Laurence Hoareau

Presence of the cannabinoid receptors, CB1 and CB2, in human omental and subcutaneous adipocytes

To investigate the expression of the endocannabinoid 1 and 2 receptors by human adipocyte cells of omental and subcutaneous fat tissue, as well as to determine whether these receptors are functional. The expression of CB1 and CB2 receptors on human adipocytes was analyzed by western blotting, immunohistology and immunocytology. We also investigated intracytoplasmic cyclic AMP level modulation following CB1 and CB2 receptor stimulation by an enzymatic immuno assay. All mature adipocytes, from visceral (epiploon) and subcutaneous fat tissue, express CB1 and CB2 on their plasma membranes. We also demonstrate in this study that adipocyte precursors (pre-adipocytes) express CB1 and CB2 on their plasma membranes and that both receptors are functional. Activation of CB1 increases intracytoplasmic cyclic AMP whilst CB2 activation leads to a cyclic AMP decrease. Here we demonstrate, for the first time, that adipocytes of human adipose tissue (mature adipocytes and pre-adipocytes) express functional plasma membrane CB1 and CB2 receptors. Their physiological role on the adipose tissue is not known. However, their major involvement in the physiology of other tissues leads us to suppose that they could play a significant role in the homeostasis of the energy balance and/or in the regulation of adipose tissue inflammation.

Role and regulation of acylethanolamides in energy balance: focus on adipocytes and β‐cells

The endocannabinoid, arachidonoylethanolamide (AEA), and the peroxisome proliferator‐activated receptor (PPAR)‐α ligand, oleylethanolamide (OEA) produce opposite effects on lipogenesis. The regulation of OEA and its anti‐inflammatory congener, palmitoylethanolamide (PEA), in adipocytes and pancreatic β‐cells has not been investigated. We report here the results of studies on acylethanolamide regulation in these cells during obesity and hyperglycaemia, and provide an overview of acylethanolamide role in metabolic control. We analysed by liquid chromatography‐mass spectrometry OEA and PEA levels in: 1) mouse 3T3F442A adipocytes during insulin‐induced differentiation, 2) rat insulinoma RIN m5F β‐cells kept in ‘low’ or ‘high’ glucose, 3) adipose tissue and pancreas of mice with high fat diet‐induced obesity (DIO), and 4) in visceral fat or blood of obese or type 2 diabetes (T2D) patients. In adipocytes, OEA levels remain unchanged during differentiation, whereas those of PEA decrease significantly, and are under the negative control of both leptin and PPAR‐γ. PEA is significantly downregulated in subcutaneous adipose tissue of DIO mice. In RIN m5F insulinoma β‐cells, OEA and PEA levels are inhibited by ‘very high’ glucose, this effect being enhanced by insulin, whereas in cells kept for 24 h in ‘high’ glucose, they are stimulated by both glucose and insulin. Elevated OEA and PEA levels are found in the blood of T2D patients. Reduced PEA levels in hypertrophic adipocytes might play a role in obesity‐related pro‐inflammatory states. In β‐cells and human blood, OEA and PEA are down‐ or up‐regulated under conditions of transient or chronic hyperglycaemia, respectively.

Presence of functional TLR2 and TLR4 on human adipocytes.

In addition to the well-known role of adipose tissue in energy metabolism, it has recently been demonstrated that this tissue can secrete a large array of molecules, including inflammatory cytokines. Furthermore, recent studies suggest that adipose cells can behave as immune cells. Therefore, the aim of this study was to determine the presence of the two most prominent ‘pattern recognition receptors’ for bacterial and fungal cell wall components, TLR2 and TLR4 on human adipose cells, as well as to assess their functionality. We demonstrated that TLR2 and TLR4 were expressed at relatively high levels (compared to a monocyte cell line) on the surface of human adipose cells. Stimulation of human adipocytes with lipopolysaccharide (LPS), or with lipoteichoic acid (LTA), two specific ligands of TLR4 and TLR2, respectively, induced a strong increase in TNFα production. The specificity of the response was demonstrated by the use of anti-TLR4 and anti-TLR2 blocking antibodies, which were able to decrease LPS- or LTA-induced TNFα secretion. Thus, it is clear that these receptors are functional in human adipocytes. This study adds weight to the argument that human fat tissue plays a potential role in innate immunity.

Signaling pathways involved in LPS induced TNFalpha production in human adipocytes.

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.

Identification of Endocannabinoids and Related Compounds in Human Fat Cells

Objective: Recently, an activation of the endocannabinoid system during obesity has been reported. More particularly, it has been demonstrated that hypothalamic levels of both endocannabinoids, 2‐arachidonoylglycerol and anandamide (N‐arachidonoylethanolamine), are up‐regulated in genetically obese rodents. Circulating levels of both endocannabinoids were also shown to be higher in obese compared with lean women. Yet, the direct production of endocannabinoids by human adipocytes has never been demonstrated. Our aim was to evaluate the ability of human adipocytes to produce endocannabinoids.

Surface protein expression between human adipose tissue-derived stromal cells and mature adipocytes

Adipose tissue contains a stroma that can be easily isolated. Thus, human adipose tissue presents an source of multipotent stromal cells. In order to determine the implication of hematopoietic markers in adipocyte biology, we have defined part of the phenotype of the human adipose tissue-derived stromal cells, and compared this to fully differentiated adipocytes. Flow cytometry demonstrates that the protein expression phenotype of both cell types are similar and includes the expression of CD10, CD13, CD34, CD36, CD55, CD59 and CD65. No significant difference between subcutaneous and omental adipose tissue could be demonstrated concerning the expression of these markers. However, the expression of CD34, CD36 and CD65 is cell-dependent. While the expression of CD36 and CD65 doubled between stromal cells and mature adipocytes, the expression of CD34 decreased, despite this protein being present on the mature adipocyte. As CD34 is described as a stem cell marker and it being unlikely to be expressed on differentiated cells, this result was confirmed by immunostaining and western blot. The clear function of this protein on the adipocyte membrane remains to be determined. The characterization of new proteins on mature adipocytes could have broad implications for the comprehension of the biology of this tissue.

Fatty acids do not pay the toll: effect of SFA and PUFA on human adipose tissue and mature adipocytes inflammation

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.

Anti-inflammatory Effect of Palmitoylethanolamide on Human Adipocytes

Obesity leads to the appearance of an inflammatory process, which can be initiated even with a moderate weight gain. Palmitoylethanolamide (PEA) is an endogenous lipid, secreted by human adipocytes, that possesses numerous anti‐inflammatory properties. The main purpose of this study was to investigate the anti‐inflammatory effect of PEA on human adipocytes, as well as in a murine model. The production of tumor necrosis factor–α (TNF‐α) by lipopolysaccharide (LPS)‐treated human subcutaneous adipocytes in primary culture and CF‐1 mice was investigated by enzyme‐linked immunosorbent assay. The effects of PEA on adipocyte TNF‐α secretion were explored as well as some suspected PEA anti‐inflammatory pathways: nuclear factor–κB (NF‐κB) pathway, peroxisome proliferator‐activated receptor–α (PPAR‐α) gene expression, and TNF‐α‐converting enzyme (TACE) activity. The effects of PEA on the TNF‐α serum concentration in intraperitoneally LPS‐treated mice were also studied. We demonstrate that the LPS induced secretion of TNF‐α by human adipocytes is inhibited by PEA. This action is neither linked to a reduction in TNF‐α gene transcription nor to the inhibition of TACE activity. Moreover, PPAR‐α is not implicated in this anti‐inflammatory activity. Lastly, PEA exhibits a wide‐reaching anti‐inflammatory action as the molecule is able to completely inhibit the strong increase in TNF‐α levels in the serum of mice treated with high doses of LPS. In view of its virtual lack of toxicity, PEA might become a potentially interesting candidate molecule in the prevention of obesity‐associated insulin resistance.

Identification of preferential protein targets for carbonylation in human mature adipocytes treated with native or glycated albumin

Oxidative modifications in proteins can participate in the regulation of cellular functions and are frequently observed in numerous states of diseases. Albumin can undergo increased glycation during diabetes. An accumulation of oxidatively modified proteins in human mature adipocytes incubated with glycated albumin has previously been described. This study herein reports the identification of specifically carbonylated targets following separation of the cell proteins by 2D gels, Western blotting and mass spectrometry analyses. It identified eight oxidatively modified proteins, two of which (ACTB and Annexin A2) appeared as significantly more carbonylated in adipocytes treated with glycated albumin than with native albumin. Intracellular stress, evaluated in SW872 cell line, showed an impairment in the protective antioxidant action exerted by native BSA after the glycation of the protein. Decreased proteasome peptidase activities were found in glycated BSA-treated mature adipocytes. The data suggest an association of oxidative damage with the progression of diabetes disorders at the adipocytes level.

Effect of centrifugation and washing on adipose graft viability: a new method to improve graft efficiency.

BACKGROUND Adipose tissue grafting is a promising method in the field of surgical filling. We studied the effect of centrifugation on fat grafts, and we propose an optimised protocol for the improvement of adipose tissue viability. METHODS Adipose tissue was subjected to different centrifugations, and the volumes of interstitial liquid and oil released were measured to choose the optimal condition. Tissue from this condition was then compared to tissue obtained from two traditional techniques: strong centrifugation (commonly 3 min at 3000 rpm/900 g), and decantation, by injecting into immunodeficient mice. The cytokine interleukin-6 (IL-6) and chemokine monocyte chemotactic protein-1 (MCP-1) were assayed 24 h post-injection, and after 1 month of grafting the state of the lipografts was evaluated through macroscopic and histological analysis, with oil gap area measurement. RESULTS Strong centrifugation (900 g, 1800 g) is deleterious for adipose tissue because it leads to until threefold more adipocyte death compared to low centrifugation (100 g, 400 g). In addition, mice injected with strong centrifuged and non-centrifuged adipose tissue have higher rates of blood IL-6 and MCP-1, compared to those grafted with soft centrifuged fat. Moreover, extensive lipid vacuoles were detectable on histological sections of the non-centrifuged lipografts, whereas lipografts from soft centrifugation contain a higher amount of connective tissue containing collagen fibres. CONCLUSION It is necessary to wash and centrifuge adipose tissue before reinjection in order to remove infiltration liquid and associated toxic molecules, which in the long term are deleterious for the graft. However, strong centrifugation is not recommended since it leads very quickly to greater adipocyte death. Thus, soft centrifugation (400 g/1 min), preceded by washings, seems to be the most appropriate protocol for the reinjection of adipose tissue.