Expressions of mRNA and encoded proteins of mitochondrial uncoupling protein genes (UCP1, UCP2, and UCP3) in epicardial and mediastinal adipose tissue and associations with coronary artery disease.

Objective: To evaluate the expression of UCP1, UCP2, and UCP3 mRNA and encoded proteins in epicardial and mediastinal adipose tissues in patients with coronary artery disease (CAD). Subjects and methods: We studied 60 patients with CAD and 106 patients undergoing valve replacement surgery (controls). Expression levels of UCP1, UCP2, and UCP3 mRNA and encoded proteins were measured by quantitative real-time PCR and Western blot analysis, respectively. Results: : We found increased UCP1, UCP2, and UCP3 mRNA levels in the epicardial adipose tissue in the CAD versus the control group, and higher UCP1 and UCP3 mRNA expression in the epicardial compared with the mediastinal tissue in the CAD group. There was also increased expression of UCP1 protein in the epicardial tissue and UCP2 protein in the mediastinum tissue in patients with CAD. Finally, UCP1 expression was associated with levels of fasting plasma glucose, and UCP3 expression was associated with levels of high-density lipoprotein cholesterol and low-density cholesterol in the epicardial tissue. Conclusion: Our study supports the hypothesis that higher mRNA expression by UCP genes in the epicardial adipose tissue could be a protective mechanism against the production of reactive oxygen species and may guard the myocardium against damage. Thus, UCP levels are essential to maintain the adaptive phase of cardiac injury in the presence of metabolic disorders.

Overexpression of UCP3 decreases mitochondrial efficiency in mouse skeletal muscle in vivo.

Uncoupling protein-3 (UCP3) is a mitochondrial transmembrane protein highly expressed in the muscle that has been implicated in regulating the efficiency of mitochondrial oxidative phosphorylation. Increasing UCP3 expression in skeletal muscle enhances proton leak across the inner mitochondrial membrane and increases oxygen consumption in isolated mitochondria, but its precise function in vivo has yet to be fully elucidated. To examine whether muscle-specific overexpression of UCP3 modulates muscle mitochondrial oxidation in vivo, rates of ATP synthesis were assessed by 31 P magnetic resonance spectroscopy (MRS), and rates of mitochondrial oxidative metabolism were measured by assessing the rate of [2-13 C]acetate incorporation into muscle [4-13 C]-, [3-13 C]-glutamate, and [4-13 C]-glutamine by high-resolution 13 C/1 H MRS. Using this approach, we found that the overexpression of UCP3 in skeletal muscle was accompanied by increased muscle mitochondrial inefficiency in vivo as reflected by a 42% reduction in the ratio of ATP synthesis to mitochondrial oxidation.

Association of UCP3 Polymorphisms with Nonalcoholic Steatohepatitis and Metabolic Syndrome in Nonalcoholic Fatty Liver Disease Brazilian Patients.

Background: We investigated the possible association of uncoupling protein 3 gene (UCP3) single nucleotide polymorphisms (SNPs) with nonalcoholic steatohepatitis (NASH) and metabolic syndrome (MetS) in nonalcoholic fatty liver disease (NAFLD) Brazilian patients. Methods:UCP3 SNPs rs1726745, rs3781907, and rs11235972 were genotyped in 158 biopsy-proven NAFLD Brazilian patients. Statistics was performed with JMP, R, and SHEsis softwares. Results: The TT genotype of rs1726745 was associated with less occurrence of MetS (P = 0.006) and with lower body mass index (BMI) in the entire NAFLD sample (P = 0.01) and in the NASH group (P = 0.02). The rs1726745-T was associated with lower values of AST (P = 0.001), ALT (P = 0.0002), triglycerides (P = 0.01), and total cholesterol (P = 0.02) in the entire NAFLD sample. Between groups, there were lower values of aminotransferases strictly in individuals with NASH (AST, P = 0.002; ALT, P = 0.0007) and with MetS (AST, P = 0.002; ALT, P = 0.001). The rs3781907-G was associated with lower GGT elevation values in the entire NAFLD sample (P = 0.002), in the NASH group (P = 0.004), and with MetS group (P = 0.003) and with protection for advanced fibrosis (P = 0.01). The rs11235972-A was associated with lower GGT values in the entire NAFLD sample (P = 0.006) and in the NASH group (P = 0.01) and with MetS group (P = 0.005), with fibrosis absence (P = 0.01) and protection for advanced fibrosis (P = 0.01). The TAA haplotype was protective for NASH (P = 0.002), and TGG haplotype was protective for MetS (P = 0.01). Conclusion:UCP3 gene variants were associated with protection against NASH and MetS, in addition to lower values of liver enzymes, lipid profile, BMI and, lesser fibrosis severity in the studied population.

EGCG Upregulates UCP3 Levels to Protect MIN6 Pancreatic Islet Cells from Interleukin-1ß-Induced Apoptosis.

OBJECTIVE: The protective effects of epigallocatechin gallate (EGCG) on interleukin-1ß (IL-1ß)-induced apoptosis were investigated in murine MIN6 pancreatic ß-cells. The role of uncoupling protein-3 (UCP3) signaling in this process was also explored. METHODS: After treatment with IL-1ß and EGCG, cells were collected and analyzed. Cell viability was measured using the CCK8 assay and the function of ß-cells was evaluated by analyzing insulin secretion. Detection of mitochondrial function in cells was performed by measuring mitochondrial membrane potential, the concentration of ATP and activity of ROS. Apoptosis was analyzed by Hochest33258 staining and flow cytometry. Expression levels of UCP3 were interrogated using immunohistochemistry, RT-PCR and Western blotting. RESULTS: Compared with the control group, IL-1ß treatment (20nM) for 24 h significantly decreased cell viability and insulin secretion, damaged mitochondrial function and increased ROS activity. Results also showed increased apoptosis and a decrease in UCP3 expression levels (p<0.01). However, treatment with low (1mM) or high (5mM) concentrations of EGCG significantly decreased IL-1ß-induced apoptosis (p<0.01), restored mitochondrial function and subsequently increased UCP3 levels in IL-1ß-induced ß-cells (p<0.01). CONCLUSION: These results suggest that EGCG protects against IL-1ß-induced mitochondrial injury and apoptosis in ß-cells through the up-regulation of UCP3.

The association of uncoupling proteins 1, 2, and 3 with weight loss variability after bariatric surgery: a systematic review.

BACKGROUND: Bariatric surgery is currently the most effective treatment for patients with severe obesity. Uncoupling proteins (UCP) 1, 2, and 3 play key roles in the regulation of energy balance and weight. Previous studies have suggested that changes in UCP1-3 genes could influence weight loss after bariatric surgery. However, it is still unclear if these UCPs are indeed involved in weight loss variability after surgery. Therefore, we performed a systematic review aiming to summarize the results of studies on this subject. METHODS: A literature search was performed for all studies that evaluated associations of UCP1-3 expressions and their polymorphisms with obesity-related outcomes after bariatric surgery. RESULTS: Twenty-six studies were eligible for inclusion in this systematic review. Among them, 18 evaluated UCP1-3 expressions while 8 studies investigated the association between UCP1-3 polymorphisms and weight loss after bariatric surgery. In general, UCP2 and UCP3 expressions in adipose tissue and skeletal muscle seem to be affected by metabolic changes of bariatric surgery, which might be influenced by the surgery type. Data on UCP1 expression in adipose tissue is still inconclusive. Only few studies investigated the association between polymorphisms in UCP1-3 genes and weight loss after bariatric surgery, with contradictory results. CONCLUSION: Available studies suggest that changes caused by bariatric surgery could influence UCP2 and UCP3 expressions in adipose tissue and muscles, consequently affecting weight loss. However, because of the reduced number of studies, further studies are needed to confirm whether these UCPs and their polymorphisms are indeed involved in weight loss after bariatric surgery.

Fitness is improved by adjustments in muscle intracellular signaling in rats with renovascular hypertension 2K1C undergoing voluntary physical exercise.

AIM: To evaluate physical fitness and cardiovascular effects in rats with renovascular hypertension, two kidneys, one clip (2K1C) submitted to voluntary exercise (ExV). MAIN METHODS: 24 h after surgery (SHAM and 2K1C) rats were submitted to ExV for one week (adaptation). ExV adherent rats were separated into exercise (2K1C-EX and SHAM-EX) or sedentary (2K1C-SED and SHAM-SED) groups. After 4 weeks, exhaustion test, plasma lactate, cardiovascular parameters were evaluated and gastrocnemius muscle was removed for evaluation of gene expression of muscle metabolism markers (PGC1α; AMPK, SIRT-1, UCP-3; MCP-1; LDH) and of the redox process. KEY FINDINGS: ExV decreased blood lactate concentration and increased SOD and CAT activity and a SIRT-1 and UCP-3 gene expression in the gastrocnemius muscle of 2K1C-ExV rats compared to 2K1C-SED rats. Gene expressions of PGC1α, UCP-3, MCT-1, AMPK were higher in 2K1C-ExV rats compared to SHAM-SED rats. Blood pressure in 2K1C-ExV was lower compared to 2K1C-SED and higher in SHAM-SED rats. Reflex bradycardia in 2K1C-EX rats increased compared to 2K1C-SED and was similar to SHAM-SED. The variation in mean blood pressure induced by ganglion blocker hexamethonium and Ang II AT1 receptor antagonist, losartan in the 2K1C-ExV rats was smaller compared to the 2K1C-SED rats and it was similar to the SHAM-SED rats. SIGNIFICANCE: O ExV induced adaptive responses in 2K1C-ExV rats by decreasing sympathetic and Ang II activities and stimulating intracellular signaling that favors redox balance and reduced blood lactate concentration. These adaptive responses, then, contribute to reduced arterial pressure, improved baroreflex sensitivity and physical fitness of 2K1C rats.

Inorganic nitrate and nitrite supplementation fails to improve skeletal muscle mitochondrial efficiency in mice and humans.

BACKGROUND: Inorganic nitrate, abundant in leafy green vegetables and beetroot, is thought to have protective health benefits. Adherence to a Mediterranean diet reduces the incidence and severity of coronary artery disease, whereas supplementation with nitrate can improve submaximal exercise performance. Once ingested, oral commensal bacteria may reduce nitrate to nitrite, which may subsequently be reduced to nitric oxide during conditions of hypoxia and in the presence of "nitrite reductases" such as heme- and molybdenum-containing enzymes. OBJECTIVE: We aimed to explore the putative effects of inorganic nitrate and nitrite on mitochondrial function in skeletal muscle. METHODS: Mice were subjected to a nitrate/nitrite-depleted diet for 2 wk, then supplemented with sodium nitrate, sodium nitrite, or sodium chloride (1 g/L) in drinking water ad libitum for 7 d before killing. Skeletal muscle mitochondrial function and expression of uncoupling protein (UCP) 3, ADP/ATP carrier protein (AAC) 1 and AAC2, and pyruvate dehydrogenase (PDH) were assessed by respirometry and Western blotting. Studies were also undertaken in human skeletal muscle biopsies from a cohort of coronary artery bypass graft patients treated with either sodium nitrite (30-min infusion of 10 µmol/min) or vehicle [0.9% (wt:vol) saline] 24 h before surgery. RESULTS: Neither sodium nitrate nor sodium nitrite supplementation altered mitochondrial coupling efficiency in murine skeletal muscle, and expression of UCP3, AAC1, or AAC2, and PDH phosphorylation status did not differ between the nitrite and saline groups. Similar results were observed in human samples. CONCLUSIONS: Sodium nitrite failed to improve mitochondrial metabolic efficiency, rendering this mechanism implausible for the purported exercise benefits of dietary nitrate supplementation. This trial was registered at clinicaltrials.gov as NCT04001283.

Pseudomonas aeruginosa Quorum Sensing Molecule Alters Skeletal Muscle Protein Homeostasis by Perturbing the Antioxidant Defense System.

Skeletal muscle function is compromised in many illnesses, including chronic infections. The Pseudomonas aeruginosa quorum sensing (QS) signal, 2-amino acetophenone (2-AA), is produced during acute and chronic infections and excreted in human tissues, including the lungs of cystic fibrosis patients. We have shown that 2-AA facilitates pathogen persistence, likely via its ability to promote the formation of bacterial persister cells, and that it acts as an interkingdom immunomodulatory signal that epigenetically reprograms innate immune functions. Moreover, 2-AA compromises muscle contractility and impacts the expression of genes involved in reactive oxygen species (ROS) homeostasis in skeletal muscle and in mitochondrial functions. Here, we elucidate the molecular mechanisms of 2-AA's impairment of skeletal muscle function and ROS homeostasis. Murine in vivo and differentiated C2C12 myotube cell studies showed that 2-AA promotes ROS generation in skeletal muscle via the modulation of xanthine oxidase (XO) activity, NAD(P)H oxidase2 (NOX2) protein level, and the activity of antioxidant enzymes. ROS accumulation triggers the activity of AMP-activated protein kinase (AMPK), likely upstream of the observed locations of induction of ubiquitin ligases Muscle RING Finger 1 (MuRF1) and Muscle Atrophy F-box (MAFbx), and induces autophagy-related proteins. The protein-level perturbation in skeletal muscle of silent mating type information regulation 2 homolog 1 (SIRT1), peroxisome proliferator-activated receptor gamma coactivator 1 (PGC-1), and uncoupling protein 3 (UCP3) is rescued by the antioxidant N-acetyl-l-cysteine (NAC). Together, these results unveil a novel form of action of a QS bacterial molecule and provide molecular insights into the 2-AA-mediated skeletal muscle dysfunction caused by P. aeruginosaIMPORTANCEPseudomonas aeruginosa, a bacterium that is resistant to treatment, causes serious acute, persistent, and relapsing infections in humans. There is increasing evidence that bacterial excreted small molecules play a critical role during infection. We have shown that a quorum sensing (QS)-regulated excreted small molecule, 2-AA, which is abundantly produced by P. aeruginosa, promotes persistent infections, dampens host inflammation, and triggers mitochondrial dysfunction in skeletal muscle. QS is a cell-to-cell communication system utilized by bacteria to promote collective behaviors. The significance of our study in identifying a mechanism that leads to skeletal muscle dysfunction, via the action of a QS molecule, is that it may open new avenues in the control of muscle loss as a result of infection and sepsis. Given that QS is a common characteristic of prokaryotes, it is possible that 2-AA-like molecules promoting similar effects may exist in other pathogens.

Bta-miR-152 affects intracellular triglyceride content by targeting the UCP3 gene.

According to our previous studies, bta-miR-152, PRKAA1 and UCP3 are differentially expressed in mammary gland tissues of high milk fat and low milk fat cows, and the trend in bta-miR-152 expression is opposite from those of PRKAA1 and UCP3. To further identify the function and regulatory mechanism of bta-miR-152 in milk fat metabolism, we investigated the effect of bta-miR-152 on cellular triglyceride content in bovine mammary epithelial cells cultured in vitro, on the basis of bta-miR-152 overexpression and inhibition assays. The target genes of bta-miR-152 were identified through qPCR, Western blotting and dual luciferase reporter gene detection. Compared with that in the control group, the expression of UCP3 was significantly lower in the bta-miR-152 mimic group, the expression of PRKAA1 was decreased, and the intracellular TAG content was significantly increased. After transfection with bta-miR-152 inhibitor, the expression of UCP3 increased significantly, and the expression of PRKAA1 decreased, but the difference was not significant; in addition, the intracellular TAG content decreased significantly. Therefore, we concluded that bta-miR-152 affects the intracellular TAG content by targeting UCP3.

Insulin-like growth factor-1 directly mediates expression of mitochondrial uncoupling protein 3 via forkhead box O4.

OBJECTIVE: The objective of our study was to examine the direct action of insulin-like growth factor-1(IGF-1) signaling on energy homeostasis in myocytes. DESIGN: We studied the IGF-1 stimulation of mitochondrial uncoupling protein 3 (UCP3) expression in the HEK 293 derived cell line TSA201, murine C2C12 skeletal muscle myoblasts, and rat L6 skeletal myoblasts. We also investigated the direct effect of IGF-1 on the Insulin/IGF-1 receptor (IGF-1R)/phosphatidylinositol 3 (PI3)-Akt/forkhead box O4 (FOXO4) pathway using a combination of a reporter assay, semi-quantitative polymerase chain reaction, western blotting, and animal experiments. RESULTS: We demonstrated that IGF-1 regulates UCP3 expression via phosphorylation of FOXO4, which is a downstream signal transducer of IGF-1. UCP3 expression increased with activated FOXO4 in a dose-dependent manner. We also examined the functional FOXO4 binding site consensus sequences and identified it as the -1922 bp site in the UCP3 promoter region. UCP3 was also found to be concomitantly expressed with IGF-1 during differentiation of C2C12 myoblasts. Our animal experiments showed that high fat diet induced IGF-1 levels which likely influenced UCP3 expression in the skeletal muscle. CONCLUSION: Our findings demonstrate that that IGF-1 directly stimulates UCP3 expression via the IGF-1/IGF-1R/PI3-Akt/FOXO4 pathway.

Fractionated whole body gamma irradiation modulates the hepatic response in type II diabetes of high fat diet model rats.

HFD animals were exposed to a low rate of different fractionated whole body gamma irradiation doses (0.5, 1 and 2 Gy, three fractions per week for two consecutive months) and the expression of certain genes involved in type 2 diabetes mellitus (T2DM) in livers and brains of HFD Wistar rats was investigated. Additionally, levels of diabetes-related proteins encoded by the studied genes were analyzed. Results indicated that mRNA level of incretin glucagon like peptite-1 receptor (GLP-1R) was augmented in livers and brains exposed to 1 and 2 Gy doses. Moreover, the mitochondrial uncoupling proteins 2 and 3 (UCP2/3) expressions in animals fed on HFD compared to those fed on normal chow diet were significantly increased at all applied doses. GLP-1R and UCP3 protein levels were up regulated in livers. Total protein content increased at 0.5 and 1 Gy gamma irradiation exposure and returned to its normal level at 2 Gy dose. Results could be an indicator of type 2 diabetes delayed development during irradiation exposure and support the importance of GLP-1R as a target gene in radiotherapy against T2DM and its chronic complications. A new hypothesis of brain-liver and intestine interface is speculated by which an increase in the hepatic GLP-1R is influenced by the effect of fractionated whole body gamma irradiation.

Cmah-dystrophin deficient mdx mice display an accelerated cardiac phenotype that is improved following peptide-PMO exon skipping treatment.

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin protein, leading to progressive muscle weakness and premature death due to respiratory and/or cardiac complications. Cardiac involvement is characterized by progressive dilated cardiomyopathy, decreased fractional shortening and metabolic dysfunction involving reduced metabolism of fatty acids-the major cardiac metabolic substrate. Several mouse models have been developed to study molecular and pathological consequences of dystrophin deficiency, but do not recapitulate all aspects of human disease pathology and exhibit a mild cardiac phenotype. Here we demonstrate that Cmah (cytidine monophosphate-sialic acid hydroxylase)-deficient mdx mice (Cmah-/-;mdx) have an accelerated cardiac phenotype compared to the established mdx model. Cmah-/-;mdx mice display earlier functional deterioration, specifically a reduction in right ventricle (RV) ejection fraction and stroke volume (SV) at 12 weeks of age and decreased left ventricle diastolic volume with subsequent reduced SV compared to mdx mice by 24 weeks. They further show earlier elevation of cardiac damage markers for fibrosis (Ctgf), oxidative damage (Nox4) and haemodynamic load (Nppa). Cardiac metabolic substrate requirement was assessed using hyperpolarized magnetic resonance spectroscopy indicating increased in vivo glycolytic flux in Cmah-/-;mdx mice. Early upregulation of mitochondrial genes (Ucp3 and Cpt1) and downregulation of key glycolytic genes (Pdk1, Pdk4, Ppara), also denote disturbed cardiac metabolism and shift towards glucose utilization in Cmah-/-;mdx mice. Moreover, we show long-term treatment with peptide-conjugated exon skipping antisense oligonucleotides (20-week regimen), resulted in 20% cardiac dystrophin protein restoration and significantly improved RV cardiac function. Therefore, Cmah-/-;mdx mice represent an appropriate model for evaluating cardiac benefit of novel DMD therapeutics.

Dark Chocolate Intake Positively Modulates Redox Status and Markers of Muscular Damage in Elite Football Athletes: A Randomized Controlled Study.

Intensive physical exercise may cause increase oxidative stress and muscular injury in elite football athletes. The aim of this study was to exploit the effect of cocoa polyphenols on oxidative stress and muscular injuries induced by intensive physical exercise in elite football players. Oxidant/antioxidant status and markers of muscle damage were evaluated in 24 elite football players and 15 controls. Furthermore, the 24 elite football players were randomly assigned to either a dark chocolate (>85% cocoa) intake (n = 12) or a control group (n = 12) for 30 days in a randomized controlled trial. Oxidative stress, antioxidant status, and muscle damage were assessed at baseline and after 30 days of chocolate intake. Compared to controls, elite football players showed lower antioxidant power and higher oxidative stress paralleled by an increase in muscle damage markers. After 30 days of dark chocolate intake, an increased antioxidant power was found in elite athletes assuming dark chocolate. Moreover, a significant reduction in muscle damage markers (CK and LDH, p < 0.001) was observed. In the control group, no changes were observed with the exception of an increase of sNox2-dp, H2O2, and myoglobin. A simple linear regression analysis showed that sNox2-dp was associated with a significant increase in muscle damage biomarker release (p = 0.001). An in vitro study also confirmed that polyphenol extracts significantly decreased oxidative stress in murine myoblast cell line C2C12-derived. These results indicate that polyphenol-rich nutrient supplementation by means of dark chocolate positively modulates redox status and reduced exercise-induced muscular injury biomarkers in elite football athletes. This trial is registered with NCT03288623.

Testosterone mediates hyperthermic response of mice to heat exposure.

AIMS: Testosterone is implicated as a potential contributing factor to heat-induced injury. We examined effects of testosterone on thermal response of mice to heat exposure. MAIN METHODS: Adult C57BL/6J male mice received gonadectomy or sham surgery subsequent vehicle (Gnx) or testosterone implants (Gnx+T). Body core temperature (Tc) of mice was recorded telemetrically during acute heat exposure. Thermal responses to heat exposure were also examined in age-matched female mice at each stage of the estrous cycle. KEY FINDINGS: Basal Tc was lower in sham male mice than females, but did not differ among sham, Gnx or Gnx+T males. No alterations in expression of uncoupling proteins 2 and 3 in the gastrocnemius muscle were found in either Gnx or Gnx+T mice compared to sham males. During heat exposure, sham male mice had a faster and greater rise in Tc, compared to females. This rapid hyperthermic response to heat was abolished in Gnx males, but not in Gnx+T males. No significant correlation was revealed between peak Tc values and plasma testosterone concentrations in Gnx+T males treated with either low- or high-dose testosterone. No effects of estrous cycle phase on the thermal response to heat exposure were detected in female mice. SIGNIFICANCE: We found that male mice were more susceptible to development of heat-induced hyperthermia than females and this was prevented by castration, not by castration with testosterone replacement. These results suggest that testosterone mediates heat-induced hyperthermia and is a heat stress susceptibility factor.

[Effect of pyrrolidine dithiocarbamate on oxidative stress and mitochondrial function of lung tissue in mice with acute lung injury].

OBJECTIVE: To investigate the effects of pyrrolidine dithiocarbamate (PDTC) on oxidative stress and mitochondrial function of lung tissue in mice with acute lung injury (ALI) induced by lipopolysaccharide (LPS). METHODS: Forty female Balb/c mice were randomly divided into normal saline (NS) control group, LPS model group, PDTC group, and PDTC+LPS group, with 10 mice in each group. The model of mice with ALI was reproduced by intraperitoneal injection of 15 mg/kg LPS. PDTC was administered intraperitoneally with 50 mg/kg PDTC 1 hour before LPS treatment in the PDTC+LPS group. The mice in NS control group was given intraperitoneal injection of 0.1 mL NS only, and those in PDTC group was given intraperitoneal injection of 50 mg/kg PDTC only. The mice were sacrificed at 24 hours after model reproduction, and the lung tissues were harvested. The total antioxidant capacity (T-AOC) of lung tissue was measured by spectrophotometric kits. The content of malondialdehyde (MDA) was determined by thiobarbituric acid reactive substances assay. The protein expressions of superoxide dismutases (SOD1, SOD2) and catalase (CAT) in lung tissue were determined by Western Blot. Mitochondria from mouse lungs were isolated, and adenosine triphosphate (ATP) synthesis was measured with a luciferase/luciferin-based approach. The mitochondrial membrane potential (ΔΨm) was estimated by using Rhodamine. The mRNA expressions of mitochondrial uncoupling proteins (UCPs) were determined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS: LPS stimulation could significantly increase oxidative stress in lung tissue of mice and lead to mitochondrial dysfunction. The results showed that the protein expressions of T-AOC and SOD1 were decreased, the level of MDA was increased, the ATP synthesis was decreased in the mitochondrial, the ΔΨm was decreased, and the mRNA expression of UCP2 was decreased. However, there was no significant change in the expressions of SOD2, CAT in lung tissue and UCP1, UCP3 in the mitochondria. Pretreatment with PDTC could obviously alleviate the increase in LPS-induced oxidative stress in lung tissue and mitigate mitochondrial dysfunction. Compared with the LPS model group, T-AOC in lung tissue of PDTC+LPS group was significantly increased (U/g: 0.35±0.08 vs. 0.31±0.07), the level of MDA was significantly decreased (µmol/mg: 13.29±1.13 vs. 17.54±1.72), the protein expression of SOD1 was significantly upregulated (SOD1 protein: 1.13±0.11 vs. 0.71±0.09), ATP synthesis was significantly increased in the mitochondrial (µmol/mg: 49.23±5.42 vs. 36.92±2.21), ΔΨm was significantly increased (mV: 226.03±11.69 vs. 194.86±7.79), and the mRNA expression of UCP2 was significantly increased (2-ΔΔCt: 0.88±0.06 vs. 0.73±0.04). The differences were statistically significant (all P < 0.05). In lung tissue of normal mice, PDTC treatment also had the effect of anti-oxidizing, reducing oxidative stress and promoting ATP synthesis in the mitochondrial. Compared with the NS control group, the level of T-AOC (U/g: 0.49±0.09 vs. 0.43±0.06) and the protein expressions of SOD2 and CAT (SOD2 protein: 1.33±0.08 vs. 1.00±0.11, CAT protein: 1.39±0.08 vs. 1.00±0.11), and ATP synthesis in the mitochondrial of PDTC group was significantly increased (µmol/mg: 61.53±4.92 vs. 53.33±3.20), MDA was significantly decreased (µmol/mg: 10.27±1.25 vs. 12.27±1.36), with statistical differences, but had no effect on the protein expression of SOD1 in lung tissue and ΔΨm and UCPs mRNA expressions in mitochondrion. CONCLUSIONS: LPS can induce ALI in mice, increased oxidative stress in lung tissue, and induce mitochondrial dysfunction by inhibiting ATP synthesis. PDTC pretreatment has anti-oxidative effect on LPS-induced ALI, and can mitigate mitochondrial dysfunction.

GH directly stimulates UCP3 expression.

OBJECTIVE: We evaluated the direct action of GH signaling in energy homeostasis in myocytes. DESIGN: We investigated the GH-induced expression of UCP3 in human embryonic kidney 293 cells, human H-EMC-SS chondrosarcoma cells, murine C2C12 skeletal muscle myoblasts, and rat L6 skeletal muscle cells, as well as its direct effect on the GHR/JAK/STAT5 pathway using a combination of a reporter assay, real-time quantitative polymerase chain reaction, and western blotting. RESULTS: We demonstrated that the regulation of energy metabolism by GH involves UCP3 via activated STAT5, a signal transducer downstream of GH. UCP3 expression increased with STAT5 in a dose-dependent manner and was higher than that of UCP2. We confirmed the functional STAT5 binding site consensus sequences at -861 and -507 bp in the UCP3 promoter region. CONCLUSION: The results suggest that GH stimulates UCP3 directly and that UCP2 and that UCP3 participate in the signal transduction pathway that functions downstream of the GHR/JAK/STAT.

Effect of speed endurance training and reduced training volume on running economy and single muscle fiber adaptations in trained runners.

The aim of the present study was to examine whether improved running economy with a period of speed endurance training and reduced training volume could be related to adaptations in specific muscle fibers. Twenty trained male (n = 14) and female (n = 6) runners (maximum oxygen consumption (VO2 -max): 56.4 ± 4.6 mL/min/kg) completed a 40-day intervention with 10 sessions of speed endurance training (5-10 × 30-sec maximal running) and a reduced (36%) volume of training. Before and after the intervention, a muscle biopsy was obtained at rest, and an incremental running test to exhaustion was performed. In addition, running at 60% vVO2 -max, and a 10-km run was performed in a normal and a muscle slow twitch (ST) glycogen-depleted condition. After compared to before the intervention, expression of mitochondrial uncoupling protein 3 (UCP3) was lower (P < 0.05) and dystrophin was higher (P < 0.05) in ST muscle fibers, and sarcoplasmic reticulum calcium ATPase 1 (SERCA1) was lower (P < 0.05) in fast twitch muscle fibers. Running economy at 60% vVO2 -max (11.6 ± 0.2 km/h) and at v10-km (13.7 ± 0.3 km/h) was ~2% better (P < 0.05) after the intervention in the normal condition, but unchanged in the ST glycogen-depleted condition. Ten kilometer performance was improved (P < 0.01) by 3.2% (43.7 ± 1.0 vs. 45.2 ± 1.2 min) and 3.9% (45.8 ± 1.2 vs. 47.7 ± 1.3 min) in the normal and the ST glycogen-depleted condition, respectively. VO2 -max was the same, but vVO2 -max was 2.0% higher (P < 0.05; 19.3 ± 0.3 vs. 18.9 ± 0.3 km/h) after than before the intervention. Thus, improved running economy with intense training may be related to changes in expression of proteins linked to energy consuming processes in primarily ST muscle fibers.

Inhibition of mitochondrial UCP1 and UCP3 by purine nucleotides and phosphate.

Mitochondrial membrane uncoupling protein 3 (UCP3) is not only expressed in skeletal muscle and heart, but also in brown adipose tissue (BAT) alongside UCP1, which facilitates a proton leak to support non-shivering thermogenesis. In contrast to UCP1, the transport function and molecular mechanism of UCP3 regulation are poorly investigated, although it is generally agreed upon that UCP3, analogous to UCP1, transports protons, is activated by free fatty acids (FFAs) and is inhibited by purine nucleotides (PNs). Because the presence of two similar uncoupling proteins in BAT is surprising, we hypothesized that UCP1 and UCP3 are differently regulated, which may lead to differences in their functions. By combining atomic force microscopy and electrophysiological measurements of recombinant proteins reconstituted in planar bilayer membranes, we compared the level of protein activity with the bond lifetimes between UCPs and PNs. Our data revealed that, in contrast to UCP1, UCP3 can be fully inhibited by all PNs and IC50 increases with a decrease in PN-phosphorylation. Experiments with mutant proteins demonstrated that the conserved arginines in the PN-binding pocket are involved in the inhibition of UCP1 and UCP3 to different extents. Fatty acids compete with all PNs bound to UCP1, but only with ATP bound to UCP3. We identified phosphate as a novel inhibitor of UCP3 and UCP1, which acts independently of PNs. The differences in molecular mechanisms of the inhibition between the highly homologous transporters UCP1 and UCP3 indicate that UCP3 has adapted to fulfill a different role and possibly another transport function in BAT.

Systematic-analysis of mRNA expression profiles in skeletal muscle of patients with type II diabetes: The glucocorticoid was central in pathogenesis.

Since the past 30 years, the prevalence of diabetes has more than doubled, making it an urgent challenge globally. We carried out systematic analysis with the public data of mRNA expression profiles in skeletal muscle to study the pathogenesis, since insulin resistance in the skeletal muscle is an early feature. We utilized three GEO datasets, containing total 60 cases and 63 normal samples. After the background removal, R package QC was utilized to finish the preprocessing of datasets. We obtained a dataset containing 2481 genes and 123 samples after the preprocessing. Quantitative quality control measures were calculated to represent the quality of these datasets. MetaDE package provides functions for conducting different systematic analysis methods for differential expression analysis. The GO term enrichment was carried out using PANTHER. Protein-protein interactions, drug-gene interactions, and genetic association of the identified differentially expressed genes were analyzed using STRING v10.0 online tool, DGIdb, and the Genetic Association Database, respectively. The datasets had good performances on IQC and EQC, which suggested that the datasets had good internal and external quality. Totally 96 differentially expressed genes were detected using 0.01 as cutoff of AW. The enriched GO terms were mainly associated with the response to glucocorticoid. There were seven genes involving in the gluconeogenesis were differentially expressed, which might be the potential treatment target for this disease. The closely connected networks and potential targets of existed drugs suggested that some of the drugs might be applied to the treatment of diabetes as well.

Association of 5-HT2C (rs3813929) and UCP3 (rs1800849) gene polymorphisms with type 2 diabetes in obese women candidates for bariatric surgery.

OBJECTIVE: Obesity can cause systemic arterial hypertension (SAH) and type 2 diabetes mellitus (DM2) factor that is also influenced by genetic variability. The present study aims to investigate the association between gene polymorphisms related with obesity on the prevalence of SAH and DM2 in the preoperative period and 1 year after Roux-en-Y gastric bypass surgery. SUBJECTS AND METHODS: In total, 351 obese women in a Brazilian cohort completed the study. The clinical diagnosis of SAH and DM2 was monitored from medical records. Twelve gene polymorphisms (rs26802; rs572169; rs7799039; rs1137101; rs3813929; rs659366; rs660339; rs1800849; rs7498665; rs35874116; rs9701796; and rs9939609) were determined using real-time polymerase chain reaction and TaqMan assay. RESULTS: In the preoperative period, prevalence of SAH and DM2 was 57% and 22%, respectively. One year postoperatively, 86.8% subjects had remission of DM2 and 99.5% had control of SAH. Subjects with T allele from the serotonin receptor gene (5-HT2C, rs3813929) had five times greater chance of DM2, and the CC genotype from uncoupling protein 3 gene (UCP3, rs1800849) had three times greater chance in the preoperative period. CONCLUSION: These findings indicate that polymorphisms rs3813929 and rs1800849 from 5-HT2C and UCP3 genes were related to DM2 prevalence among the Brazilian obese women candidates for bariatric surgery.