Danièle Daviaud

TNFα up-regulates apelin expression in human and mouse adipose tissue

We have recently identified apelin as a novel adipokine up‐regulated by insulin and obesity. Since obesity and insulin resistance are associated with chronically elevated levels of both insulin and TNFα, the present study was performed to investigate a putative regulation of apelin expression in adipocytes by TNFα. Herein, we report a tight correlation between apelin and TNFα expression in adipose tissue of lean and obese humans. Apelin regulation by TNFα was further studied in cultured explants of human adipose tissue. The endogenous expression of TNFα in adipocytes isolated from the explants was accompanied by a 6–9 h subsequent increase of apelin expression in adipocytes. This increase was reversed by inhibiting TNFα expression with 100 µM isobutylmethylxanthine. In different mouse models of obesity, expression of both TNFα and apelin was also significantly increased in adipocytes of obese mice. Furthermore, short‐term exposure to an i.p. injection of TNFα in C57Bl6/J mice induced an increase of apelin expression in adipose tissue as well as apelin plasma levels. Finally, a direct positive effect of TNFα has been shown in differentiated 3T3F442A adipocytes on apelin expression and secretion. The signaling pathways of TNFα for the induction of apelin were dependent of PI3‐kinase, c‐Jun NH2‐terminal kinase (JNK), and MAPK but not PKC activation. All together, these findings suggest that apelin might be a candidate to better understand potential links between obesity and associated disorders such as inflammation and insulin resistance.—Daviaud, D., Boucher, J., Gesta, S., Dray, C., Guigne, C., Quil‐liot, D., Ayav, A., Ziegler, O., Carpene, C., Saulnier‐Blache, J.‐S., Valet, P., Castan‐Laurell, I. TNFα up‐regulates apelin expression in human and mouse adipose tissue. FASEB J. 20, E796–E802 (2006)

Alpha2-adrenergic receptor-mediated release of lysophosphatidic acid by adipocytes. A paracrine signal for preadipocyte growth.

In the search for the existence of adrenergic regulation of the autocrine/paracrine function of the white adipose tissue, it was observed that conditioned media from isolated adipocytes or dialysates obtained by in situ microdialysis of human subcutaneous adipose tissue increased spreading and proliferation of 3T3F442A preadipocytes. These effects were amplified when an alpha2-adrenergic agonist was present during the obtention of conditioned media and microdialysates. This alpha2-adrenergic-dependent trophic activity was completely abolished by pretreatment of the conditioned media or microdialysates with the lysophospholipase, phospholipase B. Among the different lysophospholipids tested only lysophosphatidic acid (LPA) was able to induce spreading and proliferation of 3T3F442A preadipocytes. Moreover, previous chronic treatment of 3T3F442A preadipocytes with LPA which led to a specific desensitization of LPA responsiveness, abolished the alpha2-adrenergic-dependent trophic activities of the conditioned media and microdialysates. Finally, alpha2-adrenergic stimulation led to a rapid, sustained, and pertussis toxin-dependent release of [32P]LPA from [32P]-labeled adipocytes. Based upon these results it was proposed that in vitro and in situ stimulation of adipocyte alpha2-adrenergic receptors provokes the extracellular release of LPA leading, in turn, to regulation of preadipocyte growth.

Altered food consumption in mice lacking lysophosphatidic acid receptor-1.

The release of lysophosphatidic acid (LPA) by adipocytes has previously been proposed to play a role in obesity and associated pathologies such as insulin resistance and diabetes. In the present work, the sensitivity to diet-induced obesity was studied in mice lacking one of the LPA receptor subtype (LPA1R). Conversely to what was observed in wild type (WT) mice, LPA1R-KO-mice fed a high fat diet (HFD) showed no significant increase in body weight or fat mass when compared to low fat diet (LFD). In addition, in contrast to what was observed in WT mice, LPA1R-KO mice did not exhibit over-consumption of food associated with HFD. Surprisingly, when fed a LFD, LPA1R-KO mice exhibited significant higher plasma leptin concentration and higher level of adipocyte leptin mRNA than WT mice. In conclusion, LPA1R-KO mice were found to be resistant to diet-induced obesity consecutive to a resistance to fat-induced over-consumption of food that may result at least in part from alterations in leptin expression and production.

TLR7 escapes X chromosome inactivation in immune cells

Biallelic TLR7 expression in immune cells may contribute to increased autoimmune disease risk in women. The X chromosome link to lupus Nine of 10 individuals who develop systemic lupus erythematosus (SLE) are women. Furthermore, individuals with Klinefelter syndrome (47,XXY) also have increased incidence of SLE, suggesting that X chromosome dosage could be an important risk factor in SLE. Using sensitive quantification methods, Souyris et al. demonstrate that Toll-like receptor 7 (TLR7) that is encoded from the X chromosome escapes X inactivation in B cells and myeloid cells in females and Klinefelter individuals. TLR7 binds single-stranded RNA and activates type I interferon signaling, a pathway that is also activated in SLE patients. On the basis of this, the authors propose that biallelic expression of TLR7 contributes to greater SLE risk in individuals with two X chromosomes. Toll-like receptor 7 (TLR7) is critical to the induction of antiviral immunity, but TLR7 dosage is also a key pathogenic factor in systemic lupus erythematosus (SLE), an autoimmune disease with strong female bias. SLE prevalence is also elevated in individuals with Klinefelter syndrome, who carry one or more supernumerary X chromosomes, suggesting that the X chromosome complement contributes to SLE susceptibility. TLR7 is encoded by an X chromosome locus, and we examined here whether the TLR7 gene evades silencing by X chromosome inactivation in immune cells from women and Klinefelter syndrome males. Single-cell analyses of TLR7 allelic expression demonstrated that substantial fractions of primary B lymphocytes, monocytes, and plasmacytoid dendritic cells not only in women but also in Klinefelter syndrome males express TLR7 on both X chromosomes. Biallelic B lymphocytes from women displayed greater TLR7 transcriptional expression than the monoallelic cells, correlated with higher TLR7 protein expression in female than in male leukocyte populations. Biallelic B cells were preferentially enriched during the TLR7-driven proliferation of CD27+ plasma cells. In addition, biallelic cells showed a greater than twofold increase over monoallelic cells in the propensity to immunoglobulin G class switch during the TLR7-driven, T cell–dependent differentiation of naive B lymphocytes into immunoglobulin-secreting cells. TLR7 escape from X inactivation endows the B cell compartment with added responsiveness to TLR7 ligands. This finding supports the hypothesis that enhanced TLR7 expression owing to biallelism contributes to the higher risk of developing SLE and other autoimmune disorders in women and in men with Klinefelter syndrome.

α2-adronoceptor in HT29 human colon adenocarcinoma cell-line: study of [3H](−)-adrenaline binding

Abstract The HT29 human adenocarcinoma cell-line was used to investigate the binding of [ 3 H](−)-adrenaline at α 2 -adrenoceptors. All aspects of the study indicated that α 2 -adrenoceptors were specifically labeled. [ 3 H](−)-adrenaline bound with high affinity (K D = 2.28 ± 0.41 nM) to a single population of non-interacting sites. The rank order of adrenoceptor antagonists (yohimbine > phentolamine ⪢ prazosin) and agonists (UK-14,304 > clonidine > (−)-adrenaline > (−)-noradrenaline > (+)-adrenaline > (+)-noradrenaline > amidephrine) to complete with [ 3 H](−)-adrenaline binding showed that the labeled sites were α 2 -selective and stereospecific. Comparison of the binding parameters of [ 3 H](−)-adrenaline with those of [ 3 H]clonidine (partial-agonist) and [ 3 H]yohimbine (antagonist) indicated that [ 3 H](−)-adrenaline and [ 3 H]clonidine labeled a similar number of sites (156 ± 13 versus 175 ± 21 fmol/mg protein) and that [ 3 H]yohimbine (B max = 246 ± 22 fmol/mg protein) labeled more sites than the 3 H-agonists. Data on the inhibition of [ 3 H]yohimbine binding by (−)-adrenaline was better fitted to a two-site model and revealed (1) that the K iL /K iH ratio was higher for (−)-adrenaline than for clonidine (2) that both agonists recognized the same percentage of high-affinity receptors. The results from a kinetic study of [ 3 H](−)-adrenaline binding were apparently inconsistent with the equilibrium data. Both the association and dissociation were bi-exponential, suggesting a relative heterogeneity of the labeled sites. The tritiated physiological full-agonist was moreover able to induce tight-binding. The practical consequences of this property are discussed.

Adipocyte α2A-adrenoceptor is the only α2-adrenoceptor regulated by testosterone

Abstract The effects of chronic administration of testosterone of α 2 -adrenoceptor expression in male hamsters were investigated in order to explore the selectivity of testosterone regulation towards the α 2 -adrenoceptor subtypes. A homogeneous population of α 2 -adrenoceptors was identified with [ 3 H]RX821002 binding in adipocytes, colocytes and liver, whereas the α 2 -adrenoceptor sites identified in kidney and brain were heterogeneous. Competition studies with α 2 -adrenoceptor ligands characterized the presence of the α 2A -adrenoceptor subtype in adipocytes, colocytes, kidney and brain homogenates and of the α 2B -adrenoceptor subtype in kidney and liver. RNase protection assay with a selective hamster α 2A -adrenoceptor cRNA probe confirmed the expression of α 2A -adrenoceptor mRNA in adipocytes, colocytes, kidney and brain. Testosterone treatment did not modify the α 2 -adrenoceptor densities whatever the subtype, except for the adipocyte α 2A -adrenoceptor, which was significantly increased. These results demonstrate that testosterone only up-regulates the adipocyte α 2A -adrenoceptor.

Interrelationship between lymphocytes and leptin in fat depots of obese mice revealed by changes in nutritional status

The mechanisms underlying the relationships between nutritional status and immunity remain to be fully characterized. The present study was undertaken to analyze by flow cytometry, in the context of diet-induced obesity, the status of immune cells in subcutaneous, and epididymal fat depots in wild-type and immunodeficient Rag2−/− mice submitted to nutritional challenge, i.e., 48-h fasting and 1-week refeeding. In parallel, the responsiveness of mature adipocytes and immune cells in bone marrow, lymph node, and liver were also analyzed. The results show that fasting in obese wild-type mice induces a prominent lipolysis in epididymal AT and immunosuppression restricted to both subcutaneous and epididymal AT, characterized by reduced number of CD4+ T and B lymphocytes and M1/M2 macrophages associated with reduced leptin and increased FGF21 expression in mature adipocytes. One-week refeeding was sufficient to reverse the fasting-induced effects. Obese immunodeficient mice under nutritional challenge exhibited no changes in adipocyte leptin expression and no marked trafficking of AT macrophages or NK cells, while the fasted-induced upregulation of FGF21 expression was maintained as well as the lipolytic responses. The present results demonstrate that, in a context of diet-induced obesity, fasting-induced immunosuppression is restricted to fat depots in immunocompetent mice. Lack of adipocyte leptin regulation and fasting-induced immunosuppression in obese immunodeficient mice strongly suggests that lymphocytes are involved in the modulation of adipocyte leptin expression on one hand and on the other that leptin is involved in the immune changes in AT according to nutritional status.

Coupling of inhibitory receptors with Gi-proteins in hamster adipocytes: comparison between adenosine A1 receptor and α2-adrenoceptor

Adenosine A1 receptors and alpha 2-adrenoceptors are both potent inhibitors of adipocyte lipolysis when activated by their agonists. The aim of this work was to compare the coupling of these receptors to the Gi-proteins in hamster adipocytes. The adenosine A1 receptor was characterized with the antagonist [3H]dipropyl-cyclopentyl-xanthine ([3H]DPCPX) and the agonist [3H](-)-phenylisopropyladenosine ([3H]PIA). It was demonstrated by [32P]ADP-ribosylation with pertussis toxin and immunoblotting that Gi1, Gi2 and Gi3 are expressed in hamster adipocytes. Partial ADP-ribosylation of Gi-proteins by pertussis toxin, acting on the intact cells or on the adipocyte membranes, demonstrated that the adenosine A1 receptor was less sensitive to the disappearance of functional Gi-proteins than the alpha 2-adrenoceptor. These results are in accordance with the weak sensitivity of the binding of the agonist [3H]PIA to guanine nucleotides and seem to confirm that the adenosine A1 receptor is strongly and differently coupled than the alpha 2-adrenoceptor to the Gi-proteins in hamster adipocyte membranes.