Overexpression of SIRT1 protects pancreatic beta-cells against cytokine toxicity by suppressing the nuclear factor-kappaB signaling pathway

Abstract

Objective: SIRT1, a class III histone/protein deacetylase, is known to interfere with the nuclear factor-kappaB (NF-kappaB) signaling pathway and thereby has an anti-inflammatory function. Because of the central role of NF-kappaB in cytokine-mediated pancreatic beta-cell damage, we postulated that SIRT1 might work in pancreatic beta-cell damage models.

Research design and methods: RINm5F (RIN) cells or isolated rat islets were treated with interleukin-1beta and interferon-gamma. SIRT1 was activated by resveratrol, a pharmacological activator, or ectopic overexpression. The underlying mechanisms of SIRT1 against cytokine toxicity were further explored.

Results: Treatment of RIN cells with cytokines induced cell damage, and this damage was well correlated with the expression of the inducible form of nitric oxide (NO) synthase (iNOS) and NO production. However, SIRT1 overexpression completely prevented cytokine-mediated cytotoxicity, NO production, and iNOS expression. The molecular mechanism by which SIRT1 inhibits iNOS expression appeared to involve the inhibition of the NF-kappaB signaling pathway through deacetylation of p65. In addition, SIRT1 activation by either resveratrol or adenoviral-directed overexpression of SIRT1 could prevent cytokine toxicity and maintain normal insulin-secreting responses to glucose in isolated rat islets.

Conclusions: This study will provide valuable information not only into the mechanisms underlying beta-cell destruction but also into the regulation of SIRT1 as a possible target to attenuate cytokine-induced beta-cell damage.

FIG. 1.

Decreased expression of SIRT1 by cytokine in RIN cells (A) and isolated rat islets (B). RIN cells (5 × 10⁶) or islets (30) were treated with IL-1β (2 ng/ml) alone or IL-1β and IFN-γ (100 units/ml). After 48 h of incubation, Western blotting for SIRT1 was conducted. C: Protein bands were detected using the enhanced chemiluminescence detection system and analyzed by imaging densitometry. Each value represents the mean ± SE of six independent experiments. **P < 0.01 vs. untreated control.

FIG. 2.

Ectopic overexpression of SIRT1 regulates HDAC activity in RIN cells. A: RIN cells (5 × 10⁶) were transduced with retroviruses expressing WT-SIRT1 or DN-SIRT1, and RIN cell extracts prepared 48 h after viral infection were subjected to Western blot analysis with anti-SIRT1 antibody. B: Deacetylase activity of overexpressed SIRT1 was measured using fluorogenic substrate as described in research design and methods. Each value represents the mean ± SE of three independent experiments. **P < 0.01 vs. pBABE.

FIG. 3.

Effects of SIRT1 overexpression on cytokine-induced cytotoxicity. Control or SIRT1-overexpressing RIN cells (1 × 10⁵) were treated with IL-1β alone or IL-1β and IFN-γ for 48 h. The cell viability (A) and cell proliferative potential (B) were then determined using an MTT and BrdU incorporation assay, respectively. Each value represents the mean ± SE of three independent experiments. *P < 0.05, **P < 0.01 vs. untreated pBABE; ##P < 0.01 vs. IL-1β + IFN-γ–treated pBABE; $$P < 0.01 vs. untreated DN-SIRT1.

FIG. 4.

Effect of SIRT1 overexpression on cytokine-induced NO production and iNOS mRNA and protein expressions. Control or SIRT1-overexpressing RIN cells were treated with IL-1β alone or IL-1β and IFN-γ for 24 h, and nitrite production (A) and iNOS mRNA (B) and protein (C) expressions were determined. The results of three independent experiments are expressed as the means ± SE. *P < 0.05, **P < 0.01 vs. untreated pBABE; ##P < 0.01 vs. IL-1β + IFN-γ–treated pBABE; $P < 0.05, $$P < 0.01 vs. untreated DN-SIRT1.

FIG. 5.

Effect of SIRT1 overexpression on cytokine-induced NF-κB activation and translocation and acetylation of p65. Control or SIRT1-overexpressing RIN cells were treated with IL-1β alone or IL-1β and IFN-γ. After 1 h of incubation, DNA binding (A) and transcriptional (B) activities of NF-κB were analyzed by EMSA and luciferase reporter assay, respectively. C: The nuclear translocation of p65 and acetylation of p65 at K310 were determined by Western blotting. PCNA was used as loading control for nuclear protein. Each value represents the mean ± SE of three independent experiments. *P < 0.05, **P < 0.01 vs. untreated pBABE; ##P < 0.01 vs. IL-1β + IFN-γ–treated pBABE; $$P < 0.01 vs. untreated DN-SIRT1. PCNA, proliferating cell nuclear antigen; RLU, relative light unit.

FIG. 6.

Inhibition of cytokine-induced activation of the NF-κB pathway by resveratrol in RIN cells. RIN cells were treated with IL-1β and IFN-γ with or without a 3-h pretreatment with resveratrol. SIRT1 deacetylase activity was determined 1 h later (A), and nitrite production and iNOS mRNA and protein expressions were determined 24 h later (C). Transcriptional activity of NF-κB (B) and acetylation of p65 (D) were analyzed by luciferase reporter assay and Western blotting, respectively. Each value represents the mean ± SE of three independent experiments. **P < 0.01 vs. untreated control; ##P < 0.01 vs. IL-1β + IFN-γ.

FIG. 7.

Inhibition of NF-κB–mediated cytotoxic pathway and restoration of glucose-stimulated insulin secretion by resveratrol and Ad-SIRT1 in rat islets. Rat islets (30) were treated with IL-1β and IFN-γ with or without a 3-h pretreatment with resveratrol. DNA binding of NF-κB (A) was then determined 1 h later, and nitrite production and iNOS mRNA and protein expressions (B) were determined 24 h later. Rat islets (50) were infected with 1 × 10⁹ pfu Ad-SIRT1 or Ad-lacZ for 12 h and exposed to IL-1β and IFN-γ for 24 h. C: Islets extracts prepared 24 h after viral infection were subjected to Western blot analysis with anti-SIRT1 antibody. D: The effects of overexpressed SIRT1 on IL-1B–and IFN-γ–induced nitrite production, iNOS mRNA expression, and protein expression were determined. Rat islets (10) were treated with IL-1β and IFN-γ with or without a 3-h pretreatment with resveratrol or Ad-SIRT1 infection. E: After 24 h of incubation, glucose-stimulated insulin secretion was quantified. F: Islet viability was analyzed by microscopic analysis after staining with acridine orange and propidium iodide as described in research design and methods. Viable islets were counted manually and represented as percentage viability. Results of triplicate samples are expressed as the mean ± SE. **P < 0.01 vs. untreated control; ##P < 0.01 vs. IL-1β + IFN-γ.