Natsuko Mori

ANTI‐GLUCOCORTICOID EFFECTS OF MELATONIN IN YOUNG RATS

Administration of the pineal hormone melatonin to young female rats provided significant prevention of the injurious effects (decrease of body weight gain, atrophy of the thymus and adrenals, glucosuria, elevation of the blood level of glucose, free fatty acid, triglyceride, and total cholesterol) caused by three different glucocorticoids, i.e. dexamethasone, prednisolone, and hydrocortisone. Although these protective effects of melatonin were slightly more remarkable in dexamethasone‐treated rats than in prednisolone‐ or hydrocortisone‐treated rats, our hypothesis of melatonins anti‐glucocorticoid effects is said to have been confirmed rather universally and with a considerably wide range through this experiment.

Anti-hypercholesterolemic effect of melatonin in rats.

The effects on plasma lipids of daily intraperitoneal injections of 4mg of melatonin (N‐acetyl‐5‐methoxytrypt‐amine) for 10 27 day periods were examined biochemically and morphologically in rats fed regular and high‐cholesterol (1% cholesterol, 0.5% cholic acid) diets. Melatonin administration had no significant effect on plasma lipids and lipoproteins in the rats on a normal diet but blunted the effects of a high‐cholesterol diet on these parameters. No effects of melatonin on lipase activity were noted. Melatonin also diminished the fatty infiltration in the liver of animals on the high‐cholesterol diet. The high‐cholesterol diet produced major increases in VLDL and LDL cholesterol and protein content, and decreases in HDL cholesterol and protein. Melatonin decreased the extent of this plasma lipoprotein increase, although it did not completely prevent the phenomenon. Therefore, the effect is thought to be quantitative and not quantitative in nature. Acta Pathol Jpn 39: 613‐618, 1989.

Effects of human recombinant macrophage colony-stimulating factor on the secretion of lipoprotein lipase from macrophages.

The effects of human recombinant macrophage colony-stimulating factor (M-CSF) on the secretion of lipoprotein lipase were studied in rat alveolar macrophages. Five nanograms per milliliter M-CSF significantly enhanced lipoprotein lipase secretion (threefold), and the maximal effect (10-fold) of M-CSF on lipoprotein lipase secretion was observed at a dose of 200 ng/ml M-CSF. The effect of M-CSF was time dependent but was not manifested during the first 8 hours of incubation. After 24 hours, its effects were evident and dose dependent. On blot hybridization of macrophage RNAs with human cDNA of lipoprotein lipase, a remarkable and dose-dependent increase in mRNA level (7.3-fold) was found in M-CSF-treated alveolar macrophages. The secretion of lipoprotein lipase was also enhanced in human monocyte-derived macrophages (2.6-fold), whereas the secretion from either THP-1 cells, P388 cells, or J774 cells was not significantly enhanced. These results indicate that the stimulation of lipoprotein lipase secretion after M-CSF treatment was evident in rat alveolar macrophages and human monocyte-derived macrophages on the basis of both enzyme activity and mRNA level; therefore, M-CSF may be involved in lipoprotein metabolism of macrophages through modulation of the secretion of lipoprotein lipase.

Effects of melatonin on genetic hypercholesterolemia in rats

Administration of the pineal hormone melatonin to genetically hypercholesterolemic rats resulted in a decrease in plasma cholesterol levels and in an improvement of fatty changes of the liver. Thus, the antihyperlipemic effect of melatonin, which was first discovered in hypercholesterolemia produced by short- or long-term administration of glucocorticoids, has now been proved to be rather universal and not simply anti-glucocorticoidal. The mechanism of the decrease of plasma cholesterol levels remains unknown. It was also found that the pathogenesis of this so-called genetic hypercholesterolemia in rats involved biochemical nephrotic changes and histopathological changes in the kidney.

Uptake of 13-Hydroperoxylinoleic Acid by Cultured Cells

Oxidized free fatty acids have profound effects on cultured cells. However, little is known about whether these effects depend on their uptake and metabolism by cells or primarily involve their interaction with cell-surface components. We determined the uptake and metabolism of unoxidized (linoleic or oleic acid) and oxidized linoleic acid (13-hydroperoxyoctadecadienoic acid, 13-HPODE) by endothelial cells, smooth muscle cells, and macrophages. We show that 13-HPODE is poorly taken up by cells. The levels of uptake were dependent on the cell type but were independent of the expression of CD36. 13-HPODE was also poorly used by microsomal lysophosphatidylcholine acyltransferase that is involved in the formation of phosphatidylcholine. Based on these results, we suggest that most of the biological effects of 13-HPODE and other oxidized free fatty acids on cells might involve a direct interaction with cell-surface components. Alternatively, very small amounts of oxidized free fatty acids that enter the cell may have effects, analogous to those of hormones or prostanoids.

Plasma cholesterol-lowering activity of monocyte colony-stimulating factor (M-CSF).

We investigated the action of monocyte colony-stimulating factor (M-CSF) on plasma cholesterol metabolism. Recombinant human monocyte colony-stimulating factor (rhM-CSF) was intravenously injected into Watanabe heritable hyperlipidemic (WHHL) rabbits that were deficient in LDL receptor. The treated rabbits showed decreases in plasma total cholesterol levels from 493 +/- 39 to 416 +/- 45 mg/dl (about 15%) during the treatment. The decrease in total plasma cholesterol level was due to decreased levels of lipoproteins containing apo B 100 such as VLDL, IDL, and LDL. The effect of M-CSF on the LDL-receptor-deficient animal in vivo and evidence from Northern blot analysis of liver suggested that M-CSF lowers plasma cholesterol level through activated uptake of lipoproteins containing apo B 100 via pathways other than the hepatic LDL receptor. We have started a clinical study to evaluate the plasma cholesterol-lowering effect of M-CSF in patients with familial hypercholesterolemia, and have observed a decreased in total plasma cholesterol level in one of three patients treated with M-CSF. M-CSF may provide new insight into plasma cholesterol metabolism and a possible new tool to treat patients with hypercholesterolemia and atherosclerosis.

The enhanced cellular uptake of very-low-density lipoprotein enriched in apolipoprotein E

We have recently reported an increased clearance of plasma very-low-density lipoprotein (VLDL) after intravenous injection of apolipoprotein (apo) E in Watanabe heritable hyperlipidemic (WHHL) rabbits. In the present study, we have investigated the cellular uptake of VLDL enriched in apo E (VLDL-E) which had been incubated with purified rabbit apo E. VLDL-E was taken up approx. 2-fold more than VLDL in human skin fibroblast, human monocyte-derived macrophage and Hep G2 cell and its degradation was least in macrophage. To characterize the binding of VLDL-E, we performed a binding assay using hepatic endosome isolated from estradiol-treated rats and we observed both increased EDTA-sensitive and -resistant binding of VLDL-E on endosome. Ligand blotting of hepatic endosome demonstrated two major bands of LDL receptor (130 and 260 kDa protein) and a minor band of LDL receptor-related protein (580 kDa protein) with a ligand of VLDL-E. These results suggested that VLDL-E was endocytosed in liver through a similar pathway among three cell types, and enrichment of apo E in VLDL enhanced the uptake of VLDL not only via an EDTA-sensitive binding site (classical LDL receptor) but also via other binding sites including an EDTA-resistant binding site and an LDL receptor-related protein.

A newly identified null allelic mutation in the human lipoprotein lipase (LPL) gene of a compound heterozygote with familial LPL deficiency

In a Japanese patient with familial LPL deficiency, a new null allelic mutation, one base pair deletion at nucleotide position 916 was identified in exon 5 of one allele. In exon 3 of the other allele, we found the same nonsense mutation as we described previously in other Japanese kindreds. For the deletional mutant allele, we developed a simple detection method and constructed the DNA haplotype.

Immunohistochemical localization of apolipoprotein E in atherosclerotic lesions of the aorta and coronary arteries

The distribution of apolipoprotein E (apo E) immunoreactive substances (IRS) in atherosclerotic lesions and lesion-free areas of the aorta and coronary arteries obtained from 17 autopsied cases was studied using a specific anti-apo E serum and the unlabeled peroxidase-antiperoxidase (PAP) method. In fatty streak lesions of the aorta, many cells containing apo E-IRS were found in the deeper layer of the intima and diffuse staining of apo E in the extracellular spaces was also noted. In more advanced lesions apo E-positive cells could not be found. Immunohistochemical findings of coronary arteries differed distinctly from those of the aorta in that the apo-E-positive cells were absent in the deeper layer of the intima. The endothelial cells of coronary arteries, but not those of the aorta, showed positive staining for apo E.

Effect of tumor necrosis factor/cachectin on the activity of the low density lipoprotein receptor on human skin fibroblasts

We have investigated the effects of recombinant human tumor necrosis factor (TNF)/cachectin on the cellular binding of human low density lipoprotein (LDL) to human skin fibroblasts. When recombinant TNF was added to cultured cells, LDL binding doubled after 24 h of incubation. The effect of TNF was dose-dependent and its maximal effect was observed at concentrations of 1-10 ng/ml. TNF also stimulated the growth of human skin fibroblasts 1.6-fold. These results indicate that TNF increases LDL-receptor activity, which might be related to its stimulatory effect on cell growth.