Lipocalin-2 (Lcn2), a critical component of the innate immune response which binds siderophores and limits bacterial iron acquisition, can elicit spillover adverse proinflammatory effects. Here we show that holo-Lcn2 (Lcn2-siderophore-iron, 1:3:1) increases mitochondrial reactive oxygen species (ROS) generation and attenuates mitochondrial oxidative phosphorylation in adult rat primary cardiomyocytes in a manner blocked by -acetyl-cysteine or the mitochondria-specific antioxidant SkQ1. We further demonstrate using siderophores 2,3-DHBA (2,3-dihydroxybenzoic acid) and 2,5-DHBA that increased ROS and reduction in oxidative phosphorylation are direct effects of the siderophore component of holo-Lcn2 and not due to apo-Lcn2 alone. Extracellular apo-Lcn2 enhanced the potency of 2,3-DHBA and 2,5-DHBA to increase ROS production and decrease mitochondrial respiratory capacity, whereas intracellular apo-Lcn2 attenuated these effects. These actions of holo-Lcn2 required an intact plasma membrane and were decreased by inhibition of endocytosis. The hearts, but not serum, of Lcn2 knockout (LKO) mice contained lower levels of 2,5-DHBA compared with wild-type hearts. Furthermore, LKO mice were protected from ischemia/reperfusion-induced cardiac mitochondrial dysfunction. Our study identifies the siderophore moiety of holo-Lcn2 as a regulator of cardiomyocyte mitochondrial bioenergetics.

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Background: Dietary n-6 polyunsaturated fats (n-6 PUFA) like linoleic acid (LA) may worsen cardiac remodeling after injury. Results: Excess LA increased cardiac collagen I/III ratio and lysyl oxidase causing early diastolic dysfunction. In vitro experiments in fibroblasts with genetic manipulation confirmed such mechanisms. Conclusion: LA promotes noncompliant collagen and cardiac stiffening. Significance: This study demonstrates a novel, cardiac-specific lipotoxic pathway of n-6 PUFAs.

PGC-1α, a transcriptional coactivator, controls inflammation and mitochondrial gene expression in insulin-sensitive tissues following exercise intervention. However, attributing such effects to PGC-1α is counfounded by exercise-induced fluctuations in blood glucose, insulin or bodyweight in diabetic patients. The goal of this study was to investigate the role of PGC-1α on inflammation and mitochondrial protein expressions in aging db/db mice hearts, independent of changes in glycemic parameters. In 8-month-old db/db mice hearts with diabetes lasting over 22 weeks, short-term, moderate-intensity exercise upregulated PGC-1α without altering body weight or glycemic parameters. Nonetheless, such a regimen lowered both cardiac (macrophage infiltration, iNOS and TNFα) and systemic (circulating chemokines and cytokines) inflammation. Curiously, such an anti-inflammatory effect was also linked to attenuated expression of downstream transcription factors of PGC-1α such as NRF-1 and several respiratory genes. Such mismatch between PGC-1α and its downstream targets was associated with elevated mitochondrial membrane proteins like Tom70 but a concurrent reduction in oxidative phosphorylation protein expressions in exercised db/db hearts. As mitochondrial oxidative stress was predominant in these hearts, in support of our in vivo data, increasing concentrations of H2O2 dose-dependently increased PGC-1α expression while inhibiting expression of inflammatory genes and downstream transcription factors in H9c2 cardiomyocytes in vitro. We conclude that short-term exercise-induced oxidative stress may be key in attenuating cardiac inflammatory genes and impairing PGC-1α mediated gene transcription of downstream transcription factors in type 2 diabetic hearts at an advanced age.

Iron is essential for energy metabolism, and states of iron deficiency or excess are detrimental for organisms and cells. Therefore, iron and carbohydrate metabolism are tightly regulated. Serum iron and glucose levels are subjected to hormonal regulation by hepcidin and insulin, respectively. Hepcidin is a liver-derived peptide hormone that inactivates the iron exporter ferroportin in target cells, thereby limiting iron efflux to the bloodstream. Insulin is a protein hormone secreted from pancreatic β-cells that stimulates glucose uptake and metabolism via insulin receptor signaling. There is increasing evidence that systemic, but also cellular iron and glucose metabolic pathways are interconnected. This review article presents relevant data derived primarily from mouse models and biochemical studies. In addition, it discusses iron and glucose metabolism in the context of human disease.

Moderate exercise improves cardiac antioxidant status in young humans and animals with Type-2 diabetes (T2D). Given that both diabetes and advancing age synergistically decrease antioxidant expression in most tissues, it is unclear whether exercise can upregulate cardiac antioxidants in chronic animal models of T2D. To this end, 8-month-old T2D and normoglycemic mice were exercised for 3 weeks, and cardiac redox status was evaluated. As expected, moderate exercise increased cardiac antioxidants and attenuated oxidative damage in normoglycemic mice. In contrast, similar exercise protocol in 8-month-old db/db mice worsened cardiac oxidative damage, which was associated with a specific dysregulation of glutathione (GSH) homeostasis. Expression of enzymes for GSH biosynthesis [γ-glutamylcysteine synthase, glutathione reductase] as well as for GSH-mediated detoxification (glutathione peroxidase, glutathione-S-transferase) was lower, while toxic metabolites dependent on GSH for clearance (4-hydroxynonenal) were increased in exercised diabetic mice hearts. To validate GSH loss as an important factor for such aggravated damage, daily administration of GSH restored cardiac GSH levels in exercised diabetic mice. Such supplementation attenuated both oxidative damage and fibrotic changes in the myocardium. Expression of transforming growth factor beta (TGF-β) and its regulated genes which are responsible for such profibrotic changes were also attenuated with GSH supplementation. These novel findings in a long-term T2D animal model demonstrate that short-term exercise by itself can deplete cardiac GSH and aggravate cardiac oxidative stress. As GSH administration conferred protection in 8-month-old diabetic mice undergoing exercise, supplementation with GSH-enhancing agents may be beneficial in elderly diabetic patients undergoing exercise.

Lcn2 is a host defense protein induced via the innate immune response to sequester iron-loaded bacterial siderophores. However, excess or prolonged elevation of Lcn2 levels can induce adverse cellular effects, including oxidative stress and inflammation. In this work, we use Hydrogen–Deuterium eXchange (HDX) and Isothermal Titration Calorimetry (ITC) to characterize the binding interaction between Lcn2 and siderophores enterobactin and 2,3-DHBA, in the presence and absence of iron. Our results indicate a rare “Type II” interaction in which binding of siderophores drives the protein conformational equilibrium toward an unfolded state. Linking our molecular model to cellular assays, we demonstrate that this “distorted binding mode” facilitates a deleterious cellular accumulation of reactive oxygen species that could represent the molecular origin of Lcn2 pathology. These results add important insights into mechanisms of Lcn2 action and have implications in Lcn2-mediated effects including inflammation.

Background: The prevalence of type 2 diabetes, obesity and their various comorbidities have continued to rise. In skeletal muscle lipotoxicity is well known to be a contributor to the development of insulin resistance. Here it was examined if the small molecule adiponectin receptor agonist AdipoRon mimicked the effect of adiponectin to attenuate palmitate induced reactive oxygen species (ROS) production and cell death in L6 skeletal muscle cells. Methods: L6 cells were treated ±0.1 mM PA, and ± AdipoRon, then assays analyzing reactive oxygen species (ROS) production and cell death, and intracellular and extracellular levels of sphingosine-1 phosphate (S1P) were conducted. To determine the mechanistic role of S1P gain (using exogenous S1P or using THI) or loss of function (using the SKI-II) were conducted. Results: Using both CellROX and DCFDA assays it was found that AdipoRon reduced palmitate-induced ROS production. Image-IT DEAD, MTT and LDH assays all indicated that AdipoRon reduced palmitate-induced cell death. Palmitate significantly increased intracellular accumulation of S1P, whereas in the presence of AdipoRon there was increased release of S1P from cells to extracellular medium. It was also observed that direct addition of extracellular S1P prevented palmitate-induced ROS production and cell death, indicating that S1P is acting in an autocrine manner. Pharmacological approaches to enhance or decrease S1P levels indicated that accumulation of intracellular S1P correlated with enhanced cell death. Conclusion: This data indicates that increased extracellular levels of S1P in response to adiponectin receptor activation can activate S1P receptor-mediated signaling to attenuate lipotoxic cell death. Taken together these findings represent a possible novel mechanism for the protective action of adiponectin.