UCP1 Protein Expression Research Articles

Nox4 mediates skeletal muscle metabolic responses to exercise

ObjectiveThe immediate signals that couple exercise to metabolic adaptations are incompletely understood. Nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) produces reactive oxygen species (ROS) and plays a significant role in metabolic and vascular adaptation during stress conditions. Our objective was to determine the role of Nox4 in exercise-induced skeletal muscle metabolism. MethodsMice were subjected to acute exercise to assess their immediate responses. mRNA and protein expression responses to Nox4 and hydrogen peroxide (H2O2) were measured by qPCR and immunoblotting. Functional metabolic flux was measured via ex vivo fatty acid and glucose oxidation assays using 14C-labeled palmitate and glucose, respectively. A chronic exercise regimen was also utilized and the time to exhaustion along with key markers of exercise adaptation (skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase activity) were measured. Endothelial-specific Nox4-deficient mice were then subjected to the same acute exercise regimen and their subsequent substrate oxidation was measured. ResultsWe identified key exercise-responsive metabolic genes that depend on H2O2 and Nox4 using catalase and Nox4-deficient mice. Nox4 was required for the expression of uncoupling protein 3 (Ucp3), hexokinase 2 (Hk2), and pyruvate dehydrogenase kinase 4 (Pdk4), but not the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α). Global Nox4 deletion resulted in decreased UCP3 protein expression and impaired glucose and fatty acid oxidization in response to acute exercise. Furthermore, Nox4-deficient mice demonstrated impaired adaptation to chronic exercise as measured by the time to exhaustion and activity of skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase. Importantly, mice deficient in endothelial-Nox4 similarly demonstrated attenuated glucose and fatty acid oxidation following acute exercise. ConclusionsWe report that H2O2 and Nox4 promote immediate responses to exercise in skeletal muscle. Glucose and fatty acid oxidation were blunted in the Nox4-deficient mice post-exercise, potentially through regulation of UCP3 expression. Our data demonstrate that endothelial-Nox4 is required for glucose and fatty acid oxidation, suggesting inter-tissue cross-talk between the endothelium and skeletal muscle in response to exercise.

In Vitro and In Vivo Effects of Fermented Oyster-Derived Lactate on Exercise Endurance Indicators in Mice.

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.

3'-Hydroxydaidzein Improves Obesity Through the Induced Browning of Beige Adipose and Modulation of Gut Microbiota in Mice with Obesity Induced by a High-Fat Diet.

Obesity is an important health issue nowadays. 3'-Hydroxydaidzein (OHD) is a metabolite of daidzein (DAI) that can be found in fermented soybean products, such as miso. DAI has been known to affect lipid accumulation, but the effect of OHD on lipid accumulation still needs to be investigated. In this study, we investigated the effects of OHD on mice with obesity induced by a high-fat diet (HFD). The results showed that mice treated with 0.1% OHD (HOHD) significantly reduced their body weight and inguinal fat without altering their food intake compared with the HFD group. The HOHD and DAI groups' hyperlipidemia were alleviated through decreased serum triacylglycerols and total cholesterol levels. The adipocyte sizes in inguinal fat were significantly smaller in the HOHD and DAI groups compared with the HFD group. Both the HOHD and DAI groups had increased PRDM16, C/EBP β, p-p38, SIRT1, PGC1 α, and UCP1 protein expression in their inguinal adipose tissue compared with the HFD group. Moreover, the OHD and DAI groups had significantly lower amounts of Lachnospira and GCA_900066225 compared with the HFD group. Collectively, OHD can ameliorate HFD-induced obesity in mice by stimulating the browning of the white adipose tissue and modulating gut microbiota.

Cold‐induced beigeing of stem cell‐derived adipocytes is not fully reversible after return to normothermia

Beige adipocytes possess the morphological and biochemical characteristics of brown adipocytes, including the mitochondrial uncoupling protein (UCP)1. Mesenchymal stem cells (MSCs) are somatic multipotent progenitors which differentiate into lipid‐laden adipocytes. Induction of MSC adipogenesis under hypothermic culture conditions (ie 32°C) promotes the appearance of a beige adipogenic phenotype, but the stability of this phenotypic switch after cells are returned to normothermic conditions of 37°C has not been fully examined. Here, cells transferred from 32°C to 37°C retained their multilocular beige‐like morphology and exhibited an intermediate gene expression profile, with both beige‐like and white adipocyte characteristics while maintaining UCP1 protein expression. Metabolic profile analysis indicated that the bioenergetic status of cells initially differentiated at 32°C adapted post‐transfer to 37°C, showing an increase in mitochondrial respiration and glycolysis. The ability of the transferred cells to respond under stress conditions (eg carbonyl cyanide‐4‐phenylhydrazone (FCCP) treatment) demonstrated higher functional capacity of enzymes involved in the electron transport chain and capability to supply substrate to the mitochondria. Overall, MSC‐derived adipocytes incubated at 32°C were able to remain metabolically active and retain brown‐like features after 3 weeks of acclimatization at 37°C, indicating these phenotypic characteristics acquired in response to environmental conditions are not fully reversible.

Effect of Insulin-Regulated FOXC2 Expression in Adipocyte Differentiation and Insulin Resistance.

Objective1) To investigate the effect of FOXC2 on the differentiation of adipose-derived mesenchymal stem cells. 2) To analyze the mechanism between FOXC2 expression regulation in adipose differentiation and insulin resistance (IR).MethodsWe first amplified the FOXC2 promoter region-512 and cloned it into the luciferase expression vector. The reporter gene system was transfected into the adipose tissue-derived mesenchymal stem cell to study insulin-mediated FOXC2 expression. We also manipulated FOXC2 protein expression by either siRNA or overexpression and studied the differentiation capability of adipose tissue-derived mesenchymal stem cell into adipocytes, as well as the influence on several IR-related genes: GLUT4, PPARγ, UCP1 and PAI-1.Results1) Insulin effectively induced the expression of FOXC2 protein in adipose tissue-derived mesenchymal stem cells under differentiation (P<0.01). Insulin also induced FOXC2-pro-512T promoter activity significantly (P<0.01). 2) The stem cell adipose differentiation decreased in the FOXC2 overexpression group. 3) When FOXC2 was overexpressed, the expression of GLUT4, PAI-1 and UCP1 was higher than control groups (p<0.001). When FOXC2 was down-regulated by siRNA, both GLUT4 and PAI-1’s protein expression were decreased (p<0.001), and the protein expression of PPARγ was increased (p<0.001). In the presence of insulin induction, overexpression of FOXC2 led to significantly higher UCP-1 expression (p<0.001) and lower PAI-1 expression (p<0.001). The protein expression of GLUT4, PAI-1 (p<0.001) and UCP-1 (p<0.05) was decreased in cells transfected with FOXC2 siRNA.ConclusionInsulin effectively induced the expression of FOXC2 protein in adipose tissue-derived mesenchymal stem cells under differentiation, possibly through the regulation of the FOXC2-pro-512T promoter activity. The different protein expression of FOXC2 has regulatory effects on several genes related to insulin resistance. FOXC2 is an important regulatory factor in adipocyte differentiation and insulin resistance.

Sitagliptin mitigates oxidative stress and up-regulates mitochondrial biogenesis markers in Brown adipose tissues of high-fat diet fed obese mice through AMPK phosphorylation

AimsThe present study was designed to investigate the effects of Sitagliptin (SGN) on oxidative stress, 5' adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, together with mitochondrial biogenesis and thermogenic markers in intercapsular brown adipose tissue (iBAT) of Swiss albino mice fed high-fat diet for sixteen weeks. MethodsMice were distributed into five groups with last eight weeks of treatment: Normal control (NC): control diet with regular drinking water; Obese control (OC): 60% high-fat diet with 20% fructose drinking water (HFFR); Treatment-1: (OC + MET): HFFR with metformin (MET) 100 mg/kg/day; Treatment-2 (OC + SGN30): HFFR with SGN 30 mg/kg/day; Treatment-3 (OC + SGN20): HFFR with SGN 20 mg/kg/day. Fasting blood glucose, serum free fatty acid and adiponectin levels were measured. Oxidative stress, P-AMPK protein expression and mRNA expression of PPARα, PGC1-α, NRF-1, TFAM, and UCP-1 in iBAT were studied. ResultsSGN treatment for eight weeks ameliorated oxidative stress, improved P-AMPK protein expression and enhanced mitochondrial biogenesis and thermogenic mediators in iBAT. ConclusionThe findings from the present study suggest the beneficial role of SGN in ameliorating obesity associated oxidative stress and up-regulated mitochondrial biogenesis and thermogenic markers in iBAT of obese mice, possibly through AMPK activation.

The Role Of COX2 In The Regulation Of Brown Adipose Tissue In Obesity By Aerobic Exercise

PURPOSE: The aim was to explore the regulation mechanism of aerobic exercise on brown adipose tissue (BAT) in obese mice and the role of COX2. METHODS: The mice were randomly divided into the control group (C, normal diet), obesity group (H, high-fat diet to establish obesity model) and the obesity exercise group (HE, high-fat diet and medium intensity treadmill training). Blood lipid and glucose were detected after 4 weeks. Differentially expression genes in scapula BAT were determined by mRNA expression profiles, and functional annotation and signal pathway enrichment were carried out. COX2 (VEGF signaling pathway) involved in key biological processes (BP) and pathway. ADRβ3 agonist Isoprenaline (activating VEGF pathway) and NS-398 (COX2 inhibitor) were used, and the distribution of lipid droplets, the glycerol level, and the expression of mRNA and protein in VEGF pathway in brown adipocytes were detected. RESULTS: High-fat diet could induce obesity, accompanied by increased blood glucose and lipid levels (2.03±0.09 vs. 4.54±0.3), reducing UCP1 levels (1 vs. 0.71±0.1) in BAT. Aerobic exercise could significantly reduce the obese mice weight (C: 26.21±0.57; H: 27.53±0.61; HE: 26.32±0.52) and blood glucose levels (C: 8.5±0.78; H: 10.22±1.77; HE: 8.25±1.33). The BP of up-regulated genes in group H/C were mainly enriched in immune system progression, inflammatory and immune response, down-regulated genes were lipid metabolism and oxidation reduction. The up-regulated genes in group HE/H were enriched in glycolipid metabolism, while the down-regulated genes were cell death and apoptosis. VEGF signaling pathway plays an important role in this process, and COX2 in the VEGF pathway played a central role by interaction analysis. Application of Isoprenaline could increase the glycerin level (12.8±1.38 vs. 31.27±3.49) and the protein expression of VEGFa (1 vs.1.34±0.12), COX2 (1 vs.1.49±0.22) and UCP1 (1 vs. 1.27±0.18); Simultaneous application of NS-398 could inhibit the protein expression of COX2 and UCP1 in brown adipocytes. CONCLUSIONS: Obesity could lead to disorder of glycolipid metabolism and inflammation of BAT. Aerobic exercise could activate BAT activity through VEGF-COX2 pathway to reduce adverse effects of obesity. Funded by FRF for the Central Universities of China (2018GJ017)

1945-P: Pioglitazone Promotes the Function of White and Brown Adipose Tissue in Diet-Induced Obese Mice

PPARγ agonist pioglitazone is an insulin sensitizer in the treatment of type 2 diabetes. Here, we investigate the effects of pioglitazone on white and brown fat in diet-induced obese mice. After 16-week diet intervention, C57BL/6 mice were divided randomly into three groups: normal chow diet group (NC group), high-fat diet group (HFD group) and high-fat diet + pioglitazone group (HFDP group). Pioglitazone (30-40mg/kg/day) was administered in drinking water for 6 weeks. After 6-week pioglitazone treatment, there was no significant difference in both body weight and fat mass between the HFDP group and the HFD group. However, fasting blood glucose, glucose tolerance and insulin sensitivity were significantly improved in HFDP group. What’s more, the weight of liver and epididymal adipose tissue (eWAT) were not reduced by pioglitazone treatment. However, the weight of brown adipose tissue (BAT) in the HFDP group was significantly higher than HFD group. Histological staining showed that the hepatic steatosis was ameliorated by pioglitazone. Quantitative real-time PCR revealed that mRNA levels of heat-producing genes (PRDM16, FGF-21, UCP1) and mitochondrial biosynthesis and functional genes (Tfam, cidea, cox7a1, Fabp4) were upregulated in eWAT by pioglitazone. Western blotting results also showed that the protein expression of UCP1 and PGC1α was upregulated by pioglitazone both in eWAT and in BAT when compared with the HFD group. To sum up, our study revealed that pioglitazone could promote the browning of WAT and activate the function of BAT with improved hepatic steatosis in diet-induced obese mice. Disclosure F. Xu: None. W. Wang: None. X. Zheng: None. W. Guo: None. Y. Shen: None.

1498-P: A Feedback Loop between miRNA Processing Complex and miR-3975 in Western Diet–Induced Brown Adipose Tissue Dysfunction

Brown adipose tissue (BAT) is considered as promising therapeutic targets for obesity and metabolic disorder through modulation of energy homeostasis. microRNAs (miRNAs) are established as essential modulators of biological processes in many tissues, including the differentiation and function of BAT. Here we demonstrated that high-fat/high fructose associated dysfunction of BAT coincides with impaired miRNA-processing pathway in a feedback loop with miR-3975 activation. Eight-week-old male C57BL/6 mice were fed a low-fat diet (10% fat and 17% sucrose; LFD) and high fat-high fructose (45% fat, 17% sucrose, and 17% fructose; HFHFD) for 4, 12, and 20 weeks (n=6-9 per group). We evaluated expression levels of apoptosis markers, thermogenesis markers, miRNA biogenesis regulators, and miRNAs in BAT. Mice fed an HFHFD showed a gradually significance increased body weight and brown adipose tissue weight when compared to the mice fed with LFD. BAT of HFHFD fed mice has a pale appearance versus feeding period matched LFD fed mice. The protein expression of thermogenesis markers, UCP1 (P &amp;lt;0.05), and PGC1α (P &amp;lt;0.05) were shown to decrease after 12 weeks of HFHFD diet feeding. The apoptosis marker, the Bax/Bcl-2 ratio, was upregulated at 12 weeks (P &amp;lt; 0.01). Mice fed HFHFD for 12 and 20 weeks showed a significant decrease in Dicer (P &amp;lt; 0.01 for 12 and 20 weeks) and TRBP2 (P &amp;lt; 0.01 for 12 weeks and P &amp;lt; 0.0001 for 20 weeks). miR-3975, which is predicted miRNA to regulate both Dicer and TRBP2 by IPA was activated at 12 weeks (P &amp;lt; 0.01), while inactivated at 20 weeks (P &amp;lt; 0.001). Our results suggest that miR-3975 acts as a component of a negative feedback loop that blocks miRNA biogenesis machinery through the inhibition of Dicer and TRBP2 expression. Therefore, long-term HFHFD may disturb miRNA processing and contribute to brown adipose tissue dysfunction and apoptosis by modulation of miRNA expression. We are currently investigating the novel feedback loop in brown adipocyte primary cells. Disclosure M. Park: None. S. Meruvu: None. J. Zhang: None. M. Choudhury: None.

Micronutrients Selenomethionine and Selenocysteine Modulate the Redox Status of MCF-7 Breast Cancer Cells.

Selenium is a micronutrient which is found in many foods, with redox status modulation activity. Our aim was to evaluate the effects of two chemical forms of selenoamino acids, Seleno-L-methionine and Seleno-L-cystine (a diselenide derived from selenocysteine), at different concentrations on cell viability, hydrogen peroxide production, antioxidant enzymes, UCP2 protein expression, as well as lipid and protein oxidative damage in MCF-7 breast cancer cells. Results showed that Seleno-L-methionine did not cause an increase in hydrogen peroxide production at relatively low concentrations, accompanied by a rise in the antioxidant enzymes catalase and MnSOD, and UCP2 protein expression levels. Furthermore, a decrease in protein and lipid oxidative damage was observed at 10 µM concentration. Otherwise, Seleno-L-cystine increased hydrogen peroxide production from relatively low concentrations (100 nM) to a large increase at high concentrations. Moreover, at 10 µM, Seleno-L-cystine decreased UCP2 and MnSOD protein expression. In conclusion, the chemical form of selenoamino acid and their incorporation to selenoproteins could affect the regulation of the breast cancer cell redox status. Taken together, the results obtained in this study imply that it is important to control the type of selenium-enriched nutrient consumption, taking into consideration their composition and concentration.

Milk fat globule membrane and its component phosphatidylcholine induce adipose browning both in vivo and in vitro

The functional induction of brown-like adipocytes in white adipose tissue (WAT) provides a defense against obesity. The aim of this study was to analyze the effects of milk fat globule membrane (MFGM) and its component phosphatidylcholine (PC) on the brown remodeling of WAT. Male C57BL/6 J mice were fed a high-fat diet (HFD) for 8 weeks and then fed HFD for another 8 weeks with MFGM. In vitro studies were performed in C3H10T1/2 pluripotent stem cells, 3T3-L1 pre-adipocytes and differentiated inguinal WAT stromal vascular cells (SVCs) to determine the role of MFGM and PC on the formation of brown-like adipocytes. MFGM decreased fasting glucose and serum insulin levels in HFD-fed mice. MFGM improved glucose tolerance and insulin sensitivity, and induced browning of inguinal WAT. MFGM and its component PC stimulated transformation of brown-like adipocytes in C3H10T1/2 pluripotent stem cells, 3T3-L1 adipocytes and SVCs by increasing the protein expression of UCP1, PGC-1α, PRDM16 as well as the mRNA expression of other thermogenic genes and beige cell markers. MFGM and PC also increased mitochondrial DNA (mtDNA) copy number, mitochondrial density and oxygen consumption rate and up-regulated the mRNA expression of mitochondria-biogenesis-related genes in vitro. PPARα inhibitor GW6471 treatment or knockdown of PPARα using lentivirus-expressing shRNA inhibited the PC-induced increase in the protein expression of UCP1, PGC-1α and PRDM16 in C3H10T1/2 pluripotent stem cells and 3T3-L1 adipocytes, indicating the potential role of PPARα in PC-mediated brown-like adipocyte formation. In conclusion, MFGM and milk PC induced adipose browning, which has major protective effects against obesity and metabolic dysfunction.

Inhibition of Uncoupling Protein 2 Enhances the Radiosensitivity of Cervical Cancer Cells by Promoting the Production of Reactive Oxygen Species.

Objective The mechanism of enhanced radiosensitivity induced by mitochondrial uncoupling protein UCP2 was investigated in HeLa cells to provide a theoretical basis as a novel target for cervical cancer treatment. Methods HeLa cells were irradiated with 4 Gy X-radiation at 1.0 Gy/min. The expression of UCP2 mRNA and protein was assayed by real-time quantitative polymerase chain reaction and western blotting. UCP2 siRNA and negative control siRNA fragments were constructed and transfected into HeLa cells 24 h after irradiation. The effect of UCP2 silencing and irradiation on HeLa cells was determined by colony formation, CCK-8 cell viability, γH2AX immunofluorescence assay of DNA damage, Annexin V-FITC/PI apoptosis assay, and propidium iodide cell cycle assay. The effects on mitochondrial structure and function were investigated with fluorescent probes including dichlorodihydrofluorescein diacetate (DCFH-DA) assay of reactive oxygen species (ROS), rhodamine 123, and MitoTracker Green assay of mitochondrial structure and function. Results Irradiation upregulated UCP2 expression, and UCP2 knockdown decreased the survival of irradiated HeLa cells. UCP2 silencing sensitized HeLa cells to irradiation-induced DNA damage and led to increased apoptosis, cell cycle arrest in G2/M, and increased mitochondrial ROS. Increased radiosensitivity was associated with an activation of P53, decreased Bcl-2, Bcl-xl, cyclin B, CDC2, Ku70, and Rad51 expression, and increased Apaf-1, cytochrome c, caspase-3, and caspase-9 expression. Conclusions UCP2 inhibition augmented the radiosensitivity of cervical cancer cells, and it may be a potential target of radiotherapy of advanced cervical cancer.

Inhibition of Lipolysis With Acipimox Attenuates Postburn White Adipose Tissue Browning and Hepatic Fat Infiltration.

Extensive burn injuries promote an increase in the lipolysis of white adipose tissue (WAT), a complication that enhances postburn hypermetabolism contributing to hyperlipidemia and hepatic steatosis. The systemic increase of free fatty acids (FFAs) due to burn-induced lipolysis and subsequent organ fatty infiltration may culminate in multiple organ dysfunction and, ultimately, death. Thus, reducing WAT lipolysis to diminish the mobilization of FFAs may render an effective means to improve outcomes postburn. Here, we investigated the metabolic effects of Acipimox, a clinically approved drug that suppresses lipolysis via inhibition of hormone-sensitive lipase (HSL). Using a murine model of thermal injury, we show that specific inhibition of HSL with Acipimox effectively suppresses burn-induced lipolysis in the inguinal WAT leading to lower levels of circulating FFAs at 7 days postburn (P < 0.05). The FFA substrate shortage indirectly repressed the thermogenic activation of adipose tissue after injury, reflected by the decrease in protein expression of key browning markers, UCP-1 (P < 0.001) and PGC-1α (P < 0.01). Importantly, reduction of FFA mobilization by Acipimox significantly decreased liver weight and intracellular fat accumulation (P < 0.05), suggesting that it may also improve organ function postburn. Our data validate the pharmacological inhibition of lipolysis as a potentially powerful therapeutic strategy to counteract the detrimental metabolic effects induced by burn.

Garlic oil suppresses high-fat diet induced obesity in rats through the upregulation of UCP-1 and the enhancement of energy expenditure.

Garlic (Allium sativum L.) has long been used as a medicinal food. Indeed, garlic and its constituents have been shown to possess potent regulatory activities in bodily functions, including blood coagulation, lipid metabolism, immunity and xenobiotic metabolism. In this study, we aimed to examine the anti-obesity effects of garlic oil and to elucidate the possible underlying mechanisms. For this purpose, garlic oil (GO; 80 mg/kg body weight, p.o.) or corn oil alone as a vehicle-control were administered to male Sprague-Dawley rats every other day for 10 weeks. The results revealed that GO administration significantly reduced body weight gain and white adipose tissue (WAT) mass, which had been increased by feeding on the AIN-76-based high-fat diet (60% kcal fat). Expired gas analysis was performed at 9 weeks following the GO administration to calculate fuel oxidation. GO administration enhanced O2 consumption during the dark period (at night) and increased energy expenditure through fat oxidation during the light period (daytime); however, carbohydrate oxidation remained unaltered. Western blot analysis revealed that GO administration increased UCP1 protein expression in brown adipose tissue (BAT). On the whole, the findings of this study indicated that GO suppressed body weight gain and WAT mass in the rat model of high-fat diet-induced obesity by increasing UCP1 expression and by enhancing fat oxidation and energy expenditure.

Age-related sex differences in the expression of important disease-linked mitochondrial proteins in mice

The prevalence and progression of many illnesses, such as neurodegenerative and cardiovascular diseases, obesity, and cancer, vary between women and men, often in an age-dependent manner. A joint hallmark of these diseases is some type of mitochondrial dysfunction. While several mitochondrial proteins are known to be regulated by sex hormones, the levels of those proteins have not been systematically analyzed with regard to sex and age, and studies that consider sex and/or age differences in the protein expression are very rare. In this study, we compared the expression patterns of physiologically important mitochondrial proteins in female and male C57BL/6N mice of age cohorts frequently used in experiments. We found that sex-related differences in the expression of uncoupling proteins 1 and 3 (UCP1 and UCP3) occur in an age-dependent manner. The sex-specific expression of UCP1 and UCP3 in brown adipose tissue (BAT) was inversely correlated with differences in body weight. Expression of UCP4 in the brain, Complex I in the spleen, and Complex II in the brain and BAT was least affected by the sex of the mouse. We further demonstrated that there are serious limitations in using VDAC1 and actin as markers in western blot analyses, due to their sex- and age-specific fluctuations. Our results confirm that sex and age are important parameters and should be taken into account by researchers who examine the mechanistic aspects of diseases.HighlightsThe levels of UCP1 and UCP3 protein expression differ between females and males in an age-dependent manner.Pre-pubertal expression of almost all proteins tested in this study does not depend on the sex of the mouse.Expression of VDAC1 and actin, which are often used as loading control proteins in western blot analysis, is tissue-specifically influenced by sex and age.

Effect of Quercetin on Non-shivering Thermogenesis and Intestinal Microbial Populations (P06-037-19)

ObjectivesActivation of non-shivering thermogenesis in adipose tissues and alteration in intestinal microbiome have been linked with improved obese condition. With emerging evidences of dietary compounds to prevent obesity, the objective of this study was to examine whether quercetin activates non-shivering thermogenesis in adipose tissues and influences intestinal microbiome, which eventually improves obese condition. MethodsFour-week-old C57BL/6 male mice were fed either a low-fat diet (LFD) or a high-fat diet (HFD) with or without 1% quercetin (Q) for 16 weeks. On the completion of the feeding study, brown adipose tissue (BAT), white adipose tissue (WAT), and cecum were collected. Total RNA was extracted from BAT and WAT, and then cDNA was synthesized. The expression of genes that are involved in the regulation of non-shivering thermogenesis such as uncoupling protein 1 (ucp1), cell death-inducing DFFA-like effector A (cidea), peroxisome proliferator-activated receptor gamma (pparγ), pparγ-coactivator 1 alpha (pgc1α), fibroblast growth factor 21 (fgf21), positive regulatory domain containing 16 (prdm16), and T-box protein 1 (tbx1) were determined by a real-time PCR. The expression of the proteins such as UCP1 and AMP-activated protein kinase (AMPK) was assessed by western blot analysis. Microbial populations in cecum were analyzed via the Illumnia MiSeq sequencing platform and QIIME (Quantitative Insights Into Microbial Ecology) Software. ResultsMice fed HFDQ showed reduced body weight and retroperitoneal (R) WAT weight compared to mice fed HFD. Quercetin supplementation increased the expression of ucp1, prdm16, pgc1α, cidea, and tbx1 genes in BAT and RWAT of mice fed HFD. The expression of UCP1 protein and phosphorylation of AMPK were increased. However, browning effect was not observed in other WATs. Mice fed LFDQ and HFDQ exhibited higher relative abundance of Bacteroidetes than mice fed LFD and HFD whereas the relative abundance of Firmicutes was decreased. ConclusionsQuercetin may be a potential dietary compound that increases energy metabolism by activating BAT and attracting beige adipocytes in RWAT. In addition, quercetin-induced energy metabolism may have a correlation with changes of microbial populations in intestine. Funding SourcesThe work was supported by USDA.

Aerobic exercise training prevents obesity and insulin resistance independent of the renin angiotensin system modulation in the subcutaneous white adipose tissue.

We investigate the effects of aerobic exercise training (AET) on the thermogenic response, substrate metabolism and renin angiotensin system (RAS) in the subcutaneous white adipose tissue (SC-WAT) of mice fed cafeteria diet (CAF). Male C57BL/6J mice were assigned into groups CHOW-SED (chow diet, sedentary; n = 10), CHOW-TR (chow diet, trained; n = 10), CAF-SED (CAF, sedentary; n = 10) and CAF-TR (CAF, trained; n = 10). AET consisted in running sessions of 60 min at 60% of maximal speed, five days per week for eight weeks. The CAF-SED group showed higher body weight and adiposity, glucose intolerance and insulin resistance (IR), while AET prevented such damages in CAF-TR group. AET reduced the p-AKT/t-AKT ratio and increased ATGL expression in CHOW-TR and CAF-TR groups and increased t-HSL and p-HSL/t-HSL ratio in CAF-TR. AET prevented adipocyte hypertrophy in CAF-TR group and increased UCP-1 protein expression only in CHOW-TR. Serum ACE2 increased in CHOW-TR and CAF-TR groups, and Ang (1–7) increased in the CHOW-TR group. In the SC-WAT, CAF-TR group increased the expression of AT1, AT2 and Mas receptors, whereas CHOW-TR increased Ang (1–7) and Ang (1–7)/Ang II ratio in SC-WAT. No changes were observed in ACE and Ang II. Positive correlations were observed between UCP-1 and kITT (r = 0.6), between UCP-1 and Ang (1–7) concentration (r = 0.6), and between UCP-1 and Ang (1–7)/Ang II ratio (r = 0.7). In conclusion, the AET prevented obesity and IR, reduced insulin signaling proteins and increased lipolysis signaling proteins in the SC-WAT. In addition, the CAF diet precludes the AET-induced thermogenic response and the partial modulation of the RAS suggests that the protective effect of AET against obesity and IR could not be associated with SC-WAT RAS.

Comparison of UCP2 protein expression in normal RIN‐5F and UCP2 inhibited cells

Uncoupling protein 2 (UCP2) is a mitochondria integral membrane protein that transports protons across the intermembrane space. Traditionally, the function of UCP2 is to impact oxidative phosphorylation by uncoupling ATP synthesis. However, a cell signaling role for UCP2 has recently been investigated. UCP2 expression has been shown to have expression in pancreatic beta cells and the presence of UCP2 has been suggested to lead to a decrease in the release of insulin. Although amylin is co‐secreted with insulin, studies on the relationship between amylin and UCP2 have had little investigation. A known function of amylin is to block glucagon secretion, which leads to a reduction in the rate of glucose production by decreasing the rate of gastric emptying. This study analyzed the protein expression level of amylin in normal RIN‐5F cells and RIN‐5F cells that have been transfected with a UCP2 siRNA construct. The study included stimulating the cells with either 11mM or 30 mM of glucose and comparing the protein expression level in the two cell lines. Western blot analysis was conducted and the results suggest a difference in UCP2 and amylin levels in normal RIN‐5F cells in comparison to the transfected RIN‐5F cells.Support or Funding InformationNational Science Foundation Research Initiation Award #24430This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Circulating factors present in the sera of naturally skinny people may influence cell commitment and adipocyte differentiation of mesenchymal stromal cells.

BACKGROUNDResearch on physiopathology of obesity may receive new hints from studies on skinny people (SP). These are individuals who show a poor or null gaining of body weight, in spite of high-calorie intake, by far exceeding the body requirements.AIMTo evaluate how circulating factors present in the SP sera may affect adipogenesis of mesenchymal stromal cells (MSCs).METHODSWe isolated MSCs from bone marrow of healthy donors with both normal body mass index (BMI) and caloric consumption. MSC cultures were primed with sera collected from SP or normal people (NP). Then biomolecular assays were performed to evaluate effect on proliferation, apoptosis, senescence, cell commitment, and differentiation.RESULTSSP priming affected adipocyte cell commitment and reduced spontaneous adipogenesis. Moreover, an in-depth analysis of exogenous-induced adipocyte differentiation showed striking differences between differentiation in SP-primed samples compared with NP ones. In adipocytes from SP cultures we observed a reduced size of lipid droplets, an increased expression of adipose triglyceride lipase, along with high mitochondria content and ability to produce ATP in starvation condition. These data and the expression of UCP1 protein, indicated that SP pretreatment produced a bias toward brown adipocyte differentiation.CONCLUSIONOur data suggest that sera from SP may promote brown adipogenesis rather that white adipocyte differentiation. This finding could explain why SP present normal body composition in spite of an excess of caloric intake. We hypothesize that some circulating components present in the blood of these individuals may favor brown adipogenesis at expense of white adipocyte production.

Compensation for cold-induced thermogenesis during weight loss maintenance and regain.

Prevalence of obesity is exacerbated by low rates of successful long-term weight loss maintenance (WLM). In part, relapse from WLM to obesity is due to a reduction in energy expenditure (EE) that persists throughout WLM and relapse. Thus, interventions that increase EE might facilitate WLM. In obese mice that were calorically restricted to reduce body weight by ~20%, we manipulated EE throughout WLM and early relapse using intermittent cold exposure (ICE; 4°C, 90 min/day, 5 days/wk, within the last 3 h of the light cycle). EE, energy intake, and spontaneous physical activity were measured during the obese, WLM, and relapse phases. During WLM and relapse, the ICE group expended more energy during the light cycle because of cold exposure but expended less energy in the dark cycle, which led to no overall difference in total daily EE. The compensation in EE appeared to be mediated by activity, whereby the ICE group was more active during the light cycle because of cold exposure but less active during the dark cycle, which led to no overall effect on total daily activity during WLM and relapse. In brown adipose tissue of relapsing mice, the ICE group had greater mRNA expression of Dio2 and protein expression of UCP1 but lower mRNA expression of Prdm16. In summary, these findings indicate that despite robust increases in EE during cold exposures, ICE is unable to alter total daily EE during WLM or early relapse, likely due to compensatory behaviors in activity.