Tatsuya Moriyama

Soybean β-Conglycinin Diet Suppresses Serum Triglyceride Levels in Normal and Genetically Obese Mice by Induction of β-Oxidation, Downregulation of Fatty Acid Synthase, and Inhibition of Triglyceride Absorption

The purpose of this study was to discover the effects of soybean β-conglycinin (7S-globulin) and glycinin (11S-globulin) on serum lipid levels and metabolism in the livers of normal and genetically obese mice. Male normal (ICR) and obese (KK-Ay) mice were fed ad libitum high fat diets for two weeks, followed by a 2-week restriction of diet (2 g diet/mouse/day) containing 20% casein, soybean β-conglycinin, or soybean glycinin, and then sacrificed immediately. Serum triglyceride (TG), glucose, and insulin levels of β-conglycinin-fed mice were lower than in casein- and glycinin-fed mice of both strains. In order to analyze the related events to these effects, enzyme activities and relative mRNA levels of lipid metabolism-related proteins were measured. The activities of two enzymes related to fatty acid β-oxidation were higher while that of fatty acid synthase was lower in livers of β-conglycinin-fed mice than of casein-fed both mice. Messenger RNA levels of acyl-CoA oxidase (fatty acid β-oxidation related enzyme) were significantly higher in livers of β-conglycinin-fed mice than of both casein-fed mice. On the contrary, mRNA levels of SREBP-1 and 2 tended to be lowered in livers of soy protein-fed mice than of both casein-fed mice. Fecal excretion of TG was higher in β-conglycinin-fed mice than in casein-fed mice. Our results demonstrated that the soy β-conglycinin diet reduced serum TG levels by acceleration of β-oxidation, suppression of fatty acid synthase and/or increased TG fecal excretion, and also diminished serum glucose and insulin levels. Some of these events might be caused at the transcriptional levels, judged from the result that relative messenger RNA levels of lipid metabolism-related proteins were altered. These results suggest that soy β-conglycinin could be a potentially useful dietary protein source for the prevention of hypertriglyceridemia, hyperinsulinemia, and hyperglycemia, which are recognized as risk factors for atherosclerosis.

Human XTP3-B Forms an Endoplasmic Reticulum Quality Control Scaffold with the HRD1-SEL1L Ubiquitin Ligase Complex and BiP

The recognition of terminally misfolded proteins in the endoplasmic reticulum (ER) and the extraction of these proteins to the cytoplasm for proteasomal degradation are determined by a quality control mechanism in the ER. In yeast, Yos9p, an ER lectin containing a mannose 6-phosphate receptor homology (MRH) domain, enhances ER-associated degradation (ERAD) of glycoproteins. We show here that human XTP3-B (hXTP3-B), an ER lectin containing two MRH domains, has two transcriptional variants, and both isoforms retard ERAD of the human α1-antitrypsin variant null Hong Kong (NHK), a terminally misfolded glycoprotein. The hXTP3-B long isoform strongly inhibited ERAD of NHK-QQQ, which lacks all of the N-glycosylation sites of NHK, but the short transcriptional variant of hXTP3-B had almost no effect. Examination of complex formation by immunoprecipitation and by fractionation using sucrose density gradient centrifugation revealed that the hXTP3-B long isoform associates with the HRD1-SEL1L membrane-anchored ubiquitin ligase complex and BiP, forming a 27 S ER quality control scaffold complex. The hXTP3-B short isoform, however, is excluded from scaffold formation. Another MRH domain-containing ER lectin, hOS-9, is incorporated into this large complex, but gp78, another mammalian homolog of the yeast ubiquitin ligase Hrd1p, is not. Based on these results, we propose that this large ER quality control scaffold complex, containing ER lectins, a chaperone, and a ubiquitin ligase, provides a platform for the recognition and sorting of misfolded glycoproteins as well as nonglycosylated proteins prior to retrotranslocation into the cytoplasm for degradation.

Soybean β‐conglycinin as the main allergen in a patient with food‐dependent exercise‐induced anaphylaxis by tofu: food processing alters pepsin resistance

Background Food‐dependent exercise‐induced anaphylaxis (FDEIA) due to soybeans is a rare disorder. The allergen responsible for FDEIA due to soybeans has not yet been determined.

Degradation of HMG-CoA Reductase in Vitro CLEAVAGE IN THE MEMBRANE DOMAIN BY A MEMBRANE-BOUND CYSTEINE PROTEASE

We have recently shown that the endoplasmic reticulum (ER) membrane protein, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, is cleaved in isolated membrane fractions enriched for endoplasmic reticulum. Importantly, the cleavage rate is accelerated when the membranes are prepared from cells that have been pretreated with mevalonate or sterols, physiological regulators of the degradation process in vivo (McGee, T. P., Cheng, H. H., Kumagai, H., Omura, S., and Simoni, R. D. (1996)J. Biol. Chem. 271, 25630–25638). In the current study, we further characterize this in vitro cleavage of HMG-CoA reductase. E64, a specific inhibitor of cysteine-proteases, inhibits HMG-CoA reductase cleavage in vitro. In contrast, lactacystin, an inhibitor of the proteasome, inhibits HMG-CoA reductase degradation in vivo but does not inhibit the in vitro cleavage. Purified ER fractions contain lactacystin-sensitive and E64-insensitive proteasome activity as measured by succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin hydrolysis. We removed the proteasome from purified ER fractions by solubilization with heptylthioglucoside and observed that the detergent extracted, proteasome-depleted membrane fractions retain regulated cleavage of HMG-CoA reductase. This indicates that ER-associated proteasome is not involved in degradation of HMG-CoA reductase in vitro. In order to determine the site(s) of proteolysis of HMG-CoA reductasein vitro, four antisera were prepared against peptide sequences representing various domains of HMG-CoA reductase and used for detection of proteolytic intermediates. The sizes and antibody reactivity of the intermediates suggest that HMG-CoA reductase is cleaved in the in vitro degradation system near the span 8 membrane region, which links the N-terminal membrane domain to the C-terminal catalytic domain of the protein. We conclude that HMG-CoA reductase can be cleaved in the membrane-span 8 region by a cysteine protease(s) tightly associated with ER membranes.

Different Localization Patterns of Anthocyanin Species in the Pericarp of Black Rice Revealed by Imaging Mass Spectrometry

Black rice (Oryza sativa L. Japonica) contains high levels of anthocyanins in the pericarp and is considered an effective health-promoting food. Several studies have identified the molecular species of anthocyanins in black rice, but information about the localization of each anthocyanin species is limited because methodologies for investigating the localization such as determining specific antibodies to anthocyanin, have not yet been developed Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) is a suitable tool for investigating the localization of metabolites. In this study, we identified 7 species of anthocyanin monoglycosides and 2 species of anthocyanin diglycosides in crude extracts from black rice by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis. We also analyzed black rice sections by MALDI-IMS and found 2 additional species of anthocyanin pentosides and revealed different localization patterns of anthocyanin species composed of different sugar moieties. Anthocyanin species composed of a pentose moiety (cyanidin-3-O-pentoside and petunidin-3-O-pentoside) were localized in the entire pericarp, whereas anthocyanin species composed of a hexose moiety (cyanidin-3-O-hexoside and peonidin-3-O-hexoside) were focally localized in the dorsal pericarp. These results indicate that anthocyanin species composed of different sugar moieties exhibit different localization patterns in the pericarp of black rice. This is the first detailed investigation into the localization of molecular species of anthocyanins by MALDI-IMS.

Low resistin levels in adipose tissues and serum in high-fat fed mice and genetically obese mice: development of an ELISA system for quantification of resistin

Obesity is a major risk factor for insulin resistance. Resistin, an adipocyte-derived hormone-like molecule, is considered to serve as an important link between obesity and insulin resistance. However, the physiological role of resistin and the mechanism by which it neutralizes insulin action are still unclear. There are also conflicting reports that cast doubt on the cause of insulin resistance. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) system for quantification of mouse resistin levels, analyzed in relation to insulin resistance. C57BL/6J mice fed high-fat diet compared with normal diet had low resistin levels (by 70%, P<0.01) in epididymal adipose tissues. Genetically obese mice, db/db and KK-A(y), had hyperinsulinemia and hyperglycemia but low resistin levels (decreases by 83 and 90%, both P<0.01) compared with C57/BL6J mice in epididymal adipose tissues. Serum resistin levels determined by Western blotting showed a similar pattern to those in adipose tissues. Resistin levels in adipose tissues correlated with serum adiponectin concentrations positively (r=0.49). Our results indicate that the novel ELISA system is suitable for measurement of resistin levels in adipose tissues. The results do not support a role for resistin in insulin resistance.

Changes in lipid metabolism by soy β-conglycinin-derived peptides in HepG2 cells.

In this study, HepG2 cells were treated with short peptides (7S-peptides) derived from highly purified soybean beta-conglycinin (7S), which was free from lipophilic protein, and the effect of the peptide treatment on lipid metabolism was determined. 7S-peptide treatment suppressed the secretion of apolipoprotein B-100 from HepG2 cells into the medium. The 7S-peptides also suppressed the incorporation of (3)H-glycerol and (14)C-acetate into triacylglyceride but not into major phospholipids, such as phosphatidylcholine and phosphatidylethanolamine. Additionally, the synthesis of cholesterol esters was dramatically decreased for 2 h after the addition of the 7S-peptides, whereas the synthesis of cholesterol remained unchanged by 4 h and increased by 8 h after the addition of the 7S-peptides. The cleaved nuclear form of SREBP-2 increased 8 h after the addition of the 7S peptides, suggesting a decrease in intracellular cholesterol levels. Analysis of changes in mRNA expression after 7S-peptide treatment suggested that the 7S-peptides lower the level of cholesterol in the endoplasmic reticulum, increase the mRNA of genes related to beta-oxidation of fatty acids, and increase the synthesis of cholesterol. From these results, it may be concluded that the peptides derived from 7S altered the lipid metabolism to decrease secretion of apolipoprotein B-100-containing lipoprotein from HepG2 cells.

Visualization of anthocyanin species in rabbiteye blueberry Vaccinium ashei by matrix-assisted laser desorption/ionization imaging mass spectrometry

Anthocyanins are naturally occurring compounds that impart color to fruits, vegetables, and plants, and are believed to have a number of beneficial health effects in both humans and animals. Because of these properties, pharmacokinetic analysis of anthocyanins in tissue has been performed to quantify and identify anthocyanin species although, currently, no methods exist for investigating tissue localization of anthocyanin species or for elucidating the mechanisms of anthocyanin activity. Imaging mass spectrometry (IMS) is powerful tool for determining and visualizing the distribution of a wide range of biomolecules. To investigate whether anthocyanin species could be identified and visualized by IMS, we performed matrix-assisted laser desorption/ionization (MALDI)–IMS analysis, by tandem mass spectrometry (MALDI–IMS–MS), of ten anthocyanin molecular species in rabbiteye blueberry (Vaccinium ashei). The distribution patterns of each anthocyanin species were different in the exocarp and endocarp of blueberry sections. Anthocyanin species composed of delphinidin and petunidin were localized mainly in the exocarp. In contrast, those species composed of cyanidin, peonidin, and malvidin were localized in both the exocarp and the endocarp. Moreover, MALDI–IMS analysis of anthocyanidins in a blueberry section indicated that the distribution patterns of each anthocyanidin species were nearly identical with those of the corresponding anthocyanins. These results suggested that the different distribution patterns of anthocyanin species in the exocarp and endocarp depended on the aglycone rather than on the sugar moieties. This study is the first to visualize anthocyanin molecular species in fruits.

Molecular and structural analysis of electrophoretic variants of soybean seed storage proteins

Soybean (Glycine max L.) storage proteins are composed mainly of two major components, beta-conglycinin and glycinin. Electrophoretic variants of the beta subunit of beta-conglycinin and the A3 polypeptide of glycinin were detected on SDS-PAGE, and designated them as beta* and A3*, respectively. beta* and A3* exhibited higher and lower mobilities, respectively, than the common beta subunit and A3 polypeptide. The N-terminal nine and 10 amino acid sequences of beta* and A3* were completely identical to the previously reported sequences of the beta subunit and the A3 polypeptide, respectively. Analysis using concanavalin A-horseradish peroxidase and treatment with N-glycosidase indicated that glycans were not responsible for the difference in electrophoretic mobility of beta* or A3*. Furthermore, five clones of beta* or beta and three clones of A3*, respectively, were sequenced but we could not detect deletions and insertions except for a single or a few amino acid substitutions as compared with the common beta subunit and A3 polypeptide. These results indicate that a single or a few amino acid substitution affects the electrophoretic mobilities of beta* and A3*.

Activation of peroxisome proliferator-activated receptor-α (PPARα) suppresses postprandial lipidemia through fatty acid oxidation in enterocytes.

Activation of peroxisome proliferator-activated receptor (PPAR)-α which regulates lipid metabolism in peripheral tissues such as the liver and skeletal muscle, decreases circulating lipid levels, thus improving hyperlipidemia under fasting conditions. Recently, postprandial serum lipid levels have been found to correlate more closely to cardiovascular diseases than fasting levels, although fasting hyperlipidemia is considered an important risk of cardiovascular diseases. However, the effect of PPARα activation on postprandial lipidemia has not been clarified. In this study, we examined the effects of PPARα activation in enterocytes on lipid secretion and postprandial lipidemia. In Caco-2 enterocytes, bezafibrate, a potent PPARα agonist, increased mRNA expression levels of fatty acid oxidation-related genes, such as acyl-CoA oxidase, carnitine palmitoyl transferase, and acyl-CoA synthase, and oxygen consumption rate (OCR) and suppressed secretion levels of both triglycerides and apolipoprotein B into the basolateral side. In vivo experiments revealed that feeding high-fat-diet containing bezafibrate increased mRNA expression levels of fatty acid oxidation-related genes and production of CO(2) and acid soluble metabolites in enterocytes. Moreover, bezafibrate treatment suppressed postprandial lipidemia after oral administration of olive oil to the mice. These findings indicate that PPARα activation suppresses postprandial lipidemia through enhancement of fatty acid oxidation in enterocytes, suggesting that intestinal lipid metabolism regulated by PPARα activity is a novel target of PPARα agonist for decreasing circulating levels of lipids under postprandial conditions.