Uncoupling protein 3 attenuates generation of reactive oxygen species by interacting with thioredoxin 2 in the mitochondrial intermembrane space

Katsuya Hirasaka1*, Edward M Mills2, Shohei Kohno1, Tomoki Abe1, Chika Ikeda1, Tasuku Maeda1, Shigetada Kondo1, Ayako Maita1, Yuushi Okumura1 and Takeshi Nikawa1 Author Affiliations 1 Department of Nutritional Physiology, Institute of Health Biosciences, University of Tokushima, Tokushima, 770-8503, Japan 2 Division of Pharmacology/Toxicology, University of Texas at Austin, Austin, TX 78712, USAPoster presentation Uncoupling protein 3 (UCP3) is primarily expressed in the inner membrane of skeletal muscle mitochondria. It has been proposed that UCP3 reduces production of reactive oxygen species (ROS) and oxidative damage. However, the mechanisms by which UCP3 attenuates ROS production are not well understood. Here we report that UCP3 interacts with the non-processed form of thioredoxin 2 (Trx2), a redox protein that is localized in mitochondria, but not processed Trx2, which is involved in cellular responses to ROS. The hydrophilic sequences within the N-terminal tail of UCP3, which faces the intermembrane space, are necessary for binding to Trx2. In addition, Trx2 directly associated with UCP3 through a mitochondrial targeting signaling sequence, was processed in the intermembrane space, and thereby allowing redox reactions. A bimolecular fluorescence complementation analysis demonstrated that the interaction of these proteins occurs in the mitochondrial intermembrane space. Furthermore, increased UCP3 expression significantly attenuated ROS production in isolated mitochondrial without effects on membrane potential, however this effect is lost by Trx2 knock down. These results suggest that UCP3 binds to Trx2 in the mitochondrial intermembrane space and attenuates ROS production.Pharmac

Cbl-b ケッソン ニ ヨル マクロファージ ノ カッセイカ オ カイシタ タイトウノウ イジョウ

Obesity is a major cause of insulin resistance and is considered a chronic low-grade inflammatory disease. Substantial evidence has accumulated in recent years that chronic infiltration and activation of macrophages in white adipose tissue underlie the obesity-related component of these insulin resistant states. In the present study, we examined the role of Cbl-b, ubiquitin ligase, in insulin action. Elderly Cbl-b-deficient mice（Cbl-b－/－mice）developed glucose intolerance and peripheral insulin resistance. Deficiency of Cbl-b gene was associated with infiltration of macrophages into the WAT and expression of cytokines, such as tumor necrosis factor-α, interleukin-6 and monocyte chemoattractant protein-1. Furthermore, Vav1, a key factor in macrophage activation, was highly phosphorylated in peritoneal Cbl-b－/－macrophages, compared with wild type macrophages, suggesting that Cbl-b deficiency induces macrophage activation. Our results suggest that Cbl-b is a negative regulator of macrophage activation, and that macrophage activation by Cbl-b deficiency, at least in part, contributes to the peripheral insulin resistance and glucose intolerance

Awa (Tokushima) lactate-fermented tea as well as green tea enhance the effect of diet restriction on obesity in rats

Drinking tea is recommended for promoting health due to its bioactive nutrients, such as catechins and caffeine. In Tokushima area, we have a unique traditional tea, named Awa tea, which are fermented with Lactobacillus pentosus and Lactobacillus plantarum. The present study was designed to investigate anti-obesity effects of the Awa tea and compare with those of non-fermented green tea. Obese male Wistar rats (19 weeks of age) were given by low energy diets containing 3% of Awa and green tea extracts, respectively, or without any tea extracts (control), for 4 weeks. Awa tea contained smaller amount of catechins than green tea, although they contained similar amounts of polyphenols. This finding indicates that there are distinct kinds of polyphenols from catechins. The diets containing Awa and green tea extracts further decreased whole body weight, fat tissue mass and plasma leptin level, compared with control diet. In addition, their diets increased the daily amount of lipid excreted to feces and total 24-h-energy consumption, compared with the control group. However, there is no significant difference in these anti-obesity effects between Awa tea and green tea. Our results indicate that Awa lactate-fermented tea as well as green tea similarly enhance the effect of diet restriction on obesity, at least in part, through the increase in fat energy consumption and the decrease in fat absorption in rats

Mitochondrial uncoupling links lipid catabolism to Akt inhibition and resistance to tumorigenesis

To support growth, tumour cells reprogramme their metabolism to simultaneously upregulate macromolecular biosynthesis while maintaining energy production. Uncoupling proteins (UCPs) oppose this phenotype by inducing futile mitochondrial respiration that is uncoupled from ATP synthesis, resulting in nutrient wasting. Here using a UCP3 transgene targeted to the basal epidermis, we show that forced mitochondrial uncoupling inhibits skin carcinogenesis by blocking Akt activation. Similarly, Akt activation is markedly inhibited in UCP3 overexpressing primary human keratinocytes. Mechanistic studies reveal that uncoupling increases fatty acid oxidation and membrane phospholipid catabolism, and impairs recruitment of Akt to the plasma membrane. Overexpression of Akt overcomes metabolic regulation by UCP3, rescuing carcinogenesis. These findings demonstrate that mitochondrial uncoupling is an effective strategy to limit proliferation and tumorigenesis through inhibition of Akt, and illuminate a novel mechanism of crosstalk between mitochondrial metabolism and growth signalling

Polyphenols prevent clinorotation-induced expression of atrogenes in mouse C2C12 skeletal myotubes

Oxidative stress is a key factor in stimulating the expression of atrogenes, which are muscle atrophy-related ubiquitin ligases, in skeletal muscle, and it induces muscle atrophy during unloading. However, the effects of antioxidative nutrients on atrogene expression have not been demonstrated. We report on the inhibitory effects of polyphenols, such as epicatechin (EC), epicatechin gallate (ECg) and epigallocatechin gallate (EGCg) and quercetin, on atrogene expression up-regulated by three dimensional (3D)-clinorotation or glucocorticoid. These treatments markedly elevated the expression of atrogenes, including atrogin-1 and MuRF-1, in mouse C2C12 myoblasts and myotubes. Interestingly, EC, ECg, EGCg and quercetin significantly decreased the expression of atrogin-1 and MuRF-1 up-regulated by 3D-clinorotation, whereas they hardly affected atrogene expression induced by dexamethasone. ERK signaling is a well known MAPK pathway to mediate oxidative stress. Therefore, we also investigated the effect of these polyphenols on phosphorylation of ERK in C2C12 myotubes. As expected, EC, ECg, EGCg, and quercetin significantly suppressed phosphorylation of ERK, corresponding to the up-regulation of atrogenes induced by 3D-clinorotation. These results suggest that antioxidative nutrients, such as catechins and quercetin, suppress atrogene expression in skeletal muscle cells, possibly through the inhibition of ERK signaling. Thus, catechins and quercetin may prevent unloading-mediated muscle atrophy

Cbl-b DEFICIENCY AND MACROPHAGE ACTIVATION

We previously reported the potential involvement of casitas B-cell lymphoma-b (Cbl-b) in aging-related murine insulin resistance. Because obesity also induces macrophage recruitment into adipose tissue, we elucidated here the role of Cbl-b in obesity-related insulin resistance. Cbl-b+/+ and Cbl-b-/- mice were fed a high-fat diet (HFD) and then examined for obesity-related changes in insulin signaling. The HFD caused recruitment of macrophages into adipose tissue and increased inflammatory reaction in Cbl-b-/- compared with Cbl-b+/+ mice. Peritoneal macrophages from Cbl-b-/- mice and Cbl-b–overexpressing RAW264.7 macrophages were used to examine the direct effect of saturated fatty acids (FAs) on macrophage activation. In macrophages, Cbl-b suppressed saturated FA-induced Toll-like receptor 4 (TLR4) signaling by ubiquitination and degradation of TLR4. The physiological role of Cbl-b in vivo was also examined by bone marrow transplantation and Eritoran, a TLR4 antagonist. Hematopoietic cell-specific depletion of the Cbl-b gene induced disturbed responses on insulin and glucose tolerance tests. Blockade of TLR4 signaling by Eritoran reduced fasting blood glucose and serum interleukin-6 levels in obese Cbl-b-/- mice. These results suggest that Cbl-b deficiency could exaggerate HFD-induced insulin resistance through saturated FA-mediated macrophage activation. Therefore, inhibition of TLR4 signaling is an attractive therapeutic strategy for treatment of obesity-related insulin resistance

Circadian Disruption Accelerates Tumor Growth and Angio/Stromagenesis through a Wnt Signaling Pathway

Epidemiologic studies show a high incidence of cancer in shift workers, suggesting a possible relationship between circadian rhythms and tumorigenesis. However, the precise molecular mechanism played by circadian rhythms in tumor progression is not known. To identify the possible mechanisms underlying tumor progression related to circadian rhythms, we set up nude mouse xenograft models. HeLa cells were injected in nude mice and nude mice were moved to two different cases, one case is exposed to a 24-hour light cycle (L/L), the other is a more “normal” 12-hour light/dark cycle (L/D). We found a significant increase in tumor volume in the L/L group compared with the L/D group. In addition, tumor microvessels and stroma were strongly increased in L/L mice. Although there was a hypervascularization in L/L tumors, there was no associated increase in the production of vascular endothelial cell growth factor (VEGF). DNA microarray analysis showed enhanced expression of WNT10A, and our subsequent study revealed that WNT10A stimulates the growth of both microvascular endothelial cells and fibroblasts in tumors from light-stressed mice, along with marked increases in angio/stromagenesis. Only the tumor stroma stained positive for WNT10A and WNT10A is also highly expressed in keloid dermal fibroblasts but not in normal dermal fibroblasts indicated that WNT10A may be a novel angio/stromagenic growth factor. These findings suggest that circadian disruption induces the progression of malignant tumors via a Wnt signaling pathway