The familial amyotrophic lateral sclerosis-associated A4V SOD1 mutant is not able to regulate aerobic glycolysis.

Under certain stress conditions, astrocytes operate in aerobic glycolysis, a process controlled by pyruvate dehydrogenase (PDH) inhibition through its E1 α subunit (Pda1) phosphorylation. This supplies lactate to neurons, which save glucose to obtain NADPH to, among other roles, counteract reactive oxygen species. A failure in this metabolic cooperation causes severe damage to neurons. In this work, using humanized Saccharomyces cerevisiae cells in which its endogenous Cu/Zn Superoxide Dismutase (SOD1) was replaced by human ortholog, we investigated the role of human SOD1 (hSOD1) in aerobic glycolysis regulation and its implications to amyotrophic lateral sclerosis (ALS), a neurodegenerative disease. Yeast cells ferment glucose even in the presence of oxygen and switch to respiratory metabolism after glucose exhaustion. However, like cells of SOD1-knockout strain, cells expressing A4V mutant of hSOD1 growing on glucose showed a respiratory phenotype, i.e., low glucose and high oxygen consumptions and low intracellular oxidation levels in response to peroxide stress, contrary to cells expressing wild-type (WT) SOD1 (yeast or human). The A4V mutation in hSOD1 is linked to ALS. In contrast to WT SOD1 strains, PDH activity of both sod1Δ and A4V hSOD1 cells did not change in response to a metabolic shift toward oxidative metabolism, which was associated to lower Pda1 phosphorylation levels under growth on glucose. Taken together, our results suggest that A4V mutant cannot regulate aerobic glycolysis via Pda1 phosphorylation the same way WT hSOD1, which might be linked to problems observed in the motor neurons of ALS patients with the SOD1 A4V mutation.

Is Methylglyoxal a Potential Biomarker for the Warburg Effect Induced by the Lipopolysaccharide Neuroinflammation Model?

Methylglyoxal (MG) is considered a classical biomarker of diabetes mellitus and its comorbidities. However, a role for this compound in exacerbated immune responses, such as septicemia, is being increasingly observed and requires clarification, particularly in the context of neuroinflammatory responses. Herein, we used two different approaches (in vivo and acute hippocampal slice models) to investigate MG as a biomarker of neuroinflammation and the neuroimmunometabolic shift to glycolysis in lipopolysaccharide (LPS) inflammation models. Our data reinforce the hypothesis that LPS-induced neuroinflammation stimulates the cerebral innate immune response by increasing IL-1ß, a classical pro-inflammatory cytokine, and the astrocyte reactive response, via elevating S100B secretion and GFAP levels. Acute neuroinflammation promotes an early neuroimmunometabolic shift to glycolysis by elevating glucose uptake, lactate release, PFK1, and PK activities. We observed high serum and cerebral MG levels, in association with a reduction in glyoxalase 1 detoxification activity, and a close correlation between serum and hippocampus MG levels with the systemic and neuroinflammatory responses to LPS. Findings strongly suggest a role for MG in immune responses.

Dissecting the Role of Autophagy-Related Proteins in Cancer Metabolism and Plasticity.

Modulation of autophagy as an anticancer strategy has been widely studied and evaluated in several cell models. However, little attention has been paid to the metabolic changes that occur in a cancer cell when autophagy is inhibited or induced. In this review, we describe how the expression and regulation of various autophagy-related (ATGs) genes and proteins are associated with cancer progression and cancer plasticity. We present a comprehensive review of how deregulation of ATGs affects cancer cell metabolism, where inhibition of autophagy is mainly reflected in the enhancement of the Warburg effect. The importance of metabolic changes, which largely depend on the cancer type and form part of a cancer cell's escape strategy after autophagy modulation, is emphasized. Consequently, pharmacological strategies based on a dual inhibition of metabolic and autophagy pathways emerged and are reviewed critically here.

P2RX7 signaling drives the differentiation of Th1 cells through metabolic reprogramming for aerobic glycolysis.

Introduction: This study provides evidence of how Th1 cell metabolism is modulated by the purinergic receptor P2X7 (P2RX7), a cation cannel activated by high extracellular concentrations of adenosine triphosphate (ATP). Methods: In vivo analysis was performed in the Plasmodium chabaudi model of malaria in view of the great relevance of this infectious disease for human health, as well as the availability of data concerning Th1/Tfh differentiation. Results: We show that P2RX7 induces T-bet expression and aerobic glycolysis in splenic CD4+ T cells that respond to malaria, at a time prior to Th1/Tfh polarization. Cell-intrinsic P2RX7 signaling sustains the glycolytic pathway and causes bioenergetic mitochondrial stress in activated CD4+ T cells. We also show in vitro the phenotypic similarities of Th1-conditioned CD4+ T cells that do not express P2RX7 and those in which the glycolytic pathway is pharmacologically inhibited. In addition, in vitro ATP synthase blockade and the consequent inhibition of oxidative phosphorylation, which drives cellular metabolism for aerobic glycolysis, is sufficient to promote rapid CD4+ T cell proliferation and polarization to the Th1 profile in the absence of P2RX7. Conclusion: These data demonstrate that P2RX7-mediated metabolic reprograming for aerobic glycolysis is a key event for Th1 differentiation and suggest that ATP synthase inhibition is a downstream effect of P2RX7 signaling that potentiates the Th1 response.

Immunoexpression profile of hypoxia-inducible factor (HIF) targets in potentially malignant and malignant oral lesions: a pilot study

Abstract Oral potentially malignant disorders (OPMD) are associated with an increased risk of oral squamous cell carcinoma (OSCC). OSCC has an aggressive profile and is the most prevalent among different head and neck malignancies. Most OSCC patients are diagnosed with advanced stage tumors and have a poor prognosis. Cancer cells are able to reprogram their metabolism, even in the presence of oxygen, enhancing the conversion of glucose to lactate via the glycolytic pathway, a phenomenon mainly regulated by hypoxia-inducible factor (HIF) signaling. Thus, several glycometabolism-related biomarkers are upregulated. Objectives This study aimed to evaluate the immunoexpression of the HIF targets GLUT1, GLUT3, HK2, PFKL, PKM2, pPDH, LDHA, MCT4, and CAIX in OPMD and OSCC samples, in order to identify potential correlations between biomarkers' immunoexpression, clinicopathological features, and prognostic parameters. Methodology OSCC and OPMD samples from 21 and 34 patients (respectively) were retrospectively collected and stained for the different biomarkers by immunohistochemistry. Results CAIX and MCT4 expressions were significantly higher in OSCC samples when compared with OPMD samples, while the rest were also expressed by OPMD. GLUT3 and PKM2 alone, and the concomitant expression of more than four glycometabolism-related biomarkers were significantly correlated with the presence of dysplasia in OPMD. When considering OSCC cases, a trend toward increased expression of biomarkers and poor clinicopathological features was observed, and the differences regarding HK2, PFKL, LDHA and MCT4 expression were significant. Moreover, HK2 and CAIX were correlated with low survival rates. GLUT1 and GLUT3 were significantly associated with poor outcome when their expression was observed in the hypoxic region of malignant lesions. Conclusion OPMD and OSCC cells overexpress glycolysis-related proteins, which is associated with aggressive features and poor patient outcome. Further research is needed to deeply understand the glycolic phenotype in the process of oral carcinogenesis.

Hexokinase 2: The preferential target of trehalose-6-phosphate over hexokinase 1.

Cancer-related metabolic features are in part maintained by hexokinase 2 upregulation, which leads to high levels of glucose-6-phosphate (G6P) and is needed to provide energy and biomass to support rapid proliferation. Using a humanized model of the yeast Saccharomyces cerevisiae, we explored how human hexokinase 2 (HK2) behaves under different nutritional conditions. At high glucose levels, yeast presents aerobic glycolysis through a regulatory mechanism known as catabolic repression, which exerts a metabolic adaptation like the Warburg effect. At high glucose concentrations, HK2 did not translocate into the nucleus and was not able to shift the metabolism toward a highly glycolytic state, in contrast to the effect of yeast hexokinase 2 (Hxk2), which is a crucial protein for the control of aerobic glycolysis in S. cerevisiae. During the stationary phase, when glucose is exhausted, Hxk2 is shuttled out of the nucleus, ceasing catabolic repression. Cells harvested at this condition display low glucose consumption rates. However, glucose-starved cells expressing HK2 had an increased capacity to consume glucose. In those cells, HK2 localized to mitochondria, becoming insensitive to G6P inhibition. We also found that the sugar trehalose-6-phosphate (T6P) is a human HK2 inhibitor, like yeast Hxk2, but was not able to inhibit human HK1, the isoform that is ubiquitously expressed in almost all mammalian tissues. In contrast to G6P, T6P inhibited HK2 even when HK2 was associated with mitochondria. The binding of HK2 to mitochondria is crucial for cancer survival and proliferation. T6P was able to reduce the cell viability of tumor cells, although its toxicity was not impressive. This was expected as cell absorption of phosphorylated sugars is low, which might be counteracted using nanotechnology. Altogether, these data suggest that T6P may offer a new paradigm for cancer treatment based on specific inhibition of HK2.

Essential role of aerobic glycolysis in epithelial-to-mesenchymal transition during carcinogenesis.

Epithelial-to-mesenchymal transition (EMT) confers the most lethal characteristics to cancer cells i.e., metastasis and resistance to chemo-and-radio-therapy, and therefore exhibit an appealing target in the field of oncology. Research in the past decade has demonstrated the crucial role of aerobic glycolysis in EMT, which is generally credited as the glucose metabolism for the creation of biomass such as fatty acids, amino acids, and nucleotides thereby providing building blocks for limitless proliferation. In the present review, apart from discussing EMT's evident role in the metastatic process and cancer stemness, we also talked about the vital role of glycolytic enzymes viz. GLUTs, HKs, PGI, PFK-1, aldolase, enolase, PK, LDHA, etc. in the induction of the EMT process in cancerous cells.

Ras related GTP binding D promotes aerobic glycolysis of hepatocellular carcinoma.

INTRODUCTION AND OBJECTIVES: Warburg effect is attracting increasing attention as it is important for cancer progression. However, how cancer cells regulate glucose metabolism through glycolysis is still unknown. Here, we demonstrated the regulatory role of Ras related GTP binding D (RRAGD) in human hepatocellular carcinoma (HCC) cells. PATIENTS OR MATERIALS AND METHODS: Kaplan-Meier's analysis was used to analyze the correlation between RRAGD expression levels and prognosis of HCC patients from the Cancer Genome Atlas database. Two stable RRAGD knockdown HCC cell lines were created using shRNAs to investigate cancer progression and aerobic glycolysis. Western blot and quantitative reverse transcription polymerase chain reaction were performed to detect the expression levels of RRAGD and MYC. RESULTS: RRAGD expression was elevated in HCC patients with poor prognosis. RRAGD knockdown could inhibit the proliferation, invasion and migration of Huh-7 and HepG2 cells. Interestingly, silence RRAGD was able to reduce the glucose uptake, lactate production and extracellular acidification rate of HCC. RRAGD expression level was up-regulated by oncogene MYC in HCC cells. CONCLUSION: This study highlights RRAGD as an important cancer-promoting factor for cancer progression and aerobic glycolysis, and thereby it is a potential therapeutic target for HCC intervention.

Monitoring Lactate Dynamics in Individual Macrophages with a Genetically Encoded Probe.

Lactate, the product of aerobic glycolysis, plays a dual role as fuel and intercellular signal in inflammation, immune evasion, and tumor progression. The production of lactate by macrophages has been associated with their polarization and function. Here we describe imaging protocols to characterize the metabolism of cultured human macrophages using a genetically encoded fluorescent sensor-specific for lactate. By superfusing cultures with increasing lactate concentrations and pharmacological inhibitors, it is possible to estimate the kinetic parameters of monocarboxylate transporter 4 (MCT4) and lactate production. Practical advice is given regarding sensor expression, imaging, and data analysis. The spatiotemporal resolution of this technique is amenable to the study of fast events at the single-cell level in different immune and other cell types.

Protein Kinase CK2 in Cancer Energetics.

Protein kinase CK2 (formerly known as casein kinase 2) is abnormally elevated in many cancers. This may increase tumor aggressiveness through CK2-dependent phosphorylation of key proteins in several signaling pathways. In this work, we have compiled evidence from the literature to suggest that CK2 also modulates a metabolic switch characteristic of cancer cells that enhances resistance to death, due to either drugs or to a microenvironment deficient in oxygen or nutrients. Concurrently, CK2 may help to preserve mitochondrial activity in a PTEN-dependent manner. PTEN, widely recognized as a tumor suppressor, is another CK2 substrate in the PI3K/Akt signaling pathway that promotes cancer viability and aerobic glycolysis. Given that CK2 can regulate Akt as well as two of its main effectors, namely mTORC1 and ß-catenin, we comprehensively describe how CK2 may modulate cancer energetics by regulating expression of key targets and downstream processes, such as HIF-1 and autophagy, respectively. Thus, the specific inhibition of CK2 may lead to a catastrophic death of cancer cells, which could become a feasible therapeutic strategy to beat this devastating disease. In fact, ATP-competitive inhibitors, synthetic peptides and antisense oligonucleotides have been designed as CK2 inhibitors, some of them used in preclinical models of cancer, of which TBB and silmitasertib are widely known. We will finish by discussing a hypothetical scenario in which cancer cells are "addicted" to CK2; i.e., in which many proteins that regulate signaling pathways and metabolism-linked processes are highly dependent on this kinase.

Lactic Acidosis in the Presence of Glucose Diminishes Warburg Effect in Lung Adenocarcinoma Cells.

Lactic acidosis (3 to 40 mM, pH < 6.9) is a condition found in solid tumors because tumor cells have a high rate of glucose consumption and lactate production even in the presence of oxygen; nevertheless, the microenvironment might still provide a sufficient glucose supply. Lactic acidosis has been proposed to shift metabolism from aerobic glycolysis toward oxidative phosphorylation (OXPHOS). We tested if lung tumor cells cultured under lactic acidosis shift their metabolism from glycolysis to OXPHOS by consuming extracellular lactate, increasing growth rate. We analyzed lung adenocarcinoma (A-549, A-427) cell lines and non-transformed fibroblast cells (MRC-5), which were cultured using RPMI-1640 medium initially containing lactate (2 mM) and glucose (10 mM), at pH 7.2 or 6.2 and oxygen tension 21% O2 (normoxia) or 2% O2 (hypoxia). We obtained growth curves, as well as glucose consumption and lactate production rates (measured during exponential growth) for each cell line. HIF-1α (Hypoxia-inducible factor 1 α), CS (citrate synthase) and AMPK (AMP-activated protein kinase) transcript levels were analyzed using RT-qPCR. By flow cytometry, we determined: (a) expression of glucose transporters (GLUT)1 and 4; (b) lactate transporters (MCT)1 and 4; (c) cell cycle profile, and (d) protein levels of HIF-1α, total and phosphorylated AMPK (pAMPK). Mitochondrial functionality was evaluated by measuring O2 consumption in tumor cells using polarography and a Clark-type electrode. Tumor and non-transformed cells used both aerobic glycolysis and OXPHOS for obtaining energy. As of 48 h of culture, lactate levels ranged from (4.5-14 mM), thus forming a lactic environment. Lactic acidosis diminished GLUT1/GLUT4 expression and glucose consumption in A-549, but not in A-427 cells, and induced differential expression of HIF-1α, AMPK, and CS transcripts. A-427 cells increased pAMPK and HIF-1α levels and shifted their metabolism increasing OXPHOS; thus supporting cell growth. Conversely, A-549 cells increased HIF-1α protein levels, but did not activate AMPK and diminished OXPHOS. A-549 cells survived by arresting cells in G1-phase. Our findings show that lactic acidosis diminishes Warburg effect in tumor cells, but this change does not necessarily promote a shift to OXPHOS. Hence, lung adenocarcinomas show a differential metabolic response even when they are under the same microenvironmental conditions.

Emerging roles of aerobic glycolysis in breast cancer.

Altered aerobic glycolysis is a well-recognized characteristic of cancer cell energy metabolism, known as the Warburg effect. Even in the presence of abundant oxygen, a majority of tumor cells produce substantial amounts of energy through a high glycolytic metabolism, and breast cancer (BC) is no exception. Breast cancer continues to be the second leading cause of cancer-associated mortality in women worldwide. However, the precise role of aerobic glycolysis in the development of BC remains elusive. Therefore, the present review attempts to address the implication of key enzymes of the aerobic glycolytic pathway including hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK), glucose transporters (GLUTs), together with related signaling pathways including protein kinase B(PI3K/AKT), mammalian target of rapamycin (mTOR) and adenosine monophosphate-activated protein kinase (AMPK) and transcription factors (c-myc, p53 and HIF-1) in the research of BC. Thus, the review of aerobic glycolysis in BC may evoke novel ideas for the BC treatment.

A common mechanism explains the induction of aerobic fermentation and adaptive antioxidant response in Phaffia rhodozyma.

BACKGROUND: Growth conditions that bring about stress on Phaffia rhodozyma cells encourage the synthesis of astaxanthin, an antioxidant carotenoid, which protects cells against oxidative damage. Using P. rhodozyma cultures performed with and without copper limitation, we examined the kinetics of astaxanthin synthesis along with the expression of asy, the key astaxanthin synthesis gene, as well as aox, which encodes an alternative oxidase protein. RESULTS: Copper deficiency had a detrimental effect on the rates of oxygen consumption and ethanol reassimilation at the diauxic shift. In contrast, copper deficiency prompted alcoholic fermentation under aerobic conditions and had a favorable effect on the astaxanthin content of cells, as well as on aox expression. Both cultures exhibited strong aox expression while consuming ethanol, but particularly when copper was absent. CONCLUSION: We show that the induction of either astaxanthin production, aox expression, or aerobic fermentation exemplifies the crucial role that redox imbalance plays in triggering any of these phenomena. Based on our own results and data from others, we propose a mechanism that rationalizes the central role played by changes of respiratory activity, which lead to redox imbalances, in triggering both the short-term antioxidant response as well as fermentation in yeasts and other cell types.

Neuronal control of astrocytic respiration through a variant of the Crabtree effect.

Aerobic glycolysis is a phenomenon that in the long term contributes to synaptic formation and growth, is reduced by normal aging, and correlates with amyloid beta deposition. Aerobic glycolysis starts within seconds of neural activity and it is not obvious why energetic efficiency should be compromised precisely when energy demand is highest. Using genetically encoded FRET nanosensors and real-time oxygen measurements in culture and in hippocampal slices, we show here that astrocytes respond to physiological extracellular K+ with an acute rise in cytosolic ATP and a parallel inhibition of oxygen consumption, explained by glycolytic stimulation via the Na+-bicarbonate cotransporter NBCe1. This control of mitochondrial respiration via glycolysis modulation is reminiscent of a phenomenon previously described in proliferating cells, known as the Crabtree effect. Fast brain aerobic glycolysis may be interpreted as a strategy whereby neurons manipulate neighboring astrocytes to obtain oxygen, thus maximizing information processing.

Targeting Metabolic Remodeling in Triple Negative Breast Cancer in a Murine Model.

Background: Chemotherapy is the backbone of systemic treatment for triple negative breast cancer (TNBC), which is one of the most relevant breast cancers molecular types due to the ability of tumor cells to develop drug resistance, highlighting the urgent need to design newer and safer drug combinations for treatment. In this context, to overcome tumor cell drug resistance, we employed a novel combinatorial treatment including Doxorubicin, Metformin, and Sodium Oxamate (DoxMetOx). Such pharmacological combination targets indispensable hallmarks of cancer-related to aerobic glycolysis and DNA synthesis. Materials and Methods: Thirty-five female nude mice were transplanted subcutaneously with MDA-MB-231 triple negative human cancer cell line. Once tumors were visible, mice were treated with doxorubicin, metformin, oxamate or all possible pharmacologic combinations. Treatments were administered daily for 15 days and tumors were measured by calipers every day. MicroPET images were taken in three different occasions, basal state, in the middle of the treatment, and at the end of treatment. Western blot analyses, qRT-PCR, flow cytometry, and cytotoxicity assays were performed to elucidate the mechanism of cell death promoted by the drugs in vitro. Results: In this work we assessed the proof of concept of metabolic correction in solid tumors as an effective drug treatment; hence, mice bearing tumors treated with the DoxMetOx therapy showed a complete inhibition of the tumor mass growing in 15 days of treatment depicted by the micro PET images. In vitro studies displayed that the three drugs together act by inhibiting both, mTOR-phosphorylation and expression of LDH-A gene, promoting apoptosis via dependent on the caspase-3 pathway, accompanied by cleavage of PARP. Moreover, induction of autophagy process was observed by the accumulation of LC3-II, a primordial protein implicated in the conformation and elongation of the autophagolysosome. Conclusions: The lack of effective drugs to inhibit TNBC growth is the main cause of therapy failure and tumor relapse. We have showed that targeting crucial molecular pathways in cancer by the combination of Doxorubicin, Metformin, and Oxamate resulted as an efficient and rapid tumor growth inhibitor in a triple negative xenograft model. Our findings are promising for patients diagnosed with TNBC tumors, for which unfortunately there are no reliable drug therapies.

Advances in improving mammalian cells metabolism for recombinant protein production

Background: The production of recombinant proteins for therapeutic use represents a great impact on the biotechnology industry. In this context, established mammalian cell lines, especially CHO cells, have become a standard system for the production of such proteins. Their ability to properly configure and excrete proteins in functional form is an enormous advantage which should be contrasted with their inherent technological limitations. These cell systems exhibit a metabolic behaviour associated with elevated cell proliferation which involves a high consumption of glucose and glutamine, resulting in the rapid depletion of these nutrients in the medium and the accumulation of ammonium and lactate. Both phenomena contribute to the limitation of cell growth, the triggering of apoptotic processes and the loss of quality of the recombinant protein. Results: In this review, the use of alternative substrates and genetic modifications (host cell engineering) are analyzed as tools to overcome those limitations. In general, the results obtained are promising. However, metabolic and physiological phenomena involved in CHO cells are still barely understood. Thus, most of publications are focused on specific modifications rather than giving a systemic perspective. Conclusions: A deeper insight in the integrated understanding of metabolism and cell mechanisms is required in order to define complementary strategies at these two levels, so providing effective means to control nutrients consumption, reduce by-products and increase process productivity.