Autophagy-mediated insulin receptor down-regulation contributes to endoplasmic reticulum stress-induced insulin resistance

Abstract

Endoplasmic reticulum (ER) stress is associated with obesity-induced insulin resistance, yet the underlying mechanisms remain to be fully elucidated. Here we show that ER stress-induced insulin receptor (IR) down-regulation may play a critical role in obesity-induced insulin resistance. The expression levels of IR are negatively associated with the ER stress marker C/EBP homologous protein (CHOP) in insulin target tissues of db/db mice and mice fed a high-fat diet. Significant IR down-regulation was also observed in fat tissue of obese human subjects and in 3T3-L1 adipocytes treated with ER stress inducers. ER stress had little effect on IR tyrosine phosphorylation per se but greatly reduced IR downstream signaling. The ER stress-induced reduction in IR cellular levels was greatly alleviated by the autophagy inhibitor 3-methyladenine but not by the proteasome inhibitor N-benzoyloxycarbonyl (Z)-Leu-Leu-leucinal (MG132). Inhibition of autophagy prevented IR degradation but did not rescue IR downstream signaling, consistent with an adaptive role of autophagy in response to ER stress-induced insulin resistance. Finally, chemical chaperone treatment protects cells from ER stress-induced IR degradation in vitro and obesity-induced down-regulation of IR and insulin action in vivo. Our results uncover a new mechanism underlying obesity-induced insulin resistance and shed light on potential targets for the prevention and treatment of obesity-induced insulin resistance and type 2 diabetes.

Fig. 1.

Obesity induces ER stress and IR down-regulation. A, the expression levels of IRβ in WAT of 4-month-old db/db mice or lean control mice fed with normal chow diet were determined by Western blot and quantified and normalized to β-tubulin. B, proteins from visceral adipose tissue of normal weight (NW) and obese patients (OB) (Table 1) were determined by Western blot with anti-IRβ antibody. The expression levels of IRβ were quantified and normalized to β-tubulin. Data represent the mean ± S.E.M. ^*, P < 0.05.

Fig. 2.

ER stress down-regulates IR protein levels and impairs insulin signaling. A, differentiated 3T3-L1 cells were treated with TG (1 μM) for different times as indicated, followed with or without 10 nM insulin for 5 min as indicated. The expression of proteins in cell lysates was determined by Western blot with specific antibodies as indicated. B, differentiated 3T3-L1 adipocytes were treated with or without TG (1 μM) for 36 h followed by 10 nM insulin for 5 min. Cells were lysed and IRS-1 in cell lysates was immunoprecipitated using an antibody to IRS-1. The tyrosine phosphorylation and protein expression of immunoprecipitated IRS-1 were determined by Western blot analysis with specific antibodies as indicated. C, differentiated 3T3-L1 adipocytes were pretreated with or without SP600125 (10 μM) for 1 h and then with vehicle or TG (1 μM) for 36 h. Cells were then stimulated with 10 nM insulin for 5 min. Cell lysates were resolved by SDS-polyacrylamide gel electrophoresis and the phosphorylation and/or expression of IRβ, JNK1, Akt, and CHOP were determined by Western blot analysis with specific antibodies as indicated. D, differentiated 3T3L1 adipocytes were treated with 1 μM TG for different times as indicated, followed with or without 10 nM insulin stimulation for 5 min as indicated. IRβ was immunoprecipitated from cell lysates and the tyrosine phosphorylation and protein expression were determined by Western blot analysis with an antibody to phosphor-Tyr (top) and to IRβ (bottom), respectively. E, differentiated 3T3-L1 adipocytes were treated with or without TG (1 μM) for 36 h, flowed by 10 nM insulin for 5 min and then lysed. IRβ was immunoprecipitated from cell lysates. Same amount of IRβ was separated by SDS-polyacrylamide gel electrophoresis and analyzed by Western blot using an antibody to phosphor-Tyr (top) or IRβ (bottom), respectively. Data are representatives of three independent experiments with similar results.

Fig. 3.

ER stress reduces IR cellular levels by an autophagy-dependent mechanism. A, differentiated 3T3-L1 adipocytes were pretreated with or without 3-MA (5 μM) for 1 h followed by TG (0.01 μM) for different times as indicated. The cells were then treated with 10 nM insulin for 5 min and lysed. The phosphorylation and/or protein levels of IRβ, Akt, LC3, and β-tubulin were determined by Western blot with specific antibodies as indicated. Data are representatives of four independent experiments with similar results. B, the relative expression levels of IRβ were quantified and normalized to β-tubulin. Data represent the mean ± S.E.M. ^**, P < 0.01. C, 3T3-L1 adipocytes were pretreated with or without 3-MA (5 μM) for 1 h followed with TG (0.01 μM) for 36 h. Cells were then treated with insulin (10 nM) for 5 min and lysed. IRβ was immunoprecipitated from cell lysates and its tyrosine phosphorylation and protein levels were determined by Western blot using an antibody to phosphotyrosine and IRβ, respectively. Akt phosphorylation and the cellular levels of β-tubulin in cell lysates were determined by Western blot using specific antibodies. β-Tubulin was shown as a loading control. Data are representatives of three independent experiments with similar results. D, 3T3L1 adipocytes were pretreated with or without 3-MA (5 μM) for 1 h followed with TG (0.01 μM) for 36 h. Cells were then serum-starved for 2 h and treated with insulin (100 nM) or phosphate-buffered saline for 10 min. 2-deoxy-d-2-[³H]glucose (0.5 μCi/ml) and 10 M 2-deoxyglucose were added to the cells. Uptake was allowed at 37°C for 10 min. After intense washing, cells were lysed, followed by measurements of 2-deoxy-d-2-[³H]glucose radioactivity. Data represent the mean ± S.E.M. ^**, P < 0.01; n = 4.

Fig. 4.

Chemical chaperone (TUDCA) prevents ER stress-induced IR down-regulation in 3T3L1 adipocytes. A, differentiated 3T3-L1 adipocytes were pretreated with or without TUDCA (1 mM) for 1 h followed with TG (0.01 μM) for different times as indicated. The cells were stimulated with 10 nM insulin for 5 min and lysed. The expression levels of IRβ and phosphorylation of Akt (B) were determined by Western blot and quantified. Data represent the mean ± S.E.M. ^*, P < 0.05. C, 3T3-L1 adipocytes were pretreated with or without TUDCA for 1 h followed with or without TG for 36 h. Cells were then treated with 10 nM insulin for 5 min and lysed. IRβ was immunoprecipitated from cell lysates and its tyrosine phosphorylation and protein levels were determined by Western blot using an antibody to phosphor-Tyr (first blot) or to IRβ (second blot), respectively. Akt phosphorylation and expression in cell lysates were determined by Western blot with antibodies to phospho-Akt (Thr308) and to Akt, respectively (third and fourth blots). Tubulin is shown as a loading control. Data are representatives of three independent experiments with similar results. D, differentiated 3T3-L1 adipocytes were pretreated with or without TUDCA (1 mM) or 3-MA (5 μM) for 1 h, followed with or without 0.01 μM TG for 36 h. The cells were then fixed and the cellular localization of IRβ was detected by immunofluorescence studies. Scale bar, 20 μm. Data are representatives of three independent experiments with similar results.

Fig. 5.

Chemical chaperone (TUDCA) prevents obesity-induced IRβ down-regulation and improves insulin sensitivity in vivo. A, fasting blood glucose levels were measured after treating the lean mice and db/db mice with saline or TUDCA for 18 days. Data represent the mean ± S.E.M. ^*, p < 0.05. B, insulin (2 IU/kg) tolerance tests of lean control mice and db/db mice treated with vehicle (saline) or TUDCA for 22 days. Data represent the mean ± S.E.M. n = 8. ^**, p < 0.01; ^***, p < 0.001 (TUDCA versus vehicle-treated mice). C, lean mice and db/db mice treated with saline or TUDCA for 26 days followed by stimulation of insulin (2 IU/kg, 10 min) before termination as indicated. The expression levels of IRβ, LC3-I, and LC3-II as well phosphorylation of Akt (Thr³⁰⁸) or eIF2α (Ser⁵²) were determined by Western blot with specific antibodies as indicated. β-Actin was used as a loading control. The relative expression levels of IRβ (D) and phosphorylation of Akt (E) or eIF2α (F) were quantified. Data represent the mean ± S.E.M. ^**, P < 0.05. n = 8.