
factors (NRF-1 and NRF-2/GABA), transcription factors
which target nuclear subunits of the respiratory chain and
genes involved in mitochondrial DNA transcription and
replication (Wu et al. 1999). Ectopic expression of PGC-1
in a white adipocyte cell line not only induces UCP-1
mRNA levels, but also strongly elevates mRNA levels for
nuclear- (COX-IV, b-F1-ATPase) and mitochondrial-(COXII) encoded genes of the respiratory chain as well as the
copy number of mitochondrial genome per cell. Based on
these findings it was hypothesized that, in brown adipocytes

Exp Physiol 88.1

exposed to noradrenaline, increasing PGC-1 levels may


coordinate the transcriptional upregulation of mitochondrial
and nuclear genes important for mitochondrial biogenesis
by coactivation of NRF-1 as well as the nuclear receptors
controlling the Ucp1 gene (Lowell & Spiegelman, 2000).
Three subunits of cytochrome c oxidase (COX) are
transcribed from the mitochondrial genome, whereas
the remaining components are encoded on nuclear
chromosomes. The mRNA levels of mitochondrial COX-I
and COX-III are not increased in brown adipose tissue of
cold-exposed Djungarian hamsters (Fig. 2 and Klingenspor
et al. 1996b) and the COXII mRNA level is transiently
increased by 50 % in rats exposed to cold for 6 h (Martin et
al. 1993). This indicates that transcriptional control of the
mitochondrial genome is not involved in cold-induced
mitochondrial biogenesis, whereas the regulation of gene
expression of nuclear-encoded mitochondrial proteins is
essential.
The mechanisms controlling mitochondrial biogenesis in
brown adipose tissue have been investigated for the

Figure 1
Direct and indirect cAMP-dependent pathways involved in the transcriptional control of the Ucp1
enhancer at ~2.4 kb upstream of transcription start site.

Experimental Physiology :

Translation and Integration

Exp Physiol 88.1

Cold-induced mitochondrial biogenesis

mitochondrial F0F1-ATP synthetase. In mammals, assembly


of the F0F1-ATP synthetase requires six subunits for the
catalytic domain (F1) and ten subunits for the protontranslocating domain (F0). All subunits are encoded by
nuclear genes, except for two subunits of the F0-ATPase
domain (Houstek et al. 1995). In order to gain insight into
the mechanisms involved in the transcriptional regulation
of mitochondrial biogenesis, the control of the nuclearencoded mitochondrial subunit b-F1-ATPase gene (b-F1)
has been investigated during brown adipocyte differentiation
(Villena et al. 1998). The promotor activity of the b-F1
gene is increased when HIB1B preadipocytes enter
differentiation and the responsible enhancer region was
shown to primarily depend on a response element for the
nuclear respiratory factor 2 (NRF-2). The identification of
functional NRF-2 binding sites in several nuclear genes
encoding mitochondrial proteins suggests an important
role for this factor in transcriptional coordination of
mitochondrial biogenesis (Scarpulla, 2002).
However, in brown adipose tissue the mRNA levels of
b-F1, as well as several other nuclear- and mitochondrialencoded subunits do not correspond to F0F1-ATP
synthetase protein levels in mitochondria (Houstek et al.
1995). This indicates that transcriptional control may be
limited to a few genes encoding essential subunits of the
multiprotein complex, whereas other subunits may be
expressed constitutively. In brown adipose tissue mitochondria the content of F0F1-ATP synthetase in relation to
other respiratory complexes is lower than in heart and
other metabolically active tissues, and is even further
reduced during cold acclimation (Houstek et al. 1995).
Despite a low rate of F0F1-ATPase biosynthesis, the mRNA
levels of nuclear-encoded subunits in brown adipose tissue
and heart are comparable, except for the c-F0 mRNA,
which is exceptionally low in brown adipose tissue. During
cold acclimation of the rat and postnatal development of

Figure 2
Effect of 7 days cold exposure on
mitochondrial and nuclear gene
expression in brown adipose tissue of the
Djungarian hamster. The mRNA levels of
12S rRNA, COX-I and COX-III
(mitochondrial), COX-IV, COX-Va and
UCP-1 were quantified by slot blot
analysis and related to the 28S rRNA
hybridization signal. A two-sample t test
(unpaired) was applied to compare means;
* P < 0.05.

145

the golden hamster the b-F1 mRNA level is increased in


brown adipose tissue whereas the c-F0 mRNA level is
decreased (Houstek et al. 1995; Andersson et al. 1997).
Comparisons of subunit mRNA and protein levels
demonstrate that changes in the subunit c-F0 mRNA
coincide with mitochondrial ATPase concentration.
Therefore the biosynthesis of subunit c-F0, which is
essential for proton transport, may the limiting factor for
the assembly of the F0F1-ATPase complex in brown
adipose tissue. This would spare the need for complex
global control mechanisms which coordinate the
transcription of all nuclear and mitochondrial genes
encoding the multiprotein complex.
Hypertrophy of brown adipocytes
In different situations an increase of NST capacity can be
observed in the absence of hyperplasia. Treatment of rats
with the selective b3-AR agonist CL316,243 causes an
increase in mitochondrial mass, but has no effect on cell
proliferation and differentiation (Himms-Hagen et al. 1994).
b3 adrenergic stimulation of mitochondrial biogenesis
cannot be observed in primary cultured hamster brown
adipocytes in the presence of insulin and T3 (Klaus et al.
2001), but chronic T3 treatment alone or in combination
with insulin promotes the expression of UCP-1 and
increases the respiratory (thermogenic) capacity without
affecting proliferation and differentiation (Klaus et al.
1995). In the mature brown adipocytes of cold-exposed
animals the increase of intracellular T3 concentration by
DIO-2 activation may be a key hormonal signal involved in
mitochondrial biogenesis.
In Djungarian hamsters the improvement of NST capacity
during cold acclimation is primarily based on the
hypertrophy of mature brown adipocytes, whereas only a
small increase in the cellularity of the tissue occurs
(Klingenspor et al. 1996a). In fact the tissue wet weight
decreases during cold acclimation, due to a decrease in the

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M. Klingenspor

cellular triglyceride content. The mere lack of tissue


hyperplasia allows the selective study of Ucp1 gene
expression and mitochondrial biogenesis in mature brown
adipocytes during cold acclimation in vivo. After 2 weeks
of cold acclimation UCP-1 mRNA and protein levels are
~3-fold elevated and COX activity, as an index for mitochondrial protein, is increased ~2.5-fold in brown adipose
tissue (Klingenspor et al. 1996a). Surgical denervation of
brown adipose tissue, which strongly decreases the
noradrenaline content of the tissue, almost completely
diminishes the cold-induced rise in Ucp1 expression and
COX activity (Klingenspor et al. 1994) confirming the
prominent role of the sympathetic innervation for cold
acclimation.
The increase in mitochondrial biogenesis observed in
brown adipose tissue of cold-acclimated hamsters does not
involve an increase in mitochondrial DNA content per cell
(Klingenspor et al. 1996b). This is fundamentally different
from mitochondrial biogenesis in differentiating brown
preadipocytes, where replication of the mitochondrial
genome occurs. In Djungarian hamsters exposed to cold
for 7 days a strong stimulation of UCP-1 mRNA expression
was observed in brown adipose tissue. The mRNA level of
mitochondrial transcription factor A (mtTFA), which has
been shown to control replication and transcription of the
mitochondrial genome, was not increased (Klingenspor et
al. 1996b). Accordingly, the rise in COX activity was not
associated with increased mRNA levels for the mitochondrially encoded subunits COX-I and COX-III (Fig. 2).
A small but significant increase was found for the nuclearencoded COX-IV mRNA, suggesting that this gene may be
involved in the transcriptional control of COX biosynthesis
in brown adipocytes. Possibly transcriptional regulation of
only a few subunit genes, such as COX-IV, may promote
the biosynthesis of the COX multiprotein complex.
The small changes observed in COX gene expression
suggest that post-transcriptional mechanisms are of
importance for mitochondrial biogenesis. Mitochondria
isolated from brown adipose tissue of hamsters after 7 days
of cold acclimation showed a 2.5-fold higher rate of
protein synthesis (Klingenspor et al. 1996b). The higher
translation activity may be partly due to an increased total
RNA content of mitochondria, but may also involve the
regulation of mitochondrial translation efficiency.
Retrograde regulation of mitochondrial biogenesis in
adipocytes
Accumulating evidence suggests that mitochondrial
biogenesis is not exclusively regulated by external stimuli
but can be triggered by a postulated energy-sensing
mechanism in situations of decreased coupling of
mitochondrial respiration. Induction of UCP-1 expression
in HeLa cells leads to increased oxygen consumption and
results in a NRF-1-dependent rise in the activity of
d-aminolevulinate synthase, a rate-limiting enzyme for the
synthesis of haem, which is regarded as an early marker of
mitochondrial biogenesis (Li et al. 1999). In skeletal

Exp Physiol 88.1

muscle, the activation of AMP-activated protein kinase is


associated with increases of NRF-1 binding, cytochrome c
levels and muscle mitochondrial density, which suggests
that a decrease in the cellular ATP/AMP ratio may trigger
mitochondrial biogenesis (Bergeron et al. 2001).
In transgenic mice which overexpress UCP-1 in adipose
tissue, an increase in mitochondrial protein content was
found (Rossmeisl et al. 2002). This ectopic UCP-1
expression was associated with elevated COX-IV mRNA
levels and increased cytochrome content in white adipose
tissue. Ultrastructural appearance of white adipocyte
mitochondria and morphometric analysis of mitochondria
revealed a stimulatory effect on mitochondrial content and
biogenesis by the presence of UCP-1. Interestingly, treatment
of cultured 3T3-L1 adipocytes with the artificial uncoupler
2,4-dinitrophenol increased the transcript levels for COXIV as well as NRF-1, indicating that NRF-1 may initiate
coordinated expression of nuclear and mitochondrial
genes required for mitochondrial biogenesis (Rossmeisl et
al. 2002). Taken together, these findings suggest that high
expression levels of UCP-1, as such, may activate
mitochondrial biogenesis in brown adipocytes through the
proposed energy-sensing mechanism.
In conclusion, the currently limited knowledge on
mitochondrial biogenesis in brown adipose tissue during
cold acclimation suggests the involvement of transcriptional
as well as translational control mechanisms. The latter
appears to be of major importance in mature brown
adipocytes which undergo hypertrophy in response to cold
exposure, whereas transcriptional regulation is most likely
to be crucial for hyperplasia, when brown adipocytes are
recruited from preadipocytes.

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Acknowledgements
Many thanks to Gerhard Heldmaier, Carola Meyer, Christa von
Praun and Tatjana Schneider for helpful discussions and comments
on draft versions of this review. Our studies are supported by the
German Research Foundation (DFG).

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