Eunsook S. Jin

Reductive carboxylation supports growth in tumour cells with defective mitochondria

Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells. In normally functioning tumour cell mitochondria, oxidative metabolism of glucose- and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis. Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function, and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP+/NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetyl-coenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containing mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with mutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism.

Pyruvate carboxylase is required for glutamine-independent growth of tumor cells

Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in cancer. Precursors for lipids, nucleic acids, and proteins are generated in the tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many tumor cells use glutamine to feed anaplerosis. Here we studied how “glutamine-addicted” cells react to interruptions of glutamine metabolism. Silencing of glutaminase (GLS), which catalyzes the first step in glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by pyruvate carboxylase (PC), allowing the cells to use glucose-derived pyruvate rather than glutamine for anaplerosis. Although PC was dispensable when glutamine was available, forcing cells to adapt to low-glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic enzymes. Cells with high PC activity were resistant to GLS silencing and did not require glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated, glucose-dependent anaplerosis allows cells to achieve glutamine independence. Induction of PC during chronic suppression of glutamine metabolism may prove to be a mechanism of resistance to therapies targeting glutaminolysis.

Metabolic Heterogeneity in Human Lung Tumors

Non-small cell lung cancer (NSCLC) is heterogeneous in the genetic and environmental parameters that influence cell metabolism in culture. Here, we assessed the impact of these factors on human NSCLC metabolism in vivo using intraoperative (13)C-glucose infusions in nine NSCLC patients to compare metabolism between tumors and benign lung. While enhanced glycolysis and glucose oxidation were common among these tumors, we observed evidence for oxidation of multiple nutrients in each of them, including lactate as a potential carbon source. Moreover, metabolically heterogeneous regions were identified within and between tumors, and surprisingly, our data suggested potential contributions of non-glucose nutrients in well-perfused tumor areas. Our findings not only demonstrate the heterogeneity in tumor metabolism in vivo but also highlight the strong influence of the microenvironment on this feature.

Myc controls transcriptional regulation of cardiac metabolism and mitochondrial biogenesis in response to pathological stress in mice

In the adult heart, regulation of fatty acid oxidation and mitochondrial genes is controlled by the PPARgamma coactivator-1 (PGC-1) family of transcriptional coactivators. However, in response to pathological stressors such as hemodynamic load or ischemia, cardiac myocytes downregulate PGC-1 activity and fatty acid oxidation genes in preference for glucose metabolism pathways. Interestingly, despite the reduced PGC-1 activity, these pathological stressors are associated with mitochondrial biogenesis, at least initially. The transcription factors that regulate these changes in the setting of reduced PGC-1 are unknown, but Myc can regulate glucose metabolism and mitochondrial biogenesis during cell proliferation and tumorigenesis in cancer cells. Here we have demonstrated that Myc activation in the myocardium of adult mice increases glucose uptake and utilization, downregulates fatty acid oxidation by reducing PGC-1alpha levels, and induces mitochondrial biogenesis. Inactivation of Myc in the adult myocardium attenuated hypertrophic growth and decreased the expression of glycolytic and mitochondrial biogenesis genes in response to hemodynamic load. Surprisingly, the Myc-orchestrated metabolic alterations were associated with preserved cardiac function and improved recovery from ischemia. Our data suggest that Myc directly regulates glucose metabolism and mitochondrial biogenesis in cardiac myocytes and is an important regulator of energy metabolism in the heart in response to pathologic stress.

Simultaneous steady-state and dynamic 13C NMR can differentiate alternative routes of pyruvate metabolism in living cancer cells

Background: 13C hyperpolarization sensitively and non-destructively detects pyruvate-lactate exchanges in cancer cells. Results: Combining 13C hyperpolarization with isotopomer analysis allowed many pyruvate-dependent pathways to be quantified simultaneously. Conclusion: Monitoring H[13C]O3− production from hyperpolarized [1-13C]pyruvate yielded a quantitative readout of oncogene-regulated pyruvate dehydrogenase activity. Significance: This approach might enable a broader quantitative assessment of metabolic activity in tumors. Metabolic reprogramming facilitates cancer cell growth, so quantitative metabolic flux measurements could produce useful biomarkers. However, current methods to analyze flux in vivo provide either a steady-state overview of relative activities (infusion of 13C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized 13C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined 13C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-13C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-13C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[13C]O3− and [1-13C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-13C]pyruvate. Quantitation of hyperpolarized H[13C]O3− provided a single definitive metabolic rate, which was then used to convert relative rates derived from isotopomer analysis into quantitative fluxes. This revealed that H[13C]O3− appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1-13C]pyruvate metabolism, enhancing exchanges with [1-13C]lactate and suppressing H[13C]O3− formation. Furthermore, inhibiting Akt, an oncogenic kinase that stimulates glycolysis, reversed these effects, indicating that metabolism of pyruvate by both LDH and pyruvate dehydrogenase is subject to the acute effects of oncogenic signaling on glycolysis. The data suggest that combining 13C isotopomer analyses and dynamic hyperpolarized 13C spectroscopy may enable quantitative flux measurements in living tumors.

The impact of obesity, sex, and diet on hepatic glucose production in cats

Obesity is a risk factor for type 2 diabetes in cats. The risk of developing diabetes is severalfold greater for male cats than for females, even after having been neutered early in life. The purpose of this study was to investigate the role of different metabolic pathways in the regulation of endogenous glucose production (EGP) during the fasted state considering these risk factors. A triple tracer protocol using (2)H(2)O, [U-(13)C(3)]propionate, and [3,4-(13)C(2)]glucose was applied in overnight-fasted cats (12 lean and 12 obese; equal sex distribution) fed three different diets. Compared with lean cats, obese cats had higher insulin (P < 0.001) but similar blood glucose concentrations. EGP was lower in obese cats (P < 0.001) due to lower glycogenolysis and gluconeogenesis (GNG; P < 0.03). Insulin, body mass index, and girth correlated negatively with EGP (P < 0.003). Female obese cats had approximately 1.5 times higher fluxes through phosphoenolpyruvate carboxykinase (P < 0.02) and citrate synthase (P < 0.05) than male obese cats. However, GNG was not higher because pyruvate cycling was increased 1.5-fold (P < 0.03). These results support the notion that fasted obese cats have lower hepatic EGP compared with lean cats and are still capable of maintaining fasting euglycemia, despite the well-documented existence of peripheral insulin resistance in obese cats. Our data further suggest that sex-related differences exist in the regulation of hepatic glucose metabolism in obese cats, suggesting that pyruvate cycling acts as a controlling mechanism to modulate EGP. Increased pyruvate cycling could therefore be an important factor in modulating the diabetes risk in female cats.

An Electrogenerated Chemiluminescence Imaging Fiber Electrode Chemical Sensor for NADH

An electrogenerated chemiluminescence (ECL)-based imaging fiber electrode chemical sensor (IFECS) for the detection of NADH is presented. In brief, an imaging fibers distal tip was metalized with gold to serve as an electrode (i.e., an IFE). The 350-μm diameter IFE was coated with a thin, planar layer of Nafion doped with Ru(bpy)32+ to create an IFECS. An electrical contact was made to the gold layer such that Ru(bpy)32+ could be oxidized to Ru(bpy)33+ and electrochemically regenerated following the application of appropriate potentials. Following the diffusion of triethylamine or NADH into the IFE sensing layer and reaction with Ru(bpy)33+, the ECL was captured using a charge coupled device-based imaging system. The triethylamine calibration plot was linear between 0.0 and 50 mM. IFECSs represent the first demonstration whereby ECL imaging is performed through the actual sensor itself.

Influence of Liver Triglycerides on Suppression of Glucose Production by Insulin in Men

Context: The ability of insulin to suppress hepatic glucose production is impaired among subjects with increased intrahepatic triglycerides (IHTG). However, little is known about the roles of insulin on the supporting fluxes of glucose production among patients with fatty liver. Objective: To evaluate the effects of insulin on fluxes through the three potential sources of plasma glucose (glycerol, the citric acid cycle, and glycogen) among patients with fatty liver. Design, Settings, Participants, and Intervention: Nineteen men with a range of IHTG (∼0.5% to 23%) were studied after an overnight fast and during hyperinsulinemia using magnetic resonance spectroscopy and stable isotope tracers. Main Outcome Measures: IHTG, gluconeogenesis from glycerol, gluconeogenesis from the citric acid cycle, glycogenolysis, and 13C-labeled glucose produced from the citric acid cycle during hyperinsulinemia were measured. Results: Men with high IHTG had higher fluxes through all pathways contributing to glucose production during hyperinsulinemia, compared to men with low IHTG, but they had similar fluxes after the fast. Consequently, men with fatty liver had impaired insulin efficiency in suppressing total glucose production as well as fluxes through all three biochemical pathways contributing to glucose. The detection of glucose isotopomers with 13C arising from [U-13C3]propionate ingested during hyperinsulinemia demonstrated continuous gluconeogenesis from the citric acid cycle in all subjects. Conclusions: These findings challenge the concept that individual glucose production pathways are selectively dysregulated during hepatic insulin resistance. Overproduction of glucose during hyperinsulinemia in men with fatty liver results from inadequate suppression of all the supporting fluxes of glucose production in response to insulin.

Comparison of [3,4-13C2]glucose to [6,6- 2H2]glucose as a tracer for glucose turnover by nuclear magnetic resonance

A recently introduced tracer, [3,4‐13C2]glucose, was compared to the widely used tracer, [6,6‐2H2]glucose, for measurement of whole‐body glucose turnover. The rate of glucose production (GP) was measured in rats after primed infusions of [3,4‐13C2]glucose, [6,6‐2H2]glucose, or both tracers simultaneously followed by a constant infusion of tracer(s) over 90 min. Blood glucose was purified and converted into monoacetone glucose for analysis by 13C NMR (for [3,4‐13C2]glucose) or 1H and 2H NMR (for [6,6‐2H2]glucose). The values of GP measured during infusion of each single tracer were not significantly different. In rats infused with both tracers simultaneously, GP was identical as reported by each tracer, 42 ± 4 μmol/kg/min. Since 2H and 13C enrichment in glucose is typically much less than 2% for in vivo studies, [3,4‐13C2]glucose does not interfere with measurements of 13C or 2H enrichment patterns and therefore is valuable when multiple metabolic pathways are being evaluated simultaneously. Magn Reson Med 53:1479–1483, 2005.

Hepatic glucose production pathways after three days of a high-fat diet.

OBJECTIVE A three-day high-fat diet induces hepatic steatosis and hepatic insulin resistance in rats without altering fasting plasma glucose concentration or the rate of glucose production. However, as the nutrient profile available to the liver is substantially altered by a high-fat diet, we hypothesized that the relative fluxes supporting hepatic glucose production would be altered. MATERIALS/METHODS To test this hypothesis, we used multiple tracers ([3,4-(13)C(2)]glucose, (2)H(2)O, and [U-(13)C(3)]propionate) followed by NMR analysis of blood glucose to quantify net glucose production and the contributions of glycogen and key gluconeogenesis precursors in 4-5-h fasted rats. RESULTS NMR analysis demonstrated that the majority of blood glucose was derived from glycogen and the citric acid cycle, while a smaller fraction of glucose was derived from glycerol in both controls and high-fat-fed animals. High-fat feeding was associated with a two-fold increase in plasma glycerol concentration and an increase in the contribution (both fractional and absolute) of glycerol-gluconeogenesis. The increase in gluconeogenesis from glycerol tended to be balanced by a decrease in glycogenolysis. The absolute fluxes associated with the citric acid cycle including gluconeogenesis from the cycle intermediates, pyruvate cycling and the citric acid cycle flux itself, were not altered by this short high-fat diet. CONCLUSIONS A short term high-fat diet altered the specific pathways for hepatic glucose production without influencing the overall rate of glucose production or flux in the citric acid cycle.