Homeostatic and pathogenic roles of GM 3 ganglioside molecular species in TLR 4 signaling in obesity
2020; Springer Nature; Volume: 39; Issue: 12 Linguagem: Inglês
10.15252/embj.2019101732
ISSN1460-2075
AutoresHirotaka Kanoh, Takahiro Nitta, Shinji Go, Kei‐ichiro Inamori, Lucas Veillon, Wataru Nihei, Mayu Fujii, Kazuya Kabayama, Atsushi Shimoyama, Koichi Fukase, Umeharu Ohto, Toshiyuki Shimizu, Taku Watanabe, Hiroki Shindo, Sorama Aoki, Kenichi Sato, Masao Nagasaki, Yutaka Yatomi, Naoko Komura, Hiromune Ando, Hideharu Ishida, Makoto Kiso, Yoshihiro Natori, Yuichi Yoshimura, Asia Zonca, Anna Giulia Cattaneo, Marilena Letizia, Maria Grazia Ciampa, Laura Mauri, Alessandro Prinetti, Sandro Sonnino, Akemi Suzuki, Jin‐ichi Inokuchi,
Tópico(s)Helicobacter pylori-related gastroenterology studies
ResumoArticle7 May 2020Open Access Homeostatic and pathogenic roles of GM3 ganglioside molecular species in TLR4 signaling in obesity Hirotaka Kanoh Hirotaka Kanoh Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Takahiro Nitta Takahiro Nitta Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Shinji Go Shinji Go Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Kei-ichiro Inamori Kei-ichiro Inamori Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Lucas Veillon Lucas Veillon Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Wataru Nihei Wataru Nihei Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Mayu Fujii Mayu Fujii Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Kazuya Kabayama Kazuya Kabayama Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Atsushi Shimoyama Atsushi Shimoyama Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Koichi Fukase Koichi Fukase Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Umeharu Ohto Umeharu Ohto Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Toshiyuki Shimizu Toshiyuki Shimizu Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Taku Watanabe Taku Watanabe Medical and Pharmaceutical Information Science, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Hiroki Shindo Hiroki Shindo Medical and Pharmaceutical Information Science, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Sorama Aoki Sorama Aoki orcid.org/0000-0002-1148-2688 Medical and Pharmaceutical Information Science, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Kenichi Sato Kenichi Sato Medical and Pharmaceutical Information Science, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Mika Nagasaki Mika Nagasaki Department of Cardiovascular Medicine and Computational Diagnostic Radiology & Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yutaka Yatomi Yutaka Yatomi Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Naoko Komura Naoko Komura Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu, Japan Search for more papers by this author Hiromune Ando Hiromune Ando Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu, Japan Search for more papers by this author Hideharu Ishida Hideharu Ishida Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu, Japan Department of Applied Bio-organic Chemistry, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan Search for more papers by this author Makoto Kiso Makoto Kiso Organization for Research and Community Development, Gifu University, Gifu, Japan Search for more papers by this author Yoshihiro Natori Yoshihiro Natori Division of Organic and Pharmaceutical Chemistry, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Yuichi Yoshimura Yuichi Yoshimura Division of Organic and Pharmaceutical Chemistry, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Asia Zonca Asia Zonca Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Anna Cattaneo Anna Cattaneo Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Marilena Letizia Marilena Letizia Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Maria Ciampa Maria Ciampa Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Laura Mauri Laura Mauri Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Alessandro Prinetti Alessandro Prinetti Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Sandro Sonnino Sandro Sonnino Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Akemi Suzuki Akemi Suzuki Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Jin-ichi Inokuchi Corresponding Author Jin-ichi Inokuchi [email protected] orcid.org/0000-0002-0703-5746 Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Hirotaka Kanoh Hirotaka Kanoh Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Takahiro Nitta Takahiro Nitta Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Shinji Go Shinji Go Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Kei-ichiro Inamori Kei-ichiro Inamori Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Lucas Veillon Lucas Veillon Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Wataru Nihei Wataru Nihei Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Mayu Fujii Mayu Fujii Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Kazuya Kabayama Kazuya Kabayama Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Atsushi Shimoyama Atsushi Shimoyama Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Koichi Fukase Koichi Fukase Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan Search for more papers by this author Umeharu Ohto Umeharu Ohto Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Toshiyuki Shimizu Toshiyuki Shimizu Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan Search for more papers by this author Taku Watanabe Taku Watanabe Medical and Pharmaceutical Information Science, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Hiroki Shindo Hiroki Shindo Medical and Pharmaceutical Information Science, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Sorama Aoki Sorama Aoki orcid.org/0000-0002-1148-2688 Medical and Pharmaceutical Information Science, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Kenichi Sato Kenichi Sato Medical and Pharmaceutical Information Science, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Mika Nagasaki Mika Nagasaki Department of Cardiovascular Medicine and Computational Diagnostic Radiology & Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Yutaka Yatomi Yutaka Yatomi Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan Search for more papers by this author Naoko Komura Naoko Komura Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu, Japan Search for more papers by this author Hiromune Ando Hiromune Ando Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu, Japan Search for more papers by this author Hideharu Ishida Hideharu Ishida Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu, Japan Department of Applied Bio-organic Chemistry, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan Search for more papers by this author Makoto Kiso Makoto Kiso Organization for Research and Community Development, Gifu University, Gifu, Japan Search for more papers by this author Yoshihiro Natori Yoshihiro Natori Division of Organic and Pharmaceutical Chemistry, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Yuichi Yoshimura Yuichi Yoshimura Division of Organic and Pharmaceutical Chemistry, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Asia Zonca Asia Zonca Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Anna Cattaneo Anna Cattaneo Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Marilena Letizia Marilena Letizia Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Maria Ciampa Maria Ciampa Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Laura Mauri Laura Mauri Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Alessandro Prinetti Alessandro Prinetti Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Sandro Sonnino Sandro Sonnino Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy Search for more papers by this author Akemi Suzuki Akemi Suzuki Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Jin-ichi Inokuchi Corresponding Author Jin-ichi Inokuchi [email protected] orcid.org/0000-0002-0703-5746 Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan Search for more papers by this author Author Information Hirotaka Kanoh1,‡, Takahiro Nitta1,‡, Shinji Go1,†, Kei-ichiro Inamori1, Lucas Veillon1,†, Wataru Nihei1, Mayu Fujii2, Kazuya Kabayama2, Atsushi Shimoyama2, Koichi Fukase2, Umeharu Ohto3, Toshiyuki Shimizu3, Taku Watanabe4, Hiroki Shindo4, Sorama Aoki4, Kenichi Sato4, Mika Nagasaki5,†, Yutaka Yatomi6, Naoko Komura7, Hiromune Ando7, Hideharu Ishida7,8, Makoto Kiso9, Yoshihiro Natori10, Yuichi Yoshimura10, Asia Zonca11, Anna Cattaneo11, Marilena Letizia11, Maria Ciampa11, Laura Mauri11, Alessandro Prinetti11, Sandro Sonnino11, Akemi Suzuki1 and Jin-ichi Inokuchi *,1 1Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Medical and Pharmaceutical University, Sendai, Japan 2Department of Chemistry, Graduate School of Science, Osaka University, Osaka, Japan 3Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan 4Medical and Pharmaceutical Information Science, Tohoku Medical and Pharmaceutical University, Sendai, Japan 5Department of Cardiovascular Medicine and Computational Diagnostic Radiology & Preventive Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 6Department of Clinical Laboratory Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 7Center for Highly Advanced Integration of Nano and Life Sciences (G-CHAIN), Gifu University, Gifu, Japan 8Department of Applied Bio-organic Chemistry, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan 9Organization for Research and Community Development, Gifu University, Gifu, Japan 10Division of Organic and Pharmaceutical Chemistry, Tohoku Medical and Pharmaceutical University, Sendai, Japan 11Department of Medical Biotechnology and Translational Medicine, University of Milan, Milano, Italy †Present address: Department of Pathophysiology and Metabolism, Kawasaki Medical School, Okayama, Japan †Present address: Department of Bioinformatics and Computational Biology, The University of Texas MD Anderson Cancer Center, Houston, TX, USA †Present address: Nagasaki Clinic, Tokyo, Japan ‡These authors contributed equally to this work *Corresponding author. Tel: +81 22 727 0117; E-mail: [email protected] The EMBO Journal (2020)39:e101732https://doi.org/10.15252/embj.2019101732 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Innate immune signaling via TLR4 plays critical roles in pathogenesis of metabolic disorders, but the contribution of different lipid species to metabolic disorders and inflammatory diseases is less clear. GM3 ganglioside in human serum is composed of a variety of fatty acids, including long-chain (LCFA) and very-long-chain (VLCFA). Analysis of circulating levels of human serum GM3 species from patients at different stages of insulin resistance and chronic inflammation reveals that levels of VLCFA-GM3 increase significantly in metabolic disorders, while LCFA-GM3 serum levels decrease. Specific GM3 species also correlates with disease symptoms. VLCFA-GM3 levels increase in the adipose tissue of obese mice, and this is blocked in TLR4-mutant mice. In cultured monocytes, GM3 by itself has no effect on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhances TLR4 activation by LPS/HMGB1, while LCFA-GM3 and unsaturated VLCFA-GM3 suppresses TLR4 activation. GM3 interacts with the extracellular region of TLR4/MD2 complex to modulate dimerization/oligomerization. Ligand-molecular docking analysis supports that VLCFA-GM3 and LCFA-GM3 act as agonist and antagonist of TLR4 activity, respectively, by differentially binding to the hydrophobic pocket of MD2. Our findings suggest that VLCFA-GM3 is a risk factor for TLR4-mediated disease progression. Synopsis Analysis of GM3 ganglioside composition in human serum under chronic inflammation conditions reveals that the fatty acid chain length of GM3 ganglioside impacts the inflammatory activation of macrophages via direct modulation of TLR4 signaling. GM3 ganglioside in human serum is composed of a variety of fatty acids including long-chain (LCFA) and very long-chain (VLCFA). Serum VLCFA-GM3 levels increase and LCFA-GM3 levels decrease in metabolic disorders. GM3 by itself has no effects on TLR4 activation; however, VLCFA-GM3 synergistically and selectively enhanced TLR4 activation by LPS/HMGB1while LCFA- and unsaturated VLCFA-GM3 suppress TLR4 activation. GM3 interacts with extracellular regions of TLR4/MD2 complex, and modulates dimerization/oligomerization. Ligand-macromolecular docking study suggested that VLCFA- and LCFA-GM3 act as agonist and antagonist against TLR4 activation, respectively, by differentially binding to hydrophobic pocket of MD2. VLCFA-GM3 could be a risk factor for TLR4-mediated disease progression. Introduction Chronic inflammation plays critical roles in pathogenesis of a variety of human diseases, including metabolic disorders (Lumeng, 2011; Hotamisligil, 2017). Prolonged and abnormal activation of pattern recognition receptors in innate immune system, such as Toll-like receptors (TLR; Kawai & Akira, 2011; Moresco et al, 2011), causes chronic inflammation. In metabolic disorders, various ligands activate TLR4: (i) exogenous lipopolysaccharides elevated in serum (Cani et al, 2007), (ii) endogenous damage-associated molecular patterns (DAMPs), e.g., high-mobility group box-1 protein (HMGB1; Harris et al, 2012; Guzmán-Ruiz et al, 2014), free fatty acids (FFAs; Shi et al, 2006), and fetuin-A protein (Pal et al, 2012), which are released from macrophages and adipose tissue. LPS and endogenous ligands induce production of various effector molecules including proinflammatory cytokines (e.g., tumor necrosis factor-α [TNF-α], interleukin-6 [IL-6]), which contributes to insulin resistance and dysregulation of lipid and energy metabolisms (Lumeng, 2011; Hotamisligil, 2017). Gangliosides are important downstream metabolites of ceramide, a sphingolipid formed by an amide linkage between the sphingoid base and fatty acid (Bikman & Summers, 2011), and involved in a variety of cellular events (Inokuchi et al, 2018). Glycosyltransferases, UGCG and B4GALT5/6, convert ceramide into glucosylceramide (GlcCer) and lactosylceramide (LacCer), precursor glycosphingolipids (GSLs) for GM3 ganglioside. Consecutively, ST3GAL5, a GM3 synthase (GM3S), converts LacCer into GM3 by conjugating a sialic acid (N-acetylneuraminic acid) (Fig 1A), which is followed by biosynthesis of complex gangliosides. Figure 1. Molecular species of ganglioside GM3 in human serum and the acyl-chain structures Biosynthetic pathway (schematic) of GM3, from ceramide, and to complex gangliosides. TLC analysis of ganglioside species in human serum. Quantification by densitometry of major ganglioside species GM3 and GD1a in human serum. Data expressed as mean ± SD, n = 6. Detailed structures of GM3 species: sialyllactose head group, sphingoid base (d18:1), typical fatty-acid lengths (LCFA, VLCFA), and acyl-chain modifications (α-hydroxylation, ω-9 desaturation). Quantification by LC-MS/MS analysis of serum GM3 species with differing acyl-chain structures. Total abundance of 10 species was defined as 1. Data expressed as mean ± SD, n = 6. Download figure Download PowerPoint GM3 is secreted abundantly into human serum (Fig 1B), with concentration 10–15 μg/ml (~10 μM) (Fig 1C), and is circulated to all parts of the body, including insulin-sensitive organs (e.g., liver, muscle, adipose; Senn et al, 1989; Veillon et al, 2015). Fatty acids of serum GM3 are composed of long-chain fatty acid (LCFA), 16:0, 18:0, and 20:0; very-long-chain fatty acid (VLCFA), 22:0, 23:0, and 24:0; unsaturated VLCFA, 22:1 and 24:1; and α-hydroxy VLCFA including h24:0 and h24:1 (Fig 1D), in almost same abundances of LCFA, VLCFA, and unsaturated VLCFA species, and a small amount of α-hydroxy species (Fig 1E). Altered expression of various GM3 species has been observed in patients with metabolic disorders (Veillon et al, 2015); however, specific biological functions of these species are poorly understood. On the other hand, it has been suggested that GM3 on the plasma membrane plays important roles in pathogenesis of metabolic disorders (Inokuchi et al, 2018). GM3 is also a major ganglioside in adipocytes, and its expression is induced by proinflammatory cytokines derived from adipose tissue macrophages (Tagami et al, 2002; Nagafuku et al, 2015; Wentworth et al, 2016). GM3 biosynthesis occurs in Golgi, and it subsequently becomes secreted into extracellular compartment or localized in plasma membrane as a component of membrane microdomains (also called "rafts"), which are signaling platforms comprised of sphingolipids (Lingwood & Simons, 2010). GM3 on plasma membrane affects diffusion kinetics of insulin receptors and regulates signal transduction (Kabayama et al, 2007); conversely, insulin signaling is restored when GM3 biosynthesis is blocked by glycosyltransferase inhibitors, e.g., D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) and Genz-123346 (Tagami et al, 2002; Zhao et al, 2007). Knockout of GM3S diminishes not only systemic insulin resistance but also chronic inflammation in adipose tissue (Yamashita et al, 2003; Nagafuku et al, 2015), suggesting that GM3 might be involved in innate immune signaling upstream of insulin resistance; however, the molecular basis for such relationship remains unclear. In this study, we investigated how serum GM3 species, carrying different acyl chains, regulate inflammatory signaling and contribute to onset of metabolic disorders. Here, we demonstrate that GM3 acts as an endogenous TLR4 modulator. VLCFA-GM3 synergistically and selectively augmented TLR4 activation by LPS and HMGB1, and in contrast, LCFA and unsaturated VLCFA-GM3 suppressed TLR4 activation. Serum VLCFA-GM3 increased significantly and LCFA-GM3 decreased sharply in metabolic disorders. Computational approaches using artificial intelligence revealed that specific GM3 species are related to clinical symptoms. VLCFA-GM3 also increased in the adipose tissue of obese mice and the increase was attenuated in TLR4-mutant mice, implying that TLR4 signaling itself is involved in production of VLCFA-GM3. Our findings suggest that serum GM3 plays a role of rheostat for TLR4 signaling, and the increase in VLCFA-GM3 is a risk factor for TLR4-mediated disease progression. Results VLCFA-GM3 species are involved in progression of chronic inflammation in metabolic disorders To elucidate the role of GM3 species in pathophysiology of metabolic disorders, we analyzed expression patterns of serum GM3 species in human subjects (Veillon et al, 2015; Appendix Fig S1A–I). Sera were collected from human subjects, representing five pathological phases: healthy subjects (control, n = 24), visceral fat accumulation (VFA, n = 38) in presymptomatic phase, VFA with dyslipidemia (lipidemia, n = 28), VFA with hyperglycemia (glycemia, n = 15), and VFA with dyslipidemia and hyperglycemia (lipidemia + glycemia, n = 17). Scores of homeostatic model assessment for insulin resistance (HOMA-IR) and serum C-reactive protein (CRP) were evaluated as indicators of insulin resistance and chronic inflammation, respectively. HOMA-IR and CRP displayed significant correlation with each other (Appendix Fig S1J), and a gradual increase in the order: control < VFA < lipidemia < glycemia < lipidemia + glycemia (Appendix Fig S1K and L). These findings indicate that the order of the five phases corresponds to increasing severity of insulin resistance and chronic inflammation. Circulating levels of serum GM3 species were evaluated by LC-MS/MS analysis (Appendix Fig S2A–K). Heat map analysis, which summarizes properties of the ten major species, indicated progressive increase in VLCFA species and decrease in LCFA species in association with increases in HOMA-IR and serum CRP (Fig 2A). LCFA species (16:0, 18:0, 20:0) decreased sharply in VFA, lipidemia, and glycemia (Fig 2B), whereas VLCFA species (22:0, 23:0, 24:0, h24:0) largely increased (Fig 2C). Unsaturated VLCFA species were mostly constant as total (Fig 2D); 22:1 and h24:1 decreased, but 24:1 slightly increased (Fig 2A). The ratio of total VLCFA species to total LCFA/ unsaturated VLCFA species increased notably in presymptomatic and early phases of metabolic disorders (Appendix Fig S2L). Figure 2. Alterations of relative abundance of GM3 species are involved in disease progression and chronic inflammation A. Heat map analysis of serum GM3 species in various pathological phases: control (n = 24), VFA (n = 38), lipidemia (n = 28), glycemia (n = 15), and lipidemia + glycemia (n = 17). Colors indicate fold change average of each species relative to control (defined as 1), as shown in key at right. Order of pathological phases corresponds to increments of HOMA-IR and serum CRP. B–D. Properties of various GM3 species as a function of pathological phases: control (n = 24), VFA (n = 38), lipidemia (n = 28), glycemia (n = 15), and lipidemia + glycemia (n = 17). Data shown are relative abundances of total LCFA species (16:0, 18:0, 20:0) (B), total VLCFA species (22:0, 23:0, 24:0, h24:0) (C), and total unsaturated VLCFA species (22:1, 24:1, h24:1) (D) relative to total of ten major GM3 species (defined as 1) in each subject. E–H. Properties of various GM3 species as a function of BMI: LCFA-GM3 (E), VLCFA-GM3 (F), unsaturated VLCFA-GM3 (G), and α-hydroxy VLCFA-GM3 (h24:0) (H). Colors indicate disease severity: light blue, no abnormal scores (n = 25); orange, early-phase obesity (n = 74); purple, severe obesity (n = 23). I, J. Spearman's correlations for GM3 h24:0 vs. ALT (I) and vs. HOMA-IR (J). K. Plots of α-hydroxylation rate (h24:0/24:0) vs. serum CRP. Colors indicate range of CRP value (mg/dl): light blue, 0.01–0.02 (n = 21); orange, 0.03–0.09 (n = 56); gray, 0.10–0.29 (n = 29); red, 0.3–1.0 (diagnostically abnormal; n = 15). L. Association between serum GM3 species and progression of metabolic disorders (schematic). Data information: Data shown are individual values and mean ± SD, analyzed by two-tailed unpaired t-test with Bonferroni's correction. *P < 0.05, **P < 0.01, and ***P < 0.001 for comparisons between indicated groups. Download figure Download PowerPoint Early-phase increases in body mass index (BMI) (> 25) or abdominal circumference (> 85 cm) were associated with sharp reduction in LCFA species (Figs 2E and EV1A) and increase in VLCFA species (Figs 2F and EV1B). These findings suggest that increases in VLCFA-GM3 species occur in obesity, and play a role in early pathogenesis of metabolic disorders. In cases of severe obesity (BMI > 30 and/or abdominal circumference > 100 cm) and severe metabolic disorders (lipidemia + glycemia), there was moderate reduction in VLCFA-GM3 species (Figs 2F and EV1B) and significant increase in unsaturated species (Figs 2G and EV1C). These findings indicate that desaturation of VLCFA species occurs after onset of metabolic disorders. Click here to expand this figure. Figure EV1. Properties of various GM3 species as a function of clinical markers of metabolic disorders and chronic inflammation A–D. LCFA species (A), VLCFA species (B), unsaturated VLCFA species (C), and α-hydroxy VLCFA-GM3 (h24:0) (D). Colors indicate disease severity: light blue, no abnormal scores (n = 17); orange, early-phase obesity (n = 80); purple, severe obesity (n = 25). Data shown are mean ± SD, analyzed by two-tailed unpaired t-test with Bonferroni's correction. *P < 0.05, **P < 0.01, and ***P < 0.001 for comparisons between indicated groups. E–J. Spearman's correlations for GM3 h24:0 vs. BMI (E), GM3 h24:0 vs. abdominal circumference (F), total of α-hydroxy GM3 (h24:0 and h24:1) vs. HOMA-IR (G), GM3 h24:0 vs. serum CRP (H), α-hydroxylation rate (h24:0 to 24:0) vs. serum CRP (I), and α-hydroxylation rate (h24:0 and h24:1 to 24:0 and 24:1) vs. serum CRP (J). Data information: Sample sizes: (A–G), n = 122; (H-J), n = 121. Download figure Download PowerPoint Abundance of α-hydroxy VLCFA-GM3 (h24:0) showed a linear increase along with increases in BMI and abdominal circumference (Figs 2H and EV1D), with strong correlation (Fig EV1E and F). α-hydroxy VLCFA-GM3 was also strongly correlated with indicators of insulin resistance and chronic inflammation (ALT, HOMA-IR, CRP) (Figs 2I and J, and EV1G–J). In particular, the ratio of h24:0 to 24:0 was much higher in subjects with abnormal CRP value (> 0.3 mg/dl) (Figs 2K and EV1I), indicating considerable involvement of h24:0 in chronic inflammation. Relationships between these GM3 species and pathophysiology of metabolic disorders are summarized schematically in Fig 2L. In steady state, homeostasis is maintained by balance of GM3 species; in presymptomatic and early phases, VLCFA species increase in correlation with chronic inflammation and insulin resistance; in late phases, modifications such as desaturation and α-hydroxylation could occur in VLCFA species. Artificial intelligence-based approaches revealed GM3 species specific to disease symptoms To analyze more detailed relationships between GM3 species and metabolic disorders, we utilized an unbiased approach using self-organization map (SOM), a neural-network-type artificial intelligence
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