Bezafibrate, a peroxisome proliferator-activated receptor α agonist, decreases circulating CD14(+)CD16(+) monocytes in patients with type 2 diabetes.

CD14(+)CD16(+) monocytes are proinflammatory cells that produce tumor necrosis factor and interleukin (IL)-1ß. The number of circulating CD14(+)CD16(+) monocytes is increased in patients with chronic renal failure or coronary artery disease. We investigated the effect of bezafibrate, a peroxisome proliferator-activated receptor α agonist, on circulating CD14(+)CD16(+) monocytes in patients with type 2 diabetes. Using cells isolated from type 2 diabetic subjects, we also examined the in vitro expression of CD16 messenger RNA (mRNA) by mononuclear cells (MNCs) exposed to bezafibrate. The percentage of CD14(+)CD16(+) monocytes among all CD14(+) monocytes was significantly higher in subjects with impaired glucose tolerance (P < 0.01) or type 2 diabetes (P < 0.05) than in those with normal glucose tolerance. The percentage of CD14(+)CD16(+) monocytes was significantly lower in patients with type 2 diabetes who were taking bezafibrate (400 mg/d) than in patients not taking it (P < 0.01). Treatment with bezafibrate for 12 weeks significantly reduced the percentage of circulating CD14(+)CD16(+) monocytes from 45.4 ± 25.2% to 38.3 ± 21.8% (P = 0.0144). In an in vitro study, the expression of CD16 mRNA by MNCs from 6 diabetic subjects was decreased after 24 hours of treatment with 10 µg/mL of bezafibrate (P < 0.05). Expression of IL-1ß mRNA by MNCs was also decreased after 24 hours of treatment with 10 µg/mL of bezafibrate, whereas the IL-1ß level in the culture supernatant was significantly decreased after treatment of MNCs with either 1 or 10 µg/mL of bezafibrate. In conclusion, bezafibrate decreased circulating CD14(+)CD16(+) monocytes in patients with type 2 diabetes, probably by inhibiting the expression of CD16 mRNA.

Spontaneous platelet aggregation evaluated by laser light scatter in patients with type 2 diabetes: effects of short-term improved glycemic control and adiponectin.

Spontaneous platelet aggregation (SPA) is enhanced in patients with type 2 diabetes. Adiponectin may inhibit platelet aggregation. The aims of the current study were to identify factors associated with in vitro SPA measured by a laser light scattering method and to investigate the effects of short-term glycemic control and adiponectin on SPA. In study 1, we investigated platelet aggregation in 20 healthy control subjects and 82 patients with type 2 diabetes. In study 2, we evaluated the changes of SPA and serum high-molecular-weight (HMW) adiponectin after 2 weeks of improved glycemic control in 20 hospitalized diabetic patients. In study 3, using washed platelets from 10 healthy subjects, in vitro SPA was measured over 15 min in the absence or presence of recombinant adiponectin (20 µg/mL). Platelet aggregation was assessed with a laser light scatter aggregometer that measured the size of platelet aggregates. SPA was defined as formation of small aggregates under constant stirring in the absence of any agonists. The area under the curve was calculated for SPA and also for agonist-induced small, medium, and large aggregates. SPA was increased in diabetic patients compared with control subjects. In diabetic patients, SPA was correlated positively with plasma fibrinogen, fasting plasma glucose, glycated albumin, and high-sensitivity C-reactive protein. A stepwise multivariate analysis showed that plasma fibrinogen was the strongest independent determinant of SPA in diabetic patients. In 20 diabetic patients, SPA decreased significantly after 2 weeks of glycemic control. A significant negative correlation was found between changes of SPA and those of HMW adiponectin during treatment. The in vitro study showed that adiponectin inhibited the spontaneous aggregation of washed platelets. In conclusion, hyperfibrinogenemia and hyperglycemia are associated independently with SPA in patients with type 2 diabetes. SPA is reduced after even short-term improvement of glycemic control and adiponectin also inhibits SPA directly.

Calcineurin-mediated BAD Ser155 dephosphorylation in ammonia-induced apoptosis of cultured rat hippocampal neurons.

We previously reported that ammonia induced apoptosis in cultured rat hippocampal neurons with moderate increases in the intracellular calcium concentration and decreases in phospho-BAD levels. Since this suggested the involvement of calcineurin in the apoptosis, the effects of calcineurin inhibitors, 1 microM cyclosporin A and 1 microM FK506, on the ammonia-induced neuronal apoptosis were tested. Both of the inhibitors abolished the neuronal apoptosis assessed by double staining with Hoechst 33258 and anti-neurofilament antibody, and the ammonia-induced decrease in phospho-BAD Ser(155) level. Thus, calcineurin appeared to be involved in the dephosphorylation of BAD at the sites including Ser(155) in ammonia-induced apoptosis.

GABAC receptor agonist suppressed ammonia-induced apoptosis in cultured rat hippocampal neurons by restoring phosphorylated BAD level.

Ammonia-induced apoptosis and its prevention by GABAC receptor stimulation were examined using primary cultured rat hippocampal neurons. Ammonia (0.5-5 mm NH4Cl) dose-dependently induced apoptosis in pyramidal cell-like neurons as assayed by double staining with Hoechst 33258 and anti-neurofilament antibody. A GABAC receptor agonist, cis-4-aminocrotonic acid (CACA, 200 microm), but not GABAA and GABAB receptor agonists, muscimol (10 micro m) and baclofen (50 microm), respectively, inhibited the ammonia (2 mm)-induced apoptosis, and this inhibition was abolished by a GABAC receptor antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA, 15 microm). Expression of all three GABAC receptor subunits was demonstrated in the cultured neurons by RT-PCR. The ammonia-treatment also activated caspases-3 and -9 as observed in immunocytochemistry for PARP p85 and western blot. Such activation of the caspases was again inhibited by CACA in a TPMPA-sensitive manner. The anti-apoptotic effect of CACA was blocked by inhibitors for MAP kinase kinase and cAMP-dependent protein kinase, PD98059 (20 microm) and KT5720 (1 microm), suggesting possible involvement of an upstream pro-apoptotic protein, BAD. Levels of phospho-BAD (Ser112 and Ser155) were decreased by the ammonia-treatment and restored by coadministration of CACA. These findings suggest that GABAC receptor stimulation protects hippocampal pyramidal neurons from ammonia-induced apoptosis by restoring Ser112- and Ser155-phospho-BAD levels.

Continuous administration of antisense oligonucleotides to c-fos reduced the development of seizure susceptibility after ethacrynic acid-induced seizure in mice.

We previously demonstrated that seizure susceptibility developed by the 14th day post-ethacrynic acid (EA)-induced seizure in mice, with a prolonged increase in the expression of c-fos mRNA in the brain during days 10-14. To examine whether such c-fos increase contributes to the development of seizure susceptibility, we administered antisense oligodeoxynucleotide to c-fos by continuous infusion into the lateral ventricle of mice that had shown a moderate stage of EA seizure, and evaluated the seizure susceptibility to kainic acid (10 mg/kg) on the 14th day. Antisense-infused mice displayed significant reduction of the c-Fos level in the hippocampus and cerebral cortex on the 7th and 14th days, and a significant decrease in seizure severity. These findings suggest that the prolonged increase in c-fos expression after EA seizure may lead to the development of seizure susceptibility.

Regulation of the expression of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium cells.

Lysyl oxidase, an extracellular amine oxidase, controls the maturation of collagen and elastin. We examined the regulation of lysyl oxidase mRNA in cultured rabbit retinal pigment epithelium (RPE) cells in relation to the changes in subretinal fluid transport and phenotype of RPE cells. The level of the mRNA in cells grown on microporous membranes was markedly increased by application of hyperosmotic mannitol solution on the apical side (191% of control), implying that RPE cells express more lysyl oxidase in the condition which may cause the accumulation of subretinal fluid. Platelet-derived growth factor increased the mRNA level in subconfluent cells in culture (137% of control) and basic fibroblast growth factor decreased it (79% of control). In addition, exposure of cells to retinoic acid alone or in combination with dibutyryl cAMP for 22 days markedly decreased the level of lysyl oxidase mRNA (52 or 35% of control) while increasing the level of mRNA of N-acetylglucosaminidase (NAG), a marker enzyme for lysosomes (162 or 142% of control). Moreover, the level of lysyl oxidase mRNA in cells grown on microporous membranes was lower than that in cells grown on plastic dishes, while the level of NAG mRNA in the former cells was higher than that in the latter. Taken together, the expression of lysyl oxidase seemed to increase during proliferation of RPE cells and decrease toward differentiation. beta-Aminopropionitrile, an inhibitor of lysyl oxidase, significantly inhibited the contraction of collagen gels by fetal calf serum, suggesting that lysyl oxidase may be involved in pathogenesis caused by RPE cells.