Activation of protein kinase B and induction of adipogenesis by insulin in 3T3-L1 preadipocytes: contribution of phosphoinositide-3,4,5-trisphosphate versus phosphoinositide-3,4-bisphosphate.

Abstract

Ectopic expression of activated protein kinase B (PKB) induces the differentiation of confluent 3T3-L1 preadipocytes into adipocytes. PKB is regulated by the lipid products of phosphoinositide 3-kinase (PI 3-kinase), phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2], and phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3]. However, the relative contribution of each 3-phosphorylated phosphoinositide species in activating PKB remains unclear. Treatment of intact 3T3-L1 preadipocytes with synthetic 3-phosphorylated phosphoinositides revealed that only PI(3,4)P2 stimulated PKB activity. PKB was also activated by insulin, in a dose- and time-dependent manner. This activation was associated with an isolated rise in PI(3,4,5)P3, without any detectable change in PI(3,4)P2, demonstrating that this lipid was sufficient to activate PKB. Wortmannin and LY294002, inhibitors of PI 3-kinase, reduced insulin-dependent activation of PKB, whereas rapamycin, an inhibitor of p70 S6 kinase, had no effect. Platelet-derived growth factor (PDGF), which is not adipogenic, stimulated the production of both 3-phosphorylated phosphoinositide species, and this was associated with a greater activation of PKB than that observed with insulin. A low dose of PDGF (1 ng/ml), which increased the production of only PI(3,4,5)P3 and mirrored the insulin effect, was unable to induce adipocyte differentiation. In summary, insulin and PDGF differ with respect to the accumulation of 3-phosphorylated phosphoinositides and to PKB activation in 3T3-L1 preadipocytes, but these responses do not themselves explain why insulin, but not PDGF, is adipogenic.

Macrophage-conditioned medium inhibits the differentiation of 3T3-L1 and human abdominal preadipocytes

“…After 8 days, cultures were photographed with a digital camera (Coolpix 995; Nikon, Mississauga, ON, Canada) mounted on a microscope (Eclipse TS-100; Nikon). Cells were washed and triacylglycerol was extracted and quantified spectrophotometrically [15]. Proteins were solubilised in Laemmli buffer [16], quantified with the modified Lowry method using BSA as a standard (Bio-Rad, Hercules, CA, USA), and processed for immunoblot analysis.…”

Depletion of cAMP-response Element-binding Protein/ATF1 Inhibits Adipogenic Conversion of 3T3-L1 Cells Ectopically Expressing CCAAT/Enhancer-binding Protein (C/EBP) α, C/EBP β, or PPARγ2

“…siRNA-mediated depletion of cyclin D1 blocks MDI-stimulated adipogenic conversion of 3T3-L1 cells. 3 Inhibition of phosphatidylinositol 3-kinase/ Akt pathways in 3T3-L1 cells prevents adipogenesis (32), indicating the importance of this pathway to adipocyte development. In the absence of CREB, Akt levels may be insufficient to support robust adipocyte differentiation.…”

Galectin-12 Is Required for Adipogenic Signaling and Adipocyte Differentiation

“…It is known that Akt activation is critical for adipocyte differentiation (55). Inhibition of phosphatidylinositol 3-kinase, a kinase upstream of Akt, suppresses adipogenesis (61), whereas the expression of constitutively active Akt induces adipocyte differentiation (52). Mice deficient in Akt display impeded PPAR␥ expression and adipogenesis (53).…”