Insulin stimulates adipogenesis through the Akt-TSC2-mTORC1 pathway.

Hui Zhang, Jingxiang Huang, Katrin Düvel, Bernard Boback, Shulin Wu, Rachel Squillace, Chin-Lee Wu and Brendan Manning

PLOS One 2009. 4: e6189.


The signaling pathways imposing hormonal control over adipocyte differentiation are poorly understood. While insulin and Akt signaling have been found previously to be essential for adipogenesis, the relative importance of their many downstream branches have not been defined. One direct substrate that is inhibited by Akt-mediated phosphorylation is the tuberous sclerosis complex 2 (TSC2) protein, which associates with TSC1 and acts as a critical negative regulator of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). Loss of function of the TSC1-TSC2 complex results in constitutive mTORC1 signaling and, through mTORC1-dependent feedback mechanisms and loss of mTORC2 activity, leads to a concomitant block of Akt signaling to its other downstream targets. We find that, despite severe insulin resistance and the absence of Akt signaling, TSC2-deficient mouse embryo fibroblasts and 3T3-L1 pre-adipocytes display enhanced adipocyte differentiation that is dependent on the elevated mTORC1 activity in these cells. Activation of mTORC1 causes a robust increase in the mRNA and protein expression of peroxisome proliferator-activated receptor gamma (PPARgamma), which is the master transcriptional regulator of adipocyte differentiation. In examining the requirements for different Akt-mediated phosphorylation sites on TSC2, we find that only TSC2 mutants lacking all five previously identified Akt sites fully block insulin-stimulated mTORC1 signaling in reconstituted Tsc2 null cells, and this mutant also inhibits adipogenesis. Finally, renal angiomyolipomas from patients with tuberous sclerosis complex contain both adipose and smooth muscle-like components with activated mTORC1 signaling and elevated PPARgamma expression. This study demonstrates that activation of mTORC1 signaling is a critical step in adipocyte differentiation and identifies TSC2 as a primary target of Akt driving this process. Therefore, the TSC1-TSC2 complex regulates the differentiation of mesenchymal cell lineages, at least in part, through its control of mTORC1 activity and PPARgamma expression.

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This study aimed at investigating the importance of mTORC1 signaling in adipogenesis, and whether this adipogenic process is dependent upon a functional insulin and Akt signaling in mouse embryonic fibroblasts and 3T3-L1 preadipocytes.

Tsc1-/- or Tsc2-/- MEFs display enhanced mTORC1-dependent adipogenesis independent of a functional insulin signaling

As part of this analysis, the adipogenic potential of Tsc1-/- or Tsc2-/- MEFs was assessed. In Tsc1-/- or Tsc2-/- MEFs there is insulin resistance as proven by the absence of insulin induced Akt phosphorylation, as well as the absence of the Akt-mediated FOXO1 phosphorylation, and the constitutive nuclear localization of FOXO3a, a phenomenon seen with the FOXO3a having mutations at the Akt phosphorylation sites. An enhanced adipogenic potential in Tsc2-/- MEFs was proven by Oil Red O staining and measurement of intracellular triglyceride content, followed by the significant increase in mRNA expression of the maturation adipogenic markers cEBPa, and PPAR?, as well as the adipose hormones leptin and adiponectin. In the presence of rapamycin, adipogenesis was significantly reversed with Oil Red O stained triglycerides and intracellular triglyceride measurements matching those of the wild-type MEFs DMSO-treated control group.

Tsc2-/- MEFs have the ability to differentiate into adipocytes in an insulin independent manner

The lack of insulin in the adipose differentiation medium did not prevent adipogenesis in Tsc2-/- MEFs, suggesting that the adipogenic effects of insulin are greatly mediated through the mTORC1 pathway. In undifferentiated Tsc2-/- MEFs, as well as adipocytes derived from Tsc2-/- MEFs, there is no induction of Akt phosphorylation by serum or high levels of insulin. The mRNA levels of the insulin signaling molecules Inr, Irs-1, and Irs-2 were significantly reduced in Tsc2-/- MEFs, suggesting a functional negative-feedback mechanism by the mTORC1. The increase in mRNA (Irs-1, and Irs-2) and protein (IRS) levels of these molecules following a rapamycin treatment further supports the inhibitory effects of mTORC1 on insulin signaling. Ablation of the Tsc1 or Tsc2 results in Akt-independent activation of mTORC1 and concurrently a defective Akt signaling which includes the FOXO1 and FOXO3a proteins. Adipogenic induction in Tsc2-/- MEFs results in the formation of insulin resistant adipocytes as observed by the lack of Akt phosphorylation and IRS1 expression in Tsc2-/- adipocytes, as compared to the Tsc2+/+ adipocytes. These findings were corroborated using short hairpinTsc2 transfected 3T3-L1 preadipocytes where shRNA-mediated knockdown of Tsc2 enhanced adipogenesis and a decrease in insulin-stimulated Akt phosphorylation. In Tsc2-/- MEFs, the expression of glucose transporter Glut1, but not Glut4, was significantly elevated with the overacitvation of mTORC1, a state resembling the constituve expression of GLUT1 in the insulin resistant adipose tissue of obese patients.

Akt-dependent phosphorylation and inhibition of Tsc2 is necessary for adipogenic differentiation in MEFs

Mutating all of the 5 known Akt phosphorylation sites on Tsc2 (TSC2-5A mutant) results in reduced adipogenic potential of Tsc2-/- MEFs transfected with the TSC2-5A. there was a decrease in PPARy and an increase in Akt expression in the adipocytes derived from both the TSC2 and TSC2-5A transfected Tsc2-/- MEFs. Even though there was restoration of insulin signaling in Tsc2-/- MEFs by the TSC2-5A transfected cells, the inability of Akt to phosphorylate and inhibit TSC2, and consequently activate mTORC1, decreases the potential of adipocyte formation. Consequently, Akt-mediated adipogenesis depends to a great extent on its phosphorylation and inhibitory effects of TSC2.

Suggestions for additional experimental approaches to strengthen the paper: To clearly show that adipogenesis is taking place prior to de novo lipogenesis, an assessment and comparison of gene expression for both adipogenic and lipogenic markers at the various differentiation stages (preadipocyte, early adipogenic and terminal adipogenic phase) should be made. Markers involved in de novo lipogenesis (Acc, Fasn, Scd1, Srebp1, ChREBP). Since, mTORC1 is known to induce Srebp1 and loss of Tsc1 or Tsc2 result in Srebp induction, time-dependent expression of adipogenic and lipogenic markers is necessary.

Written by Nicole Poritsanos on Dec. 4, 2013.


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