Aerobic Glycolysis Metabolism Research Articles

Antifibrotic effects of vitamin D3 on human lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a progressive and fatal interstitial lung disease. Up to now, no treatment can stop the progression of IPF. Vitamin D3 (VD) reduces experimental lung fibrosis in murine models and depletion of vitamin D3 might be associated with the reduced survival of patients with IPF. In this context, we determined if VD can prevent the pro-fibrotic functions of human lung fibroblasts (HLFs) isolated from patients with IPF. IPF and control HLFs were derived from surgical lung biopsies collected from patients with IPF or with primary lung cancer, respectively. VD (3-100 nM) markedly reduced the basal and PDGF-induced proliferation of HLFs. VD also altered cell cycle by increasing the percentage of IPF HLFs arrested in the G0/G1 phase, and by downregulating the expression of various cell cycle regulatory proteins. In addition, VD barely prevented the TGF-β1-induced differentiation in HLFs. At 100 nM, VD slightly reduced the expression of the pro-fibrotic marker α-smooth muscle actin, and had no effect on fibronectin and collagen-1 expression. In contrast, 100 nM VD strongly inhibited the aerobic glycolytic metabolism induced by TGF- β1. Finally, VD reduced both the secretion of lactate, the levels of lactate deshydrogenase mRNA and the activity of intracellular LDH in IPF HLFs. In conclusion, our study shows that VD reduced pro-fibrotic functions of HLFs. These findings suggest that it might be interesting to assess the potential clinical benefits of vitamin D supplementation in patients with IPF, especially on lung function decline.

Shikonin reverses cancer-associated fibroblast-induced gemcitabine resistance in pancreatic cancer cells by suppressing monocarboxylate transporter 4-mediated reverse Warburg effect

BackgroundGemcitabine is a first-line chemotherapeutic agent for pancreatic cancer (PC); however, most patients who receive adjuvant gemcitabine rapidly develop resistance and recurrence. Cancer-associated fibroblasts (CAFs) are a crucial component of the tumor stroma that contribute to gemcitabine-resistance. There is thus an urgent need to find a novel therapeutic strategy to improve the efficacy of gemcitabine in PC cells under CAF-stimulation. PurposeTo investigate if shikonin potentiates the therapeutic effects of gemcitabine in PC cells with CAF-induced drug resistance. MethodsPC cell-stimulated fibroblasts or primary CAFs derived from PC tissue were co-cultured with PC cells to evaluate the ability of shikonin to improve the chemotherapeutic effects of gemcitabine in vitro and in vivo. Glucose uptake assay, ATP content analysis, lactate measurement, real-time PCR, immunofluorescence staining, western blot, and plasmid transfection were used to investigate the underlying mechanism. ResultsCAFs were innately resistant to gemcitabine, but shikonin suppressed the PC cell-induced transactivation and proliferation of CAFs, reversed CAF-induced resistance, and restored the therapeutic efficacy of gemcitabine in the co-culture system. In addition, CAFs underwent a reverse Warburg effect when co-cultured with PC cells, represented by enhanced aerobic glycolytic metabolism, while shikonin reduced aerobic glycolysis in CAFs by reducing their glucose uptake, ATP concentration, lactate production and secretion, and glycolytic protein expression. Regarding the mechanism underlying these sensitizing effects, shikonin suppressed monocarboxylate transporter 4 (MCT4) expression and cellular membrane translocation to inhibit aerobic glycolysis in CAFs. Overexpression of MCT4 accordingly reversed the inhibitory effects of shikonin on PC cell-induced transactivation and aerobic glycolysis in CAFs, and reduced its sensitizing effects. Furthermore, shikonin promoted the effects of gemcitabine in reducing the growth of tumors derived from PC cells and CAF co-inoculation in BALB/C mice, with no significant systemic toxicity. ConclusionThese results indicate that shikonin reduced MCT4 expression and activation, resulting in inhibition of aerobic glycolysis in CAFs and overcoming CAF-induced gemcitabine resistance in PC. Shikonin is a promising chemosensitizing phytochemical agent when used in combination with gemcitabine for PC treatment. The results suggest that disrupting the metabolic coupling between cancer cells and stromal cells might provide an attractive strategy for improving gemcitabine efficacy.

Disruption of phosphofructokinase activity and aerobic glycolysis in human bronchial epithelial cells by atmospheric ultrafine particulate matter

Exposure to ambient ultrafine particulate matter (UPM) causes respiratory disorders; however, the underlying molecular mechanisms remain unclear. In this study, we synthesized simulated UPM (sUPM) with controlled physicochemical properties using the spark-discharge method. Subsequently, we investigated the biological effects of sUPM using BEAS-2B human bronchial epithelial cells (HBECs) and a mouse intratracheal instillation model. High throughput RNA-sequencing and bioinformatics analyses revealed that dysregulation of the glycolytic metabolism is involved in the inhibited proliferation and survival of HBECs by sUPM treatment. Furthermore, signaling pathway and enzymatic analyses showed that the treatment of BEAS-2B cells with sUPM induces the inactivation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB, also known as AKT), resulting in the downregulation of phosphofructokinase 2 (PFK2) S483 phosphorylation, PFK enzyme activity, and aerobic glycolysis in HBECs in an oxidative stress-independent manner. Additionally, intratracheal instillation of sUPM reduced the phosphorylation of ERK, AKT, and PFK2, decreased proliferation, and increased the apoptosis of bronchial epithelial cells in mice. The findings of this study imply that UPM induces pulmonary toxicity by disrupting aerobic glycolytic metabolism in lung epithelial cells, which can provide novel insights into the toxicity mechanisms of UPM and strategies to prevent their toxic effects.

Capturing Transitional Pluripotency through Proline Metabolism.

In this paper, we summarize the current knowledge of the role of proline metabolism in the control of the identity of Embryonic Stem Cells (ESCs). An imbalance in proline metabolism shifts mouse ESCs toward a stable naïve-to-primed intermediate state of pluripotency. Proline-induced cells (PiCs), also named primitive ectoderm-like cells (EPLs), are phenotypically metastable, a trait linked to a rapid and reversible relocalization of E-cadherin from the plasma membrane to intracellular membrane compartments. The ESC-to-PiC transition relies on the activation of Erk and Tgfβ/Activin signaling pathways and is associated with extensive remodeling of the transcriptome, metabolome and epigenome. PiCs maintain several properties of naïve pluripotency (teratoma formation, blastocyst colonization and 3D gastruloid development) and acquire a few traits of primed cells (flat-shaped colony morphology, aerobic glycolysis metabolism and competence for primordial germ cell fate). Overall, the molecular and phenotypic features of PiCs resemble those of an early-primed state of pluripotency, providing a robust model to study the role of metabolic perturbations in pluripotency and cell fate decisions.

Nonylphenol inhibited HIF-1alpha regulated aerobic glycolysis and induced ROS mediated apoptosis in rat Sertoli cells

Nonylphenol (NP) is an endocrine disruptor with reproductive toxicity, which can induce apoptosis of Sertoli cells (SCs). SCs have a high aerobic glycolytic flux to ensure sufficient lactate for germ cells as central energy metabolite, and hypoxia-inducible factors 1alpha (HIF-1α) is a major regulator of glycolysis. This study aimed to investigate whether NP can alter HIF-1α-regulated aerobic glycolysis metabolism and thus induce apoptosis in rat SCs. The results revealed that cell viability, intracellular and extracellular lactate levels, the expression of Hk2, Ldha and Mct4, and the protein levels of HIF-1α, HK2, LDHA and MCT4 were decreased significantly when rat SCs exposed to 20 and 30 μM NP for 24 h. Compared with the 30 μM NP group, the protein levels of HIF-1α, HK2 and LDHA, the expression of Hk2 and Ldha and intracellular lactate levels were increased in 30 μM NP and 125 μM cobalt chloride (CoCl2, inhibitor of HIF-1α proteasome-mediated degradation) co-treated group. Furthermore, the elevation of reactive oxygen species (ROS) and apoptosis induced by 30 μM NP were also reversed. In summary, exposure to NP inhibited the ability of SCs to produce and secrete lactate. Meanwhile, NP exposure could lead to a decrease in HIF-1α thereby inhibiting aerobic glycolysis in rat SCs, disrupting intracellular homeostasis and further inducing ROS-mediated apoptosis. This research is the first to explore the NP toxicity on SCs function with respect to nutrition support to germ cells, and provide new evidence on the inhibition of aerobic glycolysis inducing ROS-mediated apoptosis in SCs.

Abstract 5624: PD-L1 mediates IFNγ-regulation of glucose but not of tryptophan metabolism in clear cell renal cell carcinoma

Abstract The immune checkpoint programmed death-ligand 1 (PD-L1) is expressed on the cell surface of tumor cells and is key for maintaining an immunosuppressive microenvironment through its interaction with the programmed cell death 1 (PD-1). Clear cell renal cell carcinoma (ccRCC) is a highly immunogenic cancer characterized by an aberrant aerobic glycolytic metabolism and is known to overexpress PD-L1. Multiple immunotherapies have been approved for the treatment of ccRCC, including cytokines and immune checkpoint inhibitors. Recently the intrinsic role of PD-L1 and interferon gamma (IFNγ) signaling have been studied in several types of tumor cells, yet it remains unclear how they affect the metabolism and signaling pathways of ccRCC. Using metabolomics, metabolic assays and RNAseq, we showed that IFNγ enhanced aerobic glycolysis and tryptophan metabolism in ccRCC cells in vitro and induced the transcriptional expression of signaling pathways related to inflammation, cell proliferation and cellular energetics. These metabolic and transcriptional effects were partially reversed following PD-L1 transient silencing. Aerobic glycolysis as well as signaling pathways related to inflammation were not induced by IFNγ when PD-L1 was silenced, however tryptophan metabolism and activation of Jak2 and STAT1 were maintained. Our data demonstrate that PD-L1 expression is required to mediate some of IFNγ’s effect in ccRCC cells and highlight the importance of PD-L1 signaling in regulating the metabolism of ccRCC cells in response to inflammatory signals. Citation Format: Mamatha Garige, Susmita Ghosh, ALexis Norris, Guangyuan Li, Carole Sourbier. PD-L1 mediates IFNγ-regulation of glucose but not of tryptophan metabolism in clear cell renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5624.

PD-L1 Mediates IFNγ-Regulation of Glucose but Not of Tryptophan Metabolism in Clear Cell Renal Cell Carcinoma

The immune checkpoint programmed death-ligand 1 (PD-L1) is expressed on the cell surface of tumor cells and is key for maintaining an immunosuppressive microenvironment through its interaction with the programmed death 1 (PD-1). Clear cell renal cell carcinoma (ccRCC) is a highly immunogenic cancer characterized by an aberrant aerobic glycolytic metabolism and is known to overexpress PD-L1. Multiple immunotherapies have been approved for the treatment of ccRCC, including cytokines and immune checkpoint inhibitors. Recently the intrinsic role of PD-L1 and interferon gamma (IFNγ) signaling have been studied in several types of tumor cells, yet it remains unclear how they affect the metabolism and signaling pathways of ccRCC. Using metabolomics, metabolic assays and RNAseq, we showed that IFNγ enhanced aerobic glycolysis and tryptophan metabolism in ccRCC cells in vitro and induced the transcriptional expression of signaling pathways related to inflammation, cell proliferation and cellular energetics. These metabolic and transcriptional effects were partially reversed following transient PD-L1 silencing. Aerobic glycolysis, as well as signaling pathways related to inflammation, were not induced by IFNγ when PD-L1 was silenced, however, tryptophan metabolism and activation of Jak2 and STAT1 were maintained. Our data demonstrate that PD-L1 expression is required to mediate some of IFNγ’s effect in ccRCC cells and highlight the importance of PD-L1 signaling in regulating the metabolism of ccRCC cells in response to inflammatory signals.

Focal adhesion kinase-related non-kinase ameliorates liver fibrosis by inhibiting aerobic glycolysis via the FAK/Ras/c-myc/ENO1 pathway.

BACKGROUNDHepatic stellate cell (HSC) hyperactivation is a central link in liver fibrosis development. HSCs perform aerobic glycolysis to provide energy for their activation. Focal adhesion kinase (FAK) promotes aerobic glycolysis in cancer cells or fibroblasts, while FAK-related non-kinase (FRNK) inhibits FAK phosphorylation and biological functions.AIMTo elucidate the effect of FRNK on liver fibrosis at the level of aerobic glycolytic metabolism in HSCs.METHODSMouse liver fibrosis models were established by administering CCl4, and the effect of FRNK on the degree of liver fibrosis in the model was evaluated. Transforming growth factor-β1 was used to activate LX-2 cells. Tyrosine phosphorylation at position 397 (pY397-FAK) was detected to identify activated FAK, and the expression of the glycolysis-related proteins monocarboxylate transporter 1 (MCT-1) and enolase1 (ENO1) was assessed. Bioinformatics analysis was performed to predict putative binding sites for c-myc in the ENO1 promoter region, which were validated with chromatin immunoprecipitation (ChIP) and dual-luciferase reporter assays.RESULTSThe pY397-FAK level was increased in human fibrotic liver tissue. FRNK knockout promoted liver fibrosis in mouse models. It also increased the activation, migration, proliferation and aerobic glycolysis of primary hepatic stellate cells (pHSCs) but inhibited pHSC apoptosis. Nevertheless, opposite trends for these phenomena were observed after exogenous FRNK treatment in LX-2 cells. Mechanistically, the FAK/Ras/c-myc/ENO1 pathway promoted aerobic glycolysis, which was inhibited by exogenous FRNK.CONCLUSIONFRNK inhibits aerobic glycolysis in HSCs by inhibiting the FAK/Ras/c-myc/ENO1 pathway, thereby improving liver fibrosis. FRNK might be a potential target for liver fibrosis treatment.

Identification and isolation of slow-cycling glioma stem cells.

Cancer stem cells are defined as low-abundance, quiescent cells and are considered a major cellular source of tumor recurrence following therapy, which identifies these cells as important therapeutic targets for difficult-to-treat cancers, including high-grade gliomas. By contrast to the highly proliferative bulk tumor cells, glioma stem cells (GSC) are slow-cycling, and therefore less sensitive to DNA damaging cytotoxic drugs. GSC are also less reliant on aerobic glycolytic metabolism, leading to inadequate clearing of GSC by chemotherapy and radiotherapy. The definition of GSC is based on the expression of specific stem cell protein markers. This method of GSC isolation is successful in isolating cell populations that can reliably recapitulate the tumor. However, cell populations that lack stem marker expression may also be capable of tumor recapitulation. Therefore, robust, reproducible methods for isolating GSC are required to identify and isolate cells with stem cell characteristics. Here, we provide a comprehensive and reproducible protocol for the isolation of slow-cycling GSC. Using this method, GSC isolated retain key characteristics of the cells in situ, including expression of genes associated with cell quiescence and invasive potential, compared to non-quiescent cell populations. Thus, isolation of GSC gated on cell proliferation offers a reliable alternative method for in vitro GSC identification, that adequately mirrors the physiological properties of GSC seen in vivo.

Neural is Fundamental: Neural Stemness as the Ground State of Cell Tumorigenicity and Differentiation Potential.

Tumorigenic cells are similar to neural stem cells or embryonic neural cells in regulatory networks, tumorigenicity and pluripotent differentiation potential. By integrating the evidence from developmental biology, tumor biology and evolution, I will make a detailed discussion on the observations and propose that neural stemness underlies two coupled cell properties, tumorigenicity and pluripotent differentiation potential. Neural stemness property of tumorigenic cells can hopefully integrate different observations/concepts underlying tumorigenesis. Neural stem cells and tumorigenic cells share regulatory networks; both exhibit neural stemness, tumorigenicity and pluripotent differentiation potential; both depend on expression or activation of ancestral genes; both rely primarily on aerobic glycolytic metabolism; both can differentiate into various cells/tissues that are derived from three germ layers, leading to tumor formation resembling severely disorganized or more degenerated process of embryonic tissue differentiation; both are enriched in long genes with more splice variants that provide more plastic scaffolds for cell differentiation, etc. Neural regulatory networks, which include higher levels of basic machineries of cell physiological functions and developmental programs, work concertedly to define a basic state with fast cell cycle and proliferation. This is predestined by the evolutionary advantage of neural state, the ground or initial state for multicellularity with adaptation to an ancient environment. Tumorigenesis might represent a process of restoration of neural ground state, thereby restoring a state with fast proliferation and pluripotent differentiation potential in somatic cells. Tumorigenesis and pluripotent differentiation potential might be better understood from understanding neural stemness, and cancer therapy should benefit more from targeting neural stemness.

Distinct metabolic transcriptional patterns and Warburg effect in porcine model of ischaemic cardiomyopathy

Abstract Background Despite improvements in therapy for heart failure, the prognosis remains poor. Energetic imbalances related to mitochondrial dysfunction, impaired oxidative phosphorylation and oxidative damage have been implicated in the pathogenesis or worsening of heart failure. Improved understanding of the metabolic alterations of the heart may provide new biomarkers or therapeutic targets. Purpose It has been speculated that aerobic glycolysis, known as the Warburg effect, may be advantageous for cell survival whereby there is the opportunity for simultaneous energy production and production of intermediary metabolites crucial for anabolic pathways that sustain cell division and maintenance, and protein synthesis (1,2). In this study, we studied a preclinical model of ischaemic cardiomyopathy to investigate transcriptional patterns in two defined areas; peri-infarct and remote locations. Specifically, we investigated if aerobic glycolysis may be present in this setting. Methods 18 female Yorkshire pigs were studied. 10 animals underwent balloon catheter myocardial infarction followed by termination at 4 weeks. A group of 8 healthy animals served as controls. All animals underwent cardiac MRI prior to termination confirming ventricular impairment and remodelling in the chronic animals (see Table 1). Gene expression profiles of glucose mobilisation and glucose metabolic markers (GLUT1, GLUT4, ANK2, GAPDH and LDHA), oxidative phosphorylation and mitochondrial function (ND1, TFAM, and PGC1-alpha), key markers of insulin resistance (AKT1, AS160) and a hypoxia marker (HIF1-alpha) were measured. Results Our results indicate the presence of two distinct metabolic profiles at the gene expression level, differing from control tissue but more interestingly with striking differences regionally. In the remote region compared to controls, we demonstrate a picture suggestive of increased glycolysis with significant transcriptional upregulation of GLUT1 and GLUT4 glucose transporters (p=0.01 and p=0.02 respectively), alongside a significant reduction in PGC1-alpha (p=0.04) with maintained transcriptional levels of LDHA and HIF1-alpha. Combined, these results are suggestive of reduced oxidative phosphorylation alongside compensated, or early stage, aerobic glycolytic metabolism in this region. In contrast, in peri-infarct tissue versus controls, we demonstrate a reduction in glycolysis (GLUT1 p=0.09, GLUT4 p=0.01 with a dramatic increase in GLUT1/GLUT4 ratio of 6.7 times) and reduced markers of mitochondrial function indicative of insulin-resistance with evidence of a fetal-pattern metabolic profile (see Table 2 for detailed results). Conclusions This study demonstrates distinct transcriptional profiles within hearts with ischaemic cardiomyopathy and for the first time suggests that the Warburg effect may be present in remote myocardium in this setting supporting the need for further work in this area. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): This research work was supported by grants awarded to Professor Ascione: the British Heart Foundation (BHF) (BHF IG/14/2/30991, BHF RM/13/2/30158), and the Medical Research Council (MRC) (MRC MR/L012723/1). Table 1. CMR parameters for animals studiedTable 2. Transcriptional signals

MiR-27a is a master regulator of metabolic reprogramming and chemoresistance in colorectal cancer

BackgroundMetabolic reprogramming towards aerobic glycolysis in cancer supports unrestricted cell proliferation, survival and chemoresistance. The molecular bases of these processes are still undefined. Recent reports suggest crucial roles for microRNAs. Here, we provide new evidence of the implication of miR-27a in modulating colorectal cancer (CRC) metabolism and chemoresistance.MethodsA survey of miR-27a expression profile in TCGA-COAD dataset revealed that miR-27a-overexpressing CRCs are enriched in gene signatures of mitochondrial dysfunction, deregulated oxidative phosphorylation, mTOR activation and reduced chemosensitivity. The same pathways were analysed in cell lines in which we modified miR-27a levels. The response to chemotherapy was investigated in an independent cohort and cell lines.ResultsmiR-27a upregulation in vitro associated with impaired oxidative phosphorylation, overall mitochondrial activities and slight influence on glycolysis. miR-27a hampered AMPK, enhanced mTOR signalling and acted in concert with oncogenes and tumour cell metabolic regulators to force an aerobic glycolytic metabolism supporting biomass production, unrestricted growth and chemoresistance. This latter association was confirmed in our cohort of patients and cell lines.ConclusionsWe disclose an unprecedented role for miR-27a as a master regulator of cancer metabolism reprogramming that impinges on CRC response to chemotherapy, underscoring its theragnostic properties.

Immunometabolism is a key factor for the persistent spontaneous elite control of HIV-1 infection.

BackgroundApproximately 25% of elite controllers (ECs) lose their virological control by mechanisms that are only partially known. Recently, immunovirological and proteomic factors have been associated to the loss of spontaneous control. Our aim was to perform a metabolomic approach to identify the underlying mechanistic pathways and potential biomarkers associated with this loss of control.MethodsPlasma samples from EC who spontaneously lost virological control (Transient Controllers, TC, n = 8), at two and one year before the loss of control, were compared with a control group of EC who persistently maintained virological control during the same follow-up period (Persistent Controllers, PC, n = 8). The determination of metabolites and plasma lipids was performed by GC-qTOF and LC-qTOF using targeted and untargeted approaches. Metabolite levels were associated with the polyfunctionality of HIV-specific CD8+T-cell response.FindingsOur data suggest that, before the loss of control, TCs showed a specific circulating metabolomic profile characterized by aerobic glycolytic metabolism, deregulated mitochondrial function, oxidative stress and increased immunological activation. In addition, CD8+ T-cell polyfunctionality was strongly associated with metabolite levels. Finally, valine was the main differentiating factor between TCs and PCs.InterpretationAll these metabolomic differences should be considered not only as potential biomarkers but also as therapeutic targets in HIV infection.FundThis work was supported by grants from Fondo de Investigación Sanitaria, Instituto de Salud Carlos III, Fondos FEDER; Red de Investigación en Sida, Gilead Fellowship program, Spanish Ministry of Education and Spanish Ministry of Economy and Competitiveness.

MTOR Regulation of Glycolytic Metabolism in T Cells.

T cell activation, differentiation and effector function is intrinsically linked to the regulation of metabolic pathways. Evidence has shown that inflammatory T cell responses are dependent upon the adoption of aerobic glycolytic metabolism. Furthermore, activation and regulation of the mechanistic target of rapamycin signaling pathway serves a key determinant of T cell metabolism, with subsequent effects on T cell effector responses. In this mini-review, we discuss the mechanisms underpinning the function of the Warburg effect in T cell responses and the role of mTOR in these processes.

Lactate and Immunosuppression in Sepsis.

ABSTRACTSerum lactate levels are traditionally interpreted as a marker of tissue hypoxia and often used clinically as an indicator of severity and outcome of sepsis/septic shock. Interestingly, recent studies involving the effects of tumor-derived lactate suggest that lactate itself may have an immunosuppressive effect in its local environment. This finding adds to the recent advances in immunometabolism that shed light on the importance of metabolism and metabolic intermediates in the regulation of innate immune and inflammatory responses in sepsis. In this article, we summarize recent studies, showing that the activation of immune cells requires aerobic glycolytic metabolism and that lactate produced by aerobic glycolysis may play an immunosuppressive role in sepsis.

Doxorubicin Induces Inflammatory Modulation and Metabolic Dysregulation in Diabetic Skeletal Muscle

Anti-cancer agent doxorubicin (DOX) has been demonstrated to worsen insulin signaling, engender muscle atrophy, trigger pro-inflammation, and induce a shift to anaerobic glycolytic metabolism in skeletal muscle. The myotoxicity of DOX in diabetic skeletal muscle remains largely unclear. This study examined the effects of DOX on insulin signaling, muscle atrophy, pro-/anti-inflammatory microenvironment, and glycolysis metabolic regulation in skeletal muscle of db/db diabetic and db/+ non-diabetic mice. Non-diabetic db/+ mice and diabetic db/db mice were randomly assigned to the following groups: db/+CON, db/+DOX, db/dbCON, and db/dbDOX. Mice in db/+DOX and db/dbDOX groups were intraperitoneally injected with DOX at a dose of 15 mg per kg body weight whereas mice in db/+CON and db/dbCON groups were injected with the same volume of saline instead of DOX. Gastrocnemius was immediately harvested, weighed, washed with cold phosphate buffered saline, frozen in liquid nitrogen, and stored at −80°C for later analysis. The effects of DOX on diabetic muscle were neither seen in insulin signaling markers (Glut4, pIRS1Ser636∕639, and pAktSer473) nor muscle atrophy markers (muscle mass, MuRF1 and MAFbx). However, DOX exposure resulted in enhancement of pro-inflammatory favoring microenvironment (as indicated by TNF-α, HIFα and pNFκBp65) accompanied by diminution of anti-inflammatory favoring microenvironment (as indicated by IL15, PGC1α and pAMPKβ1Ser108). Metabolism of diabetic muscle was shifted to anaerobic glycolysis after DOX exposure as demonstrated by our analyses of PDK4, LDH and pACCSer79. Our results demonstrated that there might be a link between inflammatory modulation and the dysregulation of aerobic glycolytic metabolism in DOX-injured diabetic skeletal muscle. These findings help to understand the pathogenesis of DOX-induced myotoxicity in diabetic muscle.

Protein Sialylation Regulates a Gene Expression Signature that Promotes Breast Cancer Cell Pathogenicity.

Many mechanisms have been proposed for how heightened aerobic glycolytic metabolism fuels cancer pathogenicity, but there are still many unexplored pathways. Here, we have performed metabolomic profiling to map glucose incorporation into metabolic pathways upon transformation of mammary epithelial cells by 11 commonly mutated human oncogenes. We show that transformation of mammary epithelial cells by oncogenic stimuli commonly shunts glucose-derived carbons into synthesis of sialic acid, a hexosamine pathway metabolite that is converted to CMP-sialic acid by cytidine monophosphate N-acetylneuraminic acid synthase (CMAS) as a precursor to glycoprotein and glycolipid sialylation. We show that CMAS knockdown leads to elevations in intracellular sialic acid levels, a depletion of cellular sialylation, and alterations in the expression of many cancer-relevant genes to impair breast cancer pathogenicity. Our study reveals the heretofore unrecognized role of sialic acid metabolism and protein sialylation in regulating the expression of genes that maintain breast cancer pathogenicity.

A Whole-Genome RNA Interference Screen for Human Cell Factors Affecting Myxoma Virus Replication

Myxoma virus (MYXV) provides an important model for investigating host-pathogen interactions. Recent studies have also highlighted how mutations in transformed human cells can expand the host range of this rabbit virus. Although virus growth depends upon interactions between virus and host proteins, the nature of these interactions is poorly understood. To address this matter, we performed small interfering RNA (siRNA) screens for genes affecting MYXV growth in human MDA-MB-231 cells. By using siRNAs targeting the whole human genome (21,585 genes), a subset of human phosphatases and kinases (986 genes), and also a custom siRNA library targeting selected statistically significant genes ("hits") and nonsignificant genes ("nonhits") of the whole human genome screens (88 genes), we identified 711 siRNA pools that promoted MYXV growth and 333 that were inhibitory. Another 32 siRNA pools (mostly targeting the proteasome) were toxic. The overall overlap in the results was about 25% for the hits and 75% for the nonhits. These pro- and antiviral genes can be clustered into pathways and related groups, including well-established inflammatory and mitogen-activated protein kinase pathways, as well as clusters relating to β-catenin and the Wnt signaling cascade, the cell cycle, and cellular metabolism. The validity of a subset of these hits was independently confirmed. For example, treating cells with siRNAs that might stabilize cells in G(1), or inhibit passage into S phase, stimulated MYXV growth, and these effects were reproduced by trapping cells at the G(1)/S boundary with an inhibitor of cyclin-dependent kinases 4/6. By using 2-deoxy-D-glucose and plasmids carrying the gene for phosphofructokinase, we also confirmed that infection is favored by aerobic glycolytic metabolism. These studies provide insights into how the growth state and structure of cells affect MYXV growth and how these factors might be manipulated to advantage in oncolytic virus therapy.

Right-To-Left Ventricular Differences in the Expression of Mitochondrial Hexokinase and Phosphorylation of Akt

Background/Aims: Hexokinase (HK) is a key glycolytic enzyme which promotes the maintenance of glucose homeostasis in cardiomyocytes. HK1 isoform is predominantly bound to the outer mitochondrial membrane and highly supports oxidative phosphorylation by increasing the availability of ADP for complex V of the respiratory chain. HK2 isoform is under physiological conditions predominantly localized in the cytosol and upon stimulation of PI3K/ Akt pathway associates with mitochondria and thus can prevent apoptosis. The purpose of this study was to investigate expression and subcellular localization of both HK isoforms in left (LV) and right (RV) heart ventricles of adult male Wistar rats. Methods: Real-Time RT-PCR, Western blotting, and quantitative immunofluorescence microscopy were used. Results: Our results showed a significantly higher expression of both HK1 and HK2 at mRNA and protein levels in the RV compared to the LV. These findings were corroborated by immunofluorescence staining which revealed substantially higher fluorescence signals of both HKs in the RV than in the LV. The ratios of phospho-Ser473-Akt/non-phospho-Akt and phospho-Thr308-Akt/non-phospho-Akt were also markedly higher in the RV than in the LV. Conclusion: These results suggest that the RV has a higher activity of aerobic glycolytic metabolism and may be able to respond faster and more powerfully to stressful stimuli than the LV.

Human Hepatocellular Carcinoma Metabolism: Imaging by Hyperpolarized 13C Magnetic Resonance Spectroscopy

Most cancers exhibit high levels of aerobic glycolytic metabolism with diminished levels of mitochondrial oxidative phosphorylation even in the presence of normal or near-normal levels of oxygen ("Warburg effect"). However, technical challenges have limited the development of non-invasive in vivo imaging techniques for monitoring glycolytic metabolism of hepatocellular carcinoma (HCC) and quantitatively evaluating the impact of this effect on the growth and therapy of this disease. Thus, there is a critical need to develop non-invasive techniques for longitudinal assessment of the metabolism and treatment response of patients with unresectable HCCs. This article discusses a novel method, "Hyperpolarized 13C MRS imaging", for achieving this objective and thus improving the prognosis of HCC patients. The primary objective has been to characterize in vivo metabolic biomarkers as determinants of HCC metabolism and treatment response of unresectable HCC tumors or viable HCC cells. This innovative technique capitalizes on a new technology that increases the sensitivity of MRS detection of crucial metabolites in cancer cells. It is anticipated that this innovative approach will lead to improved methods, both for the diagnosis and staging of HCCs and for the facilitation of the development of enzyme targeted therapies and other therapeutic interventions.