Effect of paraquat-induced oxidative stress on insulin regulation of insulin-like growth factor-binding protein-1 gene expression

Abstract

Oxidative stress is thought to play a role in the development of insulin resistance. In order to elucidate the molecular effect of oxidative stress on liver insulin signaling, we analyzed the effect of paraquat (1,1-dimethyl-4,4-dipyridynium; PQ)-derived oxidative stress on the expression of insulin-dependent genes and activation of liver insulin signaling pathway. Incubation of primary cultured rat hepatocytes with 2 mM PQ for 6 h impaired the suppressive effect of insulin on insulin-like growth factor-binding protein-1 (IGFBP-1) gene expression, but did not influence glucose-6-phosphatase gene expression. Insulin-dependent phosphorylation or activation of insulin receptor, insulin receptor substrate-1 and -2, phosphatidylinositol 3-kinase, Akt and forkhead in rhabdomyosarcoma were not affected by PQ pre-treatment. In contrast, PQ treatment impaired insulin-dependent phosphorylation of mammalian target of rapamycin (mTOR). These results indicate that PQ-induced oxidative stress impairs insulin-dependent mTOR activation and that this impairment probably causes inhibition of insulin-dependent repression of IGFBP-1 expression.

Keywords: IGFBP-1; hepatocyte; insulin; mTOR; paraquat.

Fig. 1

PQ treatment induced oxidative stress in primary cultured rat hepatocytes. (A) The viability of cells treated with 0, 1, or 5 mM PQ for 15 min, 6 h, 12 h and 24 h was estimated by trypan blue exclusion assay. (B) Plasma membrane damage of the cells treated with 0, 2, or 5 mM PQ for 15 min, 6 h, 12 h and 24 h was estimated by measuring LDH activity in the cell culture medium. Values shown are means ± SEM of three dishes, and expressed as relative values to cells without PQ treatment (PQ 0 mM). *, ^# Significantly different from cells without PQ treatment (p<0.05).

Fig. 2

PQ-derived oxidative stress impairs insulin regulation of the IGFBP-1 mRNA level in primary cultured rat hepatocytes. (A) Effect of PQ on insulin regulation of IGFBP-1 and G6Pase mRNA level was measured with cells treated with/without 2 mM PQ, 100 nM insulin for 6 h. Total RNA was prepared from the cells and the mRNA contents of IGFBP-1 and G6Pase were estimated by RT-PCR. Values shown are relative mRNA levels compared to cells without insulin and PQ treatment (Ins-, PQ 0 mM) and expressed as means ± SEM of three dishes. (B) Effect of NAC on PQ treatment was examined with cells treated with/without 2 mM PQ, 100 nM insulin and antioxidant NAC simultaneously for 6 h. Values shown are relative IGFBP-1 mRNA levels compared to the cells without PQ, NAC, and insulin treatment (Ins-, NAC 0 mM, PQ 0 mM) and expressed as means ± SEM of three dishes. ^#Significantly different from cells without insulin treatment (p<0.05). *Significantly different between the two groups by PQ or NAC treatment (p<0.05).

Fig. 3

PQ treatment did not affect insulin-dependent phosphorylation of IRSs and association of PI 3-kinase with IRS. (A, D) Phosphorylation of IRSs and PI 3-kinase association was estimated with primary cultured rat hepatocytes pre-treated with 0, 2, or 5 mM PQ for 6 h and then treated with/without 100 nM insulin for 1 min. After the protein lysate of the cells was immunoprecipitated with anti-IRS antibody, the IRS phosphorylation level, IRS-associated PI 3-kinase p85 subunit level, and IRS protein level were analyzed by immunoblotting with anti-phosphotyrosine (PY-20), anti-p85 and anti-IRS antibodies, respectively. (B, C, E, F) IRS phosphorylation level and the amount of IRS-associated p85 were quantified and expressed as relative values to cells treated with insulin and without PQ (Ins+, PQ 0 mM). Values are means ± SEM of three dishes. ^#Significantly different from cells without insulin treatment (p<0.05). *Significantly different between the two groups by PQ treatment (p<0.05).

Fig. 4

PQ treatment did not affect insulin-dependent activation of PI 3-kinase. Primary cultured rat hepatocytes were pre-treated with 0, 2, or 5 mM PQ for 6 h and then treated with/without 100 nM insulin for 1 min. Protein lysate was prepared from the cells, immunoprecipitated with anti-IRS-1 antibody (A) or anti-IRS-2 antibody (B), and PI 3-kinase activity was analyzed by measuring ³²P incorporation into phosphatidylinositol (upper panels). ³²P incorporated into phophatidylinositol 3,4,5-triphosphate was quantified and expressed as relative values to cells treated without insulin and PQ (Ins-, PQ 0 mM) (lower panels). Values are means ± SEM of three dishes. #Significantly different from cells without insulin treatment (p<0.05).

Fig. 5

PQ treatment did not affect insulin-dependent phosphorylation of Akt and FKHR. Primary cultured rat hepatocytes were pre-treated with 0, 2, or 5 mM PQ for 6 h and then treated with/without insulin for 1 min for Akt and 15 min for FKHR. Protein lysate was prepared from the cells, and Ser 473 phosphorylation of Akt (A) and Ser 256 phosphorylation of FKHR (B) was analyzed by Western blot analysis with anti-phospho-Akt (Ser 473) or anti-phospho-FKHR (Ser 256) antibody (upper panels). Phosphorylated levels were quantified and expressed as relative values to cells without insulin and PQ treatment (Ins-, PQ 0 mM) (lower panels). Values are means ± SEM of three dishes. ^#Significantly different from the cells without insulin treatment (p<0.05).

Fig. 6

PQ treatment impaired insulin-induced phosphorylation of mTOR. (A) phosphorylation of mTOR was analyzed with primary cultured rat hepatocytes pre-treated with/without 2 mM PQ for 6 h and then treated with/without 100 nM insulin for 10 min. Protein lysate was analyzed by Western blot analysis with anti-phospho-mTOR (Ser 2448) antibody. Phosphorylated levels were quantified and expressed as relative values to cells without insulin and PQ treatment (Ins-, PQ 0 mM). Values are means ± SEM of three dishes. ^#Significantly different from the cells without insulin treatment (p<0.05). (B) The effect of rapamycin treatment on the IGFBP-1 mRNA level was measured by RT-PCR with primary cultured rat hepatocytes treated with/without 10 nM rapamycin and 1 µM insulin for 6 h simultaneously. Values represent the repressive effect of insulin on the IGFBP-1 mRNA level (1 µM insulin/0 M insulin) and are expressed as means ± SEM of three dishes. *Significantly different between the two groups by rapamycin treatment (p<0.05).