Shigeki Nakagawa

Sterol and Triterpene Derivatives from Plants Inhibit the Effects of a Tumor Promoter, and Sitosterol and Betulinic Acid Inhibit Tumor Formation in Mouse Skin Two-Stage Carcinogenesis

A single topical application of 1 microgram of 12-O-tetradecanoylphorbol- 13-acetate (TPA) to the ears of mice was shown to induce edema, and this TPA-induced inflammation was inhibited by 4-methylsterol and triterpene derivatives. The ED50 of these compounds against TPA-induced inflammation was 0.1-3 mumol. Phytosterols had only slight inhibitory effects. Furthermore, application of 5 micrograms TPA to mouse skin rapidly caused accumulation of ornithine decarboxylase (ODC). Similarly, sitosterol and lupane-type triterpene derivatives markedly inhibited this TPA-induced ODC accumulation. In addition, 5 mumol betulinic acid markedly inhibited the promoting effect of 2.5 micrograms TPA applied twice weekly on skin tumor formation in mice initiated with 50 micrograms of 7,12-dimethylbenz[a]anthracene, and 5 mumol of sitosterol caused slight suppression. Thus, the inhibitory effects of sterol and triterpene derivatives on TPA-induced inflammation roughly parallelled their inhibitory activities against tumor promotion.

Long‐term melatonin administration reduces hyperinsulinemia and improves the altered fatty‐acid compositions in type 2 diabetic rats via the restoration of Δ‐5 desaturase activity

The objective of this study was to investigate the effect of long‐term melatonin administration on plasma levels of triglycerides, insulin and leptin, and on the fatty‐acid metabolism of plasma and hepatic lipids in type 2 diabetic rats. Otsuka Long‐Evans Tokushima Fatty (OLETF) rats, an animal model of type 2 diabetes mellitus, were divided into two groups: one untreated (n=6), and one implanted with time‐releasing melatonin pellets (1.1 mg/day for 30 wk) under the abdominal skin (n=6). Age‐matched Long‐Evans Tokushima Otsuka (LETO) rats (n=6) were used as healthy controls. The untreated diabetic rats had the increased plasma levels of triglycerides, cholesterol, insulin and leptin at 35 wk, as compared with the healthy control rats (n=6). The diabetic rats also had augmented ratios of 20:3n‐6/20:4n‐6 fatty acids, owing to diminished activity of Δ‐5 desaturase, an insulin‐permissive enzyme, in the liver. Melatonin administration to OLETF rats reduced the hypertriglyceridemia (−39%, P < 0.05), hyperinsulinemia (−33%, P < 0.01) and hyperleptinemia (–43%, P < 0.01), and restored hepatic Δ‐5 desaturase activity (148%, P < 0.005). This resulted in a return to normal ratios of 20:3n‐6/20:4n‐6 fatty acids in plasma and hepatic lipids. There was a significant correlation (r=0.64, P < 0.005) between plasma levels of insulin and the ratios of 20:3n‐6/20:4n‐6 in plasma phospholipids of all rats in the three groups. Thus, subcutaneous implantation of a melatonin‐releasing pellet thus resulted in improved lipid metabolism in diabetic rats, probably through restored insulin resistance.ALP, alkaline phosphataseGOT, glutamic oxalacetic transaminaseGPT, glutamic pyruvic transaminaseHPLC, high performance liquid chromatographyLETO rats, Long‐Evans Tokushima Otsuka ratsMUFA, monounsaturated fatty acidOLETF rats, Otsuka Long‐Evans Tokushima Fatty ratsPUFA, polyunsaturated fatty acidVLDL, very low density lipoprotein

Effect of pinealectomy on plasma levels of insulin and leptin and on hepatic lipids in type 2 diabetic rats

Abstract: We previously reported that pharmacological melatonin administration to type 2 diabetic rats reduces hyperinsulinemia and improves the altered fatty‐acid metabolism. To determine whether melatonin deficiency exacerbates diabetes‐associated conditions, we investigated the effect of pinealectomy (i.e. melatonin‐deficiency) on plasma hormone levels and lipid metabolism in type 2 diabetic Otsuka Long‐Evans Tokushima Fatty (OLETF) rats. We compared levels of insulin and leptin, and hepatic lipids in pinealectomized OLETF (PO) rats, sham‐operated OLETF (SO) rats and sham‐operated healthy Long‐Evans Tokushima Otsuka (LETO) (SL) rats 16 and 30 wk after the operation. Plasma glucose and triglycerides were increased in SO and PO rats 30 wk after operation compared with age‐matched SL rats. Pinealectomy caused an increase in free cholesterol among the plasma lipids, as compared with SO rats. Sixteen weeks after pinealectomy, typical hyperinsulinemia was observed in PO rats (3.47‐fold increase, P < 0.01) as compared with SL rats, whereas at 30 wk, the plasma levels of insulin in PO and SO rats had decreased and there was no significant difference among the three groups. Hepatic triglycerides were increased (1.54‐fold, P < 0.005) in PO rats, compared with SO rats. Hepatic acyl‐CoA synthetase (ACS) activity was significantly augmented in PO rats at 30 wk (10%, P < 0.01 versus SO group), while microsomal triglyceride transfer protein (MTP) decreased (−27% versus SO, P < 0.05); thus, the increased ACS activity and decreased MTP might have a role in the accumulation of hepatic triglycerides in PO rats. In summary, pinealectomy causes severe hyperinsulinemia and accumulation of triglycerides in the liver, probably owing to the loss of the nocturnal melatonin surge.

Gomisin A Inhibits Tumor Promotion by 12-O-Tetra-decanoylphorbol-13-Acetate in Two-Stage Carcinogenesis in Mouse Skin

Gomisin A, isolated from the fruits of Schisandra chinensis, is one of the dibenzocyclooctadiene lignans. Application of 12-O-tetradecanoylphorbol-13-acetate (TPA, 1 microgram/ear), a tumor-promoting agent, to the ears of mice induces inflammation. Among seven dibenzocyclooctadiene lignans assayed, gomisin A, gomisin J, and wuweizisu C inhibited the inflammatory activity induced by TPA in mice. The ED50 of these compounds for TPA-induced inflammation was 1.4-4.4 mumol. Gomisin A, with an ED50 of 1.4 mumol, showed the strongest inhibitory effect. Furthermore, at 5 mumol/mouse, it markedly suppressed the promotion effect of TPA (2.5 micrograms/mouse) on skin tumor formation in mice following initiation with 7,12-dimethylbenz[a]anthracene (50 micrograms/mouse). It is assumed that the inhibition of tumor promotion by gomisin A is due to its anti-inflammatory activity.

Brain natriuretic peptide is cosecreted with atrial natriuretic peptide from porcine cardiocytes

Using primary cultures of atrial cardiocytes from neonatal pig, the secretion of brain natriuretic peptide (BNP) and atrial natriuretic peptide (ANP)‐like immunoreactivities (LI) was studied in vitro. Porcine cardiocytes time‐dependently secreted both BNP‐LI and ANP‐LI into medium under a serum‐free condition, although the amount of BNP‐LI secreted was about one‐third that of ANP‐LI. Phorbol ester and Ca2+ ionophore had less stimulatory effects on secretion of BNP‐LI than that of ANP‐LI. Reverse‐phase HPLC of the conditioned medium revealed a single major BNP‐LI component corresponding to synthetic porcine BNP(1‐26). These data suggest that a small molecular weight form BNP, possibly BNP(1‐26), is cosecreted with ANP from porcine cardiocytes.

ALPHA -TOCOPHEROL PROTECTS PC12 CELLS FROM HYPEROXIA-INDUCED APOPTOSIS

A rat clonal pheochromocytoma cell line (PC12) was cultured under normoxic (21% O2) and hyperoxic (50% O2) conditions. PC12 cells underwent apoptotic cell death when they were cultured in charcoal‐stripped medium in a high‐oxygen atmosphere. Vitamin E homologs, α‐tocopherol (αT), β‐tocopherol (βT), γ‐tocopherol (γT), and δ‐tocopherol (δT), were added to the culture medium to study their biological activities. αT was more effective than γT and δT in preventing hyperoxia‐induced cell death. Addition of exogenous αT to charcoal‐treated medium prevented lactate dehydrogenase (LDH) leakage from PC12 cells and also inhibited the apoptosis, which was accompanied by DNA fragmentation. Additional αT was rapidly concentrated in PC12 cells, suggesting that it exerts antioxidant effects. Our data show that PC12 cell death under high‐oxygen conditions is due to apoptosis and that, among the vitamin E homologs, αT most effectively prevents hyperoxic apoptosis. J. Neurosci. Res. 52:184–191, 1998.

Elevated SCF levels in the serum of patients with chronic renal failure.

Serum stem cell factor (SCF) and soluble KIT (sKIT) levels were estimated in patients with chronic renal failure (CRF) and anaemia, and compared with clinical parameters of blood cells and renal function. Serum SCF levels in CRF patients were 5‐fold higher than those in healthy controls. However, serum sKIT levels in haemodialysis (HD)‐CRF patients were only slightly higher than those of healthy controls. In untreated CRF patients and healthy controls, serum SCF levels were significantly correlated with blood urea nitrogen (BUN), creatinine, haemoglobin, red blood cell (RBC) count and sKIT. In untreated CRF patients, serum SCF levels were significantly correlated with BUN, creatinine, and sKIT. These results suggest that serum SCF levels increased with the deterioration of renal function and might be related to erythropoiesis.

Expression of nerve growth factor-induced type 1 plasminogen activator inhibitor (PAI-1) mRNA is inhibited by genistein and wortmannin.

Nerve growth factor (NGF), which acts as a neurotrophic factor in a rat pheochromocytoma cell line (PC12), stimulated type 1 plasminogen activator inhibitor (PAI-1) mRNA expression from 1 to 5 h, after addition at 5 ng/ml. PAI-1 antigen in culture medium, which was measured using an enzyme linked immunosorbent assay (ELISA), was also increased dose dependently by the addition of NGF. Neither epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), phorbol myristate acetate (PMA) nor forskolin increased PAI-1 mRNA expression in PC12 cells. Genistein, an inhibitor of tyrosine protein kinase, completely inhibited NGF induced PAI-1 mRNA in the presence of 100 μM. Wortmannin, a potent and specific inhibitor of phosphatidylinositol 3-kinase (PI-3 kinase), decreased induction of PAI-1 mRNA level at doses of ≥ 10-7 M.

The effects of lidocaine on superoxide production and p47 Phox translocation in opsonized zymosan-activated neutrophils.

Superoxide is generated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that exists in the cell membrane of neutrophils and contains p47 phox in the enzyme complex. Superoxide is closely related to various diseases, including infection, myocardial infarction, and aspiration pneumonia. Therefore we performed this study to establish how lidocaine acts on NADPH oxidase. We examined the effects of lidocaine on superoxide production in neutrophils using the method of 2-methyl-6-phenyl-3,7-dihydro [1,2-a] pyrazin-3-one (CLA phenyl)-dependent chemiluminescence activated by opsonized zymosan (OZ). Treatment with lidocaine 15 &mgr;M suppressed OZ-activated CLA phenyl-dependent chemiluminescence by approximately 40% compared with that of OZ only-activated neutrophils. It was further demonstrated that the suppressive effect of lidocaine on superoxide production was dependent on the concentration of lidocaine. Translocation of p47 phox from the cytosol to the cell membrane was studied using immunoblot analysis with a polyclonal anti-p47 phox antibody. Lidocaine suppressed OZ-induced p47 phox translocation in a dose-dependent manner. Our results suggest that suppression of superoxide production by a therapeutic dose of lidocaine correlates strongly with suppression of p47 phox translocation.

Measurement of KIT ligand/stem cell factor: clinical and biochemical significance.

Stem cell factor (SCF) and its receptor KIT play an important role in various biologic phases, such as hematopoiesis, reproduction, and regeneration. It has been possible to measure both soluble SCF and soluble KIT using enzyme-linked immunosorbent assay since 1993 and 1995, respectively. Although the significance of interaction of soluble SCF with soluble KIT has not yet been elucidated, in certain diseases proteins fluctuate in human sera. We found that serum SCF levels were fivefold higher in patients with chronic renal failure than levels in healthy controls. We review the results of the analysis of SCF. In addition, possible pathologic mechanisms in various clinical abnormalities and the clinical potential for recombinant human SCF are discussed.