The recent insight that brown adipocytes and muscle cells share a common origin and in this respect are distinct from white adipocytes has spurred questions concerning the origin and molecular characteristics of the UCP1-expressing cells observed in classic white adipose tissue depots under certain physiological or pharmacological conditions. Examining precursors from the purest white adipose tissue depot (epididymal), we report here that chronic treatment with the peroxisome proliferator-activated receptor ␥ agonist rosiglitazone promotes not only the expression of PGC-1␣ and mitochondriogenesis in these cells but also a norepinephrine-augmentable UCP1 gene expression in a significant subset of the cells, providing these cells with a genuine thermogenic capacity. However, although functional thermogenic genes are expressed, the cells are devoid of transcripts for the novel transcription factors now associated with classic brown adipocytes (Zic1, Lhx8, Meox2, and characteristically PRDM16) or for myocyte-associated genes (myogenin and myomirs (muscle-specific microRNAs)) and retain white fat characteristics such as Hoxc9 expression. Co-culture experiments verify that the UCP1-expressing cells are not proliferating classic brown adipocytes (adipomyocytes), and these cells therefore constitute a subset of adipocytes ("brite" adipocytes) with a developmental origin and molecular characteristics distinguishing them as a separate class of cells.

The phenomenon of white fat "browning," in which certain white adipose tissue depots significantly increase gene expression for the uncoupling protein UCP1 and thus supposedly acquire thermogenic, fat-burning properties, has attracted considerable attention. Because the mRNA increases are from very low initial levels, the metabolic relevance of the change is unclear: is the UCP1 protein thermogenically competent in these brite/beige-fat mitochondria? We found that, in mitochondria isolated from the inguinal "white" adipose depot of cold-acclimated mice, UCP1 protein levels almost reached those in brown-fat mitochondria. The UCP1 was thermogenically functional, in that these mitochondria exhibited UCP1-dependent thermogenesis with lipid or carbohydrate substrates with canonical guanosine diphosphate (GDP) sensitivity and loss of thermogenesis in UCP1 knockout (KO) mice. Obesogenic mouse strains had a lower thermogenic potential than obesity-resistant strains. The thermogenic density (UCP1-dependent oxygen consumption per g tissue) of inguinal white adipose tissue was maximally one-fifth of interscapular brown adipose tissue, and the total quantitative contribution of all inguinal mitochondria was maximally one-third of all interscapular brown-fat mitochondria, indicating that the classical brown adipose tissue depots would still predominate in thermogenesis.

The mtDNA mutator mice have high levels of point mutations and linear deletions of mtDNA causing a progressive respiratory chain dysfunction and a premature aging phenotype. We have now performed molecular analyses to determine the mechanism whereby these mtDNA mutations impair respiratory chain function. We report that mitochondrial protein synthesis is unimpaired in mtDNA mutator mice consistent with the observed minor alterations of steady-state levels of mitochondrial transcripts. These findings refute recent claims that circular mtDNA molecules with large deletions are driving the premature aging phenotype. We further show that the stability of several respiratory chain complexes is severely impaired despite normal synthesis of the corresponding mtDNA-encoded subunits. Our findings reveal a mechanism for induction of aging phenotypes by demonstrating a causative role for amino acid substitutions in mtDNA-encoded respiratory chain subunits, which, in turn, leads to decreased stability of the respiratory chain complexes and respiratory chain deficiency.

Elucidation of the regulation of uncoupling protein 1 (UCP1) activity in its native environment, i.e. the inner membrane of brown-fat mitochondria, has been hampered by the presence of UCP1-independent, quantitatively unresolved effects of investigated regulators on the brown-fat mitochondria themselves. Here we have utilized the availability of UCP1-ablated mice to dissect UCP1-dependent and UCP1-independent effects of regulators. Using a complex-I-linked substrate (pyruvate), we found that UCP1 can mediate a 4-fold increase in thermogenesis when stimulated with the classical positive regulator fatty acids (oleate). After demonstrating that the fatty acids act in their free form, we found that UCP1 increased fatty acid sensitivity ϳ30-fold (as compared with the 1.5-fold increase reported earlier based on nominal fatty acid values). By identifying the UCP1-mediated fraction of the response, we could conclude that the interaction between purine nucleotides (GDP) and fatty acids (oleate) unexpectedly displayed simple competitive kinetics. In GDP-inhibited mitochondria, oleate apparently acted as an activator. However, only a model in which UCP1 is inherently active (i.e."activating" fatty acids cannot be included in the model), where GDP functions as an inhibitor with a K m of 0.05 mM, and where oleate functions as a competitive antagonist for the GDP effect (with a K i of 5 nM) can fit all of the experimental data. We conclude that, when examined in its native environment, UCP1 functions as a proton (equivalent) carrier in the absence of exogenous or endogenous fatty acids.

Most physiologically induced examples of recruitment of brown adipose tissue (BAT) occur as a consequence of chronic sympathetic stimulation (norepinephrine release within the tissue). However, in some physiological contexts (e.g., prenatal and prehibernation recruitment), this pathway is functionally contraindicated. Thus a nonsympathetically mediated mechanism of BAT recruitment must exist. Here we have tested whether a PPARγ activation pathway could competently recruit BAT, independently of sympathetic stimulation. We continuously treated primary cultures of mouse brown (pre)adipocytes with the potent peroxisome proliferator-activated receptor-γ (PPARγ) agonist rosiglitazone. In rosiglitazone-treated cultures, morphological signs of adipose differentiation and expression levels of the general adipogenic marker aP2 were manifested much earlier than in control cultures. Importantly, in the presence of the PPARγ agonist the brown adipocyte phenotype was significantly enhanced: UCP1 was expressed even in the absence of norepinephrine, and PPARα expression and norepinephrine-induced PGC-1α mRNA levels were significantly increased. However, the augmented levels of PPARα could not explain the brown-fat promoting effect of rosiglitazone, as this effect was still evident in PPARα-null cells. In continuously rosiglitazone-treated brown adipocytes, mitochondriogenesis, an essential part of BAT recruitment, was significantly enhanced. Most importantly, these mitochondria were capable of thermogenesis, as rosiglitazone-treated brown adipocytes responded to the addition of norepinephrine with a large increase in oxygen consumption. This thermogenic response was not observable in rosiglitazone-treated brown adipocytes originating from UCP1-ablated mice; hence, it was UCP1 dependent. Thus the PPARγ pathway represents an alternative, potent, and fully competent mechanism for BAT recruitment, which may be the cellular explanation for the enigmatic recruitment in prehibernation and prenatal states.

A physiological function of the original uncoupling protein, UCP1, is well established: UCP1 is the molecular background for nonshivering thermogenesis. The functions of the "novel" UCPs, UCP2 and UCP3, are still not established. Recent discussions imply that all UCPs may play a role in protection against reactive oxygen species (ROS). Here we examine critically the evidence that UCP1, UCP2 and UCP3 are stimulated by ROS (superoxide) or ROS products (4-hydroxy-2-nonenal), and that the UCPs actually diminish oxidative damage. We conclude that, concerning UCP1, it is unlikely that it has such a role; concerning UCP2/UCP3, most evidence for physiologically significant roles in this respect is still circumstantial.

During the recruitment process of brown adipose tissue, the mRNA level of the fatty acyl chain elongase Elovl3 is elevated more than 200-fold in cold-stressed mice. We have obtained Elovl3-ablated mice and report here that, although cold-acclimated Elovl3-ablated mice experienced an increased heat loss due to impaired skin barrier, they were unable to hyperrecruit their brown adipose tissue. Instead, they used muscle shivering in order to maintain body temperature. Lack of Elovl3 resulted in a transient decrease in the capacity to elongate saturated fatty acyl-CoAs into very long chain fatty acids, concomitantly with the occurrence of reduced levels of arachidic acid (C20:0) and behenic acid (C22:0) in brown adipose tissue during the initial cold stress. This effect on very long chain fatty acid synthesis could be illustrated as a decrease in the condensation activity of the elongation enzyme. In addition, warmacclimated Elovl3-ablated mice showed diminished ability to accumulate fat and reduced metabolic capacity within the brown fat cells. This points to ELOVL3 as an important regulator of endogenous synthesis of saturated very long chain fatty acids and triglyceride formation in brown adipose tissue during the early phase of the tissue recruitment.Brown adipose tissue is the only tissue that functions exclusively to combust fat for heat production (1-3). When mammals encounter cold, there is an induced synthesis of specific mRNA species in brown adipose tissue, which encode enzymes regulating energy expenditure and lipid metabolism (4 -6). Most notable is the increased mRNA level of the mitochondrial uncoupling protein 1 (UCP1), which can uncouple the mitochondrial respiratory chain and thereby dissipate heat instead of conserving energy in the form of ATP. The induction of processes needed for increased brown adipose tissue activity is referred to as brown fat recruitment.The process is controlled by norepinephrine release from sympathetic nerve endings found in the tissue (7, 8) and can roughly be divided into two major phases, depending on how the animal experiences the cold (4°C) (i.e. cold stress or cold acclimation). During the first days of cold exposure (i.e. cold stress), the major effect on brown adipose tissue is the initiation of hypertrophy, and a few days later, during the onset of cold acclimation, hyperplasia occurs (9, 10). After 4 days of cold exposure, there is a doubling in DNA amount, and after 3 weeks, a 3-fold increase is detected, which is the maximal level achieved with this degree of cold (11,12). The increase in the total amount of protein in cold-exposed mice occurs also as a two-phase phenomenon (5). The first phase is completed within 3 days, and the second phase of protein increase (i.e. during cold acclimation) is found in the tissue after 2-3 weeks of cold exposure. During these events, protein synthesis mainly reflects an increase in mRNA levels (5).During the process of identifying specific cDNA molecules corresponding to mRNA species that are induced in the brown adipose ti...

Mice lacking the thermogenic mitochondrial membrane protein UCP1 (uncoupling protein 1)--and thus all heat production from brown adipose tissue--can still adapt to a cold environment (4 degrees C) if successively transferred to the cold. The mechanism behind this adaptation has not been clarified. To examine possible adaptive processes in the skeletal muscle, we isolated mitochondria from the hind limb muscles of cold-acclimated wild-type and UCP1(-/-) mice and examined their bioenergetic chracteristics. We observed a switch in metabolism, from carbohydrate towards lipid catabolism, and an increased total mitochondrial complement, with an increased total ATP production capacity. The UCP1(-/-) muscle mitochondria did not display a changed state-4 respiration rate (no uncoupling) and were less sensitive to the uncoupling effect of fatty acids than the wild-type mitochondria. The content of UCP3 was increased 3-4 fold, but despite this, endogenous superoxide could not invoke a higher proton leak, and the small inhibitory effect of GDP was unaltered, indicating that it was not mediated by UCP3. Double mutant mice (UCP1(-/-) plus superoxide dismutase 2-overexpression) were not more cold sensitive than UCP1(-/-), bringing into question an involvement of reactive oxygen species (ROS) in activation of any alternative thermogenic mechanism. We conclude that there is no evidence for an involvement of UCP3 in basal, fatty-acid- or superoxide-stimulated oxygen consumption or in GDP sensitivity. The adaptations observed did not imply any direct alternative process for nonshivering thermogenesis but the adaptations observed would be congruent with adaptation to chronically enhanced muscle activity caused by incessant shivering in these mice.