Background: Endothelial cells depend on glycolysis for much of their energy production. Impaired endothelial glycolysis has been associated with various vascular pathobiologies, including impaired angiogenesis and atherogenesis. Interferon-gamma (IFN- γ )-producing CD4 + and CD8 + T-lymphocytes have been identified as the predominant pathologic cell subsets in human atherosclerotic plaques. While the immunological consequences of these cells have been extensively evaluated, their IFN- γ -mediated metabolic effects on endothelial cells remain unknown. The purpose of this study was to determine the metabolic consequences of the T-lymphocyte cytokine, IFN- γ , on human coronary artery endothelial cells (HCAEC). Methods: The metabolic effects of IFN- γ on primary HCAEC were assessed by unbiased transcriptomic and metabolomic analyses combined with real-time extracellular flux analyses and molecular mechanistic studies. Cellular phenotypic correlations were made by measuring altered endothelial intracellular cyclic guanosine monophosphate (cGMP) content, wound healing capacity, and adhesion molecule expression. Results: IFN- γ exposure inhibited basal glycolysis of quiescent primary HCAEC by 20% through the global transcriptional suppression of glycolytic enzymes resulting from decreased basal hypoxia inducible factor 1α (HIF1α) nuclear availability in normoxia. The decrease in HIF1α activity was a consequence of IFN- γ -induced tryptophan catabolism resulting in ARNT (aryl hydrocarbon receptor nuclear translocator)/HIF1β sequestration by the kynurenine-activated aryl hydrocarbon receptor (AHR). Additionally, IFN- γ resulted in a 23% depletion of intracellular NAD + in HCAEC. This altered glucose metabolism was met with concomitant activation of fatty acid oxidation, which augmented its contribution to intracellular ATP balance by over 20%. These metabolic derangements were associated with adverse endothelial phenotypic changes, including decreased basal intracellular cGMP, impaired endothelial migration, and a switch to a pro-inflammatory state. Conclusions: IFN- γ impairs endothelial glucose metabolism via altered tryptophan catabolism destabilizing HIF1, depletes NAD + , and results in a metabolic shift toward increased fatty acid oxidation. This work suggests a novel mechanistic basis for pathologic T-lymphocyte-endothelial interactions in atherosclerosis mediated by IFN- γ , linking endothelial glucose, tryptophan, and fatty acid metabolism with NAD(H) and ATP generation, and their adverse endothelial functional consequences.

Proteinuria is a major risk factor for chronic kidney disease progression. Furthermore, exposure of proximal tubular epithelial cells to excess albumin promotes tubular atrophy and fibrosis, key predictors of progressive organ dysfunction. However, the link between proteinuria and tubular damage is unclear. We propose that pathological albumin exposure impairs proximal tubular autophagy, an essential process for recycling damaged organelles and toxic intracellular macromolecules. In both mouse primary proximal tubule and immortalized human kidney cells, albumin exposure decreased the number of autophagosomes, visualized by the autophagosome-specific fluorescent markers monodansylcadaverine and GFP-LC3, respectively. Similarly, renal cortical tissue harvested from proteinuric mice contained reduced numbers of autophagosomes on electron micrographs, and immunoblots showed reduced steady-state LC3-II content. Albumin exposure decreased autophagic flux in vitro in a concentration-dependent manner as assessed by LC3-II accumulation rate in the presence of bafilomycin, an H-ATPase inhibitor that prevents lysosomal LC3-II degradation. In addition, albumin treatment significantly increased the half-life of radiolabeled long-lived proteins, indicating that the primary mechanism of degradation, autophagy, is dysfunctional. In vitro, mammalian target of rapamycin (mTOR) activation, a potent autophagy inhibitor, suppressed autophagy as a result of intracellular amino acid accumulation from lysosomal albumin degradation. mTOR activation was demonstrated by the increased phosphorylation of its downstream target, S6K, with free amino acid or albumin exposure. We propose that excess albumin uptake and degradation inhibit proximal tubule autophagy via an mTOR-mediated mechanism and contribute to progressive tubular injury.

Background: L-2-hydroxyglutarate (L2HG) couples mitochondrial and cytoplasmic energy metabolism to support cellular redox homeostasis. Under oxygen-limiting conditions, mammalian cells generate L2HG to counteract the adverse effects of reductive stress induced by hypoxia. Very little is known, however, about whether and how L2HG provides tissue protection from redox stress during low-flow ischemia (LFI) and ischemia-reperfusion injury. We examined the cardioprotective effects of L2HG accumulation against LFI and ischemia-reperfusion injury and its underlying mechanism using genetic mouse models. Methods and Results: L2HG accumulation was induced by homozygous (L2HGDH [L-2-hydroxyglutarate dehydrogenase] –/– ) or heterozygous (L2HGDH +/– ) deletion of the L2HGDH gene in mice. Hearts isolated from these mice and their wild-type littermates (L2HGDH +/+ ) were subjected to baseline perfusion or 90-minute LFI or 30-minute no-flow ischemia followed by 60- or 120-minute reperfusion. Using [ 13 C]- and [ 31 P]-NMR spectroscopy, high-performance liquid chromatography, real-time quantitative real-time polymerase chain reaction, ELISA, triphenyltetrazolium staining, colorimetric/fluorometric spectroscopy, and echocardiography, we found that L2HGDH deletion induces L2HG accumulation at baseline and under stress conditions with significant functional consequences. In response to LFI or ischemia-reperfusion, L2HG accumulation shifts glucose flux from glycolysis towards the pentose phosphate pathway. These key metabolic changes were accompanied by enhanced cellular reducing potential, increased elimination of reactive oxygen species, attenuated oxidative injury and myocardial infarction, preserved cellular energy state, and improved cardiac function in both L2HGDH –/– and L2HGDH +/– hearts compared with L2HGDH +/+ hearts under ischemic stress conditions. Conclusion: L2HGDH deletion-induced L2HG accumulation protects against myocardial injury during LFI and ischemia-reperfusion through a metabolic shift of glucose flux from glycolysis towards the pentose phosphate pathway. L2HG offers a novel mechanism for eliminating reactive oxygen species from myocardial tissue, mitigating redox stress, reducing myocardial infarct size, and preserving high-energy phosphates and cardiac function. Targeting L2HG levels through L2HGDH activity may serve as a new therapeutic strategy for cardiovascular diseases related to oxidative injury.