Obesity-induced endoplasmic reticulum stress causes chronic inflammation in adipose tissue

Adipose tissue plays a central role in maintaining metabolic homeostasis under normal conditions. Metabolic diseases such as obesity and type 2 diabetes are often accompanied by chronic inflammation and adipose tissue dysfunction. In this study, we observed that endoplasmic reticulum (ER) stress and the inflammatory response occurred in adipose tissue of mice fed a high-fat diet for a period of 16 weeks. After 16 weeks of feeding, ER stress markers increased and chronic inflammation occurred in adipose tissue. We found that ER stress is induced by free fatty acid (FFA)-mediated reactive oxygen species (ROS) generation and up-regulated gene expression of inflammatory cytokines in 3T3-L1 adipocytes. Oral administration to obese mice of chemical chaperons, which alleviate ER stress, improved chronic inflammation in adipose tissue, followed by the suppression of increased body weight and improved insulin signaling. These results indicate that ER stress plays important pathophysiological roles in obesity-induced adipose tissue dysfunction.This work was partly supported by grants from the Japan Society for the Promotion of Science KAKENHI (#22020030, #22800049), Sumitomo Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Astellas Foundation for Research on Metabolic Disorders, Takeda Science Foundation, The Pharmacological Research Foundation Tokyo, Daiichi-Sankyo Foundation of Life Science, and The Naito Foundation

High plasma concentration of beta-D-glucan after administration of sizofiran for cervical cancer

A 69-year-old woman with a history of cervical cancer was admitted to our hospital for further investigation of abnormal shadows on her chest roentgenogram. Histologic examination of transbronchial lung biopsy specimens revealed epithelioid cell granuloma, and Mycobacterium intracellulare was detected in the bronchial lavage fluid. The plasma level of (1→3)-beta-d-glucan was very high, and this elevated level was attributed to administration of sizofiran for treatment of cervical cancer 18 years previously. Therefore, in patients with cervical cancer, it is important to confirm whether or not sizofiran has been administered before measuring (1→3)-beta-d-glucan levels

Unfolded protein response, activated by OASIS family transcription factors, promotes astrocyte differentiation

OASIS is a member of the CREB/ATF family of transcription factors and modulates cell- or tissue-specific unfolded protein response signalling. Here we show that this modulation has a critical role in the differentiation of neural precursor cells into astrocytes. Cerebral cortices of mice specifically deficient in OASIS (Oasis−/−) contain fewer astrocytes and more neural precursor cells than those of wild-type mice during embryonic development. Furthermore, astrocyte differentiation is delayed in primary cultured Oasis−/− neural precursor cells. The transcription factor Gcm1, which is necessary for astrocyte differentiation in Drosophila, is revealed to be a target of OASIS. Introduction of Gcm1 into Oasis−/− neural precursor cells improves the delayed differentiation of neural precursor cells into astrocytes by accelerating demethylation of the Gfap promoter. Gcm1 expression is temporally controlled by the unfolded protein response through interactions between OASIS family members during astrocyte differentiation. Taken together, our findings demonstrate a novel mechanism by which OASIS and its associated family members are modulated by the unfolded protein response to finely control astrocyte differentiation.This work was partly supported by grants from the Japan Society for the Promotion of Science KAKENHI (#22020030, #22800049), Sumitomo Foundation, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Astellas Foundation for Research on Metabolic Disorders, Takeda Science Foundation, The Pharmacological Research Foundation Tokyo, Daiichi-Sankyo Foundation of Life Science, The Naito Foundation, Senri Life Science Foundation, Hokuto Foundation for Bioscience, and The Japan Prize Foundation

大腸杯細胞の最終分化における小胞体ストレスセンサーOASISの役割

広島大学(Hiroshima University)博士(医学)Doctor of Philosophy in Medical Sciencedoctora

A sulfated glycosaminoglycan linkage region is a novel type of human natural killer-1 (HNK-1) epitope expressed on aggrecan in perineuronal nets

Human natural killer-1 (HNK-1) carbohydrate (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-R) is highly expressed in the brain and required for learning and neural plasticity.We previously demonstrated that expression of the HNK-1 epitope is mostly abolished in knockoutmice for GlcAT-P (B3gat1), a major glucuronyltransferase required for HNK-1 biosynthesis, but remained in specific regions such as perineuronal nets (PNNs) in these mutantmice. Considering PNNs are mainly composed of chondroitin sulfate proteoglycans (CSPGs) and regulate neural plasticity, GlcAT-P-independent expression of HNK-1 in PNNs is suggested to play a role in neural plasticity. However, the function, structure, carrier glycoprotein and biosynthetic pathway for GlcAT-P-irrelevant HNK-1 epitope remain unclear. In this study, we identified a unique HNK-1 structure on aggrecan in PNNs. To determine the biosynthetic pathway for the novel HNK-1, we generated knockout mice for GlcAT-S (B3gat2), the other glucuronyltransferase required for HNK-1 biosynthesis. However, GlcAT-P and GlcAT-S double-knockout mice did not exhibit reduced HNK-1 expression compared with single GlcAT-P-knockoutmice, indicating an unusual biosynthetic pathway for the HNK-1 epitope in PNNs. Aggrecan was purified from cultured cells in which GlcAT-P and-S are not expressed and we determined the structure of the novel HNK-1 epitope using liquid chromatography/mass spectrometry (LC/MS) as a sulfated linkage region of glycosaminoglycans (GAGs), HSO3-GlcA-Gal-Gal-Xyl-R. Taken together, we propose a hypothetical model where GlcAT-I, the sole glucuronyltransferase required for synthesis of the GAG linkage, is also responsible for biosynthesis of the novel HNK-1 on aggrecan. These results could lead to discovery of new roles of the HNK-1 epitope in neural plasticity

Structural and biochemical characterization of O-mannose-linked human natural killer-1 glycan expressed on phosphacan in developing mouse brains.

The human natural killer-1 (HNK-1) carbohydrate comprising a sulfated trisaccharide (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-) is expressed on N-linked and O-mannose-linked glycans in the nervous system and involved in learning and memory functions. Although whole/core glycan structures and carrier glycoproteins for the N-linked HNK-1 epitope have been studied, carrier glycoproteins and the biosynthetic pathway of the O-mannose-linked HNK-1 epitope have not been fully characterized. Here, using mass spectrometric analyses, we identified the major carrier glycoprotein of the O-linked HNK-1 as phosphacan in developing mouse brains and determined the major O-glycan structures having the terminal HNK-1 epitope from partially purified phosphacan. The O-linked HNK-1 epitope on phosphacan almost disappeared due to the knockout of protein O-mannose β1, 2-N-acetylglucosaminyltransferase 1, an N-acetylglucosaminyltransferase essential for O-mannose-linked glycan synthesis, indicating that the reducing terminal of the O-linked HNK-1 is mannose. We also showed that glucuronyltransferase-P (GlcAT-P) was involved in the biosynthesis of O-mannose-linked HNK-1 using the gene-deficient mice of GlcAT-P, one of the glucuronyltransferases for HNK-1 synthesis. Consistent with this result, we revealed that GlcAT-P specifically synthesized O-linked HNK-1 onto phosphacan using cultured cells. Furthermore, we characterized the as-yet-unknown epitope of the 6B4 monoclonal antibody (mAb), which was thought to recognize a unique phosphacan glycoform. The reactivity of the 6B4 mAb almost completely disappeared in GlcAT-P-deficient mice, and exogenously expressed phosphacan was selectively recognized by the 6B4 mAb when co-expressed with GlcAT-P, suggesting that the 6B4 mAb preferentially recognizes O-mannose-linked HNK-1 on phosphacan. This is the first study to show that 6B4 mAb-reactive O-mannose-linked HNK-1 in the brain is mainly carried by phosphacan

<i>Campylobacter jejuni pdxA</i> Affects Flagellum-Mediated Motility to Alter Host Colonization

<div><p>Vitamin B6 (pyridoxal-5'-phosphate, PLP) is linked to a variety of biological functions in prokaryotes. Here, we report that the <i>pdxA</i> (putative 4-hydroxy-L-threonine phosphate dehydrogenase) gene plays a pivotal role in the PLP-dependent regulation of flagellar motility, thereby altering host colonization in a leading foodborne pathogen, <i>Campylobacter jejuni</i>. A <i>C. jejuni pdxA</i> mutant failed to produce PLP and exhibited a coincident loss of flagellar motility. Mass spectrometric analyses showed a 3-fold reduction in the main flagellar glycan pseudaminic acid (Pse) associated with the disruption of <i>pdxA</i>. The <i>pdxA</i> mutant also exhibited reduced growth rates compared with the WT strain. Comparative metabolomic analyses revealed differences in respiratory/energy metabolism between WT <i>C. jejuni</i> and the <i>pdxA</i> mutant, providing a possible explanation for the differential growth fitness between the two strains. Consistent with the lack of flagellar motility, the <i>pdxA</i> mutant showed impaired motility-mediated responses (bacterial adhesion, ERK1/2 activation, and IL-8 production) in INT407 cells and reduced colonization of chickens compared with the WT strain. Overall, this study demonstrated that the <i>pdxA</i> gene affects the PLP-mediated flagellar motility function, mainly through alteration of Pse modification, and the disruption of this gene also alters the respiratory/energy metabolisms to potentially affect host colonization. Our data therefore present novel implications regarding the utility of PLP and its dependent enzymes as potent target(s) for the control of this pathogen in the poultry host.</p></div

Inactivation of the <i>pdxA</i> gene impairs the biosynthesis of vitamin B6 (PLP) in <i>C. jejuni</i>.

<p>(A) A scheme for the PLP production pathway (right box) in <i>C. jejuni</i> in relation to Pse biosynthesis (left box) is illustrated based on <i>in silico</i> pathway analysis performed using PATRIC (<a href="http://patricbrc.vbi.vt.edu/portal/portal/patric/Home" target="_blank">http://patricbrc.vbi.vt.edu/portal/portal/patric/Home</a>). (B) The <i>pdxA</i> mutant produced no PLP. The <i>C. jejuni</i> 81–176 WT, <i>pdxA</i> mutant, and the complemented strains were grown in 10ml of MH broth to an OD₆₀₀ of 0.60. The suspensions were then homogenized, serially diluted, and subjected to ELISA to quantify the amounts of PLP (μg 10 ml⁻¹). The data show the mean +/− standard deviations from three independent assays.</p

Deletion of the <i>pdxA</i> gene impairs <i>in vitro</i> cellular responses and <i>in vivo</i> colonization.

<p>(A) INT407 cells were infected for 1 h with the <i>C. jejuni</i> WT, pdxA−, pdxA−/+, and flaA− strains. The number of cell-adherent bacteria was measured by counting the plates after washing three times with PBS. (B) ERK1/2 activation upon infection. Western blotting was performed to detect the levels of phosphorylated and total ERK1/2 in the lysates from infected cells. (C) IL-8 production in INT407 cells was measured at 4 h and 16 h <i>p.i. via</i> ELISA. The data are presented in sections A and C as the mean values ± standard deviations from samples run in duplicate in at least three experiments. (D) Disruption of the <i>pdxA</i> gene reduces the colonization of the chicken cecum by <i>C. jejuni</i>. Groups of 14-day-old chickens (n = 10 per group) were orally inoculated with approximately 3×10⁷ CFU of WT or <i>pdxA</i> mutant <i>C. jejuni</i>. At 1 week and 4 weeks <i>p.i.</i>, the ceca were aseptically removed from the infected animals (n = 5 for each time point) and homogenized. Serial dilutions of the suspensions were plated on mCCDA agar to count CFU numbers. The closed diamonds and open circles represent the numbers of WT and <i>pdxA</i> mutant CFUs recovered from the animals, respectively.</p