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The multifaceted roles of metabolism in invasion have been investigated across many cancers. The brain tumor glioblastoma (GBM) is a highly invasive and metabolically plastic tumor with an inevitable recurrence. The neuronal glucose transporter 3 (GLUT3) was previously reported to correlate with poor glioma patient survival and be upregulated in GBM cells to promote therapeutic resistance and survival under restricted glucose conditions. It has been suggested that the increased glucose uptake mediated by GLUT3 elevation promotes survival of circulating tumor cells to facilitate metastasis. Here we suggest a more direct role for GLUT3 in promoting invasion that is not dependent upon changes in cell survival or metabolism. Analysis of glioma datasets demonstrated that GLUT3, but not GLUT1, expression was elevated in invasive disease. In human xenograft derived GBM cells, GLUT3, but not GLUT1, elevation significantly increased invasion in transwell assays, but not growth or migration. Further, there were no changes in glycolytic metabolism that correlated with invasive phenotypes. We identified the GLUT3 C-terminus as mediating invasion: substituting the C-terminus of GLUT1 for that of GLUT3 reduced invasion. RNA-seq analysis indicated changes in extracellular matrix organization in GLUT3 overexpressing cells, including upregulation of osteopontin. Together, our data suggest a role for GLUT3 in increasing tumor cell invasion that is not recapitulated by GLUT1, is separate from its role in metabolism and survival as a glucose transporter, and is likely broadly applicable since GLUT3 expression correlates with metastasis in many solid tumors.

The production of reactive species contributes to the age-dependent accumulation of dysfunctional mitochondria and protein aggregates, all of which are associated with neurodegeneration. A putative mediator of these effects is the lipid peroxidation product 4-hydroxynonenal (4-HNE), which has been shown to inhibit mitochondrial function, and accumulate in the postmortem brains of patients with neurodegenerative diseases. This deterioration in mitochondrial quality could be due to direct effects on mitochondrial proteins, or through perturbation of the macroautophagy/autophagy pathway, which plays an essential role in removing damaged mitochondria. Here, we use a click chemistry-based approach to demonstrate that alkyne-4-HNE can adduct to specific mitochondrial and autophagy-related proteins. Furthermore, we found that at lower concentrations (5–10 μM), 4-HNE activates autophagy, whereas at higher concentrations (15 μM), autophagic flux is inhibited, correlating with the modification of key autophagy proteins at higher concentrations of alkyne-4-HNE. Increasing concentrations of 4-HNE also cause mitochondrial dysfunction by targeting complex V (the ATP synthase) in the electron transport chain, and induce significant changes in mitochondrial fission and fusion protein levels, which results in alterations to mitochondrial network length. Finally, inhibition of autophagy initiation using 3-methyladenine (3MA) also results in a significant decrease in mitochondrial function and network length. These data show that both the mitochondria and autophagy are critical targets of 4-HNE, and that the proteins targeted by 4-HNE may change based on its concentration, persistently driving cellular dysfunction.

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABAA or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.

How cellular metabolic activities regulate autophagy and determine the susceptibility to oxidative stress and ultimately cell death in neuronal cells is not well understood. An important example of oxidative stress is 4-hydroxynonenal (HNE), which is a lipid peroxidation product that is formed during oxidative stress, and accumulates in neurodegenerative diseases causing damage. The accumulation of toxic oxidation products such as HNE, is a prevalent feature of neurodegenerative diseases, and can promote organelle and protein damage leading to induction of autophagy. In this study, we used differentiated SH-SY5Y neuroblastoma cells to investigate the mechanisms and regulation of cellular susceptibility to HNE toxicity and the relationship to cellular metabolism. We found that autophagy is immediately stimulated by HNE at a sublethal concentration. Within the same time frame, HNE induces concentration dependent CASP3/caspase 3 activation and cell death. Interestingly, both basal and HNE-activated autophagy, were regulated by glucose metabolism. Inhibition of glucose metabolism by 2-deoxyglucose (2DG), at a concentration that inhibited autophagic flux, further exacerbated CASP3 activation and cell death in response to HNE. Cell death was attenuated by the pan-caspase inhibitor Z-VAD-FMK. Specific inhibition of glycolysis using koningic acid, a GAPDH inhibitor, inhibited autophagic flux and exacerbated HNE-induced cell death similarly to 2DG. The effects of 2DG on autophagy and HNE-induced cell death could not be reversed by addition of mannose, suggesting an ER stress-independent mechanism. 2DG decreased LAMP1 and increased BCL2 levels suggesting that its effects on autophagy may be mediated by more than one mechanism. Furthermore, 2DG decreased cellular ATP, and 2DG and HNE combined treatment decreased mitochondrial membrane potential. We conclude that glucose-dependent autophagy serves as a protective mechanism in response to HNE.

The full characterization of the lipid content of the cell, the lipidome, is now emerging as a new approach to understand how lipid molecules modulate cellular function. As with the proteome, the lipidome can be extended by modification of the lipid molecule post synthesis. One way in which this can occur is through the enzymatic or nonspecific oxidation of unsaturated fatty acids and cholesterol by free radicals and their by-products. The free radicals and their products that lead to these lipid modifications are called reactive oxygen and nitrogen species (ROS and RNS, respectively), since they are formed from both oxygen metabolism and the reactions of the endogenously formed signaling molecule, nitric oxide. These modifications result in the insertion of specific functional groups into lipids, which fundamentally change their receptor binding properties and biochemical reactivity. This subgroup of lipid molecules has been termed the oxy-nitroxy-lipidome. Many of these molecules are capable of modifyin...

There is little doubt that oxidative modification of low-density lipoprotein (LDL) is an important process during atherogenesis. This conclusion has been derived in a relatively short period of time since the initial descriptions of LDL oxidation with a significant contribution from Professor Esterbauer and colleagues. In this short overview, we have described the mechanisms by which copper promotes LDL oxidation focussing on the importance of lipid hydroperoxides in this process. These mechanisms are discussed in the context of the ongoing debate as to relevance of metal dependent LDL oxidation in vivo and as a model reaction for assessing antioxidants.

The ischaemic myocardium shows a number of distinct oxygen dependent responses to reperfusion. In the case of myocardial stunning and reperfusion arrhythmias there appears to be a beneficial effect of scavenging radicals in the extracellular space. This result is supported by the finding that free radicals can be detected extracellularly after reperfusion. The source of these oxidants and site of action is as yet unclear. In contrast hypoxic myocytes shown an oxygen dependent Ca2+ uptake on reoxygenation which is not affected by externally applied antioxidants. This Ca2+ uptake may in turn lead to the cell lysis characteristic of the oxygen paradox in the perfused heart. As yet there is no compelling evidence to suggest that this aspect of reperfusion damage is due to oxidative stress. It appears more likely that mitochondrial electron transport and ion movement play a central role. In the open chested dog model of ischaemia reperfusion, in which the volume of infarcted tissue is measured, considerable evidence suggests that oxidants derived from activated neutrophils contribute to cell death. This is not however the sole mechanism for cell damage in this model since an inhibitor of mitochondrial Ca2+ uptake, ruthenium red, can improve recovery after reperfusion. We conclude that a number of mechanisms may contribute to the observed oxygen dependent dysfunctions which occur on reperfusion of ischaemic tissue.