The Effects of Low-Level Helium–Neon (He–Ne) Laser Irradiation on Lipids and and Fatty Acids, and the Activity of Energetic Metabolism Enzymes and Proteome in the Blastula Stage and Underyearlings of the Atlantic Salmon Salmo salar: A Novel Approach in Salmonid Restoration Procedures in the North

The effect of He–Ne laser irradiation on fishery parameters as well as on biochemical state, including the lipids and fatty acids, the activity of energy metabolism enzymes and the proteome in the blastula stage and in underyearlings of wild Atlantic salmon after irradiation at the cleavage stage/early blastula (considered as the stages when the cell has a high potential for differentiation) was studied. Low mortality rates of eggs were determined during embryogenesis, as well as increased weight gain and lower morality rates of underyearlings in the experimental group. This is confirmed by changes in a number of interrelated indicators of lipid metabolism: a decrease in total lipids content, including diacylglycerols, triacylglycerols, cholesterol esters, and the phospholipids content remained unchanged. The embryos in the blastula stage (experimental group) had higher aerobic capacity and an increase in pentose phosphate pathway activity. The proteome profiles of eggs in the blastula stage were 131 proteins, of which 48 were significantly identified. The major protein was found to be phosvitin. The proteomes of underyearlings were represented by 2018 proteins, of which 49 were unique for the control and 39 for the experimental group. He-–Ne laser irradiation had a strong effect on the contents of histone proteins.

Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

Pentose Phosphate Pathway Regulates Tolerogenic Apoptotic Cell Clearance and Immune Tolerance

The efficient removal of apoptotic cells (ACs), a process termed as efferocytosis, is essential for immune homeostasis. While recent work has established an important interplay between efferocytosis and cellular metabolic changing, underlying mechanisms remain poorly known. Here, we discovered that pentose phosphate pathway (PPP) regulates tolerogenic ACs clearance and immune tolerance. ACs decreased levels of PPP-related genes and metabolites in macrophages. AG1, the agonist of PPP, increased the activity of PPP but greatly reduced macrophage phagocytosis of ACs and enhanced the inflammatory response during efferocytosis. miR-323-5p regulated the expression of PPP-related genes and its levels increased during efferocytosis. miR-323-5p inhibitor greatly promoted levels of PPP-related genes, reduced the macrophage phagocytosis of ACs, and increased inflammatory response during efferocytosis, suggesting that miR-323-5p was essential in regulating PPP activity and ACs clearance in macrophages. Correspondingly, the PPP agonist AG1 exacerbated the lupus-like symptoms in the AC-induced systemic lupus erythematosus (SLE) model. Our study reveals that regulating PPP-dependent metabolic reprogramming is critical for tolerogenic ACs phagocytosis and immune tolerance.

ROS/PI3K/Akt and Wnt/β-catenin signalings activate HIF-1α-induced metabolic reprogramming to impart 5-fluorouracil resistance in colorectal cancer

Background Acquired resistance of 5-fluorouracil (5-FU) remains a clinical challenge in colorectal cancer (CRC), and efforts to develop targeted agents to reduce resistance have not yielded success. Metabolic reprogramming is a key cancer hallmark and confers several tumor phenotypes including chemoresistance. Glucose metabolic reprogramming events of 5-FU resistance in CRC has not been evaluated, and whether abnormal glucose metabolism could impart 5-FU resistance in CRC is also poorly defined. Methods Three separate acquired 5-FU resistance CRC cell line models were generated, and glucose metabolism was assessed by measuring glucose and lactate utilization, RNA and protein expressions of glucose metabolism-related enzymes and changes of intermediate metabolites of glucose metabolite pool. The protein levels of hypoxia inducible factor 1α (HIF-1α) in primary tumors and circulating tumor cells of CRC patients were detected by immunohistochemistry and immunofluorescence. Stable HIF1A knockdown in cell models was established with a lentiviral system. The influence of both HIF1A gene knockdown and pharmacological inhibition on 5-FU resistance in CRC was evaluated in cell models in vivo and in vitro. Results The abnormality of glucose metabolism in 5-FU-resistant CRC were described in detail. The enhanced glycolysis and pentose phosphate pathway in CRC were associated with increased HIF-1α expression. HIF-1α-induced glucose metabolic reprogramming imparted 5-FU resistance in CRC. HIF-1α showed enhanced expression in 5-FU-resistant CRC cell lines and clinical specimens, and increased HIF-1α levels were associated with failure of fluorouracil analog-based chemotherapy in CRC patients and poor survival. Upregulation of HIF-1α in 5-FU-resistant CRC occurred through non-oxygen-dependent mechanisms of reactive oxygen species-mediated activation of PI3K/Akt signaling and aberrant activation of β-catenin in the nucleus. Both HIF-1α gene knock-down and pharmacological inhibition restored the sensitivity of CRC to 5-FU. Conclusions HIF-1α is a potential biomarker for 5-FU-resistant CRC, and targeting HIF-1a in combination with 5-FU may represent an effective therapeutic strategy in 5-FU-resistant CRC.

The POU2F1-ALDOA axis promotes the proliferation and chemoresistance of colon cancer cells by enhancing glycolysis and the pentose phosphate pathway activity

Cancer metabolic reprogramming enhances its malignant behaviors and drug resistance, which is regulated by POU domain transcription factors. This study explored the effect of POU domain class 2 transcription factor 1 (POU2F1) on metabolic reprogramming in colon cancer. The POU2F1 expression was analyzed in GEO dataset, TCGA cohorts and human colon cancer tissues by bioinformatics and immunohistochemistry. The effects of altered POU2F1 expression on proliferation, glucose metabolism and oxaliplatin sensitivity of colon cancer cells were tested. The impacts of POU2F1 on aldolase A (ALDOA) expression and malignant behaviors of colon cancer cells were examined. We found that up-regulated POU2F1 expression was associated with worse prognosis and oxaliplatin resistance in colon cancer. POU2F1 enhanced the proliferation, aerobic glycolysis and the pentose phosphate pathway (PPP) activity, but reduced oxidative stress and apoptosis in colon cancer cells, dependent on up-regulating ALDOA expression. Mechanistically, POU2F1 directly bound to the ALDOA promoter to enhance the ALDOA promoter activity in colon cancer cells. Moreover, activation of the POU2F1-ALDOA axis decreased the sensitivity to oxaliplatin in colon cancer cells. These data indicate that the POU2F1-ALDOA axis promotes the progression and oxaliplatin resistance by enhancing metabolic reprogramming in colon cancer. Our findings suggest that the POU2F1-ALDOA axis may be new therapeutic targets to overcome oxaliplatin resistance in colon cancer.

Enhanced glucose metabolism through activation of HIF-1α covers the energy demand in a rat embryonic heart primordium after heartbeat initiation

The initiation of heartbeat is an essential step in cardiogenesis in the heart primordium, but it remains unclear how intracellular metabolism responds to increased energy demands after heartbeat initiation. In this study, embryos in Wistar rats at embryonic day 10, at which heartbeat begins in rats, were divided into two groups by the heart primordium before and after heartbeat initiation and their metabolic characteristics were assessed. Metabolome analysis revealed that increased levels of ATP, a main product of glucose catabolism, and reduced glutathione, a by-product of the pentose phosphate pathway, were the major determinants in the heart primordium after heartbeat initiation. Glycolytic capacity and ATP synthesis-linked mitochondrial respiration were significantly increased, but subunits in complexes of mitochondrial oxidative phosphorylation were not upregulated in the heart primordium after heartbeat initiation. Hypoxia-inducible factor (HIF)-1α was activated and a glucose transporter and rate-limiting enzymes of the glycolytic and pentose phosphate pathways, which are HIF-1α-downstream targets, were upregulated in the heart primordium after heartbeat initiation. These results suggest that the HIF-1α-mediated enhancement of glycolysis with activation of the pentose phosphate pathway, potentially leading to antioxidant defense and nucleotide biosynthesis, covers the increased energy demand in the beating and developing heart primordium.

G6PD functions as a metabolic checkpoint to regulate granzyme B expression in tumor-specific cytotoxic T lymphocytes

BackgroundGranzyme B is a key effector of cytotoxic T lymphocytes (CTLs), and its expression level positively correlates with the response of patients with mesothelioma to immune checkpoint inhibitor immunotherapy. Whether metabolic pathways regulate Gzmb expression in CTLs is incompletely understood.MethodsA tumor-specific CTL and tumor coculture model and a tumor-bearing mouse model were used to determine the role of glucose-6-phosphate dehydrogenase (G6PD) in CTL function and tumor immune evasion. A link between granzyme B expression and patient survival was analyzed in human patients with epithelioid mesothelioma.ResultsMesothelioma cells alone are sufficient to activate tumor-specific CTLs and to enhance aerobic glycolysis to induce a PD-1hi Gzmblo CTL phenotype. However, inhibition of lactate dehydrogenase A, the key enzyme of the aerobic glycolysis pathway, has no significant effect on tumor-induced CTL activation. Tumor cells induce H3K9me3 deposition at the promoter of G6pd, the gene that encodes the rate-limiting enzyme G6PD in the pentose phosphate pathway, to downregulate G6pd expression in tumor-specific CTLs. G6PD activation increases acetyl-coenzyme A (CoA) production to increase H3K9ac deposition at the Gzmb promoter and to increase Gzmb expression in tumor-specific CTLs converting them from a Gzmblo to a Gzmbhi phenotype, thus increasing CTL tumor lytic activity. Activation of G6PD increases Gzmb+ tumor-specific CTLs and suppresses tumor growth in tumor-bearing mice. Consistent with these findings, GZMB expression level was found to correlate with increased survival in patients with epithelioid mesothelioma.ConclusionG6PD is a metabolic checkpoint in tumor-activated CTLs. The H3K9me3/G6PD/acetyl-CoA/H3K9ac/Gzmb pathway is particularly important in CTL activation and immune evasion in epithelioid mesothelioma.

Sod1 integrates oxygen availability to redox regulate NADPH production and the thiol redoxome

Cu/Zn superoxide dismutase (Sod1) is a highly conserved and abundant antioxidant enzyme that detoxifies superoxide (O2•−) by catalyzing its conversion to dioxygen (O2) and hydrogen peroxide (H2O2). Using Saccharomyces cerevisiae and mammalian cells, we discovered that a major aspect of the antioxidant function of Sod1 is to integrate O2 availability to promote NADPH production. The mechanism involves Sod1-derived H2O2 oxidatively inactivating the glycolytic enzyme, GAPDH, which in turn reroutes carbohydrate flux to the oxidative phase of the pentose phosphate pathway (oxPPP) to generate NADPH. The aerobic oxidation of GAPDH is dependent on and rate-limited by Sod1. Thus, Sod1 senses O2 via O2•− to balance glycolytic and oxPPP flux, through control of GAPDH activity, for adaptation to life in air. Importantly, this mechanism for Sod1 antioxidant activity requires the bulk of cellular Sod1, unlike for its role in protection against O2•− toxicity, which only requires <1% of total Sod1. Using mass spectrometry, we identified proteome-wide targets of Sod1-dependent redox signaling, including numerous metabolic enzymes. Altogether, Sod1-derived H2O2 is important for antioxidant defense and a master regulator of metabolism and the thiol redoxome.

Sustained Energy Deficit Following Perinatal Asphyxia: A Shift towards the Fructose-2,6-bisphosphatase (TIGAR)-Dependent Pentose Phosphate Pathway and Postnatal Development

Labor and delivery entail a complex and sequential metabolic and physiologic cascade, culminating in most circumstances in successful childbirth, although delivery can be a risky episode if oxygen supply is interrupted, resulting in perinatal asphyxia (PA). PA causes an energy failure, leading to cell dysfunction and death if re-oxygenation is not promptly restored. PA is associated with long-term effects, challenging the ability of the brain to cope with stressors occurring along with life. We review here relevant targets responsible for metabolic cascades linked to neurodevelopmental impairments, that we have identified with a model of global PA in rats. Severe PA induces a sustained effect on redox homeostasis, increasing oxidative stress, decreasing metabolic and tissue antioxidant capacity in vulnerable brain regions, which remains weeks after the insult. Catalase activity is decreased in mesencephalon and hippocampus from PA-exposed (AS), compared to control neonates (CS), in parallel with increased cleaved caspase-3 levels, associated with decreased glutathione reductase and glutathione peroxidase activity, a shift towards the TIGAR-dependent pentose phosphate pathway, and delayed calpain-dependent cell death. The brain damage continues long after the re-oxygenation period, extending for weeks after PA, affecting neurons and glial cells, including myelination in grey and white matter. The resulting vulnerability was investigated with organotypic cultures built from AS and CS rat newborns, showing that substantia nigra TH-dopamine-positive cells from AS were more vulnerable to 1 mM of H2O2 than those from CS animals. Several therapeutic strategies are discussed, including hypothermia; N-acetylcysteine; memantine; nicotinamide, and intranasally administered mesenchymal stem cell secretomes, promising clinical translation.