Effects of Endogenous PPAR Agonist Nitro-Oleic Acid on Metabolic Syndrome in Obese Zucker Rats

Nitroalkene derivatives of nitro-oleic acid (OA-NO2) are endogenous lipid products with novel signaling properties, particularly the activation of PPARs. The goal of this proposal was to examine the therapeutic potential of this OA-NO2 in treatment of obesity and obesity-related conditions in obese Zucker rats. The animals were randomly divided to receive OA-NO2, oleic acid (OA), both at 7.5 μg/kg/d, or vehicle ethanol via osmotic mini-pumps for 2 weeks. Following OA-NO2 treatment, food intake was decreased as early as the first day and this effect appeared to persist throughout the experimental period. At day 14, body weight gain was significantly reduced by OA-NO2 treatment. This treatment significantly reduced plasma triglyceride and almost normalized plasma free fatty acid and significantly increased plasma high-density lipid (HDL). The plasma TBARS and proteinuria were paralelly decreased. In contrast, none of these parameters were affected by OA treatment. After 14 days of OA-NO2 treatment, hematocrit, a surrogate of fluid retention associated with PPARγ agonists, remained unchanged. Together, these data demonstrated that OA-NO2 may offer an effective and safe therapeutic intervention for obesity and obesity-related conditions.


Introduction
The prevalence of overweight and obesity has dramatically increased during the past two decades. According to results of recent National Health and Nutrition Examination Survey (NHANES), 66.3% of adults in the United States are overweight or obese defined as a body mass index (BMI) of 25 or greater. Of these, 32.2% are obese defined as a BMI of 30 or greater [1]. The World Health Organization has estimated that worldwide, over one billion adults are overweight, with at least 300 million of them being obese [2]. Obesity is associated with a variety of chronic diseases, including endocrine disorders including metabolic syndrome, type 2 diabetes, and dislipidemia; cardiovascular disease including hypertension, congestive cardiomyopathy, stroke, and coronary heart disease; respiratory disorders including dyspnea and obstructive sleep apnea; depression, sleep disorders, musculoskeletal disorders, and gallbladder disease. Life style modification including exercise and calorie restriction remains the cornerstone of anti-obesity therapy but the long term success rate is low. In recent years, pharmacotherapy of obesity has received much attention.
Recently, nitrated free fatty acids (NO 2 -FA), notably nitroalkene derivatives of linoleic acid (nitrolinoleic acid, LNO 2 ) and oleic acid (OA-NO 2 ) are found to be endogenous molecules with several attractive signaling properties [3,4]. In particular, nitroalkenes are found to be a robust endogenous ligand for peroxisome proliferator-activated receptor γ (PPARγ) and they also activate PPARα and PPARδ at increasing concentrations [3,4]. PPARs have emerged as a novel therapeutic target for treatment of various components of metabolic syndrome. For example, synthetic PPARγ agonists, thiazolidinediones, have been widely used as insulin sensitizing agents for treatment of type 2 diabetes; synthetic PPARα agonists such as clofibrate and fenofibrate have been used in clinics for more than 30 years as lipid lowering agents [5][6][7][8]. Unfortunately, these synthetic agents are often associated with various toxicities [7,[9][10][11]. The goal of the present study was to examine the therapeutic potential of OA-NO 2 in an experimental model of obesity and metabolic syndrome.

Animals.
Four month-old male obese Zucker and lean rats were purchased from Charles River Laboratory (Wilmington, MA). All animals were housed at the University of Utah Comparative Medicine Center, maintained on a 12-hour light/dark cycle, and provided food and water ad libitum. Procedures and protocols followed guidelines set by the Laboratory Animal Care Committee at the University of Utah.

Protocols for Animal Experiments.
Under general anesthesia, obese Zucker rats were subcutaneously implanted with a mini-pump (DURECT Corporation, Cupertino, CA) delivering OA-NO 2, oleic acid (OA), both at 7.5 μg/kg/d, or vehicle ethanol for 2 weeks. Age and gender matched lean rats with vehicle treatment were used as controls. Food intake was determined periodically; body weight and collections of blood and 24-h urine samples were determined at the end of the experiments. Animals were fasted from 6:00 pm to 9:00 am before blood sampling. Blood sampling was conducted by making a small cut (∼2 mm) in the tail using razor blade. 24-hour urine was collected using metabolic cages.

Plasma Glucose and Lipids.
The following determinations were analyzed by using ROCHE, Modular P in the University Hospital Chemistry Laboratory: glucose, cholesterol, triglyceride, HDL, and LDL; non-esterided fatty acids were determined by using a colorimetric method (Cat# SFA-5, ZenBio, Research Triangle Park, NC).

Hematocrit.
Hematocrit was determined as the popular ways. Briefly, 5-10 μl of blood was collected from tail cut using a 10 μl capillary glass (Idaho Technology). One side of the tube was sealed with Hemato-Seal and then centrifuged for 4 minutes in a Thermo IEC microcentrifuge machine. The total height of sample and height of the red blood cell column were measured. The hematocrit reflects the ratio between the red blood cell column and total height.

Statistical Analyses.
All data are presented as means ± SEM. Differences between groups were analyzed by unpaired t test or 2-way ANOVA, followed by Bonferroni post hoc test using GraphPad Prism software version 3.03 (GraphPad Software, San Diego, CA). Differences within groups, before and after OA-NO 2 treatment, were analyzed by paired t test.
A probability value of < 0.05 was considered significant.

Effect of OA-NO 2 on Food Intake and Body Weight Gain.
Starting from 4 months of age, the obese Zucker rats were chronically infused for 2 weeks with vehicle, OA-NO 2 or OA each at 7.5 μg/kg/day via an osmotic mini-pump. Untreated age-matched lean rats were used as controls. As shown in Figure 1, OA-NO 2 treatment in obese Zucker rats reduced food intake as early as day 1 (Vehicle 36.6 ± 1.01 versus OA-NO 2 31.2 ± 1.04 g, P < .01), and this effect appeared to persist throughout the experiment except that only a trend was detected on day 14. Over the 14-day period, obese Zucker rats exhibited a significant increase in body weight as compared with lean controls (obese Zucker 48.0 ± 4.0 versus lean 16.7 ± 1.2 g, P < .01). The body weight gain in obese Zucker rats was reduced to 34.0 ± 1.1 g by OA-NO 2 treatment (P < .05). In contrast, a 14-day treatment with OA in obese Zucker rats at the same dose and same infusion rate did not affect food intake or body weight.   . OA treatment tended to exhibit an anti-proteuric effect but this did not reach a statistical significance. PPARγ agonists are limited by significant fluid retention as reflected by body weight gain and plasma volume expansion. The drop of hematocrit has been used as a surrogate marker of plasma volume expansion since these compounds do not influence erythropoiesis. Therefore we examined hematocrit in awake animals; hematocrit remained unchanged irrespective of OA-NO 2 or OA treatment.

Discussion
linoleic acid (nitrolinoleic acid, LNO 2 ) and oleic acid (OA-NO 2 ) are two major nitroalkene derivatives formed endogenously via NO-dependent oxidative reactions [12,13]. Studies in cell cultures have demonstrated PPAR ligand activities of these derivatives [3,4]. In this regard, LNO 2 was initially reported to be a potent an endogenous ligand for PPARγ that acts within physiological concentration ranges [3]. Subsequently, OA-NO 2 was shown to be a more robust  PPARγ activator as compared with LNO 2 and it also activated PPARα and PPARδ at increasing concentrations [3,4]. The three subtypes of PPARs are critically important for the control of glucose homeostasis and lipid metabolism. Therefore we were prompted to evaluate the therapeutic potential of nitroalkene derivatives in an animal model of obesity and metabolic syndrome. Here we demonstrate that a 14-day osmotic delivery of a low dose (7.5 μg/kg/d) of OA-NO 2 in obese Zucker rats produced beneficial effects on various components of metabolic syndrome, including obesity, hyperlipidemia, and proteinuria, accompanied by an immediate reduction of food intake. Epidemiological studies demonstrate that ingestion of polyunsaturated fatty acids (PUFA) and monounsaturated fatty acids (MUFA) but ingestion of saturated fatty acid deteriorates insulin resistance and obesity-related diseases [14]. An issue arises as to whether the beneficial effects of OA-NO 2 observed in obese Zucker rats are attributable to the native OA. This possibility is not supported by a previous study showing that chronic administration of dietary oleic acid in obese Zucker at a dose roughly 500 times higher than that in the present study fails to affect body weight, plasma glucose and lipids despite the amelioration of pancreatic islet disruption [15]. Along this line, the benefits of omega-3 polyunsaturated fatty acid (LC omega-3 PUFA) are also seen with a substantially high dose [16]. More importantly, we found that administration of OA at the same dose and with the same route did not produce any measurable effects on body weight or other metabolic parameters. It appears evident that the beneficial effects of OA-NO 2 observed in obese Zucker rats are derived from the nitration of OA but not the native fatty acid. The mechanism of action of OA-NO 2 in obese Zucker rats remains elusive but the involvement of PPARs appears conceivable. PPARα agonists (Wy-14643 and GW7647) and endogenous PPARα agonists (oleylethanolamide;OEA) treatment induces satiety and reduces body weight gain [17][18][19][20], an effect almost analogous to that of OA-NO 2 in obese Zucker rats. The rapid onset of appetite-suppressing effect (within a day) between PPARα agonists and OA-NO 2 is also similar. Furthermore, PPARα agonists are well known to reduce plasma triglyceride and increase HDL, a profile again similar to that of OA-NO 2 . Together, PPARα activity appears to explain most of metabolic effects of OA-NO 2 in obese Zucker rats. Of note, both OEA and OA-NO 2 are naturally occurring products derived from the same precursor, OA, and exert a similar role in regulation of satiety and body weight. It is an intriguing possibility that the two derivatives of OA converge on a common final pathway involving PPARα. However, the differences may exist in regulation of their production. OEA is produced by the small intestine in response to food intake [17,20] while the production of OA-NO 2 appears to be primarily regulated by NO-dependent oxidative reactions which may or may not be related to feeding.
Although OA-NO 2 is a robust PPARγ activator in an in vitro system, the involvement of PPARγ in OA-NO 2 signaling in the current experimental model might be minimal. In a sharp contrast to the appetite-suppressing and weightlowering effects of OA-NO 2 , the synthetic PPARγ agonists, thiazolidinediones, cause increases in food intake [21,22] and body weight and plasma volume expansion [10,23,24]. The lack of an effect of the PPARδ/β agonist on food intake [17] also does not support involvement of PPARδ in the anorectic action of OA-NO 2 . However, it is still possible that these two PPAR subtypes may partially contribute to some aspects of OA-NO 2 action, such as the lipid-lowering and proteinuria-lowering effects. Indeed, all three PPAR subtypes share common lipid-lowering properties [25][26][27][28], and PPARα [29][30][31][32] and PPARγ [33][34][35][36] exert similar renoprotective effects in animals or humans with diabetic nephropathy. It should be pointed out that in addition to PPARs, nitrated lipids can engage other pathways such as NO, NF-kB, and hemoxygnease-1. Whether these PPARindependent or other as yet unidentified pathways play a contributory role in mediating the metabolic actions of OA-NO 2 remains unclear.
Whatever the underlying mechanism, our data suggest that OA-NO 2 may offer therapeutic opportunities for management of obesity and obesity-related conditions. At the present, most of the anti-obesity agents target neurotransmitter receptors and are often associated with increased risk of psychiatric events. One example is rimonabant, a selective antagonist of the cannabinoid type 1 receptor, which is the most officious weight-lowering agent but causes depression and anxiety [37][38][39]. Because of the psychiatric side effects, the Committee for Medicinal Products for Human Use of the European Medicines Agency (EMEA) has recommended suspension of rimonabant as an antiobesity therapy [40]. OA-NO 2 is unique in that it is a naturally occurring product likely acting nuclear receptors, thus representing a novel class of weight-lowering agents. We demonstrated that it is not only highly effective but also well tolerated. Moreover, OA-NO 2 exhibited beneficial effects beyond weight loss. For example, it improved lipid profile, suppressed oxidative stress, and reduced proteinuria.
In summary, the present study describes novel therapeutic potential of OA-NO 2 in a rodent model of obesity and metabolic syndrome. A low-dose treatment produces anorexia and reduces body weight gain, accompanied by improved lipid profile, and reduction of oxidative stress and proteinuria, in the absence of plasma volume expansion. These effects are derived from the nitration of OA rather than the native fatty acid. As a natural product, OA-NO 2 appears to be an optimal pharmacotherapy for obesity and obesity-related conditions with a superior efficacy and safety profile.