Takuya Sakurai

A circadian clock gene, Rev-erbα, modulates the inflammatory function of macrophages through the negative regulation of Ccl2 expression.

Disruption of the circadian rhythm is a contributory factor to clinical and pathophysiological conditions, including cancer, the metabolic syndrome, and inflammation. Chronic and systemic inflammation are a potential trigger of type 2 diabetes and cardiovascular disease and are caused by the infiltration of large numbers of inflammatory macrophages into tissue. Although recent studies identified the circadian clock gene Rev-erbα, a member of the orphan nuclear receptors, as a key mediator between clockwork and inflammation, the molecular mechanism remains unknown. In this study, we demonstrate that Rev-erbα modulates the inflammatory function of macrophages through the direct regulation of Ccl2 expression. Clinical conditions associated with chronic and systemic inflammation, such as aging or obesity, dampened Rev-erbα gene expression in peritoneal macrophages from C57BL/6J mice. Rev-erbα agonists or overexpression of Rev-erbα in the murine macrophage cell line RAW264 suppressed the induction of Ccl2 following an LPS endotoxin challenge. We discovered that Rev-erbα represses Ccl2 expression directly through a Rev-erbα–binding motif in the Ccl2 promoter region. Rev-erbα also suppressed CCL2-activated signals, ERK and p38, which was recovered by the addition of exogenous CCL2. Further, Rev-erbα impaired cell adhesion and migration, which are inflammatory responses activated through the ERK- and p38-signaling pathways, respectively. Peritoneal macrophages from mice lacking Rev-erbα display increases in Ccl2 expression. These data suggest that Rev-erbα regulates the inflammatory infiltration of macrophages through the suppression of Ccl2 expression. Therefore, Rev-erbα may be a key link between aging- or obesity-associated impairment of clockwork and inflammation.

Antioxidative Effects of a New Lychee Fruit-Derived Polyphenol Mixture, Oligonol, Converted into a Low-Molecular Form in Adipocytes

In this study we investigated the antioxidative effects of Oligonol (Amino Up Chemical Co., Ltd., Sapporo, Japan), a new polyphenol, in adipocytes. The levels of reactive oxygen species (ROS) and the expression of adipokine genes decreased in HW mouse white adipocytes upon treatment with Oligonol as compared to control cells. The transcriptional activity of nuclear factor-kappaB (NF-κB) and the activation of extracellular signal-regulated kinase (ERK) 1/2 were also down-regulated by Oligonol. In addition, when C57BL/6J mice were fed a high fat diet (HFD) for 5 weeks, the levels of epididymal white adipose tissue (WAT) mass and lipid peroxidation in WAT both increased, but Oligonol intake clearly inhibited such HFD-induced increases. Furthermore, dysregulated expression of genes for adipokines in WAT of mice fed solely a HFD was attenuated by Oligonol intake. These results suggest that Oligonol has antioxidative effects and that it attenuates HFD-induced dysregulated expression of genes for adipokines in adipocytes.

Direct association between PU.1 and MeCP2 that recruits mSin3A-HDAC complex for PU.1-mediated transcriptional repression.

PU.1, a member of the Ets family of transcription factors, is implicated in hematopoietic cell differentiation through its interactions with other transcriptional factors and cofactors. To identify a novel protein(s) binding to PU.1, we carried out affinity purification using a column of Glutathione-Sepharose beads bound to GST-PU.1 fusion protein and isolated several individual proteins using murine erythroleukemia (MEL) cell extracts. Sequence analysis of these proteins revealed that one was MeCP2 a methyl CpG binding protein. GST-pull-down assay and immunoprecipitation assay showed that PU.1 bound directly to MeCP2 via its Ets domain and MeCP2 bound to PU.1 via either its amino terminal domain or trans-repression domain. MeCP2 repressed transcriptional activity of PU.1 on a reporter construct with trimerized PU.1 binding sites. This downregulation was recovered in the presence of histone deacetylase inhibitor, trichostatin A (TSA). MeCP2 was integrated in PU.1-mSin3A-HDAC complex but not in PU.1-CBP complex. Chromatin immunoprecipitation (ChIP) assays showed that PU.1 and MeCP2 were collocated at the PU.1 binding site on the reporter construct and the PU.1 binding site of the intervening sequence 2 (IVS2) region in the intron of the β-globin gene, which has been proposed to regulate expression of the gene, in undifferentiated MEL cells. The complex disappeared from the region during the course of erythroid differentiation of MEL cells. Our results suggest that MeCP2 acts as a corepressor of PU.1 probably due to facilitating complex formation with mSin3A and HDACs.

In vivo complex formation of PU.1 with HDAC1 associated with PU.1-mediated transcriptional repression

We previously reported that overexpression of PU.1, a member of the Ets family of transcription factors, induces differentiation inhibition and apoptosis associated with c-Myc down-regulation in murine erythroleukemia (MEL) cells. To understand the molecular mechanism by which c-Myc is down-regulated due to overexpression of PU.1, we performed luciferase reporter assays using the mouse c-myc promoter. PU.1 repressed the activities of not only the c-myc promoter but also several other promoters. Experiments with deletion mutants of PU.1 revealed that the C-terminal region spanning amino acids (aa) 123–272 including the PEST and ETS domains but not the activation domain was sufficient for this transcriptional repression. It was unlikely that the repression was due to sequestration of a limited amount of CBP/p300 nor pCAF, because overexpression of these co-activators did not relieve PU.1-mediated transcriptional repression. Instead, it was found that the C-terminal aa 101–272 of PU.1 formed a complex with mSin3A and HDAC1 in vivo, which was speculated to be associated with the repression. The C-terminal region of PU.1 also formed a complex with the basic transcription factor TBP in vitro and in vivo. Our results suggest that overexpression of PU.1 induces transcriptional repression in several gene promoters including the c-myc promoter which may be mediated by its complex formation with HDACs.

Oligomerized grape seed polyphenols attenuate inflammatory changes due to antioxidative properties in coculture of adipocytes and macrophages

Macrophage infiltration of white adipose tissue (WAT) is implicated in the metabolic complications of obesity. In addition, inflammatory changes through dysregulated expression of inflammation-related adipokines such as tumor necrosis factor-alpha (TNF-alpha) and monocyte chemoattractant protein-1 (MCP-1) in WAT are considered to be one of the causes of insulin resistance. Recently, enhanced oxidative stress in adipocytes has been reported to be implicated in dysregulated expression of inflammation-related adipokines. Polyphenols are well known as potent natural antioxidants in the diet. In the present study, we investigated the antioxidative effects of an oligomerized grape seed polyphenol (OGSP) on inflammatory changes in coculture of adipocytes and macrophages. Coculture of HW mouse white adipocytes and RAW264 mouse macrophages markedly increased the production of TNF-alpha, MCP-1 and plasminogen activator inhibitor-1 compared with control culture. Treatment of HW cells with OGSP significantly attenuated the dysregulated production of adipokines. Moreover, OGSP significantly suppressed coculture-induced production of reactive oxygen species (ROS). Although enhanced release of free fatty acids (FFAs) by coculture was not altered by OGSP, FFA-induced ROS production in HW cells was significantly attenuated by OGSP. Furthermore, OGSP significantly reduced increases in the transcriptional activity of nuclear factor-kappaB and activation of extracellular signal-regulated kinase by coculture. Thus, these results suggest that the antioxidative properties of OGSP attenuate inflammatory changes induced by the coculture of adipocytes and macrophages.

Exercise training decreases expression of inflammation-related adipokines through reduction of oxidative stress in rat white adipose tissue.

Increased oxidative stress in adipocytes causes dysregulated expression of inflammation-related adipokines. We have examined the effects of exercise training on oxidative stress in rat white adipose tissue (WAT), especially focusing on inflammation-related adipokines. The levels of lipid peroxidation in WAT of exercise-trained (TR) rats were lower than those in control (C) rats. The content of manganese-containing superoxide dismutase in WAT of TR rats was increased as compared with those in C rats. In contrast, the expression of the NADPH oxidase NOX2 protein in WAT was downregulated by exercise training. Moreover, the levels of inflammation-related adipokines, such as tumor necrosis factor-alpha and monocyte chemoattractant protein-1, in WAT of TR rats were lower than those in C rats. The effects of exercise training were more remarkable in visceral WAT than in subcutaneous. These results suggest that exercise training decreases the expression of inflammation-related adipokines by reducing oxidative stress in WAT.

β2‐Adrenergic receptor regulates Toll‐like receptor‐4‐induced nuclear factor‐κB activation through β‐arrestin 2

Toll‐like receptors (TLRs) play an important role in innate immunity while, β2‐adrenergic receptors (β2AR) provide the key linkages for the sympathetic nervous system to regulate the immune system. However, their role in macrophages remains uncertain. Here, we demonstrate the cross‐talk between β2AR and TLR signalling pathways. Expression of β2AR was down‐regulated by TLR4 ligand lipopolysaccharide (LPS) stimulation. To investigate the physiological consequence of this down‐regulation RAW264 cells, a macrophage cell line, were transfected with a β2AR expression vector (RAWar). Both LPS‐stimulated inducible nitric oxide synthase (NOS II) expression and NO production were markedly suppressed in the RAWar cells. The activation of nuclear factor‐κB (NF‐κB) and degradation of the inhibitor of NF‐κB (IκBα) in response to LPS were markedly decreased in these cells. The level of β‐arrestin 2, which regulates β2AR signalling, was also reduced in RAW264 cells after stimulation with LPS, but not in RAWar cells. Overexpression of β‐arrestin 2 (RAWarr2) also inhibited NO production and NOS II expression. Furthermore, we demonstrated that β‐arrestin 2 interacted with cytosolic IκBα and that the level of IκBα coimmunoprecipitated by anti‐β‐arrestin 2 antibodies was decreased in the RAW264 cells but not in RAWar or RAWarr2 cells. These findings suggest that LPS‐stimulated signals suppress β2AR expression, leading to down‐regulation of β‐arrestin 2 expression, which stabilizes cytosolic IκBα and inhibits the NF‐κB activation essential for NOS II expression, probably to ensure rapid and sufficient production of NO in response to microbial attack.

Effect of exercise training on adipocyte-size-dependent expression of leptin and adiponectin.

AIMS Our aim was to evaluate the effect of exercise training (TR) on adipocyte-size-dependent expression of leptin and adiponectin. MAIN METHODS Male Wistar rats were divided into 2 groups, sedentary control (CR) and TR group, and both monitored for 9weeks. Adipocytes isolated from epididymal, retroperitoneal, and inguinal fat depots were independently separated into 3 fractions of different cell size, and the relationships between adipocyte size and either leptin or adiponectin mRNA were determined by real-time RT-PCR analysis. KEY FINDINGS In epididymal and inguinal adipose tissue, positive relationships between adipocyte size and both leptin and adiponectin mRNA expression were found. Comparison of TR and CR rats showed no significant effect of TR on the slopes of the linear regression lines of correlation between leptin mRNA and adipocyte size in either adipose tissue, whereas the slopes of the regression line of correlation between adipocyte size and adiponectin mRNA were greater in TR group. Leptin levels per milliliter of plasma were significantly lower in TR than CR rats, whereas leptin levels adjusted to the 3 fat depots did not differ. TR did not affect adiponectin levels in plasma, whereas adiponectin levels adjusted to the 3 fat depots were significantly greater in TR than CR group. SIGNIFICANCE TR-induced reduction in leptin mRNA expression was closely associated with smaller adipocyte size. However, TR amplified the adipocyte-size-dependent expression of adiponectin mRNA, suggesting that TR-induced alterations in adiponectin mRNA may also be mediated by factor(s) other than adipocyte size.

Adaptation of macrophages to exercise training improves innate immunity.

The effects of 3-week exercise training on the functions of peritoneal macrophages from BALB/c mice were investigated. Lipopolysaccharide (LPS)-stimulated nitric oxide (NO) and proinflammatory cytokine production in macrophages from trained mice was markedly higher than those from control mice. Meanwhile, exercise training decreased the steady state level of beta(2)-adrenergic receptor (beta(2)AR) mRNA in macrophages. Overexpression of beta(2)AR in the macrophage cell line RAW264 by transfecting with beta(2)AR cDNA suppressed NO synthase (NOS) II expression but dose not influenced proinflammatory cytokine expression. When expression of transfected beta(2)AR in RAWar cells was downregulated by a tetracycline repressor-regulated mammalian expression system, NOS II mRNA expression was significantly increased; this suggested that the changes in the beta(2)AR expression level in macrophages associated with exercise training play a role in the regulation of NO production following LPS stimulation. These findings indicate that exercise training improves macrophage innate immune function in a beta(2)AR-dependent and -independent manner.

Oligonol, a new lychee fruit-derived low-molecular form of polyphenol, enhances lipolysis in primary rat adipocytes through activation of the ERK1/2 pathway

The effect of Oligonol, a phenolic product from lychee fruit polyphenol (LFP) containing catechin‐type monomers and lower oligomers of proanthocyanidin, on lipolysis in primary adipocytes was investigated in order to examine the possible mechanism underlying the regulation of in vivo metabolism in fat. Oligonol significantly increased lipolysis, which was accompanied by both activation of extracellular signaling‐related kinase 1/2 (ERK1/2) and down‐regulation of perilipin protein expression, without an increase in intracellular cAMP production. The increase in lipolysis with Oligonol was prevented completely by pretreatment with either PD98059 or U0126, selective ERK1/2 inhibitors, which also prevented the reduction in the expression of perilipin protein. Tumor necrosis factor‐α also down‐regulated the expression of perilipin protein. However, there was no significant alteration in the expression of Gαi protein with Oligonol. These findings indicate that Oligonol enhances lipolysis in primary adipocytes, independent of cAMP production, but its effect is dependent on activation of the ERK1/2 pathway, leading to down‐regulation of perilipin protein expression. Copyright