EET agonist prevents adiposity and vascular dysfunction in rats fed a high fat diet via a decrease in Bach 1 and an increase in HO-1 levels

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Abstract

Recent reports have shown interplay between EETs (epoxides) and the heme oxygenase (HO) system in attenuating adipogenesis in cell culture models; prompting an examination of the effectiveness of EET agonist on obesity and associated cardio-metabolic dysfunction. Patho-physiological effects of an EET agonist (NUDSA) were contrasted in the absence and in the presence of stannous mesoporphyrin (an HO inhibitor) in SD rats fed a high fat (58%, HF) for 16 weeks. Animals on HF diet exhibited enhanced oxidative stress, increased levels of inflammatory cytokines and decreased levels of adiponectin along with reduced vascular and adipose tissue levels of EETs, HO-1; as compared to control rats (11% dietary fat). Treatment with NUDSA not only reversed serum adiponectin and vascular and adipose tissue levels of EETs and HO-1, but also, decreased blood pressure, subcutaneous and visceral fat content and serum TNFα and IL-6 levels in rats on HF diet. Aortic endothelial function, peNOS expression and adipose tissue markers of energy homeostasis i.e. pAMPK, Sirt1 and FAS, impaired in rats fed a HF diet, were restored in animals treated with this EET agonist. That NUDSA enhanced HO-1 expression, was accompanied by increase in p-GSK-3β and pAKT levels along with attenuation of adipose tissue levels of Bach 1 – the transcriptional suppresser of HO-1 expression. Prevention of these beneficial effects of NUDSA, in animals on HF diet and concurrently exposed to NUDSA and SnMP, supports the role of EET-HO interaction in mediating such effects. Taken together, our findings suggest that the EETs stimulate HO-1 expression via suppression of Bach 1 and interplay of these two systems affords vascular and metabolic protection in diet induced obesity.

Introduction

Epoxyeicosatrienoic acids (EETs) are catalyzed from arachidonic acid (AA) by a family of enzymes belonging to the Cytochrome P450 (CYP) super family [1], [2]. Upon formation, EETs are rapidly hydrolyzed by soluble epoxide hydrolase (sEH) to their respective dihydroxyepoxytrienoic acids (DHETs) as well as to esterification products primarily to glycerophospholipids [3]. EETs exhibit potent biological effects including vasodilation, stimulation of ion transport, inhibition of inflammatory response and stimulation of epithelial cell growth [4], [5], [6]. Studies that demonstrate that the induction of CYP2C23 [7] and inhibition of sEH provide vascular protection [4], [5] further highlight the role of EETs as modulators of vascular function. In addition, emerging studies indicate a role of this ubiquitous lipid mediator in the regulation of metabolic homeostasis [8]; thereby warranting further examination of its biological role in conditions such as obesity.

The heme oxygenase (HO) isoforms, HO-1 (inducible) and HO-2 (constitutive), gene expression is known to increase signaling molecules – mediated antioxidant and anti-inflammatory properties [9]. HO-1 is a stress response protein whose expression is principally regulated by transcription factors NRF2 and Bach 1. Where electrophiles activate NRF2-depndent HO-1 expression, molecules such as heme bind to and inhibit Bach 1 which is an inhibitor of HO-1 expression in quiescent cells. Increased HO-1 expression and activity is associated with an increase in adiponectin secretion and downstream signaling resulting in the stimulation of NO bioavailability [9], [10], [11]. HO-1 derived carbon monoxide (CO) regulates vascular tone in part by decreasing CYP450-derived vasoconstriction [12]. Furthermore, activation of the heme-HO system could contribute towards the vasodilatory effects of lipid mediators such as EETs [13], [14].

Obesity, and associated metabolic syndrome, is a systemic inflection characterized by increased oxidative stress (ROS) along with inhibition of the HO-adiponectin system while increasing levels of inflammatory cytokines and insulin resistance [15], [16]. We have recently reported that epoxides attenuate the metabolic syndrome like phenotype in the HO-2 null mice via the activation of HO-1 dependent pathways [8]. In addition, EETs attenuate adipocyte hypertrophy and improve adipocyte function in bone marrow derived mesenchymal stem cells (MSCs) via an increase in HO-1 expression [17]. In light of this evidence, the present study aims to explore the effectiveness of an EET agonist on the prevention of adiposity and a possible interaction between the HO and epoxide systems, in vivo, where metabolic pathologies have been induced in rats fed a HF diet. This study corroborates the existence of an epoxide-HO axis whose stimulation via an exogenous EET agonist (NUDSA) induces Bach 1-dependent HO-1 expression and abates obesity associated vascular dysfunction and improves metabolic homeostatic markers in visceral adipose tissues, in SD rats. Prevention of these beneficial effects in HF fed rats concurrently exposed to SnMP and NUDSA exposes interplay of epoxide-HO systems in affording aforementioned vasculo-metabolic protections.

Section snippets

Materials

The EET analog, 11-(nonyloxy)undec-8(Z)-enoic acid (NUDA), is a potent vasorelaxant in mesenteric and renal arteries [18], [19]. For in vivo studies, NUDA was conjugated with l-aspartic acid to form (S)-2-(11-(nonyloxy) undec-8(Z)-enamido)succinic acid (NUDSA) to minimize β-oxidation and improve solubility in aqueous milieu.

Animal experimentation

All animal experiments followed an institutionally approved protocol in accordance with the NIH Guidelines. Forty, 8-week-old Sprague-Dawley (SD) rats were used in the

Effect of a HF diet on body weight and fat content

As seen in Fig. 1A, a HF diet for 16 weeks significantly increased body weight of SD rats as compared to age-matched controls (516 ± 9.4 vs. 407 ± 9.4 g, p < 0.01). This increase in body weight was attenuated in rats, on HF diet, treated with NUDSA for 2 weeks (p < 0.01). The decrease in body weight was reversed by the concurrent administration of SnMP and NUDSA to rats being fed a HF diet. These findings paralleled the change in subcutaneous fat content, Fig. 1B. Rats fed a HF diet showed marked

Discussion

This study demonstrates an inter-dependent relationship between CYP 450 derived epoxides and the heme-HO system in mediating the regulation of metabolic balance. Our results clearly establish the development of vascular and adipose tissue dysfunction in SD rats fed a HF diet, which is amenable to rescue by the EET agonist, NUDSA. In addition, the prevention of these beneficial effects of NUDSA by heme-HO inhibitor strongly implicates the existence of a synergistic relationship between these

Conclusion

In conclusion, as represented in the schematic, the present data suggests that HF diet in conjunction with chronic oxidative stress inhibits epoxide synthesis and attenuates heme-HO-adiponectin axis. These effects are reflected downstream as increased adiposity, vascular endothelial dysfunction, systemic inflammatory response and altered metabolic homeostasis. Administration of an EET analog, NUDSA, induces Bach 1-dependent HO-1 expression that in turn contributes towards abrogation of

Acknowledgments

This work was supported by NIH grants DK068134, HL55601 and HL34300 (NGA). All authors had full access to the data and take responsibility for its integrity. All authors have read and agree with the manuscript as written. We thank Jennifer Brown for her outstanding editorial assistance in the preparation of the manuscript.

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      Furthermore, based on our study, in sEH KO mice after CoPP administration exhibited in higher EETs levels, which illustrates the connection between HO-1 and EETs. These results are in agreement with our previous results that showed that the beneficial effects of EETs prevented in the presence of an tin mesoporphyrin (SnMP), an inhibitor of HO-activity [36]. HO-1, which is an antioxidant, decreased superoxide anions in both aorta and adipocyte tissue.

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    Both the authors contributed equally to this work.

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