Uncoupling Protein 1 Protein Research Articles

Down regulation of UCP2 expression in retinal pigment epithelium cells under oxidative stress: an in vitro study.

To evaluate the expression of uncoupling protein 2 (UCP2) in a retinal pigment epithelium cell line (ARPE-19), under oxidative stress (OS). ARPE-19 cells were divided into groups treated with various concentrations of hydrogen peroxide (H2O2; 0, 150, 300, 500, 700, and 900 µmol/L) for 24h, to induce oxidative damage and cell viability was assessed by MTT assay. UCP2 mRNA expression in cells treated with H2O2 was investigated by reverse transcription-polymerase chain reaction (RT-PCR). UCP2 protein expression was assessed by Western blotting and ROS levels analyzed by flow cytometry (FCM). Further, UCP2-siRNA treated cultures were exposed to H2O2 (0, 75, 150, and 300 µmol/L) for 2h and cell viability determined by MTT assay. Cells treated with higher concentrations of H2O2 appeared shrunken; their adhesion to adjacent cells was disrupted, and the number of dead cells increased. The results of cell viability assays demonstrated that the numbers of cells were decreased in a dose-dependent manner following treatment with H2O2. Compared with untreated controls, cell viability was significantly reduced after treatment with >300 µmol/L H2O2 (P<0.05). Cell metabolic activity was decreased with increased concentrations of H2O2 as detected by MTT assay. Levels of OS were further decreased in cells treated with UCP2-siRNA compared with those treated with H2O2 alone (P<0.05). The results of RT-PCR and Western blotting demonstrated that UCP2 expression was reduced in H2O2-treated groups compared with controls (P<0.05). FCM analysis showed that cell reactive oxygen species (ROS) levels were increased in H2O2-treated groups and further upregulated by UCP2-siRNA treatment (P<0.05). Expression levels of UCP2 are decreased in ARPE-19 cells treated with H2O2. ROS levels are further increased in cells treated with UCP2-siRNA relative to those treated with H2O2 alone. UCP2 may have a protective role in ARPE-19 cells during oxidative injury.

Caffeine exposure induces browning features in adipose tissue in vitro and in vivo

Brown adipose tissue (BAT) is able to rapidly generate heat and metabolise macronutrients, such as glucose and lipids, through activation of mitochondrial uncoupling protein 1 (UCP1). Diet can modulate UCP1 function but the capacity of individual nutrients to promote the abundance and activity of UCP1 is not well established. Caffeine consumption has been associated with loss of body weight and increased energy expenditure, but whether it can activate UCP1 is unknown. This study examined the effect of caffeine on BAT thermogenesis in vitro and in vivo. Stem cell-derived adipocytes exposed to caffeine (1 mM) showed increased UCP1 protein abundance and cell metabolism with enhanced oxygen consumption and proton leak. These functional responses were associated with browning-like structural changes in mitochondrial and lipid droplet content. Caffeine also increased peroxisome proliferator-activated receptor gamma coactivator 1-alpha expression and mitochondrial biogenesis, together with a number of BAT selective and beige gene markers. In vivo, drinking coffee (but not water) stimulated the temperature of the supraclavicular region, which co-locates to the main region of BAT in adult humans, and is indicative of thermogenesis. Taken together, these results demonstrate that caffeine can promote BAT function at thermoneutrality and may have the potential to be used therapeutically in adult humans.

Eicosapentaenoic Acid Increases Browning Markers in Subcutaneous Adipose Tissue and Primary Adipocytes from Wild Type and UCP-1 Deficient Mice (P15-007-19)

ObjectivesBrown adipose tissue (BAT) regulates energy balance through thermogenesis, in part via uncoupling protein -1 (UCP-1). White adipose tissue (WAT), namely subcutaneous adipose tissue (SAT) can convert to a beige/brite adipose tissue phenotype (browning) under thermogenic conditions such as cold. We previously reported that eicosapentaenoic acid (EPA) reduced obesity and glucose intolerance, and increased UCP-1 in BAT of B6 mice at ambient temperature (22°C); and these effects were attenuated at thermoneutral environment (28–30°C). We hypothesized that EPA exerts anti-obesity effects on SAT, including increased browning, adipocyte hypotrophy; and these effects require UCP-1. MethodsSix-week-old B6 wild type (WT) and UCP-1 knock-out (KO) male mice were maintained at thermoneutral environment and fed high fat diet (HF) with or without 36 g/kg of AlaskOmega EPA-enriched fish oil (800 mg/g) for 14 weeks; and SAT was collected for histological, gene and protein analyses. SAT was also prepared from chow diet-fed WT and KO mice at ambient environment to prepare stroma vascular cells, which were differentiated into adipocytes, treated with 100uM EPA for 48 hours then harvested for mRNA and protein analyses. ResultsKO mice fed HF diets had the highest body weight (P < 0.05) among all groups. EPA reduced fat cell size in both WT and KO mice fed the EPA diet. mRNA levels of fibroblast growth factor-21 (FGF-21) were higher in SAT of WT mice fed EPA compared to WT mice fed HF (P < 0.05), with no differences between the KO genotype. KO mice fed HF diets had lower levels of UCP-3 in SAT compared to WT mice fed HF (P < 0.05), which was rescued only in the KO mice fed EPA (P < 0.05). UCP-1 protein levels were very low in SAT tissues, and UCP-2 mRNA levels were similar across all groups in SAT. Interestingly, EPA significantly (P < 0.05) increased mRNA expression of UCP-2, UCP-3 and FGF21 in differentiated SAT adipocytes from both WT and KO compared to control. Furthermore, UCP-1 mRNA levels were significantly higher in WT adipocytes treated with EPA, compared to non-treated cells (P < 0.05). Additional mechanistic studies are currently underway to further dissect adipose depot differences in EPA effects in WT vs. KO mice. ConclusionsOur data suggest that EPA increases SAT browning, independently of UCP-1. Funding SourcesNIH/NCCIH.

Eicosapentaenoic Acid Regulates Brown Adipose Tissue Function, Independently of UCP1 (FS11-01-19)

ObjectivesBrown adipose tissue (BAT) is a critical tissue in energy expenditure through its specific uncoupling protein 1 (UCP1). We previously reported that mice fed high fat (HF) diet supplemented with eicosapentaenoic acid (EPA) reduced body weight, adiposity, and insulin resistance, and increased UCP1 protein and mRNA levels of other thermogenic markers in BAT at ambient temperature. Hence, we hypothesized that these metabolic effects of EPA on BAT are in part mediated by UCP1. MethodsTo determine the role of UCP1 in obesity and BAT regulation by EPA, wild type (WT) and UCP1 knockout (KO) B6 male mice were housed at thermoneutral conditions (30°C), previously reported to induce obesity in the KO mice. Mice were fed a high-fat diet (HF, 45% kcal fat) or HF diet supplemented with 36 g/kg of AlaskOmega EPA-enriched fish oil (800 mg/g), kindly provided by Organic Technologies, for up to 14 weeks. We metabolically phenotyped these mice and investigated metabolic and molecular changes in their interscapular BAT. Specifically, we determined effects of UCP1 deficiency and EPA on BAT thermogenic and mitochondrial markers. ResultsThe previously reported beneficial metabolic effects of EPA in WT mice at ambient, including increased UCP1 expression, were attenuated or lost at thermoneutral temperature. EPA reduced weight gain and adiposity, and improved glucose tolerance in KO mice. In both diets (HF and EPA), BAT triglyceride content was increased, while mitochondrial UCP1, COX I and COX IV protein levels were decreased in the KO compared to the WT genotype (P < 0.05). EPA also increased (P < 0.05) mitochondrial DNA/nuclear DNA ratio in the KO mice. Finally, BAT PGC1α at both gene and protein levels along with whole-body oxygen consumption were increased (P < 0.05) by EPA in KO mice. EPA did not alter the calcium cycling-related markers such as sarcoplasmic/endoplasmic reticulum calcium ATPase 2b (Serca2b) and transient receptor potential vanilloid 2 (Trpv2) in any of the genotypes. ConclusionsEPA effects on BAT and mitochondrial function are independent of UCP1, and include increased mitochondrial DNA and oxygen consumption, which may be in part relate to increased PGC1α. Additional studies are required to determine fuel or mitochondrial mechanisms by which energy expenditure is increased independently of UCP1. Funding SourcesNIH/NCCIH grant # R15AT008879-01A1.

ZW290 Increases Cold Tolerance by Inducing Thermogenesis via the Upregulation of Uncoupling Protein 1 in Brown Adipose Tissue In Vitro and In Vivo.

To provide molecular evidence on the thermogenic mechanism of primary brown adipocytes, western blot analysis was used to detect brown adipose tissue (BAT)-specific gene expressions. BAT protects the mammals from hypothermia injury with a large amount of mitochondria and high expression of uncoupling Protein 1 (UCP1), which is the vital protein to determine the heat production in BAT. In our previous study, the compound ZW290 (the structure shown in Fig. 1) was obtained by molecular docking with a UCP1 inducer. In the present study, ZW290 not only significantly upregulated the expression of UCP1 protein (p < 0.01) and its related signaling pathway in the primary brown adipocytes, but also remarkably decreased the mitochondrial membrane potential and the concentration of adenosine triphosphate (ATP) (p < 0.01). Kunming (KM) mice were kept under acute cold exposure (-20°C) to evaluate the preventive and protective effects of ZW290 on cold injury, and revealed its regulating mechanism in vitro. The rectal and body temperatures of ZW290-treated mice were significantly higher than those of the control (or model) group both at room temperature and at -20°C (p < 0.001). Hematoxylin-eosin (HE) staining and immunohistochemistry indicated that ZW290 notably decreased the size of lipid droplets in BAT and increased the content of mitochondria and the expression of UCP1 in BAT and white adipose tissue (WAT). Furthermore, the survival rate showed that ZW290 could prolong the overall survival of mice. Therefore, we obtained the conclusion that ZW290 might transform energy into heat by inhibiting ATP synthesis and increasing the expression of UCP1. Additionally, ZW290 may enhance cold tolerance by increasing heat production through increasing the content of mitochondria and the expression of UCP1 in BAT and WAT.

Intermittent fasting increases energy expenditure and promotes adipose tissue browning in mice.

Intermittent fasting (IF) may limit metabolic adaptations that reduce energy expenditure, potentially by stimulating white adipose tissue (WAT) browning. The aim of this study was to examine the effects of 8 wk of IF on energy metabolism and markers of WAT browning in lean and diet-induced obese mice and in women who were overweight or obese. Male C57 BL/6 J mice were fed chow or a high-fat diet (HFD; 43%) for 8 wk before undergoing IF (3 non-consecutive d/wk) for an additional 8 wk. Food intake, energy expenditure, and inguinal and gonadal fat pads were collected in fed or fasted conditions (22 h, IF mice only). Subcutaneous adipose tissue (SAT) was also collected at baseline, and after 8 wk of IF (in the fed state, and after a 24-h fast), in women with overweight or obesity. Uncoupling protein 1 (UCP1) was assessed by quantitative real-time polymerase chain reaction (mice and humans) and immunohistochemistry (mice). IF reduced body weight and energy intake in HFD fed mice and reduced gonadal and inguinal fat pad weights in both diet groups. IF increased energy expenditure, meal number, Ucp1 mRNA levels in inguinal and gonadal fat depots, and UCP1 protein in inguinal fat in both diet groups on fed days. In women, IF reduced body weight and fat mass, but did not alter UCP1mRNA levels. IF increased energy expenditure and promoted WAT browning in mice but did not alter UCP1 mRNA levels in SAT in women.

SIRT3 controls brown fat thermogenesis by deacetylation regulation of pathways upstream of UCP1

ObjectiveBrown adipose tissue (BAT) is important for thermoregulation in many mammals. Uncoupling protein 1 (UCP1) is the critical regulator of thermogenesis in BAT. Here we aimed to investigate the deacetylation control of BAT and to investigate a possible functional connection between UCP1 and sirtuin 3 (SIRT3), the master mitochondrial lysine deacetylase. MethodsWe carried out physiological, molecular, and proteomic analyses of BAT from wild-type and Sirt3KO mice when BAT is activated. Mice were either cold exposed for 2 days or were injected with the β3-adrenergic agonist, CL316,243 (1 mg/kg; i.p.). Mutagenesis studies were conducted in a cellular model to assess the impact of acetylation lysine sites on UCP1 function. Cardiac punctures were collected for proteomic analysis of blood acylcarnitines. Isolated mitochondria were used for functional analysis of OXPHOS proteins. ResultsOur findings showed that SIRT3 absence in mice resulted in impaired BAT lipid use, whole body thermoregulation, and respiration in BAT mitochondria, without affecting UCP1 expression. Acetylome profiling of BAT mitochondria revealed that SIRT3 regulates acetylation status of many BAT mitochondrial proteins including UCP1 and crucial upstream proteins. Mutagenesis work in cells suggested that UCP1 activity was independent of direct SIRT3-regulated lysine acetylation. However, SIRT3 impacted BAT mitochondrial proteins activities of acylcarnitine metabolism and specific electron transport chain complexes, CI and CII. ConclusionsOur data highlight that SIRT3 likely controls BAT thermogenesis indirectly by targeting pathways upstream of UCP1.

Fatty Acid‐Induced Hepatocellular Carcinoma Growth is Mediated by Decreasing Mitochondrial H2O2 Emission Coupled to Increased Glutathione Levels

Rationale and HypothesisHigh fat diets are associated with increased hepatocellular carcinoma (HCC) risk, as well as increased HCC growth rates. Accelerated HCC growth in response to fatty acid challenges has been attributed to a variety of signalling pathways, but the role of metabolic and redox flexibility in mediating a pro‐growth environment in this context has not been examined. Here we examined the direct effect of fatty acid challenges on HCC growth in relation to altered metabolic and redox homeostasis.Experimental approachThe HCC cell line HepG2 was incubated with 0μM, 50μM and 100μM palmitoylcarnitine (PCarn) for up to 48 hrs. We measured clonogenic survival, mitochondrial H2O2 emission and glutathione following PCarn incubations with and without the glutathione depleting agent buthionine sulfoximine (BSO) and inhibition of uncoupling protein‐2 (UCP2) activity by genipin.Results100μM PCarn increased clonogenic survival in HepG2 by 8% more than control (p&lt;0.05) at 48 hrs which represents a marked early effect considering the population doubling time of HepG2 cells is ~48 hrs. This was associated with an increase in both reduced and oxidized glutathione at both 24 and 48 hrs (p&lt;0.05). Depleting glutathione with BSO prevented PCarn‐stimulated growth (p&lt;0.05). In a separate experiment, acute incubations of 100μM PCarn increased H2O2 emission within the first 10 minutes (p&lt;0.05) followed by a decrease in H2O2 at 1 hr that remained lower at 24 hrs (p&lt;0.05). The acute increase in H2O2 followed by a more chronic depression in H2O2 suggested PCarn might have triggered a compensatory mechanism. In support of this notion, inhibition of UCP2 with genipin sensitized HepG2 cells to PCarn‐induced decreases in clonogenic survival (p&lt;0.05) without a change in UCP2 protein content.ConclusionCollectively, this data suggests that PCarn‐induced HCC growth is in part attributed to elevated glutathione. Increases in glutathione may be a result of UCP2 rapidly attenuating PCarn‐induced H2O2 emission which may permit greater glutathione synthetic rates and creation of a pro‐growth redox environment.Support or Funding InformationFunding was provided to C.G.R.P. by National Science and Engineering Research Council (#436138‐2013) with infrastructure supported by Canada Foundation for Innovation, Ontario Research Fund and the James H. Cummings Foundation. P. C. T. was supported by an NSERC CGS‐D scholarship.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In the absence of UCP1-mediated diet-induced thermogenesis, obesity is augmented even in the obesity-resistant 129S mouse strain.

The attractive tenet that recruitment and activation of brown adipose tissue (BAT) and uncoupling protein 1 (UCP1) could counteract the development of obesity and its comorbidities in humans has been experimentally corroborated mainly by experiments demonstrating that UCP1-ablated mice on a C57Bl/6 background (exempt from thermal stress) become more obese when fed a high-fat diet. However, concerns may be raised that this outcome of UCP1 ablation is restricted to this very special inbred and particularly obesity-prone mouse strain. Therefore, we have examined to which degree UCP1 ablation has similar metabolic effects in a mouse strain known to be obesity resistant: the 129S strain. For this, male 129S2/sv or 129SV/Pas mice and corresponding UCP1-knockout mice were fed chow or a high-fat or a cafeteria diet for 4 wk. The absence of UCP1 augmented obesity (weight gain, body fat mass, %body fat, fat depot size) in high-fat diet- and cafeteria-fed mice, with a similar or lower food intake, indicating that, when present, UCP1 indeed decreases metabolic efficiency. The increased obesity was due to a decrease in energy expenditure. The consumption of a high-fat or cafeteria diet increased total BAT UCP1 protein levels in wild-type mice, and correspondingly, high-fat diet and cafeteria diet-fed mice demonstrated increased norepinephrine-induced oxygen consumption. There was a positive correlation between body fat and total BAT UCP1 protein content. No evidence for diet-induced adrenergic thermogenesis was found in UCP1-ablated mice. Thus, the obesity-reducing effect of UCP1 is not restricted to a particular, and perhaps not representative, mouse strain.

Role of brown adipose tissue in body temperature control during the early postnatal period in Syrian hamsters and mice.

Brown adipose tissue (BAT) contributes to non-shivering thermogenesis and plays an important role in body temperature control. The contribution of BAT thermogenesis to body temperature control in a non-cold environment was evaluated using developing hamsters. Immunostaining for uncoupling protein 1 (UCP1), a mitochondrial protein responsible for BAT thermogenesis, indicated that interscapular fat tissue had matured as BAT at day 14. When pups were placed on a thermal plate kept at 23°C, the body surface temperature decreased in day 7- and 10-day-old pups but was maintained at least for 15 min in 14-day-old pups, indicating that hamsters are unable to maintain their body temperature until around day 14 even in a non-cold environment. Body temperature maintenance was also evaluated in UCP1-deficient mice. BAT analysis showed that the UCP1 protein level in Ucp1+/− Hetero mice was 61.3 ± 1.4% of that in wild-type (WT) mice and was undetected in Ucp1−/− knockout (KO) mice. When 12-day-old pups were place on a thermal plate at 23°C, body surface temperature was maintained for at least 15 min in WT and Hetero mice but gradually dropped by 2.4 ± 0.2°C in 15 min in KO mice. It is concluded that BAT thermogenesis is indispensable for body temperature maintenance in pups of hamsters and mice, even in the non-cold circumstances. The early life poikilothermy and the later acquirement of homeothermy in hamsters may be because of the postnatal development of BAT.

Impaired adrenergic agonist-dependent beige adipocyte induction in obese mice.

Brown adipocytes, which exist in brown adipose tissue (BAT), are activated by adrenergic stimulation, depending on the activity of uncoupling protein 1 (UCP1). Beige adipocytes emerge from white adipose tissue (WAT) in response to chronic adrenergic stimulation. We investigated obesity-related changes in responses of both types of adipocytes to adrenergic stimulation in mice. Feeding of mice with high-fat diets (HFD: 45%-kcal fat) for 14 weeks resulted in significantly higher body and WAT weight compared to feeding with normal diets (ND: 10%-kcal fat). Injection with β3-adrenergic receptor agonist CL316,243 (CL; 0.1 mg/kg, once a day) for one week elevated the mRNA and protein expression levels of UCP1 in BAT, irrespective of diet. In WAT, CL-induced UCP1 expression in ND mice; however, the responses to CL treatment were attenuated in HFD mice, indicating that CL-induced browning of WAT was impaired in obese mice. Flow cytometric analysis revealed a significant decrease in platelet-derived growth factor receptor (PDGFR) α-expressing beige adipocyte progenitors in WAT of HFD mice compared with those of ND mice. Expression of PDGF-B, a PDGFRα ligand, increased in WAT following CL-injection in ND mice, but not in HFD mice. Treatment of mice with a PDGFR inhibitor significantly decreased CL-dependent UCP1 protein induction in WAT. Our study demonstrates that β3-adrenergic stimulation-dependent beige adipocyte induction in WAT is impaired by obesity in mice, potentially due to obesity-dependent reduction in the number of PDGFRα-expressing progenitors and decreased PDGF-B expression.

PEDF protects human retinal pigment epithelial cells against oxidative stress via upregulation of UCP2 expression.

To investigate the protective function of pigment epithelium-derived factor (PEDF) against oxidative stress (OS) in ARPE-19 cells, ARPE-19 cells were divided into different OS groups and treated with various concentrations of H2O2 (0, 75, 150 and 200 µmol/l) for 24 h. To establish the protective group, 200 ng/ml of PEDF was administered to ARPE-19 cells. Cell Counting Kit-8 assays and cell growth curve experiments were performed to determine levels of cell viability; lactate dehydrogenase and propidium iodide (PI) staining assays were also performed. The expression levels of genes associated with apoptosis as well as uncoupling protein 2 (UCP2) were detected by reverse transcription-quantitative, or semi-quantitative polymerase chain reaction. Furthermore, an OS injury animal model was established in both C57BL/6 and BALB/c mice via injection of 5 µg of PEDF in the vitreous cavity and subsequent injection of 150 µM H2O2 following a 24 h time interval. Hematoxylin and eosin (H&E) staining, as well as UCP2 immunofluorescent labeling were also performed. One-way analysis of variance was used to determine statistically significant differences, followed by multiple comparison analysis using the Newman Keuls method. The results of cell viability assays demonstrated that the numbers of apoptotic cells were increased following treatment with H2O2 in a dose-dependent manner; however, this effect was reversed following treatment with PEDF. The expression levels of caspase 3 and B cell lymphoma (Bcl2) associated X genes associated with apoptosis were inhibited, whereas levels of the anti-apoptotic gene Bcl2 were enhanced following treatment with PEDF in different passages of ARPE-19 cells. Significant differences were demonstrated in the levels of UCP2 gene expression between the PEDF+ H2O2 treated group and cells treated with H2O2 alone. Labeling of the UCP2 detector in the confocal images demonstrated decreased UCP2 protein staining in the retinal pigment epithelium (RPE) cells and RPE layers following H2O2 injury; however, this effect was inhibited following treatment with PEDF. H&E staining was performed to investigate the thickness of the RPE layers, and the results revealed that thicknesses were significantly increased in sections treated with PEDF during OS, due to increased numbers of RPE cells. Furthermore, PEDF was demonstrated to increase UCP2 gene expression in ARPE-19 cells and animal RPE layers under OS, which suggested that PEDF may protect RPE cells and tissues during oxidative injury.

PO-119 Adrenergic Receptor β3 Up-regulates Uncoupling Protein 1 and Cyclooxygenase 2 Expressions in The Brown Adipocyte

Objective Brown adipose tissues (BAT) activation is important for losing weight as its high energy expenditure in Mammalian. Recent studies showed that exercise may also be essential for BAT activation. Uncoupling protein 1 (UCP1), specifically expressed in BAT's mitochondria, uncouples oxidative phosphorylation and dissipates energy from Free Fatty Acids into heat. Activating the Adrenergic Receptor β3 (Adrβ3) provides fuel for mitochondrial heat production and up-regulates Cyclooxygenase 2 (COX2), which is a key factor of UCP1 synthesis. Sympathetic nerve excitement stimulated by exercise can release norepinephrine as a neurotransmitter, which can affect Adrβ3. Brown adipocyte (BAC) is a kind of adipocyte in vitro as a model to study heat production. Isoprenaline Hydrochloride (ISO) is a widely used as an Adrβ agonist. In this research, we tried to figure out the response of BAC to Adrβ3 activations with different time points and whether ISO can be used as a BAC activator.&#x0D; Methods C3H10T1/2 cells were maintained in a humidified, 37°C, 5% CO2 incubator in DMEM/F12 medium with 10% fetal bovine serum (FBS). For brown adipogenesis, cells were first split into differentiation medium (DMEM/F12 containing 10% FBS, 20nM insulin, 1nM 3,3’5-Triiodo-L-thyronine(T3)) for 4 days, the medium was changed every other day. Confluent cells were treated for 2 days with brown adipose adipogenesis cocktails (differentiation medium containing 2µg/mL dexamethasone, 0.5mM isobutylmethylxanthine (IBMX), 0.125mM indomethacin and 1µM rosiglitazone) on day 4. Then the medium was replaced by differentiation medium and changed every other day. At day 10, the full differentiation adipocytes were treated with 10µM ISO for 0 (as control), 1, 3, 6, 12 and 24 hours. For the lipid droplets staining, the cells were fixed by 4% paraformaldehyde solution then stained with Oil Red O. The cells were harvested and the total cell lysates were extracted for protein analysis after each time point. The UCP1, COX2, and Adrβ3 expression levels were detected by western blot, using Actin as the internal protein. The results were expressed as the mean ± standard error of the mean (SEM). Group comparisons were performed using two-way ANOVA and LSD’s post-hoc tests.&#x0D; Results After differentiation, the cell shapes converted from fibroblastic to a spherical shape. Dispersed small lipid droplets were observed in the cells. After ISO treatment, the red color after Oil Red Staining became lighter and the size of the lipid droplets turned to smaller. The Adrβ3 protein expressions were 1.00±0.00, 1.34±0.32, 1.07±0.50, 4.65±1.84*, 2.44±0.73, and 3.43±1.09 at 0h, 1h, 3h, 6h, 12h, and 24h after ISO treatment, respectively. After introduced to ISO, the UCP1 expression levels were 1.00±0.00, 1.95±0.39, 2.72±0.57, 5.68±1.82*, 3.49±0.92, and 2.79±1.05 at 0h, 1h, 3h, 6h, 12h, and 24h, respectively. And for COX2, the protein expressions were 1.00±0.00, 2.13±0.67, 1.82±0.33, 4.67±1.82*, 2.88±0.44, and 2.65±0.54, respectively. The * means p ˂ 0.05, compared with oh controls. The proteins expressions were reached to peak after 6 hours ISO treatment from the above results.&#x0D; Conclusions UCP1 and COX2 protein expressions were increased in BAC according to Adrβ3's expression in different time points, indicating that Adrβ3 may induce adipolysis in BAC and help to burn fat and produce heat.

Effects of visfatin on brown adipose tissue energy regulation using T37i cells

The role of brown adipose tissue (BAT) in pathological states of energy homeostasis and impaired adipocyte function, such as obesity has been a major area of research interest in recent years. Herein, we sought to determine the direct effects of adipokines, visfatin and leptin on BAT thermogenesis.The effects of mouse recombinant visfatin, nicotinamide mononucleotide (NMN) and leptin with or without FK866 were studied on differentiated T37i cells. Treated cells were analyzed for key genes and proteins regulating BAT [UCP-1, PRD1-BF1-RIZ1 homologous domain-containing 16 (PRDM-16), PPARgamma-coactivator-1alpha (PGC-1α) and receptor-interacting protein 140 (RIP-140)] using quantitative PCR and western blot analysis. Data is presented as mean P-values.Both visfatin and leptin had significant concentration dependent effects on thermogenesis in brown pre-adipocytes and at physiological levels, increased uncoupling protein-1 (UCP-1) levels in brown adipocytes. These effects of visfatin were similar to that of nicotinamide mononucleotide (NMN), further strengthening the enzymatic role of visfatin. We also showed that leptin induced UCP-1 mRNA expression and protein production appears to be mediated by visfatin. High concentrations of both visfatin and leptin led to a dramatic decrease in UCP-1 protein levels, supporting the notion that visfatin levels are raised in obesity and that obese people have reduced BAT activity, plausibly through a reduction in UCP-1 levels. Additionally, we found differential regulation of key brown adipogenic genes, specifically, PRD1-BF1-RIZ1 homologous domain-containing 16 (PRDM-16), PPARgamma-coactivator-1alpha (PGC-1α) and receptor-interacting protein 140 (RIP-140) by visfatin. Our observations provide novel insights in the potential actions of visfatin in BAT.

Secretory phospholipase A2 group IIA enhances the metabolic rate and increases glucose utilization in response to thyroid hormone.

Secretory phospholipase A2 group IIA (PLA2G2A) is a phospholipase which has a role in inflammation, atherogenesis, and host defense. Previously, we found that PLA2G2A protects mice on high-fat diets from weight gain and insulin resistance. Here, we examined the regulation of PLA2G2A and the metabolic changes that occur in response to variations in thyroid status. In particular, the impact of PLA2G2A on the brown adipose tissue (BAT) thermogenic gene expression was explored. We induced hypothyroidism in C57BL/6 and PLA2G2A-overexpressing (IIA+) mice over a 10 wk period or treated them with thyroid hormone (T3) for 5 wk. There were no significant changes in PLA2G2A abundance in response to thyroid status. The energy expenditure of hypothyroid IIA+ mice did not increase; however, the energy expenditure, substrate utilization, insulin sensitivity, and glucose tolerance were all elevated in the IIA+ mice given T3. Moreover, white adipocytes from IIA+ mice were much more prone to "beiging," including increased expression of brown adipose thermogenic markers such as uncoupling protein 1 (UCP1), PR domain containing 16, and early B cell factor 2. Finally, the BAT of IIA+ mice had increased UCP1 and other proteins indicative of mitochondrial uncoupling and nonshivering adaptive thermogenesis. These data reveal a novel role for PLA2G2A on adipose tissue thermogenesis depending on thyroid status.-Kuefner, M. S., Deng, X., Stephenson, E. J., Pham, K., Park, E. A. Secretory phospholipase A2 group IIA enhances the metabolic rate and increases glucose utilization in response to thyroid hormone.

Melinjo (Gnetum gnemon L.) seed extract induces uncoupling protein 1 expression in brown fat and protects mice against diet-induced obesity, inflammation, and insulin resistance

Dietary supplementation with melinjo (Gnetum gnemon L.) seed extract (MSE) has been proposed as an anti-obesity strategy. However, it remains unclear how MSE modulates energy balance. We tested the hypothesis that dietary MSE reduces energy intake and/or increases physical activity and metabolic thermogenesis in brown and white adipose tissue (BAT and WAT) in mice. Twenty-four C57BL/6 J mice were provided with normal diet, high-fat diet (HFD), or HFD with 1% MSE added, for 17 weeks. Food intake, spontaneous locomotor activity, hepatic triglyceride (TG) content, and blood parameters were examined. Mitochondrial thermogenesis-associated molecule and inflammatory marker expression levels in BAT and WAT were examined by quantitative PCR and western blotting. Dietary MSE did not affect energy intake or spontaneous locomotor activity, but significantly suppressed HFD-induced fat accumulation, hyperglycemia, and hyperinsulinemia. Homeostasis model assessment of insulin resistance score and hepatic TG content were both lower in the MSE-supplemented HFD-fed group than in the HFD-fed group, indicating reduced insulin resistance and a less fatty liver. Dietary MSE upregulated thermogenic uncoupling protein 1 (UCP1) and mitochondrial marker cytochrome c oxidase subunit IV protein expression in BAT; this was closely associated with sirtuin 1 mRNA induction. mRNAs of adipose inflammatory markers, such as monocyte chemotactic 1 and interleukin-1, were induced by HFD but suppressed by MSE. Considering that UCP1 protein expression is the most physiologically relevant parameter to assess the thermogenic capacities of BAT, our results indicate that dietary MSE supplementation induces BAT thermogenesis and reduces obesity-associated adipose tissue inflammation, hepatic steatosis, and insulin resistance.

Abstract 2399: Loss of ubiquitin-specific peptidase 18 (USP18) destabilizes the regulator of thermogenesis uncoupling protein-1 (UCP-1) and represses lung cancer growth

Abstract USP18 is the ISG15 (Interferon-Stimulated Gene 15) deconjugase that removes ISG15 from substrate proteins. We found USP18 null mice are markedly cold sensitive versus their wild-type littermates. USP18 null mice had significantly (P &amp;lt; 0.05) greater temperature declines than wild-type mice. These mice also had lower lipolysis rates and altered fat to body weight ratios. This implicated USP18 as a regulator of lipid and fatty acid metabolism. Protein expression profiles of thermogenic regulators were examined in brown fat of USP18 null versus wild-type mice. Strikingly, the thermoregulatory protein UCP-1 was substantially repressed in brown fat of USP18 null mice. To establish that UCP-1 repression was caused by loss of USP18, stable USP18 knock-down was independently achieved in a panel of murine lung cancer cell lines using transfected small hairpin RNAs (shRNAs). USP18 down-regulation by different shRNAs reduced UCP-1 levels as compared to controls. Engineered gain of USP18 expression stabilized UCP-1 expression in these lung cancer cell lines. UCP-1 destabilization followed complex formation with the ubiquitin-like protein ISG15. Immunoprecipitation assays established that complexes formed between ISG15 and UCP-1. Prior work found that USP18 regulates lung tumorigenesis. We therefore examined UCP-1 levels in human lung adenocarcinomas using The Cancer Genome Atlas (TCGA). Lung adenocarcinomas with undetected UCP-1 expression had significantly improved survival versus cases that expressed UCP-1. Notably, gain of UCP-1 expression in both human and murine lung cancer cell lines promoted their growth. In marked contrast, UCP-1 knockdown reduced proliferation. We explored whether UCP-1 expression affected fatty acid metabolism. The beta-hydroxybutyrate (ketone body) colorimetric assay confirmed that UCP-1 overexpression promoted fatty acid beta-oxidation. Gain of UCP-1 expression also increased fatty acid beta-oxidation using seahorse assays. Thus, a direct link exists between USP18 and expression of the thermoregulator UCP-1. This affects UCP-1 stability and lung cancer growth by altering fatty acid metabolism. Together, these findings implicate the deubiquitinase USP18 as a novel molecular pharmacologic target that controls fatty acid metabolism as an energy source for lung cancer growth. Citation Format: Xi Liu, Yun Lu, Weiguo Hu, Zibo Chen, Lisa M. Mustachio, Jason Roszik, Lin Zheng, Masanori Kawakami, Yulong Chen, Sarah J. Freemantle, Ethan Dmitrovsky. Loss of ubiquitin-specific peptidase 18 (USP18) destabilizes the regulator of thermogenesis uncoupling protein-1 (UCP-1) and represses lung cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2399.

Cellular and subcellular localization of uncoupling protein 2 in the human kidney.

The uncoupling protein-2 (UCP2) is an anion transporter that plays a key role in the control of intracellular oxidative stress. In animal models UCP2 downregulation has several pathological sequelae, particularly affecting the vasculature and the kidney. Specifically, in these models kidney damage is highly favored in the absence of UCP2 in the context of experimental hypertension. Confirmations of these data in humans awaits further information, as no data are yet available concerning the cell-type and subcellular expression in the human kidney. In the present study, we aimed to characterize the UCP2 protein distribution in human kidney biopsies. In humans UCP2 is mainly localized in proximal convoluted tubule cells, with an intracytoplasmic punctate staining. UCP2 positive puncta are often localized at the interface between the endoplasmic reticulum and the mitochondria. Glomerular structures do not express UCP2 at detectable levels. The expression of UCP2 in proximal tubular cells may explain their relative propensity to damage in pathological conditions including the hypertensive disease.

Functional Role and Mechanism of UCP2 Upregulation in Pancreatic ß Cells

Uncoupling protein 2 (UCP2) is a mitochondrial protein, which uncouples the oxidative phosphorylation. UCP2 protein level is reportedly upregulated in pancreatic islets of patients with type 2 diabetes; however, the functional role and mechanism of the upregulated UCP2 remain unclear. In this study, we undertook both in vitro regulation of gene expression and in vivo transgenic approaches to assess the significance of UCP2 in β cells. The expression of UCP2 was significantly upregulated by the stimulation with glucose, glucokinase activator, or insulin in mouse isolated islets (n=3-5, p &amp;lt;0.05). The islet UCP2 expression was also enhanced by the deletion of insulin receptor substrate 2 (IRS-2) (n=3, p &amp;lt;0.05). We generated β-cell-specific UCP2 transgenic (RIP-UCP2) mice, which had 40-fold higher UCP2 expression in islets (n=5, p &amp;lt;0.01), but not in hypothalamus. RIP-UCP2 mice developed glucose intolerance (n=13, p &amp;lt;0.01) in association with tendency to decrease insulin secretion (n=4, p=0.17, at 2 or 5 min after glucose loading), without alterations in insulin sensitivity and β cell mass, compared to the control. ATP/ADP ratio of RIP-UCP2 islets was significantly decreased after glucose stimulation (n=4, p &amp;lt;0.05). Next, we fed mice with high-fat diets (HFD) for 20 weeks. HFD-fed RIP-UCP2 mice initially showed hyperglycemia compared to the control; however, the difference in blood glucose was disappeared after 8 weeks. There were no changes in glucose tolerance, insulin secretion, and insulin sensitivity in HFD-fed RIP-UCP2 mice. β cell mass tended to be increased in HFD-fed RIP-UCP2 mice (1.5-fold compared with HFD-fed control, n=4-5, p=0.15). The IRS-2 expression level was significantly higher in the islets from HFD-fed RIP-UCP2 mice (n=4, p &amp;lt;0.05). Taken together, UCP2 upregulation potentially participates in the reduction in insulin secretion and β cell mass expansion by modulating ATP production and IRS-2 expression. Disclosure R. Inoue: None. J. Shirakawa: None. Y. Togashi: None. Y. Terauchi: Research Support; Self; MSD K.K.. Speaker's Bureau; Self; MSD K.K.. Advisory Panel; Self; MSD K.K.. Research Support; Self; Ono Pharmaceutical Co., Ltd.. Speaker's Bureau; Self; Ono Pharmaceutical Co., Ltd.. Research Support; Self; Novartis Pharma K.K., Boehringer Ingelheim GmbH. Speaker's Bureau; Self; Boehringer Ingelheim GmbH. Advisory Panel; Self; Boehringer Ingelheim GmbH. Research Support; Self; Mitsubishi Tanabe Pharma Corporation. Speaker's Bureau; Self; Mitsubishi Tanabe Pharma Corporation. Advisory Panel; Self; Mitsubishi Tanabe Pharma Corporation. Research Support; Self; Daiichi Sankyo Company, Limited. Speaker's Bureau; Self; Daiichi Sankyo Company, Limited. Advisory Panel; Self; Daiichi Sankyo Company, Limited. Research Support; Self; Sanwa Kagaku Kenkyusho Co., Ltd.. Speaker's Bureau; Self; Sanwa Kagaku Kenkyusho Co., Ltd.. Research Support; Self; Novo Nordisk Inc.. Speaker's Bureau; Self; Novo Nordisk Inc.. Advisory Panel; Self; Novo Nordisk Inc.. Research Support; Self; Eli Lilly and Company. Speaker's Bureau; Self; Eli Lilly and Company. Advisory Panel; Self; Eli Lilly and Company. Research Support; Self; Sanofi. Speaker's Bureau; Self; Sanofi. Advisory Panel; Self; Sanofi. Research Support; Self; Sumitomo Dainippon Pharma Co., Ltd.. Speaker's Bureau; Self; Sumitomo Dainippon Pharma Co., Ltd.. Research Support; Self; Shionogi &amp; Co., Ltd.. Speaker's Bureau; Self; Shionogi &amp; Co., Ltd., Bayer Yakuhin, Ltd., Astellas Pharma US, Inc., AstraZeneca. Advisory Panel; Self; AstraZeneca, Teijin Pharma Limited.

Adipose tissue uncoupling protein 1 levels and function are increased in a mouse model of developmental obesity induced by maternal exposure to high-fat diet.

With brown adipose tissue (BAT) becoming a possible therapeutic target to counteract obesity, the prenatal environment could represent a critical window to modify BAT function and browning of white AT. We investigated if levels of uncoupling protein 1 (UCP1) and UCP1-mediated thermogenesis are altered in offspring exposed to prenatal obesity. Female CD-1 mice were fed a high-fat (HF) or standard-fat (SF) diet for 3 months before breeding. After weaning, all pups were placed on SF. UCP1 mRNA and protein levels were quantified using quantitative real-time PCR and Western blot analysis, respectively, in brown (BAT), subcutaneous (SAT) and visceral (VAT) adipose tissues at 6 months of age. Total and UCP1-dependent mitochondrial respiration were determined by high-resolution respirometry. A Student's t-test and Mann-Whitney test were used (significance: P<0.05). UCP1 mRNA levels were not different between the HF and SF offspring. UCP1 protein levels, total mitochondrial respiration and UCP1-dependent respiration were significantly higher in BAT from HF males (P=0.02, P=0.04, P=0.005, respectively) and females (P=0.01, P=0.04, P=0.02, respectively). In SAT, the UCP1 protein was significantly lower in HF females (P=0.03), and the UCP1-dependent thermogenesis was significantly lower from HF males (P=0.04). In VAT, UCP1 protein levels and UCP1-dependent respiration were significantly lower only in HF females (P=0.03, P=0.04, respectively). There were no differences in total respiration in SAT and VAT. Prenatal exposure to maternal obesity leads to significant increases in UCP1 levels and function in BAT in offspring with little impact on UCP1 levels and function in SAT and VAT.