ADH5-mediated NO bioactivity maintains metabolic homeostasis in brown adipose tissue.

Brown adipose tissue (BAT) thermogenic activity is tightly regulated by cellular redox status, but the underlying molecular mechanisms are incompletely understood. Protein S-nitrosylation, the nitric-oxide-mediated cysteine thiol protein modification, plays important roles in cellular redox regulation. Here we show that diet-induced obesity (DIO) and acute cold exposure elevate BAT protein S-nitrosylation, including UCP1. This thermogenic-induced nitric oxide bioactivity is regulated by S-nitrosoglutathione reductase (GSNOR; alcohol dehydrogenase 5 [ADH5]), a denitrosylase that balances the intracellular nitroso-redox status. Loss of ADH5 in BAT impairs cold-induced UCP1-dependent thermogenesis and worsens obesity-associated metabolic dysfunction. Mechanistically, we demonstrate that Adh5 expression is induced by the transcription factor heat shock factor 1 (HSF1), and administration of an HSF1 activator to BAT of DIO mice increases Adh5 expression and significantly improves UCP1-mediated respiration. Together, these data indicate that ADH5 controls BAT nitroso-redox homeostasis to regulate adipose thermogenesis, which may be therapeutically targeted to improve metabolic health.

IL-25-induced shifts in macrophage polarization promote development of beige fat and improve metabolic homeostasis in mice.

Beige fat dissipates energy and functions as a defense against cold and obesity, but the mechanism for its development is unclear. We found that interleukin (IL)-25 signaling through its cognate receptor, IL-17 receptor B (IL-17RB), increased in adipose tissue after cold exposure and ß3-adrenoceptor agonist stimulation. IL-25 induced beige fat formation in white adipose tissue (WAT) by releasing IL-4 and IL-13 and promoting alternative activation of macrophages that regulate innervation and up-regulate tyrosine hydroxylase (TH) up-regulation to produce more catecholamine including norepinephrine (NE). Blockade of IL-4Rα or depletion of macrophages with clodronate-loaded liposomes in vivo significantly impaired the beige fat formation in WAT. Mice fed with a high-fat diet (HFD) were protected from obesity and related metabolic disorders when given IL-25 through a process that involved the uncoupling protein 1 (UCP1)-mediated thermogenesis. In conclusion, the activation of IL-25 signaling in WAT may have therapeutic potential for controlling obesity and its associated metabolic disorders.

Fibroblast growth factor induced Ucp1 expression in preadipocytes requires PGE2 biosynthesis and glycolytic flux.

High uncoupling protein 1 (Ucp1) expression is a characteristic of differentiated brown adipocytes and is linked to adipogenic differentiation. Paracrine fibroblast growth factor 8b (FGF8b) strongly induces Ucp1 transcription in white adipocytes independent of adipogenesis. Here, we report that FGF8b and other paracrine FGFs act on brown and white preadipocytes to upregulate Ucp1 expression via a FGFR1-MEK1/2-ERK1/2 axis, independent of adipogenesis. Transcriptomic analysis revealed an upregulation of prostaglandin biosynthesis and glycolysis upon Fgf8b treatment of preadipocytes. Oxylipin measurement by LC-MS/MS in FGF8b conditioned media identified prostaglandin E2 as a putative mediator of FGF8b induced Ucp1 transcription. RNA interference and pharmacological inhibition of the prostaglandin E2 biosynthetic pathway confirmed that PGE2 is causally involved in the control over Ucp1 transcription. Importantly, impairment of or failure to induce glycolytic flux blunted the induction of Ucp1, even in the presence of PGE2 . Lastly, a screening of transcription factors identified Nrf1 and Hes1 as required regulators of FGF8b induced Ucp1 expression. Thus, we conclude that paracrine FGFs co-regulate prostaglandin and glucose metabolism to induce Ucp1 expression in a Nrf1/Hes1-dependent manner in preadipocytes, revealing a novel regulatory network in control of Ucp1 expression in a formerly unrecognized cell type.

Uncoupling protein-1 expression does not protect mice from diet-induced obesity.

We studied the metabolic phenotype of a novel Ucp1-LUC-iRFP713 knock-in reporter gene mouse model originally generated to monitor endogenous Ucp1 gene expression. Both reporter mice and reporter cells reliably reflected Ucp1 gene expression in vivo and in vitro. We here report an unexpected reduction in UCP1 content in homozygous knock-in (KI) reporter mice. As a result, the thermogenic capacity of KI mice stimulated by norepinephrine was largely blunted, making them more sensitive to an acute cold exposure. In return, these reporter mice with reduced UCP1 expression enabled us to investigate the physiological role of UCP1 in the prevention of weight gain. We observed no substantial differences in body mass across the three genotypes, irrespective of the type of diet or the ambient temperature, possibly due to the insufficient UCP1 activation. Indeed, activation of UCP1 by daily injection of the selective ß3-adrenergic receptor agonist CL316,243 resulted in significantly greater reduction of body weight in wild-type mice than in KI mice. Taken together, we conclude that the intact expression of UCP1 is essential for cold-induced thermogenesis but the presence of UCP1 per se does not protect mice from diet-induced obesity.NEW & NOTEWORTHY To study the functional role of UCP1-dependent brown adipose tissue thermogenesis for energy balance, new animal models are needed. By metabolic phenotyping of a novel mouse model with low UCP1 levels in brown fat, we demonstrate that the susceptibility to diet-induced obesity is not increased despite impaired cold-induced thermogenic capacity. Brown fat requires pharmacological activation to promote negative energy balance in diet-induced obese mice.

Adipose tissue growth and development: the modulating role of ambient temperature.

Adipose tissue is usually laid down in small amounts in the foetus and is characterised as possessing small amounts of the brown adipose tissue-specific mitochondrial uncoupling protein (UCP)1. In adults, a primary factor determining the abundance and function of UCP1 is ambient temperature. Cold exposure causes activation and the rapid generation of heat through the free flow of protons across the mitochondria with no requirement to convert ADP to ATP. In rodents, housing at an ambient temperature below thermoneutrality promotes the appearance of beige like adipocytes. These arise as discrete regions of UCP1 containing cells in white fat depots. There is increasing evidence to show that to gain credible translational results on brown and beige fat function in rodent models that they should be housed at thermoneutrality. This not only reflects the type of environment in which humans spend a majority of their time, but is in accord with the rise of global temperature caused by industrialisation and the uncontrolled burning of fossil fuels. There is now good evidence in adult humans, that stimulating brown fat can improve glucose homeostasis which can be achieved either by nutritional or pharmacological interventions. The challenge, therefore, is to establish credible developmental models in animals maintained at thermoneutrality which will elucidate the true impact of nutrition. The primary focus should fall specifically on the components of breast milk and how these modulate long term effects on brown or beige fat development and function.

[Formononetin stimulates thermogenesis of brown adipocytes by promoting the expression of uncoupling protein 1].

Objective To investigate the mechanism of formononetin regulating the heat production of brown adipocytes via decoupling protein 1 (UCP1). Methods The brown preadipocytes was isolated from wild-type (WT) C57BL/6J mice and differentiated into mature fat cells in vitro. Moreover, the mRNA levels of fatty acid binding protein 4 (FABP4) and adiponectin were detected by real-time quantitative PCR (RT-qPCR). To confirm formononetin could induce the expression of thermogenic genes, we first prepared WT mature brown adipocytes and treated them with DMSO and formononetin separately. The mRNA and protein levels of thermogenic genes, such as peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), peroxisome proliferators-activated receptor γ (PPARγ), UCP1 and iodothyronine deiodinase 2 (Dio2), were detected by RT-qPCR and Western blot analysis. To investigate the role of UCP1 in mediating differentiation of brown preadipocytes, Fabp4 and adiponectin mRNA levels were analyzed by RT-qPCR in WT and UCP1 mutation differentiated brown adipocytes. To determine cellular oxygen consumption, isolated WT and UCP1 mutation brown preadipocytes were plated in an XF24-well microplate and differentiated into mature brown adipocytes treated with formononetin or DMSO, followed by oxygen consumption rate (OCR) measurement using XF24 analyser. Results Both WT and UCP1 KO brown preadipocytes could be differentiated into adipocyte. The expression of thermogenic genes, including PGC-1α, Dio2, PPARγ and UCP1, induced by formononetin was similar in UCP1 KO adipocytes and WT cells. But the ability of formononetin to increase cellular respiration was inhibited in Ucp1 KO cells. Conclusion Formononetin mediated stimulation of thermogenesis and oxygen consumption via UCP1 in brown fat cells.

Intramuscular Brown Fat Activation Decreases Muscle Atrophy and Fatty Infiltration and Improves Gait After Delayed Rotator Cuff Repair in Mice.

BACKGROUND: Successful repair of large and massive rotator cuff (RC) tears remains a challenge at least partially because of secondary muscle atrophy and fatty infiltration. ß3 Adrenergic agonists are a group of drugs that promote fat resorption through "white fat browning" of intramuscular stem cells. PURPOSE: To test the role of a ß3 adrenergic receptor agonist, amibegron, in improving muscle quality and forelimb function in a delayed RC repair model via promoting brown/beige adipose tissue activation. STUDY DESIGN: Controlled laboratory study. METHODS: Three-month-old PDGFRα-GFP reporter mice, wild type C57BL/6J mice, and uncoupling protein 1 (UCP-1) knockout mice underwent unilateral supraspinatus tendon transection with a 6-week delayed tendon repair. Animals with sham surgery served as controls. Amibegron was given either immediately after tendon transection or after repair. Gait analysis was conducted to measure forelimb function at 6 weeks after tendon repair. Animals were sacrificed at 6 weeks after repair. Supraspinatus muscles were harvested and analyzed histologically. Reverse transcription polymerase chain reaction was performed to quantify gene expression related to atrophy, fibrosis, and fatty infiltration. RESULTS: Histology of PDGFRα reporter mice showed significantly increased UCP-1 expression, suggesting white fat browning in muscle after RC repair. As administered either immediately after tendon transection or after tendon repair, amibegron significantly reduced muscle atrophy and fatty infiltration and resumed normal upper extremity gait in wild type mice. However, the effect of amibegron was not present in UCP-1 knockout mice, suggesting that the effect of amibegron in treating RC muscle atrophy and fatty infiltration is through a UCP 1-dependent mechanism. CONCLUSION: Amibegron reduced muscle atrophy and fatty infiltration and improved forelimb function after delayed RC repair through a UCP 1-dependent mechanism. This may be an effective clinical treatment strategy for patients to improve muscle quality after RC repair. CLINICAL RELEVANCE: ß3 Adrenergic agonists may serve as a new pharmacologic modality to treat RC muscle atrophy and fatty infiltration to improve clinical outcome of RC repair.

The generation of immune-induced fever and emotional stress-induced hyperthermia in mice does not involve brown adipose tissue thermogenesis.

We examined the role of brown adipose tissue (BAT) for fever and emotional stress-induced hyperthermia. Wild-type and uncoupling protein-1 (UCP-1) knockout mice were injected with lipopolysaccharide intraperitoneally or intravenously, or subjected to cage exchange, and body temperature monitored by telemetry. Both genotypes showed similar febrile responses to immune challenge and both displayed hyperthermia to emotional stress. Neither procedure resulted in the activation of BAT, such as the induction of UCP-1 or peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) mRNA, or reduced BAT weight and triglyceride content. In contrast, in mice injected with a ß3 agonist, UCP-1 and PGC-1α were strongly induced, and BAT weight and triglyceride content reduced. Both lipopolysaccharide and the ß3 agonist, and emotional stress, induced UCP-3 mRNA in skeletal muscle. A ß3 antagonist did not attenuate lipopolysaccharide-induced fever, but augmented body temperature decrease and inhibited BAT activation when mice were exposed to cold. An α1 /α2b antagonist or a 5HT1A agonist, which inhibit vasoconstriction, abolished lipopolysaccharide-induced fever, but had no effect on emotional stress-induced hyperthermia. These findings demonstrate that in mice, UCP-1-mediated BAT thermogenesis does not take part in inflammation-induced fever, which is dependent on peripheral vasoconstriction, nor in stress-induced hyperthermia. However, both phenomena may involve UCP-3-mediated muscle thermogenesis.

Systems-Genetics-Based Inference of a Core Regulatory Network Underlying White Fat Browning.

Recruitment of brite/beige cells, known as browning of white adipose tissue (WAT), is an efficient way to turn an energy-storing organ into an energy-dissipating one and may therefore be of therapeutic value in combating obesity. However, a comprehensive understanding of the regulatory mechanisms mediating WAT browning is still lacking. Here, we exploit the large natural variation in WAT browning propensity between inbred mouse strains to gain an inclusive view of the core regulatory network coordinating this cellular process. Combining comparative transcriptomics, perturbation-based validations, and gene network analyses, we present a comprehensive gene regulatory network of inguinal WAT browning, revealing up to four distinct regulatory modules with key roles for uncovered transcriptional factors, while also providing deep insights into the genetic architecture of brite adipogenesis. The presented findings therefore greatly increase our understanding of the molecular drivers mediating the intriguing cellular heterogeneity and plasticity of adipose tissue.

ComBATing aging-does increased brown adipose tissue activity confer longevity?

Brown and its related beige adipose tissue (BAT) play a definitive role in maintaining body temperature by producing heat through uncoupling protein 1 (UCP1), which acts by dissociating oxidative phosphorylation from ATP production, resulting in the release of heat. Therefore, in order to maintain high thermogenic capacity, BAT must act as a metabolic sink by taking up vast amounts of circulating glucose and lipids for oxidation. This, along with the rediscovery of BAT in adult humans, has fueled the study of BAT as a putative therapeutic approach to manage the growing rates of obesity and metabolic syndromes. Notably, many of the beneficial consequences of BAT activity overlap with metabolic biomarkers of extended lifespan and healthspan. In this review, we provide background about BAT including the thermogenic program, BAT's role as a secretory organ, and differences between BAT in mice and humans. We also provide details on BAT during aging, and perspectives on the potential of targeting BAT to promote lifespan and healthspan.

Role of epicardial adipose tissue NPR-C in acute coronary syndrome.

BACKGROUND AND AIMS: It has been suggested that epicardial adipose tissue (EAT) thermogenesis plays a role in coronary artery disease (CAD). Recent evidence indicates that natriuretic peptide receptors (NPRs) are critical for thermogenesis. We determined the expression and signaling of NPRs in EAT in the context of CAD progression and their association with brown fat-related genes, such as uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator alpha (PGC1α). METHODS: NPR-A, NPR-B and NPR-C mRNA and protein expression levels were analyzed in EAT and thoracic subcutaneous adipose tissue (SAT) from non-CAD (NCAD), stable CAD and acute coronary syndrome (ACS) patients. The associations of NPRs with thermogenic genes were also evaluated. RESULTS: The EAT of ACS patients showed lower NPR-C gene and protein expression levels compared with that of stable CAD or NCAD patients. NPR-C mRNA expression in EAT also decreased as the number of injured arteries rose, and correlated positively with left ventricular ejection fraction and EAT PGC1α mRNA expression. EAT PGC1α and UCP1 gene expression levels also decreased in the ACS group. Linear and logistic regression models showed associations of EAT NPR-C mRNA levels with EAT PGC1α mRNA levels and the presence of ACS. Furthermore, the EAT of ACS patients showed reduced p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation levels, which correlated positively with NPR-C protein levels. CONCLUSIONS: The EAT of patients with ACS is characterized by decreased NPR-C, reduced UCP1 and PGC1α mRNA expression levels and reduced activation of the p38 MAPK pathway. The associations among the expression of EAT NPR-C and ACS, and brown fat markers suggest that NPR-C may play a role in ACS and in the regulation of EAT brown-like fat features in humans.

Brown and beige fat: From molecules to physiology.

The recent re-discovery of brown adipose tissue (BAT) and even more recent discovery of the browning of white adipose tissue (WAT) in humans have generated intense scientific interest in the role of adipose tissue as potential target against obesity and its metabolic complications. The purpose of this review is to: i) critically evaluate the current evidence on the physiological significance of BAT and the browning of WAT in metabolic function in humans and ii) discuss factors that have been reported to regulate BAT and/or the browning of WAT in humans. The current literature supports that BAT and the browning of WAT constitute promising emerging targets for interventions aiming to prevent and/or treat of obesity and its metabolic complications. Further research is needed to better understand the physiological significance of BAT and browning of WAT in health and disease along with the factors modulating their metabolic function in humans.

Uncoupling mechanism and redox regulation of mitochondrial uncoupling protein 1 (UCP1).

Brown adipose tissue (BAT) and brown in white (brite) adipose tissue, termed also beige adipose tissue, are major sites of mammalian nonshivering thermogenesis. Mitochondrial uncoupling protein 1 (UCP1), specific for these tissues, is the key factor for heat production. Recent molecular aspects of UCP1 structure provide support for the fatty acid cycling model of coupling, i.e. when UCP1 expels fatty acid anions in a uniport mode from the matrix, while uncoupling. Protonophoretic function is ensured by return of the protonated fatty acid to the matrix independent of UCP1. This mechanism is advantageous for mitochondrial uncoupling and compatible with heat production in a pro-thermogenic environment, such as BAT. It must still be verified whether posttranslational modification of UCP1, such as sulfenylation of Cys253, linked to redox activity, promotes UCP1 activity. BAT biogenesis and UCP1 expression, has also been linked to the pro-oxidant state of mitochondria, further endorsing a redox signalling link promoting an establishment of pro-thermogenic state. We discuss circumstances under which promotion of superoxide formation exceeds its attenuation by uncoupling in mitochondria and throughout point out areas of future research into UCP1 function.

UCP1-independent signaling involving SERCA2b-mediated calcium cycling regulates beige fat thermogenesis and systemic glucose homeostasis.

Uncoupling protein 1 (UCP1) plays a central role in nonshivering thermogenesis in brown fat; however, its role in beige fat remains unclear. Here we report a robust UCP1-independent thermogenic mechanism in beige fat that involves enhanced ATP-dependent Ca2+ cycling by sarco/endoplasmic reticulum Ca2+-ATPase 2b (SERCA2b) and ryanodine receptor 2 (RyR2). Inhibition of SERCA2b impairs UCP1-independent beige fat thermogenesis in humans and mice as well as in pigs, a species that lacks a functional UCP1 protein. Conversely, enhanced Ca2+ cycling by activation of α1- and/or ß3-adrenergic receptors or the SERCA2b-RyR2 pathway stimulates UCP1-independent thermogenesis in beige adipocytes. In the absence of UCP1, beige fat dynamically expends glucose through enhanced glycolysis, tricarboxylic acid metabolism and pyruvate dehydrogenase activity for ATP-dependent thermogenesis through the SERCA2b pathway; beige fat thereby functions as a 'glucose sink' and improves glucose tolerance independently of body weight loss. Our study uncovers a noncanonical thermogenic mechanism through which beige fat controls whole-body energy homeostasis via Ca2+ cycling.

Mitochondrial uncoupling in the melanocortin system differentially regulates NPY and POMC neurons to promote weight-loss.

OBJECTIVE: The mitochondrial uncoupling agent 2,4-dinitrophenol (DNP), historically used as a treatment for obesity, is known to cross the blood-brain-barrier, but its effects on central neural circuits controlling body weight are largely unknown. As hypothalamic melanocortin neuropeptide Y/agouti-related protein (NPY/AgRP) and pro-opiomelanocortin (POMC) neurons represent key central regulators of food intake and energy expenditure we investigated the effects of DNP on these neurons, food intake and energy expenditure. METHOD: C57BL/6 and melanocortin-4 receptor (MC4R) knock-out mice were administered DNP intracerebroventricularly (ICV) and the metabolic changes were characterized. The specific role of NPY and POMC neurons and the ionic mechanisms mediating the effects of uncoupling were examined with in vitro electrophysiology performed on NPY hrGFP or POMC eGFP mice. RESULTS: Here we show DNP-induced differential effects on melanocortin neurons including inhibiting orexigenic NPY and activating anorexigenic POMC neurons through independent ionic mechanisms coupled to mitochondrial function, consistent with an anorexigenic central effect. Central administration of DNP induced weight-loss, increased BAT thermogenesis and browning of white adipose tissue, and decreased food intake, effects that were absent in MC4R knock-out mice and blocked by the MC4R antagonist, AgRP. CONCLUSION: These data show a novel central anti-obesity mechanism of action of DNP and highlight the potential for selective melanocortin mitochondrial uncoupling to target metabolic disorders.

Adipocyte Liver Kinase b1 Suppresses Beige Adipocyte Renaissance Through Class IIa Histone Deacetylase 4.

Uncoupling protein 1+ beige adipocytes are dynamically regulated by environment in rodents and humans; cold induces formation of beige adipocytes, whereas warm temperature and nutrient excess lead to their disappearance. Beige adipocytes can form through de novo adipogenesis; however, how "beiging" characteristics are maintained afterward is largely unknown. In this study, we show that beige adipocytes formed postnatally in subcutaneous inguinal white adipose tissue lost thermogenic gene expression and multilocular morphology at the adult stage, but cold restored their beiging characteristics, a phenomenon termed beige adipocyte renaissance. Ablation of these postnatal beige adipocytes inhibited cold-induced beige adipocyte formation in adult mice. Furthermore, we demonstrated that beige adipocyte renaissance was governed by liver kinase b1 and histone deacetylase 4 in white adipocytes. Although neither presence nor thermogenic function of uncoupling protein 1+ beige adipocytes contributed to metabolic fitness in adipocyte liver kinase b1-deficient mice, our results reveal an unexpected role of white adipocytes in maintaining properties of preexisting beige adipocytes.

Mitochondrial protein UCP1 mediates liver injury induced by LPS through EKR signaling pathway.

OBJECTIVE: Mitochondria are abundant in liver. The roles of mitochondrial protein in liver injury and related signaling pathways are still unclear. UCP1 is a novel mitochondrial transmembrane protein. Its expression pattern and function in liver still needs further investigation. MATERIALS AND METHODS: A mouse model of liver injury was established by the treatment of LPS. UCP1 expression in the liver tissue was detected by Western blot and qRT-PCR. ERK signaling activity was tested by enzymatic activity kit. ATP production was evaluated by flow cytometry. Cell apoptosis was determined by Western blot and flow cytometry. ERK signaling pathway inhibitor, U0126, was used to pre-treat mice. Liver tissue from sepsis patients was collected from the surgery. RESULTS: Our data showed that the level of UCP1 was upregulated, ERK signaling was activated, ATP production was reduced, and cell apoptosis was enhanced in mice with liver injury model caused by LPS. U0126 intervention significantly suppressed UCP1 expression, inhibited ERK signaling pathway, enhanced ATP production, and restrained liver cell apoptosis in mice liver injury model. UCP1 increased, ERK signaling activated, and cell apoptosis elevated in the liver tissue of sepsis patients. CONCLUSIONS: UCP1 plays a role in the liver tissue of mouse liver injury model and sepsis patients through the modulation of mitochondrial ATP production and cell apoptosis by ERK signaling pathway.

Adaptive facultative diet-induced thermogenesis in wild-type but not in UCP1-ablated mice.

The significance of diet-induced thermogenesis (DIT) for metabolic control is still debated. Although obesogenic diets recruit UCP1 and adrenergically inducible thermogenesis, and although the absence of UCP1 may promote the development of obesity, no actual UCP1-related thermogenesis identifiable as diet-induced thermogenesis has to date been unambiguously demonstrated. Examining mice living at thermoneutrality, we have identified a process of facultative (directly elicited by acute eating), adaptive (magnitude develops over weeks on an obesogenic diet), and fully UCP1-dependent thermogenesis. We found no evidence for UCP1-independent diet-induced thermogenesis. The thermogenesis was proportional to the total amount of UCP1 protein in brown adipose tissue and was not dependent on any contribution of UCP1 in brite/beige adipose tissue, since no UCP1 protein was found there under these conditions. Total UCP1 protein amount developed proportionally to total body fat content. The physiological messenger linking obesity level and acute eating to increased thermogenesis is not known. Thus UCP1-dependent diet-induced thermogenesis limits obesity development during exposure to obesogenic diets but does not prevent obesity as such.