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It has previously been reported that the globular form of adiponectin (gAd), mature adipocyte-derived cytokine, induced generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated whether diacylglycerol kinases (DGKs), enzymes functioning in sub-cellular signalling pathways, had a role on gAd-induced ROS generation in RAW 264 cells. Administration of R59022, a specific inhibitor for DGK, reduced gAd-induced ROS generation and NO release. RAW 264 cell expressed DGKα mRNA. Depression of DGKα mRNA by RNA interference significantly reduced the ROS generation in response to gAd treatment. Interestingly, transfection with the DGKα-specific small interfering RNA attenuated the expression level of Nox1 mRNA in gAd-treated RAW 264 cells. In addition, the DGKα knockdown with siRNA suppressed gAd-induced NO release.

Adiponectin is a protein hormone produced by differentiating adipocytes and has been proposed to have anti-diabetic and immunosuppressive properties. We previously reported that the globular form of adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO), followed by caspase-dependent apoptotic cell death in RAW 264 cells. Here, we demonstrate that gAd-induced ROS generation and apoptosis were diminished by suppressor of cytokine signaling 3 (SOCS3). The phosphorylation level of signal transducer and activator of transcription (STAT) 3 detected by Western blotting was highest at 20 min in gAd-treated RAW 264 cells. This phosphorylation was inhibited by AG490, a specific inhibitor of janus-activator kinase (JAK). The gAd-induced ROS and NO were reduced by administration of AG490 and Jak-2-specific siRNA in RAW 264 cells. The gAd stimulation transiently induced SOCS3 mRNA expression and protein production. We examined SOCS3-overexpressing RAW 264 cells to investigate the role of the JAK-STAT pathway in gAd-induced ROS and NO generation. SOCS3 overexpression significantly reduced both ROS and NO generation. Additionally, gAd-induced caspase activation and apoptotic cell death were reduced in SOCS3 transfectants compared with vector control transfectants. These results suggest that the JAK-STAT pathway, which can be suppressed by SOCS3 expression, is involved in gAd-induced ROS and NO generation followed by apoptotic cell death., (© 2010 Wiley-Liss, Inc.)

Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. This study investigated the role of the mammalian target of rapamycin (mTOR) in gAd-induced ROS and NO generation. gAd stimulation induced phosphorylation of mTOR, which peaked at 20 min and dissolved rapidly. Inhibition of phosphatidylinositol 3-kinase activity with wortmannin suppressed gAd-induced phosphorylation of Akt and mTOR. Administration of rapamycin partially reduced gAd-induced generation of intracellular and mitochondrial ROS, but not release of NO. To further confirm the role of mTOR in gAd stimulation, the effect of the activators of AMP-activated protein kinase (AMPK) on gAd-induced mTOR phosphorylation was examined. Pre-treatment with three kinds of AMPK activators, AICAR, 2-deoxy-D-glucose and A-769662, suppressed gAd-induced mTOR phosphorylation. Furthermore, these AMPK activators significantly reduced gAd-evoked intracellular and mitochondrial ROS generation and NO release.

Globular adiponectin (gAd), a truncated form of adipocyte-derived cytokine, stimulates RAW 264 cells to produce reactive oxygen species (ROS), which trigger an apoptotic cascade. In this study, we investigated the generation of intracellular and mitochondrial ROS in gAd-stimulated RAW 264 cells. Treatment with gAd efficiently induced the generation of intracellular and mitochondrial ROS, as detected by dichlorodihydrofluorescein diacetate and MitoSOX fluorescence, respectively. Furthermore, gAd treatment significantly increased 8-oxoguanine, a specific indicator of oxidative DNA damage. The transfection of RAW 264 cells with iNOS- and gp91(phox)-specific small interfering RNA reduced markedly the generation of intracellular, but not mitochondrial, ROS. Quantitative PCR revealed that the expression ratio of Bcl-2 to Bax was reduced in a time-dependent manner in gAd-treated RAW 264 cells. The overexpression of Bcl-2 markedly inhibited gAd-induced apoptosis in RAW 264 cells and also reduced both the intracellular and the mitochondrial ROS generation induced by gAd treatment. Moreover, the overexpression of Bcl-2 significantly suppressed gAd-induced NO secretion and NOS activity. In addition, the inhibition of NOS activity partially reduced the oxidative DNA damage induced by gAd. Taken together, these results demonstrate that the gAd-induced apoptotic pathway acting via ROS/RNS generation involves Bcl-2.

Adiponectin, produced predominantly by differentiating adipocytes, is a protein hormone with antidiabetic and immunosuppressive properties. Here, we report evidence that treatment with globular adiponectin (gAd) induces apoptosis in murine macrophage-like RAW264 cells through the generation of reactive oxygen and/or nitrogen species (ROS/RNS). Treatment with gAd induced apoptosis and enhanced the activities of caspase-3 and -9, but not caspase-8. The gAd stimulation increased ROS generation and significantly reduced the ratio of NADPH to total NADP. Pretreatment with diphenyleneiodonium or apocynin reduced ROS and apoptosis in gAd-treated cells. In addition, transfection with p47(phox)- or gp91(phox)-specific small interfering RNA (siRNA) partially reduced ROS and apoptosis in response to gAd treatment. These results suggest that the administration of gAd induces apoptosis after ROS generation involving activation of NADPH oxidases. The gAd stimulation increased the release of NO into the culture medium, the activity of nitric oxide synthase (NOS), and the expression of inducible NOS (iNOS) mRNA in RAW264 cells. l-NAME reduced gAd-induced apoptotic cell death. In addition, transfection with an iNOS-specific siRNA markedly reduced the generation of NO and the population of apoptotic cells. Taken together, these results demonstrate that the gAd-induced apoptotic process in RAW264 cells involves ROS and RNS, which are generated by NADPH oxidases and iNOS, respectively.

Adiponectin, an adipocyte-derived cytokine, affects a number of physiological processes, including immune function and inflammation. We investigated whether globular adiponectin (gAd) affects the expression of inflammation-related genes in murine macrophages (RAW264 cells). DNA microarray analysis indicated that granulocyte colony-stimulating factor (G-CSF) showed the largest increase in expression in gAd-stimulated RAW264 cells. The gAd-induced secretion of G-CSF increased in a time- and dose-dependent manner. U0126 (MEK1/2 inhibitor) and PD98059 (MEK1 inhibitor) reduced the gAd-induced G-CSF mRNA expression and G-CSF protein production. gAd induced the phosphorylation of MEK1/2 and ERK1/2 in RAW264 cells. In addition, the gAd-induced phosphorylation of MEK1/2 and ERK1/2 was dramatically reduced by PD98059 and U0126, respectively. Collectively, these results suggest that MEK1/2-ERK1/2 signaling is involved in the adiponectin-induced secretion of G-CSF.

Abstract: We previously demonstrated that treatment with a globular type of adiponectin (gAd) induced expression of granulocyte colony-stimulating factor (G-CSF) via the MEK/ERK signaling pathway in a murine macrophage cell line, RAW 264. In the present study, we investigated whether suppressor of cytokine signaling-1 (SOCS1) has roles in the regulation of gAd-induced G-CSF generation. Intracellular G-CSF generation induced by gAd treatment peaked after 10h and then attenuated. SOCS1 mRNA and protein were expressed at 1h and 4h after gAd treatment, respectively. Overexpression of SOCS1 reduced G-CSF generation and phosphorylation of ERK, JNK, and p38 MAPK in gAd-treated cells. While gAd treatment induced the translocation of STAT3 to the nucleus under control conditions, STAT3 stayed in the cytosol when SOCS1 was overexpressed. Additionally, knockdown of SOCS1 by interfering RNA caused levels of G-CSF to continue to rise beyond 10h after gAd treatment. These results suggest that SOCS1 is involved in providing negative feedback for gAd-induced production of G-CSF. [Copyright &y& Elsevier]

Adiponectin is an adipokine with potent anti-inflammatory properties. We previously reported that a globular adiponectin (gAd) suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced nuclear factor-κB activity, suggesting an anti-inflammatory effect of gAd. In this study, we investigated whether gAd is able to modulate the effect of A. actinomycetemcomitans lipopolysaccharide on cytokine induction in a murine macrophage cell line (RAW 264). The phosphorylation of p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and IκB kinase α/β and the degradation of IκB, which were induced by A. actinomycetemcomitans lipopolysaccharide intoxication, were clearly reduced in gAd-pretreated RAW 264 cells compared with the untreated cells. Expression levels of tumor necrosis factor (TNF)-α and interleukin-10 (IL-10) mRNA were assessed by real-time PCR. Cell-free supernatants were collected after 12 h of stimulation and analyzed by enzyme-linked immunosorbent assay for TNF-α and IL-10. Pretreatment with gAd significantly inhibited the A. actinomycetemcomitans lipopolysaccharide-induced TNF-α mRNA expression and protein secretion. In contrast, pretreatment with gAd significantly enhanced the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 mRNA expression and protein secretion. These data suggest a mechanism for the anti-inflammatory activity of gAd in local inflammatory lesions, such as periodontitis. [ABSTRACT FROM AUTHOR]

Abstract: Globular adiponectin (gAd) induces the generation of reactive oxygen species (ROS) and nitric oxide (NO) in the murine macrophage cell line RAW 264. We investigated the role of Ca2+ in gAd-induced ROS and NO generation. Pretreatment with BAPTA-AM, a selective chelator of intracellular Ca2+ ([Ca2+]i), partially reduced gAd-induced generation of ROS and NO in gAd-treated RAW 264 cells. The lowest [Ca2+]i occurred 30min after gAd treatment, after which [Ca2+]i increased continually and exceeded the initial level. The mitochondrial Ca2+ ([Ca2+]m) detected by Rhod-2 fluorescence started to increase at 6h after gAd treatment. Pretreatment with a NAD(P)H oxidase inhibitor, diphenyleneiodonium, prevented the reduction of [Ca2+]i in the early phase after gAd treatment. Calcium depletion by BAPTA-AM had no effect on the gAd-induced [Ca2+]m oscillation. The administration of a specific calmodulin inhibitor, calmidazolium, significantly suppressed gAd-induced ROS and NO generation and NOS activity. [Copyright &y& Elsevier]