Basal Glycolysis Research Articles

Cellular targets of the myeloperoxidase-derived oxidant hypothiocyanous acid (HOSCN) and its role in the inhibition of glycolysis in macrophages

Myeloperoxidase (MPO) released at sites of inflammation catalyzes the formation of the oxidants hypochlorous acid (HOCl) and hypothiocyanous acid (HOSCN) from H2O2 and halide and pseudo-halide ions. HOCl, a major oxidant produced under physiological conditions reacts rapidly with many biological molecules, and is strongly linked with tissue damage during inflammatory disease. The role of HOSCN in disease is less clear, though it can initiate cellular damage by pathways involving the selective oxidation of thiol-containing proteins. Utilizing a thiol-specific proteomic approach, we explored the cellular targets of HOSCN in macrophages (J774A.1). We report that multiple thiol-containing proteins involved in metabolism and glycolysis; fructose bisphosphate aldolase, triosephosphate isomerase, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and creatine kinase, together with a number of chaperone, antioxidant and structural proteins, were modified in a reversible manner in macrophages treated with HOSCN. The modification of the metabolic enzymes was associated with a decrease in basal glycolysis, glycolytic reserve, glycolytic capacity and lactate release, which was only partly reversible on further incubation in the absence of HOSCN. Inhibition of glycolysis preceded cell death and was seen in cells exposed to low concentrations (≤25µM) of HOSCN. The ability of HOSCN to inhibit glycolysis and perturb energy production is likely to contribute to the cell death seen in macrophages on further incubation after the initial treatment period, which may be relevant for the propagation of inflammatory disease in smokers, who have elevated plasma levels of the HOSCN precursor, thiocyanate.

Poly(ADP-ribose) polymerase regulates glycolytic activity in kidney proximal tubule epithelial cells.

After renal injury, selective damage occurs in the proximal tubules as a result of inhibition of glycolysis. The molecular mechanism of damage is not known. Poly(ADP-ribose) polymerase (PARP) activation plays a critical role of proximal tubular cell death in several renal disorders. Here, we studied the role of PARP on glycolytic flux in pig kidney proximal tubule epithelial LLC-PK1 cells using XFp extracellular flux analysis. Poly(ADP-ribosyl)ation by PARP activation was increased approximately 2-fold by incubation of the cells in 10 mM glucose for 30 minutes, but treatment with the PARP inhibitor 3-aminobenzamide (3-AB) does-dependently prevented the glucose-induced PARP activation (approximately 14.4% decrease in 0.1 mM 3-AB–treated group and 36.7% decrease in 1 mM 3-AB–treated group). Treatment with 1 mM 3-AB significantly enhanced the glucose-mediated increase in the extracellular acidification rate (61.1±4.3 mpH/min vs. 126.8±6.2 mpH/min or approximately 2-fold) compared with treatment with vehicle, indicating that PARP inhibition increases only glycolytic activity during glycolytic flux including basal glycolysis, glycolytic activity, and glycolytic capacity in kidney proximal tubule epithelial cells. Glucose increased the activities of glycolytic enzymes including hexokinase, phosphoglucose isomerase, phosphofructokinase-1, glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase in LLC-PK1 cells. Furthermore, PARP inhibition selectively augmented the activities of hexokinase (approximately 1.4-fold over vehicle group), phosphofructokinase-1 (approximately 1.6-fold over vehicle group), and glyceraldehyde-3-phosphate dehydrogenase (approximately 2.2-fold over vehicle group). In conclusion, these data suggest that PARP activation may regulate glycolytic activity via poly(ADP-ribosyl)ation of hexokinase, phosphofructokinase-1, and glyceraldehyde-3-phosphate dehydrogenase in kidney proximal tubule epithelial cells.

Abstract A33: CD38 and Nampt regulate tumor cell metabolism through modulation of NAD+

Abstract In order to maintain high rates of proliferation, tumor cells must alter their metabolic machinery in favor of increased macromolecule synthesis and energy production. A key factor in tumor cell metabolism is Nicotinamide Adenine Dinucleotide, NAD+. NAD+ is used as a cofactor for catabolic reactions (NAD(H)), or after conversion to NADP(H), for anabolic reactions. In addition, NAD+ is utilized as part of the catalytic mechanism for several classes of enzymes including the sirtuins, PARPs, and CD38. Given the juxtaposition of NAD+ to cell metabolism and other critical cellular process we hypothesize a balance between synthesis and consumption of NAD+ is a primary metabolic determinant in cancer. In prostate cancer, the primary pathway by which NAD+ is synthesized is via the salvage pathway, which recycles nicotinamide into Nicotinamide Mononucleotide (NMN) via the rate-limiting enzyme Nicotinamide Phosphoribosyltransferase (Nampt). We determined the metabolic consequences of Nampt blockade in prostate cancer and glioblastoma (GBM) cell lines using the pharmacological inhibitor FK866. As expected, treatment with FK866 decreases the levels of NAD+/NADP+ and the NAD+ precursor nicotinamide. In relation to glycolysis, there were increases in metabolites upstream of glyceraldehyde-3-phosphate dehydrogenase, which requires NAD+ as a cofactor. Specifically, glucose is increased 1.6 fold in PC3 cells (p≤0.05), glucose-6-phosphate is decreased 1.7 fold SNB19s (p≤0.05), and both lines have decreased fructose-1-phosphate levels between 6 and11 fold (p≤0.05), respectively. To compliment these changes we measured the effects of Nampt inhibition on glycolysis in real time using a Seahorse XF extracellular flux analyzer. Nampt inhibition lowers basal glycolysis 40% (p<0.01) in prostate cancer (PCa) cells and glycolytic capacity 46% (p<0.01) and 64% (p<0.01) in PCa and glioblastoma (GBM) cells, respectively. Reactions requiring NAD+ as a cofactor in the TCA cycle were similarly perturbed. Specifically, in both cell lines fumarate and malate were increased between 1.5 and 2 fold (p<0.05) resulting from reduced malic enzyme activity. Corresponding to altered TCA cycle metabolites there was a 45% decrease in spare mitochondrial respiratory capacity (p<0.01) in PCa cells and a 90% decrease in GBM cells (p<0.01), relative to vehicle. Together these data suggest blockage of NAD+ synthesis is sufficient to perturb the activity of NAD+ dependent metabolic enzymes and induce energetic stress. Of the three classes of NAD+ consuming enzymes, the cADP-ribose synthase CD38 is the primary NAD'ase in cells. Western blot analysis of normal prostate epithelial cells (PrEC) and several prostate tumor cell lines demonstrates expression of CD38 is nearly non-existent in PCa. In line with this, CD38 mRNA levels are decreased 2-fold across 31 independent PCa samples when compared to mRNA from 8 normal prostate samples (p=0.007). CD38 expression levels were also determined by immunohistochemistry (IHC) in primary PCa tissue micro-arrays (TMA). In a total of 91 samples: 37 benign, 17 prostatic intraepithelial neoplasia (PIN), and 37 Cancers, CD38 staining intensity was reduced 20% in PIN samples (p≤0.001) and 50% in cancer samples (p≤0.01). In these experiments, no change in Nampt protein expression, mRNA levels, or staining intensity was detected. Induction of CD38 expression in prostate cancer cell lines resulted in blocked cell proliferation. CD38 expression also reduced NAD levels by 54% and 91% after 48 and 96 hours, respectively (p≤0.01). Concomitant with decreased NAD levels, CD38 expression also reduced spare mitochondrial respiratory capacity 24% (p=0.026), mimicking the effects of Nampt inhibition. Fatty acid synthesis was similarly inhibited following CD38 expression. When taken together, these data suggest an interplay between Nampt and CD38 regulates total NAD+ levels, ultimately supporting the metabolic reprogramming associated with prostate cancer. Citation Format: Jeffrey P. Chmielewski, Frances Wheeler, Scott Cramer, Shi Lihong, Joseph Sirintrapun, Steven J. Kridel. CD38 and Nampt regulate tumor cell metabolism through modulation of NAD+. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A33.

Abstract 12709: Dynamics of Flow-Responsive Kinase Cε Modulates Glycolytic Metabolites to Promote Vascular Repair

Hemodynamic shear stress has recently been reported to modulate vascular development and repair via canonical Wnt--catenin signaling pathway. It is recognized that protein Kinase C isoform epsilon (PKCε) signaling in mitochondria confers cardioprotection via VEGF and eNOS activation in response to ischemia/reperfusion injury. Here, we hypothesized that shear stress-responsive PKCε signaling modulates glycolytic metabolites to promote vascular repair. By performing a metabolomic analysis, both oscillatory (OSS: ±1 dynes·cm -2 at 1 Hz) and pulsatile shear stress (PSS: 23 dynes·cm -2 at 1 Hz) significantly increased endothelial glycolytic metabolites; namely, xylitol and dihydroxyacetone ( n =6, p < 0.01 vs. control), but decreased gluconeogenic metabolites; namely, aspartic acid. Both OSS and PSS up-regulated PKCε mRNA and protein expression via VEGF receptor (n=3, p < 0.05); and constitutively active PKCε increased basal glycolysis and activity by the Seahorse assay ( n =4, p <0.01 vs. dominant negative PKCε control), implicating that shear stress-responsive PKCε modulates glycolysis. To recapitulate in transgenic Tg(flk1:GFP) zebrafish embryos, we performed micro-injection of GATA-1a morpholino oligonucleotide (MO) at 1-4 cell stage to inhibit hematopoeisis, thus decreasing viscosity and fluid shear stress. In response to tail amputation, GATA-1 MO impaired and delayed vascular repair compared with the control injection ( n =20, p <0.01 vs. control) ( Figure 1 ). Co-injection of PKCε mRNA with GATA-1a MO rescued tail repair ( n =5, p <0.05). Overall, shear stress-responsive VEGFR- PKCε signaling modulates glycolytic metabolites to influence vascular repair. Thus, hemodynamic shear stress imparts both developmental and metabolic effects to promote vascular regeneration. Figure 1. Tail repair in response to injection with GATA-1 MO or rescue by co-injection with PKCε mRNA, dpf: days post-fertilization; dpa: days post-amputation.

Defining the effects of storage on platelet bioenergetics: The role of increased proton leak

The quality of platelets decreases over storage time, shortening their shelf life and potentially worsening transfusion outcomes. The changes in mitochondrial function associated with platelet storage are poorly defined and to address this we measured platelet bioenergetics in freshly isolated and stored platelets. We demonstrate that the hypotonic stress test stimulates both glycolysis and oxidative phosphorylation and the stored platelets showed a decreased recovery to this stress. We found no change in aggregability between the freshly isolated and stored platelets. Bioenergetic parameters were changed including increased proton leak and decreased basal respiration and this was reflected in a lower bioenergetic health index (BHI). Mitochondrial electron transport, measured in permeabilized platelets, showed only minor changes which are unlikely to have a significant impact on platelet function. There were no changes in basal glycolysis between the fresh and stored platelets, however, glycolytic rate was increased in stored platelets when mitochondrial ATP production was inhibited. The increase in proton leak was attenuated by the addition of albumin, suggesting that free fatty acids could play a role in increasing proton leak and decreasing mitochondrial function. In summary, platelet storage causes a modest decrease in oxidative phosphorylation driven by an increase in mitochondrial proton leak, which contributes to the decreased recovery to hypotonic stress.

Bioenergetic analysis of human peripheral blood mononuclear cells

Leucocytes respond rapidly to pathogenic and other insults, with responses ranging from cytokine production to migration and phagocytosis. These are bioenergetically expensive, and increased glycolytic flux provides adenosine triphosphate (ATP) rapidly to support these essential functions. However, much of this work is from animal studies. To understand more clearly the relative role of glycolysis and oxidative phosphorylation in human leucocytes, especially their utility in a translational research setting, we undertook a study of human peripheral blood mononuclear cells (MNCs) bioenergetics. Glycolysis was essential during lipopolysaccharide (LPS)-mediated interleukin (IL)-1β, IL-6 and tumour necrosis factor (TNF)-α production, as 2-deoxy-D-glucose decreased significantly the output of all three cytokines. After optimizing cell numbers and the concentrations of all activators and inhibitors, oxidative phosphorylation and glycolysis profiles of fresh and cryopreserved/resuscitated MNCs were determined to explore the utility of MNCs for determining the bioenergetics health profile in multiple clinical settings. While the LPS-induced cytokine response did not differ significantly between fresh and resuscitated cells from the same donors, cryopreservation/resuscitation significantly affected mainly some measures of oxidative phosphorylation, but also glycolysis. Bioenergetics analysis of human MNCs provides a quick, effective means to measure the bioenergetics health index of many individuals, but cryopreserved cells are not suitable for such an analysis. The translational utility of this approach was tested by comparing MNCs of pregnant and non-pregnant women to reveal increased bioenergetics health index with pregnancy but significantly reduced basal glycolysis and glycolytic capacity. More detailed analysis of discrete leucocyte populations would be required to understand the relative roles of glycolysis and oxidative phosphorylation during inflammation and other immune responses.

Abstract 235: Sarcolemmal Membrane Associated Protein Isoform 1: a Unique Regulator of Glucose Uptake and Metabolism in the Myocardium

As one of the leading causes of heart disease, diabetes is a problem which needs a solution. Regulation of glucose uptake and metabolism within skeletal and cardiac muscle has proven capable of altering systemic glucose levels and impacting metabolism to potentially improve patient outcomes. Unfortunately, to date, very few muscle specific metabolic regulators are known which can allow us to achieve blood glucose uptake and metabolism. Sarcolemmal Membrane Associated Protein Isoform 1 (SLMAP1) is a novel protein expressed predominantly within muscle tissue. It has been linked to diabetes through animal models, although its role in metabolism remains to be defined. Here we describe a novel role for SLMAP1 in glucose metabolism within the myocardium. We engineered a transgenic (TG) mouse with cardiac specific expression of SLMAP1. Using neonatal cardiomyocytes (NCMs) collected from these mice we performed glucose uptake assays with 2-deoxy-glucose (2DG), measured glycolytic rate using an Extracellular Flux XF24e Bioanalyzer, and analyzed glucose transporter 4 (GLUT4) trafficking using Western Blots, qPCR, and immunofluorescence imaging. NCMs extracted from TG hearts expressing SLMAP1 displayed increased levels of 2DG uptake (93% ± 25%, n=5, P<0.01), basal glycolysis (glycolysis (92 ± 40%, n=5, P<0.05), and maximal glycolysis (75 ± 31%, n=5, P<0.05) compared with wildtype littermates. Confocal microscopy revealed both increased localization of glucose transporter 4 (GLUT4) at the cell surface as well as an expansion of GLUT4 early endosomes in TG NCMs. The data here indicates SLMAP1 as a novel regulator of glucose uptake and metabolism in the myocardium. The targeted expression of SLMAP1 in a muscle specific manner may enhance systemic glycemic control and serve to limit cardiovascular disease in metabolic disorders such as diabetes.

Regional assessment of energy-producing metabolic activity in the endothelium of donor corneas.

We characterized mitochondrial respiration and glycolysis activity of human corneal endothelium, and compared metabolic activity between central and peripheral regions. Endothelial keratoplasty-suitable corneas were obtained from donors aged 50 to 75 years. The endothelium-Descemet membrane complex (EDM) was isolated, and 3-mm punches were obtained from central and peripheral regions. Endothelium-Descemet membrane punches were assayed for mitochondrial respiration (oxygen consumption) and glycolysis (extracellular acidification) using an extracellular flux analyzer. Enzymatic (citrate synthase, glucose hexokinase) and mitochondrial density (MitoTracker) assays also were performed. Ten corneas were analyzed per assay. Metabolic activity for mitochondrial respiration and glycolysis showed expected changes to assay compounds (P < 0.01, all pairwise comparisons). Basal mitochondrial respiration and glycolysis activity did not differ between regions (P > 0.99). Similarly, central versus peripheral activity after assay compound treatment showed no significant differences (P > 0.99, all time points). The intracorneal coefficient of variation for basal readings between two and four peripheral punches was 18.5% of the mean. Although peripheral samples displayed greater enzymatic activity than central samples (P < 0.05), similar to extracellular flux results, mitochondrial density did not differ between regions (P = 0.78). Extracellular flux analysis of oxygen and pH is a valid technique for characterizing metabolic activity of human corneal endothelium. This technique demonstrates high reproducibility, allows quantification of metabolic parameters using small quantities of live cells, and permits estimation of overall metabolic output. Neither oxygen consumption nor extracellular acidification differed between central and peripheral regions of transplant suitable corneas in this series. Our results show that endothelial cell health can be quantified biochemically in transplant suitable corneas.

SIRT3 Deficiency Impairs Hypoxic Signaling, Reprograms Basal Glycolytic Metabolism and Exacerbates Myocardial Ischemic Injury

Increased glycolytic metabolism is critical for maintaining homeostasis of cardiomyocytes and endothelial cells (EC) during myocardial ischemia. Sirtuin 3 (SIRT3) is involved in epigenetic regulation of variety of cellular functions. The present study investigated the role of SIRT3 in altering glycolytic metabolism and hypoxia/ischemia responses using SIRT3 knockout (SIRT3 KO) mice and SIRT3 deficient EC. In vitro, exposure of EC to hypoxia up to 24 hours resulted in a gradual increase in HIF‐2α expression in WT and SIRT3 KO‐EC. Interestingly, hypoxia‐induced HIF‐2α expression was less in SIRT3 KO‐EC compared to EC isolated from WT mice. Moreover, hypoxia‐induced VEGF expression was reduced in SIRT3 KO‐EC. Using Seahorse XFe24 analyzer, EC glycolysis and mitochondrial function were further examined. Basal glycolysis, glycolytic reserve and glycolytic capacity were dramatically reduced in Sirt3 KO‐EC compared to WT‐EC. Moreover, Sirt3KO EC exhibited higher oxygen consumption rate than WT‐EC. Intriguingly, treatment with prolyl‐hydroxylase inhibitor‐dimethyloxalylglycine (DMOG) or VEGF rescued the impaired basal glycolytic metabolism in Sirt3 KO‐EC. Treatment with DMOG resulted in a reduction of basal oxygen consumption in WT‐EC, but failed to reduce basal oxygen consumption in Sirt3 KO‐EC. In vivo, SIRT3 KO mice subjected to ischemia exhibited a larger infarct size and impaired cardiac function compared to WT mice. VEGF and glycolytic enzyme PFKFB3 levels were significantly decreased in the heart of SIRT3 KO relative to WT mice. These studies suggest that the loss of SIRT3 aggravates myocardial ischemic injury by altering hypoxic signaling, glycolytic metabolism and increasing oxygen consumption.

Aerobic glycolysis, motility, and cytoskeletal remodeling

Cancer cell motility and cytoskeletal remodeling are ubiquitous processes essential in cancer progression to metastasis. The role of metabolism in driving motility processes and cytoskeletal rearrangements is not fully understood, but abnormal usage of cellular energetics is a well-documented hallmark of cancer.1 Shiraishi and colleagues now provide evidence that energy utilized for cancer cell motility and cytoskeletal rearrangement processes is derived primarily from aerobic glycolysis and not mitochondrial oxidative phosphorylation, indicative of a deep metabolic compartmentalization program within cancer cells2 (Fig. 1). Figure 1. Energy for cancer cell movement is derived from glycolysis. The authors demonstrate that more aggressive mesenchymal prostate cancer cells (PC3-EMT) exhibit an increased level of basal aerobic glycolysis relative to less aggressive epithelial prostate cancer cells (PC3-Epi) and non-cancerous human prostate epithelial cells (PrECs). These findings are consistent with the Warburg effect, which describes a high rate of aerobic glycolysis as a major consequence of tumorigenesis.3 Mitochondrial respiration is unchanged between the aggressive and less aggressive prostate cancer cells but provides the bulk of cellular energy in both groups. This finding highlights a widely held misconception about the Warburg Effect; despite the increased rate of aerobic glycolysis in tumor cells, mitochondrial respiration still provides the majority of cellular ATP. In addition, a higher glycolytic capacity in PC3-EMT cells (maximal rate of glycolysis) relative to PC3-Epi and PrECs was observed. Complementary studies were executed with the breast cancer cell line MDA-MB-231 (mesenchymal) and MDA-MB-231-EPI (overexpressing the transcription factors OVOL1 and OVOL2 which drive a mesenchymal to epithelial transition). The stable expression of OVOL1 and OVOL2 led to a decrease in proton production rate (PPR), a surrogate measurement for aerobic glycolysis, in MDA-MB-231 cells. Consistent with previous findings, these studies highlight the metabolic shift toward aerobic glycolysis in cells that display an aggressive phenotype.4 Given the importance of actin polymerization and subsequent turnover during cellular motility and cellular invasion in cancer progression,5 cytoskeletal (CSK) rearrangement was measured by measuring contractile moment, cellular movement, and focal adhesion formation. PC3-EMT cells display a greater net contractile moment and a greater capacity to remodel their cytoskeleton than PC3-Epi cells. By tracking cellular displacement over time, PC3-EMT cells displayed greater motility relative to PC3-Epi cells. Cancer cell motility in both cancer cell lines is attenuated upon addition of 2-deoxy-D-Glucose (2-DG), a potent inhibitor of aerobic glycolysis, but not upon addition of oligomycin A, an inhibitor of mitochondrial respiration (Fig. 1). Focal adhesion formation and cellular contractile moment are also dramatically decreased upon addition of 2-DG but not oligomycin A. These findings are surprising given that actin is an ATPase and that mitochondrial respiration provides the majority of cellular ATP for these cells.2,6 Taken together, these findings indicate that aerobic glycolysis but not mitochondrial respiration is necessary for cellular movement (Fig. 1). Additionally, these studies underscore the importance of aerobic glycolysis in CSK rearrangement and focal adhesion formation. This study demonstrates a direct link between aerobic glycolysis and cellular movement consistent with previous studies that describe glycolytic enzyme co-localization at sites of active actin turnover.7 Surprisingly, the source of fuel for motility appears to be more important than the amount of energy provided by a given fuel type (Fig. 1) indicative of cellular compartmentalization of metabolic flux. These findings suggest that targeting aerobic glycolysis therapeutically could curb cancer cell motility and subsequent metastasis.

Metabolic Reprogramming of Acute Myeloid Leukemia Blasts By Bone Marrow Stroma Cells

Introduction: Acute myeloid leukemia (AML) represents the most common form of acute leukemia in adults. Despite the enormous efforts during the last decades treatment resistance is still observed at a high rate. Previous studies have shown that bone marrow stroma promotes an increased resilience of AML blasts towards chemotherapeutics. Furthermore, current data suggest that alterations of the malignant cells’ metabolism could represent a strong determinant for the disease’s (including AML) course and/or treatment resistance. In fact, a deregulated metabolism could lead to a reduced sensitivity towards therapy and it remains to be elucidated whether this is a mechanism contributing to the blast-protective effects elicited by the bone marrow stroma. Here, we sought out to characterize the impact of stroma cells on the AML blasts’ metabolism.Methods and Results: The human bone marrow stroma cell-line HS-5 was utilized for establishing the in vitro niche model. We compared in our assays AML cell lines (THP-1, OCI-AML, and KG-1) as well as primary blasts cultured on a HS-5 monolayer or alone. In line with previous observations we could detect an increased proportion of AML cells in the S-phase of the cell cycle upon co-culture with HS-5. When evaluating the cells’ metabolism we observed a shift towards glycolysis despite presence of oxygen, i.e. aerobic glycolysis or the “Warburg”-effect. Basal glycolysis as well as maximal glycolytic capacity upon blocking ATP production in the mitochondrial respiratory chain was increased. Respiration (including basal respiration, coupling efficacy, and maximal respiratory capacity) was not significantly affected. However, mitochondrial biogenesis appeared reduced.Increased glycolysis was accompanied by an increased up-take of fluorescently labeled glucose as well as an increased expression of glucose transporters. The expression of several glycolytic molecules found to be increased upon HS-5 co-culture. Noticeably, cell-to-cell contact was not a pre-requisite for the metabolic shift. Our data was further corroborated by direct observations from AML patients: AML blasts isolated from the bone marrow exhibited an up-regulated glycolysis as compared to their counterparts from the periphery collected at the same time point.Conclusion: Taken together, our data indicates a stromal cell-mediated metabolic shift in AML blasts towards aerobic glycolysis. This metabolic phenotype is linked to an unfavorable prognosis and increased chemo resistance. The underlying molecular pathways remain to be elucidated and could represent a promising target for future interventions. DisclosuresNo relevant conflicts of interest to declare.

Overexpression of mitochondrial sirtuins alters glycolysis and mitochondrial function in HEK293 cells.

SIRT3, SIRT4, and SIRT5 are mitochondrial deacylases that impact multiple facets of energy metabolism and mitochondrial function. SIRT3 activates several mitochondrial enzymes, SIRT4 represses its targets, and SIRT5 has been shown to both activate and repress mitochondrial enzymes. To gain insight into the relative effects of the mitochondrial sirtuins in governing mitochondrial energy metabolism, SIRT3, SIRT4, and SIRT5 overexpressing HEK293 cells were directly compared. When grown under standard cell culture conditions (25 mM glucose) all three sirtuins induced increases in mitochondrial respiration, glycolysis, and glucose oxidation, but with no change in growth rate or in steady-state ATP concentration. Increased proton leak, as evidenced by oxygen consumption in the presence of oligomycin, appeared to explain much of the increase in basal oxygen utilization. Growth in 5 mM glucose normalized the elevations in basal oxygen consumption, proton leak, and glycolysis in all sirtuin over-expressing cells. While the above effects were common to all three mitochondrial sirtuins, some differences between the SIRT3, SIRT4, and SIRT5 expressing cells were noted. Only SIRT3 overexpression affected fatty acid metabolism, and only SIRT4 overexpression altered superoxide levels and mitochondrial membrane potential. We conclude that all three mitochondrial sirtuins can promote increased mitochondrial respiration and cellular metabolism. SIRT3, SIRT4, and SIRT5 appear to respond to excess glucose by inducing a coordinated increase of glycolysis and respiration, with the excess energy dissipated via proton leak.

D Mitochondrial DNA Damage can Promote Atherosclerosis Independently of Reactive Oxygen Species and Correlates with Higher Risk Plaques in Humans

IntroductionMitochondrial DNA (mtDNA) damage occurs in both the vessel wall and in circulating cells in human atherosclerosis. However, whether mtDNA damage promotes atherogenesis or is a consequence of tissue damage...

Activation-specific metabolic regulation of NK cell IFN-gamma production (INC6P.334)

Abstract Many factors that stimulate NK cell functional responses have been described; however metabolic fuels that drive NK cell function are unknown. Metabolism is significantly altered in disease states and metabolic regulation of T cell function is well-described. We hypothesize that NK cells require distinct metabolic fuels for survival, proliferation, and functional responses. NK cell metabolic phenotype was determined with an extracellular flux analyzer to determine basal oxidative phosphorylation (oxphos) and glycolysis of resting and activated NK cells. NK cells were then cultured under a variety of conditions using inhibitors and nutrient deprivation to determine cellular requirements for glucose, fatty acids, and oxphos for NK cell survival, proliferation, and function. NK cells primarily utilize oxphos at baseline and, in contrast to T cells, upon proliferation. However, there is significant plasticity in the metabolic requirements for NK cell IFN-gamma production. NK cell IFN-gamma production in response to cytokine stimulation is not dependent on either glycolysis or oxphos. By contrast, NK cell IFN-gamma production in response to receptor stimulation requires oxphos and is partially dependent on glycolysis. The metabolic requirements for NK cell IFN-gamma production are not altered by priming in vitro or in vivo, but do change with proliferation. These findings suggest that the metabolic microenvironment in vivo may significantly alter NK cell function.

Transcript levels of class I GLUTs within individual tissues and the direct relationship between GLUT1 expression and glucose metabolism in Atlantic cod (Gadus morhua)

GLUTs 1-4 are sodium-independent facilitated glucose transporters and are considered to play a major role in glucose trafficking. The relative transcript levels of GLUTs 1-4 were determined in tissues of Atlantic cod (Gadus morhua). The distribution profile of GLUTs normalized to RNA is similar to mammals and with a few exceptions other fish. GLUT1 is ubiquitous, GLUT2 is relatively abundant in tissues that release glucose, GLUT3 expression is relatively strong in brain, and GLUT4 is relatively high in heart and muscle. The functionally significant level of transcript is presumably the level in the cell. Normalization of relative GLUT levels to tissue mass reveals there are extremely high levels of GLUT1 transcript in gas gland consistent with the high lactate production rates, GLUT3 is dominant in gill and head kidney as well as brain, and GLUT4 expression in gill is elevated relative to other tissues. Consideration of GLUTs within tissues reveals that GLUT1 is the dominant transcript in a group of tissues including gas gland, heart, white muscle, and RBCs. Brain, gill, and spleen display a co-dominance of GLUTs 1 and 3. There are relatively low levels of GLUT4 in most tissues, the highest being found in white muscle where GLUT4 accounts for only 12% of the total transcript level. The apparent low level of GLUT4 transcript may reflect two tissues that were not included in the current study, red muscle and adipose tissue, due to their low abundance in Atlantic cod. The rate of glucose metabolism in isolated cells prepared from gas gland, heart, and RBCs was determined by tracking the rate of (3)H2O production from [2-(3)H]-glucose. The steady-state rate of basal glycolysis in these three tissues correlates with relative transcript levels of GLUT1.

Mitochondrial energetic and AKT status mediate metabolic effects and apoptosis of metformin in human leukemic cells.

Previous reports demonstrate that metformin, an anti-diabetic drug, can decrease the risk of cancer and inhibit cancer cell growth. However, its mechanism in cancer cells is still unknown. Metformin significantly blocks cell cycle and inhibits cell proliferation and colony formation of leukemic cells. However, the apoptotic response to metformin varies. Furthermore, daily treatment with metformin induces apoptosis and reduces tumor growth in vivo. While metformin induces early and transient activation of AMPK, inhibition of AMPKα1/2 does not abrogate anti-proliferative or pro-apoptotic effects of metformin. Metformin decreases electron transport chain complex I activity, oxygen consumption and mitochondrial ATP synthesis, while stimulating glycolysis for ATP and lactate production, pentose phosphate pathway for purine biosynthesis, fatty acid metabolism, as well as anaplerotic and mitochondrial gene expression. Importantly, leukemic cells with high basal AKT phosphorylation, glucose consumption or glycolysis exhibit a markedly reduced induction of the Pasteur effect in response to metformin and are resistant to metformin-induced apoptosis. Accordingly, glucose starvation or treatment with deoxyglucose or an AKT inhibitor induces sensitivity to metformin. Overall, metformin elicits reprogramming of intermediary metabolism leading to inhibition of cell proliferation in all leukemic cells and apoptosis only in leukemic cells responding to metformin with AKT phosphorylation and a strong Pasteur effect.

Distinct energy requirements for human memory CD4 T‐cell homeostatic functions

Differentiation and activation of CD4 memory T cells (T(mem) cells) require energy from different sources, but little is known about energy sources for maintenance and surveillance activities of unactivated T(mem) cells. Mitochondrial fatty acid oxidation (FAO) in human unactivated CD4 T(mem) cells was significantly enhanced by inhibition of glycolysis, with respective means of 1.7- and 4.5-fold for subjects <45 yr and >65 yr, and by stimulation of AMP-activated protein kinase, with respective means of 1.3- and 5.2-fold. However, CCL19 and sphingosine 1-phosphate (S1P), which control homeostatic lymphoid trafficking of unactivated T(mem) cells, altered FAO and glycolysis only minimally or not at all. Inhibition of CD4 T(mem)-cell basal FAO, but not basal glycolysis, significantly suppressed CCL19- and S1P-mediated adherence to collagen by >50 and 20%, respectively, and chemotaxis by >20 and 50%. Apoptosis of unactivated T(mem) cells induced by IL-2 deprivation or CCL19 was increased significantly by >150 and 70%, respectively, with inhibition of FAO and by >110 and 30% with inhibition of glycolysis. Anti-TCR antibody activation of T(mem) cells increased their chemotaxis to CCL5, which was dependent predominantly on glycolysis rather than FAO. The sources supplying energy for diverse functions of unactivated T(mem) cells differ from that required for function after immune activation.

Small Ubiquitin-related Modifier (SUMO)-1 Promotes Glycolysis in Hypoxia

Under conditions of hypoxia, most eukaryotic cells undergo a shift in metabolic strategy, which involves increased flux through the glycolytic pathway. Although this is critical for bioenergetic homeostasis, the underlying mechanisms have remained incompletely understood. Here, we report that the induction of hypoxia-induced glycolysis is retained in cells when gene transcription or protein synthesis are inhibited suggesting the involvement of additional post-translational mechanisms. Post-translational protein modification by the small ubiquitin related modifier-1 (SUMO-1) is induced in hypoxia and mass spectrometric analysis using yeast cells expressing tap-tagged Smt3 (the yeast homolog of mammalian SUMO) revealed hypoxia-dependent modification of a number of key glycolytic enzymes. Overexpression of SUMO-1 in mammalian cancer cells resulted in increased hypoxia-induced glycolysis and resistance to hypoxia-dependent ATP depletion. Supporting this, non-transformed cells also demonstrated increased glucose uptake upon SUMO-1 overexpression. Conversely, cells overexpressing the de-SUMOylating enzyme SENP-2 failed to demonstrate hypoxia-induced glycolysis. SUMO-1 overexpressing cells demonstrated focal clustering of glycolytic enzymes in response to hypoxia leading us to hypothesize a role for SUMOylation in promoting spatial re-organization of the glycolytic pathway. In summary, we hypothesize that SUMO modification of key metabolic enzymes plays an important role in shifting cellular metabolic strategies toward increased flux through the glycolytic pathway during periods of hypoxic stress.

Abstract LB-108: Glutamine addition and glucose addiction - linking metabolic abnormalities to genetic alterations in glioblastoma SF188 and hereditary leiomyomatosis renal carcinoma UOK262

Abstract Metabolic transformations of malignant cells are essential for tumor development and progression. Oncogene Akt and c-myc stimulate glucose metabolism and glutamine metabolism, respectively, and render the cancers dependent on glucose or glutamine for growth and survival (otherwise known as glucose addiction and glutamine addiction). However, many other oncogenes, tumor suppressor genes and signaling molecules also regulate glucose and glutamine metabolism, which results in various metabolic abnormalities in different cancers. The question we want to ask is how one or more genetic mutations can lead to the ultimate metabolic abnormalities of a given cancer. Toward this end, we report here the metabolic abnormalities of two human cancer cell lines, glioblastoma SF188, which is c-myc-dependent and is addicted to glutamine, and hereditary leiomyomatosis renal carcinoma (HLRCC) UOK262, which is fumarate hydratase-deficient and is addicted to glucose. We performed our metabolic analysis using the XF24 Extracellular Flux Analyzer and pharmacological modulators of cellular metabolism. The XF24 Analyzer simultaneously monitors oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), which are indicators of mitochondrial respiration and aerobic glycolysis respectively. Oxygen consumption rate is also used as an indicator for the oxidation of glucose and glutamine when they are added to the cells after baseline measurement. We found that SF188 cells showed a small increase in acidification rate upon glucose addition, suggesting a relatively low basal glycolysis rate. We estimated the glycolytic capacity of the cells by determining the acidification rate after the addition of mitochondrial ATP syntase inhibitor oligomycin. The glycoltic capacity remained high although basal glycolysis rate was low. Glucose was not oxidized at all by these cells. Most importantly, we observed significant increases in oxygen consumption rate upon addition of glutamine indicating active uptake and oxidation of glutamine. Associated with active glutamine oxidation, SF188 cells exhibited high basal reparation rate and high respiratory capacity. Finally, SF188 cells grew and survived in medium containing glutamine but no glucose; however, they started to die in the medium containing glucose but no glutamine. In contrast with glioblastoma SF188, we found that HLRCC UOK262 cells exhibit high basal glycolysis rate, which is close to its maximal capacity. Interestingly, basal mitochondrial respiration and respiratory capacity are extremely low, which is associated with reported defective mitochondrial complex I. The growth and survival of UOK262 is dependent on a high concentration of glucose in the culture medium. Collectively, we established metabolic abnormalities of glioblastama SF188 and HLRCC UOK262, which contain genetic alterations in c-myc and fumarate hydratase, respectively, that makes them addicted to glutamine and glucose. The metabolic abnormalities we found include the preferential use of glucose and glutamine, the activation or inactivation of relevant enzymatic pathways that catabolize glucose and glutamine, and the modulation of mitochondrial bioenergetic activities in the two cancer cell lines. Most of cancer cell lines that we have studied showed metabolic abnormalities falling between the two extremes. Therefore, this type of metabolic analysis coupled with the analysis of genetic abnormalities in cancers will provides a rapid means to gain insight into the aberrant metabolism of each type of cancer and improve the efficacy of cancer therapy for a cancer with a particular set of metabolic abnormalities. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-108.

Cardiac-specific overexpression of GLUT1 prevents the development of heart failure attributable to pressure overload in mice.

Increased rates of glucose uptake and glycolysis have been repeatedly observed in cardiac hypertrophy and failure. Although these changes have been considered part of the fetal gene reactivation program, the functional significance of increased glucose utilization in hypertrophied and failing myocardium is poorly understood. We generated transgenic (TG) mice with cardiac-specific overexpression of insulin-independent glucose transporter GLUT1 to recapitulate the increases in basal glucose uptake rate observed in hypertrophied hearts. Isolated perfused TG hearts showed a greater rate of basal glucose uptake and glycolysis than hearts isolated from wild-type littermates, which persisted after pressure overload by ascending aortic constriction (AAC). The in vivo cardiac function in TG mice, assessed by echocardiography, was unaltered. When subjected to AAC, wild-type mice exhibited a progressive decline in left ventricular (LV) fractional shortening accompanied by ventricular dilation and decreased phosphocreatine to ATP ratio and reached a mortality rate of 40% at 8 weeks. In contrast, TG-AAC mice maintained LV function and phosphocreatine to ATP ratio and had <10% mortality. We found that increasing insulin-independent glucose uptake and glycolysis in adult hearts does not compromise cardiac function. Furthermore, we demonstrate that increasing glucose utilization in hypertrophied hearts protects against contractile dysfunction and LV dilation after chronic pressure overload.