Effect of the menstrual cycle on serum diamine oxidase levels in healthy women.

OBJECTIVES: Serum diamine oxidase (DAO) level is employed as a useful marker of intestinal mucosal integrity. As reported previously, the range of serum DAO levels in women is wider than that in men. We hypothesized that the menstrual cycle may affect DAO levels. DESIGN AND METHODS: Thirty-six women of Japanese descent were recruited. All participants, aged 20-29 years, were healthy. Food surveys utilized in this study were based on questionnaires validated by dietitians. Complete blood counts, biochemical parameters, female hormones, and serum DAO levels during the follicular and luteal phases were measured in each subject. RESULTS: Biochemical parameters, except for DAO levels, were comparable between the two phases. However, serum DAO levels during the luteal phase were significantly higher than those during the follicular phase. CONCLUSIONS: Serum DAO levels were influenced by the menstrual cycle. Furthermore, our findings suggest that serum DAO levels should be interpreted cautiously in premenopausal women.

Activation of AMP-activated protein kinase suppresses oxidized low-density lipoprotein-induced macrophage proliferation.

Macrophage-derived foam cells play important roles in the progression of atherosclerosis. We reported previously that ERK1/2-dependent granulocyte/macrophage colony-stimulating factor (GM-CSF) expression, leading to p38 MAPK/ Akt signaling, is important for oxidized low density lipoprotein (Ox-LDL)-induced macrophage proliferation. Here, we investigated whether activation of AMP-activated protein kinase (AMPK) could suppress macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages was assessed by [(3)H]thymidine incorporation and cell counting assays. The proliferation was significantly inhibited by the AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) and restored by dominant-negative AMPKalpha1, suggesting that AMPK activation suppressed macrophage proliferation. AICAR partially suppressed Ox-LDL-induced ERK1/2 phosphorylation and GM-CSF expression, suggesting that another mechanism is also involved in the AICAR-mediated suppression of macrophage proliferation. AICAR suppressed GM-CSF-induced macrophage proliferation without suppressing p38 MAPK/Akt signaling. GM-CSF suppressed p53 phosphorylation and expression and induced Rb phosphorylation. Overexpression of p53 or p27(kip) suppressed GM-CSF-induced macrophage proliferation. AICAR induced cell cycle arrest, increased p53 phosphorylation and expression, and suppressed GM-CSF-induced Rb phosphorylation via AMPK activation. Moreover, AICAR induced p21(cip) and p27(kip) expression via AMPK activation, and small interfering RNA (siRNA) of p21(cip) and p27(kip) restored AICAR-mediated suppression of macrophage proliferation. In conclusion, AMPK activation suppressed Ox-LDL-induced macrophage proliferation by suppressing GM-CSF expression and inducing cell cycle arrest. These effects of AMPK activation may represent therapeutic targets for atherosclerosis.

Statins activate peroxisome proliferator-activated receptor gamma through extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase-dependent cyclooxygenase-2 expression in macrophages.

Both statins and peroxisome proliferator-activated receptor (PPAR)gamma ligands have been reported to protect against the progression of atherosclerosis. In the present study, we investigated the effects of statins on PPARgamma activation in macrophages. Statins increased PPARgamma activity, which was inhibited by mevalonate, farnesylpyrophosphate, or geranylgeranylpyrophosphate. Furthermore, a farnesyl transferase inhibitor and a geranylgeranyl transferase inhibitor mimicked the effects of statins. Statins inhibited the membrane translocations of Ras, RhoA, Rac, and Cdc42, and overexpression of dominant-negative mutants of RhoA (DN-RhoA) and Cdc42 (DN-Cdc42), but not of Ras or Rac, increased PPARgamma activity. Statins induced extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) activation. However, DN-RhoA and DN-Cdc42 activated p38 MAPK, but not ERK1/2. ERK1/2- or p38 MAPK-specific inhibitors abrogated statin-induced PPARgamma activation. Statins induced cyclooxygenase (COX)-2 expression and increased intracellular 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)) levels through ERK1/2- and p38 MAPK-dependent pathways, and inhibitors or small interfering RNA of COX-2 inhibited statin-induced PPARgamma activation. Statins also activate PPARalpha via COX-2-dependent increases in 15d-PGJ(2) levels. We further demonstrated that statins inhibited lipopolysaccharide-induced tumor necrosis factor alpha or monocyte chemoattractant protein-1 mRNA expression, and these effects by statins were abrogated by the PPARgamma antagonist T0070907 or by small interfering RNA of PPARgamma or PPARalpha. Statins also induced ATP-binding cassette protein A1 or CD36 mRNA expression, and these effects were suppressed by small interfering RNAs of PPARgamma or PPARalpha. In conclusion, statins induce COX-2-dependent increase in 15d-PGJ(2) level through a RhoA- and Cdc42-dependent p38 MAPK pathway and a RhoA- and Cdc42-independent ERK1/2 pathway, thereby activating PPARgamma. Statins also activate PPARalpha via COX-2-dependent pathway. These effects of statins may explain their antiatherogenic actions.

Troglitazone inhibits oxidized low-density lipoprotein-induced macrophage proliferation: impact of the suppression of nuclear translocation of ERK1/2.

Thiazolidinediones (TZDs), which were known as novel insulin-sensitizing antidiabetic agents, have been reported to inhibit the acceleration of atherosclerotic lesions. Macrophages play important roles in the development of atherosclerosis. We previously reported that oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation through ERK1/2-dependent GM-CSF production. In the present study, we investigated the effects of two TZDs, troglitazone and ciglitazone on Ox-LDL-induced macrophage proliferation. Troglitazone significantly inhibited Ox-LDL-induced increases in [(3)H]thymidine incorporation into and proliferation of mouse peritoneal macrophages, whereas ciglitazone had no effects. Troglitazone and ciglitazone both significantly induced PPARgamma activity, suggesting that the inhibitory effect of troglitazone was not mediated by PPARgamma. Ox-LDL-induced production of GM-CSF was significantly inhibited by troglitazone, but not by ciglitazone. Troglitazone inhibited Ox-LDL-induced production of intracellular reactive oxygen species, whereas ciglitazone had no effect. The antioxidant reagents NAC and NMPG each inhibited phosphorylation of ERK1/2, whereas troglitazone and ciglitazone had no effects. However, troglitazone, NAC and NMPG all inhibited nuclear translocation of ERK1/2. In conclusion, troglitazone inhibited Ox-LDL-induced GM-CSF production by suppressing nuclear translocation of ERK1/2, thereby inhibiting macrophage proliferation. This suppression of macrophage proliferation by troglitazone may, at least in part, explain its antiatherogenic effects.

Impact of mitochondrial reactive oxygen species and apoptosis signal-regulating kinase 1 on insulin signaling.

Tumor necrosis factor (TNF)-alpha inhibits insulin action; however, the precise mechanisms are unknown. It was reported that TNF-alpha could increase mitochondrial reactive oxygen species (ROS) production, and apoptosis signal-regulating kinase 1 (ASK1) was reported to be required for TNF-alpha-induced apoptosis. Here, we examined roles of mitochondrial ROS and ASK1 in TNF-alpha-induced impaired insulin signaling in cultured human hepatoma (Huh7) cells. Using reduced MitoTracker Red probe, we confirmed that TNF-alpha increased mitochondrial ROS production, which was suppressed by overexpression of either uncoupling protein-1 (UCP)-1 or manganese superoxide dismutase (MnSOD). TNF-alpha significantly activated ASK1, increased serine phosphorylation of insulin receptor substrate (IRS)-1, and decreased insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt, and all of these effects were inhibited by overexpression of either UCP-1 or MnSOD. Similar to TNF-alpha, overexpression of wild-type ASK1 increased serine phosphorylation of IRS-1 and decreased insulin-stimulated tyrosine phosphorylation of IRS-1, whereas overexpression of dominant-negative ASK1 ameliorated these TNF-alpha-induced events. In addition, TNF-alpha activated c-jun NH(2)-terminal kinases (JNKs), and this observation was partially inhibited by overexpression of UCP-1, MnSOD, or dominant-negative ASK1. These results suggest that TNF-alpha increases mitochondrial ROS and activates ASK1 in Huh7 cells and that these TNF-alpha-induced phenomena contribute, at least in part, to impaired insulin signaling.

Biochemical properties of ordinary and dark muscle myosin from carp skeletal muscle.

Two types of myosin isolated from ordinary (fast) and dark (slow) muscles of carp were examined by ATPase and in vitro motility assays. Vmax of the ATPase activity and sliding velocity of ordinary myosin showed 1.6 and 1.5 times higher activities than those of dark myosin, whereas those of mammalian fast myosin were much higher, 3 to 10 times, than those of slow myosin. Although ordinary myosin had almost identical activities to those of mammalian fast myosin, activities of dark myosin was twice of those of mammalian slow myosin. This high motile activity of dark myosin can account for the physiological role of dark muscle in cruising of fish. By comparing Km of the actin-activated ATPase activity, ordinary myosin was appeared to have higher affinity to F-actin than dark myosin, and this was confirmed by the binding assay of HMM or S-1 of carp myosin to F-actin. Investigation of myosin assembly by electron microscopy and the centrifugation assay revealed that ordinary myosin assembled much poorly than dark myosin or mammalian fast myosin. This phenomenon may reflect characteristic cellular function of fish skeletal muscle.

Adenosine monophosphate-activated protein kinase suppresses vascular smooth muscle cell proliferation through the inhibition of cell cycle progression.

Vascular smooth muscle cell (VSMC) proliferation is a critical event in the development and progression of vascular diseases, including atherosclerosis. We investigated whether the activation of adenosine monophosphate-activated protein kinase (AMPK) could suppress VSMC proliferation and inhibit cell cycle progression. Treatment of human aortic smooth muscle cells (HASMCs) or isolated rabbit aortas with the AMPK activator 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) induced phosphorylation of AMPK and acetyl Co-A carboxylase. AICAR significantly inhibited HASMC proliferation induced by both platelet-derived growth factor-BB (PDGF-BB) and fetal calf serum (FCS). Treatment with AICAR inhibited the phosphorylation of retinoblastoma gene product (Rb) induced by PDGF-BB or FCS, and increased the expression of cyclin-dependent kinase inhibitor p21(CIP) but not that of p27(KIP). Pharmacological inhibition of AMPK or overexpression of dominant negative-AMPK inhibited both the suppressive effect of AICAR on cell proliferation and the phosphorylation of Rb, suggesting that the effect of AICAR is mediated through the activation of AMPK. Cell cycle analysis in HASMCs showed that AICAR significantly increased cell population in G0/G1-phase and reduced that in S- and G2/M-phase, suggesting AICAR induced cell cycle arrest. AICAR increased both p53 protein and Ser-15 phosphorylated p53 in HASMCs, which were blocked by inhibition of AMPK. In isolated rabbit aortas, AICAR also increased Ser-15 phosphorylation and protein expression of p53 and inhibited Rb phosphorylation induced by FCS. These data suggest for the first time that AMPK suppresses VSMC proliferation via cell cycle regulation by p53 upregulation. Therefore, AMPK activation in VSMCs may be a therapoietic target for the prevention of vascular diseases.

Statins suppress oxidized low density lipoprotein-induced macrophage proliferation by inactivation of the small G protein-p38 MAPK pathway.

Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (statins) ameliorate atherosclerotic diseases. Macrophages play an important role in the development and subsequent stability of atherosclerotic plaques. We reported previously that oxidized low density lipoprotein (Ox-LDL) induced macrophage proliferation through the secretion of granulocyte/macrophage colony-stimulating factor (GM-CSF) and the consequent activation of p38 MAPK. The present study was designed to elucidate the mechanism of the inhibitory effect of statins on macrophage proliferation. Mouse peritoneal macrophages were used in our study. Cerivastatin and simvastatin each inhibited Ox-LDL-induced [(3)H]thymidine incorporation into macrophages. Statins did not inhibit Ox-LDL-induced GM-CSF production, but inhibited GM-CSF-induced p38 MAPK activation. Farnesyl transferase inhibitor and geranylgeranyl transferase inhibitor inhibited GM-CSF-induced macrophage proliferation, and farnesyl pyrophosphate and geranylgeranyl pyrophosphate prevented the effect of statins. GM-CSF-induced p38 MAPK phosphorylation was also inhibited by farnesyl transferase inhibitor or geranylgeranyl transferase inhibitor, and farnesyl pyrophosphate and geranylgeranyl pyrophosphate prevented the suppression of GM-CSF-induced p38 MAPK phosphorylation by statins. Furthermore, we found that statin significantly inhibited the membrane translocation of the small G protein family members Ras and Rho. GM-CSF-induced p38 MAPK activation and macrophage proliferation was partially inhibited by overexpression of dominant negative Ras and completely by that of RhoA. In conclusion, statins inhibited GM-CSF-induced Ras- or RhoA-p38 MAPK signal cascades, thereby suppressing Ox-LDL-induced macrophage proliferation. The significant inhibition of macrophage proliferation by statins may also explain, at least in part, their anti-atherogenic action.

Extracellular signal-regulated kinase and p38 mitogen-activated protein kinase mediate macrophage proliferation induced by oxidized low-density lipoprotein.

We previously reported that oxidized low-density lipoprotein (Ox-LDL)-induced expression of granulocyte/macrophage colony-stimulating factor (GM-CSF) via PKC, leading to activation of phosphatidylinositol-3 kinase (PI-3K), was important for macrophage proliferation [J Biol Chem 275 (2000) 5810]. The aim of the present study was to elucidate the role of extracellular-signal regulated kinase 1/2 (ERK1/2) and of p38 MAPK in Ox-LDL-induced macrophage proliferation. Ox-LDL-induced proliferation of mouse peritoneal macrophages assessed by [3H]thymidine incorporation and cell counting assays was significantly inhibited by MEK1/2 inhibitors, PD98059 or U0126, and p38 MAPK inhibitors, SB203580 or SB202190, respectively. Ox-LDL-induced GM-CSF production was inhibited by MEK1/2 inhibitors but not by p38 MAPK inhibitors in mRNA and protein levels, whereas recombinant GM-CSF-induced macrophage proliferation was inhibited by p38 MAPK inhibitors but enhanced by MEK1/2 inhibitors. Recombinant GM-CSF-induced PI-3K activation and Akt phosphorylation were significantly inhibited by SB203580 but enhanced by PD98059. Our results suggest that ERK1/2 is involved in Ox-LDL-induced macrophage proliferation in the signaling pathway before GM-CSF production, whereas p38 MAPK is involved after GM-CSF release. Thus, the importance of MAPKs in Ox-LDL-induced macrophage proliferation was confirmed and the control of MAPK cascade could be targeted as a potential treatment of atherosclerosis.

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB.

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.