Peroxiredoxin 5 regulates adipogenesis-attenuating oxidative stress in obese mouse models induced by a high-fat diet

https://doi.org/10.1016/j.freeradbiomed.2018.05.061Get rights and content

Highlights

  • Prx5 inhibited adipogenesis through regulating the expression of adipogenic gene.

  • Prx5 decreased the generation of intracellular and mitochondrial ROS during the adipogenesis.

  • Deletion of Prx5 increased susceptibility to high fat diet induced obesity.

  • High fat diet feeding Prx5 KO mice exhibited impaired lipid metabolisms.

Abstract

Elevated levels of reactive oxygen species (ROS) are a hallmark of obesity. Peroxiredoxin 5 (Prx5), which is a cysteine-dependent peroxidase enzyme, has an intensive ROS scavenging activity because it is located in the cytosol and mitochondria. Therefore, we focused on the role of Prx5 in regulating mitochondrial ROS and adipogenesis. We demonstrated that Prx5 expression was upregulated during adipogenesis and Prx5 overexpression suppressed adipogenesis by regulating cytosolic and mitochondrial ROS generation. Silencing Prx5 promoted preadipocytes to differentiate into adipocytes accumulating lipids by activating adipogenic protein expression. Prx5-deletion mice fed on a high-fat diet (HFD) exhibited significant increase in body weight, enormous fat pads, and adipocyte hypertrophy in comparison to wild type mice. Prx5 deletion also remarkably induced adipogenesis-related gene expression in white adipose tissue. These phenotypic changes in Prx5-deletion mice were accompanied with lipid metabolic disorders, such as excessive lipid accumulation in the liver, severe hepatic steatosis, and high levels of triglyceride in the serum. These results demonstrated that Prx5 deletion increased the susceptibility to HFD-induced obesity and several of its associated metabolic disorders. In conclusion, we suggest that Prx5 inhibits adipogenesis by modulating ROS generation and adipogenic gene expression, implying that Prx5 may serve as a potential strategy to prevent and treat obesity.

Introduction

Obesity is a global health problem that leads to chronic metabolic diseases, including type 2 diabetes, insulin resistance, cardiovascular disease, and endocrine disease [1]. It is characterized by excessive fat accumulation and weight gain [2], [3], and is mainly caused by an imbalance in food intake and energy consumption, resulting in adipocyte proliferation and differentiation. Adipocytes, composed of adipose tissue, are important in maintaining energy homeostasis and insulin sensitivity [4]. White adipose tissue (WAT) is particularly associated with regulating whole-body energy by storing lipids and secreting adipokines.

In WATs, fibroblast like preadipocytes differentiate into mature adipocytes via a multi-step process called adipogenesis. This process is triggered by hormones such as insulin and several other transcriptional factors such as CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor families (PPARs) [5], [6]. During adipogenesis, insulin induces the cascade of transcriptional factors in preadipocytes. The expression of C/EBPβ and δ promotes differentiation in the early stages of adipogenesis [7]. Insulin binds to insulin receptors and subsequently induces the phosphorylation of protein kinase B (AKT) [8], [9]. Insulin-mediated phosphorylation of AKT translocates glucose transporter 4 (GLUT4) and regulates PPARγ activity in adipocytes. PPARγ and C/EBPα lead to mutual expression by forming a positive feedback loop that promotes adipocyte differentiation [10]. At the adipogenesis termination stage, PPARγ and C/EBPα express adipogenic genes such as fatty acid binding protein 4 (aP2) and GLUT4 in mature adipocytes [11].

Recent studies reported that adipogenesis was closely associated with reactive oxygen species (ROS), which promoted obesity by stimulating pre-adipocyte proliferation, adipocyte differentiation, and enlarging mature adipocytes [12], [13]. During adipogenesis, high levels of ROS play an important role in signal transduction cascades. In the early stage of adipogenesis, insulin exposure generates intracellular ROS such as hydrogen peroxide (H2O2) and stimulates the induction of mitochondrial-localized NADPH oxidase 4 (Nox4), which also accelerates adipogenesis by generating ROS [14].

Mitochondria are major sources of hydrogen peroxide generation in cells that also play a role in regulating adipocyte functions [15], [16]. ROS generated by the mitochondrial complex III particularly appears to be important in activating adipogenic gene transcription. Antioxidant enzymes are also upregulated during adipogenesis [17]. Several studies reported that the mitochondrial-targeted antioxidant treatment attenuates the intracellular H2O2, and subsequently reduces the expression of adipogenic genes such as PPARγ and C/EBPα [18], [19]. Therefore, mitochondrial ROS is considered to be the key regulator for adipogenesis, and modulating its level can regulate lipid accumulation in adipocytes.

Peroxiredoxins (Prxs) are a family of cysteine-dependent peroxidase enzymes that play an important role in scavenging peroxide and peroxynitrite in mammalian cells. Prxs are classified as Prx1–6 according to its subcellular localization. Since Prx3 and Prx5 are located within the mitochondria, mitochondrial ROS can be effectively eliminated by Prx3 and Prx5 [20]. Results from previous studies showed that Prx3 reduced H2O2 production and inhibited fat accumulation in mice [21]. However, the exact role of Prx5 in adipocytes still remains unclear. Therefore, we investigated the role of Prx5 linking mitochondrial ROS and adipogenesis in 3T3L1 adipocyte cells. In addition, we also confirmed adipogenesis and lipid accumulation in a Prx5 knockout mouse model.

In this study, we determined that Prx5 downregulated adipogenesis by scavenging mitochondrial ROS and inhibiting lipid accumulation in adipocytes. We further observed that adipogenic genes were more strongly expressed in the Prx5 knockout mouse model than in wild type (WT) mouse when obesity was induced with a high-fat diet. Our results indicate that Prx5 plays a role in regulating mitochondrial ROS and obesity.

Section snippets

Cell culture, differentiation, and treatments

We purchased 3T3L1 preadipocytes from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in 5% CO2 at 37 °C using Dulbecco's modified Eagle's medium (DMEM) containing 4500 mg/L glucose (Welgene, Gyeongsan, Korea), supplemented with 1% penicillin/streptomycin (Welgene) and 10% bovine calf serum (Gibco, New Zealand). 3T3L1 preadipocytes were differentiated as previously described [22], [23]. The cultures were allowed to grow to confluency. After 48 h, cells were

Insulin treatment enhances the expression of Prxs and generation of ROS

Insulin treatment resulted in the differentiation of preadipocytes into mature adipocytes. To examine the insulin-mediated change in expression of adipogenic and antioxidant proteins, we incubated 3T3L1 cells with insulin for 8 days. As shown in Fig. 1A, insulin treatment increased adipogenic gene expression. We confirmed that pAkt, PPARγ, and C/EBPα (associated with differentiation) exhibited high expression levels in insulin treated 3T3L1 cells. GLUT4 and aP2, expressed during the terminal

Discussion

To demonstrate the role of Prx5 in adipogenesis in vitro and in vivo, we generated Prx5 knockdown in overexpressed 3T3L1 cells and employed a novel mouse model with increased ROS generation by deletion. This model explained the role of Prx5 in correlation to ROS and adipogenesis. In this study, we provided evidence that suggested Prx5 overexpression in 3T3L1 significantly reduced adipogenesis by controlling the expression of the adipogenesis gene (Fig. 2). On the other hand, deletion of Prx5

Acknowledgments

Mi Hye Kim performed the experiment and wrote the paper. Sun-Ji Park, Jung-Hak Kim, Jung Bae Seong, Kyung-Min Kim, Hyun Ae Woo, Dong-Seok Lee designed the study and experiments.

Funding

This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government [MSIT, NRF-2017R1A5A2015391 and NRF-2017R1A2B4008176].

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