In isolated brown adipose tissue mitochondria, UCP1 is not essential for - nor involved in - the uncoupling effects of the classical uncouplers FCCP and DNP.

Recent patch-clamp studies of mitoplasts have challenged the traditional view that classical chemical uncoupling (by e.g. FCCP or DNP) is due to the protonophoric property of these substances themselves.

Enhanced ROS Production in Mitochondria from Prematurely Aging mtDNA Mutator Mice.

An increase in mitochondrial DNA (mtDNA) mutations and an ensuing increase in mitochondrial reactive oxygen species (ROS) production have been suggested to be a cause of the aging process ("the mitochondrial hypothesis of aging"). In agreement with this, mtDNA-mutator mice accumulate a large amount of mtDNA mutations, giving rise to defective mitochondria and an accelerated aging phenotype. However, incongruously, the rates of ROS production in mtDNA mutator mitochondria have generally earlier been reported to be lower - not higher - than in wildtype, thus apparently invalidating the "mitochondrial hypothesis of aging".

Elovl2-Ablation Leads to Mitochondrial Membrane Fatty Acid Remodeling and Reduced Efficiency in Mouse Liver Mitochondria.

The fatty acid elongase elongation of very long-chain fatty acids protein 2 (ELOVL2) controls the elongation of polyunsaturated fatty acids (PUFA) producing precursors for omega-3, docosahexaenoic acid (DHA), and omega-6, docosapentaenoic acid (DPA-6) in vivo. Expectedly, Elovl2-ablation drastically reduced the DHA and DPA-6 in liver mitochondrial membranes. Unexpectedly, however, total PUFAs levels decreased further than could be explained by Elovl2 ablation.

Establishing the potency of N-acyl amino acids versus conventional fatty acids as thermogenic uncouplers in cells and mitochondria from different tissues.

The possibility that N-acyl amino acids could function as brown or brite/beige adipose tissue-derived lipokines that could induce UCP1-independent thermogenesis by uncoupling mitochondrial respiration in several peripheral tissues is of significant physiological interest. To quantify the potency of N-acyl amino acids versus conventional fatty acids as thermogenic inducers, we have examined the affinity and efficacy of two pairs of such compounds: oleate versus N-oleoyl-leucine and arachidonate versus N-arachidonoyl-glycine in cells and mitochondria from different tissues. We found that in cultures of the muscle-derived L6 cell line, as well as in primary cultures of murine white, brite/beige and brown adipocytes, the N-acyl amino acids were proficient uncouplers but that they did not systematically display higher affinity or potency than the conventional fatty acids, and they were not as efficient uncouplers as classical protonophores (FCCP).

Glucocorticoid-Induced Obesity Develops Independently of UCP1.

An excess of glucocorticoids leads to the development of obesity in both mice and humans, but the mechanism for this is unknown. Here, we determine the extent to which decreased BAT thermogenic capacity (as a result of glucocorticoid treatment) contributes to the development of obesity. Contrary to previous suggestions, we show that only in mice housed at thermoneutrality (30°C) does corticosterone treatment reduce total BAT UCP1 protein.

Flow Cytometry of Mouse and Human Adipocytes for the Analysis of Browning and Cellular Heterogeneity.

Adipocytes, once considered simple lipid-storing cells, are rapidly emerging as complex cells with many biologically diverse functions. A powerful high-throughput method for analyzing single cells is flow cytometry. Several groups have attempted to analyze and sort freshly isolated adipocytes; however, using an adipocyte-specific reporter mouse, we demonstrate that these studies fail to detect the majority of white adipocytes.

The GPR120 agonist TUG-891 promotes metabolic health by stimulating mitochondrial respiration in brown fat.

Brown adipose tissue (BAT) activation stimulates energy expenditure in human adults, which makes it an attractive target to combat obesity and related disorders. Recent studies demonstrated a role for G protein-coupled receptor 120 (GPR120) in BAT thermogenesis. Here, we investigated the therapeutic potential of GPR120 agonism and addressed GPR120-mediated signaling in BAT We found that activation of GPR120 by the selective agonist TUG-891 acutely increases fat oxidation and reduces body weight and fat mass in C57Bl/6J mice.

Improved health-span and lifespan in mtDNA mutator mice treated with the mitochondrially targeted antioxidant SkQ1.

MtDNA mutator mice exhibit marked features of premature aging. We find that these mice treated from age of ≈100 days with the mitochondria-targeted antioxidant SkQ1 showed a delayed appearance of traits of aging such as kyphosis, alopecia, lowering of body temperature, body weight loss, as well as ameliorated heart, kidney and liver pathologies. These effects of SkQ1 are suggested to be related to an alleviation of the effects of an enhanced reactive oxygen species (ROS) level in mtDNA mutator mice: the increased mitochondrial ROS released due to mitochondrial mutations probably interact with polyunsaturated fatty acids in cardiolipin, releasing malondialdehyde and 4-hydroxynonenal that form protein adducts and thus diminishes mitochondrial functions.

UCP1 inhibition in Cidea-overexpressing mice is physiologically counteracted by brown adipose tissue hyperrecruitment.

Cidea is a gene highly expressed in thermogenesis-competent (UCP1-containing) adipose cells, both brown and brite/beige. Here, we initially demonstrate a remarkable adipose-depot specific regulation of Cidea expression. In classical brown fat, Cidea mRNA is expressed continuously and invariably, irrespective of tissue recruitment.

Cidea improves the metabolic profile through expansion of adipose tissue.

In humans, Cidea (cell death-inducing DNA fragmentation factor alpha-like effector A) is highly but variably expressed in white fat, and expression correlates with metabolic health. Here we generate transgenic mice expressing human Cidea in adipose tissues (aP2-hCidea mice) and show that Cidea is mechanistically associated with a robust increase in adipose tissue expandability. Under humanized conditions (thermoneutrality, mature age and prolonged exposure to high-fat diet), aP2-hCidea mice develop a much more pronounced obesity than their wild-type littermates.

Metabolically inert perfluorinated fatty acids directly activate uncoupling protein 1 in brown-fat mitochondria.

The metabolically inert perfluorinated fatty acids perfluorooctane sulfonate (PFOS) and perfluorooctanoate (PFOA) can display fatty acid-like activity in biological systems. The uncoupling protein 1 (UCP1) in brown adipose tissue is physiologically (re)activated by fatty acids, including octanoate. This leads to bioenergetically uncoupled energy dissipation (heat production, thermogenesis).

The Environmental Pollutants Perfluorooctane Sulfonate and Perfluorooctanoic Acid Upregulate Uncoupling Protein 1 (UCP1) in Brown-Fat Mitochondria Through a UCP1-Dependent Reduction in Food Intake.

The environmental pollutants perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) cause a dramatic reduction in the size of the major adipose tissue depots and a general body weight decrease when they are added to the food of mice. We demonstrate here that this is mainly due to a reduction in food intake; this reduction was not due to food aversion. Remarkably and unexpectedly, a large part of the effect of PFOA/PFOS on food intake was dependent on the presence of the uncoupling protein 1 (UCP1) in the mice.

Leydig cell steroidogenesis unexpectedly escapes mitochondrial dysfunction in prematurely aging mice.

Point mutations and deletions of mitochondrial DNA (mtDNA) accumulate in tissues during aging in animals and humans and are the basis for mitochondrial diseases. Testosterone synthesis occurs in the mitochondria of Leydig cells. Mitochondrial dysfunction (as induced here experimentally in mtDNA mutator mice that carry a proofreading-deficient form of mtDNA polymerase γ, leading to mitochondrial dysfunction in all cells types so far studied) would therefore be expected to lead to low testosterone levels.

ROS production in brown adipose tissue mitochondria: the question of UCP1-dependence.

Whether active UCP1 can reduce ROS production in brown-fat mitochondria is presently not settled. The issue is of principal significance, as it can be seen as a proof- or disproof-of-principle concerning the ability of any protein to diminish ROS production through membrane depolarization. We therefore undertook a comprehensive investigation of the significance of UCP1 for ROS production, by comparing the ROS production in brown-fat mitochondria isolated from wildtype mice (that display membrane depolarization) or from UCP1(-/-) mice (with a high membrane potential).

In vivo levels of mitochondrial hydrogen peroxide increase with age in mtDNA mutator mice.

In mtDNA mutator mice, mtDNA mutations accumulate leading to a rapidly aging phenotype. However, there is little evidence of oxidative damage to tissues, and when analyzed ex vivo, no change in production of the reactive oxygen species (ROS) superoxide and hydrogen peroxide by mitochondria has been reported, undermining the mitochondrial oxidative damage theory of aging. Paradoxically, interventions that decrease mitochondrial ROS levels in vivo delay onset of aging.

UCP1 in brite/beige adipose tissue mitochondria is functionally thermogenic.

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.

Quantification of uncoupling protein 2 reveals its main expression in immune cells and selective up-regulation during T-cell proliferation.

Uncoupling protein 2 (UCP2) is an inner mitochondrial membrane protein. Although the protein was discovered in 1997, its function and even its tissue distribution are still under debate. Here we present a quantitative analysis of mRNA and protein expression in various mice tissues, revealing that UCP2 is mainly expressed in organs and cells associated with the immune system.

Effects of the mitochondria-targeted antioxidant SkQ1 on lifespan of rodents.

The effect of the mitochondria-targeted, plastoquinone-containing antioxidant SkQ1 on the lifespan of outbred mice and of three strains of inbred mice was studied. To this end, low pathogen (LP) or specific pathogen free (SPF) vivaria in St. Petersburg, Moscow, and Stockholm were used.

Mitochondrial ('mild') uncoupling and ROS production: physiologically relevant or not?

During the last decade, the possibility that 'mild' uncoupling could be protective against oxidative damage by diminishing ROS (reactive oxygen species) production has attracted much interest. In the present paper, we briefly examine the evidence for this possibility. It is only ROS production from succinate under reverse electron-flow conditions that is sensitive to membrane potential fluctuations, and so only this type of ROS production could be affected; however, the conditions under which succinate-supported ROS production is observed include succinate concentrations that are supraphysiological.

Shikonin increases glucose uptake in skeletal muscle cells and improves plasma glucose levels in diabetic Goto-Kakizaki rats.

Background: There is considerable interest in identifying compounds that can improve glucose homeostasis. Skeletal muscle, due to its large mass, is the principal organ for glucose disposal in the body and we have investigated here if shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increases glucose uptake in skeletal muscle cells.

Methodology/principal Findings: Shikonin increases glucose uptake in L6 skeletal muscle myotubes, but does not phosphorylate Akt, indicating that in skeletal muscle cells its effect is medaited via a pathway distinct from that used for insulin-stimulated uptake.

Uncoupled respiration, ROS production, acute lipotoxicity and oxidative damage in isolated skeletal muscle mitochondria from UCP3-ablated mice.

The function of uncoupling protein 3 (UCP3) is still not established. Mitochondrial uncoupling, control of ROS production, protection against lipotoxicity and protection against oxidative stress are functions classically discussed. To establish a role for UCP3 in these functions, we have here used UCP3 (-/-) mice, backcrossed for 10 generations on a C57Bl/6 background.

Increased fatigue resistance linked to Ca2+-stimulated mitochondrial biogenesis in muscle fibres of cold-acclimated mice.

Mammals exposed to a cold environment initially generate heat by repetitive muscle activity (shivering). Shivering is successively replaced by the recruitment of uncoupling protein-1 (UCP1)-dependent heat production in brown adipose tissue. Interestingly, adaptations observed in skeletal muscles of cold-exposed animals are similar to those observed with endurance training.

Caveolin-1-ablated mice survive in cold by nonshivering thermogenesis despite desensitized adrenergic responsiveness.

Caveolin-1 (Cav1)-ablated mice display impaired lipolysis in white adipose tissue. They also seem to have an impairment in brown adipose tissue function, implying that Cav1-ablated mice could encounter problems in surviving longer periods in cold temperatures. To investigate this, Cav1-ablated mice and wild-type mice were transferred to cold temperatures for extended periods of time, and parameters related to metabolism and thermogenesis were investigated.

Uncoupling protein-1 is not leaky.

The activity of uncoupling protein-1 (UCP1) is rate-limiting for nonshivering thermogenesis and diet-induced thermogenesis. Characteristically, this activity is inhibited by GDP experimentally and presumably mainly by cytosolic ATP within brown-fat cells. The issue as to whether UCP1 has a residual proton conductance even when fully saturated with GDP/ATP (as has recently been suggested) has not only scientific but also applied interest, since a residual proton conductance would make overexpressed UCP1 weight-reducing even without physiological/pharmacological activation.