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. 2007 Aug 28;104(35):14056-61.
doi: 10.1073/pnas.0706517104. Epub 2007 Aug 20.

Identification of IRS-1 Ser-1101 as a _target of S6K1 in nutrient- and obesity-induced insulin resistance

Frédéric Tremblay  1 , Sophie Brûlé, Sung Hee Um, Yu Li, Kohei Masuda, Michael Roden, Xiao Jian Sun, Michael Krebs, Roberto D Polakiewicz, George Thomas, André Marette
Affiliations

Identification of IRS-1 Ser-1101 as a _target of S6K1 in nutrient- and obesity-induced insulin resistance

Frédéric Tremblay et al. Proc Natl Acad Sci U S A. .

Abstract

S6K1 has emerged as a critical signaling component in the development of insulin resistance through phosphorylation and inhibition of IRS-1 function. This effect can be triggered directly by nutrients such as amino acids or by insulin through a homeostatic negative-feedback loop. However, the role of S6K1 in mediating IRS-1 phosphorylation in a physiological setting of nutrient overload is unresolved. Here we show that S6K1 directly phosphorylates IRS-1 Ser-1101 in vitro in the C-terminal domain of the protein and that mutation of this site largely blocks the ability of amino acids to suppress IRS-1 tyrosine and Akt phosphorylation. Consistent with this finding, phosphorylation of IRS-1 Ser-1101 is increased in the liver of obese db/db and wild-type, but not S6K1(-/-), mice maintained on a high-fat diet and is blocked by siRNA knockdown of S6K1 protein. Finally, infusion of amino acids in humans leads to the concomitant activation of S6K1, phosphorylation of IRS-1 Ser-1101, a reduction in IRS-1 function, and insulin resistance in skeletal muscle. These findings indicate that nutrient- and hormonal-dependent activation of S6K1 causes insulin resistance in mice and humans, in part, by mediating IRS-1 Ser-1101 phosphorylation.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
S6K1 phosphorylates IRS-1. (A) Serum-deprived L6 cells were incubated in an amino acid-deprived or amino acid-containing medium for 1 h and stimulated or not with 100 nM insulin for the last 30 min of incubation as indicated, and then 0.01% DMSO vehicle or 25 nM rapamycin was added during the 1-h incubation. (B and C) Kinase activity of S6K1 was determined by using GST-fusion proteins encoding various domains of IRS-1 (as shown in A). Controls for substrate amount (GST-IRS-1) were analyzed by SDS/PAGE, followed by Coomassie blue staining. Autoradiograms are representative of at least three independent experiments. The bars in C Lower represent the means ± SE of three independent determinations of S6K1 activity by using GST-IRS-1 as a substrate. *, P < 0.01 vs. corresponding S6K1 assay by using GST-IRS-1N or GST-IRS-1M as a substrate.
Fig. 2.
Fig. 2.
S6K1 phosphorylates IRS-1 at Ser-1101. Serum-deprived L6 cells were incubated in an amino acid-deprived or amino acid-containing medium for 1 h and stimulated or not with 100 nM insulin for the last 30 min of incubation as indicated, and then 0.01% DMSO vehicle or 25 nM rapamycin was added during the 1-h incubation. (A) Kinase activity (by using cold ATP only) of S6K1 and S6K2 was determined by using GST-IRS-1C as a substrate, and then the reaction product was analyzed by SDS/PAGE by using an antibody that detects IRS-1 only when phosphorylated at Ser-1101. Controls for substrate amount (GST-IRS-1C) and immunoprecipitated kinases (S6K1 or S6K2) were analyzed by SDS/PAGE, followed by Coomassie blue staining and Western blot with specific antibodies, respectively. (B) Kinase activity of S6K1 was determined by in vitro kinase assay by using GST-IRS-1C or GST-IRS-1C (S1101A) as substrates. The means ± SE from three individual experiments are shown for B. *, P < 0.01 vs. corresponding S6K1 assay by using wild-type GST-IRS-1C as a substrate.
Fig. 3.
Fig. 3.
Phosphorylation of IRS-1 at Ser-1101 depends on nutrient availability. Serum-deprived L6 muscle cells, 3T3-L1 adipocytes, or Fao hepatoma cells were incubated either in an amino acid-containing or amino acid-deprived medium for 1 h and stimulated or not with 100 nM insulin as indicated, and then 0.01% DMSO vehicle or 25 nM rapamycin was added during the 1-h incubation. Phosphorylation of IRS-1 and S6K1 was determined by using antibodies that recognized IRS-1 and S6K1 only when phosphorylated at Ser-1101 and Thr-389, respectively. Stripped membranes were reprobed for total IRS-1 and S6K1. Results are of one representative experiment repeated at least three times.
Fig. 4.
Fig. 4.
Phosphorylation of IRS-1 at Ser-1101 causes inhibition of insulin signaling. Serum-deprived CHO cells expressing either HA-IRS-1 or HA-IRS-1 (S1101A) were incubated either in an amino acid-deprived or amino acid-containing medium for 1 h and stimulated or not with 100 nM insulin for the last 30 min of incubation as indicated. (A and B) Phosphorylation of IRS-1 was determined in anti-HA immunoprecipitates by using phosphospecific antibodies against IRS-1 Ser-307, Ser-312, Ser-636/639, and Ser-1101 (A) and phosphotyrosine (B). Levels of HA-IRS-1 recovered in anti-HA immunoprecipitates also are shown. (C) Phosphorylation and expression of Akt were determined in whole-cell extracts by using antibodies against phospho-Akt Ser-473 (Upper) and Akt (Lower). Results are of one representative experiment repeated at least three times. (D–F) Quantification of IRS-1 Ser-1101 (D), IRS-1 Tyr (E), and Akt Ser-473 (F) phosphorylation. *, P < 0.05 vs. corresponding CHO cells expressing wild-type HA-IRS-1.
Fig. 5.
Fig. 5.
Increased IRS-1 Ser-1101 phosphorylation in obesity- and nutrient-induced insulin resistance in mice and humans: role of S6K1. (A) Fourteen-week-old lean (Db/Db) and obese diabetic (db/db) mice were acutely (5 min) injected i.v. with either saline or insulin at 3.8 units/kg before the liver was collected. (B) Ten-week-old wild-type or S6K1−/− mice were maintained on a normal chow (−) or high-fat (+) diet for 14 weeks before the liver was collected. (A and B) Results are of one representative experiment repeated in at least three different animals for each group. (C and D) Lowering S6K1 by siRNA reduces IRS-1 Ser-1101 phosphorylation. Serum-deprived HeLa cells (C) or L6 myocytes (D) were stimulated with amino acids with or without 200 nM insulin for 30 min. (E) Human skeletal muscle biopsies were obtained during saline and amino acid infusion. Biopsies were sampled 120 min before insulin infusion (−) and after 30 min of prandial-like hyperinsulinemia (+). S6K1 and IRS-1 expression, as well as IRS-1 Ser-636/639 or Ser-1101 phosphorylation, were detected by Western blotting analysis. Results are of one representative experiment repeated in at least five paired subjects.
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