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 O₂⁻ 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.

Introduction

Hyperglycemia contributes to vascular complications of diabetes. High glucose has been shown to induce inflammatory cytokines, chemokines, p38 mitogen-activated protein kinase, reactive oxygen species (ROS), protein kinase C (PKC) and nuclear factor-κB (NF-κB) activity in both clinical and experimental systems [1], [2], [3], [4], [5], [6]. Several lines of evidence support a role for oxidative stress in the development of diabetes complications [7], [8]. Diabetic patients have increased O₂⁻ production in monocytes and neutrophils [2], [7], [9]. Excess accumulation of ROS can result from defects in ROS scavenging and is believed to have an impact on cellular aging and the senescence process [10]. nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is accepted as the most important mechanism for ROS generation in phagocytic cells. Previously, we have shown that p47phox, an essential component of monocyte NADPH oxidase, is required for ROS generation under high-glucose conditions [11].

Recently, SIRT1, the mammalian homologue of yeast Sir2, was identified as a key mediator that links calorie restriction and longevity in mammals. Sirtuins are a conserved family of NAD-dependent deacetylases (class III histone deacetylases) [12], [13], [14], [15], [16]. To date, seven members of sirtuin proteins (i.e., SIRT1–SIRT7) have been identified in humans. Recent studies have demonstrated that SIRT1 plays an important role in the regulation of cell death/survival and stress response in mammals. SIRT1 promotes cell survival by inhibiting apoptosis or cellular senescence induced by stresses, including DNA damage and oxidative stress [12], [13], [14], [15], [16], [17]. An increasing number of proteins have been identified as substrates of SIRT1, including p53 [18], [19], [20], [21], Forkhead box O (FOXO) transcription factors [22], [23], [24], [25], [26], [27], [28]. Improper regulation of sirtuin proteins has been reported in a number of diseases, including Bowen's disease [29], type I diabetic nephropathy [30], Alzheimer's disease and amyotrophic lateral sclerosis [31], and nonalcoholic fatty liver disease [32]. It has been suggested that loss of Sirt1 activity may be associated with metabolic diseases such as T2DM and atherosclerosis [33], [34], [35].

FOXO transcription factors FOXO1 (FKHR) [36], FOXO3a (FKHRL1) [37], FOXO4 (ARX) [38] and FOXO6 [39] are emerging as an important family of proteins that modulate the expression of genes involved in apoptosis, the cell cycle, DNA damage repair, oxidative stress, cell differentiation, glucose metabolism and other cellular functions [22], [23], [24], [26], [40]. Several studies have shown that SIRT1 can control the cellular response stress by regulate mammalian FOXO transcription factors through direct binding and/or deacetylation [12], [22], [24], [26], [28], [41]. However, the mechanism of the interactions between SIRT1 and FOXO under hyperglycemic conditions is not well understood. FOXO transcription factors regulate antioxidant expression and DNA damage repair. Among all FOXO members, FOXO3a appears to have an important role under oxidative stress. Foxo3 plays an important role in the in vivo regulation of oxidative stress in mammals. FOXO3a up-regulates transcription of the ROS scavenging enzymes superoxide dismutase 2 [SOD2, also known as manganese superoxide dismutase (MnSOD)] and catalase [42], [43], [44]. Thus, FOXO3 appears to be a critical physiological regulator of oxidative stress in mammalian cells.

One important sirtuin-activating compounds is the natural product resveratrol (3,4,5-trihydroxystilbene), a polyphenol that is synthesized by plants and is present in grapes and red wine [45]. Recently, resveratrol has been shown to improve energy balance and increase mitochondrial function in mice by activating SIRT1 [46]. Resveratrol has previously gained considerable attention because of its beneficial effects as a cardioprotective, cancer chemopreventive, and chemotherapeutic agent [47], [48], [49], [50], [51].

Previously, we have shown increased monocytic superoxide in Types 1 and 2 diabetes mellitus (T1DM and T2DM, respectively) and subsequently showed under HG condition that this is mediated via up-regulation of PKCα and p47phox [11]. However, the role of SIRT1 in regulating monocytic superoxide under HG condition is not well understood. Moreover, the effect of resveratrol, a potent SIRT1 inducer, on monocyte superoxide under HG conditions is not elucidated and is the focus of the present report. Thus, we hypothesize that resveratrol can suppress ROS production via regulatory mechanism involving FOXO3a, SIRT1 and p47phox under high-glucose conditions in human monocytes.