Interleukin-6 Induces Cellular Insulin Resistance in Hepatocytes
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Figures
- FIG. 1.
Time-dependent effects of IL-6 on tyrosine phosphorylation of the IR. Confluent HepG2 cells were serum starved overnight before treatment with 20 ng/ml IL-6 for 0–4 h. At the indicated times, cells were treated with insulin (0.5 and 100 nmol/l) or without insulin for 3 min, harvested, and lysates-protein normalized. IR was immunoprecipitated, separated by SDS-PAGE, and subjected to Western blot analysis with an antiphosphotyrosine antibody (4G10). A comparable IR immunoprecipitate from 0.5 nmol/l insulin-treated cells was probed for IR mass. Results were visualized by enhanced chemiluminescence (Amersham Pharmacia). Data are representative of three independent experiments.
- FIG. 2.
Time-dependent effects of IL-6 on tyrosine phosphorylation of IRS-1. Confluent HepG2 cells were treated as in Fig. 1. IRS-1 was immunoprecipitated, separated by SDS-PAGE, and subjected to Western blot analysis with an antiphosphotyrosine antibody (Upstate Biological). A comparable IRS-1 immunoprecipitate from 0.5 nmol/l insulin-treated cells was probed for IRS-1 mass. Results were visualized by enhanced chemiluminescence (Amersham Pharmacia). Representative blots of three independent experiments are shown. The 0.5 nmol/l blot required longer exposure times to obtain comparable signal intensity to the 100 nmol/l insulin blot. The data for 0.5 nmol/l insulin from the three experiments were quantitated by densitometry and expressed as percent of initial ± SD (no IL-6, time zero). *P < 0.01 and **P < 0.05 as compared with control (no IL-6, time zero).
- FIG. 3.
Time-dependent effects of IL-6 on association of the p85 subunit of PI 3-kinase with IRS-1. Confluent HepG2 cells were treated as in Fig. 1. IRS-1 was immunoprecipitated, separated by SDS-PAGE, and subjected to Western blot analysis with an anti-p85 antibody (Upstate Biological). A comparable lysate from 0.5 nmol/l insulin-treated cells was probed for p85 mass. Results were visualized by enhanced chemiluminescence (Amersham Pharmacia). Representative blots of three independent experiments are shown. The data for 0.5 nmol/l insulin from the three experiments were quantitated by densitometry and expressed as percent of initial ± SD (no IL-6, time zero). **P < 0.05 as compared with control (no IL-6, time zero).
- FIG. 4.
Time-dependent effects of IL-6 on insulin-dependent activation of Akt. Confluent HepG2 cells were treated as in Fig. 1. Cell lysates were separated by SDS-PAGE and subjected to Western blot analysis with an anti-phosphoAkt (ser473) antibody (Cell Signaling Technology) or an anti-Akt antibody (Cell Signaling Technology). The latter was used to probe lysates from 0.5 nmol/l insulin-treated cells to determine Akt mass. Results were visualized by enhanced chemiluminescence (Amersham Pharmacia). Representative blots of three independent experiments are shown. The data for 0.5 nmol/l insulin from the three experiments were quantitated by densitometry and expressed as percent of initial ± SD (no IL-6, time zero). **P < 0.05 as compared with control (no IL-6, time zero).
- FIG. 5.
Effect of IL-6 on sensitivity of HepG2 cells to insulin-induced IRS-1 tyrosine phosphorylation. Confluent HepG2 cells were serum starved overnight before pretreatment for 90 min with or without IL-6 (20 ng/ml) and subsequent treatment with insulin at the indicated concentrations for 3 min. IRS-1 was immunoprecipitated from lysates, separated by SDS-PAGE, and subjected to Western blot analysis with antiphosphotyrosine antibody. Results of four independent experiments were quantitated by densitometry and expressed as the fold increase over IL-6-free basal ± SD. A representative Western blot is shown.
- FIG. 6.
IL-6 inhibits insulin-induced formation of p85/IRS-1 complexes. HepG2 cells were treated as in Fig. 5. Western blots of IRS-1 were probed with anti-p85 antibody. A representative blot of four independent experiments is shown. Results of the four experiments were quantitated by densitometry and expressed as fold increase over IL-6-free basal ± SD. Inset: data were expressed as fold increase of control and IL-6-treated cells relative to their respective insulin-free basal levels.
- FIG. 7.
Effect of IL-6 on insulin-dependent Akt activation. Confluent HepG2 cells were serum starved overnight before pretreatment for 90 min with or without IL-6 (20 ng/ml) and subsequent treatment with insulin at the indicated concentrations for 3 min. Lysates were separated by SDS-PAGE and subjected to Western blot analysis with anti-pSer473-Akt antibody. A representative blot of four independent experiments is shown. Results of the four experiments were quantitated by densitometry and expressed as fold increase ± SD of control and IL-6-treated cells relative to their respective insulin-free basal levels.
- FIG. 8.
Effect of IL-6 on STAT-3 activation, insulin-dependent Akt activation, and glycogen synthesis in primary hepatocytes. Primary mouse hepatocytes were incubated overnight in serum-free media. A: Cells were treated with or without IL-6 at the indicated concentrations for 10 min. Cells were harvested, and Western blots of lysates were probed with either an anti-phosphoSTAT-3 (pY705) antibody (Cell Signaling Technology) or an anti-STAT-3 antibody (Cell Signaling Technology). B: After a 90-min pretreatment with or without IL-6 (20 ng/ml), cells were treated with insulin for 3 min. Cells were harvested, and Western blots of lysates were probed with the anti-pSer473-Akt antibody. These results are representative of three experiments. C: After a 90-min pretreatment with or without IL-6 (20 ng/ml) in low glucose media, cells were treated with [14C]d-glucose and insulin at the indicated concentrations. After 3 h, the cells were harvested and glycogen isolated as described in research design and methods. Results represent the means ± SE of four experiments, each performed in triplicate.
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