Glycogen synthase kinase-3ß inhibition of 6-(methylsulfinyl)hexyl isothiocyanate derived from wasabi (Wasabia japonica Matsum).

A new biological activity of 6-(methylsulfinyl)hexyl isothiocyanate derived from Wasabia japonica was discovered as an inhibitor of glycogen synthase kinase-3ß. The most potent isothiocyanate, 9-(methylsulfinyl)hexyl isothiocyanate, inhibited glycogen synthase kinase-3ß at a K(i) value of 10.5 µM and showed ATP competitive inhibition. The structure-activity relationship revealed an inhibitory potency of methylsulfinyl isothiocyanate dependent on the alkyl chain length and the sulfoxide, sulfone, and/or the isothiocyanate moiety.

Measurement of the rate of myofibrillar protein degradation using the arteriovenous difference in plasma 3-methylhistidine concentration of rats.

There are several methods for measuring the rate of myofibrillar protein degradation using 3-methylhistidine (3-MeHis) levels in urine, plasma and isolated muscle. However, these methods lack the accuracy of rate measurements. Therefore, it is necessary to develop a new method for determining the rate of myofibrillar protein degradation. We characterized an arteriovenous difference method using 3-MeHis plasma concentration. Rats were fasted overnight and subsequently administered leucine (135 mg/100 g BW) or fed a 20% casein diet. The rate of myofibrillar protein degradation was calculated by multiplying the femoral artery blood flow rate by the arteriovenous difference in 3-MeHis concentrations (vein-artery). The blood was collected from the femoral vein and abdominal aorta. The release of 3-MeHis from hindlimb muscle was significantly suppressed (p<0.05) in rats fed leucine or the 20% casein diet, indicating that myofibrillar protein degradation was suppressed. These results suggest that the evaluation of the rate of myofibrillar protein degradation using the arteriovenous difference method reflects nutritional conditions in a more physiological manner.

Dietary Japanese millet protein ameliorates plasma levels of adiponectin, glucose, and lipids in type 2 diabetic mice.

Millet is an important food crop in Asia and Africa, but the health benefits of dietary millet are little known. This study defined the effects of dietary Japanese millet on diabetic mice. Feeding of a high-fat diet containing Japanese millet protein concentrate (JMP, 20% protein) to type 2 diabetic mice for 3 weeks significantly increased plasma levels of adiponectin and high-density lipoprotein cholesterol (HDL cholesterol) and decreased the levels of glucose and triglyceride as compared to control. The starch fraction of Japanese millet had no effect on glucose or adiponectin levels, but the prolamin fraction beneficially modulated plasma glucose and insulin concentrations as well as adiponectin and tumor necrosis factor-alpha gene expression. Considering the physiological significance of adiponectin and HDL cholesterol levels in type 2 diabetes, insulin resistance, and cardiovascular disease, our findings imply that dietary JMP has the potential to ameliorate these diseases.

Regulation of muscle protein degradation, not synthesis, by dietary leucine in rats fed a protein-deficient diet.

The aim of this study was to elucidate the effects of long-term intake of leucine in dietary protein malnutrition on muscle protein synthesis and degradation. A reduction in muscle mass was suppressed by leucine-supplementation (1.5% leucine) in rats fed protein-free diet for 7 days. Furthermore, the rate of muscle protein degradation was decreased without an increase in muscle protein synthesis. In addition, to elucidate the mechanism involved in the suppressive effect of leucine, we measured the activities of degradation systems in muscle. Proteinase activity (calpain and proteasome) and ubiquitin ligase mRNA (Atrogin-1 and MuRF1) expression were not suppressed in animals fed a leucine-supplemented diet, whereas the autophagy marker, protein light chain 3 active form (LC3-II), expression was significantly decreased. These results suggest that the protein-free diet supplemented with leucine suppresses muscle protein degradation through inhibition of autophagy rather than protein synthesis.

Effects of dietary Korean proso-millet protein on plasma adiponectin, HDL cholesterol, insulin levels, and gene expression in obese type 2 diabetic mice.

We investigated the effect of dietary Korean proso-millet protein concentrate (PMP) on glycemic responses, plasma lipid levels, and the plasma level and gene expression of adiponectin in obese type 2 diabetic mice under normal and high-fat feeding conditions. The findings were that the feeding of PMP clearly elevated plasma high-density lipoprotein cholesterol (HDL cholesterol) and adiponectin levels and brought about effective reduction in the levels of glucose and insulin in mice under high-fat diet conditions as compared with a control diet. Gene expression study revealed that the diet up-regulated expression of adiponectin and down-regulated tumor necrosis factor-alpha (TNF-alpha). Considering the central role of adiponectin and HDL cholesterol in improving and ameliorating type 2 diabetes, obesity, and cardiovascular disease, our findings imply that PMP may have potential for therapeutic intervention in type 2 diabetes.

Inhibitory activity of linoleic acid isolated from proso and Japanese millet toward histone deacetylase.

Linoleic acid was isolated from both the methanol extracts of proso and Japanese millet as a histone deacetylase inhibitor. It showed uncompetitive inhibitory activity toward histone deacetylase (IC(50)=0.51 mM) and potent cytotoxicity toward human leukemia K562 (IC(50)=68 microM) and prostate cancer LNCaP cells (IC(50)=193 microM). Millet containing linoleic acid might have anti-tumor activity.

Supplementation with dietary leucine to a protein-deficient diet suppresses myofibrillar protein degradation in rats.

Muscle mass is regulated by the synthesis and degradation of muscle protein, which in turn are affected by aging, several catabolic diseases, and malnutrition. Amino acids, particularly leucine, are known to stimulate muscle protein synthesis and suppress muscle protein degradation, although their long-term effects are unclear. The objective of our research was to elucidate whether long-term feeding of a protein-free or low-protein diet supplemented with leucine suppresses myofibrillar protein degradation. The rate of myofibrillar protein degradation was measured by the rate of release of 3-methylhistidine (MeHis) from isolated extensor digitorum longus (EDL) muscle. The weight of gastrocnemius muscle decreased in rats fed a protein-free diet for 7 d; however, a leucine-supplemented (1.5%) diet tended to suppress this decrease. The release of MeHis from EDL muscle was increased by the protein-free diet and decreased by the feeding of a diet supplemented with leucine to the level of a 20% casein diet. When rats were fed a 5% casein diet, the gastrocnemius muscle weight decreased and MeHis release from EDL muscle increased compared to those fed a 20% casein diet. However, feeding of a 5% casein diet supplemented with leucine (1.15%) reduced muscle weight loss and MeHis release. These results suggest that long-term feeding of leucine suppresses the rate of myofibrillar protein degradation and muscle weight loss in rats fed a protein-deficient diet.

Oxidative stress induces phosphoenolpyruvate carboxykinase expression in H4IIE cells.

Oxidative stress is closely associated with diabetes and is a major cause of insulin resistance. Impairment of hepatic insulin action is thought to be responsible for perturbations in hepatic glucose metabolism. In this study, we found that oxidative stress is involved in the dysregulation of gene expression of phosphoenolpyruvate carboxykinase (PEPCK), a key gluconeogenic enzyme, by a mechanism independent of insulin. Elevation of oxidative stress by injection of ferric nitrilotriacetate in rats increased the expression of hepatic PEPCK mRNA. To examine the direct action of oxidative stress on PEPCK expression, we treated H4IIE hepatoma cells with buthionine sulfoximine (BSO), an inhibitor of glutathione synthesis. BSO increased intracellular oxidative stress and the expression of PEPCK mRNA. Inhibition of p38 mitogen-activated protein kinase (p38 MAP kinase), which mediates responses to oxidative stress, suppressed the induction of PEPCK mRNA by BSO. These results suggest that oxidative stress dysregulates hepatic PEPCK expression by an insulin-independent mechanism.

Plasma HDL subfraction levels increase in rats fed proso-millet protein concentrate.

BACKGROUND: Millet has been consumed as human food in the countries of Asia and Africa. We reported previously the effects of dietary protein concentrates from proso millet (Panicum miliaceum L.) on plasma levels of high-density lipoprotein HDL cholesterol. Of note is that of these HDL subfractions, HDL2 particles may have the more strongly protective effect against the risk of coronary heart disease than HDL3. However, it is unclear how dietary millet protein affects plasma levels of HDL subfractions. MATERIAL/METHODS: We examined the effect of feeding of proso-millet protein concentrate (PMPC) for 21 days on plasma levels of HDL cholesterol, HDL subfractions and lecithin: cholesterol acyltransferase (LCAT) activities in rats. RESULTS: Results showed a clear elevation of plasma levels of HDL cholesterol (p<0.05) without an increase in low density lipoprotein cholesterol levels and enhancement of LCAT activities (p<0.06) by the intake of a PMPC diet compared with a casein diet. This increase in HDL cholesterol levels was substantially due to the elevation of HDL2 subfractions (p<0.05). CONCLUSIONS: PMPC could have a beneficial effect on cardiovascular disease because HDL2 subfractions may have the more strongly protective effect against the risk of coronary heart disease.

Effects of dietary protein of Korean foxtail millet on plasma adiponectin, HDL-cholesterol, and insulin levels in genetically type 2 diabetic mice.

We examined the effects of intake of Korean foxtail millet protein (FMP) on plasma levels of lipid, glucose, insulin, and adiponectin in genetically type 2 diabetic KK-Ay mice. When mice were fed a normal FMP diet or a high-fat-high-sucrose diet containing FMP for 3 weeks, in both experiments plasma concentrations of high-density lipoprotein cholesterol (HDL-cholesterol) and adiponectin increased remarkably in comparison with a casein diet group, whereas concentrations of insulin decreased greatly and that of plasma glucose was comparable to that in the casein diet group. Considering the role of adiponectin, insulin, and HDL-cholesterol in diabetes, atherosclerosis, and obesity, it appears likely that FMP may improve insulin sensitivity and cholesterol metabolism through an increase in adiponectin concentration. Therefore, FMP would serve as another beneficial food component in obesity-related diseases such as type 2 diabetes and cardiovascular diseases.

Suppression of myofibrillar protein degradation after refeeding in young and adult mice.

A diet containing adequate amounts of protein rapidly suppresses myofibrillar protein degradation after refeeding in young rats and mice. However, it is unclear whether this suppression is seen in adult animals. This study was undertaken to compare dietary protein-induced suppression of myofibrillar protein degradation in young and adult mice. Reductions in rates of myofibrillar protein degradation measured by N-methylhistidine (MeHis) released from the isolated extensor digitorum longus muscle were found at 4 to 7 h after refeeding in both young (7-wk-old) and adult (8-mo-old) mice, indicating that the response time of feeding-induced suppression of myofibrillar protein degradation was the same. When young (8-wk-old) mice were fed a 20% casein diet (20C) for 1 h after 18 h starvation, the rate of myofibrillar protein degradation was significantly decreased at 4 h after refeeding; however, mice fed a 10% casein diet (10C), 5% casein diet (5C), or protein-free diet (0C) did not show suppression of myofibrillar protein degradation. Adult (8-mo-old) mice fed 20C or 10C showed a reduction in the rate of MeHis release. The plasma concentration of leucine in young mice was only higher when they were fed 20C. Adult mice fed 20C or 10C showed higher plasma concentrations of leucine. These results suggest that postprandial suppression of myofibrillar protein degradation occurs in adult mice as in young mice, but the adult mice respond to a lower amount of dietary casein compared to the young mice.

Dietary G-rutin suppresses glycation in tissue proteins of streptozotocin-induced diabetic rats.

The present study focused on examining the efficacy of feeding a rutin-glucose derivative (G-rutin) to inhibit glycation reactions that can occur in muscle, kidney and plasma proteins of diabetic rats. Both thiobarbituric acid-reactive substance levels and protein carbonyl contents in muscle and kidney were significantly (p < 0.05) reduced in streptozotocin-induced diabetic rats fed G-rutin supplemented diet, compared to diabetic rats fed control diet. The N(epsilon)-fructoselysine content in muscle and kidney, a biomarker of early glycation reaction, was markedly (p < 0.05) increased by diabetes, but significantly (p < 0.05) reduced in diabetic rats fed G-rutin. Advanced glycation end-products (AGEs) in serum and kidney protein were measured by immunoblot using anti-AGE antibody, and were also reduced in diabetic rats fed dietary G-rutin. Feeding G-rutin also slightly inhibited aldose reductase activity in these animals. These results demonstrate for the first time that dietary G-rutin consumption can provide potential health benefits that are related to the inhibition of tissue glycation reactions common to diabetes.

Inhibition of glycation reaction in tissue protein incubations by water soluble rutin derivative.

In the Maillard reaction, nonenzymatic glycation reaction reversibly produces Amadori rearrangement products which subsequently lead to the formation of irreversible advanced glycation end-product (AGE). These reactions are important in the pathogenesis of complications associated with diabetes. This study examined the antioxidant activity of rutin and related efficacy to inhibit glycation in three distinct tissue protein sources. Rutin and the rutin analogue exhibited significant antioxidant activity in a liposomal model reaction similar to quercetin. Incubation of rat muscle and kidney proteins with 50 mM glucose for 5 days resulted in the generation of N(epsilon)-fructoselysine (FL), a biomarker for initial stage glycation. The addition of G-rutin, a water soluble glucose derivative of rutin, to the incubation medium (0.1 mM) reduced (p < 0.05) FL production. AGE content in both muscle and kidney proteins was also increased (p < 0.05) with the addition of glucose in the incubation mixture, but completely suppressed by the presence of G-rutin. On the contrary, inhibition of FL and AGE formation by G-rutin was found to be comparatively less effective in bovine serum albumin than both muscle and kidney proteins. These results demonstrate that the antioxidant activity of G-rutin corresponds to a strong affinity to suppress the formation of both initial and advanced stages of Maillard reaction in tissue protein sources.

Effects of dietary protein of proso millet on liver injury induced by D-galactosamine in rats.

In this paper, we examined the effects of dietary protein from proso millet on liver injury induced by D-galactosamine or carbon tetrachloride in rats using serum enzyme activities as indices. D-galactosamine-induced elevations of serum activities of aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase were significantly suppressed by feeding the diet containing 20% protein of proso millet for 14 days as compared with those of rats fed a 20% casein diet, but not in the case of carbon tetrachloride. The results showed that proso millet protein is effective at lower dietary protein levels than that of dietary gluten reported previously. Therefore, the findings reported here may suggest that proso millet protein is considered to be another preventive food for liver injury.

Rapid suppression of protein degradation in skeletal muscle after oral feeding of leucine in rats.

A diet containing adequate amounts of protein rapidly suppresses myofibrillar protein degradation in rats and mice. This study determined whether dietary amino acids inhibit postprandial protein degradation in rat skeletal muscle. When rats fed on a 20% casein diet for 1 h after 18 h starvation, the rate of myofibrillar protein degradation measured by N(tau)-methylhistidine release from the isolated extensor digitorum longus muscle was significantly (p < 0.05) decreased at 4 h after refeeding. A diet containing an amino acid mixture which is the same composition as casein also reduced myofibrillar protein degradation at 4 h after refeeding (p < 0.05). An essential amino acid mixture (15.1%, corresponding to casein composition) and a leucine (2.9%) diets reduced the rate of myofibrillar protein degradation after refeeding (p < 0.05), whereas a protein free diet did not. Administration of leucine alone (0.135 g/100 g body weight) by a feeding tube induced a decrease in the rate of myofibrillar protein degradation at 2 h after administration (p < 0.05), whereas the serum insulin concentration was constant after leucine administration. These results suggested that leucine is one of regulating factors of myofibrillar protein degradation after refeeding of a protein diet.