Wiza C, Nascimento EB, Ouwens DM. Role of PRAS40 in Akt and mTOR signaling in health and disease. Am J Physiol Endocrinol Metab 302: E1453-E1460, 2012. First published February 21, 2012 doi:10.1152/ajpendo.00660.2011.-The proline-rich Akt substrate of 40 kDa (PRAS40) acts at the intersection of the Akt-and mammalian target of rapamycin (mTOR)-mediated signaling pathways. The protein kinase mTOR is the catalytic subunit of two distinct signaling complexes, mTOR complex 1 (mTORC1) and mTORC2, that link energy and nutrients to the regulation of cellular growth and energy metabolism. Activation of mTOR in response to nutrients and growth factors results in the phosphorylation of numerous substrates, including the phosphorylations of S6 kinase by mTORC1 and Akt by mTORC2. Alterations in Akt and mTOR activity have been linked to the progression of multiple diseases such as cancer and type 2 diabetes. Although PRAS40 was first reported as substrate for Akt, investigations toward mTOR-binding partners subsequently identified PRAS40 as both component and substrate of mTORC1. Phosphorylation of PRAS40 by Akt and by mTORC1 itself results in dissociation of PRAS40 from mTORC1 and may relieve an inhibitory constraint on mTORC1 activity. Adding to the complexity is that gene silencing studies indicate that PRAS40 is also necessary for the activity of the mTORC1 complex. This review summarizes the regulation and potential function(s) of PRAS40 in the complex Akt-and mTOR-signaling network in health and disease.proline-rich Akt substrate of 40 kDa; mammalian target of rapamycin PROLINE-RICH Akt SUBSTRATE OF 40 kDa (PRAS40) was first identified as a 14-3-3 binding protein in lysates from insulin-treated hepatoma cells (28) and is identical to the p39 protein that is phosphorylated in PC12 cells treated with nerve growth factor or epidermal growth factor (18) and the nuclear phosphoprotein Akt1 substrate 1 (AKT1S1) purified from Hela cells (3). Although originally described as substrate for Akt (28), analysis of mammalian target of rapamycin (mTOR) immunoprecipitates identified PRAS40 as a component and substrate of the mTOR complex 1 (mTORC1) (35,42,49,51).In addition to mTOR and PRAS40, the mTORC1 complex consists of regulatory-associated protein of mTOR (raptor), the mammalian ortholog of yeast lethal with Sec13 protein 8 (mLST8; also known as G␤L), and DEP domain-containing mTOR-interacting protein (deptor) (61). Within mTORC1, raptor functions as a scaffold through regulation of the assembly of the mTORC1 complex, the recruitment of substrates, and direction of the subcellular localization (61). Both PRAS40 and deptor exert an inhibitory action on mTORC1 activity (61). Activation of mTORC1, which occurs in response to nutrients and growth factors, results in phosphorylation of both PRAS40 and deptor by mTORC1 (33,61). This leads to dissociation of PRAS40 and deptor from the complex and relieves the inhibitory constraint on its activity (33, 61).

Secreted frizzled-related protein 5 (Sfrp5) is an adipokine with anti-inflammatory and insulin-sensitizing properties in mice. However, the mechanism of Sfrp5 action, especially in humans, is largely unknown. Therefore, cytokine release and insulin signaling were analyzed to investigate the impact of Sfrp5 on inflammation and insulin signaling in primary human adipocytes and skeletal muscle cells (hSkMC). Sfrp5 neither affected interleukin (IL)-6, monocyte chemoattractant protein-1 (MCP-1) and adiponectin release from human adipocytes, nor IL-6 and IL-8 release from hSkMC. In tumor necrosis factor (TNF) α-treated adipocytes, Sfrp5 reduced IL-6 release by 49% (p<0.05), but did not affect MCP-1 and adiponectin release. In MCP-1-treated hSkMC, Sfrp5 did not affect cytokine secretion. In untreated adipocytes, Sfrp5 decreased the insulin-mediated phosphorylation of Akt-Ser473, Akt-Thr308, GSK3α-Ser21 and PRAS40-Thr246 by 34% (p<0.01), 31% (p<0.05), 37% (p<0.05) and 34% (p<0.01), respectively, and the stimulation of glucose uptake by 25% (p<0.05). Incubation with TNFα increased the phosphorylation of JNK and NFκB, and impaired insulin signaling. When Sfrp5 and TNFα were combined, there was no additional effect on insulin signaling and JNK phosphorylation, but phosphorylation of NFκB was reversed to basal levels. Sfrp5 had no effect on insulin signaling in untreated or in MCP-1 treated hSkMC. Thus, Sfrp5 lowered IL-6 release and NFκB phosphorylation in cytokine-treated human adipocytes, but not under normal conditions, and decreased insulin signaling in untreated human adipocytes. Sfrp5 did not act on hSkMC. Therefore, the cellular actions of Sfrp5 seem to depend on the type of tissue as well as its inflammatory and metabolic state.

Aims/hypothesis The proline-rich Akt substrate of 40 kDa (PRAS40) is a component of the mammalian target of rapamycin complex 1 (mTORC1) and among the most prominent Akt substrates in skeletal muscle. Yet the cellular functions of PRAS40 are incompletely defined. This study assessed the function of PRAS40 in insulin action in primary human skeletal muscle cells (hSkMC). Methods Insulin action was examined in hSkMC following small interfering RNA-mediated silencing of PRAS40 (also known as AKT1S1) under normal conditions and following chemokine-induced insulin resistance. Results PRAS40 knockdown (PRAS40-KD) in hSkMC decreased insulin-mediated phosphorylation of Akt by 50% (p<0.05) as well as of the Akt substrates glycogen synthase kinase 3 (40%) and tuberous sclerosis complex 2 (32%) (both p<0.05). Furthermore, insulin-stimulated glucose uptake was reduced by 20% in PRAS40-KD myotubes (p<0.05). Exposing PRAS40-KD myotubes to chemokines caused no additional deterioration of insulin action. PRAS40-KD further reduced insulin-mediated phosphorylation of the mTORC1-regulated proteins p70S6 kinase (p70S6K) (47%), S6 (43%), and eukaryotic elongation 4E-binding protein 1 (100%), as well as protein levels of growth factor receptor bound protein 10 (35%) (all p<0.05). The inhibition of insulin action in PRAS40-KD myotubes was associated with a reduction in IRS1 protein levels (60%) (p<0.05), and was reversed by pharmacological proteasome inhibition. Accordingly, expression of the genes encoding E3-ligases Fbox protein 32 (also known as atrogin-1) and muscle RINGfinger protein-1 and activity of the proteasome was elevated in PRAS40-KD myotubes. Conclusions/interpretation Inhibition of insulin action in PRAS40-KD myotubes was found to associate with IRS1 degradation promoted by increased proteasome activity rather than hyperactivation of the p70S6K-negative-feedback loop. These findings identify PRAS40 as a modulator of insulin action.