Effect of MHY908 on body weight, plasma glucose, TG, and insulin levels in db/db mice.

<p>Mice were treated for 4/kg/day in food. Food intakes and body weights were similar in the Con group and MHY908- treated groups (n = 8/group, eight-weeks old). (A) Changes in body weights. (B) A series of plasma profiles from CR and MHY908 treated db/db mice. One-factor ANOVA was used to determine the significances of differences: *** p<0.001, ** p<0.01 and * p<0.05 versus the Con group. Lean; db/m mice, Con; db/db mice, CR; calorie restriction, 1 mg; MHY 908 1 mg/kg/day, 3 mg; MHY 908 3 mg/kg/day.</p

MHY908 improved hepatic steatosis in db/db mouse livers.

<p>(A) A histological examination based on hematoxylin-eosin staining showed marked fatty changes in the livers of db/db mice. (Original magnification 100 X) (B) Accumulated lipids in the livers of MHY908-treated db/db mice. One-factor ANOVA was used to determine the significances of differences: *** p<0.001, ** p<0.01 and * p<0.05 versus the Con group. (C) CPT-1 expression was assessed by immunohistochemistry (Original magnification 200 X). Lean; db/m mice, Con; db/db mice, CR; calorie restriction, 1 mg; MHY 908 1 mg/kg/day, 3 mg; MHY 908 3 mg/kg/day.</p

Src Tyrosine Kinase Activation by 4-Hydroxynonenal Upregulates p38, ERK/AP-1 Signaling and COX-2 Expression in YPEN-1 Cells

<div><p>4-Hydroxynonenal (4-HNE), a major end product of lipid peroxidation, is highly reactive and involved in various cellular processes, such as inflammatory signaling. However, to date, the mechanistic roles of 4-HNE in inflammatory signaling related to protein tyrosine kinases have not been elucidated. In the present study, we investigated the interaction between 4-HNE and Src (a non-receptor tyrosine kinase) for its involvement in the molecular modulation of the inflammatory signaling pathway utilizing the YPEN-1 cell system. Immunoprecipitation experiments showed that 4-HNE phosphorylates (activates) Src at Tyr416 via adduct formation. In addition, LC-MS/MS and a docking simulation model revealed an addiction site at the Cys248 residue of Src, resulting in the stimulation of downstream p38, ERK/AP-1 and cyclooxygenase-2 (COX-2) signaling in YPEN-1 cells. The role of 4-HNE-activated Src in downstream inflammatory signaling was further investigated using dasatinib (a Src inhibitor) and by siRNA knockdown of Src. p38 and ERK were directly regulated by Src, as revealed by immunoblotting of the phosphorylated forms of mitogen-activated protein kinases (MAPKs), which are key elements in the signaling transduction pathway initiated by Src. The study also shows that Src modulates the HNE-enhanced activation of AP-1 and the expression of COX-2 (a target gene of AP-1). Together, the results of this study show that 4-HNE stimulates Src tyrosine kinase in activation of the inflammation process.</p></div

MHY908 modulated serum leptin and adiponectin levels in db/db mice.

<p>(A) Serum concentrations of leptin (n = 6) (B) Serum concentrations of adiponectin (n = 6) One-factor ANOVA was used to determine the significances of differences: *** p<0.001, ** p<0.01 and * p<0.05 versus the Con group. Lean; db/m mice, Con; db/db mice, CR; calorie restriction, 1 mg; MHY 908 1 mg/kg/day, 3 mg; MHY 908 3 mg/kg/day.</p

LXR Agonism Upregulates the Macrophage ABCA1/Syntrophin Protein Complex That Can Bind ApoA‑I and Stabilized ABCA1 Protein, but Complex Loss Does Not Inhibit Lipid Efflux

Macrophage ABCA1 effluxes lipid and has anti-inflammatory activity. The syntrophins, which are cytoplasmic PDZ protein scaffolding factors, can bind ABCA1 and modulate its activity. However, many of the data assessing the function of the ABCA1–syntrophin interaction are based on overexpression in nonmacrophage cells. To assess endogenous complex function in macrophages, we derived immortalized macrophages from <i>Abca1</i>^<i>+/+</i> and <i>Abca1</i>^{<i>–/–</i>} mice and show their phenotype recapitulates primary macrophages. <i>Abca1</i>^<i>+/+</i> lines express the CD11B and F4/80 macrophage markers and markedly upregulate cholesterol efflux in response to LXR nuclear hormone agonists. In contrast, immortalized <i>Abca1</i>^{<i>–/–</i>} macrophages show no efflux to apoA-I. In response to LPS, <i>Abca1</i>^{<i>–/–</i>} macrophages display pro-inflammatory changes, including an increased level of expression of cell surface CD14, and 11–26-fold higher levels of IL-6 and IL-12 mRNA. Given recapitulation of phenotype, we show with these lines that the ABCA1–syntrophin protein complex is upregulated by LXR agonists and can bind apoA-I. Moreover, in immortalized macrophages, combined α1/β2-syntrophin loss modulated ABCA1 cell surface levels and induced pro-inflammatory gene expression. However, loss of all three syntrophin isoforms known to bind ABCA1 did not impair lipid efflux in immortalized or primary macrophages. Thus, the ABCA1–syntrophin protein complex is not essential for ABCA1 macrophage lipid efflux but does directly interact with apoA-I and can modulate the pool of cell surface ABCA1 stabilized by apoA-I

Possible mechanism of the effects of MHY908 on overnutrition-induced insulin resistance.

<p>We suggest that overnutrition induced lipid accumulation and led to insulin resistance by increasing ER stress, and that MHY908 downregulated ER stress and improved insulin signaling in the livers of db/db mice.</p

Synthesis of MHY908.

<p>Reagents and conditions: (a) Ethyl-bromoisobutyrate, 1N-NaOEt, EtOH, reflux, 14 h, 72%; (b) Na₂S₂O₅, DMF, 80°C, 11 h, 31%; (c) 1N-NaOH, 1,4-dioxane, rt, 17 h, 79%; (d) 1N-NaOH, 1,4-dioxane, rt, 4 h, 99.9%; (e) NaOAc, AcOH, reflux, 1 h, 40%.</p

Effects of Src on HNE-induced COX-2 expression in YPEN cells.

<p>(A) YPEN-1 cells were stimulated with 5 μM HNE for the indicated times (0–12 h). COX-2 protein expressions in cell lysates were analyzed by immunoblotting. (B) After pretreatment with dasatinib (300 nM) for 30 min, cells were stimulated with 5 μM 4-HNE for 9 h. (C) YPEN-1 cells were transfected with Src siRNA for 48 h and then treated with 5 μM HNE for 9 h. COX-2 protein expression levels were determined by immunoblotting. CON, control; NC, negative control; si, Src siRNA-treated cells; HNE, 4-hydroxy-2-nonenal; COX-2, cyclooxygenase-2.</p

Detailed information on the hydrogen bonding networks formed by fenofibrate, rosiglitazone, and MHY908.

<p>Detailed information on the hydrogen bonding networks formed by fenofibrate, rosiglitazone, and MHY908.</p

LXR Agonism Upregulates the Macrophage ABCA1/Syntrophin Protein Complex That Can Bind ApoA‑I and Stabilized ABCA1 Protein, but Complex Loss Does Not Inhibit Lipid Efflux

Macrophage ABCA1 effluxes lipid and has anti-inflammatory activity. The syntrophins, which are cytoplasmic PDZ protein scaffolding factors, can bind ABCA1 and modulate its activity. However, many of the data assessing the function of the ABCA1–syntrophin interaction are based on overexpression in nonmacrophage cells. To assess endogenous complex function in macrophages, we derived immortalized macrophages from <i>Abca1</i>^<i>+/+</i> and <i>Abca1</i>^{<i>–/–</i>} mice and show their phenotype recapitulates primary macrophages. <i>Abca1</i>^<i>+/+</i> lines express the CD11B and F4/80 macrophage markers and markedly upregulate cholesterol efflux in response to LXR nuclear hormone agonists. In contrast, immortalized <i>Abca1</i>^{<i>–/–</i>} macrophages show no efflux to apoA-I. In response to LPS, <i>Abca1</i>^{<i>–/–</i>} macrophages display pro-inflammatory changes, including an increased level of expression of cell surface CD14, and 11–26-fold higher levels of IL-6 and IL-12 mRNA. Given recapitulation of phenotype, we show with these lines that the ABCA1–syntrophin protein complex is upregulated by LXR agonists and can bind apoA-I. Moreover, in immortalized macrophages, combined α1/β2-syntrophin loss modulated ABCA1 cell surface levels and induced pro-inflammatory gene expression. However, loss of all three syntrophin isoforms known to bind ABCA1 did not impair lipid efflux in immortalized or primary macrophages. Thus, the ABCA1–syntrophin protein complex is not essential for ABCA1 macrophage lipid efflux but does directly interact with apoA-I and can modulate the pool of cell surface ABCA1 stabilized by apoA-I