BackgroundThe development of obesity has been linked to an inflammatory process, and the role of adipose tissue in the secretion of pro-inflammatory molecules such as IL-6 or TNFalpha has now been largely confirmed. Although TNFalpha secretion by adipose cells is probably induced, most notably by TLR ligands, the activation and secretion pathways of this cytokine are not yet entirely understood. Moreover, given that macrophagic infiltration is a characteristic of obesity, it is difficult to clearly establish the level of involvement of the different cellular types present within the adipose tissue during inflammation.MethodsPrimary cultures of human adipocytes and human peripheral blood mononuclear cells were used. Cells were treated with a pathogen-associated molecular pattern: LPS, with and without several kinase inhibitors. Western blot for p38 MAP Kinase was performed on cell lysates. TNFalpha mRNA was detected in cells by RT-PCR and TNFalpha protein was detected in supernatants by ELISA assays.ResultsWe show for the first time that the production of TNFalpha in mature human adipocytes is mainly dependent upon two pathways: NFkappaB and p38 MAP Kinase. Moreover, we demonstrate that the PI3Kinase pathway is clearly involved in the first step of the LPS-pathway. Lastly, we show that adipocytes are able to secrete a large amount of TNFalpha compared to macrophages.ConclusionThis study clearly demonstrates that the LPS induced activation pathway is an integral part of the inflammatory process linked to obesity, and that adipocytes are responsible for most of the secreted TNFalpha in inflamed adipose tissue, through TLR4 activation.

BackgroundOn the basis that high fat diet induces inflammation in adipose tissue, we wanted to test the effect of dietary saturated and polysunsaturated fatty acids on human adipose tissue and adipocytes inflammation. Moreover we wanted to determine if TLR2 and TLR4 are involved in this pathway.MethodsHuman adipose tissue and adipocytes primary cultures were treated with endotoxin-free BSA conjugated with SFA (lauric acid and palmitic acid - LA and PA) and PUFA (eicosapentaeneic acid, docosahexaenoic acid and oleic acid - EPA, DHA and OA) with or without LPS. Cytokines were then assayed by ELISA (TNF-alpha, IL-6 and MCP-1). In order to determine if TLR2 and TLR4 are activated by fatty acid (FA), we used HEK-Blue cells transfected by genes from TLR2 or TLR4 pathways associated with secreted alkaline phosphatase reporter gene.ResultsNone of the FA tested in HEK-Blue cells were able to activate TLR2 or TLR4, which is concordant with the fact that after FA treatment, adipose tissue and adipocytes cytokines levels remain the same as controls. However, all the PUFA tested: DHA, EPA and to a lesser extent OA down-regulated TNF-alpha, IL-6 and MCP-1 secretion in human adipose tissue and adipocytes cultures.ConclusionsThis study first confirms that FA do not activate TLR2 and TLR4. Moreover by using endotoxin-free BSA, both SFA and PUFA tested were not proinflammatory in human adipose tissue and adipocytes model. More interestingly we showed that some PUFA exert an anti-inflammatory action in human adipose tissue and adipocytes model. These results are important since they clarify the relationship between dietary fatty acids and inflammation linked to obesity.

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Page 1 of 18A c c e p t e d M a n u s c r i p t Diet is an important environmental factor modulating the onset of atherosclerosis. The 2 aim of this study was to evaluate the effects of different dairy-based food products on early 3 atherogenesis using both conventional and metabonomic approaches in hyperlipidemic 4 hamsters. The hamsters received up to 200 g/kg of fat as anhydrous butter or cheese made 5 from various milk fats or canola-based oil (CV), in addition to a non-atherogenic low-fat diet. 6Aortic cholesteryl ester loading was considered to be an early atherogenic point, and 7 metabolic changes linked to atherogenesis were measured using plasma 1 H-NMR-based 8 metabonomics. The lowest atherogenicity was obtained with the plant-oil cheese diet, 9followed by the dairy fat cheese diet, while the greatest atherogenicity was observed with the 10 butter diet (P < 0.05). Disease outcome was correlated with conventional plasma biomarkers 11 (total cholesterol, triglycerides, LDL cholesterol, R 2 =0.42-0.60). NMR plasma metabonomics 12 selectively captured part of the diet-induced metabotypes correlated with aortic cholesteryl 13 esters (R 2 =0.63). In these metabotypes, VLDL lipids, cholesterol, and N-acetylglycoproteins 14 (R 2 range: 0.45-0.51) were the most positively correlated metabolites, whereas a 15 multimetabolite response at 3.75ppm, albumin lysyl residues, and trimethylamine-N-oxide 16were the most negatively correlated metabolites (R 2 range: 0.43-0.63) of the aortic cholesteryl 17 esters. Collectively, these metabolites predicted 89% of atherogenic variability compared to 18 the 60% predicted by total plasma cholesterol alone. In conclusion, we show that the food 19 environment can modulate the atherogenic effect of dairy fat. This proof-of-principle study 20demonstrates the first use of plasma metabonomics for improving the prognosis of diet-21 induced atherogenesis, revealing novel potential disease biomarkers. 22Keywords: nuclear magnetic resonance spectroscopy, milk fat, atherosclerosis biomarkers, 23 hamster, metabonomics 24 25