Relationships between the expression of hepatocyte nuclear factors and factors essential for lipoprotein production in a human mesenchymal stem cell line, UE7T-13.

To clarify the mechanisms regulating lipoprotein production by hepatocyte nuclear factors (HNFs), we generated four kinds of transfectants in human bone marrow mesenchymal stem cells: UE7T-13, stably expressing FOXA2 (also known as HNF3ß), HNF4α, HNF1α or co-expressing HNF4α, and HNF1α (HNF4α/HNF1α). In HNF4α/HNF1α transfectants, cellular contents of triglycerides (TG) and cholesterol were markedly higher than in UE7T-13 cells and comparable to those in human hepatoma HepG2 cells. However, TG and cholesterol, which are secreted from cells as components of lipoproteins, were hardly detected in the medium for any of the transfectants. ApoB100 and MTP, which are essential for the formation and secretion of lipoproteins, were undetectable and detected at low levels, respectively, in HNF4α/HNF1α transfectants. We suggest that enforced co-expression of HNF4α and HNF1α is effective for cellular lipid accumulation, while additional factors are probably required for lipoprotein formation and secretion.

Fermented barley extract supplementation ameliorates metabolic state in stroke-prone spontaneously hypertensive rats.

We studied the effects of fermented barley extract P (FBEP) in stroke-prone spontaneously hypertensive rats (SHRSP). Male 10-week-old SHRSP were divided into three groups that were fed: an AIN-93M diet (control), a low dose of FBEP (4 g/kg; FBEP1), and a high dose of FBEP (20 g/kg; FBEP2) for three weeks. Hypertension was significantly improved by the use of FBEP supplementation. The FBEP diet improved plasma triglyceride, insulin sensitivity, enhanced plasma catalase, and superoxide dismutase activities, and decreased plasma 8-hydroxy-2'-deoxyguanosine levels. In addition, the FBEP diet upregulated hepatic antioxidative genes and modulated Nrf2 protein levels in the liver. Furthermore, a single oral dose of FBEP (2 g/kg body weight) was able to lower blood pressure in SHRSP. In conclusion, our data suggest that increased expression of hepatic antioxidative genes and modulation of Nrf2 may play a role in the regulation of metabolic diseases in SHRSP consuming a FBEP diet.

In silico and in vivo experiment of soymilk peptide (tetrapeptide - FFYY) for the treatment of hypertension.

Enzyme-Treated Soymilk (ETS) was produced from Commercial Soymilk (CSM) with the treatment of proteinase PROTIN SD-NY10 (Bacillus amyloliquefaciens). Previously, we have isolated novel peptides from ETS but data related to isolated-peptides are scant. In this study, bio-informatics and in vivo analysis of isolated-peptides showed strong binding affinity to the active site of the Angiotensin Converting Enzyme (ACE). Among four peptides, tetrapeptide Phe-Phe-Tyr-Tyr (FFYY) showed strong binding affinity and inhibitory activity to the ACE-enzyme (binding affinity -9.5 Kcal/mol and inhibitory concentration of 1.9 µM respectively) as well as showed less toxicity compared to other peptides. The animal experiment revealed that single oral dose of FFYY (80 µg/kg body weight/day) effectively ameliorates the systolic, diastolic and mean blood pressure in the spontaneously hypertensive rat (SHR) model. Chronic oral administration of FFYY (80 µg/kg body weight/day for 3 weeks) reduced the systolic blood pressure elevation and ACE activity without any adverse side effects on the physiological and biological parameters of SHR. In conclusion, both in silico and in vivo experiments of soymilk-isolated FFYY peptide showed a promising option as a potential alternative for hypertension treatment without adverse side effects on SHR.

Lupeol supplementation improves blood pressure and lipid metabolism parameters in stroke-prone spontaneously hypertensive rats.

Supplementation with lupeol (0.67 g·kg(-1)) of the AIN-93M-based diet fed for 7 weeks to stroke-prone spontaneously hypertensive rats caused significantly decreased blood pressure as compared with a control group. Urinary 8-hydroxy-2'-deoxyguanosine was significantly lower in the lupeol group. Finally, lupeol suppressed the hepatic mRNA expression levels of the genes involved in triglyceride and cholesterol synthesis.

Chitinase from Autographa californica multiple nucleopolyhedrovirus: rapid purification from Sf-9 medium and mode of action.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) chitinase is involved in the final liquefaction of infected host larvae. We purified the chitinase rapidly to homogeneity from Sf-9 cells infected with AcMNPV by a simple procedure using a pepstatin-aminohexyl-Sepharose column. In past studies, a recombinant AcMNPV chitinase was found to exhibit both exo- and endo-chitinase activities by analysis using artificial substrates with a fluorescent probe. In this study, however, we obtained more accurate information on the mode of action of the chitinase by HPLC analysis of the enzymatic products using natural oligosaccharide and polysaccharide substrates. The AcMNPV chitinase hydrolyzed the second ß-1,4 glycosidic linkage from the non-reducing end of the chitin oligosaccharide substrates [(GlcNAc)(n), n=4, 5, and 6], producing the ß-anomer of (GlcNAc)2. The mode of action was similar to that of Serratia marcescens chitinase A (SmChiA), the amino acid sequence of which is 60.5% homologous to that of the AcMNPV enzyme. The enzyme also hydrolyzed solid ß-chitin, producing only (GlcNAc)2. The AcMNPV chitinase processively hydrolyzes solid ß-chitin in a manner similar to SmChiA. The processive mechanism of the enzyme appears to be advantageous in liquefaction of infected host larvae.

Suppression of murine preadipocyte differentiation and reduction of visceral fat accumulation by a Petasites japonicus ethanol extract in mice fed a high-fat diet.

We investigated in this study the anti-obesity effect of an extract of Petasites japonicus (a culinary vegetable from Eastern Asia) on a murine adipocyte cell line (3T3-L1) and on diet-induced obesity-prone mice. An ethanol extract of P. japonicus. (PJET) suppressed 3T3-L1 preadipocyte differentiation; however, a hot water extract of P. japonicus (PJHW) exhibited no effect on cell differentiation. PJET significantly attenuated three adipogenetic transcription factors, peroxisome proliferator-activated receptor gamma2, CCAAT/enhancer-binding protein and sterol regulatory element-binding protein 1C, at the mRNA level and suppressed the gene expression of fatty acid synthetase. An experiment with diet-induced obesity-prone C57BL/6J mice showed that PJET lowered the body weight gain and visceral fat tissue accumulation, and ameliorated the plasma cholesterol concentration. These findings suggest that P. japonicus might be an effective food against obesity.

Reduction of blood pressure by soybean saponins, renin inhibitors from soybean, in spontaneously hypertensive rats.

The effect of commercial purified soybean saponin on renin activity and blood pressure was investigated. Soybean saponin significantly inhibited human renin in vitro with IC(50)=59.9 µg/ml. Orally administered soybean saponin at 80 mg/kg of body weight per day to spontaneously hypertensive rats for 8 weeks significantly decreased the blood pressure.

Yamabushitake mushroom (Hericium erinaceus) improved lipid metabolism in mice fed a high-fat diet.

The effects of dietary Yamabushitake mushroom (Hericium erinaceus) on lipid metabolism were examined. C57BL/6J mice were fed a high-fat diet containing hot-water extract (HW-E) and an ethanol extract (EtOH-E) of Yamabushitake mushroom. Administration of HW-E or EtOH-E with a high-fat diet for 28 d resulted in a significant decrease in body weight gain, fat weight, and serum and hepatic triacylglycerol levels. Our in vitro experiments indicated that EtOH-E acts as an agonist of peroxisome proliferator-activated receptor alpha (PPARalpha). Quantitative analyses of hepatic mRNA levels revealed that EtOH-E administration resulted in up-regulation of mRNA for a number of PPARalpha-regulating genes in spite of the fact that the gene expression of PPARalpha did not change. These results suggest that EtOH-E improves lipid metabolism in mice fed a high-fat diet, and that these effects were mediated by modulation of lipid metabolic gene expression, at least in part via activation of PPARalpha.

Plasma lipoprotein profile from nonalcoholic steatohepatitis model rats.

We recently revealed that increases in particle sizes of very-low-density lipoproteins (VLDL) are highly correlated with the progression of nonalcoholic fatty liver disease (NAFLD)/nonalcoholic steatohepatitis (NASH), and VLDL particle size may be a minimally invasive indicator of these hepatic disorders. Methionine and choline-deficient (MCD) diet fed animals are usually used as a NASH model; however, the application of this minimally invasive biomarker in MCD diet fed animals remains unclear. In the present study, we measured the levels of liver disease markers and plasma lipoprotein profiles in MCD diet fed rats, and compared them with those of normal diet fed rats. Assessing lipoprotein profiles showed marked increases in VLDL particle sizes in MCD diet fed rats with pathologically and biochemically NASH-like features.

HPLC analysis of lipoproteins in culture medium of hepatoma cells: an in vitro system for screening antihyperlipidemic drugs.

We isolated a HepG2-derived sub-clone (HepG2-Lipo), which possessed an increased lipoprotein synthesizing ability. HepG2-Lipo cells could secrete triglycerides (TG) and cholesterol at rates 9.4- and 6-fold higher, respectively, when compared to HepG2 cells. Real-time RT-PCR analysis revealed that the expression levels of sterol regulatory element-binding protein-1c and -2 were 2.9- and 1.5-fold higher than in HepG2 cells. Furthermore, two apolipoprotein (apo) genes (apoA-1 and apoB-100) in HepG2-Lipo cells were expressed at 2.8- and 1.9-fold higher levels when compared to those in parental cells. We examined the effects of three antihyperlipidemic agents on the lipoprotein profiles of HepG2-Lipo cells. Simvastatin at 5 microM selectively suppressed cholesterol secretion from HepG2-Lipo cells, and 500 microM fenofibrate inhibited both TG and cholesterol secretion from the cells.

Isolation of human renin inhibitor from soybean: soyasaponin I is the novel human renin inhibitor in soybean.

We found human renin inhibitory activity in soybean and isolated the active compound, soybean renin inhibitor (SRI). The physico-chemical data on the isolated SRI were identical with those of soyasaponin I. SRI showed significant inhibition against recombinant human renin, with an IC(50) value of 30 microg/ml. Kinetic studies with SRI indicated partial noncompetitive inhibition, with a K(i) value of 37.5 microM. On the other hand, SRI weakly inhibited pepsin, papain, and bromeline activities, but did not inhibit other proteinases, such as trypsin, kallikrein, angiotensin converting enzyme, and aminopeptidase M. Moreover, a significant (p<0.05) decrease in the systolic blood pressure of spontaneously hypertensive rats was observed when partially purified SRI was orally administrated at 40 mg/kg/d for 7 weeks. This is the first demonstration of a renin inhibitor from soybean, soyasaponin I.

Effects of nucleotides on the interaction of renin with GlcNAc 2-epimerase (renin binding protein, RnBP).

Renin binding protein (RnBP), a cellular renin inhibitor, was identified as an enzyme, GlcNAc 2-epimerase. Recombinant RnBP inhibited porcine renin activity in a dose dependent manner. However, the inhibition was neutralized by nucleotides, such as ATP, dATP, dGTP, dCTP or dTTP. Moreover, ATP inhibited the formation of hetero-complex of renin with RnBP, called high molecular weight (HMW) renin. On the other hand, N-ethylmaleimide (NEM), a SH-alkylating reagent inhibited the GlcNAc 2-epimerase activity concomitant with the decaying of the dimer to the monomer of the enzyme. The inhibition was modulated in the presence of ATP. These results indicate that nucleotides stabilize the dimeric form RnBP (GlcNAc 2-epimerase) and inhibited the formation of the renin-RnBP hetero complex, HMW renin.

Paenidase, a novel D-aspartyl endopeptidase from Paenibacillus sp. B38: purification and substrate specificity.

We discovered and characterized a novel type D-aspartyl endopeptidase (DAEP) produced extracellularly by Paenibacillus sp. B38. This bacterial DAEP of M(r) 34,798 (named paenidase) appeared to be converted into a smaller form of M(r) 34,169 by the proteolytic removal of 5 amino acid residues from the N-terminal. The intact and modified forms of the enzyme displayed essentially the same enzymatic properties. The enzyme specifically hydrolyzed succinyl-D-aspartic acid alpha-(p-nitroanilide) and succinyl-D-aspartic acid alpha-(4-methylcoumaryl-7-amide) to generate p-nitroaniline and 7-amino-4-methylcoumarin, and internally cleaved a synthetic peptide (D-A-E-F-R-H-[D-Asp]-G-S-Y) of the [D-Asp](7) amyloid beta (Abeta) protein between [D-Asp](7)-G(8). Either was totally inert to the normal Abeta peptide sequence containing L-Asp, instead of D-Asp at the 7th position. Thus, paenidase is the first DAEP from a microorganism that specifically recognizes an internal D-Asp residue to cleave [D-Asp]-X peptide bonds.

Amino acid residues conferring the nucleotide binding properties of N-acetyl-D-glucosamine 2-epimerase (renin binding protein).

Our recent studies have demonstrated that the middle domain of N-acetyl-D-glucosamine (GlcNAc) 2-epimerase participates in the specificity for and binding of nucleotides. To identify the residue conferring nucleotide binding, amino acid substitutions were introduced in the human and rat GlcNAc 2-epimerases. The mutational analyses indicate that residue 171 of GlcNAc 2-epimerase is critical for the nucleotide binding of GlcNAc 2-epimerase.

Novel effect of adenosine 5'-monophosphate on ameliorating hypertension and the metabolism of lipids and glucose in stroke-prone spontaneously hypertensive rats.

The aim of the study was to investigate the effects of adenosine 5'-monophosphate (AMP) in stroke-prone spontaneously hypertensive rats (SHRSP). Male rats (10 weeks old) were divided into three groups: a control group fed an AIN-93 M diet and two others fed supplemental AMP (17.5 and 87.5 mg/kg diet) for 3 weeks. AMP effectively improved hypertension, plasma triglyceride, and HDL-cholesterol, glucose, kidney function parameters, hepatic lipid, enhances plasma nitric oxide, and plasma adiponectin accompanied by the up-regulation of mRNA expression levels of the hepatic adiponectin receptor 2. Single and chronic oral administration of AMP affected the hepatic mRNA expression levels of genes involved in ß-oxidation, fatty acid synthesis, and AMP-activated protein kinase. Furthermore, a single oral dose of AMP (40 mg/kg body weight) improved hypertension and hyperglycemia in SHRSP. In conclusion, AMP displays a novel effect in ameliorating metabolic-related diseases in SHRSP and could be beneficial as a functional food.

Vitamin K suppresses lipopolysaccharide-induced inflammation in the rat.

Vitamin K (K) is essential for blood coagulation and bone metabolism in mammals. K acts as a cofactor in the posttranslational synthesis of gamma-carboxyglutamic acid from glutamic acid residues. In addition to the liver and bone, K is found in the brain, heart, kidney and gonadal tissue. However, the physiological role of K in these various organs is not yet fully understood. It is likely that K has functions other than its role as a cofactor of protein gamma-glutamyl carboxylation. We used in this study the DNA microarray technique to identify the effect of K status on gene expression in the rat liver. The expression of genes involved in the acute inflammation response was enhanced in rats fed with a K-deficient diet relative to the control and K1-supplemented diet groups. Moreover, dietary supplementation with K1 suppressed the inflammation induced by lipopolysaccharide administration. These results indicate that orally administrated K1 suppressed inflammation in the rat.

Purification and characterization of a novel prolyl aminopeptidase from Maitake (Grifola frondosa).

We have found a novel prolyl aminopeptidase in Grifola frondosa. The enzyme was purified by DEAE-Sepharose CL-6B, Butyl-Toyopearl, Sephacryl S-100, and Mono-Q column chromatographies. The purified enzyme exists as a dimer and gives high activity toward L-proline-p-nitroanilide. The enzyme was strongly inhibited by p-chloromercuribenzoic acid and iodoacetic acid and markedly inhibited by phenylmethylsulfonyl fluoride and arphamenin A.