Insulin Stimulates Adipogenesis through the Akt-TSC2-mTORC1 Pathway (original) (raw)
Figure 5
Akt-mediated phosphorylation of TSC2 is essential for insulin-stimulated mTORC1 signaling and is required for proper adipocyte differentiation.
(A) Model of insulin stimulation of mTORC1 signaling via Akt-mediated phosphorylation of TSC2 (Top). Akt activation downstream of IRS1/2 and PI3K leads to phosphorylation and inhibition of TSC2, thereby relieving its inhibition of mTORC1 signaling. Consequent activation of mTORC1 leads to inhibition of 4E-BP, activation of S6K, and S6K-mediated feedback inhibition of IRS1/2. Akt phosphorylates TSC2 on five sites (bottom), two of which are conserved in Drosophila (depicted in black) and three of which are specific to vertebrate TSC2 (depicted in purple). Alanine substitution mutants affecting the sites conserved in Drosophila (TSC2-2A) or all five sites (TSC2-5A) were generated. (B) Reconstitution of Tsc2 null MEFs with wild-type TSC2, TSC2-2A, or TSC2-5A restores IRS-1 protein levels and insulin responsive Akt phosphorylation. _Tsc2_−/− MEFs were reconstituted with wild-type TSC2 (WT), TSC2-2A (2A), or TSC2-5A (5A) via retroviral infection. Stable pools were serum-starved overnight and stimulated with insulin (100 nM) for 30 minutes prior to lysis and immunoblotting with the indicated antibodies. (C) Phosphorylation of both the sites conserved in Drosophila TSC2 and those specific to vertebrate TSC2 is essential for insulin stimulated mTORC1 signaling. Cells were treated as in (B). (D) The Akt phosphorylation sites on TSC2 are important for adipogenesis. _Tsc2_−/− MEFs stably reconstituted with wild-type TSC2 (WT), TSC2-2A (2A), or TSC2-5A (5A) were induced to differentiate into adipocytes for seven days and were stained with Oil Red O. Representative microscopic fields of view (scale bars = 25 µm) are shown. (E) Reduced intracellular triglyceride in Tsc2−/− cells reconstituted with wild-type TSC2 or the TSC2-5A mutant. Relative intracellular triglyceride levels, normalized to cellular protein content, were determined for the cells described in (D) and are shown from three different experiments as the mean±SEM relative to levels in the vector reconstituted cells. *P<0.01 v. vector, **P<0.001 v. vector and P<0.05 v. WT. (F) The elevated mTORC1 signaling and PPARγ expression in Tsc2−/− MEFs is reduced by reconstitution with wild-type TSC2 or the TSC2-5A mutant. The stably reconstituted pools of MEFs described in (B) were lysed at day 0 of differentiation, normalized for total protein, and immunoblotted with the indicated antibodies. Actin and eIF4E levels are provided as loading controls. Relative levels of PPARγ were determined by quantification of band intensity, normalized to actin, using the ImageJ software, and values are expressed relative to the wild-type TSC2-reconstituted cells.