Reactive oxygen species upregulate expression of muscle atrophy-associated ubiquitin ligase Cbl-b in rat L6 skeletal muscle cells.

Unloading-mediated muscle atrophy is associated with increased reactive oxygen species (ROS) production. We previously demonstrated that elevated ubiquitin ligase casitas B-lineage lymphoma-b (Cbl-b) resulted in the loss of muscle volume (Nakao R, Hirasaka K, Goto J, Ishidoh K, Yamada C, Ohno A, Okumura Y, Nonaka I, Yasutomo K, Baldwin KM, Kominami E, Higashibata A, Nagano K, Tanaka K, Yasui N, Mills EM, Takeda S, Nikawa T. Mol Cell Biol 29: 4798-4811, 2009). However, the pathological role of ROS production associated with unloading-mediated muscle atrophy still remains unknown. Here, we showed that the ROS-mediated signal transduction caused by microgravity or its simulation contributes to Cbl-b expression. In L6 myotubes, the assessment of redox status revealed that oxidized glutathione was increased under microgravity conditions, and simulated microgravity caused a burst of ROS, implicating ROS as a critical upstream mediator linking to downstream atrophic signaling. ROS generation activated the ERK1/2 early-growth response protein (Egr)1/2-Cbl-b signaling pathway, an established contributing pathway to muscle volume loss. Interestingly, antioxidant treatments such as N-acetylcysteine and TEMPOL, but not catalase, blocked the clinorotation-mediated activation of ERK1/2. The increased ROS induced transcriptional activity of Egr1 and/or Egr2 to stimulate Cbl-b expression through the ERK1/2 pathway in L6 myoblasts, since treatment with Egr1/2 siRNA and an ERK1/2 inhibitor significantly suppressed clinorotation-induced Cbl-b and Egr expression, respectively. Promoter and gel mobility shift assays revealed that Cbl-b was upregulated via an Egr consensus oxidative responsive element at -110 to -60 bp of the Cbl-b promoter. Together, this indicates that under microgravity conditions, elevated ROS may be a crucial mechanotransducer in skeletal muscle cells, regulating muscle mass through Cbl-b expression activated by the ERK-Egr signaling pathway.

The complementary and divergent roles of uncoupling proteins 1 and 3 in thermoregulation.

KEY POINTS: Both uncoupling protein 1 (UCP1) and UCP3 are important for mammalian thermoregulation. UCP1 and UCP3 in brown adipose tissue mediate early and late phases of sympathomimetic thermogenesis, respectively. Lipopolysaccharide thermogenesis requires skeletal muscle UCP3 but not UCP1. Acute noradrenaline-induced hyperthermia requires UCP1 but not UCP3. Loss of both UCP1 and UCP3 accelerate the loss of body temperature compared to UCP1KO alone during acute cold exposure. ABSTRACT: Uncoupling protein 1 (UCP1) is the established mediator of brown adipose tissue-dependent thermogenesis. In contrast, the role of UCP3, expressed in both skeletal muscle and brown adipose tissue, in thermoregulatory physiology is less well understood. Here, we show that mice lacking UCP3 (UCP3KO) have impaired sympathomimetic (methamphetamine) and completely abrogated lipopolysaccharide (LPS) thermogenesis, but a normal response to noradrenaline. By comparison, UCP1 knockout (UCP1KO) mice exhibit blunted methamphetamine and fully inhibited noradrenaline thermogenesis, but an increased febrile response to LPS. We further establish that mice lacking both UCP1 and 3 (UCPDK) fail to show methamphetamine-induced hyperthermia, and have a markedly accelerated loss of body temperature and survival after cold exposure compared to UCP1KO mice. Finally, we show that skeletal muscle-specific human UCP3 expression is able to significantly rescue LPS, but not sympathomimetic thermogenesis blunted in UCP3KO mice. These studies identify UCP3 as an important mediator of physiological thermogenesis and support a renewed focus on targeting UCP3 in metabolic physiology.

UCP3 is associated with Hax-1 in mitochondria in the presence of calcium ion.

Uncoupling protein 3 (UCP3) is known to regulate energy dissipation, proton leakage, fatty acid oxidation, and oxidative stress. To identify the putative protein regulators of UCP3, we performed yeast two-hybrid screens. Here we report that UCP3 interacted with HS-1 associated protein X-1 (Hax-1), an anti-apoptotic protein that was localized in the mitochondria, and is involved in cellular responses to Ca(2+). The hydrophilic sequences within loop 2, and the matrix-localized hydrophilic domain of mouse UCP3, were necessary for binding to Hax-1 at the C-terminal domain, adjacent to the mitochondrial inner membrane. Interestingly, interaction of these proteins occurred in a calcium-dependent manner. Moreover, the NMR spectrum of the C-terminal domain of Hax-1 was dramatically changed by removal of Ca(2+), suggesting that the C-terminal domain of Hax-1 underwent a Ca(2+)-induced conformational change. In the Ca(2+)-free state, the C-terminal Hax-1 tended to unfold, suggesting that Ca(2+) binding may induce protein folding of the Hax-1 C-terminus. These results suggested that the UCP3-Hax-1 complex may regulate mitochondrial functional changes caused by mitochondrial Ca(2+).

A novel myogenic function residing in the 5' non-coding region of Insulin receptor substrate-1 (Irs-1) transcript.

BACKGROUND: There is evidence that several messenger RNAs (mRNAs) are bifunctional RNAs, i.e. RNA transcript carrying both protein-coding capacity and activity as functional non-coding RNA via 5' and 3' untranslated regions (UTRs). RESULTS: In this study, we identified a novel bifunctional RNA that is transcribed from insulin receptor substrate-1 (Irs-1) gene with full-length 5'UTR sequence (FL-Irs-1 mRNA). FL-Irs-1 mRNA was highly expressed only in skeletal muscle tissue. In cultured skeletal muscle C2C12 cells, the FL-Irs-1 transcript functioned as a bifunctional mRNA. The FL-Irs-1 transcript produced IRS-1 protein during differentiation of myoblasts into myotubes; however, this transcript functioned as a regulatory RNA in proliferating myoblasts. The FL-Irs-1 5'UTR contains a partial complementary sequence to Rb mRNA, which is a critical factor for myogenic differentiation. The overexpression of the 5'UTR markedly reduced Rb mRNA expression, and this reduction was fully dependent on the complementary element and was not compensated by IRS-1 protein. Conversely, knockdown of FL-Irs-1 mRNA increased Rb mRNA expression and enhanced myoblast differentiation into myotubes. CONCLUSIONS: Our findings suggest that the FL-Irs-1 transcript regulates myogenic differentiation as a regulatory RNA in myoblasts.

Caenorhabditis elegans UCP4 protein controls complex II-mediated oxidative phosphorylation through succinate transport.

The novel uncoupling proteins (UCP2-5) are implicated in the mitochondrial control of oxidant production, insulin signaling, and aging. Attempts to understand their functions have been complicated by overlapping expression patterns in most organisms. Caenorhabditis elegans nematodes are unique because they express only one UCP ortholog, ceUCP4 (ucp4). Here, we performed detailed metabolic analyzes in genetically modified nematodes to define the function of the ceUCP4. The knock-out mutant ucp4 (ok195) exhibited sharply decreased mitochondrial succinate-driven (complex II) respiration. However, respiratory coupling and electron transport chain function were normal in ucp4 mitochondria. Surprisingly, isolated ucp4 mitochondria showed markedly decreased succinate uptake. Similarly, ceUCP4 inhibition blocked succinate respiration and import in wild type mitochondria. Genetic and pharmacologic inhibition of complex I function was selectively lethal to ucp4 worms, arguing that ceUCP4-regulated succinate transport is required for optimal complex II function in vivo. Additionally, ceUCP4 deficiency prolonged lifespan in the short-lived mev1 mutant that exhibits complex II-generated oxidant production. These results identify a novel function for ceUCP4 in the regulation of complex II-based metabolism through an unexpected mechanism involving succinate transport.

Astaxanthin Prevents Atrophy in Slow Muscle Fibers by Inhibiting Mitochondrial Reactive Oxygen Species via a Mitochondria-Mediated Apoptosis Pathway.

Astaxanthin (AX) is a carotenoid that exerts potent antioxidant activity and acts in the lipid bilayer. This study aimed to investigate the effects of AX on muscle-atrophy-mediated disturbance of mitochondria, which have a lipid bilayer. Tail suspension was used to establish a muscle-atrophied mouse model. AX diet fed to tail-suspension mice prevented loss of muscle weight, inhibited the decrease of myofiber size, and restrained the increase of hydrogen peroxide (H2O2) production in the soleus muscle. Additionally, AX improved downregulation of mitochondrial respiratory chain complexes I and III in the soleus muscle after tail suspension. Meanwhile, AX promoted mitochondrial biogenesis by upregulating the expressions of adenosine 5'-monophosphate-activated protein kinase (AMPK) α-1, peroxisome proliferator-activated receptor (PPAR)-γ, and creatine kinase in mitochondrial (Ckmt) 2 in the soleus muscle of tail-suspension mice. To confirm the AX phenotype in the soleus muscle, we examined its effects on mitochondria using Sol8 myotubes derived from the soleus muscle. We found that AX was preferentially detected in the mitochondrial fraction; it significantly suppressed mitochondrial reactive oxygen species (ROS) production in Sol8 myotubes. Moreover, AX inhibited the activation of caspase 3 via inhibiting the release of cytochrome c into the cytosol in antimycin A-treated Sol8 myotubes. These results suggested that AX protected the functional stability of mitochondria, alleviated mitochondrial oxidative stress and mitochondria-mediated apoptosis, and thus, prevented muscle atrophy.

Pharmacology: uncoupling the agony from ecstasy.

The recreational use of amphetamine-type stimulants can produce a marked and sometimes lethal increase in body temperature. Here we show that mice deficient in a mitochondrial protein known as UCP-3 (for 'uncoupling protein-3') have a diminished thermogenic response to the drug MDMA (3,4-methylenedioxymethamphetamine, nicknamed 'ecstasy') and so are protected against this dangerously toxic effect. Our findings indicate that UCP-3 is important in MDMA-induced hyperthermia and point to a new therapeutic direction for solving an increasing public-health problem.

The hyperthermia mediated by 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy) is sensitive to sex differences.

Female subjects have been reported to be less sensitive to the hyperthermic effects of 3,4-methylenedioxymethamine (MDMA) than males. Studies were designed to examine the cellular mechanisms involved in these sex sensitive differences. Gonadectomized female and male rats were treated with a 200 microg 100 microL(-1) of estrogen or 100 microg 100 microL(-1) of testosterone respectively every 5 days for a total of three doses. Rats were then challenged with either saline or MDMA (20 mg kg(-1), sc). Rats were then euthanized and aortas were constricted, in vitro, by serial phenylephrine (Phe) addition with or without the inhibitor of nitric oxide (NO) synthase, g-nitro-L-Arginine-Methyl Ester (L-NAME). Skeletal muscle uncoupling protein-3 (UCP3) expression was measured as well as plasma norepinephrine (NE) levels. All males but no females developed hyperthermia following MDMA treatment. The EC(50) for Phe dose response curves increased only in the females treated with MDMA and T(max) for Phe increased following L-NAME only in the females. Both males and females demonstrated an increase in plasma NE following MDMA treatment; however, males displayed a significantly greater NE concentration. Skeletal muscle UCP3 expression was 80% less in females than in males. These results suggest that the inability of MDMA to induce a thermogenic response in the female subjects may be due to four sex-specific mechanisms: 1) Female subjects have reduced sympathetic activation following MDMA challenge; 2) Female vasculature is less sensitive to alpha(1)-AR stimulation following MDMA challenge; 3) Female vasculature has an increased sensitivity to NO; 4) UCP3 expression in skeletal muscle is less in females.

Body temperature regulation and drugs of abuse.

Phenethylamine-induced hyperthermia can occur following exposure to several different types of illicit stimulants, such as amphetamine, methamphetamine, 3,4-methylenedioxymethamphetamine ("Molly"), synthetic cathinones ("bath salts"), and N-methoxybenyl ("NBOMe"), to name a few. Peripheral norepinephrine release mediated by these sympathomimetic agents induces a double-edged sword of heat accumulation through ß-adrenoreceptor-dependent activation of uncoupling protein (UCP1 and 3)-regulated thermogenesis and loss of heat dissipation through α1-adrenoreceptor-mediated vasoconstriction. Additionally, thyroid hormones are important determinants of the capacity of thermogenesis induced by phenethylamines through the regulation of free fatty acid release and the transcriptional activation of a host of metabolic genes, including adrenergic receptors and mitochondrial uncoupling proteins. Here, we review the central and peripheral mechanistic "triggers" of phenethylamine-induced hyperthermia and outline potential pharmacologic interventions for managing phenethylamine-induced hyperthermia based on these recently discovered hyperthermia mediators.

Highly sensitive and selective determination of redox states of coenzymes Q9 and Q10 in mice tissues: Application of orbitrap mass spectrometry.

Coenzyme Q (CoQ) is a redox active molecule that plays a fundamental role in mitochondrial energy generation and functions as a potent endogenous antioxidant. Redox ratio of CoQ has been suggested as a good marker of mitochondrial dysfunction and oxidative stress. Nevertheless, simultaneous measurement of redox states of CoQ is challenging owing to its hydrophobicity and instability of the reduced form. In order to improve the analytical methodology, paying special attention to this instability, we developed a highly sensitive and selective high-resolution/accurate-mass (HR/AM) UHPLC-MS/MS method for the rapid determination of redox states of CoQ9 and CoQ10 by ultra-performance liquid chromatography-hybrid quadrupole-Orbitrap mass spectrometry. CoQs were extracted using hexane with the addition of butylated hydroxytoluene to limit oxidation during sample preparation. Chromatographic separation of the analytes was achieved on a Kinetex C18 column with the isocratic elution of 5 mM ammonium formate in 2-propanol/methanol (60:40) within 4 min. A full MS/all ion fragmentation (AIF) acquisition mode with mass accuracy < 5 ppm was used for detection and determination of redox states of CoQ9 and CoQ10 in healthy mice tissues using reduced and oxidized CoQ4 as internal standards. The validated method showed good linearity (r2 ≥ 0.9991), intraday, inter-day precision (CVs ≤ 11.9%) and accuracy (RE ≤±15.2%). In contrast to existing methods, the current method offers enhanced sensitivity (up to 52 fold) with LOD and LOQ ranged from 0.01 to 0.49 ng mL-1 and 0.04-1.48 ng mL-1, respectively. Moreover, we evaluated various diluents to investigate bench top stability (at 4 °C) of targeted analytes in tissue samples during LC-MS assay up to 24 h. Ethanol was determined to be an optimum diluent without any significant oxidation of reduced CoQ up to 24 h. The developed method offers a rapid, highly sensitive and selective strategy for the measurement of redox states of CoQs in clinical studies.

The early metabolomic response of adipose tissue during acute cold exposure in mice.

To maintain core body temperature in cold conditions, mammals activate a complex multi-organ metabolic response for heat production. White adipose tissue (WAT) primarily functions as an energy reservoir, while brown adipose tissue (BAT) is activated during cold exposure to generate heat from nutrients. Both BAT and WAT undergo specific metabolic changes during acute cold exposure. Here, we use an untargeted metabolomics approach to characterize the initial metabolic response to cold exposure in multiple adipose tissue depots in mice. Results demonstrate dramatically distinct metabolic responses during cold exposure in BAT and WAT. Amino acids, nucleotide pathways, and metabolites involved in redox regulation were greatly affected 4 hours post-exposure in BAT, while no polar metabolites were observed to significantly change in WAT depots up to 6 hours post exposure. Lipid metabolism was activated early (2 hours) in both BAT and the subcutaneous WAT depots, with the most striking change being observed in the modulation of diglyceride and monoglyceride levels in BAT. Overall, these data provide a timeline of global thermogenic metabolism in adipose depots during acute cold exposure. We have highlighted differences in visceral and subcutaneous WAT thermogenic metabolism and demonstrate the distinct metabolism of BAT during cold exposure.

Chewing the fat for Akt1 inhibition and oncosuppression.

The catabolic and energy-dissipating actions of mitochondrial uncoupling proteins (UCPs) conflict with many of the bioenergetic hallmarks of malignancy. We have recently demonstrated that overexpression of mitochondrial uncoupling protein 3 (Ucp3) in the basal epidermis impedes skin tumorigenesis through a novel pathway of thymoma viral proto-oncogene 1 (Akt1) inhibition via increased mitochondrial lipid catabolism.

Potentiation of Ecstasy-induced hyperthermia and FAT/CD36 expression in chronically exercised animals.

Fatal hyperthermia as a result of 3,4-methylenedioxymethamphetamine (MDMA) use involves non-esterified free fatty acids (NEFA) and the activation of mitochondrial uncoupling proteins (UCP). NEFA gain access into skeletal muscle via specific transport proteins, including fatty acid translocase (FAT/CD36). FAT/CD36 expression is known to increase following chronic exercise. Previous studies have demonstrated the essential role of NEFA and UCP3 in MDMA-induced hyperthermia. The aims of the present study were to use a chronic exercise model (swimming for two consecutive hours per day, five days per wk for six wk) to increase FAT/CD36 expression in order to: 1) determine the contribution of FAT/CD36 in MDMA (20 mg/kg, s.c.)-mediated hyperthermia; and 2) examine the effects of the FAT/CD36 inhibitor, SSO (sulfo-N-succinimidyl oleate), on MDMA-induced hyperthermia in chronic exercise and sedentary control rats. MDMA administration resulted in hyperthermia in both sedentary and chronic exercise animals. However, MDMA-induced hyperthermia was significantly potentiated in the chronic exercise animals compared to sedentary animals. Additionally, chronic exercise significantly reduced body weight, increased FAT/CD36 protein expression levels and reduced plasma NEFA levels. The FAT/CD36 inhibitor, SSO (40 mg/kg, ip), significantly attenuated the hyperthermia mediated by MDMA in chronic exercised but not sedentary animals. Plasma NEFA levels were elevated in sedentary and exercised animals treated with SSO prior to MDMA suggesting attenuation of NEFA uptake into skeletal muscle. Chronic exercise did not alter skeletal muscle UCP3 protein expression levels. In conclusion, chronic exercise potentiates MDMA-mediated hyperthermia in a FAT/CD36 dependent fashion.

The role of the sympathetic nervous system and uncoupling proteins in the thermogenesis induced by 3,4-methylenedioxymethamphetamine.

Body temperature regulation involves a homeostatic balance between heat production and dissipation. Sympathetic agents such as 3,4-methylenedioxymethamphetamine (MDMA, ecstasy) can disrupt this balance and as a result produce an often life-threatening hyperthermia. The hyperthermia induced by MDMA appears to result from the activation of the sympathetic nervous system (SNS) and the hypothalamic-pituitary-thyroid/adrenal axis. Norepinephrine release mediated by MDMA creates a double-edged sword of heat generation through activation of uncoupling protein (UCP3) along with alpha1- and beta3-adrenoreceptors and loss of heat dissipation through SNS-mediated vasoconstriction. This review examines cellular mechanisms involved in MDMA-induced thermogenesis from UCP activation to vasoconstriction and how these mechanisms are related to other thermogenic conditions and potential treatment modalities.

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.

Attenuation of 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy)-induced rhabdomyolysis with alpha1- plus beta3-adrenoreceptor antagonists.

1. Studies were designed to examine the effects of alpha(1) (alpha(1)AR)- plus beta(3)-adrenoreceptor (beta(3)AR) antagonists on 3,4-methylenedioxymethamphetamine (MDMA, Ecstasy)-induced hyperthermia and measures of rhabdomyolysis (creatine kinase (CK)) and renal function (blood urea nitrogen (BUN) and serum creatinine (sCr)) in male Sprague-Dawley rats. 2. MDMA (40 mg x kg(-1), s.c.) induced a rapid and robust increase in rectal temperature, which was significantly attenuated by pretreatment with the alpha(1)AR antagonist prazosin (100 microg x kg(-1), i.p.) plus the beta(3)AR antagonist SR59230A (5 mg x kg(-1), i.p.). 3. CK levels significantly increased (peaking at 4 h) after MDMA treatment and were blocked by the combination of prazosin plus SR59230A. 4. At 4 h after MDMA treatment, BUN and sCr levels were also significantly increased and could be prevented by this combination of alpha(1)AR- plus beta(3)AR-antagonists. 5. The results from this study suggest that alpha(1)AR and beta(3)AR play a critical role in the etiology of MDMA-mediated hyperthermia and subsequent rhabdomyolysis.

The heat is on: Molecular mechanisms of drug-induced hyperthermia.

Thermoregulation is an essential homeostatic process in which critical mechanisms of heat production and dissipation are controlled centrally in large part by the hypothalamus and peripherally by activation of the sympathetic nervous system. Drugs that disrupt the components of this highly orchestrated multi-organ process can lead to life-threatening hyperthermia. In most cases, hyperthermic agents raise body temperature by increasing the central and peripheral release of thermoregulatory neurotransmitters that ultimately lead to heat production in thermogenic effector organs skeletal muscle (SKM) and brown adipose tissue (BAT). In many cases hyperthermic drugs also decrease heat dissipation through peripheral changes in blood flow. Drug-induced heat production is driven by the stimulation of mechanisms that normally regulate the adaptive thermogenic responses including both shivering and non-shivering thermogenesis (NST) mechanisms. Modulation of the mitochondrial electrochemical proton/pH gradient by uncoupling protein 1 (UCP1) in BAT is the most well characterized mechanism of NST in response to cold, and may contribute to thermogenesis induced by sympathomimetic agents, but this is far from established. However, the UCP1 homologue, UCP3, and the ryanodine receptor (RYR1) are established mediators of toxicant-induced hyperthermia in SKM. Defining the molecular mechanisms that orchestrate drug-induced hyperthermia will be essential in developing treatment modalities for thermogenic illnesses. This review will briefly summarize mechanisms of thermoregulation and provide a survey of pharmacologic agents that can lead to hyperthermia. We will also provide an overview of the established and candidate molecular mechanisms that regulate the actual thermogenic processes in heat effector organs BAT and SKM.

The apoptosis repressor with a CARD domain (ARC) gene is a direct hypoxia-inducible factor 1 target gene and promotes survival and proliferation of VHL-deficient renal cancer cells.

The induction of hypoxia-inducible factors (HIFs) is essential for the adaptation of tumor cells to a low-oxygen environment. We found that the expression of the apoptosis inhibitor ARC (apoptosis repressor with a CARD domain) was induced by hypoxia in a variety of cancer cell types, and its induction is primarily HIF1 dependent. Chromatin immunoprecipitation (ChIP) and reporter assays also indicate that the ARC gene is regulated by direct binding of HIF1 to a hypoxia response element (HRE) located at bp -190 upstream of the transcription start site. HIFs play an essential role in the pathogenesis of renal cell carcinoma (RCC) under normoxic conditions, through the loss of the Von Hippel-Lindau gene (VHL). Accordingly, our results show that ARC is not expressed in normal renal tissue but is highly expressed in 65% of RCC tumors, which also express high levels of carbonic anhydrase IX (CAIX), a HIF1-dependent protein. Compared to controls, ARC-deficient RCCs exhibited decreased colony formation and increased apoptosis in vitro. In addition, loss of ARC resulted in a dramatic reduction of RCC tumor growth in SCID mice in vivo. Thus, HIF-mediated increased expression of ARC in RCC can explain how loss of VHL can promote survival early in tumor formation.

Cbl-b is a critical regulator of macrophage activation associated with obesity-induced insulin resistance in mice.

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.

Unloading stress disturbs muscle regeneration through perturbed recruitment and function of macrophages.

Skeletal muscle is one of the most sensitive tissues to mechanical loading, and unloading inhibits the regeneration potential of skeletal muscle after injury. This study was designed to elucidate the specific effects of unloading stress on the function of immunocytes during muscle regeneration after injury. We examined immunocyte infiltration and muscle regeneration in cardiotoxin (CTX)-injected soleus muscles of tail-suspended (TS) mice. In CTX-injected TS mice, the cross-sectional area of regenerating myofibers was smaller than that of weight-bearing (WB) mice, indicating that unloading delays muscle regeneration following CTX-induced skeletal muscle damage. Delayed infiltration of macrophages into the injured skeletal muscle was observed in CTX-injected TS mice. Neutrophils and macrophages in CTX-injected TS muscle were presented over a longer period at the injury sites compared with those in CTX-injected WB muscle. Disturbance of activation and differentiation of satellite cells was also observed in CTX-injected TS mice. Further analysis showed that the macrophages in soleus muscles were mainly Ly-6C-positive proinflammatory macrophages, with high expression of tumor necrosis factor-α and interleukin-1ß, indicating that unloading causes preferential accumulation and persistence of proinflammatory macrophages in the injured muscle. The phagocytic and myotube formation properties of macrophages from CTX-injected TS skeletal muscle were suppressed compared with those from CTX-injected WB skeletal muscle. We concluded that the disturbed muscle regeneration under unloading is due to impaired macrophage function, inhibition of satellite cell activation, and their cooperation.