PGC-1 alpha regulates HO-1 expression, mitochondrial dynamics and biogenesis: Role of epoxyeicosatrienoic acid

https://doi.org/10.1016/j.prostaglandins.2016.07.004Get rights and content

Abstract

Background/objectives

Obesity is a risk factor in the development of type 2 diabetes mellitus (DM2), which is associated with increased morbidity and mortality, predominantly as a result of cardiovascular complications. Increased adiposity is a systemic condition characterized by increased oxidative stress (ROS), increased inflammation, inhibition of anti-oxidant genes such as HO-1 and increased degradation of epoxyeicosatrienoic acids (EETs). We previously demonstrated that EETs attenuate mitochondrial ROS. We postulate that EETs increase peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), which controls mitochondrial function, oxidative metabolism and induction of HO-1.

Methods

Cultured murine adipocytes and mice fed a high fat (HF) diet were used to assess functional relationship between EETs, HO-1 and (PGC-1α) using an EET analogue (EET-A) and lentivirus to knock down the PPARGC1A gene.

Results

EET-A increased PGC-1α and HO-1 in cultured adipocytes and increased the expression of genes involved in thermogenesis and adipocyte browning (UCP1 and PRDM16, respectively). PGC-1α knockdown prevented EET-A-induced HO-1expression, suggesting that PGC-1α is upstream of HO-1. MRI data obtained from fat tissues showed that EET-A administration to mice on a HF diet significantly reduced total body fat content, subcutaneous and visceral fat deposits and reduced the VAT: SAT ratio. Moreover EET-A normalized the VO2 and RQ (VCO2/VO2) in mice fed a HF diet, an effect that was completely prevented in PGC-1α deficient mice. In addition, EET-A increased mitochondrial biogenesis and function as measured by OPA1, MnSOD, Mfn1, Mfn2, and SIRT3, an effect that was inhibited by knockdown of PGC-1α.

Conclusion

Taken together, our findings show that EET-A increased PGC-1α thereby increasing mitochondrial viability, increased fusion potential thereby providing metabolic protection and increased VO2 consumption in HF-induced obesity in mice, thus demonstrating that the EET-mediated increase in HO-1 levels require PGC-1α expression.

Introduction

Obesity is a global epidemic and a major risk factor in the development of metabolic syndrome and diabetes and associated complications that include cardiovascular disease, increased oxidized HDL levels, kidney disease, hypertension, and neuropathies [1], [2], [3], [4]. Epoxyeicosatrienoic acids (EETs) are arachidonic acid derived metabolites generated by a family of cytochrome P450 (CYP) monooxygenases and epoxygenases [5], [6]. Although rapidly subjected to hydrolysis by soluble epoxide hydrolase (sEH) to their respective dihydroxyepoxytrienoic acids as well as by esterification primarily to glycerophospholipids, their vasodilatory, anti-inflammatory, anti-adipogenic and antiapoptotic actions are well established and sEH-inhibition increases cellular and circulating EET levels [7], [8], [9]. EET agonists prevent both adiposity and vascular complications in vitro and in vivo and obesity-induced adipose tissue expansion impairs the CYP epoxygenase pathway and the generation of EET in vivo [10], [11], [12], [13].

Heme oxygenase (HO) consists of an inducible and a constitutive form (HO-1 and HO-2, respectively) that catalyzes the degradation of heme into equimolar amounts of biliverdin/bilirubin, carbon monoxide (CO) and iron (Fe2+ ion) [14], [15]. HO-2 maintains normal metabolic cellular functions, such as vascular tone, renal channel function and activity [15], as well as control of body weight, insulin sensitivity, and blood pressure [16]. HO-2 deficiency is associated with oxidative stress, chronic inflammation, and reduced protection against diabetes-induced renal injury [17], [18]. HO-1, a stress responsive enzyme, offers increased protection against high fat diet (HF)-induced obesity [19], [20], [21] and regulates adipocyte stem cell differentiation [22], [23], attributable to the potent antioxidant and anti-inflammatory properties of biliverdin/bilirubin.

Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) is a major regulator of mitochondrial function, oxygen consumption and oxidative phosphorylation [24], [25]. PGC-1α along with the transcriptional regulator PR Domain Containing 16 (PRDM16), are the major regulators of adipocyte browning and thermogenic activation of brown fat [26], [27], [28]. Transgenic mice with mildly elevated muscle PGC-1α levels are resistant to age-related obesity and diabetes and have a prolonged lifespan suggesting that PGC-1α stimulates the secretion of factors from skeletal muscle that affect the function of other tissues [29]. In fact, PGC-1α and several of its responsive genes involved in oxidative phosphorylation display reduced protein and mRNA expression levels in muscle and adipose tissue of patients with chronic heart failure and diabetes mellitus type 2 (DM2) [30], [31], [32], [33], [34]. Furthermore mice lacking PGC-1α in adipose tissue fed a HF diet develop insulin resistance and increased circulating lipid levels [35]. Additionally, the uncoupling proteins are intramembranous of the mitochondrial proteins that play a key role in thermogenesis. UCP1 is present exclusively in brown adipose tissue [36] and myokines drive UCP1 in a PGC-1-dependent manner to increase energy expenditure [36] and possibly oxygen consumption. These studies suggest that PGC-1α is a key moderator of energy metabolism and in preventing the development of metabolic syndrome and DM2. As a transcriptional co-activator, PGC-1α enhances the expression of many transcription factors and can bind to members of the nuclear receptor family, including nuclear respiratory factor (NRF)-1 and -2 [35], [37].

Adipocyte differentiation is associated with an increase in ROS and inflammatory cytokine levels and a concomitant decrease in mitochondrial function. Although ROS are being generated in several cellular compartments, the bulk of ROS (about 90%) contribute to mitochondrial energy metabolism [38]. Sirtuin 3 (SIRT3), one of the seven mammalian sirtuins, is a major mitochondrial deacetylase and has recently been discovered to be the target of PGC-1α and is important in mitochondrial processes, such as suppression of ROS, mitochondrial biogenesis, and energy metabolism [38], including mitochondrial fatty-acid oxidation [39]. The mitochondrial network morphology is tightly linked to energy and metabolic demands as well as viability and depends greatly on quality control, involving mitochondrial fusion (the merge of dysfunctional to functional) and fission (budding and isolation of dysfunctional mitochondria, orchestrated by the dynamin-related protein 1 (DRP1) and the fission, mitochondrial 1 (Fis1) proteins [40], [41]). The autosomal dominant optic atrophy 1 (OPA1) protein is situated on the mitochondrial inner membrane that, along with the mitochondrial fusion proteins, Mitofusin 1 and 2 (Mfn 1 and 2), located on the mitochondrial outer membrane, facilitate the mitochondrial fusion process [42], [43]. That the dynamics of the mitochondrial fusion/fission processes need to be tightly regulated is exemplified by the link between the reduction of mitochondrial fusion and the development of obesity and insulin resistance [41], [44], [45]. Conversely, reduced hepatic steatosis, insulin resistance, and increased mitochondrial function and fusion potential is present in hepatocyte mitochondria of rats fed high-fish-oil diet as compared to rats fed a high-lard diet [41].

We recently reported that EETs increase HO-1 expression and mitochondrial viability, and exert their function on adipocyte differentiation via activation of PGC-1α in vitro [46]. The present report examines whether the EET-mediated regulation of adiposity is due to activation of PGC-1α and an increase in HO-1 levels in cultured adipocytes in vitro and in mice fed a high fat diet in vivo and investigates the mechanistic EET-mediated actions on mitochondrial function in reference to mitochondrial viability and fusion mediators mitochondrial superoxide dismutase (MnSOD/SOD2), SIRT3, Mfn1, Mfn2, and OPA1. We further examined the impact of HF, EETs, and lack of PGC-1α on metabolic alterations and on VO2 and respiratory quotient (RQ). We demonstrate that EET is upstream of the PGC-1α signaling pathway responsible for increased levels of HO-1 and insulin phosphorylation and further that the EET-mediated induction of HO-1 is dependent on PGC-1 α. Our results indicate that the EET-PGC1α-mediated reduction in adiposity in mice fed a HF diet involves an increase in VO2. This study highlights the existence of an EET-PGC-1α axis that is associated with increased levels of HO-1 that, concomitantly, upregulates mitochondrial function, MnSOD and SIRT3, and fusion mediators, Mfn1 and Mfn2, and OPA1, which, together, serve to regulate adipocyte cell differentiation and obesity-induced hypertension and improve metabolic homeostasis.

Section snippets

Cell culture

3T3-L1 murine pre-adipocytes were purchased from ATCC (ATCC, Manassas, VA). After thawing, 3T3-L1 cells were cultured in α-minimal essential medium (α-MEM, Invitrogen, Carlsbad CA) supplemented with 10% heat inactivated fetal bovine serum (FBS, Invitrogen, Carlsbad, CA) and 1% antibiotic/antimycotic solution (Invitrogen, Carlsbad, CA). The cultures were maintained at 37 °C in a 5% CO2 incubator and the medium was changed after 48 h and every 3–4 days thereafter as described previously [10]. For

EET mediated induction of PGC1-α, UCP1, PRDM16 and adiponectin

Western blot analysis and real-time PCR demonstrated a significant (p < 0.05) increase in PGC-1α protein and mRNA expression after treatment of adipocyte cells with EET-A (Fig. 1A–C). Concomitant treatment with EET-A and SnMP decreased PGC-1α protein and mRNA expression when compared to EET-A treated cells (Fig. 1A–C). Uncoupling protein 1 (UCP1) was significantly upregulated in cells treated with EET-A (p < 0.05), an increase that was abrogated in cells treated with SnMP along with EET-A (Fig. 1A

Discussion

This study demonstrates that EET is located upstream of PGC-1α in its signaling cascade and, in turn, that PGC-1α is crucial for the induction of HO-1. Together these factors play an important role in the regulation of the adipocyte stage of differentiation. We show that EET is a powerful inducer of PGC-1α-mediated downstream signaling and mitochondrial viability and function, including the processes of oxidative phosphorylation, fusion, and mitochondrial quality control, resulting in improved

Conflict of interest

There are no conflicts of interest among the authors.

Funding

This work was supported by National Institutes of Health grants HL55601 and HL34300 (NGA).

Author contributions

S.P.S performed experiments; L.B. performed experiments, collected literature citations, and edited manuscript; J.S. conducted ELISA assay and edited manuscript; N.G.A. planned and designed the experimental protocol, and wrote the first draft of the manuscript; J.C. designed the PGC-1α sequence.

Acknowledgments

This work was supported by National Institutes of Health grant HL34300 and The Brickstreet Foundation and The Huntington Foundation (NGA). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. The authors wish to thank Ms. Jennifer Brown, New York Medical College for her outstanding editorial assistance in the preparation of the manuscript.

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