Immunometabolic aspect of C5L2 and C5aR in adiposity : physical, functional and physiological interactions

L’obésité est maintenant reconnue comme présentant une inflammation chronique et est caractérisée par une augmentation de l’infiltration de macrophages dans le tissu adipeux. Cette infiltration est réflètée par l’activation du système du complément qui agit comme élément déclencheur et précurseur aux autres fonctions immunitaires. Le C5L2 (C5aR-like receptor 2) a récemment été identifié comme récepteur pour la protéine stimulant l’acylation (ASP) ainsi que pour le facteur inflammatoire C5a (capable quant à lui d’aussi se lier au C5aR). Cette thèse porte sur: (i) l’interaction entre C5L2 et C5aR suite à l’activation de ceux-ci par leurs ligands respectifs dans des lignées cellulaires adipocytaires (3T3-L1) et de macrophages (J774), (ii) la contribution du C5aR au métabolisme et à la réponse immunitaire d’adipocytes provenant de modèles murins et (iii) l’association entre C5L2 et C5aR avec des facteurs reliés à l’obésité chez l’humain. Les récepteurs C5L2 et C5aR s’associent entre-eux de façon constitutive en homo- et hétérodimères et l’ajout de ligand à des cultures d’adipocytes 3T3-L1 ou de macrophages J774 augmente cette colocalisation. Autant l’ASP que le C5a ont réussi à induire une réponse fonctionnelle chez des adipocytes primaires. Lorsque des adipocytes primaires provenaient de souris invalidées pour le C5aR, les effets d’un traitement avec C5a étaient perturbés tandis que les effets médiés par un traitement à l’ASP étaient maintenus. De plus, l’addition de C5a bloquait la signalisation et la réponse fonctionnelle causées par un traitement à l’ASP chez les adipocytes primaires provenant de souris C5aRKO et de type sauvage. Finalement, l’expression des gènes C5L2 et C5aR dans le tissu adipeux de femmes obèses morbides était associée avec une adiposité croissante. De façon interessante, les ratios ASP/C5L2 et C5L2/C5aR augmentaient de façon significative avec l’obésité abdominale. Lorsque pris ensemble, l’interaction fonctionelle, physiologique et la proximité physique des récepteurs C5L2 et C5aR chez les adipocytes suggèrent un rôle potentiel de ceux-ci sur l’immunométabolisme du tissu adipeux. De surcroit, cette interaction met en valeur les liens qui existent entre le tissu adipeux et les protéines et récepteurs du complément tout en démontrant comment une réponse excessive au niveau immuno-métabolique pourrait exacerber le développement du niveau d’adiposité chez l’individu.From the convergence of metabolism and immune research has emerged a new research field, termed “Immunometabolism”. Obesity, an immunometabolic disease, is associated with a state of low-grade inflammation and is characterized by increased infiltration of macrophages into adipose tissue. Complement activation can act as an early trigger and precursor of other immune functions. C5aR-like receptor 2 (C5L2) has been identified as a receptor for Acylation Stimulating Protein (ASP) and the inflammatory factor C5a (which can also bind C5aR). This thesis sequentially evaluates (i) ligand-induced C5L2 and C5aR interaction in cultured 3T3-L1 adipocytes and J774 macrophages, (ii) the C5aR contribution in adipocyte metabolic and immune responses in mouse models, (iii) as well as C5L2 and C5aR association with obesity-related factors in humans. The immunometabolic receptors, C5L2 and C5aR, constitutively self-associate into homo-/heterodimers and ligand treatment of 3T3-L1 adipocytes and J774 macrophages increased their colocalization. Both C5a and ASP directly induced primary adipocyte signaling and function. However, in C5aRKO primary adipocytes, C5a effects were disrupted, while stimulatory effects of ASP were mostly maintained. Moreover, addition of C5a completely blocked ASP signaling and activity in both C5aRKO and WT primary adipocytes. Finally, C5L2 and C5aR expression in adipose tissue from morbidly obese women was associated with increased adiposity. Interestingly, ASP/C5L2 and C5L2/C5aR ratio markedly increased with abdominal obesity. Taken together, the closely linked physical, functional and physiological interaction between C5L2 and C5aR in adipocytes suggests a potential role in adipose tissue immunometabolism. This further highlights the important new links between adipose tissue and complement proteins/receptors and demonstrates how excessive immunometabolic responses may exacerbate adiposity

C5a Receptor Deficiency Alters Energy Utilization and Fat Storage

Objective: To investigate the impact of whole body C5a receptor (C5aR) deficiency on energy metabolism and fat storage. Design: Male wildtype (WT) and C5aR knockout (C5aRKO) mice were fed a low fat (CHOW) or a high fat high sucrose diet-induced obesity (DIO) diet for 14 weeks. Body weight and food intake were measured weekly. Indirect calorimetry, dietary fatload clearance, insulin and glucose tolerance tests were also evaluated. Liver, muscle and adipose tissue mRNA gene expression were measured by RT-PCR. Results: At week one and 12, C5aRKO mice on DIO had increased oxygen consumption. After 12 weeks, although food intake was comparable, C5aRKO mice had lower body weight (−7% CHOW, −12% DIO) as well as smaller gonadal (−38% CHOW, −36% DIO) and inguinal (−29% CHOW, −30% DIO) fat pads than their WT counterparts. Conversely, in WT mice, C5aR was upregulated in DIO vs CHOW diets in gonadal adipose tissue, muscle and liver, while C5L2 mRNA expression was lower in C5aRKO on both diet. Furthermore, blood analysis showed lower plasma triglyceride and non-esterified fatty acid levels in both C5aRKO groups, with faster postprandial triglyceride clearance after a fatload. Additionally, C5aRKO mice showed lower CD36 expression in gonadal and muscle on both diets, while DGAT1 expression was higher in gonadal (CHOW) and liver (CHOW and DIO) and PPARγ was increased in muscle and liver. Conclusion: These observations point towards a role (either direct or indirect) for C5aR in energy expenditure and fat storage, suggesting a dual role for C5aR in metabolism as well as in immunity

α/β-Hydrolase Domain 6 Deletion Induces Adipose Browning and Prevents Obesity and Type 2 Diabetes

SummarySuppression of α/β-domain hydrolase-6 (ABHD6), a monoacylglycerol (MAG) hydrolase, promotes glucose-stimulated insulin secretion by pancreatic β cells. We report here that high-fat-diet-fed ABHD6-KO mice show modestly reduced food intake, decreased body weight gain and glycemia, improved glucose tolerance and insulin sensitivity, and enhanced locomotor activity. ABHD6-KO mice also show increased energy expenditure, cold-induced thermogenesis, brown adipose UCP1 expression, fatty acid oxidation, and white adipose browning. Adipose browning and cold-induced thermogenesis are replicated by the ABHD6 inhibitor WWL70 and by antisense oligonucleotides targeting ABHD6. Our evidence suggests that one mechanism by which the lipolysis derived 1-MAG signals intrinsic and cell-autonomous adipose browning is via PPARα and PPARγ activation, and that ABHD6 regulates adipose browning by controlling signal competent 1-MAG levels. Thus, ABHD6 regulates energy homeostasis, brown adipose function, and white adipose browning and is a potential therapeutic target for obesity and type 2 diabetes

Association of Immune and Metabolic Receptors C5aR and C5L2 with Adiposity in Women

Adipose tissue receptors C5aR and C5L2 and their heterodimerization/functionality and interaction with ligands C5a and acylation stimulating protein (ASP) have been evaluated in cell and rodent studies. Their contribution to obesity factors in humans remains unclear. We hypothesized that C5a receptors, classically required for host defense, are also associated with adiposity. Anthropometry and fasting blood parameters were measured in 136 women divided by body mass index (BMI): normal/overweight (≤30 kg/m2; n = 34), obese I (≤45 kg/m2; n = 33), obese II (≤51 kg/m2; n = 33), and obese III (≤80 kg/m2; n = 36). Subcutaneous and omental adipose tissue C5aR and C5L2 expression were analysed. C5L2 expression was comparable between subcutaneous and omental across all BMI groups. Plasma ASP and ASP/omental C5L2 expression increased with BMI (P < 0.001 and P < 0.01, resp.). While plasma C5a was unchanged, C5aR expression decreased with increasing BMI in subcutaneous and omental tissues (P < 0.01 and P < 0.05, resp.), with subcutaneous omental depots. Omental C5L2/C5aR ratio increased with BMI (P < 0.01) with correlations between C5L2/C5aR and waist circumference, HDL-C, and adiponectin. Tissue and BMI differences in receptors and ligands, particularly in omental, suggest relationship to metabolic disturbances and highlight adipose-immune interactions

Paradoxical Glucose-Sensitizing yet Proinflammatory Effects of Acute ASP Administration in Mice

Acylation stimulating protein (ASP) is an adipokine derived from the immune complement system, which stimulates fat storage and is typically increased in obesity, type 2 diabetes, and cardiovascular disease. Using a diet-induced obesity (DIO) mouse model, the acute effects of ASP on energy metabolism and inflammatory processes in vivo were evaluated. We hypothesized that ASP would specifically exert proinflammatory effects. C57Bl/6 wild-type mice were put on a high-fat-high-sucrose diet for 12 weeks. Mice were then subjected to both glucose and insulin tolerance tests, each manipulation being preceded by recombinant ASP or vehicle (control) bolus injection. ASP supplementation increased whole-body glucose excursion, and this was accomplished with reduced concomitant insulin levels. However, ASP did not directly alter insulin sensitivity. ASP supplementation induced a proinflammatory phenotype, with higher levels of cytokines including IL-6 and TNF-α in plasma and in adipose tissue, liver, and skeletal muscle mRNA. Additionally, ASP increased M1 macrophage content of these tissues. ASP exerted a direct concentration-dependent role in the migration and M1 activation of cultured macrophages. Altogether, the in vivo and in vitro experiments demonstrate that ASP plays a role in both energy metabolism and inflammation, with paradoxical whole-body glucose-sensitizing yet proinflammatory effects

Downregulation of complement C3 and C3aR expression in subcutaneous adipose tissue in obese women.

The central component of the complement system, C3, is associated with obesity, metabolic syndrome and cardiovascular disease however the underlying reasons are unknown. In the present study we evaluated gene expression of C3, the cleavage product C3a/C3adesArg and its cognate receptor C3aR in subcutaneous and omental adipose tissue in women.Women (n = 140, 21-69 years, BMI 19.5-79 kg/m2) were evaluated for anthropometric and blood parameters, and adipose tissue gene expression.Subjects were separated into groups (n = 34-36) according to obesity: normal/overweight (≤30 kg/m2), obese I (≤45 kg/m2), obese II (≤51 kg/m2), and obese III (≤80 kg/m2). Overall, while omental expression remained unchanged, subcutaneous C3 and C3aR gene expression decreased with increasing adiposity (2-way ANOVA, p<0.01), with a concomitant decrease in SC/OM ratio (p<0.001). In subcutaneous adipose, both C3 and C3aR expression correlated with apoB, and apoA1 and inversely with waist circumference and blood pressure, while C3aR also correlated with glucose (p<0.05-0.0001). While omental C3aR expression did not correlate with any factor, omental C3 correlated with waist circumference, glucose and apoB (all p<0.05). Further, while plasma C3a/C3adesArg increased and adiponectin decreased with increasing BMI, both correlated (C3a negatively and adiponectin positively) with subcutaneous C3 and C3aR expression (p<0.05-0.001) or less).The obesity-induced down-regulation of complement C3 and C3aR which is specific to subcutaneous adipose tissue, coupled to the strong correlations with multiple anthropometric, plasma and adipokine variables support a potential role for complement in immunometabolism

α/β-Hydrolase domain-6 and saturated long chain monoacylglycerol regulate insulin secretion promoted by both fuel and non-fuel stimuli

Objective: α/β-Hydrolase domain-6 (ABHD6) is a newly identified monoacylglycerol (MAG) lipase. We recently reported that it negatively regulates glucose stimulated insulin secretion (GSIS) in the β cells by hydrolyzing lipolysis-derived MAG that acts as a metabolic coupling factor and signaling molecule via exocytotic regulator Munc13-1. Whether ABHD6 and MAG play a role in response to all classes of insulin secretagogues, in particular various fuel and non-fuel stimuli, is unknown. Methods: Insulin secretion in response to various classes of secretagogues, exogenous MAG and pharmacological agents was measured in islets of mice deficient in ABHD6 specifically in the β cell (BKO). Islet perifusion experiments and determinations of glucose and fatty acid metabolism, cytosolic Ca2+ and MAG species levels were carried out. Results: Deletion of ABHD6 potentiated insulin secretion in response to the fuels glutamine plus leucine and α-ketoisocaproate and to the non-fuel stimuli glucagon-like peptide 1, carbamylcholine and elevated KCl. Fatty acids amplified GSIS in control and BKO mice to the same extent. Exogenous 1-MAG amplified insulin secretion in response to fuel and non-fuel stimuli. MAG hydrolysis activity was greatly reduced in BKO islets without changes in total diacylglycerol and triacylglycerol lipase activity. ABHD6 deletion induced insulin secretion independently from KATP channels and did not alter the glucose induced rise in intracellular Ca2+. Perifusion studies showed elevated insulin secretion during second phase of GSIS in BKO islets that was not due to altered cytosolic Ca2+ signaling or because of changes in glucose and fatty acid metabolism. Glucose increased islet saturated long chain 1-MAG species and ABHD6 deletion caused accumulation of these 1-MAG species at both low and elevated glucose. Conclusion: ABHD6 regulates insulin secretion in response to fuel stimuli at large and some non-fuel stimuli by controlling long chain saturated 1-MAG levels that synergize with other signaling pathways for secretion