Treatment with a Catalytic Superoxide Dismutase (SOD) Mimetic Improves Liver Steatosis, Insulin Sensitivity, and Inflammation in Obesity-Induced Type 2 Diabetes.

Oxidative stress and persistent inflammation are exaggerated through chronic over-nutrition and a sedentary lifestyle, resulting in insulin resistance. In type 2 diabetes (T2D), impaired insulin signaling leads to hyperglycemia and long-term complications, including metabolic liver dysfunction, resulting in non-alcoholic fatty liver disease (NAFLD). The manganese metalloporphyrin superoxide dismustase (SOD) mimetic, manganese (III) meso-tetrakis (N-ethylpyridinium-2-yl) porphyrin (MnP), is an oxidoreductase known to scavenge reactive oxygen species (ROS) and decrease pro-inflammatory cytokine production, by inhibiting nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. We hypothesized that targeting oxidative stress-induced inflammation with MnP would assuage liver complications and enhance insulin sensitivity and glucose tolerance in a high-fat diet (HFD)-induced mouse model of T2D. During 12 weeks of feeding, we saw significant improvements in weight, hepatic steatosis, and biomarkers of liver dysfunction with redox modulation by MnP treatment in HFD-fed mice. Additionally, MnP treatment improved insulin sensitivity and glucose tolerance, while reducing serum insulin and leptin levels. We attribute these effects to redox modulation and inhibition of hepatic NF-κB activation, resulting in diminished ROS and pro-inflammatory cytokine production. This study highlights the importance of controlling oxidative stress and secondary inflammation in obesity-mediated insulin resistance and T2D. Our data confirm the role of NF-κB-mediated inflammation in the development of T2D, and demonstrate the efficacy of MnP in preventing the progression to disease by specifically improving liver pathology and hepatic insulin resistance in obesity.

Conjugated Linoleic Acid Modulates Clinical Responses to Oral Nitrite and Nitrate.

Dietary NO3- (nitrate) and NO2- (nitrite) support ˙NO (nitric oxide) generation and downstream vascular signaling responses. These nitrogen oxides also generate secondary nitrosating and nitrating species that react with low molecular weight thiols, heme centers, proteins, and unsaturated fatty acids. To explore the kinetics of NO3-and NO2-metabolism and the impact of dietary lipid on nitrogen oxide metabolism and cardiovascular responses, the stable isotopes Na15NO3 and Na15NO2 were orally administered in the presence or absence of conjugated linoleic acid (cLA). The reduction of 15NO2- to 15NO was indicated by electron paramagnetic resonance spectroscopy detection of hyperfine splitting patterns reflecting 15NO-deoxyhemoglobin complexes. This formation of 15NO also translated to decreased systolic and mean arterial blood pressures and inhibition of platelet function. Upon concurrent administration of cLA, there was a significant increase in plasma cLA nitration products 9- and 12-15NO2-cLA. Coadministration of cLA with 15NO2- also impacted the pharmacokinetics and physiological effects of 15NO2-, with cLA administration suppressing plasma NO3-and NO2-levels, decreasing 15NO-deoxyhemoglobin formation, NO2-inhibition of platelet activation, and the vasodilatory actions of NO2-, while enhancing the formation of 9- and 12-15NO2-cLA. These results indicate that the biochemical reactions and physiological responses to oral 15NO3-and 15NO2-are significantly impacted by dietary constituents, such as unsaturated lipids. This can explain the variable responses to NO3-and NO2-supplementation in clinical trials and reveals dietary strategies for promoting the generation of pleiotropic nitrogen oxide-derived lipid signaling mediators. Clinical Trial Registration- URL: http://www.clinicaltrials.gov . Unique identifier: NCT01681836.

Nitrite and nitrate-dependent generation of anti-inflammatory fatty acid nitroalkenes.

A gap in our understanding of the beneficial systemic responses to dietary constituents nitrate (NO3(-)), nitrite (NO2(-)) and conjugated linoleic acid (cLA) is the identification of the downstream metabolites that mediate their actions. To examine these reactions in a clinical context, investigational drug preparations of (15)N-labeled NO3(-) and NO2(-) were orally administered to healthy humans with and without cLA. Mass spectrometry analysis of plasma and urine indicated that the nitrating species nitrogen dioxide was formed and reacted with the olefinic carbons of unsaturated fatty acids to yield the electrophilic fatty acid, nitro-cLA (NO2-cLA). These species mediate the post-translational modification (PTM) of proteins via reversible Michael addition with nucleophilic amino acids. The PTM of critical target proteins by electrophilic lipids has been described as a sensing mechanism that regulates adaptive cellular responses, but little is known about the endogenous generation of fatty acid nitroalkenes and their metabolites. We report that healthy humans consuming (15)N-labeled NO3(-) or NO2(-), with and without cLA supplementation, produce (15)NO2-cLA and corresponding metabolites that are detected in plasma and urine. These data support that the dietary constituents NO3(-), NO2(-) and cLA promote the further generation of secondary electrophilic lipid products that are absorbed into the circulation at concentrations sufficient to exert systemic effects before being catabolized or excreted.

Effects of metalloporphyrins on reducing inflammation and autoimmunity.

SIGNIFICANCE: High levels of reactive oxygen species can facilitate DNA and protein damage beyond the control of endogenous antioxidants, resulting in oxidative stress. Oxidative stress then triggers inflammation, which can lead to pathological conditions. In genetically susceptible individuals, the conglomeration of oxidative stress and inflammation can enhance autoreactive immune cell activation, causing beta-cell destruction in autoimmune type 1 diabetes. As a means of shielding pancreatic islets, manganese porphyrin (MnP) oxidoreductant treatment has been tested in a number of reported studies. RECENT ADVANCES: MnP affects both innate and adaptive immune cell responses, blocking nuclear factor kappa-B activation, proinflammatory cytokine secretion, and T helper 1 T-cell responses. As a result, MnP treatment protects against type 1 diabetes onset in nonobese diabetic mice and stabilizes islets for cellular transplantation. CRITICAL ISSUES: MnP displays global immunosuppressive properties, exemplified by decreased cytokine production from all T-helper cell subsets. This quality may impact infection control in the setting of autoimmunity. Nonetheless, because of their cytoprotective and immunomodulatory function, MnPs should be considered as a safer alternative to other clinical immunosuppressive agents (i.e., rapamycin) for transplantation. FUTURE DIRECTIONS: Although MnP likely affects only redox-sensitive targets, the mechanism behind global T-cell immunosuppression and the outcome on infection clearance will have to be elucidated. Based on the increased primary engraftment seen with MnP use, protection against primary nonfunction in porcine to human xenotransplants would likely be enhanced. Further, a better understanding of MnP oxidoreductase function may allow for its use in other chronic inflammatory conditions.

Redox-dependent anti-inflammatory signaling actions of unsaturated fatty acids.

Unsaturated fatty acids are metabolized to reactive products that can act as pro- or anti-inflammatory signaling mediators. Electrophilic fatty acid species, including nitro- and oxo-containing fatty acids, display salutary anti-inflammatory and metabolic actions. Electrophilicity can be conferred by both enzymatic and oxidative reactions, via the homolytic addition of nitrogen dioxide to a double bond or via the formation of α,ß-unsaturated carbonyl and epoxide substituents. The endogenous formation of electrophilic fatty acids is significant and influenced by diet, metabolic, and inflammatory reactions. Transcriptional regulatory proteins and enzymes can sense the redox status of the surrounding environment upon electrophilic fatty acid adduction of functionally significant, nucleophilic cysteines. Through this covalent and often reversible posttranslational modification, gene expression and metabolic responses are induced. At low concentrations, the pleiotropic signaling actions that are regulated by these protein targets suggest that some classes of electrophilic lipids may be useful for treating metabolic and inflammatory diseases.

Changing the energy of an immune response.

The breakdown of nutrients into the critical energy source ATP is the general purpose of cellular metabolism and is essential for sustaining life. Similarly, the immune system is composed of different cell subsets that are indispensable for defending the host against pathogens and disease. The interplay between metabolic pathways and immune cells leads to a plethora of different signaling pathways as well as cellular activities. The activation of T cells via glycolysis-mediated upregulation of surface markers, for example, is necessary for an appropriate effector response against an infection. However, tight regulation of immune cell metabolism is required for protecting the host and resuming homeostasis. An imbalance of immunological metabolic function and/or metabolic byproducts (reactive oxygen species) can oftentimes lead to diseases. In the case of cancer, overactive glucose metabolism can lead to hyperproliferation of cells and subsequent decreases in cytotoxic T cell activity, which attack and destroy the tumor. For this reason and many more, targeting metabolism in immune cells may be a novel therapeutic strategy for treatment of disease. The metabolic pathways of immune cells and the possibilities of immunometabolic therapies will be discussed.

Mn porphyrin regulation of aerobic glycolysis: implications on the activation of diabetogenic immune cells.

AIMS: The immune system is critical for protection against infections and cancer, but requires scrupulous regulation to limit self-reactivity and autoimmunity. Our group has utilized a manganese porphyrin catalytic antioxidant (MnTE-2-PyP(5+), MnP) as a potential immunoregulatory therapy for type 1 diabetes. MnP has previously been shown to modulate diabetogenic immune responses through decreases in proinflammatory cytokine production from antigen-presenting cells and T cells and to reduce diabetes onset in nonobese diabetic mice. However, it is unclear whether or not MnP treatment can act beyond the reported inflammatory mediators. Therefore, the hypothesis that MnP may be affecting the redox-dependent bioenergetics of diabetogenic splenocytes was investigated. RESULTS: MnP treatment enhanced glucose oxidation, reduced fatty acid oxidation, but only slightly decreased overall oxidative phosphorylation. These alterations occurred because of increased tricarboxylic acid cycle aconitase enzyme efficiency and were not due to changes in mitochondrial abundance. MnP treatment also displayed decreased aerobic glycolysis, which promotes activated immune cell proliferation, as demonstrated by reduced lactate production and glucose transporter 1 (Glut1) levels and inactivation of key signaling molecules, such as mammalian target of rapamycin, c-myc, and glucose-6-phosphate dehydrogenase. INNOVATION: This work highlights the importance of redox signaling by demonstrating that modulation of reactive oxygen species can supplant complex downstream regulation, thus affecting metabolic programming toward aerobic glycolysis. CONCLUSION: MnP treatment promotes metabolic quiescence, impeding diabetogenic autoimmune responses by restricting the metabolic pathways for energy production and affecting anabolic processes necessary for cell proliferation.