Lactate Metabolism and Its Effects on Glucose Metabolism in an Excised Neural Tissue

Abstract: Chains of lumbar sympathetic ganglia, excised from 15-day-old chicken embryos, were incubated for 4 h at 36°C in a bicarbonate-buffered physiological salt solution containing 5.5 mM glucose and equilibrated with 5% CO₂–95% O₂. [U-¹⁴C]Glucose and [U-¹⁴C]lactate were used as tracers to measure the products of glucose and lactate metabolism, respectively, including CO₂, lactate, and constituents of the tissue. When 5 mM lactate was added to bathing solution containing 5.5 mM glucose, lactate carbon displaced 50–70% of the glucose carbon otherwise used for CO₂ production and provided about three times as much carbon for CO₂ as did glucose. The lactate addition increased the total carbon incorporated into CO₂ and into constituents of the tissue above those observed with glucose alone and also increased the lactate released to the bathing solution from [U-¹⁴C]-glucose. The latter increase was evidently due to an interference with reuptake of the lactate released from the ganglion cells, not to an increase in the cellular release itself. When the volume of bathing solution was increased 10-fold relative to that of the tissue, the average output of CO₂ from [U-¹⁴C]glucose during a 4-h incubation was decreased by 50% when 5 mM lactate was present but was not affected significantly in the absence of added lactate. It is concluded that the effect of changing volume in the presence of lactate was due to the effects of lactate on glucose metabolism described above and resulted from a lower average lactate concentration in the smaller volume than in the larger one, due to metabolic depletion of the added lactate. Consumable substrates other than lactate, such as glutamine and certain amino acids, also affected glucose metabolism.     

Simulation of shoot chloride accumulation: separation of physical and biochemical processes governing plant salt tolerance

The effect of growth under saline condition (100 mol m^-3 NaCl in the nutrient solution) on the influx and the efflux of glucose from roots of cotton plants was analysed utilising the non metabolisable glucose analogue [¹⁴C]-3-O-methylglucose ([¹⁴C]3-OMG). Apical segments (1 cm long) excised from cotton roots took up [¹⁴C]3-OMG. At each tested concentration (5–500 mmol m^-3), the influx was completely inhibited by the presence of the protonophore carbonylcyanide-m-chlorophenyl hydrazone (CCCP) indicating that it is mediated by a H⁺-coupled co-transport mechanism. The CCCP-sensitive [¹⁴C]3-OMG influx was lower in the root segments excised from plants grown on saline solution than in the controls, and this was particularly evident at lower external concentrations. This difference was not due to a lower H⁺ apoplastic availability. In fact, the saline condition did not affect the pH of the rhizosphere and indeed the H⁺–ATPase activity, evaluated in plasma membrane vesicles purified from saline-treated plants, was higher (+23%) than in the controls. The lower uptake of [¹⁴C]3-OMG into saline treated root segments was related to an enhanced value of the apparent K_m of the carrier for the glucose analogue. This effect is discussed in relation to either the more positive value of the transmembrane electric potential difference (ΔΨ) measured in these root segments, or a competitive inhibition of Na⁺ on the H⁺ binding site of the carrier. Growth in saline solution slightly affected the efflux of the [¹⁴C]3-OMG preloaded in root segments, changing the membrane permeability to the molecule. The results strongly suggest that the higher (2.5 fold) net exudation of glucose, observed in short-term (4 h) collection experiments, from roots of cotton plants grown in saline condition, is mainly due to an effect of the saline growth condition on the system involved in the reabsorption of the hexose rather than on its efflux. This revised version was published online in June 2006 with corrections to the Cover Date.     

Blood–Brain Glucose Transport in the Conscious Rat: Comparison of the Intravenous and Intracarotid Injection Methods

Abstract: The unidirectional transfer of d -glucose from blood to parietal cortex tissue of the brain of awake rats was measured by single intravenous injection of tracer glucose, as well as by single intracarotid injection according to the method of Oldendorf. The maximal unidirectional blood–brain glucose transfer rate (T_max) was 407 μ mol (100 g)^–1 min^–1 when measured by intravenous injection, and 352 μ mol (100 g)^–1 min^–1 when measured by intracarotid injection. The half–saturation constants (K_m) were 7.8 mm and 16.8 HIM, respectively. The comparison shows that the two methods give similar results when cerebral perfusion is assessed accurately.     

Lactate Uptake and Release in the Presence of Glucose by Sympathetic Ganglia of Chicken Embryos and by Neuronal and Nonneuronal Cultures Prepared from These Ganglia

Abstract: Uptake and output of lactate were measured in lumbar sympathetic chains excised from embryos of white leghorn chickens, 14–15 days old. The chains, typically containing 30–40 μg of protein, were incubated in Eagle's minimum essential medium containing bicarbonate buffer, 6–17 mM glucose, various concentrations of lactate, and either [U-¹⁴C]lactate, [1-¹⁴C]glucose, or [6-¹⁴C]glucose. The average rate of uptake of labeled lactate was measured with incubations of 5–6 h, starting with various external lactate concentrations. From these data the instantaneous relation between lactate uptake rate and concentration was deduced with a simple computerized model. The instantaneous uptake rate increased with the concentration according to a relation that fit the Michaelis-Menten equation, with V_max = 360 μmol/g protein/h and K_m = 4.8 mM. Substantial fractions of the lactate carbon were recovered from tissue constituents and in several nonvolatile products in the medium, as well as in CO₂. Glucose uptake averaged about 108 μmol/g protein/h and did not vary greatly with external lactate concentration, although the metabolic partitioning of glucose carbon was considerably affected. Regardless of initial concentration, the lactate concentration in the medium tended to change towards approximately 0.6 mM, showing that uptake equaled output at this level, with rates at about 40 μmol/g protein/h. With the steady-state concentration of 0.6 mM lactate, about 20% of the glucose carbon was shunted out into the medium before it was reabsorbed and metabolized into various products. Lactate uptakes by neuronal and nonneuronal cultures prepared from the ganglia did not differ consistently from one another or from uptake by undissociated ganglia. The neuronal cultures tended to oxidize a greater fraction of the consumed lactate to CO₂ and to convert a smaller fraction of the lactate to products in the medium than did the nonneuronal cultures. Computer modeling, using known parameters for blood-brain transport of lactate in the adult rat and data on uptake by the ganglia, suggests that lactate may supply substantial fuel to the brain, even in the presence of abundant glucose, when the lactate concentration in the blood is raised to levels commonly observed in exercising humans, such as 10–20 mM. This is in agreement with the findings of several investigators in hypoglycemic humans and in animals with intermediate blood lactate concentrations.     

Metabolic profile and quantification of deoxyribose synthesis pathways in HepG2 cells

Traditional tracer studies of cell proliferation fail to distinguish between label enrichment due to increased DNA repair versus DNA replication. We used the emerging stable (non-radiating) isotope-based dynamic metabolic profiling technique on HepG2 cells to determine synthesis pathways of nucleic acids from glucose and rates of proliferation using CG-MS assay of RNA and DNA enrichment. Comparing the isotopic enrichment curve in DNA with the theoretical curve based on cell growth, we observed that the measured tracer enrichment was significantly higher, indicating that surplus label was acquired during DNA repair. In particular, after the first duplication (3 days), 80.13% of the total enrichment observed corresponds to duplication and 19.87% corresponds to DNA repair as calculated from the [1, 2-¹³C₂]-glucose incorporation curve. Our data indicate contemporary measurements of cell proliferation rates relying on tracer incorporation may be overestimated. ¹³C label was distributed between m1 (m1/Σm = 80) and m2 (m2/Σm = 14) of deoxyribose, indicating that most of the glucose carbon was acquired via direct glucose oxidation in the pentose cycle. The stable isotope technique distinguishes rates of DNA synthesis and repair via the oxidative and non-oxidative pentose cycle, separately, in one test, without inhibition of either process. The contribution of DNA repair in malignant cells to isotope accumulation in deoxyribose remains to be investigated.     

Cerebrospinal fluid glucose and lactate: age-specific reference values and implications for clinical practice

Cerebrospinal fluid (CSF) analysis is an important tool in the diagnostic work-up of many neurological disorders, but reference ranges for CSF glucose, CSF/plasma glucose ratio and CSF lactate based on studies with large numbers of CSF samples are not available. Our aim was to define age-specific reference values. In 1993 The Nijmegen Observational CSF Study was started. Results of all CSF samples that were analyzed between 1993 and 2008 at our laboratory were systematically collected and stored in our computerized database. After exclusion of CSF samples with an unknown or elevated erythrocyte count, an elevated leucocyte count, elevated concentrations of bilirubin, free hemoglobin, or total protein 9,036 CSF samples were further studied for CSF glucose (n = 8,871), CSF/plasma glucose ratio (n = 4,516) and CSF lactate values (n = 7,614). CSF glucose, CSF/plasma glucose ratio and CSF lactate were age-, but not sex dependent. Age-specific reference ranges were defined as 5–95^th percentile ranges. CSF glucose 5^th percentile values ranged from 1.8 to 2.9 mmol/L and 95^th percentile values from 3.8 to 5.6 mmol/L. CSF/plasma glucose ratio 5^th percentile values ranged from 0.41 to 0.53 and 95^th percentile values from 0.82 to 1.19. CSF lactate 5^th percentile values ranged from 0.88 to 1.41 mmol/L and 95^th percentile values from 2.00 to 2.71 mmol/L. Reference ranges for all three parameters were widest in neonates and narrowest in toddlers, with lower and upper limits increasing with age. These reference values allow a reliable interpretation of CSF results in everyday clinical practice. Furthermore, hypoglycemia was associated with an increased CSF/plasma glucose ratio, whereas hyperglycemia did not affect the CSF/plasma glucose ratio.     

MECHANISMS FOR THE EFFLUX OF [14C]DOPA AND [14C]DOPAMINE FROM THE CSF OF RHESUS MONKEYS

—Clearance of [¹⁴C]DOPA and [¹⁴C]dopamine from CSF was investigated in anaesthetized rhesus monkeys (M. Mulatta) subjected to ventriculocisternal perfusion. The efflux coefficients, k_VE, at tracer concentrations (3–5 m ) in the perfusate were 0.0487 ml/min and 0.0325 ml/min for [¹⁴C]DOPA and [¹⁴C]dopamine, respectively. Carrier DOPA (10 mm ) in the perfusate decreased the efflux of [¹⁴C]DOPAsignificantly, but carrier dopamine had no appreciable effect on the clearance of [¹⁴C]dopamine. These findings suggest that DOPA is cleared from CSF in part by a saturable mechanism which may be located in the choroid plexus, whereas dopamine leaves the ventricular system by passive diffusion. Radioactivity in the caudate nucleus immediately adjacent to the perfused ventricle averaged 15.5 % and 12.6% of the radioactivity in the perfusates with [¹⁴C]DOPA or [¹⁴C]dopamine, respectively. These distribution percentages were similar to those found for various extracellular indicators after ventriculocisternal perfusion and may indicate that the efflux of intraventricularly-administered exogenous DOPA and dopamine occurs in part through extracellular channels.     

An estimation of pyruvate recycling during gluconeogenesis in the perfused rat liver

To estimate the degree of recycling of pyruvate during gluconeogenesis, an isotope tracer procedure was employed. Using the isolated, perfused rat liver with pyruvate-2-¹⁴C in the perfusion fluid, the 3-carbon acids lactate and pyruvate were isolated and the distribution of ¹⁴C in each carbon was assayed. It can be shown that the degree of recycling can be approximated as twice the sum of ¹⁴C in carbons 1 and 3. Glucose, acetoacetate, and β-hydroxybutyrate were also determined, and their ¹⁴C distribution estimated by appropriate degradation procedures. In livers from fasted rats, recycling of pyruvate during 1 hr incubation occurred at a rate of 0.21 μmoles ± 0.02 (SE)/min/g while gluconeogenesis occurred at a rate of 0.49 ± 0.11 μmoles pyruvate-2-¹⁴C/min/g. In livers from carbohydrate-fed rats, the ratio was reversed, with 0.35 ± 0.06 μmoles pyruvate-2-¹⁴C recycled and only 0.09 ± 0.03 μmoles converted to glucose. These patterns were not affected by the simultaneous presence of octanoate in the perfusion, during which ketone body production was greatly increased. Only about 20% of the ketone bodies formed were derived from pyruvate, much less with octanoate present, and over 95% of the total radioactivity was in carbons 1 and 3 of acetoacetate as anticipated from the degree of pyruvate recycling. The glucose invariably had 3–4% of its total activity in carbons 3 and 4 and the remainder distributed approximately equally in carbons 1, 2, 5, and 6. The radioactivity in respired CO₂ indicated that about 13–25% of the total O₂ uptake was due to pyruvate oxidation to CO₂.     

Manganese increases high mannose glycoform on monoclonal antibody expressed in CHO when glucose is absent or limiting: Implications for use of alternate sugars

Alternate sugars such as galactose and fructose are metabolized at a slower rate than glucose and result in lower accumulation of lactate. While low lactate accumulation is desirable, we report that complete substitution of glucose with these sugars results in an increase in M5 high mannose glycans. Surprisingly, this increase is much higher when the culture is supplemented with manganese: for example, when cells are cultured with galactose, M5 high mannose glycan content increased from 5% at 1 nM Mn²⁺ in the basal medium to 32% with 16 µM Mn²⁺ supplementation. When galactose is supplemented with glucose maintained at low concentrations, a small reduction in high mannose glycans is seen. In control cultures with glucose, the high mannose content was however <2% in this range of Mn²⁺ concentration. By varying Mn²⁺ and glucose supplementation levels, with or without galactose, we systematically demonstrate that Mn²⁺ concentration and glucose availability, together, significantly affect the high mannose glycan content. To our knowledge, this is the first report that shows that the effect of Mn²⁺ on high mannose glycan content depends on glucose availability. At each Mn²⁺ supplementation level evaluated, galactosylation percentages were highest for cultures where galactose was supplemented with glucose at non‐limiting concentration. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:460–467, 2015     

Energy substrates in bovine oviduct and uterine fluid and blood plasma during the oestrous cycle

Up to 40 percent of cattle embryos die within 3 weeks of fertilization but there is little or no published information on the composition of the oviduct and uterine fluids essential for their survival during this time. We have measured the concentrations of the energy substrates, glucose, lactate, and pyruvate in cattle oviduct fluid on Days 0, 2, 4, and 6 and uterine fluid on Days 6, 8, and 14 of the oestrous cycle and corresponding blood samples. Oviduct and uterine fluids were collected in situ. Glucose concentrations in oviduct and uterine fluids were similar on all days and lower than in plasma (P < 0.05). Oviduct lactate concentration was up to eightfold higher than uterine or plasma concentration (P < 0.01). Oviduct pyruvate concentrations were similar on all days and lower than plasma concentrations on Days 0 and 2 (P < 0.005). Pyruvate concentrations were similar in the uterus and in plasma except on Day 14 when the concentration in plasma was higher (P < 0.05). There were no associations between systemic progesterone or oestradiol and glucose, lactate or pyruvate. There was a linear positive relationship (P < 0.001) between oviduct fluid secretion rate and oviduct glucose concentration and a linear negative relationship (P < 0.001) between oviduct fluid secretion rate and oviduct lactate, but no association between uterine fluid secretion rate and energy substrates. The different concentrations and associations between the energy substrates in oviduct and uterine fluids and blood plasma indicate a differential regulation of the secretion of these energy substrates by the oviduct and uterine epithelium.     

Bonded Cumomer Analysis of Human Melanoma Metabolism Monitored by 13C NMR Spectroscopy of Perfused Tumor Cells

A network model for the determination of tumor metabolic fluxes from ¹³C NMR kinetic isotopomer data has been developed and validated with perfused human DB-1 melanoma cells carrying the BRAF V600E mutation, which promotes oxidative metabolism. The model generated in the bonded cumomer formalism describes key pathways of tumor intermediary metabolism and yields dynamic curves for positional isotopic enrichment and spin-spin multiplets. Cells attached to microcarrier beads were perfused with 26 mm [1,6-¹³C₂]glucose under normoxic conditions at 37 °C and monitored by ¹³C NMR spectroscopy. Excellent agreement between model-predicted and experimentally measured values of the rates of oxygen and glucose consumption, lactate production, and glutamate pool size validated the model. ATP production by glycolytic and oxidative metabolism were compared under hyperglycemic normoxic conditions; 51% of the energy came from oxidative phosphorylation and 49% came from glycolysis. Even though the rate of glutamine uptake was ∼50% of the tricarboxylic acid cycle flux, the rate of ATP production from glutamine was essentially zero (no glutaminolysis). De novo fatty acid production was ∼6% of the tricarboxylic acid cycle flux. The oxidative pentose phosphate pathway flux was 3.6% of glycolysis, and three non-oxidative pentose phosphate pathway exchange fluxes were calculated. Mass spectrometry was then used to compare fluxes through various pathways under hyperglycemic (26 mm) and euglycemic (5 mm) conditions. Under euglycemic conditions glutamine uptake doubled, but ATP production from glutamine did not significantly change. A new parameter measuring the Warburg effect (the ratio of lactate production flux to pyruvate influx through the mitochondrial pyruvate carrier) was calculated to be 21, close to upper limit of oxidative metabolism.     

Ventriculo–cisternal perfusion of twelve amino acids in the rabbit

The clearances of twelve amino acids from the ventricles during ventriculocisternal perfusion in the rabbit have been measured; uptake by the brain was also measured and this permitted the separate computation of loss to brain and loss to blood during the perfusion. Clearance under carrier-free conditions was greater than when a concentration of 5mM unlabeled amino acid was present in the perfusion fluid. Brain uptake was also usually reduced by the presence of unlabeled amino acid due presumably to suppression of accumulation by brain cells. Reduction of transport across the blood-brain barrier would tend to increase brain uptake, and there was some evidence for a balance between the two opposing tendencies. Inhibition of clearance of a given labeled amino acid could be brought about by unlabeled amino acids of different molecular species. In general, the amino acids fell into three categories: neutral, acidic, and basic, and there was some overlap between them; of the neutral amino acids the A- and L-classification of Christensen was valid, although once again there was some overlap. If, during ventriculo-cisternal perfusion of a labeled amino acid, the activity of this labeled amino acid in the blood was raised well above that in the inflowing perfusion fluid, the labeled amino acid continued to be cleared from the perfusion fluid, suggesting uphill transport. On this basis it was suggested that the normally low concentrations of amino acids in the cerebrospinal fluid (CSF), by comparison with those in plasma, were due to an active transport from the CSF to the blood. Substrate-facilitated transport, whereby the penetration of labeled amino acid into the perfusion fluid from blood could be accelerated by adding unlabeled amino acid to the perfusion fluid, or vice versa, was demonstrated.     

The relationship between glucose concentration and rate of lactate production by human erythrocytes in an open perfusion system

A thermodynamically open system, based on an assembly of capillaries with semi-permeable walls was constructed in order to study glycolysis in human erythrocytes in high haematocrit suspensions. A phenomenological expression for the rate of lactate production as a function of glucose concentration was obtained. The rate was measured under steady-state conditions with low substrate concentrations (approx. 50 μmol/l). In a corresponding closed system, this concentration of glucose would be exhausted within a few minutes. A mathematical model of the whole system consisted of five differential equations, and involved parameters relating to flow rates, volumes of reaction chambers, the rates of lactate efflux from erythrocytes and the expression for the rate of lactate production by red cells. The binding of [¹⁴C]pyruvate to haemoglobin and the rate of efflux of [¹⁴C]lactate from red cells were measured to yield additional information for the model. The concentrations of ATP and 2,3-bisphosphoglycerate were measured during the perfusion experiments, and a detailed analysis of a model of red cell hexokinase was carried out; the former two compounds inhibit hexokinase and alter the apparent K_m and V_max for glucose in vivo. These steady-state parameters were similar to the glucose concentration at the half-maximal rate of lactate production and the maximal rate, respectively. These findings are consistent with the known high control-strength for hexokinase in glycolysis in human red cells. The practical and theoretical validation of this perfusion system indicates that it will be valuable for NMR-based studies of red cell metabolism using a flow-cell in the spectrometer.     

Glucose Metabolism in Sediments of a Eutrophic Lake: Tracer Analysis of Uptake and Product Formation

The uptake of glucose and the formation of end products from glucose catabolism have been measured for sediments of eutrophic Wintergreen Lake with a combination of tritiated and ¹⁴C-labeled tracers. Time course analyses of the loss of [³H]glucose from sediments were used to establish rate constants for glucose uptake at natural substrate concentrations. Turnover times from these analyses were about 1 min for littoral and profundal sediments. No seasonal or site differences were noted in turnover times. Time course analyses of [U-¹⁴C]glucose uptake and ¹⁴C-labeled end product formation indicated that glucose mass flow could not be calculated from end product formation since the specific activity of added [¹⁴C]glucose was significantly diluted by pools of intracellular glucose and glucose metabolites. Mass flow could only be accurately estimated by use of rates of uptake from tracer studies. Intermediate fermentation end products included acetate (71%), propionate (15%), lactate (9%), and only minor amounts of butyrates or valerates. Addition of H₂ to sediments resulted in greater production of lactate (28%) and decreased formation of acetate (50%), but did not affect glucose turnover. Depth profiles of glucose uptake indicated that rates of uptake decreased with depth over the 0- to 18-cm interval and that glucose uptake accounted for 30 to 40% of methanogenesis in profundal sediments.     

Intestinal handling of a glucose gavage by the rat

An oral gavage of either 3, 1 or 0.1 mmoles of ¹⁴C-labelled glucose was given to rats under standard feeding conditions or food deprived for 24 hr. The fate of the glucose label was determined at 10, 15, 30 and 60 min after gavage; at 60 min 40% of the glucose was absorbed in fed rats (60% in food deprived). The portal vein blood flows were determined and the levels of glucose, lactate, alanine and pyruvate, and their radioactivity, as well as that of CO, were measured in both portal and arterial blood.The net computed glucose and 3-carbon carriers (lactate, alanine and pyruvate) actually released into the portal system by the intestine was lower than the amount of glucose taken up from the intestinal lumen in one hour. Oxidation to ¹⁴CO₂ accounted for a 12–15% of the absorbed glucose. The size of the gavage deeply affected the proportion of glucose released into the portal blood (c. 50% with a 3 mmoles gavage and practically nil with a 0.1 mmoles gavage), but it affected much less the generation of lactate and other 3 C carriers. In fed rats, the net intestinal balance of non-radioactive glucose was negative, and that of lactate positive; when radioactive glucose was considered, the pattern was inverted. In starved rats, both glucose and lactate were released in large proportions by the intestine, but alanine efflux was lower.It can be concluded that the intestine consumes a considerable proportion of glucose in the fed state. Glucose handling by the intestine is compartmentalized in two functional circuits: glucose is taken up from the arterial blood and used for intestinal metabolism and lactate production, luminal glucose is absorbed mainly unaltered and transferred to the portal blood. Thus, the generation of lactate is mainly related to the availability of arterial glucose. In addition to the release of the ingested glucose as 3 C carriers or glucose, an extraportal pathway for glucose transfer into the bloodstream is postulated.     

Effects of Metformin and Vanadium on Leptin Secretion from Cultured Rat Adipocytes

Objective: We have reported that glucose utilization regulates leptin expression and secretion from isolated rat adipocytes. In this study, we employed two antidiabetic agents that act to increase glucose uptake by peripheral tissues, metformin and vanadium, as pharmacological tools to examine the effects of altering glucose utilization on leptin secretion in primary cultures of rat adipocytes. Research Methods and Procedures: Isolated adipocytes (100 μL of packed cells per well) were anchored in a defined matrix of basement membrane components (Matrigel) with media containing 5.5 mM glucose and incubated for 96 hours with metformin or vanadium. Leptin secretion, glucose utilization, and lactate production were assessed. Results: Metformin (0.5 and 1.0 mM) increased glucose uptake in the presence of 0.16 nM insulin by 37 ± 10% (p < 0.005) and 62 ± 8% (p < 0.0001) over insulin alone, respectively. Metformin from 0.5 to 5.0 mM increased lactate production by 105 ± 43% (p < 0.025) to 202 ± 52% (p < 0.0025) and at 1.0 and 5.0 mM increased the proportional rate of glucose conversion to lactate by 78 ± 18% (p < 0.005) and 166 ± 41% (p < 0.0025), respectively. At concentrations less than 0.5 mM, metformin did not affect leptin secretion, but at 0.5 mM, the only concentration that significantly increased glucose utilization without increasing glucose conversion to lactate, leptin secretion was modestly stimulated (by 20 ± 9%; p < 0.05). Concentrations from 1.0 to 25 mM inhibited leptin secretion by 25 ± 8% (p < 0.005) to 89 ± 4% (p < 0.0001). Across metformin doses, leptin secretion was inversely related to the percentage of glucose taken up and released as lactate (r = ?0.74; p < 0.0001). Vanadium (5 to 20 μM) increased glucose uptake from 20 ± 7% (p < 0.01) to 34 ± 13% (p < 0.02) and increased lactate production at 5 μM by 17 ± 8% (p < 0.025) and 10 μM by 61 ± 20% (p < 0.02) but did not alter the conversion of glucose to lactate. Vanadium (5 to 50 μM) inhibited leptin secretion by 33 ± 6% (p < 0.0025) to 61 ± 8% (p < 0.0001). Discussion: Both metformin and vanadium increase glucose uptake and inhibit leptin secretion from cultured adipocytes. The inhibition of leptin secretion by metformin is related to an increase in the metabolism of glucose to lactate. The inhibition by vanadium most likely involves direct effects on cellular phosphatases. We hypothesize that the effect of glucose utilization to stimulate leptin production involves the metabolism of glucose to a fate other than anaerobic lactate production, possibly oxidation or lipogenesis.     

Differentiation between serum stimulation of ouabain-resistant and sensitive Rb influx in quiescent NIH 3T3 cells

Summary The addition of serum to quiescent NIH 3T3 mouse cell cultures resulted in a 10- to 20-fold increase of Rb influx which was resistant to ouabain, and only a three- to fourfold activation of ouabain-sensitive Rb influx. Stimulation of the ouabain-resistant Rb influx following serum addition reached its maximum within 2 min. The stimulation of ouabain-resistant Rb influx was a result ofV _m increase while theK _m for Rb was unchanged. Ouabain-resistant Rb influx, after serum addition, was resistant to amiloride and sensitive to ethacrinic acid. Replacing chloride in the medium by NO₃ ^–, CO₃ ^– and CH₃COO^– resulted in a drastic decrease in the ouabain-resistant Rb influx. It appeared, therefore, that the ouabain-resistant Rb influx in NIH 3T3 cells was Cl^–-dependent.     

Novel model of neuronal bioenergetics: postsynaptic utilization of glucose but not lactate correlates positively with Ca2+ signalling in cultured mouse glutamatergic neurons

We have previously investigated the relative roles of extracellular glucose and lactate as fuels for glutamatergic neurons during synaptic activity. The conclusion from these studies was that cultured glutamatergic neurons utilize glucose rather than lactate during NMDA (N-methyl-d-aspartate)-induced synaptic activity and that lactate alone is not able to support neurotransmitter glutamate homoeostasis. Subsequently, a model was proposed to explain these results at the cellular level. In brief, the intermittent rises in intracellular Ca²⁺ during activation cause influx of Ca²⁺ into the mitochondrial matrix thus activating the tricarboxylic acid cycle dehydrogenases. This will lead to a lower activity of the MASH (malate–aspartate shuttle), which in turn will result in anaerobic glycolysis and lactate production rather than lactate utilization. In the present work, we have investigated the effect of an ionomycin-induced increase in intracellular Ca²⁺ (i.e. independent of synaptic activity) on neuronal energy metabolism employing ¹³C-labelled glucose and lactate and subsequent mass spectrometric analysis of labelling in glutamate, alanine and lactate. The results demonstrate that glucose utilization is positively correlated with intracellular Ca²⁺ whereas lactate utilization is not. This result lends further support for a significant role of glucose in neuronal bioenergetics and that Ca²⁺ signalling may control the switch between glucose and lactate utilization during synaptic activity. Based on the results, we propose a compartmentalized CiMASH (Ca²⁺-induced limitation of the MASH) model that includes intracellular compartmentation of glucose and lactate metabolism. We define pre- and post-synaptic compartments metabolizing glucose and glucose plus lactate respectively in which the latter displays a positive correlation between oxidative metabolism of glucose and Ca²⁺ signalling.     

Kinetics of Transport and Phosphorylation of 2-Fluoro-2-Deoxy-d-Glucose in Rat Brain

Abstract: The kinetics of transport across the blood-brain barrier and metabolism in brain (hemisphere) of [¹⁴C]2-fluoro-2-deoxy-d -glucose (FDG) were compared to that of [³H]2-deoxy-d -glucose (DG) and d -glucose in the pentobarbital-anesthetized adult rat. Saturation kinetics of transport were measured with the brain uptake index (BUI) method. The BUI for FDG was 54.3 ± 5.6. Nonlinear regression analysis gave a K_m of 6.9 ± 1.1 mM and a V_max of 1.70 ± 0.32 μmol/min/g. The K₁ for glucose inhibition of FDG transport was 10.7 ± 4.4 mM. The kinetic constants of influx (k₁) and efflux (K₂) for FDG were calculated from the K_m, V_max, and glucose concentrations of the hemisphere and plasma (2.3 ± 0.2 μmol/g and 9.9 ± 0.4 mM, respectively). The transport coefficient (k_{1 FDG}/k₁glucose) was 1.67 ± 0.07 and the phosphorylation constant was 0.55 ± 0.16. The predicted lumped constant for FDG was 0.89, whereas the measured hexose utilization index for FDG was 0.85 ± 0.16. Conclusion: The value for the lumped constant can be predicted on the basis of the known kinetic constants of FDG and glucose transport and metabolism, as well as brain and plasma glucose levels. Knowledge of the lumped constant is crucial in interpreting data obtained from ¹⁸FDG analysis of regional glucose utilization in human brain in pathological states. We propose that the lumped constant will rise to a maximum equal to the transport coefficient for FDG under conditions of transport limitation (hypoglycemia) or elevated glycolysis (ischemia, seizures), and will fall to a minimum equal to the phosphorylation coefficient during phosphorylation limitation (extreme hyperglycemia).     

Influence of Perfusate Glucose Concentration on Dialysate Lactate, Pyruvate, Aspartate, and Glutamate Levels Under Basal and Hypoxic Conditions: A Microdialysis Study in Rat Brain

Abstract: The aim of this study was to evaluate the influence of perfusion media with different glucose concentrations on dialysate levels of lactate, pyruvate, aspartate (Asp), and glutamate (Glu) under basal and hypoxic conditions in rat brain neocortex. Intracerebral microdialysis was performed with the rat under general anesthesia using bilateral probes (o.d. 0.3 mm; membrane length, 2 mm) perfused with artificial CSF containing 0.0 and 3.0 m M glucose, respectively. Basal dialysate levels were obtained 2 h after probe implantation in artificially ventilated animals. Dialysate levels of glucose were also measured for the two different perfusion fluids. The mean absolute extracellular concentration of glucose was estimated by a modification of the no-net-flux method to be 3.3 mmol/L, corresponding to an average in vivo recovery of 6% for glucose. Hypoxia was induced by lowering the inspired oxygen concentration to 3%. Hypoxia caused a disturbance of cortical electrical activity, evidenced by slower frequency and lower amplitudes on the electroencephalogram compared with prehypoxic conditions. This was associated with significant elevations of lactate, Asp, and Glu levels. There were no statistically significant differences in dialysate metabolite levels between the two perfusion fluids, during either normal or hypoxic conditions. We conclude that microdialysis with glucose-free perfusion fluid does not drain brain extracellular glucose in anesthetized rats to the extent that the dialysate lactate, pyruvate, Asp, and Glu levels during basal or hypoxic conditions are altered.