Our group has recently demonstrated (Gesta, S., Simon, M., Rey, A., Sibrac, D., Girard, A., Lafontan, M., Valet, P., and Saulnier-Blache, J. S. (2002) J. Lipid Res. 43, 904 -910) the presence, in adipocyte conditioned-medium, of a soluble lysophospholipase D-activity (LPLDact) involved in synthesis of the bioactive phospholipid lysophosphatidic acid (LPA). In the present report, LPLDact was purified from 3T3F442A adipocyte-conditioned medium and identified as the type II ecto-nucleotide pyrophosphatase phosphodiesterase, autotaxin (ATX). A unique ATX cDNA was cloned from 3T3F442A adipocytes, and its recombinant expression in COS-7 cells led to extracellular release of LPLDact. ATX mRNA expression was highly up-regulated during adipocyte differentiation of 3T3F442A-preadipocytes. This up-regulation was paralleled by the ability of newly differentiated adipocytes to release LPLDact and LPA. Differentiation-dependent up-regulation of ATX expression was also observed in a primary culture of mouse preadipocytes. Treatment of 3T3F442A-preadipocytes with concentrated conditioned medium from ATX-expressing COS-7 cells led to an increase in cell number as compared with concentrated conditioned medium from ATX non-expressing COS-7 cells. The specific effect of ATX on preadipocyte proliferation was completely suppressed by co-treatment with a LPA-hydrolyzing phospholipase, phospholipase B. Finally, ATX expression was found in mature adipocytes isolated from mouse adipose tissue and was substantially increased in genetically obesediabetic db/db mice when compared with their lean siblings. In conclusion, the present work shows that ATX is responsible for the LPLDact released by adipocytes and exerts a paracrine control on preadipocyte growth via an LPA-dependent mechanism. Up-regulations of ATX expression with adipocyte differentiation and genetic obesity suggest a possible involvement of this released protein in the development of adipose tissue and obesity-associated pathologies.Because of its ability to store extra energy as triacylglycerol (lipogenesis) and to release fatty acids and glycerol (lipolysis), adipose tissue plays a crucial role in energy balance. In obesity, excessive accumulation of triacylglycerol in adipocytes (hypertrophy) results from an alteration in the balance between lipogenic and/or lipolytic activities of the adipocytes.It is now recognized that, beside their involvement in lipid homeostasis, adipocytes also produce and secrete numerous factors. Among them are endocrine peptides (leptin, adiponectin, angiotensinogen, etc.) which may play an important role in the development of morbid complications of obesity such as cardiovascular diseases, hypertension, diabetes, and cancer. Other adipocyte-secreted factors (tumor necrosis factor, fatty acids, eicosanoids, lysophosphatidic acid, etc.) are produced locally and may influence adipose tissue development and/or metabolism by exerting autocrine/paracrine effects on the different cells composing adipose tissue (adipocytes, preadipocytes, and endothelial...

Autotaxin catalyzes the transformation of lyso-phosphatidylcholine in lyso-phosphatidic acid (LPA). LPA is a phospholipid possessing a large panel of activity, in particular as a motility factor or as a growth signal, through its G-protein coupled seven transmembrane receptors. Indirect evidence strongly suggests that autotaxin is the main, if not the only source of circulating LPA. Because of its central role in pathologic conditions, such as oncology and diabetes/obesity, the biochemical properties of autotaxin has attracted a lot of attention, but confirmation of its role in pathology remains elusive. One way to validate and/or confirm its central role, is to find potent and selective inhibitors. A systematic screening of several thousand compounds using a colorimetric assay and taking advantage of the phosphodiesterase activity of autotaxin that requires the enzymatic site than for LPA generation, led to the discovery of a potent nanomolar inhibitor, [4-(tetradecanoylamino)benzyl]phosphonic acid (S32826). This compound was inhibitory toward the various autotaxin isoforms, using an assay measuring the [ 14 C]lyso-phosphatidylcholine conversion into [ 14 C]LPA. We also evaluated the activity of S32826 in cellular models of diabesity and oncology. Nevertheless, the poor in vivo stability and/or bioavailability of the compound did not permit to use it in animals. S32826 is the first reported inhibitor of autotaxin with an IC 50 in the nanomolar range that can be used to validate the role of autotaxin in various pathologies in cellular models.

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Current melatonin research is essentially based on the finding of new molecular tools, including synthetic or natural agonists and antagonists for the melatonin receptors and synthetic inhibitors of the enzymes involved in its biosynthesis. Indeed, the use of these compounds will improve our understanding of some of the numerous mechanisms of action of melatonin. The present report deals with the establishment and description of a new cell line expressing in a stable manner human arylalkylamine-N-acetyltransferase (AANAT, E.C.2.3.1.87). This new cellular system permits one to check the capacity of newly discovered inhibitors to penetrate the cell and reach their target. Some emphasis is put on inhibitors of the bromoacetyltryptamine family since these precursor compounds form in situ bisubstrate inhibitors with strong affinity for the human enzyme. AANAT is known to undergo complex and rapid regulation by a subtle balance between extremely fast catabolism and protection against it, both due to serine phosphorylation. In the present report, this phosphorylation is shown to occur in vitro after incubation with several kinases (rho-kinase, chk-1, protein kinase A) but not with protein kinase C. Phosphorylation enhances the specific activity of the enzyme by a factor of two to five. This phosphorylation is also shown to occur after treatment of the cell with compounds such as forskolin and rolipram that enhance or protect the intracellular pool of cAMP or the cell-permeable cAMP analogue, dioctanoyl-cAMP. The specificity of the cellular model was assessed using a series of substrates and inhibitors of AANAT already described in the literature, and the characteristics of this cellular system are shown to correspond with those reported for the purified enzyme. This cell line was used to screen libraries of compounds in a living system and led to the discovery of several potent specific and non-toxic AANAT inhibitors.