Oxidative stress is a mediator of glucose toxicity in insulin-secreting pancreatic islet cell lines

Pancreatic beta cells secrete insulin in response to changes in the extracellular glucose. However, prolonged exposure to elevated glucose exerts toxic effects on beta cells and results in beta cell dysfunction and ultimately beta cell death (glucose toxicity). To investigate the mechanism of how increased extracellular glucose is toxic to beta cells, we used two model systems where glucose metabolism was increased in beta cell lines by enhancing glucokinase (GK) activity and exposing cells to physiologically relevant increases in extracellular glucose (3.3-20 mm). Exposure of cells with enhanced GK activity to 20 mm glucose accelerated glycolysis, but reduced cellular NAD(P)H and ATP, caused accumulation of intracellular reactive oxygen species (ROS) and oxidative damage to mitochondria and DNA, and promoted apoptotic cell death. These changes required both enhanced GK activity and exposure to elevated extracellular glucose. A ROS scavenger partially prevented the toxic effects of increased glucose metabolism. These results indicate that increased glucose metabolism in beta cells generates oxidative stress and impairs cell function and survival; this may be a mechanism of glucose toxicity in beta cells. The level of beta cell GK may also be critical in this process.     

Pyruvate kinase triggers a metabolic feedback loop that controls redox metabolism in respiring cells

In proliferating cells, a transition from aerobic to anaerobic metabolism is known as the Warburg effect, whose reversal inhibits cancer cell proliferation. Studying its regulator pyruvate kinase (PYK) in?yeast, we discovered that central metabolism is?self-adapting to synchronize redox metabolism when respiration is activated. Low PYK activity activated yeast respiration. However, levels of reactive oxygen species (ROS) did not increase, and cells gained resistance to oxidants. This adaptation was attributable to accumulation of the PYK substrate phosphoenolpyruvate (PEP). PEP acted as feedback inhibitor of the glycolytic enzyme triosephosphate isomerase (TPI). TPI inhibition stimulated the pentose phosphate pathway, increased antioxidative metabolism, and prevented ROS accumulation. Thus, a metabolic feedback loop, initiated by PYK, mediated by its substrate and acting on TPI, stimulates redox metabolism in respiring cells. Originating from a single catalytic step, this autonomous reconfiguration of central carbon metabolism prevents oxidative stress upon shifts between fermentation and respiration.     

High glucose-induced mitochondrial respiration and reactive oxygen species in mouse cerebral pericytes is reversed by pharmacological inhibition of mitochondrial carbonic anhydrases: Implications for cerebral microvascular disease in diabetes

Hyperglycemia-induced oxidative stress leads to diabetes-associated damage to the microvasculature of the brain. Pericytes in close proximity to endothelial cells in the brain microvessels are vital to the integrity of the blood–brain barrier and are especially susceptible to oxidative stress. According to our recently published results, streptozotocin-diabetic mouse brain exhibits oxidative stress and loose pericytes by twelve weeks of diabetes, and cerebral pericytes cultured in high glucose media suffer intracellular oxidative stress and apoptosis. Oxidative stress in diabetes is hypothesized to be caused by reactive oxygen species (ROS) produced during hyperglycemia-induced enhanced oxidative metabolism of glucose (respiration). To test this hypothesis, we investigated the effect of high glucose on respiration rate and ROS production in mouse cerebral pericytes. Previously, we showed that pharmacological inhibition of mitochondrial carbonic anhydrases protects the brain from oxidative stress and pericyte loss. The high glucose-induced intracellular oxidative stress and apoptosis of pericytes in culture were also reversed by inhibition of mitochondrial carbonic anhydrases. Therefore, we extended our current study to determine the effect of these inhibitors on high glucose-induced increases in pericyte respiration and ROS. We now report that both the respiration and ROS are significantly increased in pericytes challenged with high glucose. Furthermore, inhibition of mitochondrial carbonic anhydrases significantly slowed down both the rate of respiration and ROS production. These data provide new evidence that pharmacological inhibitors of mitochondrial carbonic anhydrases, already in clinical use, may prove beneficial in protecting the brain from oxidative stress caused by ROS produced as a consequence of hyperglycemia-induced enhanced respiration.     

Oxidative stress,mitochondrial DNA mutation,and impairment of antioxidant enzymes in aging

Mitochondria do not only produce less ATP, but they also increase the production of reactive oxygen species (ROS) as by-products of aerobic metabolism in the aging tissues of the human and animals. It is now generally accepted that aging-associated respiratory function decline can result in enhanced production of ROS in mitochondria. Moreover, the activities of free radical-scavenging enzymes are altered in the aging process. The concurrent age-related changes of these two systems result in the elevation of oxidative stress in aging tissues. Within a certain concentration range, ROS may induce stress response of the cells by altering expression of respiratory genes to uphold the energy metabolism to rescue the cell. However, beyond the threshold, ROS may cause a wide spectrum of oxidative damage to various cellular components to result in cell death or elicit apoptosis by induction of mitochondrial membrane permeability transition and release of apoptogenic factors such as cytochrome c. Moreover, oxidative damage and large-scale deletion and duplication of mitochondrial DNA (mtDNA) have been found to increase with age in various tissues of the human. Mitochondria act like a biosensor of oxidative stress and they enable cell to undergo changes in aging and age-related diseases. On the other hand, it has recently been demonstrated that impairment in mitochondrial respiration and oxidative phosphorylation elicits an increase in oxidative stress and causes a host of mtDNA rearrangements and deletions. Here, we review work done in the past few years to support our view that oxidative stress and oxidative damage are a result of concurrent accumulation of mtDNA mutations and defective antioxidant enzymes in human aging.     

Alterations in Cellular Energy Metabolism Associated with the Antiproliferative Effects of the ATM Inhibitor KU-55933 and with Metformin

KU-55933 is a specific inhibitor of the kinase activity of the protein encoded by Ataxia telangiectasia mutated (ATM), an important tumor suppressor gene with key roles in DNA repair. Unexpectedly for an inhibitor of a tumor suppressor gene, KU-55933 reduces proliferation. In view of prior preliminary evidence suggesting defective mitochondrial function in cells of patients with Ataxia Telangiectasia (AT), we examined energy metabolism of cells treated with KU-55933. The compound increased AMPK activation, glucose uptake and lactate production while reducing mitochondrial membrane potential and coupled respiration. The stimulation of glycolysis by KU-55933 did not fully compensate for the reduction in mitochondrial functions, leading to decreased cellular ATP levels and energy stress. These actions are similar to those previously described for the biguanide metformin, a partial inhibitor of respiratory complex I. Both compounds decreased mitochondrial coupled respiration and reduced cellular concentrations of fumarate, malate, citrate, and alpha-ketogluterate. Succinate levels were increased by KU-55933 levels and decreased by metformin, indicating that the effects of ATM inhibition and metformin are not identical. These observations suggest a role for ATM in mitochondrial function and show that both KU-55933 and metformin perturb the TCA cycle as well as oxidative phosphorylation.     

Cancer cells metabolically “fertilize” the tumor microenvironment with hydrogen peroxide,driving the Warburg effect: Implications for PET imaging of human tumors

Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a “weapon” to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the “reverse Warburg effect.” To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection). Mitochondrial activity, glucose uptake and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis. Conversely, co-cultured cancer cells show significant increases in mitochondrial activity and corresponding reductions in both glucose uptake and GLUT1 expression. Pre-treatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress and potently induces the death of cancer cells, likely via starvation. Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.Key words: tumor stroma, microenvironment, hydrogen peroxide, aerobic glycolysis, mitochondrial oxidative phosphorylation, glucose uptake, oxidative stress, reactive oxygen species (ROS), cancer associated fibroblasts, PET imaging, the field effect, caveolin-1     

The Warburg effect: Evolving interpretations of an established concept

Metabolic reprogramming and altered bioenergetics have emerged as hallmarks of cancer and an area of active basic and translational cancer research. Drastically upregulated glucose transport and metabolism in most cancers regardless of the oxygen supply, a phenomenon called the Warburg effect, is a major focuses of the research. Warburg speculated that cancer cells, due to defective mitochondrial oxidative phosphorylation (OXPHOS), switch to glycolysis for ATP synthesis, even in the presence of oxygen. Studies in the recent decade indicated that while glycolysis is indeed drastically upregulated in almost all cancer cells, mitochondrial respiration continues to operate normally at rates proportional to oxygen supply. There is no OXPHOS-to-glycolysis switch but rather upregulation of glycolysis. Furthermore, upregulated glycolysis appears to be for synthesis of biomass and reducing equivalents in addition to ATP production. The new finding that a significant amount of glycolytic intermediates is diverted to the pentose phosphate pathway (PPP) for production of NADPH has profound implications in how cancer cells use the Warburg effect to cope with reactive oxygen species (ROS) generation and oxidative stress, opening the door for anticancer interventions taking advantage of this. Recent findings in the Warburg effect and its relationship with ROS and oxidative stress controls will be reviewed. Cancer treatment strategies based on these new findings will be presented and discussed.     

Cancer cells metabolically "fertilize" the tumor microenvironment with hydrogen peroxide,driving the Warburg effect

Previously, we proposed that cancer cells behave as metabolic parasites, as they use targeted oxidative stress as a “weapon” to extract recycled nutrients from adjacent stromal cells. Oxidative stress in cancer-associated fibroblasts triggers autophagy and mitophagy, resulting in compartmentalized cellular catabolism, loss of mitochondrial function, and the onset of aerobic glycolysis, in the tumor stroma. As such, cancer-associated fibroblasts produce high-energy nutrients (such as lactate and ketones) that fuel mitochondrial biogenesis, and oxidative metabolism in cancer cells. We have termed this new energy-transfer mechanism the “reverse Warburg effect.” To further test the validity of this hypothesis, here we used an in vitro MCF7-fibroblast co-culture system, and quantitatively measured a variety of metabolic parameters by FACS analysis (analogous to laser-capture micro-dissection). Mitochondrial activity, glucose uptake, and ROS production were measured with highly-sensitive fluorescent probes (MitoTracker, NBD-2-deoxy-glucose, and DCF-DA). Interestingly, using this approach, we directly show that cancer cells initially secrete hydrogen peroxide that then triggers oxidative stress in neighboring fibroblasts. Thus, oxidative stress is contagious (spreads like a virus) and is propagated laterally and vectorially from cancer cells to adjacent fibroblasts. Experimentally, we show that oxidative stress in cancer-associated fibroblasts quantitatively reduces mitochondrial activity, and increases glucose uptake, as the fibroblasts become more dependent on aerobic glycolysis. Conversely, co-cultured cancer cells show significant increases in mitochondrial activity, and corresponding reductions in both glucose uptake and GLUT1 expression. Pre-treatment of co-cultures with extracellular catalase (an anti-oxidant enzyme that detoxifies hydrogen peroxide) blocks the onset of oxidative stress, and potently induces the death of cancer cells, likely via starvation. Given that cancer-associated fibroblasts show the largest increases in glucose uptake, we suggest that PET imaging of human tumors, with Fluoro-2-deoxy-D-glucose (F-2-DG), may be specifically detecting the tumor stroma, rather than epithelial cancer cells.     

Tumor Cells Switch to Mitochondrial Oxidative Phosphorylation under Radiation via mTOR-Mediated Hexokinase II Inhibition - A Warburg-Reversing Effect

A unique feature of cancer cells is to convert glucose into lactate to produce cellular energy, even under the presence of oxygen. Called aerobic glycolysis [The Warburg Effect] it has been extensively studied and the concept of aerobic glycolysis in tumor cells is generally accepted. However, it is not clear if aerobic glycolysis in tumor cells is fixed, or can be reversed, especially under therapeutic stress conditions. Here, we report that mTOR, a critical regulator in cell proliferation, can be relocated to mitochondria, and as a result, enhances oxidative phosphorylation and reduces glycolysis. Three tumor cell lines (breast cancer MCF-7, colon cancer HCT116 and glioblastoma U87) showed a quick relocation of mTOR to mitochondria after irradiation with a single dose 5 Gy, which was companied with decreased lactate production, increased mitochondrial ATP generation and oxygen consumption. Inhibition of mTOR by rapamycin blocked radiation-induced mTOR mitochondrial relocation and the shift of glycolysis to mitochondrial respiration, and reduced the clonogenic survival. In irradiated cells, mTOR formed a complex with Hexokinase II [HK II], a key mitochondrial protein in regulation of glycolysis, causing reduced HK II enzymatic activity. These results support a novel mechanism by which tumor cells can quickly adapt to genotoxic conditions via mTOR-mediated reprogramming of bioenergetics from predominantly aerobic glycolysis to mitochondrial oxidative phosphorylation. Such a “waking-up” pathway for mitochondrial bioenergetics demonstrates a flexible feature in the energy metabolism of cancer cells, and may be required for additional cellular energy consumption for damage repair and survival. Thus, the reversible cellular energy metabolisms should be considered in blocking tumor metabolism and may be targeted to sensitize them in anti-cancer therapy.     

Glucose restriction extends Caenorhabditis elegans life span by inducing mitochondrial respiration and increasing oxidative stress

Increasing cellular glucose uptake is a fundamental concept in treatment of type 2 diabetes, whereas nutritive calorie restriction increases life expectancy. We show here that increased glucose availability decreases Caenorhabditis elegans life span, while impaired glucose metabolism extends life expectancy by inducing mitochondrial respiration. The histone deacetylase Sir2.1 is found here to be dispensable for this phenotype, whereas disruption of aak-2, a homolog of AMP-dependent kinase (AMPK), abolishes extension of life span due to impaired glycolysis. Reduced glucose availability promotes formation of reactive oxygen species (ROS), induces catalase activity, and increases oxidative stress resistance and survival rates, altogether providing direct evidence for a hitherto hypothetical concept named mitochondrial hormesis or "mitohormesis." Accordingly, treatment of nematodes with different antioxidants and vitamins prevents extension of life span. In summary, these data indicate that glucose restriction promotes mitochondrial metabolism, causing increased ROS formation and cumulating in hormetic extension of life span, questioning current treatments of type 2 diabetes as well as the widespread use of antioxidant supplements.     

Understanding the tumor metabolic phenotype in the genomic era

Now, at the beginning of a new century, 80 years after Warburg's Nobel prize winning discoveries, we are beginning to make sense of the underlying causes of the well known metabolic phenotype of tumor cells. Building on decades of research to understand the interrelationships between respiration and glycolysis in cancer, the tumor metabolic phenotype can now begin to be understood in a genomic context. With the discovery of hypoxia inducible factor-1 (HIF-1), which is widely overexpressed across a broad range of cancers, modern molecular tools have allowed us to put together the pattern of events that might explain the metabolic differences between tumor and normal cells. HIF-1 controls cellular and systemic responses to oxygen availability and coordinates up-regulation of genes involved in many pathways concerned with tumour growth and metabolism including angiogenesis, glucose and energy metabolism, cellular proliferation, differentiation and viability, apoptosis, pH regulation and matrix metabolism. These findings begin to explain how glucose uptake and glycolysis could be up-regulated in cancer cells (through binding to a core DNA recognition sequence) in a co-ordinated and constitutive fashion that may also allow us to elucidate new targets for tumor therapy.     

PFKFB3 Regulates Oxidative Stress Homeostasis via Its S-Glutathionylation in Cancer

Whereas moderately increased cellular oxidative stress is supportive for cancerous growth of cells, excessive levels of reactive oxygen species (ROS) are detrimental to their growth and survival. We demonstrated that high ROS levels, via increased oxidized glutathione (GSSG), induce isoform-specific S-glutathionylation of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) at residue Cys206, which is located near the entrance to the 6-phosphofructo-2-kinase catalytic pocket. Upon this ROS-dependent, reversible, covalent modification, a marked decrease in its catalytic ability to synthesize fructose-2,6-bisphosphate (Fru-2,6-P₂), the key glycolysis allosteric activator, was observed. This event was coupled to a decrease in glycolytic flux and an increase in glucose metabolic flux into the pentose phosphate pathway. This shift, in turn, caused an increase in reduced glutathione (GSH) and, ultimately, resulted in ROS detoxification inside HeLa cells. The ability of PFKFB3 to control the Fru-2,6-P₂ levels in an ROS-dependent manner allows the PFKFB3-expressing cancer cells to continue energy metabolism with a reduced risk of excessive oxidative stress and, thereby, to support their cell survival and proliferation. This study provides a new insight into the roles of PFKFB3 as switch that senses and controls redox homeostasis in cancer in addition to its role in cancer glycolysis.     

Evaluation of mitochondrial function and metabolic reprogramming during tumor progression in a cell model of skin carcinogenesis

Metabolic reprogramming from mitochondrial aerobic respiration to aerobic glycolysis is a hallmark of cancer. However, whether it is caused by a dysfunction in the oxidative phosphorylation pathway is still under debate. In this work, we have analyzed the bioenergetic cellular (BEC) index and the relative cell ability to grow in the presence of either galactose or glucose as sources of sugar (Gal/Glu index) of a system formed by four epidermal cell lines with increasing tumorigenic potentials, ranging from nontumorigenic to highly malignant. We find that the BEC index gradually decreases whereas the Gal/Glu index increases with tumorigenicity, indicating that a progressive metabolic adaptation to aerobic glycolysis occurs in tumor cells associated with malignancy. Interestingly, this metabolic adaptation does not appear to be caused by damaged respiration, since the expression and activity of components of the respiratory chain complexes were unchanged in the cell lines. Moreover, the corresponding mitochondrial ATP synthetic abilities of the cell lines were found similar. The production of reactive oxygen species was also measured. A shift in ROS generation was found when compared nontumorigenic with tumorigenic cell lines, the latter exhibiting about threefold higher ROS levels than nontumorigenic cells. This result indicates that oxidative stress is an early event during tumor progression.     

Oxidative stress-alleviating strategies to improve recombinant protein production in CHO cells

Large scale biopharmaceutical production of biologics relies on the overexpression of foreign proteins by cells cultivated in stirred tank bioreactors. It is well recognized and documented fact that protein overexpression may impact host cell metabolism and that factors associated with large scale culture, such as the hydrodynamic forces and inhomogeneities within the bioreactors, may promote cellular stress. The metabolic adaptations required to support the high-level expression of recombinant proteins include increased energy production and improved secretory capacity, which, in turn, can lead to a rise of reactive oxygen species (ROS) generated through the respiration metabolism and the interaction with media components. Oxidative stress is defined as the imbalance between the production of free radicals and the antioxidant response within the cells. Accumulation of intracellular ROS can interfere with the cellular activities and exert cytotoxic effects via the alternation of cellular components. In this context, strategies aiming to alleviate oxidative stress generated during the culture have been developed to improve cell growth, productivity, and reduce product microheterogeneity. In this review, we present a summary of the different approaches used to decrease the oxidative stress in Chinese hamster ovary cells and highlight media development and cell engineering as the main pathways through which ROS levels may be kept under control.     

Dilation of human atria: Increased diffusion restrictions for ADP,overexpression of hexokinase 2 and its coupling to oxidative phosphorylation in cardiomyocytes

Cardiac energy metabolism with emphasis on mitochondria was addressed in atrial tissue from patients with overload-induced atrial dilation. Structural remodeling of dilated (D) atria manifested as intracellular accumulation of fibrillar aggregates, lipofuscin, signs of myolysis and autophagy. Despite impaired complex I dependent respiration and increased diffusion restriction for ADP, no changes regarding adenylate and creatine kinase occurred. We observed 7-fold overexpression of HK2 gene in D atria with concomitant 2-fold greater activation of mitochondrial oxygen consumption by glucose, which might represent an adaption to increased energy requirements and impaired mitochondrial function by effectively joining glycolysis and oxidative phosphorylation.     

Metabolic alterations in yeast lacking copper-zinc superoxide dismutase

Yeast lacking copper-zinc superoxide dismutase (sod1?) have a number of oxygen-dependent defects, including auxotrophies for lysine and methionine and sensitivity to oxygen. Here we report additional defects in metabolic regulation. Under standard growth conditions with glucose as the carbon source, yeast undergo glucose repression in which mitochondrial respiration is deemphasized, energy is mainly derived from glycolysis, and ethanol is produced. When glucose is depleted, the diauxic shift is activated, in which mitochondrial respiration is reemphasized and stress resistance increases. We find that both of these programs are adversely affected by the lack of Sod1p. Key events in the diauxic shift do not occur and sod1? cells do not utilize ethanol and stop growing. The ability to shift to growth on ethanol is gradually lost as time in culture increases. In early stages of culture, sod1? cells consume more oxygen and have more mitochondrial mass than wild-type cells, indicating that glucose repression is not fully activated. These changes are at least partially dependent on the activity of the Hap2,3,4,5 complex, as indicated by CYC1-lacZ reporter assays. These changes may indicate a role for superoxide in metabolic signaling and regulation and/or a role for glucose derepression in defense against oxidative stress.     

Proteomic evolution of a wine yeast during the first hours of fermentation

The inoculation of active dry wine yeast (ADWY) is one of the most common practices in winemaking. This inoculation exposes the yeast cells to strong osmotic, acidic and thermal stresses, and adaptation to the new medium is crucial for successful fermentation. We have analysed the changes that occur in the ADWY protein profile in the first hours after inoculation under enological-like conditions at a low temperature. Protein changes mainly included enzymes of the nitrogen and carbon metabolism and proteins related to the cellular stress response. Most of the enzymes of the lower part of the glycolysis showed an increase in their concentration 4 and 24 h after inoculation, indicating an increase in glycolytic flux and in ATP production. However, the shift from respiration to fermentation was not immediate in the inoculation because some mitochondrial proteins involved in oxidative metabolism were induced in the first hours after inoculation. Inoculation in this fresh medium also reduced the cellular concentration of stress proteins produced during industrial production of the ADWY. The only exception was Cys3p, which might be involved in glutathione synthesis as a response to oxidative stress. A better understanding of the yeast stress response to rehydration and inoculation will lead to improvements in the handling efficiency of ADWY in winemaking and presumably to better control of fermentation startup.