Differential modulation of glucose,lactate, and pyruvate oxidation by insulin and dichloroacetate in the rat heart

Despite the fact that lactate and pyruvate are potential substrates for energy production in vivo, our understanding of the control and regulation of carbohydrate metabolism is based principally on studies where glucose is the only available carbohydrate. Therefore, the purpose of this study was to determine the contributions of lactate, pyruvate, and glucose to energy production in the isolated, perfused rat heart over a range of insulin concentrations and after activation of pyruvate dehydrogenase with dichloroacetate (DCA). Hearts were perfused with physiological concentrations of [1-13C]glucose, [U-13C]lactate, [2-13C]pyruvate, and unlabeled palmitate for 45 min. Hearts were freeze clamped, and 13C NMR glutamate isotopomer analysis was performed on tissue extracts. Glucose, lactate, and pyruvate all contributed significantly to myocardial energy production; however, in the absence of insulin, glucose contributed only 25-30% of total pyruvate oxidation. Even under conditions where carbohydrates represented >95% of substrate entering the tricarboxylic acid (TCA) cycle, we found that glucose contributed at most 50-60% of total carbohydrate oxidation. Despite being present at only 0.1 mM, pyruvate contributed between approximately 10% and 30% of total acetyl-CoA entry into the TCA cycle. We also found that insulin and DCA not only increased glucose oxidation but also exogenous pyruvate oxidation; however, lactate oxidation was not increased. The differential effects of insulin and DCA on pyruvate and lactate oxidation provide further evidence for compartmentation of cardiac carbohydrate metabolism. These results may have important implications for understanding the mechanisms underlying the beneficial effects of increasing cardiac carbohydrate metabolism.     

Glucose metabolism in perfused skeletal muscle. Effects of starvation, diabetes, fatty acids, acetoacetate, insulin and exercise on glucose uptake and disposition.

1. The regulation of glucose uptake and disposition in skeletal muscle was studied in the isolated perfused rat hindquarter. 2. Insulin and exercise, induced by sciatic-nerve stimulation, enhanced glucose uptake about tenfold in fed and starved rats, but were without effect in rats with diabetic ketoacidosis. 3. At rest, the oxidation of lactate (0.44 mumol/min per 30 g muscle in fed rats) was decreased by 75% in both starved and diabetic rats, whereas the release of alanine and lactate (0.41 and 1.35 mumol/min per 30 g respectively in the fed state) was increased. Glycolysis, defined as the sum of lactate+alanine release and lactate oxidation, was not decreased in either starvation or diabetes. 4. In all groups, exercise tripled O2 consumption (from approximately 8 to approximately 25 mumol/min per 30 g of muscle) and increased the release and oxidation of lactate five- to ten-fold. The differences in lactate release between fed, starved and diabetic rats observed at rest were no longer apparent; however, lactate oxidation was still several times greater in the fed group. 5. Perfusion of the hindquarter of a fed rat with palmitate, octanoate or acetoacetate did not alter glucose uptake or lactate release in either resting or exercising muslce; however, lactate oxidation was significantly inhibited by acetoacetate, which also increased the intracellular concentration of acetyl-CoA. 6. The data suggest that neither that neither glycolysis nor the capacity for glucose transport are inhbitied in the perfused hindquarter during starvation or perfusion with fatty acids or ketone bodies. On the other hand, lactate oxidation is inhibited, suggesting diminished activity of pyruvate dehydrogenase. 7. Differences in the regulation of glucose metabolism in heart and skeletal muscle and the role of the glucose/fatty acid cycle in each tissue are discussed.     

Fuel homeostasis in the harbor seal during submerged swimming

1. The turnover rates and oxidation rates of plasma glucose, lactate, and free fatty acids (FFA) were measured in three harbor seals (average mass = 40 kg) at rest or during voluntary submerged swimming in a water flume at 35% (1.3 m.s-1) and 50% (2 m.s-1) of maximum oxygen consumption (MO2max). 2. For seals resting in water, the total turnover rates for glucose, lactate, and FFA were 23.2, 26.2, and 7.5 mumols.min-1.kg-1, respectively. Direct oxidation of these metabolites accounted for approximately 7%, 27%, and 33% of their turnover and 3%, 7%, and 18% of the total ATP production, respectively. 3. For swimming seals, MO2max was achieved at a drag load equivalent to a speed of 3 m.s-1 and averaged 1.85 mmol O2.min-1.kg-1, which is 9-fold greater than resting metabolism in water at 18 degrees C. 4. At 35% and 50% MO2max, glucose turnover and oxidation rates did not change from resting levels. Glucose oxidation contributed about 1% of the total ATP production during swimming. 5. At 50% MO2max, lactate turnover and anaerobic ATP production doubled, but the steady state plasma lactate concentration remained low at 1.1 mM. Lactate oxidation increased 63% but still contributed only 4% of the total ATP production. Anaerobic metabolism contributed about 1% of the total ATP production at rest and during swimming. 6. The plasma FFA concentration and turnover rate increased only 24% and 37% over resting levels, respectively, at 50% MO2max. However, the oxidation rate increased almost 3.5-fold and accounted for 85% of the turnover.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of Lactate in the Rat Brain During the Early Neonatal Period

The metabolism of lactate in isolated cells from early neonatal rat brain has been studied. In these circumstances, lactate was mainly oxidized to CO2, although a significant portion was incorporated into lipids (78% sterols, 4% phosphatidylcholine, 2% phosphatidylethanolamine, and 1% phosphatidylserine). The rate of lactate incorporation into CO2 and lipids was higher than those found for glucose and 3-hydroxybutyrate. Lactate strongly inhibited glucose oxidation through the pyruvate dehydrogenase-catalyzed reaction and the tricarboxylic acid cycle while scarcely affecting glucose utilization by the pentose phosphate pathway. Lipogenesis from glucose was strongly inhibited by lactate without relevant changes in the rate of glycerol phosphate synthesis. These results suggest that lactate inhibits glucose utilization at the level of the pyruvate dehydrogenase-catalyzed reaction, which may be a mechanism to spare glucose for glycerol and NADPH synthesis. The effect of 3-hydroxybutyrate inhibiting lactate utilization only at high concentrations of 3-hydroxybutyrate suggests that before ketogenesis becomes active, lactate may be the major fuel for the neonatal brain. (-)-Hydroxycitrate and aminooxyacetate markedly inhibited lipogenesis from lactate, suggesting that the transfer of lactate carbons through the mitochondrial membrane is accomplished by the translocation of both citrate and N-acetylaspartate.     

Leg and arm lactate and substrate kinetics during exercise

To study the role of muscle mass and muscle activity on lactate and energy kinetics during exercise, whole body and limb lactate, glucose, and fatty acid fluxes were determined in six elite cross-country skiers during roller-skiing for 40 min with the diagonal stride (Continuous Arm + Leg) followed by 10 min of double poling and diagonal stride at 72-76% maximal O(2) uptake. A high lactate appearance rate (R(a), 184 +/- 17 micromol x kg(-1) x min(-1)) but a low arterial lactate concentration ( approximately 2.5 mmol/l) were observed during Continuous Arm + Leg despite a substantial net lactate release by the arm of approximately 2.1 mmol/min, which was balanced by a similar net lactate uptake by the leg. Whole body and limb lactate oxidation during Continuous Arm + Leg was approximately 45% at rest and approximately 95% of disappearance rate and limb lactate uptake, respectively. Limb lactate kinetics changed multiple times when exercise mode was changed. Whole body glucose and glycerol turnover was unchanged during the different skiing modes; however, limb net glucose uptake changed severalfold. In conclusion, the arterial lactate concentration can be maintained at a relatively low level despite high lactate R(a) during exercise with a large muscle mass because of the large capacity of active skeletal muscle to take up lactate, which is tightly correlated with lactate delivery. The limb lactate uptake during exercise is oxidized at rates far above resting oxygen consumption, implying that lactate uptake and subsequent oxidation are also dependent on an elevated metabolic rate. The relative contribution of whole body and limb lactate oxidation is between 20 and 30% of total carbohydrate oxidation at rest and during exercise under the various conditions. Skeletal muscle can change its limb net glucose uptake severalfold within minutes, causing a redistribution of the available glucose because whole body glucose turnover was unchanged.     

Effect of dichloroacetate on mechanical performance and metabolism of compromised diaphragm muscle.

We investigated the effect of dichloroacetate (DCA) on tension generation and carbohydrate metabolism of the rat diaphragm in vitro. Isolated diaphragms were placed in individual organ chambers and were hooked to force-displacement transducers. Net lactate production and glucose and lactate oxidation were measured in vitro. Diaphragmatic fatigue was precipitated by in vivo endotoxemic shock, by in vitro hypoxia, or by in vitro repetitive tetanic stimulation. In diaphragms isolated from endotoxemic rats, DCA increased tension generation by 30 and 20% at stimulation frequencies of 20 and 100 Hz, respectively. Associated with changes in mechanical performance, DCA reduced net lactate production by 53% after 60 min of incubation and increased glucose oxidation 54% but had no effect on lactate oxidation. During in vitro hypoxia, DCA reduced net diaphragmatic lactate production by 30% and increased glucose oxidation by 45% but did not attenuate hypoxic fatigue. DCA had no effect on tension generation during repetitive tetanic stimulation. We conclude that DCA improves in vitro diaphragmatic fatigue due to endotoxicosis but not due to hypoxia or repetitive stimulation.     

Impaired sensitivity to insulin of rat livers perfused with blood of diminished haematocrit.

1. In livers from fed rats perfused with homologous whole blood of a haematocrit value of 37%, insulin decreased the perfusate concentrations of glucose and amino acids, production of ketone bodies (3-hydroxybutyrate + acetoacetate) and increased bile flow. 2. Perfusion with blood diluted with buffer to a haematocrit value of 17% decreased hepatic O2 consumption by 40-50%. Perfusate concentrations of glucose and lactate, the rate of ketogenesis and the ratios [lactate]/[pyruvate] and [3-hydroxybutyrate]/[acetoacetate] were all increased. 3. In livers perfused with blood of diminished haematocrit, effects of insulin on perfusate glucose an amino acids, ketogenesis and bile flow were abolished.     

Physiological stress responses of two species of coral trout (Plectropomus leopardus and Plectropomus maculatus)

The physiological responses of two species of coral trout (Plectropomus maculatus and Plectropomus leopardus) to capture, shallow water and low salinity stressors were investigated. The responses of P. maculatus and P. leopardus to capture stress were characterised by rapid and transient increases in glucose, haemoglobin, haematocrit and lactate, as well as an equally dramatic but delayed increase in cortisol levels that persisted for at least 72 h. The magnitude and duration of the response to capture stress was very similar in both species. In contrast, the levels of cortisol, glucose, lactate, haemoglobin and haematocrit were generally elevated sooner and to higher levels in P. maculatus than in P. leopardus after exposure to shallow water stress. Coral trout exposed to reduced salinity showed minimal changes in cortisol, glucose, lactate, haemoglobin and haematocrit, but such changes were not characteristic of a non-specific response to stress. Thus, the physiological stress responses of coral trouts are species-specific and dependent on the nature of the stressor. This observation probably reflects different cortical processes in the brains of P. maculatus and P. leopardus-a result that may be related to the differential variability of the respective environments in which the two species habit.     

Glucose metabolism in isolated brown adipocytes under beta-adrenergic stimulation. Quantitative contribution of glucose to total thermogenesis.

To quantify the potential of brown adipose tissue as a target organ for glucose oxidation, O2 consumption and glucose metabolism in isolated rat brown adipocytes were measured in the presence and absence of insulin, by using the beta-agonists isoprenaline or Ro 16-8714 to stimulate thermogenesis. Basal metabolic rate (278 mumol of O2/h per g of lipid) was maximally stimulated with isoprenaline (20 nm) and Ro 16-8714 (20 microM) to 1633 and 1024 mumol of O2/h per g respectively, whereas insulin had no effect on O2 consumption. Total glucose uptake, derived from the sum of [U-14C]glucose incorporation into CO2 and total lipids and lactate release, was enhanced with insulin. Isoprenaline and Ro 16-8714 had no effect on insulin-induced glucose uptake, but promoted glucose oxidation while inhibiting insulin-dependent lipogenesis and lactate production. A maximal value for glucose oxidation was obtained under the combined action of Ro 16-8714 and insulin, which corresponded to an equivalent of 165 mumol of O2/h per g of lipid. This makes it clear that glucose is a minor substrate for isolated brown adipocytes, fuelling thermogenesis by a maximum of 16%.     

Influence of some mononucleotides and their corresponding nucleosides on the metabolism of carbohydrates in the isolated rat diaphragm muscle.

1. The influence of ATP on glucose metabolism was studied in the isolated rat diaphragm; it was shown that ATP increases the oxidation of glucose and the aerobic conversion of glucose into lactate, whereas it decreases glycogen synthesis. There was no influence of ATP on the anaerobic formation of lactate from glucose. 2. A maximum effect of ATP on the oxidation of glucose (about 160% increase) was obtained in the presence of 10mm-ATP; in the presence of 2mm-ATP the effect was about 65%, and was approximately constant from 10 to 90min. incubation period. 3. In a phosphate-free tris-buffered medium the oxidation of glucose was considerably decreased, but the percentage stimulation by ATP was about the same as in a phosphate-buffered medium. 4. ATP was shown to increase the oxidation of fructose, glucose 6-phosphate, glucose 1-phosphate, fructose 1,6-diphosphate and, to a much smaller extent, pyruvate. 5. ADP stimulated the oxidation of glucose to the same extent as ATP at a concentration of 2mm and the effect with AMP was only slightly less; IMP and adenosine had only a small stimulatory effect at this concentration, whereas inosine had no effect.     

Glucose, glutamine and ketone body utilisation by resting and concanavalin A activated rat splenic lymphocytes

The utilisation of glucose, glutamine, acetoacetate and D-3-hydroxybutyrate were investigated over 72 h of incubation of rat splenic lymphocytes, with and without concanavalin A. Lymphocytes consumed both ketone bodies; acetoacetate was consumed preferentially. The ketone bodies reduced glucose consumption by 30-50%, but had little effect on lactate production. Glutamine uptake was concentration dependent up to 4 mM, and consumption was increased in the presence of concanavalin. Glutamine stimulated glucose consumption and lactate production in both resting and activated cells. Complete oxidation contributed 65% of glucose-derived ATP, but less than 40% of glutamine-derived ATP. Glutamine metabolism makes only a minor contribution to lymphocyte ATP generation.     

Effect of acetoacetate on glucose metabolism in the soleus and extensor digitorum longus muscles of the rat.

1. The effect of acetoacetate on glucose metabolism was compared in the soleus, a slow-twitch red muscle, and the extensor digitorum longus, a muscle composed of 50% fast-twitch red and 50% white fibres. 2. When incubated for 2h in a medium containing 5 mM-glucose and 0.1 unit of insulin/ml, rates of glucose uptake, lactate release and glucose oxidation in the soleus were 19.6, 18.6 and 1.47 micronmol/h per g respectively. Acetoacetate (1.7 mM) diminished all three rates by 25-50%; however, it increased glucose conversion into glycogen. In addition, it caused increases in tissue glucose, glucose 6-phosphate and fructose 6-phosphate, suggesting inhibition of phosphofructokinase. The concentrations of citrate, an inhibitor of phosphofructokinase, and of malate were also increased. 3. Rates of glucose uptake and lactate release in the extensor digitorum longus were 50-80% of those in the soleus. Acetoacetate caused moderate increases in tissue glucose 6-phosphate and possibly citrate, but it did not decrease glucose uptake or lactate release. 4. The rate of glycolysis in the soleus was approximately five times that previously observed in the perfused rat hindquarter, a muscle preparation in which acetoacetate inhibits glucose oxidation, but does not alter glucose uptake or glycolysis. A similar rate of glycolysis was observed when the soleus was incubated with a glucose-free medium. Under these conditions, tissue malate and the lactate/pyruvate ratio in the medium were decreased, and acetoacetate did not decrease lactate release or increase tissue citrate or glucose 6-phosphate. An intermediate rate of glycolysis, which was not decreased by acetoacetate, was observed when the soleus was incubated with glucose, but not insulin. 5. The data suggest that acetoacetate glucose inhibits uptake and glycolysis in red muscle under conditions that resemble mild to moderate exercise. They also suggest that the accumulation of citrate in these circumstances is linked to the rate of glycolysis, possibly through the generation of cytosolic NADH and malate formation.     

Comparative study of some haematological and biochemical blood parameters in fishes from the Skagerrak

Marine fishes caught in the Skagerrak, 27 different species representing various groups of fishes (Cyclostomi, Holocephali, Elasmobranchii and Teleostei), were examined for the following haematological and biochemical blood parameters: haematocrit, haemoglobin, mean corpuscle haemoglobin concentration, total plasma protein, blood glucose and blood lactate. Interspecies variations as well as variations within some species were observed. The haemoglobin values for all species showed a positive correlation to the corresponding haematocrit values. Relatively low values for haematocrit and haemoglobin were found in cyclostomes, holocephaleans and elasmobranchii compared to the majority of teleosts. Within the teleost group, the haematocrit and haemoglobin levels were positively correlated with the activity of the fish species. The cyclostome Myxine glutimsa L. had a total plasma protein content in the same range as most teleosts, whereas holocephaleans, elasmobranchii and the deep-water teleost Coryphaenoides rupestris Gunnerus showed comparatively low values. Among teleosts some relationship seemed to exist between the total plasma protein level and the activity of the fish species. In addition, a correlation between plasma protein content and levels of blood lipids were noted. Values for blood glucose and blood lactate were found to be lower in cyclostomes, holocephaleans and elasmobranchii than in most teleosts. Higher blood glucose levels were observed in the more active teleost species.     

Glucose and lactate oxidation rates in the fetal lamb

Both glucose and lactate are nutrients of the ovine fetus. Each may be used by the fetus as a fuel for oxidation or as a source of carbon for energy storage and net tissue accretion. The present report describes the oxidation rates of glucose and lactate in vivo for the fetal lamb over a relatively short time period. The fraction of fetal glucose or lactate oxidized was defined as the ratio of 14CO2 excretion across the umbilical circulation to the net entry of [14C]glucose or [14C]lactate into fetal tissues. The fraction of glucose oxidized over a 3-hr study averaged 61.2%, accounting for 2.55 mg X min-1 X kg-1 of glucose oxidized and for 28% of the simultaneous net oxygen uptake. The fraction of lactate oxidized averaged 71.5%, accounting for 4.12 mg X min-1 X kg-1 of lactate oxidized. Oxidation fractions and rates for both glucose and lactate increased with their concentrations in fetal blood suggesting sparing of other fuels for oxidation at higher glucose and lactate concentrations.     

Effects of haematocrit value and glucagon on the metabolism of perfused rat liver

Liver from adult male rats were perfused in situ for 30 min with either undiluted, defibrinated rat blood (haematocrit value 38%) or the same blood diluted with buffer to give a haematocrit of 20%. Perfusion with diluted blood lowered the PO2 of the effluent perfusate but this was insufficient to prevent the fall in O2 consumption due to the reduction in haematocrit. Glucagon (5 X 10(-9) M) increased hepatic O2 consumption with whole blood but not with diluted blood. perfusate K+ was increased by perfusion with diluted blood and glucagon. Bile flow was depressed and biliary K+ increased by glucagon but only in experiments with whole blood. Perfusate glucose was raised by lowering of hepatic O2 consumption but the hormonal stimulation of glucose output was the same at both haematocrits. Net ketogenesis was increased with perfusion with diluted blood and by glucagon. In the absence of glucagon there was a net secretion of triacylglycerols which was depressed by lowering of the haematocrit. Glucagon inhibited triacylglycerol secretion and the effect was greater with whole blood so that there was net uptake. While effects of glucagon were obtained during perfusion at a lower haematocrit, it would appear that whole blood was the medium that allowed their fullest expression.     

O2 dependence of insulin stimulation of glucose uptake by perfused rat liver: effects of carboxyhaemoglobin and haematocrit

Livers from fed male rats were perfused in a non-recirculating system with undiluted rat blood containing 14 mM glucose. In these experiments there was a substantial uptake of glucose which was stimulated by insulin. Perfusion with blood containing carboxyhaemoglobin at a concentration of 40% of total haemoglobin lowered O2 consumption and abolished hepatic glucose uptake in control and insulin-infused livers, respectively. In experiments with rat erythrocytes resuspended in buffer to haematocrit values of 38 and 22%, O2 consumption and control and insulin-stimulated rates of glucose uptake were similar to corresponding perfusions with undiluted blood and blood containing carboxyhaemoglobin. It is concluded that serum factors are of relatively small importance and that hepatic glucose uptake is dictated by O2 supply.     

Contribution of pH-sensitive metabolic processes to pH homeostasis in isolated rat kidney tubules.

The metabolism of isolated rat kidney tubules suspended in calcium-free physiological saline buffered with phosphate was found to be sensitive to changes in the pH of the suspending medium. Lowering the pH from 7.8 to 6.4 brought about increases in the rates of oxidation of added succinate, glutamate or glutamine as well as in the production of glucose from lactate, glutamine, succinate and fructose. The cellular ATP level was also higher in tubules incubated at pH 6.4 In contrast, the utilization of added glucose was greater at pH 7.8 than at pH 6.4, a substantial amount of lactate being produced at the higher pH. When glucose and either lactate or glutamine were provided as co-substrates glucose was the preferred fuel at pH 7.8 but the alternative substrate was the more readily utilized at pH 6.4. As a consequence of the metabolic activities of the tubules the pH of the suspending medium changed, utilization of lactate, glutamate or glutamine causing a rise in pH while conversion of glucose to lactate caused a fall in pH. In cases where two substrates were metabolized concurrently over a period of 3 h the extracellular pH tended towards a plateau level of approximately pH 7.4. It is proposed that pH-sensitive metabolism in isolated kidney tubules contributes to pH homeostasis in the cellular environment.     

Contribution of pH-sensitive metabolic processes to pH homeostasis in isolated rat kidney tubules

The metabolism of isolated rat kidney tubules suspended in calcium-free physiological saline buffered with phosphate was found to be sensitive to changes in the pH of the suspending medium. Lowering the pH from 7.8 to 6.4 brought about increases in the rates of oxidation of added succinate, glutamate or glutamine as well as in the production of glucose from lactate, glutamine, succinate and fructose. The cellular ATP level was also higher in tubules incubated at pH 6.4. In contrast, the utilization of added glucose was greater at pH 7.8 than at pH 6.4, a substantial amount of lactate being produced at the higher pH. When glucose and either lactate or glutamine were provided as co-substrates glucose was the preferred fuel at pH 7.8 but the alternative substrate was the more readily utilized at pH 6.4. As a consequence of the metabolic activities of the tubules the pH of the suspending medium changed, utilization of lactate, glutamate or glutamine causing a rise in pH while conversion of glucose to lactate caused a fall in pH. In cases where two substrates were metabolized concurrently over a period of 3 h the extracellular pH tended towards a plateau level of approximately pH 7.4. It is proposed that pH-sensitive metabolism in isolated kidney tubules contributes to pH homeostasis in the cellular environment.     

Aerobic glycolysis and lymphocyte transformation

1. The role of enhanced aerobic glycolysis in the transformation of rat thymocytes by concanavalin A has been investigated. Concanavalin A addition doubled [U-(14)C]glucose uptake by rat thymocytes over 3h and caused an equivalent increased incorporation into protein, lipids and RNA. A disproportionately large percentage of the extra glucose taken up was converted into lactate, but concanavalin A also caused a specific increase in pyruvate oxidation, leading to an increase in the percentage contribution of glucose to the respiratory fuel. 2. Acetoacetate metabolism, which was not affected by concanavalin A, strongly suppressed pyruvate oxidation in the presence of [U-(14)C]glucose, but did not prevent the concanavalin A-induced stimulation of this process. Glucose uptake was not affected by acetoacetate in the presence or absence of concanavalin A, but in each case acetoacetate increased the percentage of glucose uptake accounted for by lactate production. 3. [(3)H]Thymidine incorporation into DNA in concanavalin A-treated thymocyte cultures was sensitive to the glucose concentration in the medium in a biphasic manner. Very low concentrations of glucose (25mum) stimulated DNA synthesis half-maximally, but maximum [(3)H]thymidine incorporation was observed only when the glucose concentration was raised to 1mm. Lactate addition did not alter the sensitivity of [(3)H]-thymidine uptake to glucose, but inosine blocked the effect of added glucose and strongly inhibited DNA synthesis. 4. It is suggested that the major function of enhanced aerobic glycolysis in transforming lymphocytes is to maintain higher steady-state amounts of glycolytic intermediates to act as precursors for macromolecule synthesis.     

CYTOCHROME REDOX POTENTIAL DEPENDENCE ON SUBSTRATE IN RAT CEREBRAL CORTEX SLICES: IMPORTANCE OF CYTOPLASMIC NAD(P)H AND POTASSIUM

—Using dual-wavelength absorbance spectrophotometry, the ability of various substrates to produce and maintain a redox potential in the cytochrome chain of rat cerebral cortex slices was studied. In general, the ability to reduce the cytochromes parallels previously reported capabilities of the substrates to support metabolic responses to stimulation. The steady-state kinetics of cytochrome reduction by glucose or lactate displayed a very sharp dependency upon concentration in the regions of 1 or 3 mm , respectively. This was in contrast to a near linear reduction of the cytochromes with concentrations of pyruvate over a range of 1–10 mm . The production and maintenance of a cytochrome redox potential was found to be at least partially dependent upon the presence of potassium (3 mm in the incubation media). Reduction of the cytochromes attributable to potassium was inhibited by ouabain, indicating that intracellular potassium was the important variable. Addition of glucose or lactate to 'starved’ tissues was found to result in a complex cycle of oxidation and reduction of tissue NAD(P)H. A small initial reduction of NAD(P) was followed by an oxidation of NAD(P)H which occurred in an all-or-none fashion with reduction of the cytochromes. The oxidation of NAD(P)H and reduction of cytochrome b appeared to occur with a similar time course. Respiratory changes following addition of glucose were complex in time course, but established a new steady-state rate 0.41 μmol/g per min above the preaddition rate in 10–12 min. Despite a similar level of reduction in the cytochrome chain, stimulation of respiration by pyruvate was only about 50% of the rate observed with addition of glucose. However, stimulation of respiration by addition of equim concentrations of pyruvate and lactate was found to be additive, producing a 0.48 μmol/g per min increase in the steady-state rate of oxygen consumption. These data seem to support the conclusion that the cytoplasmic reducing equivalent derived from the initial oxidation of glucose or lactate plays an important, perhaps regulatory, role in the respiration of cerebral tissues.