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. To examine this question, we have here established optimal conditions for studying the bioenergetics of wild-type and UCP1-/- brown-fat mitochondria, analysing UCP1-mediated differences in parallel preparations of brown-fat mitochondria from both genotypes. Comparing different substrates, we find that pyruvate (or palmitoyl-L-carnitine) shows the largest relative coupling by GDP. Comparing albumin concentrations, we find the range 0.1-0.6% optimal; higher concentrations are inhibitory. Comparing basic medium composition, we find 125 mM sucrose optimal; an ionic medium (50-100 mM KCl) functions for wild-type but is detrimental for UCP1-/- mitochondria. Using optimal conditions, we find no evidence for a residual proton conductance (not a higher post-GDP respiration, a lower membrane potential or an altered proton leak at highest common potential) with either pyruvate or glycerol-3-phosphate as substrates, nor by a 3-4-fold alteration of the amount of UCP1. We could demonstrate that certain experimental conditions, due to respiratoty inhibition, could lead to the suggestion that UCP1 possesses a residual proton conductance but find that under optimal conditions our experiments concur with implications from physiological observations that in the presence of inhibitory nucleotides, UCP1 is not leaky.

Inflammasomes are multimeric protein complexes involved in the processing of IL-1β through Caspase-1 cleavage. NLRP3 is the most widely studied inflammasome, which has been shown to respond to a large number of both endogenous and exogenous stimuli. Although studies have begun to define basic pathways for the activation of inflammasome and have been instrumental in identifying therapeutics for inflammasome related disorders; understanding the inflammasome activation at the molecular level is still incomplete. Recent functional studies indicate that microRNAs (miRs) regulate molecular pathways and can lead to diseased states when hampered or overexpressed. Mechanisms involving the miRNA regulatory network in the activation of inflammasome and IL-1β processing is yet unknown. This report investigates the involvement of miR-133a-1 in the activation of inflammasome (NLRP3) and IL-1β production. miR-133a-1 is known to target the mitochondrial uncoupling protein 2 (UCP2). The role of UCP2 in inflammasome activation has remained elusive. To understand the role of miR-133a-1 in regulating inflammasome activation, we either overexpressed or suppressed miR-133a-1 in differentiated THP1 cells that express the NLRP3 inflammasome. Levels of Caspase-1 and IL-1β were analyzed by Western blot analysis. For the first time, we showed that overexpression of miR-133a-1 increases Caspase-1 p10 and IL-1β p17 cleavage, concurrently suppressing mitochondrial uncoupling protein 2 (UCP2). Surprisingly, our results demonstrated that miR-133A-1 controls inflammasome activation without affecting the basal expression of the individual inflammasome components NLRP3 and ASC or its immediate downstream targets proIL-1β and pro-Caspase-1. To confirm the involvement of UCP2 in the regulation of inflammasome activation, Caspase-1 p10 and IL-1β p17 cleavage in UCP2 of overexpressed and silenced THP1 cells were studied. Suppression of UCP2 by siRNA enhanced the inflammasome activity stimulated by H2O2 and, conversely, overexpression of UCP2 decreased the inflammasome activation. Collectively, these studies suggest that miR-133a-1 suppresses inflammasome activation via the suppression of UCP2.

Non-shivering thermogenesis in mammalian brown adipose tissue depends on thermogenic uncoupling protein 1. Its activity is triggered by free fatty acids while purine nucleotides mediate inhibition. During activation, it is thought that free fatty acids overcome purine-mediated inhibition. We measured the cellular concentration and the release of purine nucleotide metabolites to uncover a possible role of purine nucleotide degradation in uncoupling protein 1 activation.

Mitochondrial uncoupling protein 1 (UCP1) gives brown adipose tissue of mammals its specialized ability to burn calories as heat for thermoregulation. When activated by fatty acids, UCP1 catalyzes the leak of protons across the mitochondrial inner membrane, short-circuiting the mitochondrion to generate heat, bypassing ATP synthesis. In contrast, purine nucleotides bind and inhibit UCP1, regulating proton leak by a molecular mechanism that is unclear. We present the cryo-electron microscopy structure of the GTP-inhibited state of UCP1, which is consistent with its nonconducting state. The purine nucleotide cross-links the transmembrane helices of UCP1 with an extensive interaction network. Our results provide a structural basis for understanding the specificity and pH dependency of the regulatory mechanism. UCP1 has retained all of the key functional and structural features required for a mitochondrial carrier-like transport mechanism. The analysis shows that inhibitor binding prevents the conformational changes that UCP1 uses to facilitate proton leak.

Chronic adrenergic activation leads to the emergence of beige adipocytes in some depots of white adipose tissue in mice. Despite their morphological similarities to brown adipocytes and their expression of uncoupling protein 1 (UCP1), a thermogenic protein exclusively expressed in brown adipocytes, the beige adipocytes have a gene expression pattern distinct from that of brown adipocytes. However, it is unclear whether the thermogenic function of beige adipocytes is different from that of classical brown adipocytes existing in brown adipose tissue. To examine the thermogenic ability of UCP1 expressed in beige and brown adipocytes, the adipocytes were isolated from the fat depots of C57BL/6J mice housed at 24°C (control group) or 10°C (cold-acclimated group) for 3 weeks. Morphological and gene expression analyses revealed that the adipocytes isolated from brown adipose tissue of both the control and cold-acclimated groups consisted mainly of brown adipocytes. These brown adipocytes contained large amounts of UCP1 and increased their oxygen consumption when stimulated with norepinephirine. Adipocytes isolated from the perigonadal white adipose tissues of both groups and the inguinal white adipose tissue of the control group were white adipocytes that showed no increase in oxygen consumption after norepinephrine stimulation. Adipocytes isolated from the inguinal white adipose tissue of the cold-acclimated group were a mixture of white and beige adipocytes, which expressed UCP1 and increased their oxygen consumption in response to norepinephrine. The UCP1 content and thermogenic ability of beige adipocytes estimated on the basis of their abundance in the cell mixture were similar to those of brown adipocytes. These results revealed that the inducible beige adipocytes have potent thermogenic ability comparable to classical brown adipocytes.

Coenzyme Q (CoQ, aka ubiquinone) is a key component of the mitochondrial electron transport chain (ETC) and membrane-incorporated antioxidant. CoQ10 deficiencies encompass a heterogeneous spectrum of clinical phenotypes and can be caused by hereditary mutations in the biosynthesis pathway or result from pharmacological interventions such as HMG-CoA Reductase inhibitors, and statins, which are widely used to treat hypercholesterolemia and prevent cardiovascular disease. How CoQ deficiency affects individual tissues and cell types, particularly mitochondrial-rich ones such as brown adipose tissue (BAT), has remained poorly understood. Here we show that pharmacological and genetic models of BAT CoQ deficiency show altered respiration that can only in part be explained by classical roles of CoQ in the respiration chain. Instead, we found that CoQ strongly impacts brown and beige adipocyte respiration via the regulation of uncoupling protein 1 (UCP1) expression. CoQ deficiency in BAT robustly decreases UCP1 protein levels and uncoupled respiration unexpectedly, resulting in increased inner mitochondrial membrane potential and decreased ADP/ATP ratios. Suppressed UCP1 expression was also observed in a BAT-specific in vivo model of CoQ deficiency and resulted in enhanced cold sensitivity. These findings demonstrate an as yet unappreciated role of CoQ in the transcriptional regulation of key thermogenic genes and functions.

Background The redox-sensitive chaperone DJ -1 and uncoupling protein 2 are protective against mitochondrial oxidative stress. We previously reported that renal-selective depletion and germline deletion of DJ -1 increases blood pressure in mice. This study aimed to determine the mechanisms involved in the oxidative stress-mediated hypertension in DJ -1 -/- mice. Methods and Results There were no differences in sodium excretion, renal renin expression, renal NADPH oxidase activity, and serum creatinine levels between DJ -1 -/- and wild-type mice. Renal expression of nitro-tyrosine, malondialdehyde, and urinary kidney injury marker-1 were increased in DJ -1 -/- mice relative to wild-type littermates. mRNA expression of mitochondrial heat shock protein 60 was also elevated in kidneys from DJ -1 -/- mice, indicating the presence of oxidative stress. Tempol-treated DJ -1 -/- mice presented higher serum nitrite/nitrate levels than vehicle-treated DJ -1 -/- mice, suggesting a role of the NO system in the high blood pressure of this model. Tempol treatment normalized renal kidney injury marker-1 and malondialdehyde expression as well as blood pressure in DJ -1 -/- mice, but had no effect in wild-type mice. The renal Ucp2 mRNA expression was increased in DJ -1 -/- mice versus wild-type and was also normalized by tempol. The renal-selective silencing of Ucp2 led to normalization of blood pressure and serum nitrite/nitrate ratio in DJ -1 -/- mice. Conclusions The deletion of DJ -1 leads to oxidative stress-induced hypertension associated with downregulation of NO function, and overexpression of Ucp2 in the kidney increases blood pressure in DJ -1 -/- mice. To our knowledge, this is the first report providing evidence of the role of uncoupling protein 2 in blood pressure regulation.

Uncoupling protein 1 (UCP1) catalyses mitochondrial proton leak in brown adipose tissue to facilitate nutrient oxidation for heat production, and may combat metabolic disease if activated in humans. During the adrenergic stimulation of brown adipocytes, free fatty acids generated from lipolysis activate UCP1 via an unclear interaction. Here, we set out to characterise activator binding to purified UCP1 to clarify the activation process, discern novel activators and the potential to target UCP1.

Uncoupling protein 1 (UCP1) located at the mitochondrial inner membrane serves an important role in adaptive non-shivering thermogenesis. Previous data has demonstrated that membrane lipids regulate the biological functions of membrane proteins. However, how mitochondrial lipids interact with UCP1 still remains elusive. In this study, the interactions between UCP1 and membrane lipids were investigated, using bioinformatic approaches due to the limitations associated with experimental techniques. A total of 8 UCP1 peptide regions with α-helices were identified and related to functional sites of UCP1. These were all novel peptide sequences compared with the known protein-lipid interactions. Among several types of UCP1-binding molecules, cardiolipin appeared to serve as a key interacting molecule of the 8 lipid-binding α-helix regions of UCP1. Two cardiolipin-binding lysines (K175 and K269) of UCP1 may be crucial for this UCP1-cardiolipin recognition and UCP1 function. The present findings provide novel insight into the associations of UCP1 with lipids and the potential drug targets in UCP1-associated diseases.

Inflammation and endoplasmic reticulum (ER) stress are hallmarks of metabolic syndrome. While these metabolic derangements have been well-investigated in white adipose tissue, their existence and etiology in brown adipose tissue (BAT) are poorly understood. Here, we aimed to investigate ER homeostasis and the inflammatory status and of BAT lacking uncoupling protein-1 (UCP1), a protein required for BAT thermogenesis. H&E staining illustrated lipid accumulation and crown-like structures surrounding adipocytes in BAT of UCP1-/- mice housed at room temperature compared to control mice. Further, immunohistological evaluation of F4/80 and gene expression studies demonstrated BAT macrophage infiltration and robust elevation of pro-inflammatory markers in UCP1-/- BAT. ER stress was also present in BAT of UCP1-/- mice, as evidenced by elevated gene expression and post-translational modifications of unfolded protein response components. After four weeks of thermoneutral housing, UCP1-/- mice did not exhibit elevated BAT inflammation and ER stress gene expression compared to WT mice, but depot expansion persisted. Collectively, we demonstrate that the effects of UCP1 deficiency in BAT are not restricted to mitochondrial uncoupling. We conclude that brown adipose tissue of UCP1-/- mice exhibits pro-inflammatory immune cell infiltration and perturbations in ER homeostasis and that this phenotype is driven by cold exposure rather than lipid accumulation.

A strong bidirectional link between metabolic and psychiatric disorders exists; yet, the molecular basis underlying this interaction remains unresolved. Here we explored the role of the brown adipose tissue (BAT) as modulatory interface, focusing on the involvement of uncoupling protein 1 (UCP-1), a key metabolic regulator highly expressed in BAT, in the control of emotional behavior. Male and female constitutive UCP-1 knock-out (KO) mice were used to investigate the consequences of UCP-1 deficiency on anxiety-related and depression-related behaviors under mild thermogenic (23°C) and thermoneutral (29°C) conditions. UCP-1 KO mice displayed a selective enhancement of anxiety-related behavior exclusively under thermogenic conditions, but not at thermoneutrality. Neural and endocrine stress mediators were not affected in UCP-1 KO mice, which showed an activation of the integrated stress response alongside enhanced fibroblast-growth factor-21 (FGF-21) levels. However, viral-mediated overexpression of FGF-21 did not phenocopy the behavioral alterations of UCP-1 KO mice and blocking FGF-21 activity did not rescue the anxiogenic phenotype of UCP-1 KO mice. No effects of surgical removal of the intrascapular BAT on anxiety-like behavior or FGF-21 levels were observed in either UCP-1 KO or WT mice. We provide evidence for a novel role of UCP-1 in the regulation of emotions that manifests as inhibitory constraint on anxiety-related behavior, exclusively under thermogenic conditions. We propose this function of UCP-1 to be independent of its activity in the BAT and likely mediated through a central role of UCP-1 in brain regions with converging involvement in energy and emotional control.SIGNIFICANCE STATEMENT In this first description of a temperature-dependent phenotype of emotional behavior, we propose uncoupling protein-1 (UCP-1), the key component of the thermogenic function of the brown adipose tissue, as molecular break controlling anxiety-related behavior in mice. We suggest the involvement of UCP-1 in fear regulation to be mediated through its expression in brain regions with converging roles in energy and emotional control. These data are important and relevant in light of the largely unexplored bidirectional link between metabolic and psychiatric disorders, which has the potential for providing insight into novel therapeutic strategies for the management of both conditions.

Brown/beige adipocytes, which are derived from skeletal muscle/smooth muscle-lineage cells, consume excess energy as heat through the expression of mitochondrial uncoupling protein 1 (UCP1). Previous studies have shown that forced expression of PR/SET domain (PRDM)-16 or early B-cell factor (EBF)-2 induced UCP1-positive adipocytes in C2C12 myogenic cells. Here, we explored the culture conditions to induce Ucp1 expression in C2C12 cells without introducing exogenous genes. Treatment with rosiglitazone (a peroxisome proliferator-activated receptor (PPAR)-γ agonist), GW501516 (a PPARδ agonist), and bone morphogenetic protein (BMP)-7 for 8 days efficiently increased Ucp1 expression in response to treatment with forskolin, an activator of the protein kinase A pathway. BMP7 dose-dependently increased forskolin-induced Ucp1 expression in the presence of rosiglitazone and GW501516; however, GW501516 was not required for Ucp1 induction. Additionally, the structurally related proteins, BMP6 and BMP9, efficiently increased forskolin-induced Ucp1 expression in rosiglitazone-treated cells. UCP1 protein was localized in cells with lipid droplets, but adipocytes were not always positive for UCP1. Continuous treatment with BMP7 was needed for the efficient induction of Ucp1 by forskolin treatment. Significant expression of Prdm16 was not detected, irrespective of the treatment, and treatment with rosiglitazone, GW501516, and BMP7 did not affect the expression levels of Ebf2. Fibroblast growth factor receptor (Fgfr)-3 expression levels were increased by BMP9 in rosiglitazone-treated cells, and molecules that upregulate Fgfr3 transcription partly overlapped with those that stimulate Ucp1 transcription. The present results provide basic information on the practical differentiation of myogenic cells to brown adipocytes.

Brown adipocytes are highly specialized cells with the unique metabolic ability to dissipate chemical energy in the form of heat. We determined and inferred the flux of a number of key catabolic metabolites, their changes in response to adrenergic stimulation, and the dependency on the presence of the thermogenic uncoupling protein 1 and/or oxidative phosphorylation. This study provides reference values to approximate flux rates from a limited set of measured parameters in the future and thereby allows to evaluate the plausibility of claims about the capacity of metabolic adaptations or manipulations. From the resulting model, we delineate that in brown adipocytes (1) free fatty acids are a significant contributor to extracellular acidification, (2) glycogen is the dominant glycolytic substrate source in the acute response to an adrenergic stimulus, and (3) the futile cycling of free fatty acids between lipolysis and re-esterification into triglyceride provides a mechanism for uncoupling protein 1-independent, non-shivering thermogenesis in brown adipocytes.

The number of brown adipocytes residing within murine white fat depots (brite adipocytes) varies a lot by depot, strain and physiological condition. Several endocrine fibroblast growth factors are implicated in the regulation of brite adipocyte abundance. The family of fibroblast growth factors can be categorized by their site of action into endocrine, paracrine and intracellular peptides. We here screened paracrine fibroblast growth factors for their potential to drive brite adipogenesis in differentiating epididymal white adipocytes and identified fibroblast growth factor 8b to induce uncoupling protein 1 expression, but at the same time to interfere in adipogenesis. In an in vivo trial, fibroblast growth factor 8b released into the epididymal fat depot failed to robustly increase the number of brite adipocytes. The specific action of fibroblast growth factor 8b on the uncoupling protein 1 promoter in cultured epididymal adipocytes provides a model system to dissect specific gene regulatory networks.

Uncoupling protein 1 (UCP1) plays an important role in increasing energy expenditure; thus, it is being considered as a new target for preventing obesity and metabolic complications. In this study, we investigated the effect of quercetin, a naturally occurring flavonoid, on UCP1 expression in white/brown adipose tissues (WAT/BAT).

Uncoupling protein 1 (UCP1) is predominantly found in brown adipose tissue mitochondria, and mediates energy dissipation to generate heat rather than ATP via functional mitochondrial uncoupling. However, little is known about its expression and function in kidney.

Angiopoietin-1 (Ang1) is a ligand for the receptor tyrosine kinase Tie2 and has key roles in the development of the vascular system and vascular protection. In a screen to define signalling pathways regulated by Ang1 in endothelial cells we found the RNA-binding protein hnRNP-K to be phosphorylated in response to Ang1. The ligand stimulated both tyrosine phosphorylation of hnRNP-K and recruitment of the tyrosine kinase Src to the RNA-binding protein. In endothelial cells hnRNP-K was found bound to mRNA encoding the mitochondrial protein uncoupling protein-2 (UCP2). Ang1 stimulation of cells resulted in the release of UCP2 mRNA from hnRNP-K. Using in vitro assays we confirmed direct binding between hnRNP-K and UCP2 mRNA. Furthermore Src induced phosphorylation of purified hnRNP-K and prevented UCP2 mRNA binding. Tyrosine 458 in the RNA-binding protein was found to be required for suppression of UCP2 mRNA binding by Src phosphorylation. In addition to releasing UCP2 mRNA from hnRNP-K, Ang1 induced an increase in UCP2 protein expression in endothelial cells without affecting total UCP2 mRNA levels. Consistent with the known effects of UCP2 to suppress generation of reactive oxygen species, Ang1 limited ROS production in endothelium stimulated with tumour necrosis factor-α. Taken together these data suggest that UCP2 mRNA is present in endothelial cells bound to hnRNP-K, which holds it in a translationally inactive state, and that Ang1 stimulates Src interaction with hnRNP-K, phosphorylation of the RNA-binding protein, release of these transcripts and upregulation of UCP2 protein expression. This study demonstrates a new mechanism for post-transcriptional regulation of UCP2 by the vascular protective ligand Ang1. The ability to rapidly upregulate UCP2 protein expression may be important in protecting endothelial cells from excessive generation of potentially damaging reactive oxygen species.

Accumulating evidence suggests that brown adipose tissue (BAT) is a potential therapeutic target for managing obesity and related diseases. PGAM family member 5, mitochondrial serine/threonine protein phosphatase (PGAM5), is a protein phosphatase that resides in the mitochondria and regulates many biological processes, including cell death, mitophagy, and immune responses. Because BAT is a mitochondria-rich tissue, we have hypothesized that PGAM5 has a physiological function in BAT. We previously reported that PGAM5-knockout (KO) mice are resistant to severe metabolic stress. Importantly, lipid accumulation is suppressed in PGAM5-KO BAT, even under unstressed conditions, raising the possibility that PGAM5 deficiency stimulates lipid consumption. However, the mechanism underlying this observation is undetermined. Here, using an array of biochemical approaches, including quantitative RT-PCR, immunoblotting, and oxygen consumption assays, we show that PGAM5 negatively regulates energy expenditure in brown adipocytes. We found that PGAM5-KO brown adipocytes have an enhanced oxygen consumption rate and increased expression of uncoupling protein 1 (UCP1), a protein that increases energy consumption in the mitochondria. Mechanistically, we found that PGAM5 phosphatase activity and intramembrane cleavage are required for suppression of UCP1 activity. Furthermore, utilizing a genome-wide siRNA screen in HeLa cells to search for regulators of PGAM5 cleavage, we identified a set of candidate genes, including phosphatidylserine decarboxylase (PISD), which catalyzes the formation of phosphatidylethanolamine at the mitochondrial membrane. Taken together, these results indicate that PGAM5 suppresses mitochondrial energy expenditure by down-regulating UCP1 expression in brown adipocytes and that its phosphatase activity and intramembrane cleavage are required for UCP1 suppression.

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). We have examined here the possibility that PFOA/PFOS can directly (re)activate UCP1 in isolated mouse brown-fat mitochondria. In wild-type brown-fat mitochondria, PFOS and PFOA overcame GDP-inhibited thermogenesis, leading to increased oxygen consumption and dissipated membrane potential. The absence of this effect in brown-fat mitochondria from UCP1-ablated mice indicated that it occurred through activation of UCP1. A competitive type of inhibition by increased GDP concentrations indicated interaction with the same mechanistic site as that utilized by fatty acids. No effect was observed in heart mitochondria, i.e., in mitochondria without UCP1. The stimulatory effect of PFOA/PFOS was not secondary to non-specific mitochondrial membrane permeabilization or to ROS production. Thus, metabolic effects of perfluorinated fatty acids could include direct brown adipose tissue (UCP1) activation. The possibility that this may lead to unwarranted extra heat production and thus extra utilization of food resources, leading to decreased fitness in mammalian wildlife, is discussed, as well as possible negative effects in humans. However, a possibility to utilize PFOA-/PFOS-like substances for activating UCP1 therapeutically in obesity-prone humans may also be envisaged.

Obesity is one of the main causes of morbidity and mortality worldwide. More than 120 genes have been shown to be associated with obesity related phenotypes. The aim of this study was to determine the effect of selected genetic polymorphisms in Uncoupling protein 1 (UCP1) and Niemann-Pick C1 (NPC1) genes in an obese population in Saudi Arabia.