Katsutoshi Fujie

Effect of coffee consumption on bone metabolism

The effects of coffee on bone metabolism are still controversial, although several studies have suggested that caffeine and/or heavy coffee consumption is associated with a significant increase in risk of fracture, osteoporosis, and periodontal disease. Therefore, we sought to clarify the relationship between coffee consumption and bone metabolism using male Wistar rats. Forty-eight male Wistar rats were assigned to three treatment groups including a control-diet group (control, n = 16, coffee-free diet), a 0.62% coffee-diet group (low caffeine, n = 16, diet supplemented with 6.2 g/kg of the control diet), and a 1.36% coffee-diet group (high caffeine, n = 16, diet supplemented with 13.6 g/kg of the control diet), and animals were maintained on an experimental diet for 140 days. Although caffeine in serum was not detected in rats fed the control diet, low-intake coffee for 140 days led to an increase in caffeine concentration to 0.53 +/- 0.11 microg/mL and high-intake coffee led to an increase of 1.77 +/- 0.22 microg/mL. No significant differences in body weight change, serum and urinary biochemical markers of bone metabolism, and bone histomorphometry were found between the coffee-diet groups and the control-diet group, except that urinary phosphorus excretion after 140 days of both coffee diets was significantly increased compared with controls (p < 0.05). In addition, the coffee diets were not associated with differences in tumor necrosis factor-alpha and interleukin-6, which have been implicated in the pathogenesis of bone loss together with interleukin-1beta. In conclusion, the present study strongly indicates that coffee does not stimulate bone loss in rats.

Inhibition of PGE2 production in macrophages from vitamin E-treated rats

Rat peritoneal macrophages from vitamin E-treated rats (5 mg per rat for 6 successive days) contained 403.3 +/- 90.7 ng alpha-tocopherol/10(6) cells, whereas control macrophages contained 1.2 +/- 0.4 ng. PGE2 production in the macrophages from vitamin E-treated rats was significantly suppressed when stimulated with PMA and calcium ionophore A23187. The mechanism of vitamin E inhibition of PGE2 production in macrophages was investigated. The release of (14C)-arachidonic acid from pre-labeled macrophages and the conversion of (14C)-arachidonic acid to PGE2 by the homogenate of the cells were remarkably reduced. These results strongly suggested that the inhibition of PGE2 production by vitamin E results from the inhibition of the activities of both phospholipase A2 and cyclooxygenase.

EFFECT OF VITAMIN E ON EXPRESSION OF CYCLOOXYGENASE-2 IN LIPOPOLYSACCHARIDE-STIMULATED RAT MACROPHAGES

To clarify the role of vitamin E (alpha-tocopherol) for the induction of cyclooxygenase-2 (COX-2) in rat macrophages stimulated by lipopolysaccharide (LPS), vitamin E-enriched macrophages were prepared by intraperitoneal injection of vitamin E for 6 days at a rate of 5 mg per day. The production of PGE2 was increased in dose- and time-dependent manners by addition of LPS in both control and vitamin E-enriched peritoneal macrophages. The maximum effect of LPS was observed in 12 h at concentration of 5 micrograms/ml. By analyzing COX-2 mRNA level by Northern blot and COX-2 enzyme mass and phosphotyrosine by Western blot, it was revealed that the increase of PGE2 production reflected the induction of COX-2 expression through activation of tyrosine kinase. Vitamin E failed to inhibit PGE2 production in LPS-stimulated macrophages; however, genistein, a tyrosine kinase inhibitor, completely inhibited the production at 100 microM. These results suggest that vitamin E does not inhibit COX-2 expression via LPS-mediated tyrosine kinase signal transduction pathway.

Vitamin E inhibits PGE2 and O2− production in rat peritoneal macrophages

In order to examine the possible role of vitamin E on the modulation of macrophages, we investigated the effect of vitamin E on O2- and PGE2 production in macrophages. The production of both PGE2 and O2- in rat peritoneal macrophages was dose-dependently stimulated by the addition of PMA and calcium ionophore A23187. However, the macrophages obtained after intraperitoneal injection of vitamin E for six successive days showed less PGE2 and O2- production when stimulated with PMA or A23187 as compared to those of control macrophages. O2- production in control macrophages stimulated with 139 nM PMA and 1 microM A23187 as 4.2 +/- 0.3 and 3.0 +/- 0.2 nmol/min per 10(6) cells, respectively. On the other hand, O2- production by the macrophages from vitamin E-treated rats was 1.5 +/- 0.4 nmol/min per 10(6) cells when stimulated with the PMA, and was not detectable when stimulated with A23187. As for the production of PGE2, control macrophages produced 2.59 +/- 0.70 ng/30 min per 10(6) cells when stimulated with PMA and 8.96 +/- 3.26 ng/30 min per 10(6) cells with the A23187, whereas PGE2 production by the macrophages from vitamin E-treated rats was reduced to 12-20% of the control. By analyzing alpha-tocopherol content and intracellular concentration of calcium ion [( Ca2+]i) in the macrophages isolated from control and vitamin E-treated rats, vitamin E treatment augmented alpha-tocopherol content (384.7 +/- 76.1 vs. 1.2 +/- 0.4 ng/10(6) cells) and decreased free [Ca2+]i when stimulated with A23187 (652 +/- 14 vs. 1201 +/- 223 nM).

Inhibition of macrophage migration inhibitory factor secretion from macrophages by vitamin E

Macrophage migration inhibitory factor (MIF) was identified in rat peritoneal macrophages by Western blot analysis and its secretion into culture medium by enzyme-linked immunosorbent assay. We investigated the effect of vitamin E on MIF production in macrophages in response to phorbol 12-myristate-13-acetate (PMA), calcium ionophore A23187, and lipopolysaccharide (LPS). Intraperitoneal injections of vitamin E (5 mg per rat) for 6 successive days resulted in a significant increase of alpha-tocopherol content in peritoneal macrophages (478.3+/-90.7 ng/106 cells) compared with the control (1.5+/-0.5 ng/10(6) cells). For the control macrophages, MIF content of the medium (2.5x10(6) cells/18 ml) without stimulation was 2.27+/-0.20 ng/ml after 14 h culture, whereas stimulation with calcium ionophore A23187 (400 nM) and LPS (5.0 microg/ml) induced the elevation of MIF content to 3. 66+/-0.41 and 4.12+/-0.58 ng/ml, respectively. On the other hand, vitamin E-enriched macrophages without stimulation showed less MIF content (0.77+/-0.23 ng/ml) than the control. Similarly, the increase of MIF of vitamin E-treated macrophages was significantly suppressed after stimulation with calcium ionophore A23187 or LPS, compared with the control macrophages. From analysis of intracellular MIF content by Western blot, we found no alteration of intracellular MIF content of vitamin E-macrophages, in contrast to the decreased content of control stimulated-macrophages. Taken together, these results indicate that vitamin E may contribute to the regulation of immune responses through regulation of MIF secretion.

Effect of vitamin E on arachidonic acid-release in rat peritoneal macrophages

We have previously reported on suppression of the PGE2 production in PMA- and calcium ionophore A23187-stimulated macrophages isolated from vitamin E-treated rats. To further study the mechanism, we examined the effect of vitamin E on phospholipase A2 activity in both intact macrophages and cell-free homogenates measuring the release of [14C]arachidonic acid. In macrophages from vitamin E-treated rats, arachidonic acid release in intact cells as stimulated with PMA and calcium ionophore A23187 was hardly detected. In the cell-free homogenates, increase in phospholipase A2 activity of cytosol and particulate fractions by PMA and A23187 was partially suppressed. In unstimulated macrophages, most of phospholipase A2 was recovered in the cytosol fraction. The partially purified cytosolic phospholipase A2 showed a molecular mass 95 kDa on TSK gel G3000SW gel-filtration and on Western blot analysis using anti-rabbit platelet phospholipase A2 monoclonal antibody RHY-5. The activity of cytosolic 95 kDa phospholipase A2 was not inhibited in vitro by vitamin E. From these results, it was suggested that vitamin E needs intact macrophages to suppress arachidonic acid release.

Coffee increases levels of urinary 8-hydroxydeoxyguanosine in rats

We examined whether coffee or chlorogenic acid inhibits 8-hydroxydeoxyguanosine (8-OHdG), one of the major forms of oxidative DNA damage, in vivo and in vitro. Forty-eight male Wistar rats were assigned to three treatment groups: a control-diet group (n=16; coffee-free diet), a 0.62% coffee-diet group (n=16, dose of coffee consumed 125 mg/day), and a 1.36% coffee-diet group (n=16, dose of coffee consumed 275 mg/day) and were maintained on an experimental diet for 130 days. The coffee-diet resulted in significantly increased excretion of urinary chlorogenic acid, with the 0.62 and 1.36% coffee-diets resulting in 14.00+/-0.94 and 15.80+/-0.41 ng/mg creatinine, respectively, whereas in control rats it was not detected. Using monoclonal antibody to measure 8-OHdG, it was revealed that coffee led to a significant increase in excretion of urinary 8-OHdG on day 130 (46.62+/-13.42 ng/mg creatinine in 0.62% coffee-diet group and 64.58+/-20.15 ng/mg creatinine in 1.36% coffee-diet group, P<0.05 vs. control; control group 10.89+/-2.59 ng/mg creatinine). Furthermore, to clarify the mechanism of 8-OHdG formation by coffee, we investigated the in vitro effect of chlorogenic acid on 8-OHdG formation in human placental DNA. Chlorogenic acid alone did not lead to an increase of 8-OHdG formation, but dramatically increased it in the presence of cupric chloride and H(2)O(2). However, chlorogenic acid and cupric chloride decreased the formation of 8-OHdG in the presence of H(2)O(2). Based on these results, a possible mechanism of 8-OHdG formation in vivo by chlorogenic acid is discussed.

Secretion of macrophage migration inhibitory factor differs from interleukin-6 in hydrogen peroxide- and LPS-stimulated human fibroblasts

To investigate the mechanism for secretion of macrophage migration inhibitory factor (MIF) in cultured human fibroblasts, we compared it with the secretion of interleukin-6 (IL-6) after stimulation with lipopolysaccharides (LPS) and H2O2. MIF content of the medium of 2.0 x 10(6) cells/20 ml after 20 h culture of nonstimulated fibroblasts was 0.30 +/- 0.06 ng/ml, whereas LPS-stimulation (10 microg/ml) only led to a 1.5-fold increase as compared with the nonstimulated cells. In contrast, a significant increase of IL-6 was induced by LPS-stimulation (6048 +/- 488 pg/ml in LPS-stimulated cells vs. 58 +/- 36 pg/ml in control cells). On the other hand, higher concentrations of H2O2 (0.6-1.2 mM) caused an increase of MIF secretion into the culture medium irrespective of LPS-stimulation; with 1.2 mM H2O2-stimulation for 20 h, it was increased to 40-fold as compared with the nonstimulated cells. However, lower concentrations (0.1-0.4 mM) did not cause this. Interestingly, H2O2-stimulation not only failed to increase IL-6 production from fibroblasts, but also repressed induction of IL-6 by LPS-stimulation in a dose-dependent manner. Genistein, a tyrosine kinase inhibitor, and H-7, a protein kinase C inhibitor, also inhibited IL-6 secretion but not MIF secretion in both LPS- and H2O2-stimulated fibroblasts. From analysis of trypan blue exclusion, formazan formation, morphological changes, and intracellular MIF content by Western blotting, we found that MIF secretion by H2O2 seemed to be mainly due to cell death and subsequent leakage of intracellular MIF. Taken together, these results suggest that MIF secretion differs from IL-6 via LPS-mediated signaling pathways.

Relationship of Coffee Consumption with Risk Factors of Atherosclerosis in Rats

Aims: In experimental animals we investigated the relationship of coffee consumption with risk factors of atherosclerosis such as cholesterol, homocysteine, oxidative stress and inflammatory cytokines. Methods: Forty-eight male Wistar rats were assigned to 3 treatment groups (a control diet group, 0.62% coffee diet group, and 1.36% coffee diet group), and animals were maintained on the experimental diets for 140 days. Results: Coffee diets led to an increase in the caffeine concentration to 0.53 ± 0.11 and 1.77 ± 0.22 µg/ml, respectively, although caffeine in serum was not detected in rats fed the control diet. It also led to slightly increased total serum levels of homocysteine and cholesterol, but no significant differences were found between the control and coffee diet groups. Coffee intake did not affect the production of IL-6 and TNF-α induced by LPS, which contributes to the atheroma-promoting effect of recurrent bacterial infection. Regarding the biomarkers of oxidative stress, the serum level of 15-isoprostane F2t, which was significantly increased by LPS injection, was not altered by coffee intake. In contrast, urinary 8-hydroxy-2-deoxyguanosine was significantly increased in the coffee diet groups (p < 0.05). On the other hand, serum glutathione peroxidase (GPx) activity tended to decrease in the coffee groups compared with the control group, but no significant difference was found between the control and coffee diet groups. Interestingly, a significant negative correlation was observed between GPx activity and homocysteine levels in the sera from control and coffee diet groups (r = –0.403, p < 0.05). Conclusions: This report is the first animal study on the relationship of coffee consumption with risk factors for atherosclerosis. From these results, we conclude that moderate coffee intake is not a risk factor for atherogenesis.

Degradation of T-kininogen by cathepsin D and matrix metalloproteinases

Cathepsin D, matrix metalloproteinase (MMP)-2, MMP-3 (stromelysin), and MMP-9 were isolated from rat granulomatous tissues. HT1080 human fibrosarcoma cells and rheumatoid synovial cell CM. At acidic conditions, cathepsin D cleaved T-kininogen into small peptides and released Met-T-kinin-Leu (kinin precursor), but failed to release kinin. MMP-3 cleaved T-kininogen into a 57 kDa fragment as measured by SDS-PAGE and Western blot analysis using anti-T-kininogen antiserum. On the other hand, no degradation of T-kininogen occurred during incubation with MMP-2 or MMP-9100/1) at pH 7.5 for 7 h.