Within brown-fat cells, UCP1-mediated fatty acid-induced uncoupling is independent of fatty acid metabolism.

In the present investigation, we have utilized the availability of UCP1(-/-) mice to examine a wide range of previously proposed lipid activators of Uncoupling Protein 1 (UCP1) in its native environment, i.e. in the brown-fat cells. A non-metabolizable fatty acid analogue, beta,beta cent-methyl-substituted hexadecane alpha,omega-dicarboxylic acid (Medica-16) is a potent UCP1 (re)activator in brown-fat cells, despite its bipolar structure. All-trans-retinoic acid activates UCP1 within cells, whereas beta-carotene only does so after metabolism. The UCP1-dependent effects of fatty acids are positively correlated with their chain length. Medium-chain fatty acids are potent UCP1 activators in cells, despite their lack of protonophoric properties in mitochondrial membranes. Thus, neither the ability to be metabolized nor an innate uncoupling/protonophoric ability is a necessary property of UCP1 activators within brown-fat cells.

Decreased brown adipocyte recruitment and thermogenic capacity in mice with impaired peroxisome proliferator-activated receptor (P465L PPARgamma) function.

Mice with a dominant-negative peroxisome proliferator-activated receptor gamma (PPARgamma) mutation (P465L) unexpectedly had normal amounts of adipose tissue. Here, we investigate the adipose tissue of the PPARgamma P465L mouse in detail. Microscopic analysis of interscapular adipose tissue of P465L PPARgamma mice revealed brown adipocytes with larger unilocular lipid droplets, indicative of reduced thermogenic capacity. Under conditions of cold exposure, the brown adipose tissue of the PPARgamma P465L mice was less active, a fact reflected in decreased uncoupling protein 1 levels. Analysis of the white adipocytes confirmed their normal cytoarchitecture and development, yet classical white adipose depots of the P465L PPARgamma mice had a striking reduction in brown adipocyte recruitment, a finding supported by reduced expression of UCP1 in the perigonadal adipose depot. Taken together, these data suggest that whole animal impairment of PPARgamma alters the cellular composition of the adipose organ to a more "white" adipose phenotype. Physiologically, this impairment in brown adipocyte recruitment is associated with decreased nonshivering thermogenic capacity after cold acclimation as revealed by norepinephrine responsiveness. Our results indicate that maintenance of oxidative brown-like adipose tissue is more dependent on PPARgamma function for development than white adipose tissue, an observation that may be relevant when considering PPARgamma-dependent strategies for the treatment of obesity.

ELOVL3 is an important component for early onset of lipid recruitment in brown adipose tissue.

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, warm-acclimated 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.

SOD2 overexpression: enhanced mitochondrial tolerance but absence of effect on UCP activity.

We have created P1 artificial chromosome transgenic mice expressing the human mitochondrial superoxide dismutase 2 (SOD2) and thus generated mice with a physiologically controlled augmentation of SOD2 expression leading to increased SOD2 enzyme activities and lowered superoxide levels. In the transgenic mice, effects on mitochondrial function such as enhanced oxidative capacity and greater resistance against inducers of mitochondrial permeability were observed. Superoxide in the mitochondrial matrix has been proposed to activate uncoupling proteins (UCPs), thus providing a feedback mechanism that will lower respiratory chain superoxide production by increasing a proton leak across the inner mitochondrial membrane. However, UCP1 and UCP3 activities and mitochondrial ATP production rates were not altered in isolated mitochondria from SOD2 transgenic mice, despite lowered superoxide levels. Globally, the transgenic mice displayed normal resting metabolic rates, indicating an absence of effect on any UCP activities, and normal oxygen consumption responses after norepinephrine injection. These results strongly suggest that endogenously generated matrix superoxide does not regulate UCP activity and in vivo energy expenditure.

A human-specific role of cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA) in adipocyte lipolysis and obesity.

Elevated circulating fatty acid concentration is a hallmark of insulin resistance and is at least in part attributed to the action of adipose tissue-derived tumor necrosis factor-alpha (TNF-alpha) on lipolysis. Cell death-inducing DFFA (DNA fragmentation factor-alpha)-like effector A (CIDEA) belongs to a family of proapoptotic proteins that has five known members in humans and mice. The action of CIDEA is unknown, but CIDEA-null mice are resistant to obesity and diabetes. We investigated CIDEA in adipose tissue of obese and lean humans and mice. The mRNA was expressed in white human fat cells and in brown mouse adipocytes. The adipose mRNA expression of CIDEA in mice was not influenced by obesity. However, CIDEA expression was decreased twofold in obese humans and normalized after weight reduction. Low adipose CIDEA expression was associated with several features of the metabolic syndrome. Human adipocyte depletion of CIDEA by RNA interference stimulated lipolysis and increased TNF-alpha secretion by a posttranscriptional effect. Conversely, TNF-alpha treatment decreased adipocyte CIDEA expression via the mitogen-activated protein kinase c-Jun NH(2)-terminal kinase. We propose an important and human-specific role for CIDEA in lipolysis regulation and metabolic complications of obesity, which is at least in part mediated by cross-talk between CIDEA and TNF-alpha.

Inhibitory effects of halothane on the thermogenic pathway in brown adipocytes: localization to adenylyl cyclase and mitochondrial fatty acid oxidation.

Volatile anesthetics such as halothane efficiently inhibit nonshivering thermogenesis as well as the cellular manifestation of that phenomenon: norepinephrine-induced respiration in brown adipocytes. To identify the molecular site(s) of action of such anesthetics, we have examined the effect of halothane on the sequential intracellular steps from the interaction of norepinephrine with isolated brown adipocytes to the stimulation of mitochondrial respiration (=thermogenesis). We did not identify an inhibition at the level of the adrenergic receptors, but a first site of inhibition was identified as the generation of cAMP by adenylyl cyclase; this led to inhibition of norepinephrine-induced expression of the uncoupling protein-1 (UCP1) gene and reduced norepinephrine-induced lipolysis as secondary effects. Although an inhibition of lipolysis in itself would inhibit thermogenesis, circumvention of this inhibition revealed that a second, postlipolytic, site of inhibition existed: halothane also inhibited the stimulatory effect of exogenous fatty acids on cellular respiration. This inhibition was independent of the presence of UCP1 in the mitochondria of the cells and was thus not directly on the thermogenic uncoupling mechanism. Since not only fatty acid oxidation but also pyruvate oxidation were inhibited by halothane in isolated mitochondria, whereas glycerol-3-phosphate oxidation was not, the second site of action of halothane, evident when cyclase/lipolytic inhibition was circumvented, was located to the respiratory chain, complex I. The results thus explain the inhibition of nonshivering thermogenesis by identifying two sites of action of halothane in brown adipocytes. In addition, the results may open for new formulations of the molecular background to anesthesia.