Effect of TRB3 on Insulin and Nutrient-stimulated Hepatic p70 S6 Kinase Activity

Yasuo M. Tsutsumi,Rie Matsushima,Nicholas J.G. Webster,Yutaka Nakaya,Nagakatsu Harada

doi:10.1074/jbc.m511636200

Abstract

Insulin and nutrients activate hepatic p70 S6 kinase (S6K1) to regulate protein synthesis. Paradoxically, activation of S6K1 also leads to the development of insulin resistance. In this study, we investigated the effect of TRB3, which acts as an endogenous inhibitor of Akt, on S6K1 activity in vitro and in vivo. In cultured cells, overexpression of TRB3 completely inhibited insulin-stimulated S6K1 activation by mammalian target of rapamycin, whereas knockdown of endogenous TRB3 increased both basal and insulin-stimulated activity. In C57BL/6 mice, adenoviral overexpression of TRB3 inhibited insulin-stimulated activation of hepatic S6K1. In contrast, overexpression of TRB3 did not inhibit nutrient-stimulated S6K1 activity. We also investigated the effect of starvation, feeding, or insulin treatment on TRB3 levels and S6K1 activity in the liver of C57BL/6 and db/db mice. Both insulin and feeding activate S6K1 in db/db mice, but only insulin activates in the C57BL/6 strain. TRB3 levels were 3.5-fold higher in db/db mice than C57BL/6 mice and were unresponsive to feeding or insulin, whereas both treatments reduced TRB3 in C57BL/6 mice. Akt was activated by insulin alone in the C57BL/6 strain and but not in db/db mice. Both insulin and feeding activated mammalian target of rapamycin similarly in these mice; however, feeding was unable to activate the downstream target S6K1 in C57BL/6 mice. These results suggest that the nutrient excess in the hyperphagic, hyperinsulinemic db/db mouse primes the hepatocyte to respond to nutrients resulting in elevated S6K1 activity. The combination of elevated TRB3 and constitutive S6K1 activity results in decreased insulin signaling via the IRS-1/phosphatidylinositol 3-kinase/Akt pathway.

Highlights

The p70 ribosomal protein S6 kinase 1 (S6K1)2 participates in a variety of intracellular signaling events, including mRNA translation, gene transcription, and cell cycle control [1,2,3]
We investigated the effect of TRB3, which acts as an endogenous inhibitor of Akt, on S6K1 activity in vitro and in vivo
Akt was activated by insulin alone in the C57BL/6 strain and but not in db/db mice. Both insulin and feeding activated mammalian target of rapamycin in these mice; feeding was unable to activate the downstream target S6K1 in C57BL/6 mice. These results suggest that the nutrient excess in the hyperphagic, hyperinsulinemic db/db mouse primes the hepatocyte to respond to nutrients resulting in elevated S6K1 activity

Summary

EXPERIMENTAL PROCEDURES

Stimulation of Liver Cells with Insulin, Pervanadate, Amino. 424), p-S6 ribosomal protein (Ser-235/236), p-4E-BP1 (Ser-65), phosphatases that causes activation of Akt. For amino acid and p-GSK3␣/␤ (Ser-21/9) and antibodies against Akt, mTOR, stimulation, subconfluent HepG2 cells were starved for 24 h in. HepG2 cells were transfected with the siRNAs indicated 24 h prior to treatment without (Ϫ) or with (ϩ) insulin (100 nM) for 15 min. HepG2 cells were transfected with the siRNAs indicated and were stimulated with insulin, as described above. Mice were euthanized in HepG2 Cells—HepG2 cells were transiently co-transfected 7 days following virus injection. (Sigma) and protein A-Sepharose beads (Amersham Bio- All mice were sacrificed for blood and liver collection at the sciences). Some of the mice were subjected to insulin injection (0.75 units/kg body weight, 10 min) prior to sacrifice. Statistical Analysis—Data are expressed as means Ϯ S.D. and were analyzed using analysis of variance and Bonferroni multiple comparison tests. p Ͻ 0.05 was accepted as statistically significant

RESULTS

Glucose Fed

Glucose level