Activation of the cardiac mTOR/p70^S6K pathway by leucine requires PDK1 and correlates with PRAS40 phosphorylation

Abstract

Like insulin, leucine stimulates the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) axis in various organs. Insulin proceeds via the canonical association of phosphatidylinositol 3-kinase (PI3K), phosphoinositide-dependent protein kinase-1 (PDK1), and protein kinase B (PKB/Akt). The signaling involved in leucine effect, although known to implicate a PI3K mechanism independent of PKB/Akt, is more poorly understood. In this study, we investigated whether PDK1 could also participate in the events leading to mTOR/p70S6K activation in response to leucine in the heart. In wild-type hearts, both leucine and insulin increased p70S6K activity whereas, in contrast to insulin, leucine was unable to activate PKB/Akt. The changes in p70S6K activity induced by insulin and leucine correlated with changes in phosphorylation of Thr389, the mTOR phosphorylation site on p70S6K, and of Ser2448 on mTOR, both related to mTOR activity. Leucine also triggered phosphorylation of the proline-rich Akt/PKB substrate of 40 kDa (PRAS40), a new pivotal mTOR regulator. In PDK1 knockout hearts, leucine, similarly to insulin, failed to induce the phosphorylation of mTOR and p70S6K, leading to the absence of p70S6K activation. The loss of leucine effect in absence of PDK1 correlated with the lack of PRAS40 phosphorylation. Moreover, the introduction in PDK1 of the L155E mutation, which is known to preserve the insulin-induced and PKB/Akt-dependent phosphorylation of mTOR/p70S6K, suppressed all leucine effects, including phosphorylation of mTOR, PRAS40, and p70S6K. We conclude that the leucine-induced stimulation of the cardiac PRAS40/mTOR/p70S6K pathway requires PDK1 in a way that differs from that of insulin.

A-769662 potentiates the effect of other AMP-activated protein kinase activators on cardiac glucose uptake

“…At the end of the procedure, the hearts were immediately freeze clamped in liquid nitrogen and stored at Ϫ80°C. Frozen hearts were homogenized at 4°C in 9 volumes (vol/wt) of homogenization buffer that contained phosphatases and protease inhibitors (34,45). Supernatants were stored at Ϫ80°C after centrifugation (10,000 g, 30 min, 4°C).…”

Protein Phosphatase 2A (PP2A)-specific Ubiquitin Ligase MID1 Is a Sequence-dependent Regulator of Translation Efficiency Controlling 3-Phosphoinositide-dependent Protein Kinase-1 (PDPK-1)

“…80 g/ml cycloheximide was added, and cells were harvested at each time point (0, 1,3,8,24, and 32 h). PDPK1 protein was detected on Western blot by using anti-PDPK1 (Santa Cruz Biotechnology, sc-17765), and the amount of PDPK1 protein was densitometrically analyzed using anti-GAPDH (Millipore, MAB374) as loading control.…”

“…These control loops for mTOR signaling are further potentiated by the synergistic influence of the MID1 protein complex on the protein synthesis of PDPK-1. PDPK-1 is an efficient mTOR agonist inducing mTOR activity in two different manners, directly in a leucine-dependent way and indirectly via the activation of Akt (23,24). By stimulating PDPK-1 protein synthesis, MID1 is therefore further increasing mTOR signaling.…”

Pulsatile delivery of a leucine supplement during long-term continuous enteral feeding enhances lean growth in term neonatal pigs

“…Furthermore, we found that the abundance of Deptor, an inhibitor of both mTORC1 and mTORC2 (65), was suppressed by leucine administration. However, leucine pulses had no effect on the abundance or phosphorylation of PRAS40, a specific inhibitor of mTORC1 (81), although others have reported that leucine administration in rodents enhances PRAS40 phosphorylation in skeletal (45) and cardiac muscles (68).…”