Resveratrol inhibits insulin responses in a SirT1-independent pathway

Abstract

Resveratrol mimics calorie restriction to extend lifespan of Caenorhabditis elegans, yeast and Drosophila, possibly through activation of Sir2 (silent information regulator 2), a NAD+-dependent histone deacetylase. In the present study, resveratrol is shown to inhibit the insulin signalling pathway in several cell lines and rat primary hepatocytes in addition to its broad-spectrum inhibition of several signalling pathways. Resveratrol effectively inhibits insulin-induced Akt and MAPK (mitogen-activated protein kinase) activation mainly through disruption of the interactions between insulin receptor substrates and its downstream binding proteins including p85 regulatory subunit of phosphoinositide 3-kinase and Grb2 (growth factor receptor-bound protein 2). The inhibitory effect of resveratrol on insulin signalling is also demonstrated at mRNA level, where resveratrol reverses insulin effects on phosphoenolpyruvate carboxykinase, glucose-6-phosphatase, fatty acid synthase and glucokinase. In addition, RNA interference experiment shows that the inhibitory effect of resveratrol on insulin signalling pathway is not weakened in cells with reduced expression of SirT1, the mammalian counterpart of Sir2. These observations raise the possibility that resveratrol may additionally modulate lifespan through inhibition of insulin signalling pathway, independently of its activation of SirT1 histone deacetylase. Furthermore, the present study may help to explain a wide range of biological effects of resveratrol, and provides further insight into the molecular basis of calorie restriction.

Figures

Figure 1. Resveratrol treatment inhibits insulin signalling pathway at transcriptional level

(A) Resveratrol treatment abolished insulin effects on downstream target genes in rat primary hepatocytes. Rat primary hepatocytes were isolated and set up as described in the Experimental section and treated as indicated in the Figure. For combined treatments of insulin and resveratrol, cells were pre-incubated with 100 nM resveratrol for 30 min before they were challenged with insulin (100 nM) for another 4 h. Total RNA was extracted using TRIzol® reagents according to the manufacturer's instructions. Equal amount of RNA (16 μg/lane) was used for Northern-blot analysis using probes for PEPCK, GK, glucose-6-phosphatase and IGFBP-1. The ApoE mRNA level was used as the loading control. (B) Dose–response study of resveratrol effect on insulin signalling pathway. Rat primary hepatocytes were pre-incubated with resveratrol at 0, 10, 50 and 200 μM for 30 min before they were challenged with 100 nM insulin for another 16 h. Total RNA was extracted using TRIzol® reagents, and equal amount of RNA (16 μg/lane) was used for Northern-blot analysis using probes for PEPCK and FAS. Again, ApoE mRNA level was measured as the loading control. (C) MTT study of H4IIE cells treated with resveratrol. H4IIE cells were seeded at 2×106/60 mm dish in DMEM supplemented with 5% fetal bovine serum on day 1. Cells were serum-starved overnight before they were changed into serum-free DMEM supplemented with 1 μM dexamethasone and treated with resveratrol at 0, 10, 50 and 100 μM for another 24 h. The amount of cells was measured using an MTT based CGD-1 kit from Sigma. Results shown are representative of three independent experiments. (D) Resveratrol reversed insulin effects on PEPCK and FAS mRNA levels in H4IIE cells. H4IIE cells were set up as in (C), and treated with insulin (100 nM) and resveratrol (100 μM) for another 16 h. Total RNA was extracted using TRIzol® reagent according to the manufacturer's instruction. The expression levels of FAS and PEPCK were analysed using Northern-blot analysis. The expression level of cyclophilin was used as the loading control.

Figure 2. Resveratrol reverses insulin effects on MAPK and Akt phosphorylation

(A) Dose–response study of resveratrol effects on insulin-induced MAPK and Akt phosphorylation. H4IIE cells were seeded at 2×106/60 mm dish in DMEM supplemented with 5% fetal bovine serum, and serum-deprived overnight before they were changed into plain DMEM supplemented with 1 μM dexamethasone for further treatments. Cells were pre-incubated with resveratrol ranging from 1 to 200 μM or DMSO for 30 min and then treated with 100 nM insulin for another 10 min. Total cell lysates were prepared by harvesting cells in ice-cold PBS and resuspended in lysis buffer supplemented with protease and phosphatase inhibitors. Equal amounts of whole cell lysates were subjected to Western-blot analysis using antibodies against phosphorylated forms of ERK p42/44 and Akt (Ser473). Total protein amounts of Akt and MAPK were also measured as the loading control. In the right panel, the band intensity of MAPK and Akt phosphorylation was quantified from three independent experiments, and the average of three experiments was plotted against the concentration of resveratrol. (B) Pre-incubation of resveratrol was required for its maximum inhibition of insulin-stimulated Akt and MAPK activation. H4IIE cells were set up as described in (A), with or without pre-incubation of 100 μM resveratrol as indicated in the Figure before they were treated with 100 nM insulin for another 10 min. Whole cell lysates were prepared for Western-blot analysis. (C) Resveratrol reversed insulin effects on Akt phosphorylation in rat primary hepatocytes. Rat primary hepatocytes were isolated and set up as described in Figure 1(A); cells were changed into M199 supplemented with 100 nM dexamethasone and pre-incubated with 100 μM resveratrol for 30 min before they were challenged with 100 nM insulin for another 10 min. Total cell lysates were prepared for Western-blot analysis. (D) HepG2 cells were set up at 50% confluence in DMEM supplemented with 5% fetal bovine serum for 1 day before they were serum-starved overnight and changed into plain DMEM supplemented with 1 μM dexamethasone. Cells were pre-incubated with 100 μM resveratrol for 30 min before they were treated with 100 nM insulin for another 10 min. Total cell lysates were prepared for Western-blot analysis.

Figure 3. Resveratrol treatment has no effect on insulin-induced tyrosine phosphorylations of insulin receptor, IRS-1 and IRS-2

Cells were set up as described in the legend of Figure 2(A) and pre-incubated with 100 μM resveratrol for 10 min before they were challenged with 100 nM insulin for 10 min. Total cell lysates were prepared for both Western-blot analysis and immunoprecipitation. (A) Resveratrol treatment inhibited insulin-induced MAPK and Akt activation. (B) Resveratrol treatment had no effect on insulin-induced tyrosine phosphorylations of insulin receptor (IR) β, IRS-1 and IRS-2. Insulin receptor β, IRS-1 and IRS-2 were immunoprecipitated (IP) from total cell lysates prepared in (A), using their respective antibodies. Tyrosine phosphorylation statuses of insulin receptor β, IRS-1 and IRS-2 were analysed using a phosphotyrosine antibody (4G10; pY) from Upstate Biotechnology. The amounts of immunoprecipitated insulin receptor β, IRS-1 and IRS-2 were also analysed using Western-blot analysis.

Figure 4. Resveratrol inhibits insulin signalling pathway through disruption of insulin-induced IRS complexes

(A) Resveratrol disrupts insulin-induced association between p85 regulatory unit of PI3K and IRSs (IRS-1 and IRS-2). H4IIE cells were set up and treated as described in the legend of Figure 3, and both IRS-1 and IRS-2 were immunoprecipitated from total cell lysates using their respective antibodies. The protein levels of p85 associated with IRSs were analysed using p85 antibody from Cell Signaling Technology. The left panel shows a typical Western-blot analysis, and the right panel shows the protein levels of p85 associated with either IRS-1 or IRS-2 respectively under conditions indicated in the Figure. The results shown are averages of three independent experiments, quantified by ImageJ (NIH Image) program. The results were also analysed using ANOVA analysis. *P<0.05, **P<0.01. (B) Dose–response study of resveratrol effect on insulin-induced IRS-1 complex. H4IIE cells were set up as in (A), and pretreated with resveratrol at 1, 10 and 100 μM for 10 min before they were challenged with 100 nM insulin for another 10 min. IRS-1 was immunoprecipitated from total cell lysates to measure the protein levels of tyrosine-phosphorylated form of IRS-1, total IRS-1, p85 and Grb2 using their respective antibodies. In the meantime, the total cell lysates were also used to measure the phosphorylated Akt and MAPK levels under these conditions. (C) Insulin-induced MAPK activation is partially dependent on the PI3K activation. H4IIE cells were set up as described in Figure 2, and pre-incubated with either wortmannin at 5, 25 and 100 nM for 2 h or resveratrol at 10, 50 and 200 μM for 10 min, before they were treated with insulin for another 10 min. Total cell lysates were prepared for Western-blot analysis. (D) Resveratrol directly inhibits insulin-induced association between IRS-1 and p85 subunit of PI3K in vitro. Insulin-induced IRS-1 complex was immunoprecipitated from total cell lysates prepared using insulin-treated H4IIE cells and divided equally, and washed with lysis buffer before they were incubated with DMSO or resveratrol at 5 and 50 μM for 20 min at 37 °C in lysis buffer without Triton X-100. These treated IRS-1 complexes were washed briefly three times with lysis buffer and the p85 protein levels were analysed using Western-blot analysis.

Figure 5. Biological effects of resveratrol result mainly from its inhibition of insulin signalling pathway in liver cells

(A) HEK-293 cells were set up in DMEM supplemented with 5% fetal bovine serum for 1 day and serum-deprived overnight prior to the treatments. Cells were then changed into DMEM supplemented with 1 μM dexamethasone and incubated with 100 μM resveratrol for 30 min before they were challenged with insulin (100 nM, 10 min), IGF-1 (50 ng/ml, 15 min), EGF (50 ng/ml, 30 min) and 5% fetal bovine serum (30 min). Total cell lysates were prepared for Western-blot analysis using antibodies against both total and phosphorylated forms of Akt and MAPK. The protein amount of phosphorylated MAPK and Akt were also quantified using ImageJ (NIH Image) program, and the results from three independent experiments were averaged and plotted relative to the basal level. The significance of resveratrol inhibition of MAPK and Akt activations elicited by these growth factors was analysed using Student's t test. *P<0.05. (B) Insulin signalling pathway is an essential pathway leading to MAPK and Akt activations in liver cells. H4IIE cells were set up as described in Figure 2(A). Cells were changed into DMEM supplemented with 1 μM dexamethasone and pre-incubated with 100 μM resveratrol for 20 min and then challenged with various growth factors, including insulin (100 nm, 10 min), IGF-1 (100 ng/ml, 30 min), EGF (50 ng/ml, 30 min) and NGF (50 ng/ml, 30 min). Whole cell lysates were prepared for Western-blot analysis under these conditions.

Figure 6. Resveratrol inhibits insulin-induced MAPK and Akt phosphorylations in a SirT1-independent pathway

HEK-293 cells were seeded at 5×105/60 mm dish on day 0. Cells were transfected with 400 pmol of RNA oligonucleotide sequence against luciferase sequence in control group and same amount of RNA oligonucleotides against human SirT1 sequence in anti-SirT1 group using Oligofectamine reagent. Cells were transfected with RNA oligonucleotides for 30 h before they were serum-starved overnight. Both control and anti-SirT1 groups were treated with either DMSO or 100 μM resveratrol for 10 min before they were treated with 100 nM insulin for another 10 min. Total cell lysates were prepared for Western-blot analysis. (A) Western-blot analysis of the phosphorylation statuses of MAPK and Akt in response to insulin treatment in control cells or cells transfected with RNA oligonucleotides against SirT1. (B) The protein levels of SirT1 in cells described in (A) were analysed using Western-blot analysis. The results were also quantified using ImageJ (NIH Image) program. The average amount of SirT1 in control cells (lanes 1–4) were compared with those in cells transfected with RNA oligonucleotides against SirT1 (lanes 5–8) using Student's t test. ***P<0.001.