Stimulation of hamster sperm capacitation and acrosome reaction in vitro by glucose and lactate and inhibition by the glycolytic inhibitor α-chlorohydrin

Studies were made of the effects of D(+)-glucose, L-lactate and pyruvate on in vitro capacitation and acrosome reactions (AR) of hamster sperm using a more “defined” medium that that used in previous similar studies. In the absence of glucose or lactate, sperm underwent very few AR and activation (whiplash-like motility characteristic of capacitated hamster sperm) was reduced compared to those events in sperm preincubated in the presence of glucose plus lactate plus pyruvate. Glucose and pyruvate supported more AR than glucose alone, but less than glucose, lactate, and pyruvate. The glycolytic inhibitor α-chlorohydrin (10 μm) inhibited AR by 50% and reduced activation by less. When glucose was added to sperm incubated 2 hr with pyruvate and lactate, the number of AR observed after 4 hr was the same as that obtained when glucose was present throughout the incubation. When glucose was added after 3.5 hr, AR were delayed for 1 hr and lower numbers of sperm underwent AR. In the presence of lactate and pyruvate, 0.38 mM glucose was able to support activation and AR as well as 3.24 mM glucose. These results indicate that exogenous glucose and lactate are necessary for in vitro capacitation and AR of hamster sperm; only low levels of exogenous glucose are required; exogenous glucose is not required during the first 2 hr of capacitation; and glycolytic activity is necessary for capacitation and the AR.     

Lactate transport and glycolytic activity in the freshly isolated rabbit cornea.

Studies on the intact avascular cornea reveal two types of lactate effluxes: exogenous glucose-elicited and spontaneous. The former type exhibits characteristics resembling the proton-lactate symport system previously found in tumor cells and erythrocytes, including an enhanced lactate efflux at a higher extracellular pH and in the presence of H+ and K+ ionophores, and an inhibition by mersalyl with subsequent lactate accumulation in the tissue and cessation of glycolytic activity. The latter type occurs immediately following the incubation of freshly isolated cornea in a medium containing no exogenous glucose, with a rate about 10 times that of exogenous glucose-elicited lactate efflux. It is insensitive to 10 mM iodoacetate and lacks the characteristics of the proton-lactate symport system. Findings reveal that about 50% of corneal glucose utilization occurs in the epithelium, with the stroma and endothelium sharing the other 50% approximately equally. Of the glucose utilized, the lactate formation to pyruvate oxidation rate ratios are approximately 1:1 in the epithelium, 2:1 in the stroma, and 1:2 in the endothelium. About 79% of total tissue lactate is formed in the epithelium and stroma, and in vivo, this is probably pumped into the stromal extracellular space (about 90% of total tissue volume) via the proton-lactate symport system, with spontaneous release into the aqueous humor via a simple diffusion process. The H+ and K+ ionophores facilitate lactate efflux at the expense of the cellular pyruvate pool, without significant effect on the glucose uptake and glycolytic activity. These findings suggest that the ionophore-mediated lactate efflux favors the reduction of low pyruvate concentration in the tissue, rather than parallel increases in glycolytic activity.     

Lactate release from isolated rat adipocytes: influence of cell size, glucose concentration, insulin and epinephrine

Release of lactate was studied during in vitro incubations with isolated fat cells. Lactate release increased (approximately 3-fold) with increasing medium glucose concentration (from 3 to 12 mM) in both large and small fat cells. Large fat cells from older, fatter rats, however, released 3-4 times more lactate per cell than small fat cells from young rats when incubated with 3, 6 or 12 mM glucose. Insulin and epinephrine produced a marked stimulation of lactate release in small fat cells, but these hormones had no effect in large fat cells. Lactate accounted for only 10-15% of the glucose metabolized by small fat cells under all incubation conditions but was nearly 40% of glucose utilized by large fat cells at glucose concentrations greater than 6 mM. In conclusion, lactate is a major metabolite of glucose in adipocytes, particularly in the large fat cells. Adipose tissue may therefore be a major site of lactate production, particularly in states of altered glucose metabolism (i.e., hyperglycemia) and obesity.     

Glucose and amino acid metabolism in an established line of skeletal muscle cells

The suitability of an established myogenic line (L₆) for the study of skeletal muscle intermediary metabolism was investigated. Myoblasts were grown in tissue culture for ten days at which time they had differentiated into multinucleated myotubes. Myotube preparations were then incubated for up to 96 hours in 10 ml of Dulbecco's modified Eagle medium containing 10% fetal calf serum. Glucose was utilized at a nearly linear rate, 3.0 nmol/min/mg protein. Intracellular glucose was detectable throughout the incubation, even when medium glucose was as low as 16 mg%. During the initial 28 hours of incubation, when net lactate production was observed, only 35% of the glucose utilized was converted to lactate. Alanine was produced in parallel to lactate at an average rate of 0.6 nmol/min/mg protein. In concert with active glutamine utilization, high rates of ammoniagenesis were observed as medium glutamine decreased from 3.3 mM to 0.49 mM and medium ammonia increased from 2.3 mM to 6.2 mM, between zero time and 96 hours of incubation, respectively. The cells maintained stable ATP and citrate levels, and physiologic intracellular lactate/pyruvate ratios (10–24) throughout 96 hours of incubation. These results suggest (1) glucose utilization by skeletal muscle in tissue culture is limited by phosphorylation, not transport; (2) as much as 50% of glucose-derived pyruvate enters mitochondrial pathways; (3) glutamine carbon may be utilized simultaneously with glucose consumption and this process accounts for high rates of ammoniagenesis.     

Schistosoma mansoni: effects of in vitro serotonin (5-HT) on aerobic and anaerobic carbohydrate metabolism

The effect of Serotonin on carbohydrate metabolism, excreted end products, and adenine nucleotide pools in Schistosoma mansoni was determined following 60 min in vitro incubations under air (= 21% O2) and anaerobic (95% N2:5% CO2) conditions. In the presence of 0.25 mM Serotonin, glucose uptake increased by 82-84% and lactate excretion increased by 77-78%; levels of excreted lactate were significantly higher under aerobic than under anaerobic conditions. The tissue pools of glucose, hexosephosphates, fructose 1,6-bisphosphate, pyruvate, and lactate were significantly increased under anaerobic conditions compared to air incubation; the presence of Serotonin decreased tissue glucose pools and increased the size of the pyruvate and lactate tissue pools. The glycolytic carbon pool was significantly greater under anaerobic than under aerobic conditions, irrespective of Serotonin. Serotonin increased adenosine 5'-diphosphate and adenosine 5'-monophosphate levels under aerobic conditions; neither Serotonin nor gas phase significantly affected total adenine nucleotide levels or the adenylate energy charge. Serotonin increased energy requirements by S. mansoni due to increased muscle contractions; demand was met by enhanced rates of carbohydrate metabolism. Irrespective of gas phase, 74-78% of available carbohydrate was converted to lactate. In the presence of Serotonin, conversion of glucose to lactate was reduced to 63-67%. In view of the requirements by S. mansoni for an abundant supply of glycoprotein and glycolipid precursors for surface membrane renewal, it is suggested that carbohydrate (glucose and glycogen) that was not converted to lactate may have been incorporated into biosynthetic processes leading to membrane synthesis.     

Lactate prevents the alterations in tissue amino acids, decline in ATP, and cell damage due to aglycemia in retina

Under conditions of energy impairment, CNS tissue can utilize substrates other than glucose to maintain energy metabolism. Retinas produce large amounts of lactate, although it has not been shown that lactate can be utilized by retina to prevent the cell damage associated with hypoglycemia. To investigate this, intact, isolated retinas were subjected to aglycemic conditions in the presence or absence of 20 mM lactate. Retinas incubated in the absence of glucose for 60 min showed a threefold elevation in tissue aspartate and 60% decreases in tissue glutamate and glutamine, demonstrating a mobilization of carbon from glutamine and glutamate to the tricarboxylic acid cycle. Lactate prevented these changes in tissue amino acids, indicating metabolism of lactate with sparing of tissue glutamate and glutamine. Tissue ATP was 20 and 66% of control values with zero glucose or zero glucose plus lactate, respectively. Consistent with previous findings, incubation of retinas in the absence of glucose caused acute swelling of retinal neurons and release of GABA into the medium at 60 min. These acute toxic affects caused by the absence of glucose were completely prevented by the presence of lactate. At 24 h of recovery following 60 min of zero glucose, many pyknotic profiles were observed and lactate dehydrogenase (LDH) release into the medium was elevated sevenfold, indicating the extent of cell death. In contrast, no elevation in LDH was found and histology appeared normal in retinas exposed to zero glucose in the presence of lactate. alpha-Cyano-4-hydroxy cinnamate (4-CIN; 0.5 mM), an inhibitor of the monocarboxylic acid transporter and mitochondrial pyruvate carrier, blocked the ability of lactate to maintain ATP and protect retinas from aglycemia but had no effect on ATP or toxicity per se. Derangements in tissue aspartate, glutamate, and glutamine, which were prevented by lactate during zero glucose incubation, were again observed with lactate plus zero glucose in the presence of 4-CIN. However, 0.5 mM 4-CIN alone in the presence of glucose produced similar increases in aspartate and decreases in glutamate and glutamine as observed with zero glucose while having only modest inhibitory effects on [U-(14)C]lactate uptake, suggesting the mitochondrial pyruvate carrier as the main site of action. The above findings show that lactate is readily utilized by the chick retina during glucose deprivation to prevent derangements in tissue amino acids and ATP and retinal neuronal cell death.     

The role of glucose,pyruvate and lactate in ATP production by rat spermatocytes and spermatids

The ATP content of pachytene spermatocytes and round spermatids, isolated from rat testes, was not maintained during incubation of the germ cells in the presence of glucose. Glucose was metabolized via glycolysis at a considerable rate, but the rate of oxidation of the resulting endogenous pyruvate in the mitochondria was too low to support fully ATP production. Exogenous pyruvate (0.25 mM) or exogenous l-lactate (3–6 mM), however, were effective energy substrates. The lactate dehydrogenase reaction in isolated germ cells favoured the rapid conversion of pyruvate to lactate, at the expense of reducing equivalents from mitochondrial NADH. Hence, to support ATP production by the germ cells via mitochondrial metabolism of endogenous pyruvate, a relatively high concentration of exogenous lactate may be essential. In the spermatogenic microenvironment in vivo, such high concentrations of lactate could result from the net production of lactate by Sertoli cells. The mitochondria of the isolated germ cells produced ATP probably at a close to maximal rate, and spermatogenesis therefore may be extremely sensitive to compounds which interfere with mitochondrial energy metabolism and respiratory control.     

Respiration rates and sugar utilization by marsupial spermatozoa

This paper reports the first metabolic study of marsupial spermatozoa. The oxidative metabolism of the spermatozoa of the Australian brush-tailed possum (Trichosurus vulpecula) was examined using a micro Warburg system. Semen was collected by electro-ejaculation and washed twice in Ca²⁺ free Krebs-Ringer-phosphate buffer containing antibiotics (KRPA). Washed spermatozoa suspended in fresh KRPA, were then incubated for 3 hours at 37° C in the presence and absence of added substrates (4 mM). The exogenous substrates tested were N-acetylglucosamine, glucosamine, and glucose. Small quantities of radioactively labeled [¹⁴C] substrates were included in the incubation media to allow measurement of substrate oxidation. Although the respiratory rate varied considerably between semen pools (replicate experiments), the relationship between total oxygen consumption (measured manometrically), and oxygen consumption accounted for by exogenous substrate utilization (calcuated from radioactivity recovered in the respiratory CO₂) was remarkably consistent. Oxidation of exogenous substrate accounted for 49–54% of the oxygen consumption, depending on the substrate used. There was, however, no evidence that addition of these substrates stimulated the respiratory rate over that found when no substrate was added. Lactate formation accounted for the greater part of exogenous substrate consumed.     

Effect of glucose on ATP dephosphorylation in rat spermatids

Round spermatids were isolated from rat testes and the effects of different energy-yielding substrates on the cellular ATP content were estimated. The ATP content was constant and high (6-8 nmol/10(6) cells) during metabolism of exogenous lactate. During incubation for 30 min in the absence of exogenous lactate, there was a remarkably slow decline of the ATP content, indicating ATP production from other substrates. It was shown that this could reflect beta-oxidation of fatty acids, but not the mobilization of an endogenous pool of acetylcarnitine. Glucose metabolism in the absence of exogenous lactate resulted in a rapid decline of the ATP content. This effect of glucose was correlated with a high fructose 1,6-biphosphate content (6-7 nmol/10(6) cells) and could be prevented by the addition of lactate. It is suggested that metabolism of glucose (and also mannose and fructose, but not galactose) in the absence of exogenous lactate can result in ATP dephosphorylation.     

Quantitative analysis of growth and volatile fatty acid production by the anaerobic ruminal bacterium Megasphaera elsdenii T81

Megasphaera elsdenii T81 grew on either dl-lactate or d-glucose at similar rates (0.85 h^?1) but displayed major differences in the fermentation of these substrates. Lactate was fermented at up to 210-mM concentration to yield acetic, propionic, butyric, and valeric acids. The bacterium was able to grow at much higher concentrations of d-glucose (500 mM), but never removed more than 80 mM of glucose from the medium, and nearly 60 % the glucose removed was sequestered as intracellular glycogen, with low yields of even-carbon acids (acetate, butyrate, caproate). In the presence of both substrates, glucose was not used until lactate was nearly exhausted, even by cells pregrown on glucose. Glucose-grown cultures maintained only low extracellular concentrations of acetate, and addition of exogenous acetate increased yields of butyrate, but not caproate. By contrast, exogenous acetate had little effect on lactate fermentation. At pH 6.6, growth rate was halved by exogenous addition of 60 mM propionate, 69 mM butyrate, 44 mM valerate, or 33 mM caproate; at pH 5.9, these values were reduced to 49, 49, 18, and 22 mM, respectively. The results are consistent with this species’ role as an effective ruminal lactate consumer and suggest that this organism may be useful for industrial production of volatile fatty acids from lactate if product tolerance could be improved. The poor fermentation of glucose and sensitivity to caproate suggests that this strain is not practical for industrial caproate production.     

Lactate Utilization by Isolated Cells from Early Neonatal Rat Brain

The utilization of lactate, glucose, 3-hydroxybutyrate, and glutamine has been studied in isolated brain cells from early newborn rats. Isolated brain cells actively utilized these substrates, showing saturation at concentrations near physiological levels during the perinatal period. The rate of lactate utilization was 2.5-fold greater than that observed for glucose, 3-hydroxybutyrate, or glutamine, suggesting that lactate is the main metabolic substrate for the brain immediately after birth. The apparent Km for glucose utilization suggested that this process is limited by the activity of hexokinase. However, lactate, 3-hydroxybutyrate, and glutamine utilization seems to be limited by their transport through the plasma membrane. The presence of fatty acid-free bovine serum albumin (BSA) in the incubation medium significantly increased the rate of lipogenesis from lactate or 3-hydroxybutyrate, although this was balanced by the decrease in their rates of oxidation in the same circumstances. BSA did not affect the rate of glucose utilization. The effect of BSA was due not to the removal of free fatty acid, but possibly to the binding of long-chain acyl-CoA, resulting in the disinhibition of acetyl-CoA carboxylase and citrate carrier.     

Metabolic activities of Listeria monocytogenes in the presence of sodium propionate, acetate, lactate and citrate

The effects of sodium propionate, acetate, lactate and citrate on cell proliferation, glucose and oxygen consumption, and ATP production in Listeria monocytogenes were investigated in growing and resting cells. Media pH was 6.7-6.8. Growth inhibition increased while glucose consumption continued in the presence of ≥ 1% propionate, ≥ 3% acetate and ≥ 5% lactate in broth during incubation at 35°C, indicating that glucose consumption was uncoupled from cell proliferation. Acetate and propionate were the most effective antilisterials, whereas citrate (5%) was only slightly inhibitory. Of the four salts, only lactate supported growth, oxygen consumption and ATP production. While concentrations of 1 and 5% propionate, acetate and citrate did not have an effect on oxygen consumption, they inhibited ATP production. ATP production in the presence of the four salts was consistently lower at pH 6.0 than at neutral pH. Lactate served as an alternative energy source for L. monocytogenes in the absence of glucose but became toxic to the organism in the presence of the carbohydrate.     

Substrate Preference in a Strain of Megasphaera elsdenii, a Ruminal Bacterium, and Its Implications in Propionate Production and Growth Competition

The NIAH 1102 strain of Megasphaera elsdenii utilized lactate in preference to glucose when the two substrates were present. Even when lactate was supplied to cells fermenting glucose, the cells switched substrate utilization from glucose to lactate and did not utilize glucose until lactate decreased to a low concentration (1 to 2 mM). Since substrate utilization was shifted gradually without intermittence, typical diauxic growth was not seen. The cyclic AMP content did not rise markedly with the shift in substrate utilization, suggesting that this nucleotide is not involved in the regulation of the shift. It was unlikely that propionate was produced from glucose, which was explicable by the fact that lactate racemase activity dropped rapidly with the exhaustion of lactate and cells actively fermenting glucose did not possess this enzyme. A coculture experiment indicated that M. elsdenii NIAH 1102 is overcome by Streptococcus bovis JB1 in the competition for glucose, mainly because M. elsdenii NIAH 1102 is obliged to utilize lactate produced by S. bovis JB1; i.e., glucose utilization by M. elsdenii NIAH 1102 is suppressed by the coexistence of S. bovis JB1.     

Glycolytic end products of the adult dog heartworm, Dirofilaria immitis.

1. Adult dog heartworms remained alive and motile for 24 hr without oxygen present and with only glucose available as a substrate. 2. Lactate accounted for 55% of the carbon from the 1-14C-glucose utilized in 1 hr and 14CO2 for 1.9%. 3. Only traces of 14C were found in glycogen and no net accumulation of acetate was demonstrated. 4. Dirofilaria immitis resembles Litomosoides carinii in the percent of utilized glucose appearing as lactate but is more akin to Brugia pahangi and Dipetalonema viteae in survival under anaerobic conditions and in negligible acetate production.     

The special behavior of equine erythrocytes connected with the methemoglobin regulation

Erythrocytes from thoroughbred horses were submitted to total (80-90%) and partial (25-40%) oxidation of hemoglobin by sodium nitrite. The ability of these cells to reduce methemoglobin to hemoglobin in the presence of either glucose, glucose plus methylene blue or lactate was investigated. The results were compared with those ones obtained for human erythrocytes. Under total oxidation: the horse erythrocytes need longer incubation time with glucose or glucose plus methylene blue than human erythrocytes for reducing the methemoglobin; methylene blue did not enhance methemoglobin reduction in the equine erythrocytes, as occurred in human erythrocytes; for horses, lactate was a more efficient substrate in promoting methemoglobin reduction. The reduction of methemoglobin by equine erythrocytes under partial oxidation was very quick in any of the incubation media. The results can explain the incongruity between the previously reported inability of equine erythrocytes to reduce methemoglobin and the lack of methemoglobinemias in equine veterinary practice.     

Lactate formation by rat small intestine in vitro

The formation of lactic acid by mucosal slices, rings and muscle from rat jejunum has been studied for periods of up to 8 min. Lactate output by mucosal slices incubated in the absence of glucose was characterised by two phases: a rapid, initial phase of release lasting about 1 min, followed by a much slower phase extending over the remainder of the incubation period. Glucose addition at 30 s initiated a second rapid phase of lactate release into the medium which was again followed by a slower rate of lactate output up to 8 min. The time course of lactate output suggested that there was a negative Pasteur effect in mucosal slices, which could not be reversed by the addition of ADP or glucose 6-phosphate. By contrast, the rate of lactate formation by rings and muscle from rat jejunum increased steadily over the incubation period, indicating a positive Pasteur effect. When Na⁺ in the incubating medium were replaced by K⁺, lactate formation by mucosal slices and rings was considerably reduced. Measurements of tissue lactate content before and during incubation revealed that about three-quarters of the lactate released by mucosal slices during the first 30 s of incubation was present initially in the tissue. After the first 30 s the tissue lactate remained constant both in the presence and absence of glucose so that the lactate released into the incubation medium is equivalent to the lactate formed by the slices. The role of the various tissue components of the small intestine in lactate formation is discussed in relation to sites of glucose entry.     

Evidence for a functional ATP-citrate lyase:NADP-malate dehydrogenase pathway in bovine adipose tissue: Enzyme and metabolite levels

Activities of key lipogenic and glycolytic enzymes were determined in extracts of crude homogenates to elucidate the rate-limiting step(s) for lipogenesis from lactate and glucose in bovine subcutaneous adipose tissue. The enzymes ATP-citrate lyase, NADP-malate dehydrogenase, and pyruvate carboxylase were shown to have enough activity to account for the rates of in vitro lipogenesis from 10 mm lactate with or without 2 mm glucose. Glucose utilization for fatty acid synthesis appears to be limited by the low activities of key glycolytic enzymes, especially hexokinase. Attempts were also made to estimate enzyme activities in bovine subcutaneous adipose tissue being incubated in vitro by relating primary substrate levels to kinetic characteristics for the enzymes. ATP-citrate lyase was estimated to be operating at levels equivalent to the rates of lactate incorporation into fatty acids in the absence or presence of 2 mm glucose in the incubation media. Additionally, metabolite levels were measured in rapidly frozen samples of bovine subcutaneous adipose tissue to estimate the relative importance of key lipogenic enzymes in vivo. At the citrate and malate levels measured in vivo, ATP-citrate lyase would be operating at levels that approximate those estimated in vitro.     

Regulation of Liver Glycogen Synthesis from [14C] Glucose and [14C] Lactate by Portal Arterial Glucose Difference in the Perfused Rat Liver

To study effects of the portal-arterial glucose difference on the hepatic glycogenesis, the liver was isolated from fasted rats and was bivascularly perfused. Thirty-five milliliters of Krebs-Ringer buffer (pH 7.4) with 2 mM glucose, 3 mM lactate, 20 ng/ml insulin, and [1-¹⁴C]glucose or [U-¹⁴C]lactate was recirculated at flow rates of 14 ml/min via the portal vein and 7 ml/min via the hepatic artery. Glucose was continuously infused at a rate of 27.75 μmol/min into the portal (P experiment) and the arterial cannula (A experiment), and the portal-arterial glucose gradients were + 1.98 and −3.96 mM. Perfusate glucose concentration was not different between the P and A experiments within 20 min. Perfusate lactate level was higher in the P experiment than in the A experiment at 20 min. Incorporation of radioactivity from [¹⁴C]glucosc into glycogen was higher in the P experiment than in the A experiment (0.245 ± 0.014%/20 min vs 0.175 ± 0.022%/20 min, P < 0.01), and not influenced by the addition of insulin. Incorporation of ¹⁴C from [¹⁴C]lactate into glycogen was not different between the P and A experiments, and was significantly increased with the addition of insulin. This activity, in the presence of insulin, was higher in the P experiment than in the A experiment (0.490 ± 0,028%/20 min vs 0.406 ± 0.025%/20 min, P < 0.05). These results suggest that the portal-arterial glucose difference has an important role in the regulation of hepatic glycogenesis from exogenous glucose and gluconeogenesis.     

Cellular energization protects the photosynthetic machinery against salt-induced inactivation in Synechococcus

The effects of the energization of cells by light and by exogenous glucose on the salt-induced inactivation of the photosynthetic machinery were investigated in the cyanobacterium Synechococcus sp. PCC 7942. The incubation of the cyanobacterial cells in a medium supplemented with 0.5 M NaCl induced a rapid decline with a subsequent slow decline, in the oxygen-evolving activity of Photosystem (PS) II and in the electron-transport activity of PSI. Light and exogenous glucose each protected PSII and PSI against the second phase of the NaCl-induced inactivation. The protective effects of light and glucose were eliminated by an uncoupler of phosphorylation and by lincomycin, an inhibitor of protein synthesis. Light and glucose had similar effects on the NaCl-induced inactivation of Na⁺/H⁺ antiporters. After photosynthetic and Na⁺/H⁺-antiport activities had been eliminated by the exposure of cells to 0.5 M NaCl in the darkness, both activities were partially restored by light or exogenous glucose. This recovery was prevented by lincomycin. These observations suggest that cellular energization by either photosynthesis or respiration, which is necessary for protein synthesis, is important for the recovery of the photosynthetic machinery and Na⁺/H⁺ antiporters from inactivation by a high level of NaCl.     

Tricarboxylic acid cycle inhibition by Li+ in the human neuroblastoma SH-SY5Y cell line: a 13C NMR isotopomer analysis

Li+ effects on glucose metabolism and on the competitive metabolism of glucose and lactate were investigated in the human neuroblastoma SH-SY5Y cell line using 13C NMR spectroscopy. The metabolic model proposed for glucose and lactate metabolism in these cells, based on tcaCALC best fitting solutions, for both control and Li+ conditions, was consistent with: (i) a single pyruvate pool; (ii) anaplerotic flux from endogenous unlabelled substrates; (iii) no cycling between pyruvate and oxaloacetate. Li+ was shown to induce a 38 and 53% decrease, for 1 and 15 mM Li+, respectively, in the rate of glucose conversion into pyruvate, when [U-13C]glucose was present, while no effects on lactate production were observed. Pyruvate oxidation by the tricarboxylic acid cycle and citrate synthase flux were shown to be significantly reduced by 64 and 84% in the presence of 1 and 15 mM Li+, respectively, suggesting a direct inhibitory effect of Li+ on tricarboxylic acid cycle flux. This work also showed that when both glucose and lactate are present as energetic substrates, SH-SY5Y cells preferentially consumed exogenous lactate over glucose, as 62% of the acetyl-CoA was derived from [3-13C]lactate while only 26% was derived from [U-13C]glucose. Li+ did not significantly affect the relative utilisation of these two substrates by the cells or the residual contribution of unlabelled endogenous sources for the acetyl-CoA pool.