Naohito Aoki

The up-regulation of microRNA-335 is associated with lipid metabolism in liver and white adipose tissue of genetically obese mice.

MicroRNAs (miRNAs) are short non-coding RNA that post-transcriptionally regulates gene expression. Some miRNAs have been proposed to be associated with obesity. However, miRNAs, which are related to the development of obesity in vivo remains unknown. Here in, we found the up-regulation of miR-335 in obesity using microarray analysis for miRNA. The expressions of miR-335 in liver and white adipose tissue (WAT) were up-regulated in obese mice including ob/ob, db/db, and KKAy mice. Increased miR-335 expressions were associated with an elevated body, liver and WAT weight, and hepatic triglyceride and cholesterol. Furthermore, miR-335 levels were closely correlated with expression levels of adipocyte differentiation markers such as PPARgamma, aP2, and FAS in 3T3-L1 adipocyte. These findings provide the first evidence that the up-regulated expressions of miR-335 in liver and WAT of obese mice might contribute to the pathophysiology of obesity.

Adipocyte-derived microvesicles contain RNA that is transported into macrophages and might be secreted into blood circulation.

We have recently found that 3T3-L1 adipocytes secrete microvesicles, known as adipocyte-derived microvesicles (ADMs), with angiogenic activity. In this study, we found that ADMs contain RNA without typical 28S and 18S ribosomal RNA inside the vesicles. Microarray analysis revealed that ADMs contain approximately 7000 mRNAs and 140 microRNAs. Most of transcripts for adipocyte-specific and dominant genes were contained in the ADMs, and their abundance was mostly correlated with that in the donor cells. Abundance of adipocyte-related microRNAs was also mostly correlated with that in the donor cells. ADMs mediated transport of adiponectin and resistin gene transcripts into RAW264.7 macrophages. Moreover, adipocyte-specific gene transcripts such as adiponectin, resistin, and PPARgamma2 were found in microvesicles isolated from rat serum. Thus, ADM might play a role as a novel intercellular communication tool by transporting RNA in paracrine and possibly endocrine manners.

Regulation and functional relevance of milk fat globules and their components in the mammary gland.

Mammary gland and epithelial cells are unique to mammals and are under the control of lactogenic hormones such as prolactin. Recent findings indicated that major components of milk fat globule membrane (MFGM) are under the control of lactogenic hormones, and that the major components butyrophilin and xanthine oxidoreductase are indispensable for milk fat secretion. Further, prolactin signaling is negatively controlled by two highly related protein tyrosine phosphatases, PTP1B and TC-PTP. Milk fat globule EGF factor 8 (MFG-E8) is one of the major components of MFGM and is upregulated during lactation. MFG-E8 is further upregulated in the involuting mammary gland. MFG-E8 on exosome-like membrane vesicles in the milk recovered from post-weaning but not lactating mammary glands exhibits higher binding activity to phosphatidylserine and apoptotic mammary epithelial cells, and serves as a link between apoptotic mammary epithelial cells and phagocytes. Recent reports using MFG-E8 deficient mice support the view that MFG-E8 is indispensable for eliminating apoptotic mammary epithelial cells during involution.

Receptor-Type Protein Tyrosine Phosphatase ε (PTPεM) is a Negative Regulator of Insulin Signaling in Primary Hepatocytes and Liver

Abstract Impaired insulin receptor (IR) signaling leads to insulin resistance and type 2 diabetes mellitus. Several inhibitors of the IR tyrosine kinase activity have recently been described and associated with human insulin resistance. Among these negative regulators, protein tyrosine phosphatases (PTPs) are likely to play a pivotal role in IR signaling. Transgenic studies revealed that PTP1B and TCPTP are primary candidates but IR of these animals can be finally dephosphorylated, suggesting that other PTPs are also involved in the dephosphorylation of IR. In this study, we showed that receptor-type PTPϵ (PTPϵM) dephosphorylated IR in rat primary hepatocytes and tyrosines 972, 1158, 1162 and 1163 were primary targets of PTPϵM. Wild type as well as substrate-trapping DA forms of PTPϵM suppressed phosphorylation of IR downstream enzymes such as Akt, extracellular regulated kinase (ERK) and glycogen synthase kinase 3 (GSK3). It was also demonstrated that PTPϵM suppressed insulin-induced glycogen synthesis and inhibited insulin-induced suppression of phosphoenol pyruvate carboxykinase (PEPCK) expression in primary hepatocytes. Furthermore, adenovirally introduced PTPϵM also exhibited inhibitory activity against suppression of PEPCK expression in mouse liver. These results suggest that PTPϵM is a negative regulator of IR signaling and involved in insulin-induced glucose metabolism mainly through direct dephospho-rylation and inactivation of IR in hepatocytes and liver.

Morphological and biochemical changes of isolated chicken egg-envelope during sperm penetration: degradation of the 97-kilodalton glycoprotein is involved in sperm-driven hole formation on the egg-envelope.

Abstract The chicken egg-envelope is made of two major glycoprotein components, which are designated as gp97 and gp42 (after their molecular masses). To elucidate how these two components are involved in macromolecular organization of the chicken egg-envelope, the isolated egg-envelope was characterized by immunochemical and biochemical methods. The gp97 was suggested to be a homologue of mouse ZPB based on the similarities of N-terminal and internal sequences. Immunoblotting using anti-gp97 monoclonal antibodies and two-dimensional gel electrophoresis with or without mercaptoethanol treatment revealed that gp97 formed a homodimer through disulfide bonds, whereas gp42 did not. Under indirect immunofluorescence microscopy, the anti-gp97 antibody visualized indistinct, small spots on the egg-envelope, whereas the anti-gp42 antibody showed a meshwork of blurry, fibrous structures. The hole formation on the egg-envelope by in vitro sperm penetration was completely inhibited by two anti-gp97 monoclonal antibodies. Interestingly, the anti-gp97 monoclonal antibodies blocked the proteolysis not only of gp97 but also of gp42 during incubation of the egg-envelope with either sperm or the crude chicken acrosin. Taken together, these results indicate that gp97 may play pivotal roles not only in constitution of the macromolecular organization of the egg-envelope but also in triggering hydrolysis of the egg-envelope during sperm penetration.

A food‐derived synergist of NGF signaling: identification of protein tyrosine phosphatase 1B as a key regulator of NGF receptor‐initiated signal transduction

Neurotrophins, such as the nerve growth factor (NGF), play an essential role in the growth, development, survival and functional maintenance of neurons in the central and peripheral systems. They also prevent neuronal cell death under various stressful conditions, such as ischemia and neurodegenerative disorders. NGF induces cell differentiation and neurite outgrowth by binding with and activating the NGF receptor tyrosine kinase followed by activation of a variety of signaling cascades. We have investigated the NGF‐dependent neuritogenesis enhancer potential of a food‐derived small molecule contained in Brassica vegetables and identified the protein tyrosine phosphatase (PTP) 1B as a key regulator of the NGF receptor‐initiated signal transduction. Based on an extensive screening of Brassica vegetable extracts for the neuritogenic‐promoting activity in the rat pheochromocytoma cell line PC12, we found the Japanese horseradish, wasabi (Wasabia japonica, syn. Eutrema wasabi), as the richest source and identified 6‐methylsulfinylhexyl isothiocyanate (6‐HITC), an analogue of sulforaphane isolated from broccoli, as one of the major neuritogenic enhancers in the wasabi. 6‐HITC strongly enhanced the neurite outgrowth and neurofilament expression elicited by a low‐concentration of NGF that alone was insufficient to induce neuronal differentiation. 6‐HITC also facilitated the sustained‐phosphorylation of the extracellular signal‐regulated kinase and the autophosphorylation of the NGF receptor TrkA. It was found that PTP1B act as a phosphatase capable of dephosphorylating Tyr‐490 of TrkA and was inactivated by 6‐HITC in a redox‐dependent manner. The identification of PTP1B as a regulator of NGF signaling may provide new clues about the chemoprotective potential of food components, such as isothiocyanates.

Development and evaluation of transgenic rice seeds accumulating a type II-collagen tolerogenic peptide.

Type II collagen (CII) in joint cartilage is known to be a major auto-antigen in human rheumatoid arthritis. Several animal model- and clinical-studies on tolerance-based immunotherapy for the arthritis have been conducted by administrating synthetic immunodominant peptides through an oral route. In the present study, to produce a tolerogenic peptide with therapeutic potential in transgenic rice plants, a gene construct producing glutelin fusion protein with tandem four repeats of a CII250–270 peptide (residues 250–270) (GluA-4XCII250–270) containing a human T-cell epitope was introduced with a selection marker, hygromycin phosphotransferase gene (hygromycin-resistance gene) (hph), by co-transformation. Several transgenic plants with high and stable expression of gluA-4XCII250–270, but no hph, were selected based on both DNA and protein analyses. The GluA-4XCII250–270 fusion proteins were detected as both precursor and processed forms mainly in a glutelin fraction of rice endosperm protein extracts and in protein-body rich fractions prepared by density gradient ultracentrifugation. The amount of accumulated CII250–270 peptide was immunochemically estimated to be about 1 μg per seed. Feeding DBA/1 mice the transgenic rice seeds (25 μg of the peptide per mouse a day) for 2 weeks showed tendencies lowering and delaying serum specific-IgG2a response against subsequent and repeated intraperitoneal-injection of type II collagen. Taken these together, the CII-immunodominant peptide could effectively be produced and accumulated as a glutelin-fusion protein in the transgenic rice seeds, which might be useful as pharmaceutical materials and functional food for prevention and therapy for anti-CII autoimmune diseases like human rheumatoid arthritis.

Absorption, Migration and Kinetics in Peripheral Blood of Orally Administered Ovalbumin in a Mouse Model

Intestinal absorption of food proteins is well known, whereas its physiological significance remains to be investigated. Various amounts (1, 10 and 50 mg) of ovalbumin were orally administered to mice and the blood kinetics were subsequently analyzed by two-site ELISA. The blood ovalbumin concentration consistently reached its maximum (7–90 ng/ml) about 20 min after the oral administration and then gradually decreased in a dose-dependent manner. Only intact (45 kDa) and truncated (40 kDa) ovalbumins were always detected in the blood independently of the administration site, intra-stomach or intra-intestine, while various fragments of the protein were observed in the gastrointestinal lumen after the oral administration. Recognition by a specific monoclonal antibody and an acidic shift of its pI value suggested that the 40-kDa truncated ovalbumin was produced by intracellular limited proteolysis at its C-terminus. Such stable absorption and blood kinetics of undigested ovalbumin in normal mice suggest some sort of physiological significance for the intestinal uptake of intact food proteins.

Binding of Norovirus virus-like particles (VLPs) to human intestinal Caco-2 cells and the suppressive effect of pasteurized bovine colostrum on this VLP binding.

Noroviruses (NoVs), which cannot be grown in cell culture, are a major infectious agent of gastroenteritis. An in vitro assay system was established for the evaluation of NoV binding to enterocytes using virus-like particles (VLPs) produced in a baculovirus system expressing a NoV VP1 capsid protein. After confirmation of the purity by MS analysis, VLPs were incubated with human intestinal Caco-2 cells. NoV VLPs were detected clearly by confocal laser microscopy only on a certain population of Caco-2 cells, and were semi-quantified by immunoblotting of cell lysates. Then the suppressive effect of pasteurized bovine colostrum was analyzed on the VLP binding to Caco-2 cells by immunoblotting. The colostrum reduced VLP binding in a dose-dependent manner, at about 50% suppression with 12.5 μg of the colostral proteins. Furthermore, the colostrum contained IgG antibodies reacting to VLPs, suggesting that cross-reactive antibodies in the bovine colostrums block human NoV binding to intestinal cells.

Heterocomplex Formation and Cell-Surface Accumulation of Hen's Serum Zona Pellucida B1 (ZPB1)with ZPC Expressed by a Mammalian Cell Line (COS-7): A Possible Initiating Step of Egg-Envelope Matrix Construction

Abstract The egg envelope, referred to as zona pellucida (ZP) in mammalian eggs, is a fibrous and noncollagenous extracellular matrix surrounding vertebrate eggs, and composed of three to four homologous glycoproteins with a common ZP domain. In birds, a liver-derived ZP glycoprotein (ZP1/ZPB1) is transported through the bloodstream to ovarian follicles and joins the egg-envelope matrix construction together with the other ZP glycoproteins, such as ZPC and ZPD/ZPX2, both secreted from follicular granulosa cells. We report here that, through its ZP domain, ZPB1 specifically associates with ZPC, which might lead to the construction of egg-envelope matrix. The ZPB1 in laying hens serum specifically bound to ZPC, but not to ZPX2, separated by SDS-PAGE and blotted on a membrane. Hemagglutinin (HA)-tagged ZPC expressed in a mammalian cell line (COS-7) cells was processed and secreted as a mature-form into the culture medium. From the culture supernatant of ZPC-expressing transfectants cultured in the presence of ZPB1, both ZPB1 and ZPC were recovered as heterocomplexes by immunoprecipitation using either anti-HA or anti-ZPB1 antibody. Interestingly, a monoclonal antibody, 8E1, which immunoprecipitated free ZPB1, did not immunoprecipitate the ZPB1-ZPC heterocomplexes. An 8E1 epitope was mapped on a C-terminal region of the ZP domain in a ZPB1 molecule by identifying an 8E1-positive peptide using mass spectroscopy. Furthermore, by laser scanning confocal microscopy, ZPB1 and ZPC were observed to colocalize on the surface of ZPC-expressing transfectants cultured in the presence of ZPB1, whereas almost no ZPC was detected on the surface of the transfectants cultured in the absence of ZPB1. Taken together, these results suggest that ZPB1 transported into ovarian follicles encounters and associates with ZPC secreted from granulosa cells, resulting in the formation of heterocomplexes around an oocyte. In addition, it appears that such ZPB1-ZPC complexes accumulated on the oocyte surface act as a scaffold for subsequent matrix construction events including ZPX2 association.