Resveratrol up-regulates SIRT1 and inhibits cellular oxidative stress in the diabetic milieu: mechanistic insights

Abstract

Several lines of evidence support a role for oxidative stress in diabetic complications. Diabetic patients have increased O(2)(-) production in monocytes. Loss of SIRT1 activity may be associated with metabolic diseases such as diabetes. Several studies have shown that SIRT1 can regulate mammalian FOXO transcription factors through direct binding and/or deacetylation. However, interactions between SIRT1 and FOXO under diabetic conditions are unclear. The phytochemical resveratrol has recently gained attention for its protection against metabolic disease. Resveratrol has been shown to increase mitochondrial function by activating SIRT1. In this study, we tested the protective effect of resveratrol on cellular oxidative stress through the SIRT1-FOXO pathway under high-glucose conditions. Human monocytic (THP-1) cells were cultured in the presence of mannitol (osmolar control) or normoglycemic (NG, 5.5 mmol/l glucose) or hyperglycemic (HG, 25 mmol/l glucose) conditions in absence or presence of resveratrol (3 and 6 μmol/l) for 48 h. We first examined SIRT1 activity and oxidative stress in monocytes of Type 1 diabetes mellitus (T1DM) patients compared with healthy controls. In T1DM patients, monocytic SIRT1 expression was significantly decreased and p47phox expression was increased compared with controls. Under HG in vitro, SIRT1 and FOXO3a were significantly decreased compared with NG, and this was reversed by resveratrol treatment, concomitant with reduction in HG-induced superoxide production and p47phox. Under HG, SIRT1 small interfering RNA (siRNA) inhibited FOXO3a, and there was no beneficial effect of resveratrol in siRNA-treated HG-induced cells. Thus, resveratrol decreases HG-induced superoxide production via up-regulation of SIRT1, induction of FOXO3a and inhibition of p47phox in monocytes.

Figure 1. Decreased SIRT1 expression and increased p47phox expression in T1DM subjects compared to normal subjects

Monocytes were isolated from fasting blood obtained from T1DM and normal subjects as described in Methods. For the western blot analysis, protein was subjected to SDS-PAGE and used SIRT1 and p47phox antibodies (1:1000 dilutions) as detailed in Materials and Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for TATA binding protein (TBP) and β-actin. The immunoblot shown here are representative of 8 experiments with similar results. Densitometric ratios to TBP and Beta actin are provided in the lower panel. *p<0.01 compared to controls

Figure 2. A) Resveratrol treatment significantly induced SIRT1 gene expression and HDAC activity in HG-induced THP-1 cells in vitro (B) Induced HDACs activity by resveratrol treatment in HG-induced THP-1 cells. (C) Resveratrol treatment induces FOXO3a gene expression in HG-induced THP-1 cells

(A and C) For the western blot analysis, protein was subjected to SDS-PAGE and used SIRT1, FOXO3a and FOXO1a antibodies (1:1000 dilutions) as detailed in Materials and Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for TATA binding protein (TBP). The immunoblot shown here are representative of three independent experiments with similar results. (B) Following treatment of cells with 3 and 6 µmol/L of reveratrol for 48 h as described in Methods, cells were harvested and nuclear lysates were prepared. 10 µg of nuclear lysate protein from each group were taken for determination of HDACs activity. The experiment was done according to the manufacturer's instructions. Absorbance was taken at 405 nm and 440 nm by using ELISA reader. Results were shown as mean ± SD of 5 different experiments. *a: p<0.05 compared to NG; *p<0.05; **p<0.01 compared to HG

Figure 3. Resveratrol treatment induced FOXO3a in HG-induced monocytes by immunofluorescence

The cells (50–60% confluent) were treated with 3–6 µmol/L resveratrol for 48 h. Cells were fixed with 4% formaldehyde for 30 min at 4°C and stained overnight at 4°C with SIRT1 and FOXO3a as described in Methods. Data from a typical experiment of 5 are shown; Magnification ×400.

Figure 4. Effect of resverarol on superoxide production and p47phox gene expression level in HG –induced THP-1 cells

(A) NG, HG and HG-resveratrol treated cells were incubated with 5 µmol/L dihydroethidium (DHE). Cells (1 × 10⁵) were stained with 5 µmol/L dihydroyethidium for 30 minutes at 37°C and were washed and resuspended in PBS. The oxidative conversion of dehydroxyethidium to ethidium was measured by flow cytometry. (B) For the western blot analysis, protein was subjected to SDS-PAGE and used p47phox antibody (1:1000 dilution) as detailed in Materials and Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblot shown here are representative of three independent experiments with similar results.

Figure 5. Modulation of Foxo3a and p47hox using SiRNA-SIRT1 transfection under high-glucose conditions

Cell were transfected using lipofectamine with prevalidated siRNA-SIRT1. Subsequently, human monocytic cells (1 × 10⁵ cells/ml) were cultured in presence of NG and HG conditions in absence or presence of resverarol (3–12 µmol/L) for 48 h. Then, cells were washed with phosphate-buffered saline (PBS) and then harvested. (A) NG, HG and HG-resveratrol treated cells (transfected with siRNA to SIRT1) were incubated with 5 µmol/L dihydroethidium (DHE). Cells (1 × 10⁵) were stained with 5 µmol/L dihydroyethidium for 30 minutes at 37°C and were washed and resuspended in PBS. The oxidative conversion of dehydroxyethidium to ethidium was measured by flow cytometry. (B) For the western blot analysis, protein was subjected to SDS-PAGE and used p47phox antibody (1:1000 dilution) as detailed in Materials and Methods Section. Equal loading of protein was confirmed by stripping the immunoblot and reprobing it for β-actin. The immunoblot shown here are representative of three independent experiments with similar results.

Figure 6

Proposed mechanisms by which resveratrol regulates SIRT1-FOXO3a leading to decreased p47phox expression and ROS production. High glucose suppresses SIRT1-FOXO3a pathway leading to increaesed p47phox gene expression and ROS production. Resveratrol treatment of HG-induced cells activates the HDACs activity and induces SIRT1 and FOXO3a expression, subsequently suppressing production of ROS and p47phox activation.