Nitrated fatty acids: from diet to disease

doi:10.1016/j.cophys.2019.04.013

Current Opinion in Physiology

Volume 9, June 2019, Pages 67-72

https://doi.org/10.1016/j.cophys.2019.04.013 Get rights and content

Highlights

•
Formation of nitro-fatty acids.
•
Signaling of nitro-fatty acids.
•
Anti-fibrotic effects of nitro-fatty acids.

Fatty acids not only provide caloric energy in our diets and building blocks of lipids but are also precursors of potent signaling molecules. Fatty acids can undergo enzymatic and non-enzymatic transformations to form autocrine and paracrine signaling molecules that regulate energy balance and metabolic homeostasis. A new class of lipid signaling mediators known as nitro-fatty acids (NO₂-FAs) have recently been identified. These NO₂-FAs are generated endogenously through non-enzymatic reactions of secondary products of nitrite and nitric oxide and are readily detected in human plasma and urine. NO₂-FAs are potent anti-inflammatory and antioxidant cell signaling mediators and exert protective effects in numerous pre-clinical animal models of disease including cardiovascular, pulmonary and renal fibrosis. Chronic unresolved inflammation is a common key feature underlying most fibrotic disorders. Two signaling pathways that converge on inflammation and oxidative stress are nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and nuclear factor kappa B (NF-κB). NO₂-FAs are pleiotropic signaling modulators that target both of these pathways providing a therapeutic strategy directed toward an integrated decrease in inflammation. This review summarizes the latest findings and understanding of the formation, signaling and anti-fibrotic effects of NO₂-FA.

Introduction

The need for fatty acids goes far beyond the obligatory role of building blocks for fat and dietary sources for fuel in our bodies. The discovery of leukotrienes and prostaglandins, as enzymatic oxygenation products of unsaturated fatty acids, advanced the field demonstrating that fatty acid mediators are potent autocrine and paracrine signaling molecules. The technical advances provided by mass spectrometry-based approaches led to renewed interest and discoveries in this area of research. It has become clear that this large group of newly discovered bioactive molecules derived from polyunsaturated fatty acids executes physiological responses that are central to tissue homeostasis. Among them, NO₂-FAs are formed that participate in anti-inflammatory and tissue protective actions through activation of Nrf2-dependent-signaling and concomitant inhibition of NF-κB-derived inflammation. The latest advances are provided in areas of NO₂-FA formation and role in fibrotic disorders with a focus on unresolved inflammation, which appears to be a common feature of fibrosis across all tissues, and examining the signaling actions of NO₂-FAs as a novel therapeutic.

Section snippets

Formation of nitro-fatty acids

Dietary interventions have been shown to be effective in a broad range of pathologies including diabetes and metabolic syndrome [1]. In addition to providing the nutrients to support metabolism, certain dietary components modulate metabolic processes. Moreover, these can be further metabolized and processed by the microbiome, providing a new set of modified molecules with unique signaling properties that regulate metabolism. These microbial transformations are important [2,3^•], the microbiome

Stomach as a bioreactor

The acidic conditions of the stomach during digestion result in its protonation and the secondary generation of nitric oxide (NO) through reductive reactions, the nitrosating species dinitrogen trioxide (N₂O₃) and nitrating species nitrogen dioxide (NO₂) [9]. Initial studies evaluating the intake effects of high doses of nitrite demonstrated the formation of these reactive species in the stomach to be responsible for the formation of tumorigenic nitrosamines [10], prompting stringent standards

Endogenous NO₂-FA

While NO₂-CLA detection is well established and its levels are modulated by diet and inflammation, the remaining classes of NO₂-FAs still need to be explored [13^••,14]. Nitro-oleic acid (NO₂-OA), which has long been used as a surrogate to investigate the actions of endogenous NO₂-FA [15], has been reported in the subnanomolar level in plasma [16] but is usually below the limit of detection and/or quantification. In contrast to NO₂-CLA, NO₂-OA and its metabolites are normally not detected in

Nitro-fatty acid reactivity

The interest in the physiology and pharmacology of NO₂-FA is fueled by the investigation of their anti-inflammatory and protective actions reported in several preclinical animal models of disease by NO₂-OA, which is used as a surrogate to interrogate the effects of endogenous NO₂-FA [15,18]. NO₂-FAs are electrophilic molecules that contain a nitroalkene group that reacts mainly with cysteines through Michael addition reactions [19]. While cysteine adducts are the most relevant to NO₂-FA

Nitro-fatty acid signaling

The reversibility of the NO₂-FA reaction is central to their pleiotropic signaling activity. While initially nuclear factor (erythroid-derived 2)-like 2 (Nrf2), heat shock response (HSR) activation and nuclear factor kappa B (NF-κB) inhibition were proposed as main drivers of their signaling mechanisms, emerging evidence reveals new pathways that are inhibited, specifically STING, epoxide hydrolase and angiotensin II receptor [23, 24, 25]. The current understanding of the signaling of NO₂-FA

Complexities of fibrosis

Fibrosis is a complex and vital program to repair injured tissue. It occurs after repetitive insults to the epithelium and is defined by the accumulation of extracellular matrix (ECM) molecules such as collagen and fibronectin [30]. Under normal conditions that lead to wound healing following an injury, the fibrotic ECM is degraded, the epithelium is repaired and fibrosis is resolved. In a fibrotic state, however, the normal repair and resolution mechanisms are dysfunctional leading to scarring

The need for novel therapeutics: NO₂-FA

Electrophilic drugs inhibit pro-inflammatory signaling mediators and have been shown to provide a valuable approach in several pre-clinical fibrosis models. NO₂-OA and other electrophiles, such as dimethyl fumarate and 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), protect against kidney and pulmonary animal models of fibrosis [32, 33, 34, 35]. In cardiovascular disease, NO₂-OA reverses hypoxia-induced right ventricular (RV) pressure and fibrotic RV remodeling in a pulmonary arterial

NF-κB inhibition

Under basal conditions, NF-κB is inactive as it is complexed with inhibitory κB (IκB)-α subunit, which contains a strong nuclear export signal, in the cytoplasm. In the canonical pathway, most stress signals activate NF-κB by controlling the redox-sensitive serine-specific IkB kinase (IKK). The activation of the IKK complex facilitates IκB-α phosphorylation (Ser-32 and Ser-36) by IκB kinase β (IKK-β) leading to IκB-α ubiquitination and proteasomal degradation resulting in NF-κB transactivation [

Nrf2 activation

NO₂-FA derivatives activate Nrf2-dependent gene transcription by alkylating two functionally significant cysteine residues (Cys-273 and Cys-288) of Kelch-like ECH-associated protein (Keap)-1 in the cytoplasm. This inhibits Keap1-dependent Nrf2 degradation and results in nuclear translocation of de novo synthesized Nrf2 protein, binding to antioxidant response element and transactivation of gene signaling [41,42]. Nrf2-regulated genes are critical in protecting against oxidative stress, by

Crosstalk between Nrf2 and NF-κB pathways

Not only does Nrf2 activate antioxidant gene expression but Nrf2 directly limits inflammation by binding to promoter regions of pro-inflammatory cytokines [46]. Additionally, Nrf2 inhibits RNA polymerase II recruitment to the transcription start site of IL-6 and IL-1β genes without altering the required recruitment of NF-κB [46]. In this case, Nrf2 and NF-κB signaling appear to be acting individually. However, there is considerable crosstalk between these two redox-regulated transcription

Summary and perspective

Unresolved inflammation is the backbone of many diseases, especially during the progression to fibrosis. Electrophilic NO₂-FAs have been proven safe in Phase I clinical trials and provide new dietary and pharmacological approaches to target inflammatory and fibrotic disorders by reversibly regulating signaling pathways. The decrease of inflammation and oxidative stress through Nrf2 and NF-κB pathways provides the conditions to return to homeostasis. The use of electrophiles is not free of

Conflict of interest statement

F.J.S. has financial interest in Complexa Inc. N.K.H.K. has no conflicts of interest to declare.

References and recommended reading

Papers of particular interest, published within the period of review, have been highlighted as:

• of special interest
•• of outstanding interest

Acknowledgements

We would like to thank Ms. Sonia Salvatore for input and graphic design. This work was supported by R01-GM125944 and R01-DK112854 (F.J.S), AHA17GRN33660955 (F.J.S), P01-HL103455 (N.K.H.K.) and University of Pittsburgh Medical Center Competitive Medical Research Fund Award (N.K.H.K.).

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Regulation of immunomodulatory networks by Nrf2-activation in immune cells: Redox control and therapeutic potential in inflammatory diseases
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Inflammatory diseases present a serious health challenge due to their widespread prevalence and the severe impact on patients' lives. In the quest to alleviate the burden of these diseases, nuclear factor erythroid 2-related factor 2 (Nrf2) has emerged as a pivotal player. As a transcription factor intimately involved in cellular defense against metabolic and oxidative stress, Nrf2's role in modulating the inflammatory responses of immune cells has garnered significant attention. Recent findings suggest that Nrf2's ability to alter the redox status of cells underlies its regulatory effects on immune responses. Our review delves into preclinical and clinical evidence that underscores the complex influence of Nrf2 activators on immune cell phenotypes, particularly in the inflammatory milieu. By offering a detailed analysis of Nrf2's role in different immune cell populations, we cast light on the potential of Nrf2 activators in shaping the immune response towards a more regulated state, mitigating the adverse effects of inflammation through modeling redox status of immune cells. Furthermore, we explore the innovative use of nanoencapsulation techniques that enhance the delivery and efficacy of Nrf2 activators, potentially advancing the treatment strategies for inflammatory ailments. We hope this review will stimulate the development and expansion of Nrf2-targeted treatments that could substantially improve outcomes for patients suffering from a broad range of inflammatory diseases.
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Nitrated fatty acids are important anti-inflammatory and protective lipids formed in the gastric compartment, with conjugated linoleic acid (rumenic acid, RA, 9Z,11E-18:2) being the primary substrate for lipid nitration. The recently reported identification of nitrated rumelenic acid (NO₂-RLA) in human urine has led to hypothesize that rumelenic acid (RLA, 9Z,11E,15Z-18:3) from dairy fat is responsible for the formation of NO₂-RLA. To evaluate the source and mechanism of NO₂-RLA formation, ¹⁵N labeled standards of NO₂-RLA were synthesized and characterized. Afterward, milk fat with different RA and RLA levels was administered to mice in the presence of nitrite, and the appearance of nitrated fatty acids in plasma and urine followed. We confirmed the formation of NO₂-RLA and defined the main metabolites in plasma, urine, and tissues. In conclusion, RLA obtained from dairy products is the main substrate for forming this novel electrophilic lipid reported to be present in human urine.
Electrophilic metabolites targeting the KEAP1/NRF2 partnership
2024, Current Opinion in Chemical Biology
Numerous electrophilic metabolites are formed during cellular activity, particularly under conditions of oxidative, inflammatory and metabolic stress. Among them are lipid oxidation and nitration products, and compounds derived from amino acid and central carbon metabolism. Here we focus on one cellular target of electrophiles, the Kelch-like ECH associated protein 1 (KEAP1)/nuclear factor erythroid 2 p45-related factor 2 (NRF2) partnership. Many of these reactive compounds modify C151, C273 and/or C288 within KEAP1. Other types of modifications include S-lactoylation of C273, N-succinylation of K131, and formation of methylimidazole intermolecular crosslink between two KEAP1 monomers. Modified KEAP1 relays the initial signal to transcription factor NRF2 and its downstream targets, the ultimate effectors that provide means for detoxification, adaptation and survival. Thus, by non-enzymatically covalently modifying KEAP1, the electrophilic metabolites discussed here serve as chemical signals connecting metabolism with stress responses.
Fatty acid nitroalkenes – Multi-target agents for the treatment of sickle cell disease
2023, Redox Biology
Sickle cell disease (SCD) is a hereditary hematological disease with high morbidity and mortality rates worldwide. Despite being monogenic, SCD patients display a plethora of disease-associated complications including anemia, oxidative stress, sterile inflammation, vaso-occlusive crisis-related pain, and vasculopathy, all of which contribute to multiorgan dysfunction and failure. Over the past decade, numerous small molecule drugs, biologics, and gene-based interventions have been evaluated; however, only four disease-modifying drug therapies are presently FDA approved. Barriers regarding effectiveness, accessibility, affordability, tolerance, and compliance of the current polypharmacy-based disease-management approaches are challenging. As such, there is an unmet pharmacological need for safer, more efficacious, and logistically accessible treatment options for SCD patients. Herein, we evaluate the potential of small molecule nitroalkenes such as nitro-fatty acid (NO₂-FA) as a therapy for SCD. These agents are electrophilic and exert anti-inflammatory and tissue repair effects through an ability to transiently post-translationally bind to and modify transcription factors, pro-inflammatory enzymes and cell signaling mediators. Preclinical and clinical studies affirm safety of the drug class and a murine model of SCD reveals protection against inflammation, fibrosis, and vascular dysfunction. Despite protective cardiac, renal, pulmonary, and central nervous system effects of nitroalkenes, they have not previously been considered as therapy for SCD. We highlight the pathways targeted by this drug class, which can potentially prevent the end-organ damage associated with SCD and contrast their prospective therapeutic benefits for SCD as opposed to current polypharmacy approaches.
Nitro-fatty acids as novel Virgin olive oil quality markers
2023, Journal of Food Composition and Analysis
Extra Virgin Olive oil (EVOO) consumption exerts beneficial health effects. Nitro-fatty acids (NO₂-FA) are endogenously formed in EVOO as well as in gastric conditions, exhibiting pleiotropic anti-inflammatory responses. In this review, we analyzed the conditions that favor the formation of EVOO-derived NO₂-FA. Firstly, will discuss the formation of NO₂-FA in contrasting varieties of EVOO and the relationship between the formation of NO₂-FA and the type of cultivar. Also, the variation of NO₂-FA levels with the stage of maturation will be discussed. It should be considered that EVOO has other key bioactive components such as polyphenols. We will discuss how polyphenols play a role in modulating NO₂-FA formation and how all these components would be acting synergistically to exert antioxidant/anti-inflammatory protection. Finally, will discuss how EVOO supplementation is capable of improving cellular respiration in mitochondria from liver disease, mainly due to the presence of NO₂-FA. Updated data collection strongly supports the proposal of NO₂-FA as novel EVOO quality markers. The information reviewed here can be useful to make recommendations about which cultivars to use and at what stages of maturation the oil should be extracted to maximize the concentration of these compounds that are so beneficial to human health after consumption.
Endogenous anti-tumorigenic nitro-fatty acids inhibit the ubiquitin-proteasome system by directly targeting the 26S proteasome
2023, Cell Chemical Biology
Nitro-fatty acids (NFAs) are endogenous lipid mediators causing a spectrum of anti-inflammatory effects by covalent modification of key proteins within inflammatory signaling pathways. Recent animal models of solid tumors have helped demonstrate their potential as anti-tumorigenic therapeutics. This study evaluated the anti-tumorigenic effects of NFAs in colon carcinoma cells and other solid and leukemic tumor cell lines. NFAs inhibited the ubiquitin-proteasome system (UPS) by directly targeting the 26S proteasome, leading to polyubiquitination and inhibition of the proteasome activities. UPS suppression induced the unfolded protein response, resulting in tumor cell death. The NFA-mediated effects were substantial, specific, and enduring, representing a unique mode of action for UPS suppression. This study provides mechanistic insights into the biological actions of NFAs as possible endogenous tumor-suppressive factors, indicating that NFAs might be key structures for designing a novel class of direct proteasome inhibitors.

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Nitrated fatty acids: from diet to disease

Highlights

Introduction

Section snippets

Formation of nitro-fatty acids

Stomach as a bioreactor

Endogenous NO2-FA

Nitro-fatty acid reactivity

Nitro-fatty acid signaling

Complexities of fibrosis

The need for novel therapeutics: NO2-FA

NF-κB inhibition

Nrf2 activation

Crosstalk between Nrf2 and NF-κB pathways

Summary and perspective

Conflict of interest statement

References and recommended reading

Acknowledgements

Free Radic Biol Med

J Biol Chem

Annu Rev Nutr

Cancer Lett

Gut

Redox Biol

Free Radic Biol Med

J Chromatogr B Analyt Technol Biomed Life Sci

Methods Mol Biol

Nitric Oxide

J Biol Chem

Proc Natl Acad Sci U S A

Proc Natl Acad Sci U S A

Drug Des Devel Ther

Am J Physiol Renal Physiol

Cardiovasc Res

Free Radic Biol Med

Nat Commun

Nat Rev Drug Discov

Biochem Soc Trans

Whole grain-rich diet reduces body weight and systemic low-grade inflammation without inducing major changes of the gut microbiome: a randomised cross-over trial

Gut

Gut microbiota metabolites, amino acid metabolites and improvements in insulin sensitivity and glucose metabolism: the POUNDS lost trial

Gut

Gut microbial metabolites in obesity, NAFLD and T2DM

Nat Rev Endocrinol

Role of nitrite, urate and pepsin in the gastroprotective effects of saliva

Redox Biol

Systematic evaluation on the effectiveness of conjugated linoleic acid in human health

Crit Rev Food Sci Nutr

Metabolic effects of dietary nitrate in health and disease

Cell Metab

Endogenous NO₂-FA

The need for novel therapeutics: NO₂-FA