Protein-tyrosine phosphatases are involved in interferon resistance associated with insulin resistance in HepG2 cells and obese mice

Abstract

Insulin resistance is a risk factor for non-response to interferon/ribavirin therapy in patients with chronic hepatitis C. The aim of this study was to determine the role played by protein-tyrosine phosphatases (PTPs) in the absence of interferon-α (IFNα) response associated with insulin resistance. We induced insulin resistance by silencing IRS-2 or by treating HepG2 cells with tumor necrosis factor-α (TNFα) and analyzed insulin response by evaluating Akt phosphorylation and IFNα response by measuring Stat-1 tyrosine phosphorylation and 2',5'-oligoadenylate synthase and myxovirus resistance gene expression. The response to IFNα was also measured in insulin-resistant obese mice (high fat diet and ob/ob mice) untreated and treated with metformin. Silencing IRS-2 mRNA induces insulin resistance and inhibits IFNα response. Likewise, TNFα suppresses insulin and IFNα response. Treatment of cells with pervanadate and knocking down PTP-1B restores insulin and IFNα response. Both silencing IRS-2 and TNFα treatment increase PTP and PTP-1B activity. Metformin inhibits PTP and improves IFNα response in insulin-resistant cells. Insulin-resistant ob/ob mice have increased PTP-1B gene expression and activity in the liver and do not respond to IFNα administration. Treatment with metformin improves this response. In HepG2 cells, insulin resistance provokes IFNα resistance, which is associated with an increased PTP-1B activity in the liver. Inhibition of PTP-1B activity with pervanadate and metformin or knocking down PTP-1B reestablishes IFNα response. Likewise, metformin decreases PTP-1B activity and improves response to IFNα in insulin-resistant obese mice. The use of PTP-1B inhibitors may improve the response to IFNα/ribavirin therapy.

FIGURE 1.

Silencing IRS-2 inhibits the response of cultured hepatocytes to insulin and IFNα. A, in HepG2 cells, IRS-2 protein expression was silenced, and RT-PCR was performed for IRS-2 (IRS-2 cDNA) and GAPDH (GAPDH cDNA) transcripts. Decreased IRS-2 protein expression was examined by Western blot. B, control (siRNA Control) and insulin-resistant cells (siRNA IRS-2) were treated with 100 nm insulin for 15 min in the absence or presence of 0.1 mm pervanadate (PV). Whole cellular proteins were analyzed by Western blot using specific antibodies against Akt or serine-phosphorylated Akt (pAkt). Control, untreated cells. C, control and insulin-resistant cells were treated with 250 units/ml IFNα for 60 min in the absence or presence of 0.1 mm pervanadate. Cellular proteins were immunoprecipitated (IP) with anti-Stat-1 antibody, and total and tyrosine phosphorylated Stat-1 levels were analyzed by Western blot (WB). D and E, 2′5′OAS (D) and Mx (E) gene expression was measured by quantitative RT-PCR using endogenous GAPDH as reference. Control cells and cells with silenced IRS-2 were treated with IFNα in the presence or absence of 0.1 mm pervanadate. These blots are representative of experiments that were repeated three times. ***, p < 0.001; NS, not significant.

FIGURE 2.

TNFα induces insulin and IFNα resistance in HepG2 cells. A, effects of 100 nm insulin on Akt phosphorylation (p-Akt) in the absence or presence of 20 ng/ml TNFα for 18 h are shown. The blot is representative of one experiment that was repeated four times with similar results. B, shown is the effect of insulin on tyrosine (pY-IRS-2) and serine (pSer-IRS-2) phosphorylation of IRS-2 in the absence or presence of TNFα. Blots are representative of one experiment that was repeated four times with similar results. IP, immunoprecipitation. WB, Western blot. C, shown are the effects of insulin on tyrosine and serine phosphorylation of IRS-1 in the absence or presence of TNFα. Experimental conditions and procedures were identical to those described for IRS-2. D, shown are the effects of IFNα on tyrosine phosphorylation of Stat-1 (pY-Stat-1) in HepG2 cells in the absence or presence of TNFα. U, units. E and F, shown are the effects of IFNα on 2′5′OAS (E) and Mx (F) gene expression in the presence or absence of TNFα or pervanadate (PV). ***, p < 0.001; NS, not significant.

FIGURE 3.

Inhibition or absence of PTP1B restored the response to IFNα in cells treated with TNFα. A, HepG2 cells were exposed to 250 units/ml IFNα for 60 min, 20 ng/ml TNFα for 18 h, or both in the absence or presence of 0.1 mm pervanadate added to the cells 60 min before treatment with IFNα. Cellular proteins were immunoprecipitated (IP) as indicated in Fig. 1C, and tyrosine-phosphorylated Stat-1 was analyzed (pY-Stat1) by Western blot (WB). B, HepG2 cells were exposed to 100 nm insulin (Ins) for 15 min, 20 ng/ml TNFα for 18 h, or both in the absence or presence of 0.1 mm pervanadate added to the cells before insulin. Cellular proteins were immunoprecipitated with anti-IRS-2 antibody analyzed by Western blot using specific antibody against IRS-2 and antiphosphotyrosine. pY-IRS-2, IRS-2 tyrosine-phosphorylated. C, neonatal PTP-1B-deficient hepatocytes (KO PTP1B) and their counterpart wild-type cells were cultured (WT PTP1B) in the presence or absence of 20 ng/ml TNFα for 18 h and treated with 100 nm insulin for 15 min. Total and serine phosphorylated Akt was analyzed by Western blot. D, total and tyrosine-phosphorylated Stat-1 were measured in neonatal wild-type and PTP-1B-deficient hepatocytes cultured in the conditions described in panel C and treated with 250 units/ml IFNα instead of insulin. E and F, neonatal PTP-1B-deficient hepatocytes were treated as indicated in panel D. 2′5′ OAS (E) and Mx (F) gene expression were measured by quantitative RT-PCR using endogenous GAPDH as reference. ***, p < 0.001; NS, not significant.

FIGURE 4.

Silencing IRS-2 and treatment of cells with TNFα increase PTP activity in HepG2 cells. A, PTP activity was measured in HepG2 cells with or without silenced IRS-2 treated with 250 units/ml IFNα for 60 min in the absence or presence of 0.1 mm pervanadate (PV) or 10 μg/ml metformin (MTF) added to the cells 60 or 30 min, respectively, before IFNα. ***, p < 0.001 as compared with not silenced control cells. NS, not significant. B, shown is a time- and dose-dependent effect of metformin on PTP activity. *, p < 0.05; **, p < 0.01; ***, p < 0.001; NS, not significant. C, PTP-1B enzyme activity was measured in HepG2 cells in the same experimental conditions as indicated in panel A. Proteins were extracted, and PTP-1B and β-actin were analyzed by Western blot. **, p < 0.01; ***, p < 0.001 as compared with not silenced control cells. NS, not significant. D, PTP activity was measured in cells treated with 20 ng/ml TNFα for 18 h, 250 units/ml IFNα for 60 min, or both in the absence or presence of 0.1 mm pervanadate or 10 μg/ml metformin. **, p < 0.01; ***, p < 0.001 as compared with cells untreated with pervanadate or metformin. NS, not significant. E, PTP1B activity was measured under the same conditions indicated in panel D. F, HepG2 cells were treated with TNFα in the absence and presence of pervanadate or metformin. After treatment, proteins were immunoprecipitated (IP) using a specific antibody either against JAK1 or Stat-1. Immunoprecipitated proteins were analyzed by Western blot (WB) using specific antibodies against JAK1, Stat-1, or PTP1B. Blots are representative of three separate experiments.

FIGURE 5.

Metformin prevents IFNα and insulin resistance induced by silencing IRS-2 or TNFα treatment. A, control cells and cells with silenced IRS-2 protein expression were treated with 250 units/ml IFNα for 30 min in the absence or presence of 10 μg/ml metformin (MTF). After treatment, cellular proteins were analyzed as described in Fig. 1C. The blot is representative of one experiment that was repeated three times. Control, untreated cells. IP, immunoprecipitation; WB, Western blot. B, control cells and cells with silenced IRS-2 were treated with 250 units/ml IFNα in the presence or absence of 10 μg/ml metformin. 2′,5′-Oligoadenylate synthase-1a and Mx gene expression was measured by quantitative RT-PCR using endogenous GAPDH as reference. These blots are representative of experiments that were repeated three times. C, cells with silenced IRS-2 were treated with 100 nm insulin for 15 min in the absence or presence of 10 μg/ml metformin. After treatment, cellular proteins were analyzed as described in Fig. 1B. Shown is a representative blot of an experiment that was repeated twice. D, HepG2 cells were exposed to 250 units/ml IFNα for 60 min, 20 ng/ml TNFα for 18 h, or both in the absence or presence of 10 μg/ml metformin added to the cells 30 min before the addition of IFNα. After treatment, cellular proteins were extracted and immunoprecipitated as indicated in Fig. 1C. Blots are representative of one experiment that was repeated three times with similar results. pY-Stat-1, Stat-1-phosphorylated at tyrosine. E, shown are the effects of IFNα on 2′,5′ oligoadenyl synthase and Mx gene expression in the presence or absence of TNFα or metformin. ***, p < 0.001; NS, not significant.

FIGURE 6.

Obese mice are resistant to insulin and IFNα. Treatment of ob/ob mice with metformin (MTF) decreased PTP-1B activity and restored insulin and IFNα sensitivity. C57BL/6J, ob/ob mice, and mice fed with a high fat diet (HFD) were treated with IFNα2a, metformin, or both as indicated under “Experimental Procedures.” A, shown are concentrations of TNFα in liver tissue of the 12 groups of mice. B, shown is total Akt and serine phosphorylated Akt (pAkt) in the liver before (basal) and 15 min after injecting 0.5 units/Kg insulin intraperitoneally. C, shown is 2′,5′OAS gene expression. D, shown is Mx gene expression. E, shown is PTP-1B gene expression. F, shown is PTP-1B activity. *, p<0.05; ***, p < 0.001 as compared with control mice. NS, not significant.