OBESITY AND THE ADIPOCYTE Molecular mechanisms of adipocyte differentiation

Cells of clonal lines fall into three categories (i) totipotent embryonic stem cells generating all lineages (ES cells), (ii) multipotent stem cells following myogenic, chondrogenic and adipogenic pathways (clonal lines 1246, C1, RCJ3·1, TA1, 30A5, azaCyd CHEF-18), and (iii) cells that have undergone determination to the adipose lineage (adipoblasts) and which have been used in most studies related to adipocyte differentiation (clonal lines 3T3-L1, 3T3-F442A, Ob17, BFC-1, ST13, A31T, .) (1). Growth arrest of adipoblasts at the G1/S phase of the cell cycle appears to be required for the commitment of adipoblasts to preadipose cells which is accompanied by the emergence of early markers (collagen type VI, lipoprotein lipase, fatty acid translocase, .) and the disappearance of Pref-1 as an adipoblast marker. The necessity of a growth arrest for adipose cell differentiation is clearly illustrated when preadipose cells are transformed by the middle-Tonly gene of polyoma virus. Among the different clones obtained, there is an inverse relationship in culture between their potentiality to overproliferate at low serum concentration and their potentiality to convert into adipose cells (2). After a singleor double-thymidine block of actively growing cells, i.e. in the absence of intercellular contacts, the emergence of early markers is rapid and adipoblasts are committed to preadipose cells (3). In rodent but not in human cells, a limited growth resumption takes place during terminal differentiation of preadipose to adipose cells. In both cases, the emergence of late and very late markers (adipocyte fatty acid-binding protein, glycerophosphate dehydrogenase (GPDH), adipsin, leptin, angiotensinogen, . ) is taking place. The receptor composition changes as a function of differentiation of preadipose to adipose cells. These modifications in hormonal responses favour the hypothesis of a critical ‘window of action’ for adipogenic hormones which are required for terminal differentiation only. The responsiveness of preadipose cells to external signals that induce terminal differentiation may vary according to differences in the stage of adipose lineage at which clonal lines have been established and that at which cells of a given line are exposed to various agents (4). Moreover, the requirement for adipogenic hormones shows differences between preadipose cells from established clonal lines or in primary culture. The most common requirements appear to be insulin-like growth factor I, insulin and glucocorticoids. Cells from clonal lines also require growth hormone and tri-iodothyronine. Glucocorticoids, including the synthetic glucocorticoid dexamethasone, stimulate terminal differentiation of Ob1771 preadipose cells and that of human preadipocytes (5, 6). Besides promoting and/or increasing the expression of numerous genes, glucocorticoids specifically stimulate the production of prostacyclin, a paracrine/autocrine adipogenic hormone, in Ob17 cells (7) (see below). Insulin modulates the differentiation programme positively by affecting the expression of various proteins involved in triacylglycerol accumulation such as GPDH (1). Under serum-free, chemically defined culture conditions, prostacyclin (acting as a cAMPelevating and a Ca mobilizing agent) (8) played a cardinal role in triggering terminal differentiation of preadipose Ob17 cells as well as modulating positively the terminal differentiation of rat and human preadipocytes. The Ca transient is (i) elicited by the natural prostaglandin, prostacyclin (PGI2), (ii) independent of the presence of extracellular Ca2+, suggesting a mobilization of Ca intracellular pools, and (iii) unaffected by cAMP elevating agents. The potential to generate simultaneously two synergistic intracellular signals allowed us to ascribe to prostacyclin a key and specific role in the differentiation of adipose precursor cells in vitro.Recent evidence shows that prostacyclin is also directly or indirectly active in preadipose and adipose cells at the gene level presumably by means of peroxisome proliferator-activated receptors (PPARs) a and/or a2 (9, 10). In fact, PGI2 appears as the only known effector of PPARs which is synthetized by preadipose and adipose cells, and its role is emphasized by its unique ability to allow a ‘cross-talk’ between preadipose and adipose cells in vitro and possibly in vivo (11 and C Darimont, P Saint-Marc, R Negrel & G Ailhaud, unpublished results). The salient feature of this regulation by carbacyclin (cPGI2), a stable analogue of PGI2, is its clear dissociation from events controlled by the ‘classical’ cell surface receptor of PGI2 (IP receptor) (12). Three arguments are in favour of this conclusion: (i) the inability of two prostaglandins to induce the expression of genes encoding for angiotensinogen (AT) and adipocyte-fatty acid-binding protein (a-FABP), namely prostaglandin E1 (PGE1) and 6-keto-PGE1, despite the fact that both are 201