Glucose metabolism and the channeling of glycolytic intermediates in permeabilized L-929 cells

L-929 cells (mouse fibroblasts) permeabilized with dextran sulfate (DSP cells) carry out vigorous and linear rates of glycolysis when supplied with a suitable incubation medium. Glycolysis in DSP cells is pH dependent, being strongly inhibited at pH 6.5. Compared to their nonpermeabilized counterparts, DSP cells exhibit faster glycolytic rates, but tend to convert a smaller proportion of the glucose utilized to lactate. [14C]Glucose is converted to lactate by DSP cells without dilution from endogenous substrates. When exogenous 12C-labeled glycolytic intermediates (12C-I) are added to glycolyzing DSP cells the [14C]lactate produced from [14C]glucose is diluted to varying extents, depending on the intermediate. However, the extent of that dilution (reduced specific activity) is not that expected from the complete mixing of exogenous 12C-I with their corresponding 14C-labeled intermediates coming from [14C]-glucose. DSP cells also respire and convert glucose to CO2. The amount of 14CO2 produced from [14C]glucose is also reduced by addition of most 12C-I, an interesting exception being pyruvate, which had no measurable effect on 14CO2 production and caused only a modest stimulation of respiration in glycolyzing DSP cells. These results suggest that channeling, or some other form of coupling, takes place between the glycolytic production of pyruvate and its further oxidation. These observations confirm previously published data and add further support to the proposition that channeling of glycolytic intermediates occurs in DSP cells but is of the "leaky" type. Although abundant evidence in the literature indicates that various glycolytic enzymes associate with F-actin, as well as other elements of the cytomatrix, we observed no effect of cytochalasin D on lactate production even at very high concentrations of this compound. Our results are compared with those from other laboratories and discussed in the context of metabolic organization.     

The role of phosphoenolpyruvate carboxykinase in muscle alanine synthesis.

3-Mercaptopicolinic acid (3-MPA) is reportedly a specific inhibitor of phosphoenolpyruvate (PEP) carboxykinase and has hitherto been used accordingly to elucidate the metabolic role of PEP carboxykinase in vitro and in vivo. We show that 3-MPA has multiple effects on intermediary metabolism in hemidiaphragms from 40 h-starved rats. It decreases the release of lactate + pyruvate and alanine in hemidiaphragms provided with no added substrate or with valine, leucine or isoleucine. Moreover, irrespective of the substrate provided (none, valine, leucine, isoleucine, glucose, acetate, oleate), 3-MPA decreases the [lactate]/[pyruvate] ratio. 3-MPA is without effect on 14CO2 production from [U-14C]valine, [1-14C]valine, [1-14C]leucine, [U-14C]isoleucine or [1-14C]oleate, but stimulates 14CO2 production from [U-14C]glucose and [1-14C]pyruvate and inhibits 14CO2 production from [1-14C]acetate. Glycolytic flux (measured as 3H2O formation from [5-3H]glucose) is stimulated by 3-MPA. It is concluded that 3-MPA has site(s) of actions other than PEP carboxykinase and that the putative role of PEP carboxykinase in alanine synthesis de novo in skeletal muscle from tricarboxylic acid-cycle intermediates and related amino acids requires reappraisal.     

The permissive effects of glucocorticoid on hepatic gluconeogenesis. Glucagon stimulation of glucose-suppressed gluconeogenesis and inhibition of 6-phosphofructo-1-kinase in hepatocytes from fasted rats

Production of [14C]glucose from [14C]lactate in the perfused livers of 24-h fasted adrenalectomized rats was not stimulated by 1 nM glucagon but was significantly increased by 10 nM hormone. Crossover analysis of glycolytic intermediates in these livers revealed a significant reduction in glucagon action at site(s) between fructose 6-phosphate and fructose 1,6-bisphosphate as a result of adrenalectomy. Site(s) between pyruvate and P-enolpyruvate was not affected. In isolated hepatocytes, adrenalectomy reduced glucagon response in gluconeogenesis while not affecting glucagon inactivation of pyruvate kinase. A distinct lack of glucagon action on 6-phosphofructo-1-kinase activity was noted in these cells. When hepatocytes were incubated with 30 mM glucose, lactate gluconeogenesis was greatly stimulated by glucagon. A reduction in both sensitivity and responsiveness to the hormone in gluconeogenesis was seen in the adrenalectomized rat. These changes were well correlated with similar impairment in glucagon action on 6-phosphofructo-1-kinase activity and fructose 2,6-bisphosphate content in hepatocytes from adrenalectomized rats incubated with 30 mM glucose. These results suggest that adrenalectomy impaired the gluconeogenic action of glucagon in livers of fasted rats at the level of regulation of 6-phosphofructo-1-kinase and/or fructose 2,6-bisphosphate content.     

Sorting of metabolic pathway flux by the plasma membrane in cerebrovascular smooth muscle cells

We used-escin-permeabilized pig cerebral microvessels (PCMV) to study theorganization of carbohydrate metabolism in the cytoplasm of vascularsmooth muscle (VSM) cells. We have previously demonstrated (Lloyd PGand Hardin CD. Am J Physiol Cell Physiol 277: C1250-C1262,1999) that intact PCMV metabolize the glycolytic intermediate[1-¹³C]fructose 1,6-bisphosphate (FBP) to[1-¹³C]glucose with negligible production of[3-¹³C]lactate, while simultaneouslymetabolizing [2-¹³C]glucose to[2-¹³C]lactate. Thus gluconeogenic andglycolytic intermediates do not mix freely in intact VSM cells(compartmentation). Permeabilized PCMV retained the ability tometabolize [2-¹³C]glucose to[2-¹³C]lactate and to metabolize[1-¹³C]FBP to[1-¹³C]glucose. The continued existence ofglycolytic and gluconeogenic activity in permeabilized cells suggeststhat the intermediates of these pathways are channeled (directlytransferred) between enzymes. Both glycolytic and gluconeogenic flux inpermeabilized PCMV were sensitive to the presence of exogenous ATP andNAD. It was most interesting that a major product of[1-¹³C]FBP metabolism in permeabilized PCMV was[3-¹³C]lactate, in direct contrast to ourprevious findings in intact PCMV. Thus disruption of the plasmamembrane altered the distribution of substrates between the glycolyticand gluconeogenic pathways. These data suggest that organization of theplasma membrane into distinct microdomains plays an important role insorting intermediates between the glycolytic and gluconeogenic pathwaysin intact cells.

     

Regulation of gluconeogenesis from pyruvate and lactate in the isolated perfused rat liver

The effects of glucagon and the alpha-adrenergic agonist, phenylephrine, on the rate of 14CO2 production and gluconeogenesis from [1-14C]lactate and [1-14C]pyruvate were investigated in isolated perfused livers of 24-h-fasted rats. Both glucagon and phenylephrine stimulated the rate of 14CO2 production from [1-14C]lactate but not from [1-14C]pyruvate. Neither glucagon nor phenylephrine affected the activation state of the pyruvate dehydrogenase complex in perfused livers derived from 24-h-fasted rats. 3-Mercaptopicolinate, an inhibitor of the phosphoenolpyruvate carboxykinase reaction, inhibited the rates of 14CO2 production and glucose production from [1-14C]lactate by 50% and 100%, respectively. Furthermore, 3-mercaptopicolinate blocked the glucagon- and phenylephrine-stimulated 14CO2 production from [1-14C]lactate. Additionally, measurements of the specific radioactivity of glucose synthesized from [1-14C]lactate, [1-14C]pyruvate and [2-14C]pyruvate indicated that the 14C-labeled carboxyl groups of oxaloacetate synthesized from 1-14C-labeled precursors were completely randomized and pyruvate----oxaloacetate----pyruvate substrate cycle activity was minimal. The present study also demonstrates that glucagon and phenylephrine stimulation of the rate of 14CO2 production from [1-14C]lactate is a result of increased metabolic flux through the phosphoenolpyruvate carboxykinase reaction, and phenylephrine-stimulated gluconeogenesis from pyruvate is regulated at step(s) between phosphoenolpyruvate and glucose.     

Acetoacetate and Glucose as Lipid Precursors and Energy Substrates in Primary Cultures of Astrocytes and Neurons from Mouse Cerebral Cortex

Primary cultures of astrocytes and neurons derived from neonatal and embryonic mouse cerebral cortex, respectively, were incubated with [3-14C]acetoacetate or [2-14C]glucose. The utilization of glucose and acetoacetate, the production of lactate, D-3-hydroxybutyrate, and 14CO2, and the incorporation of 14C and of 3H from 3H2O into lipids and lipid fractions were measured. Both cell types used acetoacetate as an energy substrate and as a lipid precursor; lactate was the major product of glucose metabolism. About 60% of the acetoacetate that was utilized by neurons was oxidized to CO2, whereas this was only approximately 20% in the case of cultured astrocytes. This indicates that the rate at which 14C-labeled Krebs cycle intermediates exchange with pools of unlabeled intermediates is much higher in astrocytes than in neurons. Acetoacetate is a better precursor for the synthesis of fatty acids and cholesterol than glucose, presumably because it can be used directly in the cytosol for these processes; preferential incorporation into cholesterol was not observed in these in vitro systems. We conclude that ketone bodies can be metabolized both by the glial cells and by the neuronal cells of developing mouse brain.     

The effect of pretreatment with pentobarbital on the extent of [14C] incorporation from [U-14C]glucose into various rat brain glycolytic intermediates: relevance to regulation at hexokinase and phosphofrucktokinase

In the present investigation we monitored the incorporation of [¹⁴C] from [U-¹⁴C]glucose into various rat brain glycolytic intermediates of conscious and pentobarbital-anesthetized animals. Labeled glucose was delivered to brain by single bolus intracarotid injection and brain tissue was subsequently prepared at 15, 30 and 45 sec by freeze-blowing. Glycolytic intermediates were then separated by column chromatography. Our results showed a gradual decrease with time of¹⁴C-labeled glucose which gave a calculated rate for glucose metabolism of 0.86 mol/min/g and 0.56 mol/min/g in conscious and anesthetized animals, respectively. Compared to the results obtained using conscious animals the administration of pentobarbital not only resulted in a significant attenuation of the rate of glucose metabolism but also caused a similar reduction in the amount of¹⁴C incorporated into several glycolytic intermediates. These intermediates included: glucose 6-phosphate, fructose 6-phosphate, fructose, 1,6 diphosphate, dihydroxyacetone phosphate and post glycolytic compounds. In addition, pretreatment with pentobarbital resulted in a 75% increase in the endogenous concentration of glucose, 10% increase in glucose 6-phosphate, no change in fructose 6-phosphate and 42% decrease in lactate compared to levels in brains obtained from conscious animals. These results are discussed in relation to control of glycolysis through coupled regulation at hexokinase-phosphofructokinase.     

Exchange-mediated dilution of brain lactate specific activity: implications for the origin of glutamate dilution and the contributions of glutamine dilution and other pathways

The magnitude of metabolic activation is greatly underestimated in autoradiographic studies using [1- or 6-¹⁴C]glucose compared to parallel assays with [¹⁴C]deoxyglucose indicating that most of the label corresponding to the additional [¹⁴C]glucose consumed during activation compared to rest is quickly released from activated structures. Label could be lost by net release of [¹⁴C]lactate from brain or via lactate exchange between blood and brain. These possibilities were distinguished by comparison of glucose and lactate specific activities in arterial blood and brain before, during, and after generalized sensory stimulation and during spreading cortical depression. Over a wide range of brain lactate concentrations, lactate specific activity was close to the theoretical maximum, i.e. half that of [6-¹⁴C]glucose, indicating that exchange-mediated dilution of lactate is negligible and that efflux of [¹⁴C]lactate probably accounts for most of the label loss. Low lactate dilution also indicates that dilution of glutamate C4 fractional enrichment in [¹³C]glucose studies, currently ascribed predominantly to lactate exchange, arises from other unidentified pathways or factors. Alternative explanations for glutamate dilution (presented in Supporting Information) include poorly labeled amino acid pools and oxidative metabolism of minor substrates in astrocytes to first dilute the astrocytic glutamine pool, followed by dilution of glutamate via glutamate–glutamine cycling.     

Glucose turnover in compensated hepatic cirrhosis

Glucose turnover and recycling from glucose derived 3-carbon intermediates were examined in overnight fasted patients with compensated hepatic cirrhosis and in age- and weight-matched normal control subjects. Fasting blood concentrations of glucose, lactate and glycerol were similar in both groups but blood pyruvate (60 +/- 10 vs. 80 +/- mumol/l, P less than 0.05), blood alanine (0.23 +/- 0.02 vs 0.34 +/- 0.02 mmol/l, P less than 0.01) were decreased and serum insulin increased (19 [13-24]v 7 [4-11] mU/l, P less than 0.01) in cirrhotic subjects. Absolute glucose turnover, assessed by analysis of decay of [3H]-3-glucose specific activity was decreased in cirrhotic patients (8.1 +/- 0.6 v 12.1 +/- 0.7 mol/kg-1 min-1). Glucose "recycling", assessed by the difference between absolute glucose turnover and that given by [14C]-1-glucose data, was normal in cirrhotic patients suggesting that Cori cycle (glucose-lactate-glucose) activity was normal. These data support previous findings of decreased peripheral glucose utilisation and insulin resistance in cirrhotic patients.     

Patterns of carbon flow from glucose to methane in a thermophilic anaerobic bioreactor

[U-¹⁴C]Glucose, added carrier-free to sludge from a thermophilic anaerobic bioreactor being fed a lignocellulose waste, was rapidly turned over with less than one-third of the original radiolabel remaining as glucose after 5 s of incubation. The primary labeled products found were [¹⁴C]acetate and ¹⁴CO₂, which were in a ratio near 2:1. Further incubation resulted in the disappearance of [¹⁴C]acetate and the appearance of an equivalent amount of label as ¹⁴CH₄ and ¹⁴CO₂. No significant production of [¹⁴C]propionate, butyrate, lactate, or ethanol was detected from [¹⁴C]glucose, even if these potential intermediates (unlabeled) were added to the sludge at a concentration of 1 mM to trap any label entering their pools. Addition of 0.8 atm (80 kPa) of H₂ to the headspace over sludge resulted in some accumulation of [¹⁴C]lactate and a corresponding decrease in [¹⁴C]acetate produced from [¹⁴C]glucose. Production of [¹⁴C]propionate, butyrate and ethanol were still not significant in the presence of H₂. Incubation of sludge for 1 h in the presence of hydrogen resulted in increases in the lactate and formate concentrations, but not those of propionate, butyrate, or ethanol. These results demonstrate that glucose was metabolized directly to acetate, CO₂, and H₂ by the microbial populations in the bioreactor with little carbon from glucose flowing through other intermediates, indicating a high degree of coupling between glucose fermentation and hydrogen uptake. The short-term response of these microbial populations to elevated H₂ partial pressures was to increase lactate production.     

Gluconeogenesis in the kidney cortex. Flow of malate between compartments

1. Kidney-cortex slices from starved rats were incubated with l-[U-(14)C]lactate or l-[U-(14)C]malate plus unlabelled acetate and the specific radioactivity of the glucose formed was determined. In parallel experiments the specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate and l-malate was determined. 2. By analytical methods the major products formed from the substrates were measured. The glucose formed was purified by paper chromatography for determination of specific radioactivity. 3. The specific radioactivity of the glucose formed from l-[U-(14)C]lactate agrees with predictions of a model based on interaction of the gluconeogenic and the oxidative pathways. 4. The specific radioactivity of the glucose formed from l-[U-(14)C]malate agrees with the predicted value if rapid malate exchange between the cytosol and mitochondria is assumed. 5. The rate of malate exchange between compartments was estimated to be rapid and at least several times the rate of glucose formation. 6. The specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate or l-malate agrees with the predictions from the model, again assuming rapid malate exchange between compartments. 7. Malate exchange between compartments together with reversible malate dehydrogenase activity in the mitochondria and cytosol also tends to equilibrate isotopically the NADH pool in these compartments. (3)H from compounds such as l-[2-(3)H]lactate, which form NAD(3)H in the cytosol, appears in part in water; and (3)H from dl-beta-hydroxy[3-(3)H]butyrate, which forms NAD(3)H in the mitochondria, appears in part in glucose, largely on C-4.     

Effect of lactate on the effectiveness of glucose and succinate oxidation by myocardium homogenates

Lactate (20 mM) was studied for its effect on the intensity of [1-6-14C] glucose and [1,4-14C] succinate oxidation by the rat myocardial homogenates. It is established that lactate induces specific suppression of aerobic glucose oxidation. A moderate reduction of the succinate and lactate oxidation in their combined incubation is of a non-specific character.     

Extracellular Intermediates of Glucose Metabolism: Fluxes of Endogenous Lactate and Alanine Through Extracellular Pools in Embryonic Sympathetic Ganglia

The flux rates of lactate and alanine in and out of the cells of an intact tissue, which cannot be measured directly because some of the released materials are reabsorbed, were determined by computer analysis of uptakes and outputs by the whole tissue in the presence of various concentrations of these substances. The outputs of labeled lactate and alanine from [U-14C]glucose and the uptakes of [U-14C]lactate and [U-14C]alanine were measured on intact sympathetic ganglia excised from 15-day-old chicken embryos. The volume and time constant of the extracellular space were measured using labeled lactate, alanine, and sucrose. Models, which mathematically described the cellular uptakes and outputs as functions of the extracellular concentrations, were used to predict the exchanges that would be observed on the whole tissue, and their parameters were adjusted for best fit to the actual observations. The fitted models were then used to calculate the fluxes in and out of the cells and the concentrations in the extracellular space. The following results were obtained: (1) Cellular uptakes of lactate and alanine were both well described by familiar Michaelis-Menten kinetics. (2) The cellular output of [14C]-lactate from [14C]glucose declined with increase in the extracellular lactate concentration, whereas the cellular output of [14C]alanine from [14C]glucose rose with the extracellular alanine concentration. (3) Half-saturation values for cellular uptake, determined from the fitted equations, were 0.45 mM for lactate and 1.17 mM for alanine, both several-fold lower than less relevant estimates for the whole tissue made directly from the uptake observations. (4) As much as 45% of the carbon in the glucose consumed was released into the extracellular space as lactate and alanine, but much of this was reabsorbed. Implications for brain metabolism are discussed.     

Effects of modulation of glycerol kinase expression on lipid and carbohydrate metabolism in human muscle cells.

Glycerol is taken up by human muscle in vivo and incorporated into lipids, but little is known about regulation of glycerol metabolism in this tissue. In this study, we have analyzed the role of glycerol kinase (GlK) in the regulation of glycerol metabolism in primary cultured human muscle cells. Isolated human muscle cells exhibited lower GlK activity than fresh muscle explants, but the activity in cultured cells was increased by exposure to insulin. [U-(14)C]Glycerol was incorporated into cellular phospholipids and triacylglycerides (TAGs), but little or no increase in TAG content or lactate release was observed in response to changes in the medium glycerol concentration. Adenovirus-mediated delivery of the Escherichia coli GlK gene (AdCMV-GlK) into muscle cells caused a 30-fold increase in GlK activity, which was associated with a marked rise in the labeling of phospholipid or TAG from [U-(14)C]glycerol compared with controls. Moreover, GlK overexpression caused [U-(14)C]glycerol to be incorporated into glycogen, which was dependent on the activation of glycogen synthase. Co-incubation of AdCMV-GlK-treated muscle cells with glycerol and oleate resulted in a large accumulation of TAG and an increase in lactate production. We conclude that GlK is the limiting step in muscle cell glycerol metabolism. Glycerol 3-phosphate is readily used for TAG synthesis but can also be diverted to form glycolytic intermediates that are in turn converted to glycogen or lactate. Given the high levels of glycerol in muscle interstitial fluid, these finding suggest that changes in GlK activity in muscle can exert important influences on fuel deposition in this tissue.     

"Pyruvate recycling" and its influence on the estimation of the pentose pathway in intact liver and Morris hepatoma 5123TC cells

The phenomenon of "pyruvate recycling" is demonstrated in perfused rat liver, rabbit liver in situ and in Morris Hepatoma 5123TC cells and quantitatively measured using [2-14C]pyruvate and the method of Friedmann et al. (1971). Various metabolites, viz. lactate, DHAP, glucose, glucose 6-P and fructose 6-P were isolated and degraded following the metabolism of [2-14C]pyruvate and [2-14C]glycerol in order to assess the 14C-distributions imparted by "pyruvate recycling" reactions. The labelling of DHAP, lactate, glucose and glucose 6-P showed 14C randomizations consistent with the operation and the quantitative extent of "pyruvate recycling". These findings support the proposal that the actions of "pyruvate recycling" may account for the failure to find significant levels of 14C isotope at C-1 of glucose 6-P following the metabolism of [4,5,6-14C]- or [6-14C]glucose by L-type pentose pathway metabolism in aerobic intact tissues. "Pyruvate recycling" diminishes the measured value of the L-type pentose cycle in intact tissues and qualifies one of the mechanistic predictions of the L-type pentose pathway which was unravelled by tracing its reactions with labelled ribose 5-P and liver enzymes (Horecker et al., 1954; Williams et al., 1978a,b) in vitro. The demonstration of an association of L-type pentose pathway reactions with "pyruvate recycling" by way of the common reactions of their triose-P intermediates qualifies the superficial acceptance of the predictions of the L-type pathway in vitro for the distribution of isotopic labels by aerobic tissues in vivo.     

Glucose infused through the portal vein enhances liver gluconeogenesis and glycogenesis from [3-14C]pyruvate in the starved rat

After a pulse of [3-14C]pyruvate, 24 hr starved rats were infused through the portal vein with two different doses of glucose (7.8 or 20.8 mg/min) or the medium, and blood was collected from the inferior cava vein at the level of the suprahepatic veins. The highest dose of glucose enhanced the appearance of [14C]glucose in blood from the 2nd to the 20th min after tracer delivery. It also enhanced production of [14C]glycogen and concentration of glycogen in the liver after 5 and 20 min. At 20 min of glucose infusion the appearance of [14C]glyceride glycerol in liver as well as liver lactate concentration and lactate/pyruvate ratio were increased. The low dose of glucose used enhanced liver values of [14C]glycogen, [14C]glycogen specific activity and glycogen concentration. Our results support the hypothesis that in the starved rat glucose is converted into C3 units prior to being deposited as liver glycogen and based on the liver zonation model (Jungermann et al., 1983) it is proposed that glucose stimulated gluconeogenesis by shifting the liver to the cytosolic redox state as a secondary consequence of increased glycolytic activity.     

Glucose metabolism in the newborn rat. Temporal studies in vivo

1. The concentrations of plasma d-glucose, l-lactate, free fatty acids and ketone bodies and of liver glycogen were measured in caesarian-delivered newborn rats at time-intervals up to 4h after delivery. Glucose and lactate concentrations decreased markedly during the first hours after delivery, but there was a delay of 60-90min before significant glycogen mobilization occurred. 2. The specific radioactivity of plasma d-glucose was measured as a function of time for up to 75min after the intraperitoneal injection of d-[6-(14)C]glucose and d-[6-(3)H]glucose into caesarian-delivered rats at 0, 1 and 2h after delivery. Calculations revealed that there was an appreciable rate of glucose formation at all ages studied, but immediately after delivery this was exceeded by the rate of glucose utilization. Around 2h post partum the rate of glucose utilization decreased dramatically and this coincided with a reversal of the immediately postnatal hypoglycaemia. 3. The specific radioactivity of plasma l-lactate and the incorporation of (14)C into plasma d-glucose and liver glycogen was measured as a function of time after the intraperitoneal injection of l-[U-(14)C]lactate into rats immediately after delivery. The logarithm of the specific radioactivity of plasma l-[U-(14)C]lactate decreased linearly with time for at least 60min after injection and the calculated rate of lactate utilization exceeded the rate of lactate formation. 4. (14)C incorporation into plasma d-glucose was maximal from 30-60min after injection of l-[U-(14)C]lactate and the amount incorporated at 60min was 23% of that present in plasma lactate. Although (14)C was also incorporated into liver glycogen the amount was always less than 3% of that present in plasma glucose. 5. The results are discussed in relationship to the adaptation of the newly born rat to the extra-uterine environment and the possible involvement of gluconeogenesis at this time before feeding is established.     

Pyruvate carboxylation as an anaplerotic mechanism in the isolated perfused rat heart.

1. The role of pyruvate carboxylation in the net synthesis of tricarboxylic acid-cycle intermediates during acetate metabolism was studied in isolated rat hearts perfused with [1-14C]pyruvate. 2. The incorporation of the 14C label from [1-14C]pyruvate into the tricarboxylic acid-cycle intermediates points to a carbon input from pyruvate via enzymes in addition to pyruvate dehydrogenase and citrate synthase. 3. On addition of acetate, the specific radioactivity of citrate showed an initial maximum at 2 min, with a subsequent decline in labelling. The C-6 of citrate (which is removed in the isocitrate dehydrogenase reaction) and the remainder of the molecule showed differential labelling kinetics, the specific radioactivity of C-6 declining more rapidly. Since this carbon is lost in the isocitrate dehydrogenase reaction, the results are consistent with a rapid inactivation of pyruvate dehydrogenase after the addition of acetate, which was confirmed by measuring the 14CO2 production from [1-14C]pyruvate. 4. The results can be interpreted to show that carboxylation of pyruvate to the C4 compounds of the tricarboxylic acid cycle occurs under conditions necessitating anaplerosis in rat myocardium, although the results do not identify the enzyme involved. 5. The specific radioactivity of tissue lactate was too low to allow it to be used as an indicator of the specific radioactivity of the intracellular pyruvate pool. The specific radioactivity of alanine was three times that of lactate. When the hearts were perfused with [1-14C]lactate, the specific radioactivity of alanine was 70% of that of pyruvate. The results suggest that a subcompartmentation of lactate and pyruvate occurs in the cytosol.     

Lactate metabolism in the perfused rat hindlimb.

A preparation of isolated rat hindleg was perfused with a medium consisting of bicarbonate buffer containing Ficoll and fluorocarbon, containing glucose and/or lactate. The leg was electrically prestimulated to deplete partially muscle glycogen. The glucose was labelled uniformly with 14C and with 3H in positions 2, 5 or 6, and lactate uniformly with 14C and with 3H in positions 2 or 3. Glucose carbon was predominantly recovered in glycogen, and to a lesser extent in lactate. The 3H/14C ration in glycogen from [5-3H,U-14C]- and [6-3H,U-14C]-glucose was the same as in glucose. Nearly all the utilized 3H from [2-3H]glucose was recovered as water. Insulin increased glucose uptake and glycogen synthesis 3-fold. When the muscle was perfused with a medium containing 10 mM-glucose and 2 mM-lactate, there was little change in lactate concentration. 14C from lactate was incorporated into glycogen. There was a marked exponential decrease in lactate specific radioactivity, much greater with [3H]- than with [14C]-lactate. The 'apparent turnover' of [U-14C]lactate was 0.28 mumol/min per g of muscle, and those of [2-3H]- and [3-3H]-lactate were both about 0.7 mumol/min per g. With 10 mM-lactate as sole substrate, there was a net uptake of lactate, at a rate of about 0.15 mumol/min per g, and the apparent turnover of [U-14C]lactate was 0.3 mumol/min per g. The apparent turnover of [3H]lactate was 3-5 times greater. When glycogen synthesis was low (no prestimulation, no insulin), the incorporation of lactate carbon into glycogen exceeded that from glucose, but at high rates of glycogen deposition the incorporation of lactate carbon was much less than that of glucose. Lactate incorporation into glycogen was similar in fast-twitch white and fast-twitch red muscle, but was very low in slow-twitch red fibres. We find that (a) pyruvate in muscle is incorporated into glycogen without randomization of carbon, and synthesis is not inhibited by mercaptopicolinate or cycloserine; (b) there is extensive lactate turnover in the absence of net lactate uptake, and there is a large dilution of 14C-labelled lactate from endogenous supply; (c) there is extensive detritiation of [2-3H]- and [3-3H]-lactate in excess of 14C utilization.     

Short-term insulin infused through the portal vein enhances liver gluconeogenesis and glycogenesis from [3-14C]pyruvate in the starved rat

Insulin infusion through the portal vein immediately after a pulse of [3-14C]pyruvate in 24 hr starved rats enhanced the appearance of [14C]glucose at 2, 5 and 10 min and glucose specific activity at 1, 2 and 20 min in blood collected from the cava vein at the level of the suprahepatic veins. Insulin infusion for 5 min decreased liver pyruvate concentration and enhanced both liver and plasma lactate/pyruvate ratio, and it decreased the plasma concentration of all amino acids. When insulin was infused together with glucose, [14C]glucose levels and glucose specific activity decreased in blood but there was a marked increase in liver [14C]glycogen, glycogen specific activity and glycogen concentration, and an increase in liver lactate/pyruvate ratio. The effect of insulin plus glucose infusion on plasma amino acids concentration was smaller than that found with insulin alone. It is proposed that insulin effect enhancing liver gluconeogenesis is secondary to its effect either enhancing liver glycolysis which modifies the liver's cytoplasmic oxidoreduction state to its more reduced form, increasing liver amino acids consumption or both. In the presence of glucose, products of gluconeogenesis enhanced by insulin are diverted into glycogen synthesis rather than circulating glucose. This together with results of the preceding paper (Soley et al., 1985), indicates that glucose enhances liver glycogen synthesis from C3 units in the starved rat, the process being further enhanced in the presence of insulin.