Naoto Koyama

Ketogenic Essential Amino Acids Modulate Lipid Synthetic Pathways and Prevent Hepatic Steatosis in Mice

Background Although dietary ketogenic essential amino acid (KAA) content modifies accumulation of hepatic lipids, the molecular interactions between KAAs and lipid metabolism are yet to be fully elucidated. Methodology/Principal Findings We designed a diet with a high ratio (E/N) of essential amino acids (EAAs) to non-EAAs by partially replacing dietary protein with 5 major free KAAs (Leu, Ile, Val, Lys and Thr) without altering carbohydrate and fat content. This high-KAA diet was assessed for its preventive effects on diet-induced hepatic steatosis and whole-animal insulin resistance. C57B6 mice were fed with a high-fat diet, and hyperinsulinemic ob/ob mice were fed with a high-fat or high-sucrose diet. The high-KAA diet improved hepatic steatosis with decreased de novo lipogensis (DNL) fluxes as well as reduced expressions of lipogenic genes. In C57B6 mice, the high-KAA diet lowered postprandial insulin secretion and improved glucose tolerance, in association with restored expression of muscle insulin signaling proteins repressed by the high-fat diet. Lipotoxic metabolites and their synthetic fluxes were also evaluated with reference to insulin resistance. The high-KAA diet lowered muscle and liver ceramides, both by reducing dietary lipid incorporation into muscular ceramides and preventing incorporation of DNL-derived fatty acids into hepatic ceramides. Conclusion Our results indicate that dietary KAA intake improves hepatic steatosis and insulin resistance by modulating lipid synthetic pathways.

Early nocturnal meal skipping alters the peripheral clock and increases lipogenesis in mice

BackgroundIn humans, skipping meals, especially breakfast, has been associated with obesity and other related syndromes. Recent studies in rodents suggest that fasting and feeding times are potential factors that affect the peripheral circadian clocks and metabolism. However, the link between fasting and obesity in rodents has yet to be fully demonstrated.MethodWe conducted early nocturnal fasting (ENF) from zeitgeber time (ZT) 12 to 18 for 4 consecutive days in C57B6 mice. The first set of experiments was performed under ad libitum conditions, where ENF and free-feeding (FF) control groups were compared. The second set was performed under isocaloric adjustment by restricting the diet to 90% of the basal intake of ENF mice. Calorie-restricted ENF (ENF-CR) mice were then compared with isocaloric controls (IC-control). Body weight, food intake, core body temperature, activity, adiposity, and clock-related gene expression levels in the liver and adipose tissues were investigated. A stable isotopic analysis was also conducted to estimate de novo lipogenesis fluxes.ResultsIn the ad libitum condition, the ENF mice ate more during the day, increased their overall daily food intake and gained more weight than FF-control mice. The amplitude of the body core temperature rhythm in ENF mice was also lower than in the FF-controls. Under isocaloric conditions, ENF-CR attenuated the CR-induced body weight loss, compared with the IC-control. ENF-CR also altered the acrophase time of the expression of the clock genes, which is associated with time-shift of genes involved in lipid metabolism and increased lipogenesis, compared with the IC-control.ConclusionsENF in nocturnal mice disturbs the peripheral clock and increases de novo lipid synthesis and results in a predisposition to obesity.

Effects of safflower seed extract supplementation on oxidation and cardiovascular risk markers in healthy human volunteers

We previously demonstrated that safflower seed extract (SSE) and its major antioxidant constituents, serotonin hydroxycinnamic acid amides, suppressed LDL oxidation in vitro, decreased plasma autoantibody titres to oxidized LDL and attenuated atherosclerotic lesion formation in apoE-deficient mice. In this report, we examined whether SSE, rich in serotonin derivatives, could affect markers of oxidative stress, inflammation and aortic stiffness in healthy human subjects. Twenty Japanese male volunteers were studied at baseline, after 2.1 g SSE supplementation daily (providing 290 mg serotonin derivatives/d) for 4 weeks, and after a 4-week washout period. Significant reductions in circulating oxidized LDL, autoantibody titres to malondialdehyde-modified LDL, the soluble form of vascular cell adhesion molecule-1 (sVCAM-1), and urinary 8-isoprostane were observed after a 4-week intervention. Although there were no statistically significant differences in blood pressure or brachial-ankle pulse wave velocity (baPWV), an index of arterial stiffness, baPWV was lower than baseline in eleven of twenty subjects and was accompanied by a reduction in blood pressure. Statistically significant negative correlations were observed between the extent of initial cardiovascular risk markers (autoantibody titres, 8-isoprostane, sVCAM-1 and baPWV) and the effect of intervention. This suggested that individuals with elevated oxidative stress, inflammation, and/or arterial stiffness may receive more benefit from SSE supplementation.

Safflower seed polyphenols ( N -( p -coumaroyl)serotonin and N -feruloylserotonin) ameliorate atherosclerosis and distensibility of the aortic wall in Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits

Pulse wave velocity (PWV) has been used clinically as a direct measure of arterial stiffness. We investigated the inhibitory effects of defatted safflower seed extract (SSE) and serotonin derivatives (N-(p-coumaroyl)serotonin, N-feruloylserotonin; CS+FS), which are the active components in SSE, on hypercholesterolemia and atherosclerosis, using PWV in Kurosawa and Kusanagi-hypercholesterolemic (KHC) rabbits. SSE and CS+FS were supplemented with a commercial diet containing 0.5% cholesterol for 8 weeks in male KHC rabbits, aged 2 months. Pulse waves were recorded at different aortic positions using two catheters with micromanometers under pentobarbital anesthesia. The atherosclerotic lesioned area in the aorta was significantly reduced in the SSE and CS+FS groups, without significant changes in serum cholesterol and triglyceride levels among the three groups after supplementation. Local PWV (LPWV) in the middle thoracic and distal abdominal aortas was significantly smaller in the SSE and CS+FS groups than in the control group. PWV in the entire aorta was also significantly lower in the SSE and CS+FS groups, compared with that in the control group. Pressure–strain elastic modulus, an index of wall distensibility, was significantly lower in the middle thoracic and middle abdominal aortas in the SSE and CS+FS groups than in the control group. Wall thickness was also significantly smaller in the middle thoracic aorta in the SSE and CS+FS groups compared with that in the control group. Serotonin derivatives inhibited the progress of atherosclerosis and ameliorated wall distensibility, which contributed, in part, to the lowering of LPWV. Serotonin derivatives may be beneficial in improving vascular distensibility and in reducing cardiovascular risk.

Effect of N-(p-coumaroyl)serotonin and N-feruloylserotonin, major anti-atherogenic polyphenols in safflower seed, on vasodilation, proliferation and migration of vascular smooth muscle cells

SCOPE The objective of this study is to investigate a vascular effect of N-(p-coumaroyl)serotonin (CS) and N-feruloylserotonin (FS), major antioxidative indolic polyphenols in safflower seeds with anti-atherogenic properties, with emphasis on effects on vascular smooth muscle cells (VSMCs). METHODS AND RESULTS Both CS and FS (each 10 to 100 μM) relaxed rat femoral arteries, which were pre-contracted by 10(-5) M phenylephrine or 50 mM KCl, independently of their endothelium. Both CS and FS also concentration-dependently inhibited the increase of cytosolic free Ca(2+) concentration ([Ca(2+) ](i) ) that was induced by KCl or 5-hydroxytryptamine in cultured rat VSMCs. Next, we examined the effects of CS and FS on platelet-derived growth factor (PDGF)-BB-evoked proliferation and migration of the VSMCs. Both CS and FS inhibited PDGF-BB-evoked proliferation and migration of the VSMCs in a concentration-dependent manner. They also inhibited PDGF-BB-induced phosphorylation of PDGF receptor β and ERK1/2, and Ca(2+) release from sarcoplasmic reticulum in the VSMCs in a concentration-dependent fashion. CONCLUSION These results indicated a possible vascular effect of CS/FS to inhibit the activation of VSMCs by blocking the increase of [Ca(2+) ](i) and/or blocking PDGF signaling. These may explain a part of anti-atherogenic mechanism that underlies their ability to improve vascular distensibility and to inhibit aortic hyperplasia.

Effects of safflower seed extract on arterial stiffness

Safflower seed extract (SSE) contains characteristic polyphenols and serotonin derivatives (N-( p-coumaroyl) serotonin and N-feruloylserotonin), which are reported to inhibit oxidation of low-density lipoprotein (LDL), formation of atherosclerotic plaques, and improve arterial stiffness as assessed by pulse wave analysis in animal models. The effects of long-term supplementation with SSE on arterial stiffness in human subjects were evaluated. This doubleblind, placebo-controlled study was conducted in 77 males (35–65 years) and 15 postmenopausal females (55–65 years) with high-normal blood pressure or mild hypertension who were not undergoing treatment. Subjects received SSE (70 mg/day as serotonin derivatives) or placebo for 12 weeks, and pulse wave measurements, ie, second derivative of photoplethysmogram (SDPTG), augmentation index, and brachial-ankle pulse wave velocity (baPWV) were conducted at baseline, and at weeks 4, 8, and 12. Vascular age estimated by SDPTG aging index improved in the SSE-supplemented group when compared with the placebo group at four (P = 0.0368) and 12 weeks (P = 0.0927). The trend of augmentation index reduction (P = 0.072 versus baseline) was observed in the SSE-supplemented group, but reduction of baPWV by SSE supplementation was not observed. The SSE-supplemented group also showed a trend towards a lower malondialdehyde-modified-LDL autoantibody titer at 12 weeks from baseline. These results suggest long-term ingestion of SSE in humans could help to improve arterial stiffness.

Enhancement of postoperative recovery by preoperative oral co-administration of the amino acids, cystine and theanine, in a mouse surgical model.

BACKGROUND & AIMS Glutathione (GSH) is important in the control of immune responses, and its levels decline following trauma. We previously reported that the oral administration of cystine/theanine (CT) increased GSH synthesis and that CT intake inhibited intense exercise-induced inflammation. Based on these results, we hypothesised that CT inhibits surgically induced inflammation and promotes postoperative recovery. Our aim was to confirm this hypothesis using a mouse surgical model. METHODS CT or a vehicle (V) was administered orally to mice once a day for 5 days, until the day of surgery. On the day of surgery, a sham operation or an intestinal manipulation was performed 2 h after the oral administration of CT or V. Levels of IL-6 in the blood and GSH in the intestine were analysed 2 h after surgery. Behavioural analysis was also undertaken after surgery. RESULTS Treatment with CT inhibited the manipulation-induced increase in IL-6 in the blood and decrease in GSH in the intestine. There was a significant negative correlation between IL-6 in the blood and GSH in the intestine. In addition, behavioural analysis revealed that CT administration improved locomotor activity and food intake after surgery. CONCLUSION These results suggest that CT suppresses inflammatory responses by inhibiting the surgically induced decrease in GSH in the small intestine and promotes postoperative recovery.

Effective prevention of sorafenib-induced hand–foot syndrome by dried-bonito broth

Background Sorafenib (SOR) is a molecular medicine that prolongs the survival of patients with hepatocellular carcinoma (HCC). Therefore, the management of side effects is essential for the longer period of continuous medication. Among the various side effects, hand–foot syndrome (HFS) is the most common, occurring in 30%–50% of patients, and often results in discontinuation of the SOR medication. However, its mechanism has not been clarified, and no effective prevention method has been reported for the symptoms. Therefore, this study aimed to analyze its mechanism and to develop an effective prevention regimen for the symptoms. Materials and methods To assess the mechanism of SOR-induced HFS, the peripheral blood flow in the hand and foot was carefully monitored by Doppler ultrasound, thermography, and laser speckle flowgraphy in the cases treated with SOR and its contribution was assessed. Then, the effect of dried-bonito broth (DBB), which was reported to improve peripheral blood flow, on the prevention of the symptom was examined by monitoring its occurrence and the peripheral blood flow. Results A total of 25 patients were enrolled in this study. In all, eight patients developed HFS, and all cases showed a significant decrease in the peripheral blood flow. DBB contributed to an increase in the flow (p = 0.009) and significantly decreased occurrence of HFS (p = 0.005) than control. Multivariable analysis showed that the ingestion of DBB is a significant independent contributor to HFS-free survival period (p = 0.035). Conclusion The mechanism of SOR-induced HFS involves a decrease in the peripheral blood flow, and the ingestion of DBB effectively prevents the development of the syndrome by maintaining the flow.

Effect of histidine on Sorafenib-induced vascular damage: Analysis using novel medaka fish model

BACKGROUND Sorafenib (SFN) is an anti-angiogenic chemotherapeutic that prolongs survival of patients with hepatocellular carcinoma (HCC); its side effects, including vascular damages such as hand-foot syndrome (HFS), are a major cause of therapy discontinuation. We previously reported that maintenance of peripheral blood flow by intake of dried bonito broth (DBB) significantly prevented HFS and prolonged the administration period. The amino acids contained in DBB probably contribute to its effects, but the mechanism has not been clarified. We hypothesized that histidine, the largest component among the amino acids contained in DBB, has effects on SFN-induced vascular damage, and evaluated this possibility using a novel medaka fish model. METHODS The fli::GFP transgenic medaka fish model has a fluorescently visible systemic vasculature. We fed the fish with SFN with and without histidine to compare blood flow and vascular structure among the differently fed models. The vascular cross-sectional area of each fish was measured to determine vascular diameter changes. RESULTS Our results demonstrated that SFN-fed medaka developed a narrower vascular diameter. In addition, this narrowing was counteracted by addition of histidine to the medaka diet. We observed no positive effect of histidine on regeneration of cut vessels or on cell growth of endothelial cells and HCC cell lines. CONCLUSION We proved the efficacy of the medaka model to assess vascular changes after administration of specific chemicals. And our results suggest that SFN causes vascular damage by narrowing peripheral vessel diameter, and that histidine effectively counteracts these changes to maintain blood flow.

EDF (Erythroid Differentiation Factor = Activin A) Production in Recombinant CHO Cells

Serum-free growth and product formation by CHO/EDF (recombinant CHO cell line expressing human Erythroid Differentiation Factor=activin A)were studied. ASF-104, an autoclavable serum-free medium, was modified to give ASF-CHO, in which recombinant CHO cells showed the same growth kinetics as shown in serum supplemented medium. For evaluation of the feasibility of large-scale performance in this medium, cells were propagated in a microcarrier system and conventional suspension system. To achieve cell attachment to microcarriers without serum, pre-treatment of beads with fibronectin was found to be effective. For suspension culture, we also established useful one-step selection method to obtain anchorage-, and serum-independent sub-line of CHO/EDF. It was shown that subpopulation of the cell line could be divided into 2 groups, compact-type and diffuse-type colony forming clones. The latter clones were considered to be more adaptable to suspension culture. As ASF-CHO was found to support clonal growth of CHO cells in soft agar, an anchorage-, and serum-independent sub-clone, termed C11-5, was isolated by selecting a diffuse-type colony-forming clone in ASF-CHO soft agar. C11-5 showed the same growth rate and productivity in serum-free suspension culture as shown in serum-supplemented monolayer culture.