Fumie Hashimoto

Changes in polyamine-oxidizing capacity of peroxisomes under various physiological conditions in rats

Rat liver peroxisomal polyamine oxidase activity was determined under various physiological conditions by using the peroxidase method with phenol and 4-aminoantipyrine. N1-Acetylpolyamines such as N1-acetylspermine and N1-acetylspermidine were better substrates than the free polyamines. The polyamine oxidase activity in rat peroxisomes increased significantly when cell proliferation was high. The activity began to appear in fetal liver at the 16th approximately 18th day of pregnancy and peaked in neonatal liver on the first day (approx. 1.7-times higher than in adult liver). In regenerating rat liver, only polyamine oxidase activity among the peroxisomal enzymes tested was increased considerably 12 h after partial hepatectomy (approx. 2.8-fold over the control liver). Finally, the enzyme activity was significantly increased by administration of clofibrate, a peroxisome proliferator, which also causes hepatomegaly. In all cases, the increase in polyamine oxidase activity was not more than 3-fold. Since the level of polyamine oxidase activity in the normal liver is more than adequate in relation to the level of the substrates, the slight but significant increase under conditions of cell proliferation may have a role in modulating levels of polyamines in the proliferating liver tissue.

Significance of catalase in peroxisomal fatty acyl-CoA β-oxidation

Abstract Catalase activity was inhibited by aminotriazole administration to rats in order to evaluate the influence of catalase on the peroxisomal fatty acyl-CoA β-oxidation system. 2 h after the administration of aminotriazole, peroxisomes were prepared from rat liver, and the activities of catalase, the β-oxidation system and individual enzymes of β-oxidation (fatty acyl-CoA oxidase, crotonase, β-hydroxybutyryl-CoA dehydrogenase and thiolase) were determined. Catalase activity was decreased to about 2% of the control. Among the individual enzymes of the β-oxidation system, thiolase activity was decreased to 67%, but the activities of fatty acyl-CoA oxidase, crotonase and β-hydroxybutyryl-CoA dehydrogenase were almost unchanged. The activity of the peroxisomal β-oxidation system was assayed by measuring palmitoyl-CoA-dependent NADH formation, and the activity of the purified peroxisome preparation was found to be almost unaffected by the administration of aminotriazole. The activity of the system in the aminotriazole-treated preparation was, however, significantly decreased to 55% by addition of 0.1 mM H2O2 to the incubation mixture. Hydrogen peroxide (0.1 mM) reduced the thiolase activity of the aminotriazole-treated peroxisomes to approx. 40%, but did not affect the other activities of the system. Thiolase activity of the control preparation was decreased to 70% by addition of hydrogen peroxide (0.1 mM). The half-life of 0.1 mM H2O2 added to the thiolase assay mixture was 2.8 min in the case of aminotriazole-treated peroxisomes, and 4 s in control peroxisomes. The ultraviolet spectrum of acetoacetyl-CoA (substrate of thiolase) was clearly changed by addition of 0.1 mM H2O2 to the thiolase assay mixture without the enzyme preparation; the absorption bands at around 233 nm (possibly due to the thioester bond of acetoacetyl-CoA) and at around 303 nm (due to formation of the enolate ion) were both significantly decreased. These results suggest that H2O2 accumulated in peroxisomes after aminotriazole treatment may modify both thiolase and its substrate, and consequently suppress the fatty acyl-CoA β-oxidation. Therefore, catalase may protect thiolase and its substrate, 3-ketoacyl-CoA, by removing H2O2, which is abundantly produced during peroxisomal enzyme reactions.

Effect of gemfibrozil on lipid biosynthesis from acetyl-CoA derived from peroxisomal β-oxidation

The effect of gemfibrozil, a peroxisome proliferator, on lipid biosynthesis from acetyl-CoA derived from peroxisomal beta-oxidation was studied. The specific activity of the peroxisomal fatty acyl-CoA beta-oxidation system of rats fed a chow containing 0.2% gemfibrozil for 2 weeks was approximately five times higher than that of control rats. When [1-14C]lignoceric acid, a very-long-chain fatty acid which is degraded exclusively by the peroxisomal beta-oxidation system at first, was injected into rats treated with gemfibrozil, radioactivity and content of bile acid in the bile were enhanced to approximately 2.2 and 3.5 times the control, respectively. Gemfibrozil increased the radioactivity and content of chenodeoxycholic acid more than that of cholic acid. The incorporation of radioactivity into cholesterol in the bile was as much as 4.5 times greater than the control, and content was 2.6 times greater. In the liver, incorporation of [14C]lignoceric acid into the simple lipids phosphatidylethanolamine and phosphatidylcholine was unaffected by gemfibrozil. The radioactivity and content of cholesterol separated from the simple lipids were also virtually unaffected. However, the specific activities of 3-hydroxy-3-methylglutararyl-CoA reductase (rate-limiting enzyme of cholesterol synthesis) of peroxisomes and microsomes were remarkably stimulated by gemfibrozil treatment. These results suggest that biosyntheses of cholesterol and bile acid from acetyl-CoA derived from peroxisomal beta-oxidation are stimulated by gemfibrozil, due at least in part to activation of the peroxisomal beta-oxidation system and 3-hydroxy-3-methylglutaryl-CoA reductase of peroxisomes and/or microsomes. Most peroxisomal proliferators (e.g. clofibrate) have been known to inhibit 3-hydroxy-3-methylglutaryl-CoA reductase activity. Therefore, gemfibrozil is expected to be a very useful tool for elucidating the relationship between peroxisomes and the biosyntheses of cholesterol and bile acid.

Effect of liquid crystals with cyclodextrin on the bioavailability of a poorly water-soluble compound, diosgenin, after its oral administration to rats

Diosgenin, found in wild yam (Dioscorea villosa), has been shown to ameliorate diabetes and hyperlipidemia, increase cell proliferation in a human 3D skin model, and inhibits melanin production in B16 melanoma cells. It is also an active element in cosmeceutical and dietary supplements. Although the bioavailability of diosgenin is low due to its poor solubility and intestinal permeability, it was subsequently improved using a β-cyclodextrin (β-CD) inclusion complex. Recently liquid crystals (LCs) were shown to enhance the bioavailability of poorly water-soluble drugs. The purpose in the present study was to prepare diosgenin LCs and investigate the interaction between LC and β-CD in order to improve its bioavailability of diosgenin. Crystallinity and particle diameters of LCs in water were determined by small angle X-ray scattering (SAXS) and Zetasizer. Pharmacokinetic parameters were calculated using the plasma content of diosgenin after its oral administration to Wistar rats. Regarding the formation of glyceryl monooleate (GMO) and phytantriol (PHY) LC, SAXS patterns showed the hexagonal and cubic phases, respectively. Bioavailability was significantly enhanced after oral administration of LCs prepared by GMO than after diosgenin alone. The bioavailability was further improved with the combination of LC and β-CD than LC and water.

Effect of topically applied sphingomyelin-based liposomes on the ceramide level in a three-dimensional cultured human skin model

Sphingomyelin-based liposomes were prepared and applied to the stratum corneum side or basal layer side of a three-dimensional (3D) cultured human skin model, and the increase in the type II ceramide (ceramide II) content of the cultured skin model was evaluated. The sphingomyelin-based liposomes were prepared by a high-pressure emulsification method, and the obtained liposomes were characterized; the particle diameter and zeta potential of the liposomes were 155.3 nm and −11.4 mV, respectively. Their spherical shape and lamella structure were observed by transmission electron microscopy. The sphingomyelin-based liposomes or saline were applied to the cultured skin model, and ceramide II was extracted from the skin model. The extracted ceramide II was separated by high-performance thin-layer chromatography and quantified by a densitometer. The amount of ceramide II in the cultured skin model was significantly increased by the application of the sphingomyelin-based liposomes, compared with the nonapplication group. Thus, sphingomyelin-based liposomes are useful for enriching the ceramide level in 3D cultured skin models.

Effects of soybean peptide and collagen peptide on collagen synthesis in normal human dermal fibroblasts.

The collagen present in the dermis of the skin is a fibrous protein that fills the gaps between cells and helps maintain tissue flexibility. Effectively increasing the collagen present in the skin is an important goal for cosmetic research. Recent research has shown that soybean peptide (SP) has anti-fatigue activity, antioxidant activity, and the ability to increase type I collagen, while collagen peptide (CP) has the ability to enhance corneal moisture content and viscoelasticity, as well as to increase levels of hyaluronic acid synthesizing enzymes in human skin. Little documented research, however, has been conducted on collagen formation in relation to these peptides. Therefore, this research applied SP and CP with molecular weights primarily around 500 and preparations containing both SP and CP to normal human dermal fibroblasts together with magnesium ascorbyl phosphate (VC-PMg), and used real-time PCR to determine the gene expression of type I collagen (COL1A1), which contributes to collagen synthesis, and Smad7, which contribute to collagen breakdown. In addition, enzyme linked immuno sorbent assay (ELISA) was used to measure collagen content in the media. COL1A1 gene expression at 24 h after sample addition showed higher tendency in all samples and increased with time at 4, 8 and 24 h after addition. Smad7 gene expression was not substantially different at 4 h after addition. matrix metalloproteinase-1 gene expression was higher following SP addition, but was lower after the addition of CP and SP+CP. Medium collagen content was higher in all samples and increased with time at 8 h after addition. Collagen levels were higher when SP and CP were added together.

Effect of hyaluronan tetrasaccharides on epidermal differentiation in normal human epidermal keratinocytes

Hyaluronan (HA) plays a role in keratinocyte proliferation and differentiation. In addition, HA has been shown to have different biological activities depending on its molecular weight. It has been reported that HA‐mediated CD44 activation regulates keratinocyte differentiation. Therefore, the aim of this study was to investigate the influence of HA tetrasaccharides (HA4) on the regulation of keratinocyte differentiation, CD44 gene expression and CD44‐phosphorylated protein in human keratinocytes, and compare HA4 with high molecular weight HA.

Changes in isoprenoid lipid synthesis by gemfibrozil and clofibric acid in rat hepatocytes

We studied whether gemfibrozil and clofibric acid alter isoprenoid lipid synthesis in rat hepatocytes. After incubation of the cells with the agent for 74 hr, [(14)C]acetate or [(3)H]mevalonate was added, and the cells were further incubated for 4 hr. Gemfibrozil and clofibric acid increased ubiquinone synthesis from [(14)C]acetate and [(3)H]mevalonate. The effect of gemfibrozil was greater than that of clofibric acid. Also, gemfibrozil decreased dolichol synthesis from [(14)C]acetate and [(3)H]mevalonate. However, clofibric acid increased dolichol synthesis from [(3)H]mevalonate. Gemfibrozil decreased cholesterol synthesis from [(14)C]acetate and [(3)H]mevalonate. Clofibric acid decreased cholesterol synthesis from [(14)C]acetate, but did not affect synthesis from [(3)H]mevalonate. These results suggest that both agents, at different rates, activate the synthetic pathway of ubiquinone, at least from mevalonate. Gemfibrozil may inhibit the synthetic pathway of dolichol, at least from mevalonate. Contrary to gemfibrozil, clofibric acid may activate the synthetic pathway of dolichol from mevalonate. Gemfibrozil may inhibit the synthetic pathway of cholesterol from mevalonate in addition to the pathway from acetate to mevalonate inhibited by both agents.

Study on Membrane Fluidity of Liver Peroxisomes

The membrane fluidity of liver peroxisomes from normal and clofibrate-treated rats was investigated by using a fluorescence probe, 1-anilinonaphthalene-8-sulfonate (ANS). The excitation maximum of the probe was changed from 347 nm to 380 nm and the emission maximum from 493 nm to 463 nm in the presence of peroxisomes, and fluorescence intensity was enhanced more than 50 times. These results suggest that ANS was bound to the peroxisomal membrane. Fluorescence depolarization (P-value) was determined with such ANS- labeled peroxisomes. The P-values of peroxisomes from both normal and clofibrate-treated gradually decreased with increasing temperature, but that of clofibrate-treated peroxisomes was always smaller than that of normal peroxisomes. P-values were observed at 37° C for 60 min. The average P-value of normal peroxisomes was 0.270, while that of clofibrate-treated peroxisomes was 0.248. These results indicate that the membrane fluidity of liver peroxisomes is increased by the treatment with clofibrate. treatment of rats with another inducer of liver peroxisome proliferaton, di (2- ethylhexyl)phthalate, had an effect similar to that of clofibrate. However, alloxan treated (diabetic) rats showed no change of peroxisomal membrane fluidity.

Increase in Ceramide Level after Application of Various Sizes of Sphingomyelin Liposomes to a Cultured Human Skin Model

Sphingomyelin-based liposomes (SPM-L) that were sized (or not) by extrusion through a filter with pores of 100, 200, or 400 nm were applied to a three-dimensional cultured human skin model in order to evaluate which size of SPM-L was most effective at increasing its ceramide level. The diameters of the SPM-L in PBS were 102.7, 181.0, 224.0, and 380.1 nm. The diameters of the liposomes in the culture medium were 117.5, 199.2, 242.1, and 749.8 nm. The diameter of the small liposomes (<200 nm in diameter) did not change much, at least for 7 days. SPM-L in saline or culture medium were applied to the basal layer side or stratum corneum side of the cultured skin model, and ceramide II, III, V, and VI were then extracted from it. The extracted ceramide molecules were separated by HPTLC, and the concentration of each type of ceramide was quantified using a densitometer. When the small SPM-L (110 or 190 nm in diameter) were applied to the basal layer side, the levels of ceramide III and V were increased. When they were applied to the stratum corneum side, the levels of ceramide II, III, V, and VI were significantly increased compared to those of the PBS group, especially after the application of the small SPM-L (110 nm in diameter). Thus, the application of small SPM-L was useful for increasing the ceramide II, III, V, and VI levels of a cultured human skin model.