Effets de la protéine stimulant l'acylation dans l'interaction des adipocytes et des macrophages

L'obésité est maintenant reconnue comme un état inflammatoire chronique de faible ampleur caractérisé par une surproduction anormale de facteurs inflammatoires par le tissu adipeux. La protéine stimulant l'acylation (ASP) stimule la synthèse des triglycérides et le transport du glucose via son récepteur, le C5L2. L'objectif de ce projet était d'examiner les effets de l'ASP dans un contexte inflammatoire relié à l'obésité. Les résultats ont démontré une augmentation de la sécrétion de cytokines pro-inflammatoires après un traitement avec l'ASP chez les adipocytes mais non chez les macrophages. Parallèlement, le C5a (une anaphylatoxine se liant également au C5L2) inhibe en général la sécrétion des adipokines autant chez les adipocytes que chez les macrophages. Un effet synergique de l'ASP sur la production de MCP-1 et de l'adiponectine est observé lorsque les macrophages sont mis en contact direct avec les adipocytes. En contrepartie, l'ajout de l'ASP dans du milieu conditionné de macrophages n'a pas démontré d'effet additionnel sur la sécrétion des adipokines chez les adipocytes. Nos données ont également permis de démontrer que la réponse inflammatoire induite par l'ASP est en partie régulée par les voies de signalisation NFKB et PI3Kinase chez les adipocytes. Ces résultats suggèrent que l'ASP jouerait un rôle amplificateur dans la réponse inflammatoire des adipocytes, mais pas des macrophages, en stimulant la sécrétion de certains médiateurs inflammatoires via NFKB et PBKinase. Une compréhension détaillée des implications physiologiques de l'ASP permettrait éventuellement de saisir son potentiel thérapeutique

Deficiency of C5L2 Increases Macrophage Infiltration and Alters Adipose Tissue Function in Mice

<div><p>Background</p><p>Obesity is considered as a systemic chronic low grade inflammation characterized by increased serum pro-inflammatory proteins and accumulation of macrophages within white adipose tissue (WAT) of obese patients. C5L2, a 7-transmembrane receptor, serves a dual function, binding the lipogenic hormone acylation stimulating protein (ASP), and C5a, involved in innate immunity.</p><p>Aim</p><p>We evaluated the impact of C5L2 on macrophage infiltration in WAT of wildtype (Ctl) and C5L2 knock-out (C5L2^−/−) mice over 6, 12 and 24 weeks on a chow diet and moderate diet-induced obesity (DIO) conditions.</p><p>Results</p><p>In Ctl mice, WAT C5L2 and C5a receptor mRNA increased (up to 10-fold) both over time and with DIO. By contrast, in C5L2^−/−, there was no change in C5aR in WAT. C5L2^−/− mice displayed higher macrophage content in WAT, varying by time, fat depot and diet, associated with altered systemic and WAT cytokine patterns compared to Ctl mice. However, in all cases, the M1 (pro-) vs M2 (anti-inflammatory) macrophage proportion was unchanged but C5L2^−/− adipose tissue secretome appeared to be more chemoattractant. Moreover, C5L2^−/− mice have increased food intake, increased WAT, and altered WAT lipid gene expression, which is reflected systemically. Furthermore, C5L2^−/− mice have altered glucose/insulin metabolism, adiponectin and insulin signalling gene expression in WAT, which could contribute to development of insulin resistance.</p><p>Conclusion</p><p>Disruption of C5L2 increases macrophage presence in WAT, contributing to obesity-associated pathologies, and further supports a dual role of complement in WAT. Understanding this effect of the complement system pathway could contribute to targeting treatment of obesity and its comorbidities.</p></div

Systemic and adipose tissue cytokine profile in mice.

<p>Mouse cytokine profiler analysis in pooled plasma: (A) Data are expressed as mean±SEM of n = 8 Ctl and C5L2^−/− mice for each time point and for each diet. (B) Plasma keratinocyte derived chemokine (KC) analysis of Ctl (circle and dotted line) and C5L2^−/− (square and solid line) of mice on chow diet (white circle or square) or DIO (black circle or square) over 6, 12 and 24 weeks. Retroperirenal adipose tissue mRNA expression of interleukin 6 (IL-6) (C) and matrix metalloproteinase 3 (MMP3) (D) in Ctl (circle and dotted line) and C5L2^−/− (square solid line) mice on chow diet (white circle or square) or DIO (black circle or square) over 6, 12 and 24 weeks. Data are expressed as relative expression (re) mean±SEM (n = 4–5 mice per group) compared to 6 weeks chow diet Ctl set at 1. Evaluation of monocyte chemoattractant protein-1 (MCP-1) (E) and KC (F) in adipocyte primary cell culture media for 24 h isolated from perigonadal adipose tissue after 6, 12 and 24 weeks on chow diet (white circle or square) and DIO (black circle or square) of Ctl (circle and dotted line) and C5L2^−/− mice (square and solid line). Data are presented as means±SEM (3–10 adipose tissue media per group) over time and expressed as pg/mL or ng/mL per gram (g) of tissue used. Paired t-test for each plasma cytokine value where * <i>p</i><0.05 for C5L2^−/− vs. Ctl (A) and two-way (2-way) ANOVA for genotype × time between Ctl and C5L2^−/− for each diet or adipose tissue or media with a Bonferroni post-test where * <i>p</i><0.05 and ** <i>p</i><0.01 and *** <i>p</i><0.001 for C5L2^−/− vs. Ctl on same diet or adipose tissue or media. Abbreviations: Il-12(p40), subunit beta of interleukin 12; G-CSF, granulocyte colony stimulating factor; RANTES, Regulated upon Activation, Normal T-cell Expressed and Secreted or CCL5.</p

Chemoattractant receptors and related complement proteins in mouse adipose tissue.

<p>Retroperirenal adipose tissue mRNA gene expression of complement receptors C5L2 (A), C5aR (B) and C3aR (C) analysis of wild-type control (Ctl) (circle and dotted line) and C5L2^−/− (square and solid line) mice on chow diet (white circle or square) or diet induced obesity (DIO) (black circle or square) diet over 6, 12 and 24 weeks. The results are expressed as relative expression (re) compared to 6 weeks chow diet Ctl set as 1. Complement C5a production by primary isolated adipocytes of inguinal (D) and perigonadal (E) adipose tissue in Ctl (white bars) and C5L2^−/− (black bars) mice on chow diet (plain bars) or DIO (hatched bars). Data are presented as mean±SEM (n = 4–10 mice per group) with two-way (2-way) ANOVA for genotype × time between Ctl and C5L2^−/− for each diet with a Bonferroni post-test where * <i>p</i><0.05 and *** <i>p</i><0.001 for Ctl vs. C5L2^−/− for the same diet.</p

Adipose tissue lipid metabolism gene expression.

<p>Retroperirenal adipose tissue mRNA gene expression for genes involved in lipid metabolism in Ctl (circle and dotted line) and C5L2^−/− (square and solid line) mice on chow diet (white circle or square) and DIO (black circle or square). Adipogenesis-related genes (A–B); triglyceride synthesis-related genes (C–D); cell membrane fatty acid transport-related genes (E–F); thermogenesis-related genes (G–H) and mitochondrial long chain fatty acid transport-related genes (I). Data are expressed as relative expression (re) mean±SEM (n = 4–5 mice per group) compared to 6 weeks chow diet Ctl set as 1 where two-way (2-way) ANOVA for genotype × time between Ctl and C5L2^−/− for each diet or adipose tissue with a Bonferroni post-test where * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001 for C5L2^−/− vs. Ctl. Abbreviations: CEBPA, CCAAT/enhancer-binding protein α; PPARG, peroxisome proliferator-activated receptor γ; DGAT1 and 2, diacylglycerol O- acyltransferase 1 and 2; FABP3, fatty acid binding protein 3; CD36 or FAT, fatty acid transporter; UCP1 and 3, uncoupling protein 1 and 3; CPT1B, carnitine palmitoyltransferase 1B.</p

Insulin metabolism and insulin resistance markers in mice.

<p>Evaluation of adiponectin in Ctl (white bars) and C5L2^−/− (black bars) adipocyte primary cell culture media for 24 h isolated from inguinal (A) and perigonadal (B) adipose tissue after 6, 12 and 24 weeks on chow diet and DIO. Data are presented as means±SEM (3–10 adipose tissue media per group) over time and expressed as pg/mL or ng/mL per gram (g) of tissue used. Retroperirenal adipose tissue mRNA gene expression for genes related to insulin resistance (C–D) from Ctl (circle and dotted line) and C5L2^−/− (square and solid line) mice on chow diet (white circle or square) and DIO (black circle or square). Data are expressed as relative expression (re) mean±SEM (n = 4–5 mice per group) compared to 6 weeks chow diet Ctl set as 1. Plasma levels of adiponectin (E), glucose (F) and insulin in Ctl (circle and dotted line) and C5L2^−/− (square and solid line) mice on chow diet (white circle or square) and DIO (black circle or square). Data are presented as mean±SEM (n = 8–10 mice per group) over time with two-way (2-way) ANOVA for genotype × time between Ctl and C5L2^−/− for each diet or adipose tissue with a Bonferroni post-test where * <i>p</i><0.05, ** <i>p</i><0.01 and *** <i>p</i><0.001 for C5L2^−/− vs. Ctl. Abbreviations: SOCS3, suppressor of cytokine signaling 3; NOS2, nitric oxide synthase 2.</p

Flow cytometry analysis of F4/80 and CD11c (M1 pro-inflammatory) macrophage markers in mouse inguinal and perigonadal adipose depots.

<p>Percentage of macrophages based on F4/80 (A and B) and CD11c (C and D) markers in inguinal (A–C) and perigonadal (B–D) stromal vascular fraction (SVF) of adipose tissue after 6, 12 and 24 weeks of chow diet (white circle or square) and DIO (black circle or square) for Ctl (circle and dotted line) and C5L2^−/− (square and solid line) mice. Data are presented as percentage of F4/80 positive cells (total macrophage) over total cells of the SVF (A–B) or as CD11c percent of positive cells relative to total macrophages (C–D) as mean±SEM (n = 4–8 adipose tissues per group). All data were analysed with two-way (2-way) ANOVA with genotype × time between Ctl and C5L2^−/− for each adipose tissue with a Bonferroni post-test where * <i>p</i><0.05 and ** <i>p</i><0.01 for Ctl vs. C5L2^−/− for the same diet in the same tissue. Additional 2-way ANOVA for diet × time for % macrophages indicates a diet effect in inguinal (A) for Ctl (<i>p</i> = 0.028) and C5L2^−/− (<i>p</i> = NS) and in perigonadal (B) for Ctl (<i>p</i> = 0.05) and C5L2^−/− (<i>p</i> = 0.01).</p

Gene expression of macrophage type distribution in mouse retroperirenal adipose depot and monocyte infiltration response to adipose tissue conditionated-media adipokine.

<p>Retroperirenal adipose tissue mRNA expressions of F4/80 (total macrophage, A), CD11c (type M1, B) and CD163 (type M2, C) in Ctl (circle and dotted line) and C5L2^−/− (square and solid line) on chow diet (white circle or square) or DIO (black circle of square) over 6, 12 and 24 weeks. Data are expressed as relative expression (re) mean±SEM (n = 4–5 mice per group) compared to 6 weeks, chow diet Ctl set as 1. All data were analysed with two-way (2-way) ANOVA for genotype × time between Ctl and C5L2^−/− for each diet or adipose tissue with a Bonferroni post-test where * <i>p</i><0.05 and ** <i>p</i><0.001 for Ctl vs. C5L2^−/− for the same diet or tissue. Monocyte migration was assessed in bone marrow monocytes isolated from Ctl and C5L2^−/− mice. Cells were incubated without treatment (Basal) or with conditioned media (CM) derived from gonadal adipose tissue from Ctl (Ctl CM) or C5L2^−/− (C5L2^−/− CM) mice (D). Data are expressed as relative expression (re) of monocyte migration mean±SEM (n = 3–5 migration experiments per condition). The data were analysed with regular one-way ANOVA with Bonferroni multiple comparisons test, where “&” indicates significant difference from basal condition, “#” indicates significant difference from Ctl CM and “*” indicate significant difference between the genotype of monocyte for the same condition, all <i>p</i><0.05. Additional 2-way ANOVA for diet × time for F4/80 expression indicates a diet effect in retroperirenal (A) for Ctl (<i>p</i> = 0.0001) and C5L2^−/− (<i>p</i><0.0001).</p

Lung stiffness of C57BL/6 versus BALB/c mice

Abstract This study was undertaken to determine whether a smaller lung volume or a stiffer lung tissue accounts for the greater lung elastance of C57BL/6 than BALB/c mice. The mechanical properties of the respiratory system and lung volumes were measured with the flexiVent and compared between male C57BL/6 and BALB/c mice (n = 9). The size of the excised lung was also measured by volume liquid displacement. One lobe was then subjected to sinusoidal strains in vitro to directly assess the mechanical properties of the lung tissue, and another one was used to quantify the content of hydroxyproline. In vivo elastance was markedly greater in C57BL/6 than BALB/c mice based on 5 different readouts. For example, respiratory system elastance was 24.5 ± 1.7 vs. 21.5 ± 2.4 cmH2O/mL in C57BL/6 and BALB/c mice, respectively (p = 0.007). This was not due to a different lung volume measured by displaced liquid volume. On the isolated lobes, both elastance and the hydroxyproline content were significantly greater in C57BL/6 than BALB/c mice. These results suggest that the lung elastance of C57BL/6 mice is greater than BALB/c mice not because of a smaller lung volume but because of a stiffer lung tissue due to a greater content of collagen