Reiko Urade

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.

Gene expression in response to endoplasmic reticulum stress in Arabidopsis thaliana

Eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). In this case, so‐called unfolded protein response (UPR) genes are induced. We determined the transcriptional expression of Arabidopsis thaliana UPR genes by fluid microarray analysis of tunicamycin‐treated plantlets. Two hundred and fifteen up‐regulated genes and 17 down‐regulated ones were identified. These genes were reanalyzed with functional DNA microarrays, using DNA fragments cloned through fluid microarray analysis. Finally, 36 up‐regulated and two down‐regulated genes were recognized as UPR genes. Among them, the up‐regulation of genes related to protein degradation (HRD1, SEL‐1L/HRD3 and DER1), regulation of translation (P58IPK), and apoptosis (BAX inhibitor‐1) was reconfirmed by real‐time reverse transcriptase‐PCR. The induction of SEL‐1L protein in an Arabidopsis membrane fraction on tunicamycin‐treatment was demonstrated. Phosphorylation of initiation factor‐2α, which was inhibited by P58IPK, was decreased in tunicamycin‐treated plantlets. However, regulatory changes in translation caused by ER stress were not detected in Arabidopsis. Plant cells appeared to have a strategy for overcoming ER stress through enhancement of protein folding activity, degradation of unfolded proteins, and regulation of apoptosis, but not regulation of translation.

Quantitative analysis of polyenoic phospholipid molecular species by high performance liquid chromatography

The quantitative analysis of phospholipid molecular species containing polyenoic fatty acids is described. Dinitrobenzoyl derivatives of diacylglycerols prepared from phospholipids were separated into individual molecular species by reversed-phase high performance liquid chromatography (HPLC) using a combination of two solvent systems and were quantified at 254 nm. Thirty-six molecular species were resolved from the phosphatidylcholines of rat hearts, human platelets and Chinese hamster V79-R cells. The derivatives of alkenylacyl molecular species from platelet phosphatidylethanolamine were resolved concomitantly with diacyl molecular species.

Cellular response to unfolded proteins in the endoplasmic reticulum of plants.

Secretory and transmembrane proteins are synthesized in the endoplasmic reticulum (ER) in eukaryotic cells. Nascent polypeptide chains, which are translated on the rough ER, are translocated to the ER lumen and folded into their native conformation. When protein folding is inhibited because of mutations or unbalanced ratios of subunits of hetero‐oligomeric proteins, unfolded or misfolded proteins accumulate in the ER in an event called ER stress. As ER stress often disturbs normal cellular functions, signal‐transduction pathways are activated in an attempt to maintain the homeostasis of the ER. These pathways are collectively referred to as the unfolded protein response (UPR). There have been great advances in our understanding of the molecular mechanisms underlying the UPR in yeast and mammals over the past two decades. In plants, a UPR analogous to those in yeast and mammals has been recognized and has recently attracted considerable attention. This review will summarize recent advances in the plant UPR and highlight the remaining questions that have yet to be addressed.

The endoplasmic reticulum stress signaling pathways in plants

The unfolded protein response (UPR) is an evolutionarily conserved mechanism by which all eukaryotic cells preserve the homeostasis of the endoplasmic reticulum (ER) in the face of accumulation of unfolded proteins in the ER. Plants possess at least two signaling pathways specific for UPR. ER membrane‐bound ER stress sensor/transducers, AtbZIP60 and AtbZIP28, are basic leucine zipper transcription factors that are activated by regulated intramembrane proteolysis systems and regulate transcription of the UPR genes. These signaling pathways play important roles not only in the UPR but also in other biological processes such as the response to pathogens and heat stress.

Inhibition by acidic phospholipids of protein degradation by ER-60 protease, a novel cysteine protease, of endoplasmic reticulum

A protein (ER60) with sequence similarity to phosphoinositide‐specific phospholipase C‐α purified from rat liver endoplasmic reticulum (ER) degraded ER resident proteins and is really a protease [(1992) J. Biol. Chem. 265, 15152‐15159]. Therefore, ER60 is called ER‐60 protease. We now show that negatively charged phospholipids, phosphatidylinositol, phosphatidylinositol 4,5‐bisphosphate and phosphatidylserine inhibit ER protein degradation by ER‐60 protease. Phosphatidylcholine and phosphatidylethanolamine show no effect on the activity of ER‐60 protease. With the use of protease inhibitors, ER‐60 protease is shown to be a novel cysteine protease distinct from those of the cylosol and lysosomes.

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.

Disaggregation and Reaggregation of Gluten Proteins by Sodium Chloride

This study showed that gluten proteins were extracted with distilled water from dough prepared in the presence of NaCl. To elucidate the interrelationship of NaCl and gluten proteins in dough, the extracted proteins were characterized. These proteins were primarily found to be soluble gliadin monomers by N-terminal amino acid sequencing and analytical ultracentrifugation. Extracted proteins were aggregated by the addition of NaCl at concentrations of >10 mM. A decrease in beta-turn structures, which expose tryptophan residues to an aqueous environment in the presence of NaCl, was revealed by Fourier transform infrared analysis and scanning of fluorescence spectra. In addition, cross-linking experiments with disuccinimidyl tartrate showed that a large amount of protein was cross-linked in the dough only in the presence of NaCl. These results suggest that both interactions and distances between proteins were altered by the addition of NaCl.

Protein disulfide isomerase family proteins involved in soybean protein biogenesis

Protein disulfide isomerase family proteins are known to play important roles in the folding of nascent polypeptides and the formation of disulfide bonds in the endoplasmic reticulum. In this study, we cloned two similar protein disulfide isomerase family genes from soybean leaf (Glycine max L. Merrill cv. Jack) mRNA by RT‐PCR using forward and reverse primers designed from the expressed sequence tag clone sequences. The cDNA encodes a protein of either 364 or 362 amino acids, named GmPDIS‐1 or GmPDIS‐2, respectively. The nucleotide and amino acid sequence identities of GmPDIS‐1 and GmPDIS‐2 were 68% and 74%, respectively. Both proteins lack the C‐terminal, endoplasmic reticulum‐retrieval signal, KDEL. Recombinant proteins of both GmPDIS‐1 and GmPDIS‐2 were expressed in Escherichia coli as soluble folded proteins that showed both an oxidative refolding activity of denatured ribonuclease A and a chaperone activity. Their domain structures were identified as containing two thioredoxin‐like domains, a and a′, and an ERp29c domain by peptide mapping with either trypsin or V8 protease. In cotyledon cells, both proteins were shown to distribute to the endoplasmic reticulum and protein storage vacuoles by confocal microscopy. Data from coimmunoprecipitation and crosslinking experiments suggested that GmPDIS‐1 associates with proglycinin, a precursor of the seed storage protein glycinin, in the cotyledon. Levels of GmPDIS‐1, but not of GmPDIS‐2, were increased in cotyledons, where glycinin accumulates during seed development. GmPDIS‐1, but not GmPDIS‐2, was induced under endoplasmic reticulum‐stress conditions.