Extracellular vesicles (EVs) are biological vectors that can modulate the metabolism of target cells by conveying signalling proteins and genomic material. The level of EVs in plasma is significantly increased in cardiometabolic diseases associated with obesity, suggesting their possible participation in the development of metabolic dysfunction. With regard to the poor definition of adipocyte-derived EVs, the purpose of this study was to characterise both qualitatively and quantitatively EVs subpopulations secreted by fat cells. Adipocyte-derived EVs were isolated by differential centrifugation of conditioned media collected from 3T3-L1 adipocytes cultured for 24 h in serum-free conditions. Based on morphological and biochemical properties, as well as quantification of secreted EVs, we distinguished two subpopulations of adipocyte-derived EVs, namely small extracellular vesicles (sEVs) and large extracellular vesicles (lEVs). Proteomic analyses revealed that lEVs and sEVs exhibit specific protein signatures, allowing us not only to define novel markers of each population, but also to predict their biological functions. Despite similar phospholipid patterns, the comparative lipidomic analysis performed on these EV subclasses revealed a specific cholesterol enrichment of the sEV population, whereas lEVs were characterised by high amounts of externalised phosphatidylserine. Enhanced secretion of lEVs and sEVs is achievable following exposure to different biological stimuli related to the chronic low-grade inflammation state associated with obesity. Finally, we demonstrate the ability of primary murine adipocytes to secrete sEVs and lEVs, which display physical and biological characteristics similar to those described for 3T3-L1. Our study provides additional information and elements to define EV subtypes based on the characterisation of adipocyte-derived EV populations. It also underscores the need to distinguish EV subpopulations, through a combination of multiple approaches and markers, since their specific composition may cause distinct metabolic responses in recipient cells and tissues.

15Nicotinic acetylcholine receptors (nAChRs) are the main target of neonicotinoid insecticides, 16 which are widely used in crop protection against insect pests. Electrophysiological and 17 molecular approaches have demonstrated the presence of several nAChR subtypes with 18 different affinities for neonicotinoid insecticides. However, the precise mode of action of 19 neonicotinoids on insect nAChRs remains to be elucidated. Radioligand binding studies with 20 [ 3 H]-α-bungarotoxin and [ 3 H]-imidacloprid have proved instructive in understanding ligand 21 binding interactions between insect nAChRs and neonicotinoid insecticides. The precise 22 binding site interactions have been established using membranes from whole body and specific 23 tissues. In this review, we discuss findings concerning the number of nAChR binding sites 24 against neonicotinoid insecticides from radioligand binding studies on native tissues. We 25 summarize the data available in the literature and compare the binding properties of the most 26 commonly used neonicotinoid insecticides in several insect species. Finally, we demonstrate 27 that neonicotinoid-nAChR binding sites are also linked to biological samples used and insect 28 species. 29 30 31 32 33 Nicotinic acetylcholine receptors (nAChRs) are involved in rapid neurotransmission in 34 both insect and mammalian nervous systems and play major roles in learning and memory [1-35 3]. Because of these central roles, they are the main target of neonicotinoid insecticides which 36are used as a chemical method worldwide to control insect pest [4]. However, this has led to 37 the evolution of resistance resulting in a reduction in effectiveness [5][6][7][8], environmental 38 concerns linked to the accumulation of these compounds and potential effects on non-target 39 insects such as pollinators [9][10][11][12]. Currently, binding studies are used to monitor and analyze 40 the mode of action of neonicotinoid insecticides on insect native nAChRs in order to understand 41 the levels of resistance. Binding studies, as well as the use of electrophysiology, have proven 42 instructive in identifying different nAChR subtypes as well as providing insights into their 43 pharmacological properties. For instance, studies using the patch clamp method demonstrated 44 that imidacloprid (IMI), the forerunner of neonicotioid insecticides, is a partial agonist of insect 45 nAChRs [13-16] while clothianidin (CLT) and acetamiprid (ACE) appear to be full agonists 46 [17]. Moreover, as it is the case with vertebrates, it is possible to identify insect α-bungarotoxin 47 (α-Bgt)-sensitive and -insensitive nAChR subtypes through binding studies [16,[18][19][20]. α-Bgt 48 is a snake toxin commonly used in vertebrates to characterize homomeric nAChRs such as α7 49 receptors [21-23] even though several studies have demonstrated that it can bind to heteromeric 50 α9α10 and homomeric α8 receptors [24,25]. In insect species, CLT binds to both α-Bgt-51 sensitive and -insensitive receptors expressed in the cockroach d...

Neonicotinoid insecticides act on nicotinic acetylcholine receptor and are particularly effective against sucking pests. They are widely used in crops protection to fight against aphids, which cause severe damage. In the present study we evaluated the susceptibility of the pea aphid Acyrthosiphon pisum to the commonly used neonicotinoid insecticides imidacloprid (IMI), thiamethoxam (TMX) and clothianidin (CLT). Binding studies on aphid membrane preparations revealed the existence of high and low-affinity binding sites for [3H]-IMI (Kd of 0.16±0.04 nM and 41.7±5.9 nM) and for the nicotinic antagonist [125I]-α-bungarotoxin (Kd of 0.008±0.002 nM and 1.135±0.213 nM). Competitive binding experiments demonstrated that TMX displayed a higher affinity than IMI for [125I]-α-bungarotoxin binding sites while CLT affinity was similar for both [125I]-α-bungarotoxin and [3H]-IMI binding sites. Interestingly, toxicological studies revealed that at 48 h, IMI (LC50 = 0.038 µg/ml) and TMX (LC50 = 0.034 µg/ml) were more toxic than CLT (LC50 = 0.118 µg/ml). The effect of TMX could be associated to its metabolite CLT as demonstrated by HPLC/MS analysis. In addition, we found that aphid larvae treated either with IMI, TMX or CLT showed a strong variation of nAChR subunit expression. Using semi-quantitative PCR experiments, we detected for all insecticides an increase of Apisumα10 and Apisumβ1 expressions levels, whereas Apisumβ2 expression decreased. Moreover, some other receptor subunits seemed to be differently regulated according to the insecticide used. Finally, we also demonstrated that nAChR subunit expression differed during pea aphid development. Altogether these results highlight species specificity that should be taken into account in pest management strategies.