Metabolic state of glioma stem cells and nontumorigenic cells

Gliomas contain a small number of treatment-resistant glioma stem cells (GSCs), and it is thought that tumor regrowth originates from GSCs, thus rendering GSCs an attractive target for novel treatment approaches. Cancer cells rely more on glycolysis than on oxidative phosphorylation for glucose metabolism, a phenomenon used in 2-[(18)F]fluoro-2-deoxy-D-glucose positron emission tomography imaging of solid cancers, and targeting metabolic pathways in cancer cells has become a topic of considerable interest. However, if GSCs are indeed important for tumor control, knowledge of the metabolic state of GSCs is needed. We hypothesized that the metabolism of GSCs differs from that of their progeny. Using a unique imaging system for GSCs, we assessed the oxygen consumption rate, extracellular acidification rate, intracellular ATP levels, glucose uptake, lactate production, PKM1 and PKM2 expression, radiation sensitivity, and cell cycle duration of GSCs and their progeny in a panel of glioma cell lines. We found GSCs and progenitor cells to be less glycolytic than differentiated glioma cells. GSCs consumed less glucose and produced less lactate while maintaining higher ATP levels than their differentiated progeny. Compared with differentiated cells, GSCs were radioresistant, and this correlated with a higher mitochondrial reserve capacity. Glioma cells expressed both isoforms of pyruvate kinase, and inhibition of either glycolysis or oxidative phosphorylation had minimal effect on energy production in GSCs and progenitor cells. We conclude that GSCs rely mainly on oxidative phosphorylation. However, if challenged, they can use additional metabolic pathways. Therefore, targeting glycolysis in glioma may spare GSCs.     

Mitochondria mediate amplification of luteinizing hormone action by adenosine in luteal cells

Adenosine markedly amplifies the response of isolated rat and human luteal cells to LH via an intracellular site of action that is associated with an increase in cell ATP levels. This effect of adenosine is maximal in midstage cells and minimal at the onset of functional regression in late stage luteal cells. The objective of the present studies was to evaluate the role of mitochondria in mediating the action of adenosine in isolated rat luteal cells and to assess whether mitochondrial function may be compromised in regressing luteal cells. The present studies show that adenosine produced a significant increase in luteal cell levels of ADP and ATP, but had no effect on cell levels of GTP. Since ADP stimulates oxidative phosphorylation, we evaluated the role of mitochondria in mediating the amplification of LH action by adenosine in luteal cells with two mitochondrial inhibitors, oligomycin and dinitrophenol. Both inhibitors markedly reduced, in a dose-dependent manner, LH-stimulated cAMP accumulation in the presence or absence of adenosine. In parallel, both inhibitors decreased basal and adenosine-elevated ATP levels in a dose-related manner. Although late stage luteal cells showed a marked reduction in adenosine amplification of LH-stimulated cAMP accumulation, no change in adenosine-dependent elevation of cell levels of ATP was seen. We conclude that amplification of LH action and elevation of ATP levels in midstage cells by adenosine requires an increase in oxidative phosphorylation that is stimulated by an increase in cell levels of ADP. However, attenuation of adenosine amplification of LH action in late stage luteal cells is not due to impaired ATP production.     

Inhibition of insulin-stimulated glycogen synthesis by 5-aminoimidasole-4-carboxamide-1-beta-d-ribofuranoside-induced adenosine 5'-monophosphate-activated protein kinase activation: interactions with Akt, glycogen synthase kinase 3-3alpha/beta, and glycogen synthase in isolated rat soleus muscle

The aim of this study was to investigate the effects of 5-aminoimidasole-4-carboxamide-1-beta-d-ribofuranoside (AICAR)-induced AMP-activated protein kinase activation on glycogen metabolism in soleus (slow twitch, oxidative) and epitrochlearis (fast twitch, glycolytic) skeletal muscles. Isolated soleus and epitrochlearis muscles were incubated in the absence or presence of insulin (100 nM), AICAR (2 mM), and AICAR plus insulin. In soleus muscles exposed to insulin, glycogen synthesis and glycogen content increased 6.4- and 1.3-fold, respectively. AICAR treatment significantly suppressed ( approximately 60%) insulin-stimulated glycogen synthesis and completely prevented the increase in glycogen content induced by insulin. AICAR did not affect either basal or insulin-stimulated glucose uptake but significantly increased insulin-stimulated ( approximately 20%) lactate production in soleus muscles. Interestingly, basal glucose uptake was significantly increased ( approximately 1.4-fold) in the epitrochlearis muscle, even though neither basal nor insulin-stimulated rates of glycogen synthesis, glycogen content, and lactate production were affected by AICAR. We also report the novel evidence that AICAR markedly reduced insulin-induced Akt-Thr308 phosphorylation after 15 and 30 min exposure to insulin, which coincided with a marked reduction in glycogen synthase kinase 3 (GSK)-3alpha/beta phosphorylation. Importantly, phosphorylation of glycogen synthase was increased by AICAR treatment 45 min after insulin stimulation. Our results indicate that AICAR-induced AMP-activated protein kinase activation caused a time-dependent reduction in Akt308 phosphorylation, activation of glycogen synthase kinase-3alpha/beta, and the inactivation of glycogen synthase, which are compatible with the acute reduction in insulin-stimulated glycogen synthesis in oxidative but not glycolytic skeletal muscles.     

Glucose flux rate regulates onset of ischemic contracture in globally underperfused rat hearts

This study analyzes the importance of the source and rate of ATP production (glucose flux, glycogenolysis, and oxidative phosphorylation) in the prevention of ischemic contracture in isolated rat hearts. Ischemic contracture was initiated at about 10 minutes by buffer perfusion with nonglycolytic substrates whereas the addition of 11 mM glucose prevented contracture for 2 hours. Tissue values of ATP, phosphocreatine, and lactate could be dissociated from onset of ischemic contracture. In hearts perfused with acetate or free fatty acid, with 11 mM glucose, glycolytic ATP production was 2.3-2.8 mumol/g fresh wt/min; as initial rates of glycogenolysis fell, glycolysis was maintained by a steady increase of glucose flux to values in excess of 2 mumol ATP/g fresh wt/min. Decreasing the glucose flux by lowering the perfusate glucose or by the addition of 2-deoxyglucose precipitated ischemic contracture. When oxidative phosphorylation was further reduced by hypoxia, glucose still prevented ischemic contracture; however, when oxidative phosphorylation dropped to near zero (near-anoxic) rates, glycolysis was inhibited, and glucose could only delay ischemic contracture to about 45 minutes. Combined ATP production rates could be dissociated from contracture. The metabolic parameter that correlated best with prevention or delay of ischemic contracture was the rate of glycolytic flux from glucose, which in this model of global low-flow ischemia had to accelerate to provide a rate of ATP production from glucose in excess of 2 mumol/g fresh wt/min within 30 minutes of the start of ischemia to prevent ischemic contracture.     

Energy Metabolism After Ischemic Preconditioning in Streptozotocin-Induced Diabetic Rat Hearts

Objectives. The aim of this study was to compare the cardioprotective effects of preconditioning in hearts from streptozotocin-induced diabetic rats with its effects in normal rat hearts.Background. The protective effect of ischemic preconditioning against myocardial ischemia may come from improved energy balance. However, it is not known whether preconditioning can also afford protection to diabetic hearts.Methods. Isolated perfused rat hearts were either subjected (preconditioned group) or not subjected (control group) to preconditioning before 30 min of sustained ischemia and 30 min of reperfusion. Preconditioning was achieved with two cycles of 5 min of ischemia followed by 5 min of reperfusion.Results. In the preconditioned groups of both normal and diabetic rats, left ventricular developed pressure, high energy phosphates, mitochondrial adenosine triphosphatase and adenine nucleotide translocase activities were significantly preserved after ischemia-reperfusion; cumulative creatine kinase release was smaller during reperfusion; and myocardial lactate content was significantly lower after sustained ischemia. However, cumulative creatine kinase release was less in the preconditioned group of diabetic rats than in the preconditioned group of normal rats. Under ischemic conditions, more glycolytic metabolites were produced in the diabetic rats (control group) than in the normal rats, and preconditioning inhibited these metabolic changes to a similar extent in both groups.Conclusions. The present study demonstrates that in both normal and diabetic rats, preservation of mitochondrial oxidative phosphorylation and inhibition of glycolysis during ischemia can contribute to preconditioning-induced cardioprotection. Furthermore, our data suggest that diabetic myocardium may benefit more from preconditioning than normal myocardium, possibly as a result of the reduced production of glycolytic metabolites during sustained ischemia and the concomitant attenuation of intracellular acidosis.     

On the role of actomyosin ATPases in regulation of ATP turnover rates during intense exercise.

Actomyosin ATPase is the dominant ATP sink during muscle work. Its catalytic capacities in fast-twitch oxidative glycolytic fibers have long been known to exceed by about 3-fold those of slow-twitch oxidative fibers, but the relative contributions to control of metabolic rates during exercise have never been closely examined. We compared fast-twitch oxidative glycolytic and slow-twitch oxidative fibers that displayed similar mitochondrial abundance (similar activities of mitochondrial marker enzymes). During short-term, but near maximum, aerobic exercise, fast-twitch oxidative glycolytic fibers displayed ATP turnover rates that were 2-4 times higher than for slow-twitch oxidative fibers (despite similar mitochondrial metabolic capacities), implying a large ATPase contribution to control of maximum metabolic rate. Fluxes through the ATP in equilibrium ADP + Pi cycle were extremely well regulated; at the lower limit, the forward flux exceeded the backward flux by only 0.06%, whereas at the upper limit, ATPase rates exceeded ATP synthesis rates by 0.12%. This very high precision of energy coupling could not be easily explained by standard metabolic regulation models.     

Shaking up glycolysis: Sustained, high lactate flux during aerobic rattling

Substantial ATP supply by glycolysis is thought to reflect cellular anoxia in vertebrate muscle. An alternative hypothesis is that the lactate generated during contraction reflects sustained glycolytic ATP supply under well-oxygenated conditions. We distinguished these hypotheses by comparing intracellular glycolysis during anoxia to lactate efflux from muscle during sustained, aerobic contractions. We examined the tailshaker muscle of the rattlesnake because of its uniform cell properties, exclusive blood circulation, and ability to sustain rattling for prolonged periods. Here we show that glycolysis is independent of the O(2) level and supplies one-third of the high ATP demands of sustained tailshaking. Fatigue is avoided by rapid H(+) and lactate efflux resulting from blood flow rates that are among the highest reported for vertebrate muscle. These results reject the hypothesis that glycolysis necessarily reflects cellular anoxia. Instead, they demonstrate that glycolysis can provide a high and sustainable supply of ATP along with oxidative phosphorylation without muscle fatigue.     

The relationship between glycolysis,fatty acid metabolism and membrane integrity in neonatal myocytes

The glycolytic rate of cultured neonatal rat cardiac cells was manipulated by varying the concentration of glucose, l-lactate and 2-deoxyglucose supplementing the normal growth medium under anoxic and normoxic conditions. The leakage of lactate dehydrogenase from the cells was found to show good inverse correlation with glycolytic rate under both normoxic and anoxic incubation conditions. Enzyme release correlated inversely with intracellular ATP concentrations in anoxia but not in normoxia.Treatment of cultures with iodoacetate caused enzyme leakage but interference with mitochondrial energy production using cyanide or 2,4-dinitrophenol was without effect on enzyme release.Lactate inhibited fatty acid oxidation in a concentration-dependent manner, but 2-deoxyglucose was without effect. Treatment of cultures with lactate or 2-deoxyglucose caused an increase and a decrease in intracellular fatty acid accumulation, respectively. Elevation of the medium glucose concentration promoted free fatty acid accumulation. Although lactate and 2-deoxyglucose influence fatty acid metabolism by the cardiac cell in different ways they both cause enzyme release, an effect which correlates well with their inhibition of glycolysis even under normoxic conditions.     

Energy demand, supply, and utilization in hypoxia, and force recovery after reoxygenation in rabbit heart muscle

In rabbit papillary muscle contracting at 20 degrees C in nitrogen at 0.2 Hz, glycolytic ATP formation is just enough to support the diminished contractile activity. Basal metabolism, important to maintain cellular function and integrity, is strongly inhibited. In the present study, we address the question of whether the inhibition of basal processes in hypoxia determines redevelopment of force in reoxygenation. By not stimulating the muscle during hypoxia, we try to make more ATP available for basal processes. Isometric force of papillary muscles (0.2-Hz stimulation) is measured before, during, and after 40 minutes of hypoxia. ATP formation and utilization in hypoxia are estimated from lactate production and changes in nucleotides and creatine compounds. After reoxygenation, muscles stimulated during hypoxia produce a steady-state force of 78% of the aerobic control; resting muscles recover to 94%. In contrast to expectation, lactate production in hypoxic resting muscles is only 30% of that in contracting ones. The findings indicate that basal metabolic rate of hypoxic muscles at rest is 14% of that of quiescent, well-oxygenated myocardium. We conclude that in hypoxic myocardium little ATP is available for basal metabolism, irrespective of the energy demand of the contractile system. It is therefore unlikely that the lower force found after reoxygenation in muscles stimulated during hypoxia is related to the degree of inhibition of basal processes.     

Protective effects of amiodarone pretreatment on mitochondrial function and high energy phosphates in ischaemic rat heart

The effects of the antianginal and antiarrhythmic drug amiodarone on mitochondrial function and high-energy phosphate content were assessed during normothermic ischaemic cardiac arrest and reperfusion in Langendorff-perfused rat heart. Total ischaemia for 30 min at 37 degrees C produced highly significant changes in mitochondrial oxidative phosphorylation and high-energy phosphate content. Pretreatment of the rats with one single dose of amiodarone (20 mg/kg i.v., 30 min before killing) markedly attenuated the deleterious effect of ischaemia on mitochondrial function and slightly reduced ATP depletion. In normally perfused hearts, amiodarone pretreatment did not modify any parameter of mitochondrial respiratory function nor did it influence high-energy phosphate or glycogen content. After reperfusion for 15 min, amiodarone-treated hearts showed improved recovery of mitochondrial oxidative phosphorylation and tissue high-energy phosphate content as compared to control hearts. Pretreatment of hearts with amiodarone did not reduce ischaemia-induced leakage of total adenylic nucleotides but highly significantly reduced lactate dehydrogenase release during reperfusion. These results indicate that amiodarone could exert substantial protection on the infarcting myocardium.     

Bioenergetic pattern of turtle brain and resistance to profound loss of mitochondrial ATP generation.

The adaptations in the freshwater turtle that permit survival despite prolonged loss of mitochondrial ATP generation were investigated by comparing the bioenergetics of turtle brain slices with rat brain slices. Aerobic turtle brain shows no significant difference in basal levels of total ATP generation compared to rat brain; levels in turtle brain and rat brain were 18.4 +/- 2.8 (SD) and 19.4 +/- 2.2 mumol (100 mg of tissue)-1 hr-1, respectively. However, in turtle brain, a significantly greater fraction of ATP is derived from glycolysis both under aerobic and anaerobic conditions [aerobic turtle (24%) and rat (13%), P less than 0.02; anaerobic, turtle (28%) and rat (18%), P less than 0.05]. The increased glycolytic capacity is related to high levels of rate-limiting glycolytic enzymes, such as pyruvate kinase (EC 2.7.1.40). Turtle brain operates close to glycolytic capacity even under aerobic conditions, and no Pasteur effect can be demonstrated. Quantitatively, anaerobic glycolysis accounts for a maximum of 28% of basal aerobic ATP generation, suggesting that prolonged diving is also accompanied by a reduction in brain energy requirements. The adaptation subserving short-term (natural) diving is an increase in brain glycolytic capacity. The adaptation subserving prolonged diving (days to weeks) may be a reduction in the energy requirements of brain (and other cells).     

Different responses of astrocytes and neurons to nitric oxide: The role of glycolytically generated ATP in astrocyte protection

It was recently proposed that in Jurkat cells, after inhibition of respiration by NO, glycolytically generated ATP plays a critical role in preventing the collapse of mitochondrial membrane potential (Deltapsi(m)) and thus apoptotic cell death. We have investigated this observation further in primary cultures of rat cortical neurons and astrocytes-cell types that differ greatly in their glycolytic capacity. Continuous and significant ( approximately 85%) inhibition of respiration by NO (1.4 microM at 175 microM O(2)) generated by [(z)-1-[2-aminoethyl]-N-[2-ammonioethyl]amino]diazen-1-ium-1,2 diolate (DETA-NO) initially (10 min) depleted ATP concentrations by approximately 25% in both cell types and increased the rate of glycolysis in astrocytes but not in neurons. Activation of glycolysis in astrocytes, as judged by lactate production, prevented further ATP depletion, whereas in neurons, which do not invoke this mechanism, there was a progressive decrease in ATP concentrations over the next 60 min. During this time, there was a persistent mitochondrial hyperpolarization and absence of apoptotic cell death in astrocytes, whereas in the neurons there was a progressive fall in Deltapsi(m) and increased apoptosis. After glucose deprivation or treatment with inhibitors of the F(1)F(0)-ATPase and adenine nucleotide translocase, astrocytes responded to NO with a fall in Deltapsi(m) and apoptotic cell death similar to the response in neurons. Finally, although treatment of astrocytes with NO partially prevented staurosporin-induced collapse in Deltapsi(m) and cell death, NO and staurosporin synergized in decreasing Deltapsi(m) and inducing apoptosis in neurons. These results demonstrate that although inhibition of cellular respiration by NO leads to neurotoxicity, it may also result in initial neuroprotection, depending on the glycolytic capacity of the particular cell.     

Skeletal muscle oxidative capacity, fiber type, and metabolites after lung transplantation.

Lung transplant (LTx) recipients have a low peak work rate, peak oxygen consumption (V O2peak), and early lactate threshold on incremental exercise. We hypothesized that LTx recipients have reduced oxidative function and altered fiber type proportion in peripheral skeletal muscle. Seven stable LTx recipients and seven age- and sex-matched control subjects were studied. Incremental exercise testing with arterialized venous sampling and a resting quadriceps femoris punch muscle biopsy were performed. Muscle specimens were analyzed for fiber type proportion, metabolites, oxidative and glycolytic enzyme activities, and mitochondrial ATP production rate (MAPR) using standard techniques. The results showed that mean V O2peak in LTx recipients was 52% of control subjects. Compared with the control subjects, LTx skeletal muscle exhibited: (1) a lower MAPR; (2) lower activity of the mitochondrial enzymes glutamate dehydrogenase (GDH), citrate synthase (CS), 2-oxogluterate dehydrogenase (OGDH), and 3-hydroxyacyl-CoA-dehydrogenase (HAD). There was no difference in the activities of anaerobic enzymes, except for higher phosphofructokinase activity; (3) a lower proportion of type I fibers; (4) a higher lactate and inosine monophosphate (IMP) content and a lower ATP content at rest indicating a high reliance on anaerobic metabolism. The reduced type I fiber proportion and severely reduced mitochondrial oxidative capacity may play an important role in exercise limitation after LTx.     

Mediation of spontaneous knee osteoarthritis by progressive chondrocyte ATP depletion in Hartley guinea pigs

OBJECTIVE: Because articular chondrocytes reside in a hypoxic milieu, anaerobic glycolysis is central in generating ATP to support chondrocyte matrix synthesis and viability, with mitochondrial oxidative phosphorylation possibly providing physiologic reserve ATP generation. Nitric oxide (NO) potently suppresses mitochondrial oxidative phosphorylation. Because enhanced cartilage NO generation occurs in osteoarthritis (OA), we systematically tested for mitochondrial dysfunction in the pathogenesis of OA. METHODS: We assessed chondrocytes for ATP depletion and for in situ changes in mitochondrial ultrastructure prior to and during the evolution of spontaneous knee OA in male Hartley guinea pigs, a model in which chondrocalcinosis also supervenes. RESULTS: Spontaneous NO release from knee cartilage samples in organ culture doubled between ages 2 months and 8 months as knee OA developed. Concomitantly, chondrocyte intracellular ATP levels declined by approximately 50%, despite a lack of mitochondrial ultrastructure abnormalities in knee chondrocytes. As ATP depletion progressed with aging in knee chondrocytes, an increased ratio of lactate to pyruvate was observed, consistent with an adaptive augmentation of glycolysis to mitochondrial dysfunction. Furthermore, we observed progressive elevation of chondrocyte ATP-scavenging nucleotide pyrophosphatase/phosphodiesterase (NPP) activity and extracellular levels of the NPP enzymatic end product inorganic pyrophosphate (PPi), which stimulate chondrocalcinosis. CONCLUSION: Profound chondrocyte ATP depletion develops in association with heightened NO generation in guinea pig knee OA. Increased NPP activity and concordant increases in extracellular PPi, which are strongly associated with human aging-associated degenerative arthropathy and directly stimulate chondrocalcinosis, may be primarily driven by chondrocyte ATP depletion. Our findings implicate a decreased mitochondrial bioenergetic reserve as a pathogenic factor in both degenerative arthropathy and chondrocalcinosis in aging.     

PKCepsilon activation augments cardiac mitochondrial respiratory post-anoxic reserve--a putative mechanism in PKCepsilon cardioprotection

Modest cardiac-overexpression of constitutively active PKCepsilon (aPKCepsilon) in transgenic mice evokes cardioprotection against ischemia. As aPKCepsilon interacts with mitochondrial respiratory-chain proteins we hypothesized that aPKCepsilon modulates respiration to induce cardioprotection. Using isolated cardiac mitochondria wild-type and aPKCepsilon mice display similar basal mitochondrial respiration, rate of ATP synthesis and adenosine nucleotide translocase (ANT) functional content. Conversely, the aPKCepsilon mitochondria exhibit modest hyperpolarization of their inner mitochondrial membrane potential (DeltaPsi(m)) compared to wild-type mitochondrial by flow cytometry. To assess whether this hyperpolarization engenders resilience to simulated ischemia, anoxia-reoxygenation experiments were performed. Mitochondria were exposed to 45 min anoxia followed by reoxygenation. At reoxygenation, aPKCepsilon mitochondria recovered ADP-dependent respiration to 44 +/- 3% of baseline compared to 28 +/- 2% in WT controls (P = 0.03) in parallel with enhanced ATP synthesis. This preservation in oxidative phosphorylation is coupled to greater ANT functional content [42% > concentration of atractyloside for inhibition in the aPKCepsilon mitochondria vs. WT control (P < 0.0001)], retention of mitochondrial cytochrome c and conservation of DeltaPsi(m). These data demonstrate that mitochondria from PKCepsilon activated mice are intrinsically resilient to anoxia-reoxygenation compared to WT controls. This resilience is in part due to enhanced recovery of oxidative phosphorylation coupled to maintained ANT activity. As maintenance of ATP is a prerequisite for cellular viability we conclude that PKCepsilon activation augmented mitochondrial respiratory capacity in response to anoxia-reoxygenation may contribute to the PKCepsilon cardioprotective program.     

Role of mitochondrial oxidative phosphorylation in the maintenance of intracellular pH.

The oxidative phosphorylation of isolated rabbit heart mitochondria and the accompanying pH changes were simultaneously measured in the same sample. The nearly constant extramitochondrial proton concentration during State 4 respiration decreased considerably under conditions of oxidative phosphorylation. On the other hand, the proton concentration increased when ATP was hydrolysed by the mitochondrial ATPase. The lysis of mitochondria by Triton X-100 equilibrating the pH difference across the inner membrane did not cause significant change in the proton concentration of the sample neither before nor after State 3 respiration. The accumulation of protons during the extramitochondrial hexokinase reaction was reduced by the mitochondrial oxidative phosphorylation. It is suggested that in the normoxic myocardial cell, the protons generated by the extramitochondrial hydrolysis of ATP are utilized during mitochondrial oxidative phosphorylation.     

The in vitro effect of adenine on pyruvate kinase-deficient red cells

In pyruvate kinase (PK)-deficient blood with high levels of reticulocytes, the degree of haemolysis after incubation at 37°C for 48 h was halved by the inclusion of adenine in the medium. The decrease in haemolysis was associated with a higher ATP level but was not related to the pH of the incubation mixture. The incorporation of adenine into nucleotides studied over a 3 h period was similar in normal and PK-deficient blood. The observed increase in cellular ATP was equivalent to that shown by radioactive measurements to have been synthesized from added adenine. Inhibition of mitochondrial oxidative phosphorylation in the reticulocytes of the PK-deficient blood by KCN reduced the amount of adenine taken up by the cells by a factor of 3 and altered the pattern of incorporation into the nucleotides. Only 25% of the adenine which entered the blood cells was converted into ATP compared with 85% in the absence of cyanide. Despite the synthesis of small amounts of ATP from labelled adenine, the overall ATP content fell to less than 50% of its original level. It is suggested that incubation with adenine increased the ATP level in the reticulocytes by virtue of mitochondrial oxidative phosphorylation thereby reducing the haemolysis of these cells.     

High aerobic glycolysis of rat hepatoma cells in culture: Role of mitochondrial hexokinase

A tumorigenic anchorage-dependent cell line (H-91) was established in culture from an azo-dye-induced rat ascites hepatoma. When grown in a glucose-containing medium the cells exhibit high rates of lactic acid production characteristic of rapidly growing tumor cells. However, when glucose is replaced with galactose the cells grow equally well but exhibit only moderately elevated rates of lactic acid production. The molecular basis for this observation cannot be attributed to differences in permeability because initial rates of glucose and galactose entry into hepatoma cells are identical. Rather, the activity of hexokinase (ATP:D-hexose 6-phosphotransferase, EC 2.7.1.1) is found to be high in hepatoma cells, about 20-fold higher than that of control and regenerating rat liver. Moreover, tumor hexokinase activity is not inhibited by low concentrations (<0.6 mM) of the reaction product glucose 6-phosphate. Additionally, 50% of the hexokinase activity of hepatoma cells is found associated with the mitochondrial fraction. This fraction is 3-fold enriched in hexokinase activity relative to the homogenate and 4-fold enriched relative to the nuclear and postmitochondrial fractions. Tumor mitochondrial hexokinase appears to be coupled directly to oxidative phosphorylation, because addition of glucose to respiring hepatoma mitochondria (after a burst of ATP synthesis) results in stimulation of respiration. In contrast, glucose has no effect on the respiration of mitochondria from control and regenerating liver. These results suggest that the high glycolytic capacity of H-91 hepatoma cells is due, at least in part, to an elevated form of hexokinase concentrated in the mitochondrial fraction of the cell.