Tsuyoshi Harada

Highly purified eicosapentaenoic acid prevents the progression of hepatic steatosis by repressing monounsaturated fatty acid synthesis in high-fat/high-sucrose diet-fed mice.

Eicosapentaenoic acid (EPA) is a member of the family of n-3 polyunsaturated fatty acids (PUFAs) that are clinically used to treat hypertriglyceridemia. The triglyceride (TG) lowering effect is likely due to an alteration in lipid metabolism in the liver, but details have not been fully elucidated. To assess the effects of EPA on hepatic TG metabolism, mice were fed a high-fat and high-sucrose diet (HFHSD) for 2 weeks and were given highly purified EPA ethyl ester (EPA-E) daily by gavage. The HFHSD diet increased the hepatic TG content and the composition of monounsaturated fatty acids (MUFAs). EPA significantly suppressed the hepatic TG content that was increased by the HFHSD diet. EPA also altered the composition of fatty acids by lowering the MUFAs C16:1 and C18:1 and increasing n-3 PUFAs, including EPA and docosahexaenoic acid (DHA). Linear regression analysis revealed that hepatic TG content was significantly correlated with the ratios of C16:1/C16:0, C18:1/C18:0, and MUFA/n-3 PUFA, but was not correlated with the n-6/n-3 PUFA ratio. EPA also decreased the hepatic mRNA expression and nuclear protein level of sterol regulatory element binding protein-1c (SREBP-1c). This was reflected in the levels of lipogenic genes, such as acetyl-CoA carboxylase alpha (ACCalpha), fatty acid synthase, stearoyl-CoA desaturase 1 (SCD1), and glycerol-3-phosphate acyltransferase (GPAT), which are regulated by SREBP-1c. In conclusion, oral administration of EPA-E ameliorates hepatic fat accumulation by suppressing TG synthesis enzymes regulated by SREBP-1 and decreases hepatic MUFAs accumulation by SCD1.

The protective effect of dietary eicosapentaenoic acid against impairment of spatial cognition learning ability in rats infused with amyloid β(1-40).

BACKGROUND Amyloid beta (Abeta) peptide (1-40) can cause cognitive impairment. EXPERIMENTAL DESIGN We investigated whether dietary preadministration of eicosapentaenoic acid (EPA) is conducive to cognition learning ability and whether it protects against the impairment of learning ability in rats infused with Abeta peptide (1-40) into the cerebral ventricle. RESULTS Dietary EPA administered to rats for 12 weeks before the infusion of Abeta into the rat brain significantly decreased the number of reference memory errors (RMEs) and working memory errors (WMEs), suggesting that chronic administration of EPA improves cognition learning ability in rats. EPA preadministered to the Abeta-infused rats significantly reduced the increase in the number of RMEs and WMEs, with concurrent proportional increases in the levels of corticohippocampal EPA and docosahexaenoic acid (DHA) and in the DHA/arachidonic acid molar ratio. Decrease in oxidative stress in these tissues was evaluated by determining the reactive oxygen species and lipid peroxide levels. cDNA microarray analysis revealed that altered genes included those that control synaptic signal transduction, cell communication, membrane-related vesicular transport functions, and enzymes and several other proteins. CONCLUSION The present study suggests that EPA, by acting as a precursor for DHA, ameliorates learning deficits associated with Alzheimers disease and that these effects are modulated by the expression of proteins involved in neuronal plasticity.

Effects of eicosapentaenoic acid on synaptic plasticity, fatty acid profile and phosphoinositide 3-kinase signaling in rat hippocampus and differentiated PC12 cells.

Placebo-controlled clinical studies suggest that intake of n-3 polyunsaturated fatty acids improves neurological disorders such as Alzheimers disease, Huntingtons disease and schizophrenia. To evaluate the impact of eicosapentaenoic acid (EPA), we orally administered highly purified ethyl EPA (EPA-E) to rats at a dose of 1.0 mg/g per day and measured long-term potentiation of the CA1 hippocampal region, a physiological correlate of synaptic plasticity that is thought to underlie learning and memory. The mean field excitatory postsynaptic potential slope of the EPA-E group was significantly greater than that of the control group in the CA1 region. Gene expression of hippocampal p85alpha, one of the regulatory subunits of phosphatidylinositol 3-kinase (PI3-kinase), was increased with EPA-E administration. Investigation of fatty acid profiles of neuronal and glia-enriched fractions demonstrated that a single administration of EPA-E significantly increased neuronal and glial EPA content and glial docosahexaenoic acid content, clearly suggesting that EPA was indeed taken up by both neurons and glial cells. In addition, we investigated the direct effects of EPA on the PI3-kinase/Akt pathway in differentiated PC12 cells. Phosphorylated-Akt expression was significantly increased in EPA-treated cells, and nerve growth factor withdrawal-induced increases in cell death and caspase-3 activity were suppressed by EPA treatment. These findings suggest that EPA protects against neurodegeneration by modulating synaptic plasticity and activating the PI3-kinase/Akt pathway, possibly by its own functional effects in neurons and glial cells and by its capacity to increase brain docosahexaenoic acid.

Eicosapentaenoic acid inhibits interleukin-6 production in interleukin-1β-stimulated C6 glioma cells through peroxisome proliferator-activated receptor-gamma

Epidemiological studies suggest that intake of omega-3 polyunsaturated fatty acids improves neurological disorders such as Alzheimers disease which exhibit inflammatory pathology. We therefore investigated the anti-inflammatory effects of eicosapentaenoic acid (EPA) on interleukin (IL)-1beta-stimulated C6 glioma cells. In the present study, EPA inhibited pro-inflammatory cytokine IL-6 production, a characteristic of certain neurodegenerative disorders, in IL-1beta-stimulated C6 glioma cells in dose-dependent fashion. EPA down-regulated the expression of IL-6 at mRNA level, indicating that the effect of EPA occurs at the transcriptional level. In addition, peroxisome proliferator-activated receptor (PPAR) gamma antagonists abolished the inhibitory effect of EPA on IL-1beta-induced IL-6 production, whereas PPARalpha antagonist did not block the inhibitory effect of EPA. EPA might thus contribute to the regulation of pro-inflammatory cytokine production in astrocytes through interaction with PPARgamma. Among the PPARgamma ligands tested in this study, ciglitazone, a synthetic agonist of PPARgamma, effectively inhibited IL-6 production, but while neither rosiglitazone nor 15-deoxy-Delta(12,14)-prostaglandin J2 did. These findings indicate that the coordination of PPAR gamma ligands is important in inhibiting the production of IL-6 in C6 glioma cells.

Changes in hematology, biochemical values, and restraint ECG of rhesus monkeys (Macaca mulatta) following 6‐month laboratory acclimation

Abstract:  The aim of this study was to determine if 6‐month acclimation would enable accurate evaluation of hematological, biochemical data, and ECG recorded under restraint for conscious rhesus monkeys of both sexes. Periodic evaluation of these parameters was made during the 6‐month period of acclimation. The platelet count, alkaline phosphatase, glucose, and sodium levels significantly decreased, whereas creatinine increased, compared with pre‐acclimation values. The heart rate was significantly reduced compared with pre‐acclimation values. QT–RR relation followed the square root regression function, which means modification of Bazetts QTc formula can be applied even if the ECG is recorded under restraint. In conclusion, 6‐month acclimation was effective for stabilizing the blood data and for allowing accurate evaluation of the ECG even under restraint. Current results show that an acclimation period at least 3 months may be necessary prior to using rhesus monkeys for chronic studies.

Use of a telemetry system to examine recovery of the cardiovascular system after excitement induced by handling stress in a conscious cynomolgus monkey (Macaca fascicularis).

Abstract:  The aim of this study was to determine the time required for the cardiovascular system of a conscious cynomolgus monkey, in which a telemetry had been implanted, to recover from excitement induced by handling stress. With enforcement of guidelines regarding safety pharmacological studies, cardiovascular studies in primates have become more important. However, as macaque monkeys are promptly excited under experimental procedures, it is often difficult to evaluate the drug effects on the cardiovascular system. Therefore, we tested monkey chair restraint and intravenous injection of saline. After monkey chair restraint and intravenous injection, approximately 30 minutes were required for recovery of both heart rate and blood pressure to their pre‐treatment level; however, ECG parameters such as PR, QRS interval, and QTc did not drastically change. Based on our current results and with sufficient consideration of autonomic nervous effects, accurate evaluation of drug effects on the cardiovascular system should now be possible.

Suppression of hepatic fat accumulation by highly purified eicosapentaenoic acid prevents the progression of D-galactosamine-induced hepatitis in mice fed with a high-fat/high-sucrose diet

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis. BALB/cA mice were fed with a standard diet (STD) or a high-fat and high-sucrose diet (HFHSD) for 14 days followed by intraperitoneal injection of d-galactosamine (DGalN) or vehicle. After 20-21 h, plasma and liver tissue were collected and analyzed. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in plasma were increased significantly in HFHSD-fed mice treated with DGalN compared to STD-fed mice treated with DGalN. This exacerbation by the HFHSD was also observed in the plasma soluble tumor necrosis factor receptor (sTNFR) levels, and hepatic levels of reactive oxygen species (ROS) and the fibrogenic gene expression, such as tissue inhibitor of matrix metalloproteinase 1 (TIMP-1), connective tissue growth factor (CTGF) and osteopontin (OPN) in HFHSD-fed mice treated with DGalN. The triglyceride contents of the liver were significantly increased by the HFHSD. When eicosapentaenoic acid (EPA), a suppressor of sterol regulatory element binding protein 1 (SREBP-1), was administered to HFHSD-fed mice, the sensitivity of DGalN, as a result of plasma ALT and AST levels, was suppressed accompanied by reduced plasma sTNFR2 level and hepatic levels of triglyceride, ROS, and fibrogenic parameters, and by increased plasma adiponectin levels. These data suggest that the progression of steatotic liver injury closely depends on the accumulation of fat in the liver and is prevented by EPA through the suppression of the fatty liver change.