Marie-Paule Gonthier

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.

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.

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.

The endocannabinoid system: Its roles in energy balance and potential as a target for obesity treatment

Obesity and cardiometabolic risk continue to be major public health concerns. A better understanding of the physiopathological mechanisms leading to obesity may help to identify novel therapeutic targets. The endocannabinoid system discovered in the early 1990s is believed to influence body weight regulation and cardiometabolic risk factors. This article aims to review the literature on the endocannabinoid system including the biological roles of its major components, namely, the cannabinoid receptors, their endogenous ligands the endocannabinoids and the ligand-metabolising enzymes. The review also discusses evidence that the endocannabinoid system constitutes a new physiological pathway occurring in the central nervous system and peripheral tissues that has a key role in the control of food intake and energy expenditure, insulin sensitivity, as well as glucose and lipid metabolism. Based on the important finding that there is a close association between obesity and the hyperactivity of the endocannabinoid system, interest in blocking stimulation of this pathway to aid weight loss and reduce cardiometabolic risk factor development has become an important area of research. Among the pharmacological strategies proposed, the antagonism of the cannabinoid receptors has been particularly investigated and several clinical trials have been conducted. One challenging pharmacological task will be to target the endocannabinoid system in a more selective, and hence, safe way. As the management of obesity also requires lifestyle modifications in terms of healthy eating and physical activity, the targeting of the endocannabinoid system may represent a novel approach for a multifactorial therapeutic strategy.

Evaluation of antioxidant properties of major dietary polyphenols and their protective effect on 3T3-L1 preadipocytes and red blood cells exposed to oxidative stress

Abstract Obesity has been associated with a marked risk of metabolic diseases and requires therapeutic strategies. Changes in redox status with increased oxidative stress in adipose tissue have been linked with obesity-related disorders. Thus, the biological effect of antioxidants such as polyphenols is of high interest. We aimed to measure antioxidant capacities of 28 polyphenols representative of main dietary phenolic acids, flavonoids, stilbenes and curcuminoids. Then, 14 molecules were selected for the evaluation of their effect on 3T3-L1 preadipocytes and human red blood cells exposed to oxidative stress. Analysis of reducing and free radical-scavenging capacities of compounds revealed antioxidant properties related to their structure, with higher activities for flavonoids such as quercetin and epicatechin. Their effects on preadipocytes’ viability also depended on their structure, dose and time of exposure. Interestingly, most of the compounds exhibited a protective effect on preadipocytes exposed to oxidative stress, by reversing H2O2-induced anti-proliferative action and reactive oxygen species production. Polyphenols also exerted an anti-inflammatory effect on preadipocytes exposed to H2O2 by reducing IL-6 secretion. Importantly, such antioxidant and anti-inflammatory effects were observed in co-exposition (polyphenol and prooxidant during 24 h) or pretreatment (polyphenol during 24 h, then prooxidant for 24 h) conditions. Moreover, compounds protected erythrocytes from AAPH radical-induced lysis. Finally, these results led to demonstrate that antioxidant and anti-inflammatory properties of polyphenols may depend on structure, dose, time of exposure and cell conditioning with oxidative stress. Such findings should be considered for a better understanding of polyphenols’ benefits in strategies aiming to prevent obesity-related diseases.

Antioxidant polyphenol-rich extracts from the medicinal plants Antirhea borbonica, Doratoxylon apetalum and Gouania mauritiana protect 3T3-L1 preadipocytes against H2O2, TNFα and LPS inflammatory mediators by regulating the expression of superoxide dismutase and NF-κB genes

BackgroundAdipose cells responsible for fat storage are the targets of reactive oxygen species (ROS) like H2O2 and pro-inflammatory agents including TNFα and LPS. Such mediators contribute to oxidative stress and alter inflammatory processes in adipose tissue, leading to insulin resistance during obesity. Thus, the identification of natural compounds such as plant polyphenols able to increase the antioxidant and anti-inflammatory capacity of the body is of high interest. We aimed to evaluate the biological properties of polyphenol-rich extracts from the medicinal plants A. borbonica, D. apetalum and G. mauritiana on preadipocytes exposed to H2O2, TNFα or LPS mediators.MethodsMedicinal plant extracts were analysed for their polyphenol contents by Folin-Ciocalteu and UPLC-ESI-MS methods as well as for their free radical-scavenging activities by DPPH and ORAC assays. To assess the ability of polyphenol-rich extracts to protect 3T3-L1 preadipocytes against H2O2, TNFα or LPS mediators, several parameters including cell viability (MTT and LDH assays), ROS production (DCFH-DA test), IL-6 and MCP-1 secretion (ELISA) were evaluated. Moreover, the expression of superoxide dismutase, catalase and NF-κB genes was explored (RT-QPCR).ResultsAll medicinal plants exhibited high levels of polyphenols with free radical-scavenging capacities. Flavonoids such as quercetin, kaempferol, epicatechin and procyanidins, and phenolic acids derived from caffeic acid including chlorogenic acid, were detected. Polyphenol-rich plant extracts did not exert a cytotoxic effect on preadipocytes but protected them against H2O2 anti-proliferative action. Importantly, they down-regulated ROS production and the secretion of IL-6 and MCP-1 pro-inflammatory markers induced by H2O2, TNFα and LPS mediators. Such a protective action was associated with an increase in superoxide dismutase antioxidant enzyme gene expression and a decrease in mRNA levels of NF-κB pro-inflammatory transcription factor.ConclusionThis study highlights that antioxidant strategies based on polyphenols derived from medicinal plants tested could contribute to regulate adipose tissue redox status and immune process, and thus participate to the improvement of obesity-related oxidative stress and inflammation.

Dietary polyphenols preconditioning protects 3T3-L1 preadipocytes from mitochondrial alterations induced by oxidative stress☆

Numerous studies indicate that an increase in reactive oxygen species (ROS) significantly affects white adipose tissue biology and leads to an inflammatory profile and insulin resistance, which could contribute to obesity-associated diabetes and cardiovascular diseases. Mitochondria play a key role in adipose tissue energy metabolism and constitute the main source of cellular ROS such as H(2)O(2). Polyphenols constitute the most abundant antioxidants provided by the human diet. Indeed, they are widely distributed in fruits, vegetables and some plant-derived beverages such as coffee and tea. Thus, the biological effects of dietary polyphenols that may increase the antioxidant capacity of the body against obesity-induced oxidative stress are of high interest. Here, we studied the capacity of polyphenols to modulate the impact of oxidative stress on the mitochondria of preadipocytes, which are important cells governing the adipose tissue development for energy homeostasis. Whereas H(2)O(2) treatment induces a proliferation arrest associated with an increase in mitochondrial content in 3T3-L1 preadipocytes, preconditioning with some major dietary polyphenols totally or partially protects the cells against oxidative stress consequences. This article is part of a Directed Issue entitled: Bioenergetic dysfunction, adaptation and therapy.