Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 21 (15), 5050-62

Mammalian Target of Rapamycin Pathway Regulates Insulin Signaling via Subcellular Redistribution of Insulin Receptor Substrate 1 and Integrates Nutritional Signals and Metabolic Signals of Insulin

Affiliations

Mammalian Target of Rapamycin Pathway Regulates Insulin Signaling via Subcellular Redistribution of Insulin Receptor Substrate 1 and Integrates Nutritional Signals and Metabolic Signals of Insulin

A Takano et al. Mol Cell Biol.

Abstract

A pathway sensitive to rapamycin, a selective inhibitor of mammalian target of rapamycin (mTOR), down-regulates effects of insulin such as activation of Akt (protein kinase B) via proteasomal degradation of insulin receptor substrate 1 (IRS-1). We report here that the pathway also plays an important role in insulin-induced subcellular redistribution of IRS-1 from the low-density microsomes (LDM) to the cytosol. After prolonged insulin stimulation, inhibition of the redistribution of IRS-1 by rapamycin resulted in increased levels of IRS-1 and the associated phosphatidylinositol (PI) 3-kinase in both the LDM and cytosol, whereas the proteasome inhibitor lactacystin increased the levels only in the cytosol. Since rapamycin but not lactacystin enhances insulin-stimulated 2-deoxyglucose (2-DOG) uptake, IRS-1-associated PI 3-kinase localized at the LDM was suggested to be important in the regulation of glucose transport. The amino acid deprivation attenuated and the amino acid excess enhanced insulin-induced Ser/Thr phosphorylation and subcellular redistribution and degradation of IRS-1 in parallel with the effects on phosphorylation of p70 S6 kinase and 4E-BP1. Accordingly, the amino acid deprivation increased and the amino acid excess decreased insulin-stimulated activation of Akt and 2-DOG uptake. Furthermore, 2-DOG uptake was affected by amino acid availability even when the degradation of IRS-1 was inhibited by lactacystin. We propose that subcellular redistribution of IRS-1, regulated by the mTOR-dependent pathway, facilitates proteasomal degradation of IRS-1, thereby down-regulating Akt, and that the pathway also negatively regulates insulin-stimulated glucose transport, probably through the redistribution of IRS-1. This work identifies a novel function of mTOR that integrates nutritional signals and metabolic signals of insulin.

Figures

FIG. 1
FIG. 1
Effects of rapamycin, the PI 3-kinase inhibitors, and the MEK inhibitor on insulin-induced subcellular redistribution of IRS-1. 3T3-L1 adipocytes were serum starved for 16 h, incubated with the indicated concentration of rapamycin (A), wortmannin (B), LY294002 (C), or PD98059 (D) for 30 min, and then stimulated with 20 nM insulin for 1 h (+). The cells were homogenized and subjected to subcellular fractionation to yield the LDM and cytosol fractions. Proteins in each fraction were separated by SDS-PAGE and immunoblotted (IB) with anti-IRS-1 antibody (α IRS-1) (upper panels). Proteins in the whole-cell lysates were separated by SDS-PAGE and immunoblotted with anti-phospho-Thr389-p70 S6 kinase [α p-p70 S6K (Thr389)], anti-4E-BP1 [α 4E-BP1], anti-phospho-Thr308-Akt [α p-Akt (Thr308)], or anti-phospho-Thr202/Thr204-p44/42 MAP kinase antibody [α p-p44/42 MAPK (Thr202/Tyr204)2] (lower panels).
FIG. 2
FIG. 2
Effects of rapamycin and lactacystin on subcellular distribution of IRS-1 and IRS-2 after prolonged insulin stimulation. 3T3-L1 adipocytes were serum starved for 16 h, incubated with vehicle (0.1% dimethyl sulfoxide [DMSO]), 20 nM rapamycin, or 10 μM lactacystin for 30 min, and stimulated with 20 nM insulin for 4 h (+). Cells were homogenized and subjected to subcellular fractionation to yield the LDM and cytosol fractions. Proteins in the whole-cell lysates or each fraction were separated by SDS-PAGE and immunoblotted (IB) with anti-IRS-1 (α IRS-1) (A) or IRS-2 (α IRS-2) (B) antibody. Data were analyzed by densitometry and expressed as fold increase compared with the values for each fraction in control cells. Results are means ± standard errors for four independent experiments. Proteins in each fraction were separated by SDS-PAGE and immunoblotted with anti-mTOR (α mTOR) or anti-Akt (α Akt) antibody (C).
FIG. 3
FIG. 3
Effects of rapamycin and lactacystin on subcellular distribution of IRS-1 and IRS-2 in p110CAAX-expressing cells. 3T3-L1 adipocytes were infected with either Ad5-CT or Ad5-p110CAAX at a multiplicity of infection of 50 PFU/cell. At 48 h after infection, cells were incubated in serum-free medium with vehicle (0.1% dimethyl sulfoxide [DMSO]), 20 nM rapamycin, or 10 μM lactacystin for another 16 h. Cells were homogenized and subjected to subcellular fractionation to yield the LDM and cytosol fractions. Proteins in the whole-cell lysates or each fraction were separated by SDS-PAGE and immunoblotted (IB) with anti-IRS-1 (α IRS-1) (A) or IRS-2 (α IRS-2) (B) antibody. Data were analyzed by densitometry and expressed as fold increase compared with the values for each fraction in control cells infected with Ad5-CT. Results are means ± standard errors for three independent experiments. Proteins in each fraction were separated by SDS-PAGE and immunoblotted with anti-mTOR (α mTOR) or anti-Akt (α Akt) antibody (C).
FIG. 4
FIG. 4
Effects of rapamycin and lactacystin on subcellular distribution of tyrosine-phosphorylated IRS-1 and IRS-1-associated p85 after prolonged insulin stimulation. 3T3-L1 adipocytes were serum starved for 16 h, incubated with vehicle (0.1% dimethyl sulfoxide [DMSO]), 20 nM rapamycin, or 10 μM lactacystin for 30 min, and stimulated with 20 nM insulin for 4 h (+). The cells were homogenized and subjected to subcellular fractionation to yield the LDM and cytosol fractions. Proteins in each fraction were immunoprecipitated (IP) with anti-IRS-1 antibody (α IRS-1), separated by SDS-PAGE, and immunoblotted (IB) with antiphosphotyrosine antibody (α PY) (A) or anti-p85 antibody (α p85) (B). Data were analyzed by densitometry and expressed as fold increase compared with the values for each fraction in the cells stimulated with insulin in the presence of vehicle only. Results are means ± standard errors for three independent experiments.
FIG. 5
FIG. 5
Effects of amino acid availability on the insulin-induced phosphorylation of p70 S6 kinase and 4E-BP1 and on electrophoretic mobility shift, proteasomal degradation, and subcellular redistribution of IRS-1. 3T3-L1 adipocytes were serum starved for 16 h, incubated in serum-free MEM without amino acids (0) or with standard concentrations of amino acids (1X) or with fourfold excess (4X) of amino acids for 1 h, and stimulated with 20 nM insulin for the times indicated in the figure. (A) Proteins in the whole-cell lysates were separated by SDS-PAGE and analyzed by immunoblotting (IB) with antibody against nonphosphospecific p70 S6 kinase [α p70 S6K], p70 S6 kinase with Thr389 or Ser411 phosphorylated [α p70 S6K(Thr389) or (Ser411)], 4E-BP1 [α 4E-BP1], or IRS-1 [α IRS-1]. (B) Cells were homogenized and subjected to subcellular fractionation to yield the LDM and cytosol fractions. Proteins in each fraction were separated by SDS-PAGE and immunoblotted with anti-IRS-1 antibody. Representative immunoblots for three independent experiments are shown.
FIG. 6
FIG. 6
Effect of amino acid availability on insulin-stimulated phosphorylation of Akt. 3T3-L1 adipocytes were serum starved for 16 h, incubated in serum-free MEM without amino acids (0) or with standard concentrations of amino acids (1X) or with 4-fold excess of amino acids (4X) for 1 h, and stimulated with 20 nM insulin for 4 h (+). Cells were pretreated with vehicle (0.1% dimethyl sulfoxide [DMSO]), 20 nM rapamycin, or 10 μM lactacystin for 30 min prior to the insulin stimulation. Proteins in the whole-cell lysates were separated by SDS-PAGE and analyzed by immunoblotting (IB) with anti-IRS-1 antibody (α IRS-1) or antibody against Akt (Akt phosphorylated at Ser473 or Thr308) [α p-Akt (Ser473) or (Thr308)]. Representative immunoblots for three independent experiments are shown.
FIG. 7
FIG. 7
Effects of rapamycin and lactacystin on insulin-induced translocation of Akt to the PM after prolonged insulin stimulation. 3T3-L1 adipocytes were serum starved for 16 h, incubated with vehicle (0.1% dimethyl sulfoxide [DMSO]), 20 nM rapamycin, or 10 μM lactacystin for 30 min, and stimulated with 20 nM insulin for 4 h (+). Cells were homogenized and subjected to subcellular fractionation to yield the PM fraction. Proteins in the fraction were separated by SDS-PAGE and immunoblotted (IB) with anti-Akt antibody (α Akt). Data were analyzed by densitometry and expressed as fold increase compared with the values in control cells. Results are means ± standard errors for three independent experiments.
FIG. 8
FIG. 8
Effect of amino acid availability on insulin-stimulated 2-DOG uptake. 3T3-L1 adipocytes were incubated in serum-free MEM without amino acids (0) and with standard concentration of amino acids (1X) or with fourfold excess (4X) of amino acids for 3 h and pretreated with vehicle (0.1% dimethyl sulfoxide [DMSO]), 20 nM rapamycin, or 10 μM lactacystin for 30 min, and stimulated with 20 nM insulin for the indicated times. [3H]2-DOG uptake was measured as described in Materials and Methods. Results are shown as means ± standard errors for four independent experiments. (*, P < 0.05; **, P < 0.01)

Similar articles

See all similar articles

Cited by 80 articles

See all "Cited by" articles

Publication types

MeSH terms

Substances

Feedback