Junya Ito

Jacaric acid, a linolenic acid isomer with a conjugated triene system, has a strong antitumor effect in vitro and in vivo

In this study, we compared the cytotoxic effects of natural conjugated linolenic acids (CLnAs) on human adenocarcinoma cells (DLD-1) in vitro, with the goal of finding CLnA isomers with strong cytotoxic effects. The antitumor effect of the CLnA with the strongest cytotoxic effect was then examined in mice. The results showed that all CLnA isomers have strong cytotoxic effects on DLD-1 cells, with jacaric acid (JA) having the strongest effect. Examination of the mechanism of cell death showed that CLnAs induce apoptosis in DLD-1 cells via lipid peroxidation. The intracellular levels of incorporated CLnAs were measured to examine the reason for differences in cytotoxic effects. These results showed that JA was taken into cells efficiently. Collectively, these results suggest that the cytotoxic effect of CLnAs is dependent on intracellular incorporation and induction of apoptosis via lipid peroxidation. JA also had a strong preventive antitumor effect in vivo in nude mice into which DLD-1 cells were transplanted. These results suggest that JA can be used as a dietary constituent for prevention of cancer.

Pre-treatment of sperm reduces success of ICSI in the pig

In pigs, although ICSI is a feasible fertilization technique, its efficiency is low. In general, injected pig sperm are insufficient to induce oocyte activation and embryonic development. Pretreatments for disrupting sperm membranes have been applied to improve the fertility of ICSI oocytes; however, we hypothesize that such pretreatment(s) may reduce the ability of the sperm to induce oocyte activation. We first evaluated the effects of sperm pretreatments (sonication (SO) to isolate the sperm heads from the tails, Triton X-100 (TX), and three cycles of repeated freezing/thawing (3×-FT) for disrupting sperm membranes) on the rate of pronucleus (PN) formation after ICSI. We found that oocytes injected with control (whole) sperm had higher rates of PN formation than those obtained after subjecting the sperm to SO, TX, and 3×-FT. The amounts of phospholipase Cζ (PLCζ), which is thought to be the oocyte-activating factor in mammalian sperm, in sperm treated by each method was significantly lower than that in whole untreated sperm. Furthermore, using immunofluorescence, it was found that in pig sperm, PLCζ was localized to both the post-acrosomal region and the tail area. Thus we demonstrated for the first time that sperm pretreatment leads to a reduction of oocyte-activating capacity. Our data also show that in addition to its expected localization to the sperm head, PLCζ is also localized in the tail of pig sperm, thus raising the possibility that injection of whole sperm may be required to attain successful activation in pigs.

Continuous intake of a high-fat diet beyond one generation promotes lipid accumulation in liver and white adipose tissue of female mice

Lipid metabolism in a child may be altered when the mother has a high-fat diet (HFD), but it is unclear whether the lipid metabolism of future offspring (grandchildren) is also changed under these circumstances. In this study, we examined the influence of intake of an HFD beyond one generation on offspring in normal mice. Parent mice fed an HFD were bred and the resultant second and third generations were also fed an HFD. The diets used in the study had approximately 20% more energy than a standard chow diet. Changes in lipid metabolism were examined in each generation. Intake of an HFD from generation to generation promoted lipid accumulation in the white adipose tissue of female mice, increased lipid, glucose and insulin levels in the serum, increased the activities of enzymes associated with fatty acid metabolism in the liver, promoted lipid accumulation in hepatocytes and adipocytes and increased the mRNA levels of Cdkn1a in the liver and white adipose tissue. These results suggest that activation of Cdkn1a promoted lipid accumulation in the liver and white adipose tissue of third-generation female mice that were offspring from earlier generations fed HFDs. Moreover, intake of a high-energy diet beyond one generation led to offspring with obesity, fatty liver and hyperinsulinemia.

Increased Monocytic Adhesion by Senescence in Human Umbilical Vein Endothelial Cells

We investigated whether replicative senescence of endothelial cells contributed to the pathogenesis of atherosclerosis in human umbilical vein endothelial cells (HUVECs). HUVECs at a population-doubling level of 30 (PDL30) divided much more slowly than those at PDL9. The percentage of SA-β-Gal-positive cells and the mRNA expression levels of PAI-1 and p21 at PDL30 were significantly higher than those at PDL9. The changes induced by aging were evaluated according to the mRNA expression level of genes related to the endothelial cell function. The expression level of many adhesion molecules promoting monocytic adhesion was significantly increased, and monocytic adhesion on HUVECs was found to be significantly promoted by aging. Monocytic adhesion is an essential early event in the development of atherosclerosis, and our results suggest that replicative senescence of the vascular endothelial cells induced increased expression of adhesion molecules. The consequent increase in monocytic adhesion may then promote the pathogenesis of atherosclerosis.

jacaric acid, a linolenic acid isomer with a conjugated triene system, reduces stearoyl-CoA desaturase expression in liver of mice.

Conjugated fatty acid is a collective term used for fatty acids with conjugated double bond systems. Seed oils from certain plants include conjugated linolenic acids, which have a conjugated triene system and are geometrical and positional isomers of α-linolenic acid. One of these isomers, jacaric acid (JA, 8c, 10t, 12c-18:3), has not been examined widely. Therefore, we investigated the absorption and metabolism of JA in normal animals (ICR mice). An oral dose of JA of 5 mg/day for 1 week had no effects on body weight, food intake and tissue weight of mice. JA was detected in the serum, kidney, liver, lung and epididymal white adipose tissue. Analysis of the fatty acid composition in liver and white adipose tissue showed a tendency to increase levels of saturated fatty acids (SFAs) such as palmitic acid (16:0) and stearic acid (18:0) and to decrease levels of monounsaturated fatty acids (MUFAs) such as palmitoleic acid (16:1) and oleic acid (18:1). Thus, JA treatment decreased the desaturation index (16:1/16:0, 18:1/18:0) in liver and white adipose tissue. This index is used as an indicator of the activity of stearoyl coenzyme A desaturase (SCD), an endoplasmic reticulum enzyme that catalyzes the biosynthesis of MUFAs from SFAs. The change in this index indicates that JA inhibited SCD activity in ICR mice, and further experiments showed that JA also decreased the expression level of SCD-1 mRNA. Inhibition of SCD activity may have anti-obesity and anti-diabetes effects, and therefore the findings in this study suggest that JA may be effective for preventing obesity and diabetes.

Aging decreases antioxidant effects and increases lipid peroxidation in the Apolipoprotein E deficient mouse.

In this study, to study the effect of aging and Apolipoprotein E (ApoE) deficiency on antioxidant ability in mice, we examined whether lipid peroxidation is promoted by aging in ApoE deficient (ApoE−/−) mice, which have a shorter lifespan than normal mice. The levels of thiobarbituric acid-reactive substances (TBARS), a biomarker of lipid peroxidation, were measured in plasma and liver in ApoE−/− mice aged 12 weeks (young) and 52 weeks (early stage of senescence). TBARS in plasma and liver were significantly increased by aging. Next, we examined the reasons why lipid peroxidation was promoted by aging, based on measurement of protein and mRNA levels for antioxidant enzymes (superoxide dismutase, catalase, and glutathione peroxidase) in liver in ApoE−/− mice aged 12 and 52 weeks. The levels of superoxide dismutase 1 and 2 in liver were significantly decreased by aging. The mRNA level of catalase was also significantly decreased and the mRNA levels of superoxide dismutase 1, superoxide dismutase 2 and glutathione peroxidase 1 all showed a tendency to decrease with age. These results suggest that lipid peroxidation is caused by reduction of antioxidant activity with aging and that this promotes senescence and shortens lifespan in ApoE−/− mice.

The combination of maternal and offspring high-fat diets causes marked oxidative stress and development of metabolic syndrome in mouse offspring

Maternal overnutrition (e.g., high-fat (HF) diet) during pregnancy and lactation is believed to cause oxidative stress and increase the risk of metabolic syndrome in offspring. In the present study, we investigated the effects of both maternal and offspring HF diets on metabolic syndrome risk and oxidative stress profiles in mice. Dams of the C57BL/6J mouse strain were fed a HF or control (CO) diet during gestation and lactation. Offspring were weaned at 3weeks of age. The female offspring were sacrificed at weaning, while the males were maintained on the HF or CO diet until 11weeks of age. Tissue samples, including those from liver, were collected from offspring at 3 and 11weeks of age, and lipids, phosphatidylcholine hydroperoxide (PCOOH, an oxidative stress marker), and gene expression were evaluated. Accumulation of lipids, but not PCOOH, was found in the livers of 3-week-old offspring from dams fed the HF diet. When the offspring were maintained on a HF diet until 11weeks of age, marked accumulation of both liver lipids and PCOOH was observed. PCOOH manifestation was supported by the expression of genes such as Gpx4, encoding a PCOOH degrading enzyme. These results suggest that the combination of maternal and offspring overnutrition causes marked oxidative stress in offspring, which accelerates metabolic syndrome. The present findings in offspring from infancy to adulthood may be useful for better understanding the cause-and-effect relationships between oxidative stress and metabolic syndrome development.

Direct separation of the diastereomers of phosphatidylcholine hydroperoxide bearing 13-hydroperoxy-9Z,11E-octadecadienoic acid using chiral stationary phase high-performance liquid chromatography

Increasing evidence suggests that phospholipid peroxidation plays important roles in the pathogenesis of various diseases, such as atherosclerosis. With regard to the biochemical processes that initiate phospholipid peroxidation in vivo, enzymatic conversion of phosphatidylcholine to phosphatidylcholine hydroperoxide (PCOOH) by lipoxygenase (LOX) may play a crucial role. This will become clear if we can analyze PCOOH bearing hydroperoxy fatty acids with S-stereoconfiguration. In this study, we therefore attempted such an analysis. Initially, we used LOX, linoleic acid and Lyso phosphatidylcholine, and synthesized PCOOH bearing 13S-hydroperoxy-9Z,11E-octadecadienoic acid (13(S)-9Z,11E-HPODE). PCOOH bearing racemic 13-9Z,11E-HPODE was also prepared. We used liquid chromatography equipped with CHIRALPAK OP (+) (poly (o-pyridyl diphenylmethacrylate) coated on silica), a UV detector and a quadrupole-time-of-flight mass spectrometer, and achieved diastereomer separation of PCOOH stereoisomers with excellent resolution and peak shape. This is the first study reporting the diastereomer separation of PCOOH. The present method will be beneficial in developing a better understanding of the biochemical processes that initiate oxidative stress (PCOOH formation) in vivo, which may lead to further elucidation of the involvement of PCOOH in the development of diseases. In addition to clinical applications, the present method may also be effective in the evaluation of enzymatic oxidative food deterioration.

Tandem Mass Spectrometry Analysis of Linoleic and Arachidonic Acid Hydroperoxides via Promotion of Alkali Metal Adduct Formation

Recently, we demonstrated that tandem mass spectrometry (MS/MS) analysis in the presence of sodium ions was useful for identification of the position of the hydroperoxy group in phosphatidylcholine hydroperoxide (PCOOH). Likewise, MS/MS may enable identification of the hydroperoxy group position in various lipid hydroperoxides (LOOHs). To this end, we prepared major LOOHs, namely hydroperoxyoctadecadienoic acid (HPODE) and hydroperoxyeicosatetraenoic acid (HPETE), and analyzed them by quadrupole-time-of-flight MS/MS in both the absence and presence of alkali metals. Photo-oxidation (singlet oxygen-induced oxidation) of linoleic acid (LA) was used to prepare 9-10E,12Z-HPODE, 9-10E,12E-HPODE, 10-8E,12Z-HPODE, 12-9Z,13E-HPODE, 13-9Z,11E-HPODE, and 13-9E,11E-HPODE. Each isomer was analyzed under various MS/MS conditions (e.g., absence and presence of sodium). We found that in the presence of alkali metals, especially sodium, collision-induced dissociation (CID) of all HPODE isomers yielded structure-diagnostic fragment ions that were highly useful in identifying the position of the hydroperoxy group. For instance, CID spectra of sodiated 13-9Z,11E-HPODE revealed a neutral loss of 88 Da arising from fragmentation of the hydroperoxy group. Similar results were observed for HPETE isomers. Following oxidation of LA (or arachidonic acid) by lipoxygenase, the hydroperoxy group position of the resultant HPODE (or HPETE) was easily identified using this method, without any chromatographic separation processes. As information on the position of the hydroperoxy group provides insight into the processes that initiate lipid peroxidation (e.g., enzymatic oxidation, auto-oxidation and singlet oxygen-induced oxidation), the proposed method may be useful in elucidating the involvement and mechanism of lipid peroxidation in food deterioration and pathophysiological processes.

Atopic dermatitis causes lipid accumulation in the liver of NC/Nga mouse

Various factors have been reported to influence lipid metabolism and cause metabolic syndrome. However, the influence of allergy on the liver that plays important role of lipid metabolism has not been clarified. The aim of this study was to examine the influence of allergy on lipid metabolism of liver. A model of atopic dermatitis was developed in the NC/Nga mouse using picryl chloride to induce allergy. Lipid metabolism parameters were measured and the mechanism of changes in these parameters was examined using DNA microarray analysis and quantitative reverse transcriptase PCR. Triacylglycerol accumulation was promoted in the liver in the mouse atopic dermatitis model despite reductions in food intake, body weight gain, and serum glucose. As this mechanism, it was thought that atopic dermatitis caused the suppression of fatty acid β-oxidation. These results suggest that atopic dermatitis causes lipid accumulation in the liver.