Unlabeled Lactate Research Articles

Abstract 283: Western Diet Induces Diastolic Dysfunction Linked to Impaired Cardiac Lipid Storage Dynamics That Predate Systolic Dysfunction

Western diet (WD), containing high fat and high refined sugar, is associated with increased risk of cardiovascular disease. However, mechanisms linking WD to cardiomyopathy are far from understood. We hypothesize that WD induces cardiac dysfunction by dysregulation of cardiac lipid dynamics. Adult C57BL/6J mice were fed with either standard chow (CON, fat 4.5 %, sucrose 0%) or western diet (WD, fat 45%, sucrose 21%). At 20 weeks, WD mice exhibited higher body mass, reduced glucose tolerance, and increased isovolumetric relaxation time (IVRT), with no systolic dysfunction vs. CON. However, at 24 weeks, WD caused both systolic and diastolic dysfunction with decreases in left ventricular ejection fraction (66±2% CON vs. 54±2 WD, P <0.05) and fractional shortening (35±2% CON vs. 27±1 WD), with elevated IVRT and E/E’ ratio vs CON. Real time, dynamic-mode 13 C NMR and endpoint LC/MS of isolated perfused hearts supplied 13 C palmitate and 13 C oleate, with unlabeled glucose and lactate, revealed elevated TG content (28% increase vs. CON) with reduced TG turnover in WD hearts (35% decrease vs. CON), concurrent with early diastolic dysfunction at 20 weeks and preceding systolic dysfunction at 24 weeks. WD induced lower content of the TG lipase, ATGL (48% vs. CON), with no change in the TG synthase, DGAT1, suggesting elevated cardiac TG and lower TG turnover are due to reduced TG hydrolysis and not TAG synthesis. Impaired lipid storage dynamics can cause increased generation of potentially lipotoxic acyl intermediates, leading to cardiac dysfunction. The mitochondrial long chain fatty acid transporter, CPT1b was reduced (50%), with no change in the lesser, co-expressed CPT1a isoform in WD hearts. Despite reduced CPT1b in WD hearts, contributions from LCFAs to acetyl CoA production in the citric acid cycle were unchanged, suggesting that the WD provided sufficient LCFAs for mitochondrial oxidation and that the reduced CPT1b was not limiting. In conclusion, western diet expands the TG pool but impairs fat storage dynamics via low ATGL, leading to early diastolic dysfunction and eventual systolic dysfunction. Nutrient overload from WD leads to impaired cardiac lipid dynamics with early diastolic dysfunction that is distinguished from HFpEF due to later systolic dysfunction.

Abstract C113: The monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 triggers MCT4-dependent lactate accumulation and blocks pyruvate-lactate exchange in human cancer cells

Abstract Background: Monocarboxylate transporters (MCTs) are key modulators of lactate homeostasis and represent promising metabolic targets for molecular cancer therapeutics. The MCT1 inhibitor AZD3965 is now in clinical trial and understanding the impact of this drug on tumour cell metabolism may enable the discovery of pharmacodynamic (PD) biomarkers of target inhibition that will support the clinical development of such agents. Since MCT1 mediates the bidirectional transport of lactate and other monocarboxylates including pyruvate, here we use NMR spectroscopy to investigate the effect of AZD3965 on a) intracellular lactate levels and b) hyperpolarized 13C-pyruvate-lactate exchange, as biomarkers for MCT1 inhibition in human cancer cells with varying MCT4 expression (predictive of resistance to AZD3965). Materials and Methods: Human Raji (MCT4-) and Hut78 (MCT4 low (+)) lymphoma as well as HT29 (MCT4 high (+++)) colon carcinoma cells were treated with either 5nM or 500nM AZD3965 for 24h and levels of intracellular lactate determined by 1H NMR of cell extracts. Raji cells were also exposed to additional AZD3965 concentrations spanning 1nM-500nM. For 13C-pyruvate-lactate exchange studies, Raji cells were treated with either 5nM or 25nM AZD3965 for 24h. Cells were then incubated at 37°C in FBS-free medium and dynamic 13C NMR spectra acquired for 4 minutes with 2s intervals immediately after the addition of 10mM hyperpolarised [1-13C]pyruvic acid and 10mM unlabelled lactate. The ratio of the area under the curve for the summed lactate and pyruvate signals (LacAUC/PyrAUC) was determined to estimate pyruvate-lactate exchange. Data represent mean±SE. Results: 24h exposure to a low concentration of AZD3965 (5nM) led to increased intracellular lactate in MCT4- Raji and MCT4+ Hut78 human lymphoma cells to 2.65-fold and 10-fold respectively (p = ≤0.02) while the effect in MCT4+++ HT29 human colon carcinoma cells was insignificant (167±31% of controls, p = 0.1). Exposure to a high concentration of AZD3965 (500nM) increased intracellular lactate accumulation in HT29 cells albeit to a lesser degree than in Raji and Hut78 cells (4-fold (0.02) vs. 14 to 15-fold (p≤0.01) in the lymphoma lines). This effect is consistent with the expected blockade of lactate release in cells with low or no MCT4 expression following MCT1 inhibition. Lactate build-up in Raji cells was AZD3965 concentration-dependent being observed with as little as 1nM (up 1.7-fold), reaching a maximum at 25nM (12-fold) and plateauing thereafter. Analysis of the hyperpolarized 13C NMR data showed a significant decrease in LacAUC/PyrAUC to 31±6% in 5nM and 19±2% in 25nM AZD3965-treated Raji cells relative to controls (p<0.001). These results are consistent with MCT1-mediated transport of 13C-pyruvate being a rate limiting step in the 13C NMR-observed pyruvate-lactate exchange. Conclusions: Our data show that AZD3965 triggers intracellular lactate accumulation in a concentration- and MCT4 expression-dependent manner and inhibits 13C-pyruvate-lactate exchange (via blockade of 13C-pyruvate uptake). Intracellular lactate and hyperpolarized 13C-pyruvate-lactate exchange measurements are translatable to in vivo imaging studies and are therefore promising non-invasive metabolic biomarkers for AZD3965 and potentially other MCT1 inhibitors. Citation Format: Mounia Beloueche-Babari, Slawomir Wantuch, Markella Koniordou, Harry G. Parkes, Vaitha Arunan, Thomas R. Eykyn, Paul D. Smith, Martin O. Leach. The monocarboxylate transporter 1 (MCT1) inhibitor AZD3965 triggers MCT4-dependent lactate accumulation and blocks pyruvate-lactate exchange in human cancer cells. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C113.

Abstract 1158: Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay

Abstract Objective: The purpose of this study was to evaluate the feasibility of monitoring lactate uptake and utilization in intact human cancer cells using a dynamic 1H-NMR assay and [3-13C]-lactate. This would provide a simple, real-time method for monitoring enzyme kinetics in cells as an alternative to spectrophotometry - which cannot be applied to intact cells - or dynamic nuclear polarisation-NMR, which requires complex mathematical modelling. The ability to better understand and measure lactate metabolism in cancer cells could be applied to monitor MCT1 inhibition. Methods: 3×10^7 MDA-MB-231 human breast cancer cells were suspended in 400μl serum-free DMEM plus 100μl D2O in a 5mm NMR tube. A mixture of 10mM [3-13C]-lactate combined with varying concentrations of unlabelled 12C-pyruvate (0mM, 5mM or 10mM) was added and proton spectra were acquired every minute for 30 minutes on a 500MHz Bruker spectrometer. For comparison, cells were also incubated with 10mM [3-13C]-pyruvate and either 0mM or 10mM 12C-lactate. Results: The presence of 13C-pyruvate was detected in real-time following addition of [3-13C]-lactate indicating that 13C-label had been exchanged from lactate to pyruvate via the enzyme lactate dehydrogenase. The optimisation of reagent concentrations, however, revealed that this observation was dependent on the addition of unlabelled 12C-pyruvate and the apparent rate of 13C-label exchange was proportional to the concentration of 12C-pyruvate added. The data show that no 13C-pyruvate signal is detected when 10mM 13C-lactate is added alone. However, 6% of the 10mM 13C-lactate is exchanged with 13C-pyruvate when 5mM 12C-pyruvate is added (rate = 1.1×10-3mM s-1). This increases to 12% when the 12C-pyruvate concentration was increased to 10mM (rate = 2.2×10-3 mM s-1). The total pyruvate concentration (12C + 13C-pyruvate) remained constant, which suggests that there is no net production of pyruvate. In comparison, the 13C-pyruvate experiment was less dependent on the addition of unlabelled 12C-lactate for 13C-label exchange to be observed. Conclusion: The fact that unlabelled pyruvate was required for a reaction to be observed following addition of 13C-lactate suggests that the position of the isotopic equilibrium favours the label to remain in the lactate pool. Therefore a large amount of unlabelled pyruvate is required for the exchange process to be observed. This would also explain why the addition of unlabelled lactate was not required in the 13C-pyruvate experiment, since to reach isotopic equilibrium, a large proportion of the 13C-pyruvate needs to immediately exchange with the naturally larger lactate pool. Nevertheless, these findings demonstrate the feasibility of using this 1H-NMR assay to measure [3-13C]-lactate uptake in real-time. This could inform on metabolic pathway activity in response to MCT1 inhibition in intact cells. Citation Format: Emily G. Wholey, Harold G. Parkes, Paul Workman, Martin O. Leach, Mounia Beloueche-Babari. Real-time assessment of uptake and utilization of lactate in intact human breast cancer cells using a 1H-NMR-based assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1158. doi:10.1158/1538-7445.AM2015-1158

Probing lactate dehydrogenase activity in tumors by measuring hydrogen/deuterium exchange in hyperpolarized l-[1-(13)C,U-(2)H]lactate.

13C magnetic resonance spectroscopy and spectroscopic imaging measurements of hyperpolarized 13C label exchange between exogenously administered [1-13C]pyruvate and endogenous lactate, catalyzed by lactate dehydrogenase (LDH), has proved to be a powerful approach for probing tissue metabolism in vivo. This experiment has clinical potential, particularly in oncology, where it could be used to assess tumor grade and response to treatment. A limitation of the method is that pyruvate must be administered in vivo at supra-physiological concentrations. This problem can be avoided by using hyperpolarized [1-13C]lactate, which can be used at physiological concentrations. However, sensitivity is limited in this case by the relatively small pyruvate pool size, which would result in only low levels of labeled pyruvate being observed even if there was complete label equilibration between the lactate and pyruvate pools. We demonstrate here a more sensitive method in which a doubly labeled lactate species can be used to measure LDH-catalyzed exchange in vivo. In this experiment exchange of the C2 deuterium label between injected hyperpolarized l-[1-13C,U-2H]lactate and endogenous unlabeled lactate is observed indirectly by monitoring phase modulation of the spin-coupled hyperpolarized 13C signal in a heteronuclear 1H/13C spin–echo experiment.

Quantification of in vivo metabolic kinetics of hyperpolarized pyruvate in rat kidneys using dynamic 13C MRSI

With signal‐to‐noise ratio enhancements on the order of 10,000‐fold, hyperpolarized MRSI of metabolically active substrates allows the study of both the injected substrate and downstream metabolic products in vivo. Although hyperpolarized [1‐13C]pyruvate, in particular, has been used to demonstrate metabolic activities in various animal models, robust quantification and metabolic modeling remain important areas of investigation. Enzyme saturation effects are routinely seen with commonly used doses of hyperpolarized [1‐13C]pyruvate; however, most metrics proposed to date, including metabolite ratios, time‐to‐peak of metabolic products and single exchange rate constants, fail to capture these saturation effects. In addition, the widely used small‐flip‐angle excitation approach does not correctly model the inflow of fresh downstream metabolites generated proximal to the target slice, which is often a significant factor in vivo. In this work, we developed an efficient quantification framework employing a spiral‐based dynamic spectroscopic imaging approach. The approach overcomes the aforementioned limitations and demonstrates that the in vivo 13C labeling of lactate and alanine after a bolus injection of [1‐13C]pyruvate is well approximated by saturatable kinetics, which can be mathematically modeled using a Michaelis–Menten‐like formulation, with the resulting estimated apparent maximal reaction velocity Vmax and apparent Michaelis constant KM being unbiased with respect to critical experimental parameters, including the substrate dose, bolus shape and duration. Although the proposed saturatable model has a similar mathematical formulation to the original Michaelis–Menten kinetics, it is conceptually different. In this study, we focus on the 13C labeling of lactate and alanine and do not differentiate the labeling mechanism (net flux or isotopic exchange) or the respective contribution of various factors (organ perfusion rate, substrate transport kinetics, enzyme activities and the size of the unlabeled lactate and alanine pools) to the labeling process. Copyright © 2011 John Wiley & Sons, Ltd.

The antidiabetic metformin is toxic to liver mitochondria of Zucker Diabetic Fatty (ZDF) rats: a cellular metabolomic study with 13C‐lactate and carbon 13 NMR

Metformin decreases hyperglycemia in diabetic subjects and animals. This effect results in part from the inhibition of hepatic gluconeogenesis. The objective of this study was to investigate in vitro the effect of metformin on hepatic lactate gluconeogenesis by using an innovative approach.Precision‐cut liver slices from adult male ZDF rats were incubated with variously 13C‐labeled lactates (5 mM) or with 5 mM unlabeled lactate plus 25 mM 13C‐bicarbonate. Substrate removal and product formation were measured by enzymatic and 13C‐NMR methods.In slices from fasted lean ZDF rats, metformin dose‐dependently inhibited 13C‐glucose synthesis from [2‐13C]‐lactate (IC50 2.3 mM). The labeling patterns of the glucoses synthesized indicated that lactate gluconeogenesis involved not only the passage of carbons through pyruvate carboxylase and the reversible equilibration of oxaloacetate with fumarate but also the entire tricarboxylic acid cycle.In slices from fed diabetic ZDF rats, 2 mM metformin inhibited lactate consumption, glucose production, the synthesis of all the 13C‐labeled glucose carbons, the production of 13CO2 and reduced the cellular ATP level. By contrast, metformin dramatically increased lactic acid production and ketogenesis.These results strongly suggest that a compound found to be toxic to liver mitochondria in vitro should not necessarily be withdrawn from drug development.

Metabolic profiling of heat or anoxic stress in mouse C2C12 myotubes using multinuclear magnetic resonance spectroscopy

In the present study, the metabolic effects of heat and anoxic stress in myotubes from the mouse cell line C2C12 were investigated by using a combination of 13C, 1H, and 31P nuclear magnetic resonance (NMR) spectroscopy and enrichment with [ 13C]-glucose. Both the 13C and the 1H NMR spectra showed reduced levels of the amino acids alanine, glutamate, and aspartate after heat or anoxic stress. The decreases were smallest at 42°C, larger at 45°C, and most pronounced after anoxic conditions. In addition, in both the 1H and the 31P NMR spectra, decreases in the high-energy phosphate compounds adenosine triphosphate and phosphocreatine with increasing severity of stress were identified. At anoxic conditions, an increase in 13C-labeled lactate and appearance of glycerol-3-phosphate were observed. Accumulation of lactate and glycerol-3-phosphate is in agreement with a shift to anaerobic metabolism due to inhibition of the aerobic pathway in the mitochondria. Conversely, lower levels of unlabeled ( 12C) lactate were apparent at increasing severity of stress, which indicate that lactate is released from the myotubes to the medium. In conclusion, the metabolites identified in the present study may be useful markers for identifying severity of stress in muscles.

Abstract A72: Assessment of pyuvate dehydrogenase kinase inhibition by dichloroacetate in human colon carcinoma cells by dynamic hyperpolarized 13C MRS and steady state 1H MRS

Abstract Dichloroacetate (DCA) is an inhibitor of pyruvate dehydrogenase kinase (PDK), which causes the activation of pyruvate dehydrogenase and increases glucose oxidation by promoting the influx of acetyl-CoA into the mitochondria and the Krebs cycle [1]. The aim of this work was to use hyperpolarised 1-13C pyruvate 13C-magnetic resonance spectroscopy (MRS) and conventional 1H MRS to measure lactate formation/production in real-time and in steady state, respectively, in order to study the mechanism of action of DCA and to develop a non-invasive biomarker for response following PDK inhibition. Inhibition of PDK by DCA should cause a drop in lactate production. The hyperpolarised 13C experiment measures the exchange of labeled pyruvate for labeled lactate which has been shown to be proportional to lactate dehydrogenase activity and the availability of substrates as well as cellular NADH, the enzyme co-factor [2]. Conversely, steady state measurements of eupolarised (i.e. unlabelled) lactate production report on the overall flux through glycolysis. Dynamic nuclear polarization (DNP) was used to hyperpolarise labelled 1-13C pyruvate, and to measure lactate formation in real-time in intact cells by 13C-MRS. Intact human HT29 (colon) carcinoma cells were studied after 24 hours of DCA treatment (100mM). Culture media and extracts from the DCA-treated and control cells were analysed by 1H-MRS. Cell cycle analysis was also performed. DCA treatment of HT29 cells caused a drop in cell number (∼40% of controls, p<0.0001) and G1 arrest (p<0.0001). A dramatic drop in lactate formation (0.69+/−0.06 nmol/s/106 cells in control versus 0.06+/−0.01 nmol/s/106 cells in treated cells; p<0.0001) was measured in real-time by DNP 13C-MRS. This could be due to the combined effect of: i) a drop in cellular NADH due to apoptosis, as DCA is known to induce apoptosis [1] which causes NADH loss [2]; and/or ii) an increase in glucose oxidation causing a decrease in NADH available for further metabolism of pyuvate to lactate. 1H spectra of the culture media of DCA treated cells also showed a reduction in steady state eupolarized lactate production (38% of controls; p<0.0001), increased alanine uptake (p<0.0001) and no difference in glucose uptake when compared with controls. Increases in alanine (p<0.0001), glucose (p=0.02), free choline (p=0.02) and glycerophosphocholine (p<0.0001) and decrease in phosphocholine (p<0.0001) were found in DCA-treated cell extracts. The phospholipid changes could be associated with modulation in membrane turnover, as DCA is known to reduce proliferation in cancer cells [1]. DCA treatment resulted in reduced lactate formation/production in HT29 cells, as shown by both real time and steady-state measurements. These changes are consistent with the mechanism of action and cellular response to PDK inhibition. DCA treatment also altered phospholipid metabolism, which could also be used as biomarkers of response. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):A72.

L-3-11C-Lactate as a PET Tracer of Myocardial Lactate Metabolism: A Feasibility Study

Lactate is a key myocardial energy source. Lactate metabolism is altered in a variety of conditions, such as exercise and diabetes mellitus. However, to our knowledge, noninvasive quantitative measurements of myocardial lactate metabolism have never been performed because of the lack of an adequate radiotracer. In this study we tested L-3-(11)C-lactate ((11)C-lactate) as such a tracer. Twenty-three dogs were studied under a wide range of metabolic interventions. (11)C-Lactate and (13)C-lactate were injected as boluses and PET data were acquired for 1 h. Concomitant arterial and coronary sinus (ART/CS) blood samples were collected to identify (13)C-lactate metabolites and to measure fractional myocardial extraction/production of (11)C metabolite fractions ((11)C acidic: (11)CO(2) and (11)C-lactate; (11)C basic: (11)C-labeled amino acids; and (11)C neutral: (11)C-glucose). Lactate metabolism was quantified using 2 PET approaches: monoexponential clearance analysis (oxidation only) and kinetic modeling of PET (11)C-myocardial curves. Arterial (11)C acidic, neutral, and basic metabolites were identified as primarily (11)C-labeled lactate + pyruvate, glucose, and alanine, respectively. Despite a significant contribution of (11)C-glucose (23%-45%) and (11)C-alanine (<11%) to total arterial (11)C activity, both were minimally extracted(+)/produced(-) by the heart (1.7% +/- 1.0% and -0.12% +/- 0.84%, respectively). Whereas extraction of (11)C-lactate correlated nonlinearly with that of unlabeled lactate extraction (r = 0.86, P < 0.0001), (11)CO(2) production correlated linearly with extraction of unlabeled lactate (r = 0.89, P < 0.0001, slope = 1.20 +/- 0.13). In studies with physiologic free fatty acids (FFA) (415 +/- 216 nmol/mL), (11)C-lactate was highly extracted (32% +/- 12%) and oxidized (26% +/- 14%), and PET monoexponential clearance and kinetic modeling analyses resulted in accurate estimates of lactate oxidation and metabolism. In contrast, supraphysiologic levels of plasma FFA (4,111 +/- 1,709 nmol/mL) led to poor PET estimates of lactate metabolism due to negligible lactate oxidation (1% +/- 2%) and complete backdiffusion of unmetabolized (11)C-lactate into the vasculature (28% +/- 22%). Under conditions of net lactate extraction, L-3-(11)C-lactate faithfully traces myocardial metabolism of exogenous lactate. Furthermore, in physiologic substrate environments, noninvasive measurements of lactate metabolism are feasible with PET using myocardial clearance analysis (oxidation) or compartmental modeling. Thus, L-3-(11)C-lactate should prove quite useful in widening our understanding of the role that lactate oxidation plays in the heart and other tissues and organs.

Isotopomer subspaces as indicators of metabolic-pathway structure

The relative abundances and rates of formation of particular isotopic isomers (isotopomers) of metabolic intermediates from 13C-labelled substrates in living cells provide information on the routes taken by the initial 13C-atoms. When a primary substrate such as [U, 13C] d-glucose is added to human erythrocytes, the pattern of labels in terminal metabolites is determined by a set of carbon-group exchange reactions in both glycolysis and the pentose phosphate pathway (PPP). Of a given terminal metabolite, not all possible isotopomers will be produced from each possible primary substrate isotopomer. There are only 8 different 13C-isotopomers of lactate but not all of these are produced when one of the 64 possible 13C-isotopomers of glucose is used as the input substrate; thus a subset of all 63 glucose isotopomers×8 lactate isotopomers+1 unlabelled glucose×1 unlabelled lactate=505 pattern associations, would be produced if a complete experimental analysis were performed with all the glucose variants. The pattern of labelling in this isotopomer subspace reflects the nature of the re-ordering reactions that ‘direct’ the metabolism. Predicting the combinatorial rearrangements for particular sets of reactions and comparing these with real data should enable conclusions to be drawn about which enzymes are involved in the real metabolic system. An example of the glycolysis–PPP system is discussed in the context of a debate that occurred around the F- and L-type PPPs and which one actually operates in the human RBC. As part of this discussion we introduce the term ‘ combinatorial deficit’ of all possible isotopomers and we show that this deficit is less for the F- than the L-type pathway.

Red blood cell lactate transport in sickle disease and sickle cell trait

This study determined and compared rates and mechanisms of lactate transport in red blood cells (RBCs) of persons with 1) sickle cell disease (HbSS), 2) sickle cell trait (HbAS), and 3) a control group (HbAA). Blood samples were drawn from 30 African-American volunteers (10 HbSS, 10 HbAS, 10 HbAA). Lactate influx into RBCs was measured by using [14C]lactate at six (2, 5, 10, 15, 25, and 40 mM) unlabeled lactate concentrations. The monocarboxylate transporter pathway was blocked by p-chloromercuriphenylsulfonic acid to determine its percent contribution to total lactate influx. Generally, total lactate influx into RBCs from the HbSS group was significantly greater than influx into RBCs from HbAS or HbAA, with no difference between HbAS and HbAA. Faster influx into HbSS RBCs was attributed to increased monocarboxylate transporter activity [increased apparent Vmax (V'max)]. V'max (4.7 +/- 0.6 micromol x ml(-1) x min(-1)) for HbSS RBCs was significantly greater than V'max of HbAS RBCs (2.9 +/- 1.5 micromol x ml(-1) x min(-1)) and HbAA RBCs (2.0 +/- 0.5 micromol x ml(-1) x min(-1)). Km (42.8 +/- 8 mM) for HbSS RBCs was significantly greater than Km (27 +/- 12 mM) for HbAA RBCs. We suspect that elevated erythropoietin levels in response to chronic anemia and/or pharmacological treatment (erythropoietin injections, hydroxyurea ingestion) is the underlying mechanism for increased lactate transport capacity in HbSS RBCs.

Lactate is a preferential oxidative energy substrate over glucose for neurons in culture.

The authors investigated concomitant lactate and glucose metabolism in primary neuronal cultures using 13C- and 1H-NMR spectroscopy. Neurons were incubated in a medium containing either [1-13C]glucose and different unlabeled lactate concentrations, or unlabeled glucose and different [3-13C]lactate concentrations. Overall, 13C-NMR spectra of cellular extracts showed that more 13C was incorporated into glutamate when lactate was the enriched substrate. Glutamate 13C-enrichment was also found to be much higher in lactate-labeled than in glucose-labeled conditions. When glucose and lactate concentrations were identical (5.5 mmol/L), relative contributions of glucose and lactate to neuronal oxidative metabolism amounted to 21% and 79%, respectively. Results clearly indicate that when neurons are in the presence of both glucose and lactate, they preferentially use lactate as their main oxidative substrate.

Dichloroacetate effects on glucose and lactate oxidation by neurons and astroglia in vitro and on glucose utilization by brain in vivo.

Neuronal cultures in vitro readily oxidized both D-[(14)C]glucose and l-[(14)C]lactate to (14)CO(2), whereas astroglial cultures oxidized both substrates sparingly and metabolized glucose predominantly to lactate and released it into the medium. [(14)C]Glucose oxidation to (14)CO(2) varied inversely with unlabeled lactate concentration in the medium, particularly in neurons, and increased progressively with decreasing lactate concentration. Adding unlabeled glucose to the medium inhibited [(14)C]lactate oxidation to (14)CO(2) only in astroglia but not in neurons, indicating a kinetic preference in neurons for oxidation of extracellular lactate over intracellular pyruvatelactate produced by glycolysis. Protein kinase-catalyzed phosphorylation inactivates pyruvate dehydrogenase (PDH), which regulates pyruvate entry into the tricarboxylic acid cycle. Dichloroacetate inhibits this kinase, thus enhancing PDH activity. In vitro dichloroacetate stimulated glucose and lactate oxidation to CO(2) and reduced lactate release mainly in astroglia, indicating that limitations in glucose and lactate oxidation by astroglia may be due to a greater balance of PDH toward the inactive form. To assess the significance of astroglial export of lactate to neurons in vivo, we attempted to diminish this traffic in rats by administering dichloroacetate (50 mgkg) intravenously to stimulate astroglial lactate oxidation and then examined the effects on baseline and functionally activated local cerebral glucose utilization (lCMR(glc)). Dichloroacetate raised baseline lCMR(glc) throughout the brain and decreased the percent increases in lCMR(glc) evoked by functional activation. These studies provide evidence in support of the compartmentalization of glucose metabolism between astroglia and neurons but indicate that the compartmentalization may be neither complete nor entirely obligatory.

Metabolic phenotyping of the diseased rat heart using 13C-substrates and ex vivo perfusion in the working mode.

The objective of the present study was to compare energy substrate fluxes through metabolic pathways leading to mitochondrial citrate synthesis and release in normal and diseased rat hearts using 13C-substrates and mass isotopomer analysis by gas chromatography-mass spectrometry (GCMS). This study was prompted by our previous finding of a modulated citrate release by perfused rat hearts and by the possibility that a dysregulated myocardial citrate release represents a specific chronic alteration of energy metabolism in cardiac patients. The 15-week-old spontaneously hypertensive rat (SHR) was chosen as our animal model of disease and the Wistar-Kyoto (WKY) rat as its matched control. Ex vivo work-performing hearts were perfused with a semi-recirculating buffer containing physiological concentrations of unlabeled (glucose) and 13C-labeled ([U-13C3](lactate + pyruvate) and/or [1-(13)C]oleate) substrates. In parallel to the continuous monitoring of indices of the heart's functional and physiological status, the following metabolic parameters were documented: (i) citrate release rates and citric acid cycle intermediate tissue levels, (ii) the contribution of fatty acids as well as pyruvate decarboxylation and carboxylation to citrate synthesis, and (iii) lactate and pyruvate uptake and efflux rates. Working hearts from both rat species showed a similar percent contribution of carbohydrates for citrate synthesis through decarboxylation (70%) and carboxylation (10%). SHR hearts showed the following metabolic alterations: a higher citrate release rate, which was associated with a parallel increase in its tissue level, a lower contribution of oleate beta-oxidation to citrate synthesis, and an accelerated efflux rate of unlabeled lactate from glycolysis. These metabolic changes were not explained by differences in myocardial oxygen consumption, cardiac performance or efficiency, nor correlated with indices of tissue necrosis or ischemia. This study demonstrates how the alliance between ex vivo semi-recirculating working perfused rat hearts with 13C-substrates and mass isotopomer analysis by GCMS, can provide an unprecedented insight into the metabolic phenotype of normal and diseased rat hearts. The clinical relevance of metabolic alterations herein documented in the SHR heart is suggested by its resemblance to those reported in cardiac patients. Taken altogether, our results raise the possibility that the increased citrate release of diseased hearts results from an imbalance between citrate synthesis and utilization rates, which becomes more apparent underconditions of substrate abundance.

Lactate carbon does not enter the sugars of lipopolysaccharide when gonococci are grown in a medium containing glucose and lactate: implications in vivo

In media containing glucose, lactate stimulates the metabolism of gonococci at concentrations that simulate conditions in vivo. Nuclear magnetic resonance (NMR) spectroscopy of 13C-labelled lipids obtained from gonococci grown in a synthetic medium with 13C-labelled lactate and unlabelled glucose (culture A), 13C-labelled glucose alone (culture B) or 13C-labelled glucose and unlabelled lactate (culture C) showed lactate carbon was not present in glycerol/ethanolamine residues of lipids from culture A. This indicated that, in the presence of glucose, lactate gluconeogenesis is shut down. Hence, the stimulation of metabolism could result from the production of extra energy because lactate is used solely for conversion to acetyl-CoA, the precursor of fatty acid synthesis and the components of the tricarboxylic acid cycle. In this paper, additional evidence for lack of gluconeogenesis has been sought using a different approach. The carbohydrate moieties of lipopolysaccharide (LPS) have been examined for lactate carbon after gonococci were grown with lactate and glucose. Two methods were used: NMR spectroscopy of 13C-labelled lipopolysaccharide purified from the three cultures described above showed that, in the presence of glucose, lactate carbon, in contrast to glucose carbon, was not in the carbohydrate moiety. Also, 14C-labelled lactate was added to a culture containing unlabelled glucose and lactate (culture A) and [ 14C]glucose to cultures containing unlabelled glucose without unlabelled lactate (culture B) and with unlabelled lactate (culture C). When LPS samples purified from these cultures were subjected to hydrazinolysis, the ratio of the radioactivity of water-soluble products (carbohydrate moieties) to those of chloroform-soluble products (fatty acids) was much lower when [ 14C]lactate was used in culture A, than when [ 14C]glucose was used in cultures B and C. Thus, in the presence of glucose, lactate carbon, unlike glucose carbon, is incorporated predominantly into fatty acids of LPS, not into its carbohydrate moieties. There is no doubt, therefore, that gluconeogenesis is shut off when lactate is present with glucose and there is a consequent stimulation of metabolism. This probably occurs in vivo on mucous surfaces, where gonococci are surrounded by a mixture of glucose and lactate in the secretions.

Peroxisome Proliferator-activated Receptor α (PPARα) Influences Substrate Utilization for Hepatic Glucose Production

The hypoglycemia seen in the fasting PPARalpha null mouse is thought to be due to impaired liver fatty acid beta-oxidation. The etiology of hypoglycemia in the PPARalpha null mouse was determined via stable isotope studies. Glucose, lactate, and glycerol flux was assessed in the fasted and fed states in 4-month-old PPARalpha null mice and in C57BL/6 WT maintained on standard chow using a new protocol for flux assessment in the fasted and fed states. Hepatic glucose production (HGP) and glucose carbon recycling were estimated using [U-(13)C(6)]glucose, and HGP, lactate, and glycerol turnover was estimated utilizing either [U-(13)C(3)]lactate or [2-(13)C]glycerol infused subcutaneously via Alza miniosmotic pumps. At the end of a 17-h fast, HGP was higher in the PPARalpha null mice than in WT by 37% (p < 0.01). However, recycling of glucose carbon from lactate back to glucose was lower in the PPARalpha null than in WT (39% versus 51%, p < 0.02). The lack of conversion of lactate to glucose was confirmed using an [U-(13)C(3)]lactate infusion. In the fasted state, HGP from lactate and lactate production were decreased by 65 and 55%, respectively (p < 0.05) in PPARalpha null mice. In contrast, when [2-(13)C]glycerol was infused, glycerol production and HGP from glycerol increased by 80 and 250%, respectively (p < 0.01), in the fasted state of PPARalpha null mice. The increased HGP from glycerol was not suppressed in the fed state. While little change was evident for phosphoenolpyruvate carboxykinase (PEPCK) expression, pyruvate kinase expression was decreased 16-fold in fasted PPARalpha null mice as compared with the wild-type control. The fasted and fed insulin levels were comparable, but blood glucose levels were lower in the PPARalpha null mice than in controls. In conclusion, PPARalpha receptor function creates a setpoint for a metabolic network that regulates the rate and route of HGP in the fasted and fed states, in part, by controlling the flux of glycerol and lactate between the triose-phosphate and pyruvate/lactate pools.

Agreement between capillary and arterial lactate in the newborn

Arterial blood lactate is a reliable indicator of tissue oxygen debt and is of value in expressing the degree and prognosis of circulatory failure as a result of various diseases. Therefore, the practical issue of whether capillary lactate measurements might be of equal value was investigated in newborns. In total, 193 simultaneous measurements of capillary and arterial blood lactate concentrations were performed in 25 newborn babies with an indwelling umbilical arterial catheter. A strong linear correlation was found between capillary and arterial lactate concentration (Lcap= 1.02 Lart+ 0.04; r= 0.98; p &lt; 0.001). The mean difference was −0.08 mmol/1 and the limits of agreement (± 2 SD) were ± 0.69 mmol/1 (−0.77 to 0.61 mmol/1). Conclusion: Our data show that capillary blood lactate measurements in newborn babies yield lactate concentrations equivalent to arterial measurements over a large concentration range.

Evidence of separate pathways for lactate uptake and release by the perfused rat heart.

The simultaneous release and uptake of lactate by the heart has been observed both in vivo and ex vivo; however, the pathways underlying these observations have not been satisfactorily explained. Consequently, the purpose of this study was to test the hypothesis that hearts release lactate from glycolysis while simultaneously taking up exogenous lactate. Therefore, we determined the effects of fatty acids and diabetes on the regulation of lactate uptake and release. Hearts from control and 1-wk diabetic animals were perfused with 5 mM glucose, 0.5 mM [3-(13)C]lactate, and 0, 0.1, 0.32, or 1.0 mM palmitate. Parameters measured include perfusate lactate concentrations, fractional enrichment, and coronary flow rates, which enabled the simultaneous, but independent, measurements of the rates of 1) uptake of exogenous [(13)C]lactate and 2) efflux of unlabeled lactate from metabolism of glucose. Although the rates of lactate uptake and efflux were both similarly inhibited by the addition of palmitate, (i.e., the ratio of lactate uptake to efflux remained constant), the ratio of lactate uptake to efflux was significantly higher in the controls compared with the diabetic group (1.00 +/- 0.14 vs. 0.50 +/- 0.07, P < 0.002). These data, combined with heterogeneous (13)C enrichment of tissue lactate, pyruvate, and alanine, suggest that glycolytically derived lactate production and oxidation of exogenous lactate operate as functionally separate metabolic pathways. These results are consistent with the concept of an intracellular lactate shuttle.

Multiple compartments with different metabolic characteristics are involved in biosynthesis of intracellular and released glutamine and citrate in astrocytes.

The metabolism of glucose and lactate was investigated in cultured mouse cerebellar astrocytes, a culture preparation consisting of a homogeneous population of cells, by incubating the cells in a medium containing either [U-(13)C]glucose or [U-(13)C]lactate in combination with unlabeled lactate and glucose, respectively. After the incubation, cell extracts and media were analyzed by GC/MS (gas chromatography/mass spectrometry) for labeling patterns in aspartate, glutamate, and glutamine, as well as the tricarboxylic acid (TCA) cycle constituents citrate and fumarate. Triple labeling of extracellular citrate exceeded that of double labeling from [U-(13)C]glucose. This was not the case when lactate was the labeled precursor. These results indicate that pyruvate carboxylation in biosynthesis of releasable citrate was less prominent from lactate compared with glucose. As observed in the case of extracellular citrate, triple labeling of intracellular aspartate was higher than double labeling when [U-(13)C]glucose was the precursor, but not with [U-(13)C]lactate as precursor. The pattern of labeling in citrate was different intra- and extracellularly and the extent of labeling extracellularly was 10 times higher using [U-(13)C]glucose compared with [U-(13)C]lactate. However, the intracellular citrate labeling from [U-(13)C]glucose only exceeded that originating from labeled lactate by a factor of two. This is in contrast to the labeling pattern obtained for glutamine, since intracellularly this was equally prominent using [U-(13)C]glucose and [U-(13)C]lactate as substrates. Moreover, extracellularly the labeling was only slightly higher when using [U-(13)C]glucose compared with [U-(13)C]lactate. Intracellular glutamate and extracellular glutamine exhibited similar incorporation patterns with regard to double compared with triple labeling and the extent of incorporation of label from [U-(13)C]lactate compared with [U-(13)C]glucose. It should be noted that the main intracellular pools of citrate and glutamine were compartmentalized; i.e., releasable citrate and glutamine exhibited a labeling pattern distinctly different from that of their intracellular pools. Moreover, carboxylation of pyruvate using glucose as the precursor was more important for biosynthesis of releasable glutamine and citrate, compared with their intracellular pools. Based on the results a model of multiple compartments is suggested. The compartments differ with regard to utilization of lactate and glucose, synthetic pathways for TCA cycle intermediates and amino acids, particularly citrate and glutamine, as well as the contents of different metabolites.

In a medium containing glucose, lactate carbon is incorporated by gonococci predominantly into fatty acids and glucose carbon incorporation is increased: implications regarding lactate stimulation of metabolism

The reason for stimulation by lactate of metabolism of gonococci growing in a medium containing glucose, which enhances pathogenicity by increasing growth rate, lipopolysaccharide (LPS) synthesis and protein formation, has been investigated. Tricine dodecylpolyacrylamide gel electrophoresis (SDS-PAGE) and thin layer chromatography (TLC) on homogenates of gonococci grown in this medium with [ 14C]lactate showed that lactate carbon was preferentially incorporated into lipid and LPS. Nuclear magnetic resonance (NMR) spectroscopy on lipid extracted from gonococci grown in the glucose containing medium with [ 13C]lactate showed that lactate carbon was incorporated into fatty acid moieties and not into ethanolamine or glycerol moieties. In contrast, NMR on lipid from gonococci grown with [ 13C]glucose indicated glucose carbon in both moieties. When unlabelled lactate was added, lipid synthesis from [ 13C]glucose was stimulated and small amounts of different fatty acids were formed. The NMR data shows that gluconeogenesis from lactate carbon does not occur in the presence of glucose, suggesting that lactate is used solely for rapid production, via pyruvate, of acetyl CoA, the precursor not only for fatty acid synthesis but also for the constituents and products of the citric acid cycle, including ATP. The rapid formation of a high level of acetyl CoA is the probable reason for the stimulation of metabolism and oxygen uptake by lactate. 14C label on LPS was detected in its fatty acids. Most proteins that stained with silver in tricine SDS- PAGE were not significantly labelled by [ 14C]lactate in the glucose-containing medium. Two of three appreciably labelled proteins were identified by N-terminal sequencing as GroEL and porin 1B, and one of the two less labelled proteins was similar to peroxiredoxin type proteins. There were no signs of specific induction of these proteins by lactate and their labelling was consistent with fatty acids in attached lipid.