Mitogen-Activated Protein Kinase Activation Is Not Necessary for, but Antagonizes, 3T3-L1 Adipocytic Differentiation

Abstract

In 3T3-L1 fibroblasts, Ras proteins mediate both insulin-induced differentiation to adipocytes and its activation of cytosolic serine/threonine kinases, including Raf-1 kinase, mitogen-activated protein kinase (MAPK), and Rsk. Here, we report that insulin-and Ras-induced activation of MAPK is not required for the differentiation process and in fact antagonizes it. The treatment of 3T3-L1 preadipocytes with MEK-specific inhibitor PD98059 blocked insulin-and Ras-induced MAPK activation but had no effect on or slightly enhanced adipocytic differentiation. Tumor necrosis factor alpha (TNF-␣), an inhibitor of insulin-stimulated adipogenesis, activated MAPK in 3T3-L1 cells. PD98059 treatment blocked MAPK activation by TNF-␣ and reversed the blockade of adipogenesis mediated by low (1 ng/ml) TNF-␣ concentrations. 3T3-L1 transfectants containing hyperactivated MEK1 or overexpressed MAPK displayed impaired adipocytic differentiation. PD98059 treatment also reversed the blockade of differentiation in MEK1 transfectants. These results indicate that MAPK does not promote but can contribute to inhibition of the process of adipocytic differentiation of 3T3-L1 cells.Adipocytic differentiation is a complex process regulated by many hormones, growth factors, and cytokines. Whereas some of those signals stimulate differentiation to adipocytes, others inhibit this event (13,20), presumably based on the ability to ultimately alter gene expression. Preadipocytic cell lines, such as 3T3-L1, undergo differentiation after continuous exposure to pharmacological doses of insulin or physiologic doses of insulin-like growth factor 1 and are additionally induced by glucocorticoids and fatty acids (27,37,41,43). In contrast, tumor necrosis factor alpha (TNF-␣), a cytokine secreted by macrophages and adipose tissue, inhibits adipocytic differentiation (44, 45). However, the mechanisms that mediate the actions of insulin and other ligands on differentiation have yet to be clearly defined.We demonstrated previously that overexpression of Ras induces adipocytic differentiation of 3T3-L1 cells (3, 34), implicating Ras proteins as obligatory signaling intermediates in insulin-stimulated differentiation pathways. Our prior studies also showed that Ras proteins are necessary and sufficient for insulin activation of the cytosolic kinases Raf-1, mitogen-activated protein kinase (MAPK), and Rsk (35, 36); furthermore, Raf-1 participates downstream of Ras in the signaling cascade resulting in adipocytic differentiation. However, in contrast to its role in proliferating cells (39, 51, 54), Raf-1 kinase activation in differentiating 3T3-L1 cells is completely dissociated from activation of MAPK by insulin (35,36), suggesting that at least two separate signals emerge from Ras after insulin stimulation, regulating either growth or differentiation depending on the state of the cell or the presence of other environmental factors. Dissociation between Raf-1 kinase and MAPK has also previously been described for other cell types (15,39,(55)(56)(57).To further de...

Prostaglandin F2α inhibits adipocyte differentiation via a Gαq‐Calcium‐Calcineurin‐Dependent signaling pathway

“…While our data clearly highlights the critical role played by the calcium-calcineurin signaling pathway in mediating the inhibitory effects of PGF2a on adipocyte differentiation, it should be noted that PGF2a has also been shown to activate a variety of other downstream signaling pathways, several of which have themselves been previously shown to be capable of inhibiting adipocyte differentiation under certain circumstances (e.g., MAPK and the beta-catenin/T cell factor signaling pathways) [Font de Mora et al, 1997;Ross et al, 2000]. Indeed, a previous report has specifically implicated a role for the MAPK signaling pathway in mediating the anti-adipogenic effects of PGF2a on 3T3-L1 preadipocytes by directly phosphorylating PPARg and inhibiting its transcriptional activity [Reginato et al, 1998].…”

Specific Inhibitors of p38 Mitogen-activated Protein Kinase Block 3T3-L1 Adipogenesis

“…The inhibition of p42/44 MAP kinase allows cells to differentiate even in the presence of TNF␣, suggesting that TNF␣'s antiadipogenic effect requires activation of the p42/44 MAP kinase pathway (59). Due to the strong antiadipogenic effect of the p38 MAP kinase inhibitors, the analogous experiment cannot be performed with the these inhibitors.…”

Insulin-like Growth Factor-1/Insulin Bypasses Pref-1/FA1-mediated Inhibition of Adipocyte Differentiation

“…Antisense-mediated elimination of p42/p44 MAPK expression was reported to prevent insulin-induced adipocyte differentiation and DNA synthesis in 3T3-L1 cells (49), and similarly, terminal differentiation of 3T3-F442A induced by insulin/T 3 / EGF was shown to require p42/p44 MAPK activation (50). By contrast, administration of the MEK1 inhibitor PD98059 was reported to block insulin-induced p42/p44 MAPK activation without affecting adipocyte differentiation of 3T3-L1 preadipocytes, whereas overexpression of either a constitutively active MEK1 or p42 MAPK impaired differentiation (51). Recently, it has been shown that inhibition of the MEK1-p42/p44 MAPK pathway in 3T3-L1 preadipocytes significantly attenuated the expression of PPAR␥ and C/EBP␣ and inhibited adipocyte differentiation (53).…”