The effect of acute ethanol treatment on rates of oxygen uptake, ethanol oxidation and gluconeogenesis in isolated rat hepatocytes.

In hepatocytes isolated from fed rats, acute ethanol pretreatment (at a dose of 5.0 g/kg body wt.) did not change rates of O2 uptake. In cells from starved animals, acute ethanol pretreatment increased O2 uptake by 17-29%. The increased O2 uptake in hepatocytes from starved rats was not accompanied by increased rates of ethanol oxidation, but was accompanied by increased rates of gluconeogenesis under some conditions. The provision of ethanol (10 mM) as a substrate to cells from fed or starved rats decreased O2 uptake in the absence of other substrates or in the presence of lactate, and increased it in the presence of pyruvate or lactate and pyruvate. The results of this study show that the acute effects of ethanol on liver O2 uptake are dependent on the physiological state of the liver. Previously reported large (2-fold) increases in O2 uptake after acute ethanol pretreatment may have been an artefact owing to low control uptake rates (approximately 1.8 micromol/min per g wet wt. of cells) in the liver preparation used. The ATP contents (2.4-2.6 micromol/g wet wt. of cells) and rates of O2 uptake (2.5-5.0 micromol/min per g wet wt. of cells) of cells used in the present study were the same as values reported under conditions close to those in vivo. Therefore the increase in O2 uptake in cells from starved rats after acute ethanol pretreatment is likely to be of physiological significance.     

Carbohydrate metabolism in the isolated perfused rat kidney

1. Anaerobic formation of lactate from glucose by isolated perfused rat kidney (411mumol/h per g dry wt.) was three times as fast as in aerobic conditions (138mumol/h per g). 2. In aerobic or in anaerobic conditions, the ratio of lactate production to glucose utilization was about 2. 3. Starvation or acidosis caused a decline of about 30% in the rate of aerobic glycolysis. 4. The rate of formation of glucose from lactate by perfused kidney from a well-fed rat, in the presence of 5mm-acetoacetate (83mumol/h per g dry wt.), was of the same order as the rate of aerobic glycolysis. 5. During perfusion with physiological concentrations of glucose (5mm) and lactate (2mm) there were negligible changes in the concentration of either substrate. 6. Comparison of kidneys perfused with lactate, from well-fed or starved rats, showed no major differences in contents of intermediates of gluconeogenesis. 7. The tissue concentrations of hexose monophosphates and C(3) phosphorylated glycolytic intermediates (except triose phosphate) were decreased in anaerobic conditions. 8. Aerobic metabolism of fructose by perfused kidney was rapid: the rate of glucose formation was 726mumol/h per g dry wt. and of lactate formation 168mumol/h per g (dry wt.). Glycerol and d-glyceraldehyde were also released into the medium. 9. Aerobically, fructose generated high concentrations of glycolytic intermediates. 10. Anaerobic production of lactate from fructose (74mumol/h per g dry wt.) was slower than the aerobic rate. 11. In both anaerobic and aerobic conditions the ratio [lactate]/[pyruvate] in kidney or medium was lower during perfusion with fructose than with glucose. 12. These results are discussed in terms of the regulation of renal carbohydrate metabolism.     

Inhibition of hepatic gluconeogenesis by ethanol

1. Gluconeogenesis from 10mm-lactate in the perfused liver of starved rats is inhibited by ethanol. The degree of inhibition reached a maximum of 66% at 10mm-ethanol under the test conditions and decreased at higher ethanol concentrations. The concentration-dependence of the inhibition is paralleled by the concentration-dependence of the activity of alcohol dehydrogenase. The enzyme is also inhibited by ethanol concentrations above 10mm. 2. Gluconeogenesis from pyruvate is not inhibited by ethanol. 3. The degree of the inhibition of gluconeogenesis from lactate by ethanol depends on the concentration of lactate and other oxidizable substances, e.g. oleate, in the perfusion medium. 4. Ethanol also inhibits, to different degrees, gluconeogenesis from glycerol, dihydroxyacetone, proline, serine, alanine, fructose and galactose. 5. The inhibition of gluconeogenesis from lactate by ethanol is reversed by acetaldehyde. 6. Pyrazole, a specific inhibitor of alcohol dehydrogenase, also reverses the inhibition of gluconeogenesis by ethanol. 7. Gluconeogenesis in kidney cortex, where the activity of alcohol dehydrogenase is very low, is not inhibited by ethanol. 8. Kidney cortex, testis, ovary, uterus and certain tissues of the alimentary tract were the only rat tissues, apart from the liver, that showed measurable alcohol dehydrogenase activity. 9. The concentrations of pyruvate in the liver were decreased to about one-fifth by ethanol. 10. The concentration of lactate in the perfused liver was about 3mm below that of the perfusion medium 30min. after the addition of 10mm-lactate. 11. The great majority of the findings support the view that the inhibition of gluconeogensis by ethanol is caused by the alcohol dehydrogenase reaction, which decreases the [free NAD(+)]/[free NADH] ratio. The decrease lowers the concentration of pyruvate and this is the immediate cause of the inhibition of gluconeogenesis from lactate, alanine and serine: the fall in the concentration of pyruvate lowers the rate of the pyruvate carboxylase reaction, one of the rate-limiting reactions of gluconeogenesis. The cause of the inhibition of gluconeogenesis from other substrates is discussed.     

Gluconeogenesis from serine in rabbit hepatocytes

L-Serine alone is not gluconeogenic in isolated rabbit hepatocytes, whereas in rat liver this amino acid has been reported to yield as much glucose as does L-lactate itself. The current study has been an investigation into the explanation of the difference between the two species. Hepatocytes were isolated from 48-h-starved, 750- to 1000-g male rabbits, and the viability of each preparation was judged by ATP levels (2.4 +/- 0.2 mumol/g wet wt) at the beginning and end of the incubation as well as gluconeogenesis from 10 mM L-lactate (0.83 +/- 0.08 mumol/min/g wet wt). L-Serine alone produced virtually no glucose or pyruvate accumulation above baseline. Hydroxypyruvate, however, did appear in the incubation mixture. When L-serine and pyruvate were combined to test the functional activity of L-serine:pyruvate aminotransferase (EC 2.6.1.51), however, gluconeogenesis remained at the rate produced by pyruvate alone (0.61 +/- 0.04 mumol/min/g wet wt). On the other hand, the combination of L-serine and L-lactate produced rates of glucose accumulation 35% above that of L-lactate alone. The combination of L-lactate plus hydroxypyruvate produced nearly maximal rates (1.39 +/- 0.08 mumol/min/g wet wt), approaching those achieved by a physiologic ratio (10:1) of L-lactate and pyruvate. Hydroxypyruvate itself was only moderately gluconeogenic (0.44 +/- 0.04 mumol/min/g wet wt). That a reduction of the cytoplasmic free [NAD+]/[NADH] ratio by L-lactate was not its only contribution to L-serine utilization was suggested by the fact that ethanol completely eliminated gluconeogenesis from virtually all precursors (or combinations) tested, with the exception of hydroxypyruvate. It has been concluded from the data that, probably in contrast to the rat, the major pathway for the entrance of L-serine into gluconeogenesis in rabbit hepatocytes is through the pathway initiated by L-serine: pyruvate aminotransferase and that L-lactate is an important participant (i) by generating cytoplasmic reducing equivalents (NADH), (ii) by supplying pyruvate for the transaminating reaction itself, and, perhaps, (iii) by preventing hydroxypyruvate from being reduced by L-lactate dehydrogenase (EC 1.1.1.27) to L-glycerate.     

Hepatic redox state and gluconeogenesis from lactate in vivo in the rat

1. To examine the role of the hepatic redox state on the rate of gluconeogenesis the effects of sodium crotonate injection (6mmol/kg body wt.) on rat liver metabolite concentrations and gluconeogenesis from lactate were studied in vivo. 2. Crotonate caused a marked oxidation of cytoplasmic and mitochondrial redox couples; decreases were observed in the ratios of [lactate]/[pyruvate], [glycerol 3-phosphate]/[dihydroxyacetone phosphate], [hydroxybutyrate]/[acetoacetate] and measured [NAD(+)]/[NADH]. 3. Increases occurred in the liver concentrations of all gluconeogenic intermediates from pyruvate through to glucose 6-phosphate, but there was no change in lactate concentration. 4. To determine whether gluconeogenesis from lactate was altered by the more-oxidized hepatic redox state l-[2-(14)C]lactic acid was infused into the inferior vena cava (50mumol/min per kg body wt.) and the incorporation of radioactivity into blood glucose was measured. 5. Administration of crotonate transiently decreased the rate of lactate incorporation into glucose but within a few minutes the rate of incorporation returned to that of the controls. 6. The results indicate that in these experiments alteration of the NAD(+)-NADH systems of cytoplasm and mitochondria to a more-oxidized state did not change the rate of gluconeogenesis.     

Localization and role of pyruvate kinase isoenzymes in the regulation of carbohydrate metabolism and pyruvate recycling in rat kidney cortex

This work was performed to gain more information on the role of pyruvate kinase isoenzymes in the regulation of renal carbohydrate metabolism. Immunohistochemically, pyruvate kinase type L is shown to be localized in the proximal tubule of the nephron and pyruvate kinase type M2 in the distal tubule and the collecting duct. a tight relationship between gluconeogenesis and pyruvate recycling was found. The rate of gluconeogenesis (8 mumol/g wet wt. per 30 min) was of the same order of magnitude as the rate of pyruvate recycling (10.92 mumol/g wet wt. per 30 min). Stimulation of gluconeogenesis from 20 mM lactate in kidney cortex slices of 24-h-starved rats by dibutyryl-cAMP, alanine and parathyroid hormone was connected with a decrease in pyruvate recycling; inhibition of gluconeogenesis due to a lack of Ca2+ in the incubation medium was linked with an increase in pyruvate recycling. The degradation of [6-14C]glucose to lactate, pyruvate, ketone bodies and CO2 and of [2-14C]lactate was unaffected by dibutyryl-cAMP, alanine, epinephrine, vasopressin or the omission of Ca2+ from the incubation medium. 1 mM dibutyryl-cAMP or 5 mM alanine did not alter the activities of oxaloacetate decarboxylase, 'malic' enzyme and malate dehydrogenase from rat kidney cortex. Since aerobic glycolysis in the distal tubules and the collecting ducts is not influenced by hormones, dibutyryl-cAMP and Ca2+, pyruvate kinase type M2 residing in this tissue is unlikely to be a control point of glycolysis. Since this tissue degrades only one-seventh of the glucose formed via gluconeogenesis, it does not contribute significantly to pyruvate recycling. Therefore, the decrease of pyruvate recycling in the presence of dibutyryl-cAMP and alanine in rat kidney cortex slices, leading to increased renal gluconeogenesis, has to be ascribed to the regulation of pyruvate kinase type L.     

Lactate production in the perfused rat liver

1. In aerobic conditions the isolated perfused liver from well-fed rats rapidly formed lactate from endogenous glycogen until the lactate concentration in the perfusion medium reached about 2mm (i.e. the concentration of lactate in blood in vivo) and then production ceased. Pyruvate was formed in proportion to the lactate, the [lactate]/[pyruvate] ratio remaining between 8 and 15. 2. The addition of 5mm- or 10mm-glucose did not affect lactate production, but 20mm- and 40mm-glucose greatly increased lactate production. This effect of high glucose concentration can be accounted for by the activity of glucokinase. 3. The perfused liver released glucose into the medium until the concentration was about 6mm. When 5mm- or 10mm-glucose was added to the medium much less glucose was released. 4. At high glucose concentrations (40mm) more glucose was taken up than lactate and pyruvate were produced; the excess of glucose was probably converted into glycogen. 5. In anaerobic conditions, livers of well-fed rats produced lactate at relatively high rates (2.5mumol/min per g wet wt.). Glucose was also rapidly released, at an initial rate of 3.2mumol/min per g wet wt. Both lactate and glucose production ceased when the liver glycogen was depleted. 6. Addition of 20mm-glucose increased the rate of anaerobic production of lactate. 7. d-Fructose also increased anaerobic production of lactate. In the presence of 20mm-fructose some glucose was formed anaerobically from fructose. 8. In the perfused liver from starved rats the rate of lactate formation was very low and the increase after addition of glucose and fructose was slight. 9. The glycolytic capacity of the liver from well-fed rats is equivalent to its capacity for fatty acid synthesis and it is pointed out that hepatic glycolysis (producing acetyl-CoA in aerobic conditions) is not primarily an energy-providing process but part of the mechanism converting carbohydrate into fat.     

Inhibition of testosterone biosynthesis by ethanol. Relation to hepatic and testicular acetaldehyde, ketone bodies and cytosolic redox state in rats.

In experiments in which liver and testis freeze-stops were performed on pentobarbital-anaesthetized rats, ethanol (1.5 g/kg body wt.) reduced plasma testosterone concentration from 13.1 to 3.2 nmol/litre. 4-Methylpyrazole abolished the ethanol-induced hepatic and testicular increase in the lactate/pyruvate ratio, and the testicular acetaldehyde level, but did not diminish the reduction in plasma testosterone concentration. In testes, but not in liver, ethanol decreased the 3-hydroxybutyrate/acetoacetate ratio, and 4-methylpyrazole did not prevent this effect. In experiments in which freeze-stop was performed after cervical dislocation, ethanol decreased the testis testosterone concentration from 590 to 220 pmol per g wet wt. The effects of ethanol and 4-methylpyrazole on testis acetaldehyde, lactate/pyruvate and 3-hydroxybutyrate/acetoacetate ratios were the same as found during anaesthesia. The NAD+-dependent ethanol oxidation capacity in testis ranged from 0.1 to 0.2 mumol/min per g wet wt. and seemed to be inhibited by 4-methylpyrazole both in vivo and in vitro. In additional experiments, ethanol doses between 0.3 and 0.9 g/kg body wt. did not alter the plasma testosterone concentration in rats treated, or not treated, with cyanamide, which induced elevated acetaldehyde levels in blood and testes. The results suggest that ethanol-induced inhibition of testosterone biosynthesis was not caused by extratesticular redox increases, or by extra- or intra-testicular acetaldehyde per se. The inhibition is accompanied by changes in testicular ketone-body metabolism.     

Ethanol administration and the relationship of malonyl-coenzyme A concentrations to the rate of fatty acid synthesis in rat liver

1. The effect of ethanol on liver fatty acid synthesis was studied in vivo in 24h-starved and ;meal-fed' rats (i.e. fed for 3h per day and not ad libitum). 2. In the fed animal (3)H(2)O was incorporated into fat at a rate of 0.46mumol of C(2) units/min per g wet wt. of liver. Administration of either ethanol (3.2g/kg) or equicaloric amounts of glucose had no effect on the rate of (3)H(2)O incorporation into lipid. 3. In the 24h-starved animal, administration of the same dose of ethanol produced an increase in the rate of (3)H(2)O incorporation from 0.06 to 0.12mumol of C(2) units/min per g fresh wt. after 3h whereas [malonyl-CoA] increased from 0.006 to 0.009mumol/g. Glucose given in amounts equicaloric to ethanol was significantly more lipogenic, increasing both the (3)H(2)O incorporation from 0.06 to 0.20mumol of C(2) units/min per g and the malonyl-CoA content from 0.006 to 0.013 mumol/g wet wt. at 3h. 4. The decrease in the redox state of free cytoplasm NAD or NADP couples or the changes in content of citrate, glucose 6-phosphate and pyruvate of liver after ethanol administration had no measurable effect on the rate of fatty acid synthesis in vivo. 5. Under the conditions of the experiments there was no significant difference, among any of the groups, in the activity of liver fatty acid synthetase measured in vitro. A double-reciprocal plot of the rate of (3)H(2)O incorporation and the total tissue malonyl-CoA concentrations showed a striking relationship. It has been concluded that the rate of fatty acid synthesis in vivo is determined principally by the V(max.) of fatty acid synthetase and the concentration of free malonyl-CoA. 6. It has also been concluded that under the conditions of the present study, the synthesis of fatty acids de novo is unlikely to be an important factor in the increased liver lipid content associated with ethanol administration.     

Ethanol metabolism and lipid synthesis by isolated liver cells from fed rats.

1. The fatty acid synthesis in isolated liver cells from fed rats was studied with tritiated water as the radioactive precursor. The cells incorporated 3H20 at a rate of 1.26 mumol per min per g packed cells. 2. Addition of ethanol caused a 20% decrease in the incorporation of tritium into fatty acids. The decrease was correlated to the increase in the NAD-redox level. Probably, the decreased tritium incorporation into fatty acids during ethanol metabolism is due to a decrease in the specific activity of the NADPH used for the synthesis of fatty acids, rather than to a real inhibition of the fatty acid synthesis. 3. Ethanol oxidation via NADPH-consuming pathways and ethanol per se at a concentration of 80 mM had no effect upon the incorporation of tritium into fatty acids. 4. Fructose in a concentration of 15 mM inhibited the fatty acid synthesis by 75%, and this inhibition was further augmented by ethanol. 5. The ioslated rat liver cells oxidized ethanol at a rate of 2.72, 2.93 and 3.48 mumol per min per g packed cells at 5, 20 and 80 mM ethanol, respectively. Fructose had no effect upon ethanol oxidation neither at low nor at high concentrations of ethanol. 6. Ethanol oxidation via the non alcohol dehydrogenase pathway(s) may involve a transfer of reducing equivalents from mitochondrial NADH to cyctosolic NADP+ as judged from measurements of metabolite levels. This conclusion is supported by determinations of 14C yield in glucose from [1-14C] ethanol, and the results are taken as evidence for the presence of hydrogen shuttle activity during metabolism of ethanol, catalyzed by the NAD-dependent alcohol dehydrogenase. A metabolic scheme is proposed to account for the observed changes at low and high concentrations of ethanol.     

Application of high-field 1H-NMR spectroscopy for the study of perifused amphibian and excised mammalian muscles

Frog sartorius and gastrocnemius muscles were perifused at 20 degrees C, the intracellular pH (pHi) and the concentration of phosphocreatine were determined in the resting muscle by 1H-NMR spectroscopy at 470 MHz; values of pHi = 7.31 +/- 0.05 (n = 7) and concentration of phosphocreatine = 20.4 +/- 1.1 mumol/g wet wt. (n = 6) were found. The hydrolysis of phosphocreatine and the simultaneous increase in lactate upon perifusion with 10 mM caffeine (in Ringer's solution) was followed with a time resolution of 1 min. Lactate increased at a rate of 1.0 mumol/g per min, but no pHi change was recorded during the time monitored. The lower limit for the buffering capacity of the muscle cytosol was estimated to be 16.7 mumol/g muscle per pH unit from the uncertainty in pHi determination (+/- 0.03 pH units) and from the amount of lactate produced and phosphocreatine hydrolyzed. Changes in pHi, lactate concentration and fatty acyl chain intensity were monitored by 1H-NMR spectroscopy at 361 MHz in ischemic rat skeletal muscle, excised and stored at 20 degrees C. The resonances in the 1H-NMR spectrum of a human skeletal muscle perchloric acid extract are reported and tentatively assigned.     

The reversibility of cytosolic dehydrogenase reactions in hepatocytes from starved and fed rats. Effect of fructose.

The metabolism of [2-3H]lactate was studied in isolated hepatocytes from fed and starved rats metabolizing ethanol and lactate in the absence and presence of fructose. The yields of 3H in ethanol, water, glucose and glycerol were determined. The rate of ethanol oxidation (3 mumol/min per g wet wt.) was the same for fed and starved rats with and without fructose. From the detritiation of labelled lactate and the labelling pattern of ethanol and glucose, we calculated the rate of reoxidation of NADH catalysed by lactate dehydrogenase, alcohol dehydrogenase and triosephosphate dehydrogenase. The calculated flux of reducing equivalents from NADH to pyruvate was of the same order of magnitude as previously found with [3H]ethanol or [3H]xylitol as the labelled substrate [Vind & Grunnet (1982) Biochim. Biophys. Acta 720, 295-302]. The results suggest that the cytoplasm can be regarded as a single compartment with respect to NAD(H). The rate of reduction of acetaldehyde and pyruvate was correlated with the concentration of these metabolites and NADH, and was highest in fed rats and during fructose metabolism. The rate of reoxidation of NADH catalysed by lactate dehydrogenase was only a few per cent of the maximal activity of the enzymes, but the rate of reoxidation of NADH catalysed by alcohol dehydrogenase was equal to or higher than the maximal activity as measured in vitro, suggesting that the dissociation of enzyme-bound NAD+ as well as NADH may be rate-limiting steps in the alcohol dehydrogenase reaction.     

Cortisol induces perinatal hepatic gluconeogenesis in the lamb.

To examine the influence of a prenatal increase in plasma cortisol concentration on perinatal initiation of hepatic gluconeogenesis, we infused cortisol into seven fetal sheep at 137-140 days gestation. 14C-Lactate provided tracer substrate for estimation of gluconeogenesis. We measured hepatic blood flow using radionuclide-labeled microspheres. After delivery, fetal arterial blood glucose concentration (1.33 +/- 0.4 mmol/l) increased transiently, but returned to fetal levels within 1 h after delivery. Substantial hepatic gluconeogenesis was induced in the fetus after cortisol infusion, averaging 23.4 +/- 12.2 mumol/min/100 g liver (7.8 +/- 4.4 mumol/min/kg fetal weight). Fetal hepatic glucose output was 44.4 +/- 17.7 mumol/min/100 g liver. Hepatic glucose output did not change after delivery; estimated gluconeogenesis decreased immediately, then increased by 6 h after delivery. Lactate supply to the liver fell substantially, from 1.1 +/- 0.4 mmol/min/100 g in the fetus to 0.24 +/- 0.09 at 1 h after delivery. Lactate flux across the liver decreased from 75.3 +/- 23 mumol/min/100 g in the fetus to 20.2 +/- 15.7 at 1 h after delivery. Hepatic lactate flux was significantly related to gluconeogenesis (r = 0.734, P = 0.0001). We conclude that cortisol induces substantial hepatic gluconeogenesis in fetal sheep near term. After delivery, there appears to be a transient decline in gluconeogenesis from lactate, which may be secondary to limited hepatic oxygen and substrate supply. Onset of gluconeogenesis in the fetus fails to sustain increases in either fetal or postnatal blood glucose concentrations.     

A system for the study in situ of the pentose phosphate pathway in rabbit liver

A surgical procedure for the isolation of the liver from the systemic circulation of the anaesthetized rabbit is described. The technique allowed the metabolism in situ of intraportally infused substrates to be followed for periods up to 5min, free from the contaminating influences of metabolism by other body tissues. Details of the procedures necessary to achieve the uniform infusion, homogeneous distribution and containment of (14)C-labelled glucose substrates in the liver by haemostasis are described. Changes in pO(2), pCO(2), pH and the concentrations of NADP(+), NADPH and glucose during each minute interval of the total 5min period of metabolism are given. Reactant ratios of the lactate dehydrogenase system and the adenine nucleotide system have been calculated from the concentrations of the pertinent metabolites for the same period of metabolism. Glucose production by rabbit liver in situ proceeded at the rate of 1.08mumol/min per g wet wt. of liver during the 5min metabolic interval. The presence of the oxidative reactions of the pentose phosphate pathway of glucose metabolism was inferred from the quotient oxidation of [1-(14)C]glucose/oxidation of [6-(14)C]glucose=1.8.     

Effect of verapamil on glycogenolysis and gluconeogenesis in the perfused rat liver

In perfused livers from fed rats, rates of glucose production (glycogenolysis) were 133 +/- 12 mumol/g/hr. Infusion of 2 microM verapamil into these livers decreased the rates of glucose production significantly to 97 +/- 15 mumol/g/hr within 10 min. Conversely, rates of production of lactate plus pyruvate (glycolysis) of 64 +/- 6 mumol/g/hr were not significantly altered by verapamil (60 +/- 3 mumol/g/hr). When 50 microM verapamil was infused, however, rates of both glycogenolysis and glycolysis were diminished to 56 +/- 11 and 43 +/- 5 mumol/g/hr, respectively. In perfused livers from fasted rats, infusion of 20 mM fructose increased the rates of production of glucose (gluconeogenesis) significantly from 11 +/- 7 to 121 +/- 17 mumol/g/hr. These rates reached 138 +/- 7 mumol/g/hr upon the simultaneous infusion of verapamil (2 microM). In these livers, fructose also increased rates of production of lactate from 6 +/- 2 to 132 +/- 11 mumol/g/hr, which were further increased to 143 +/- 8 mumol/g/hr when 2 microM verapamil was infused. The results show that calcium-dependent processes involved in hepatic carbohydrate metabolism respond differently to the calcium channel blocker verapamil. Low concentrations of verapamil inhibited glycogenolysis significantly while having no effect on either glycolysis or gluconeogenesis. These data suggest that these two processes have different sensitivities to changes in intracellular calcium concentrations and/or different sources of regulatory calcium.     

The estimation of rates of utilization of glucose and ketone bodies in the brain of the suckling rat using compartmental analysis of isotopic data

The brains of 18-day-old rats utilize glucose and ketone bodies. The rates of acetyl-CoA formation from these substrates and of glycolysis were determined in vivo from the labelling of intermediary metabolites after intraperitoneal injection of d-[2-(14)C]glucose, l(+)-[3-(14)C]- and l(+)-[U-(14)C]-lactate and d(-)-3-hydroxy[(14)C]butyrate. Compartmental analysis was used in calculating rates to allow for the rapid exchange of blood and brain lactate, the presence in brain of at least two pools each of glucose and lactate, and the incomplete equilibration of oxaloacetate with aspartate and of 2-oxoglutarate with glutamate. Results were as follows. 1. Glucose and ketone bodies labelled identical pools of tricarboxylate-cycle metabolites, and were in every way alternative substrates. 2. The combined rate of oxidation of acetyl-CoA derived from pyruvate (and hence glucose) and ketone bodies was 1.05mumol/min per g. 3. Ketone bodies contributed 0.11-0.53mumol/min per g in proportion to their concentration in blood, with a mean rate of 0.30mumol/min per g at 1.24mm. 4. Pyruvate and ketone bodies were converted into lipid at 0.018 and 0.008mumol/min per g respectively. 5. Glycolysis, at 0.48mumol/min per g, was more rapid in most rats than pyruvate utilization by oxidation and lipid synthesis, resulting in a net output of lactate from brain to blood. 6. Rates of formation of brain glutamate, glutamine and aspartate were also measured. Further information on the derivation of the models has been deposited as Supplementary Publication SUP 50034 (18 pages) at the British Library, Lending Division (formerly the National Lending Library for Science and Technology), Boston Spa, Yorks. LS23 7QB, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1973) 131, 5.     

Gluconeogenesis by isolated guinea-pig liver parenchymal cells

1. Guinea-pig hepatocytes were prepared by collagenase digestion of the perfused liver. 2. The highest rates of gluconeogenesis were obtained from fructose, followed by pyruvate, xylitol and lactate, glycerol and propionate in that order. Maximum rates of gluconeogenesis were attained at 6-10mm substrate. 3. An initial 15-min lag period occurred during gluconeogenesis from lactate. This lag was abolished by preincubating the cells or by preincubation plus the addition of NH(4)Cl or lysine. 4. The lactate/pyruvate and 3-hydroxybutyrate/acetoacetate ratios were increased during the lag and adjusted to values favouring rapid gluconeogenesis from lactate after 15min. 5. The data suggest that the low glucose synthesis during the lag resulted from a limitation of the glutamate-aspartate shuttle and from the unusual redox state of the NAD(+) couple prevailing during this period. 6. At 0.1mm, amino-oxyacetate, a transaminase inhibitor, decreased gluconeogenesis from lactate by 80%, but had a negligible effect on glucose production from pyruvate. Gluconeogenesis from lactate was also inhibited (20%) by 10mm-dl-3-hydroxybutyrate.     

Effect of insulin on gluconeogenesis and the metabolism of lactate in sheep

Owing to the fermentative nature of their digestion, ruminant animals are highly dependent upon gluconeogenesis to meet their glucose needs. The role of hormones in regulating this process is not clear. The purpose of this study was to examine the effect of insulin on the utilization of lactate in glucose synthesis in sheep. The euglycemic model was used in sheep. [U-14C]Lactate and [6-3H]glucose were infused to monitor lactate and glucose fluxes. Hepatic metabolism was measured using radioisotopic and venoarterial concentration difference techniques. Insulin concentrations increased from basal concentrations of 16 +/- 2 to 95 +/- 9 microU/mL. Insulin reduced the net hepatic utilization of lactate (303 +/- 43 vs. 120 +/- 27 mumol/min), hepatic extraction efficiency of lactate (29 +/- 4 vs. 9 +/- 2%), hepatic output of glucose (338 +/- 33 vs. 103 +/- 21 mumol/min), and incorporation of lactate into glucose (90 +/- 5 vs. 46 +/- 8 mumol/min). Insulin at physiological levels can inhibit hepatic gluconeogenesis in ruminants.     

Acute effects of ethanol on the perfused rat liver. Studies on lipid and carbohydrate metabolism, substrate cycling and perfusate amino acids

1. Livers from fed rats were perfused in situ with whole rat blood containing glucose labelled uniformly with (14)C and specifically with (3)H at positions 2, 3 or 6. 2. When ethanol was infused at a concentration of 24mumol/ml of blood the rate of utilization was 2.8mumol/min per g of liver. 3. Ethanol infusion raised perfusate glucose concentrations and caused a 2.5-fold increase in hepatic glucose output. 4. Final blood lactate concentrations were decreased in ethanol-infused livers, but the mean uptake of lactate from erythrocyte glycolysis was unaffected. 5. Production of ketone bodies (3-hydroxybutyrate+3-oxobutyrate) and the ratio [3-hydroxybutyrate]/[3-oxobutyrate] were raised by ethanol. 6. Formation of (3)H(2)O from specifically (3)H-labelled glucoses increased in the order [6-(3)H]<[3-(3)H]<[2-(3)H]. Production of (3)H(2)O from [2-(3)H]glucose was significantly greater than that from [3-(3)H]glucose in both control and ethanol-infused livers. Ethanol significantly decreased (3)H(2)O formation from all [(3)H]glucoses. 7. Liver glycogen content was unaffected by ethanol infusion. 8. Production of very-low-density lipoprotein triacylglycerols was inhibited by ethanol and there was a small increase in liver triacylglycerols. Very-low-density-lipoprotein secretion was negatively correlated with the ratio [3-hydroxybutyrate]/[3-oxobutyrate]. Perfusate fatty acid concentrations and molar composition were unaffected by perfusion with ethanol. 9. Ethanol decreased the incorporation of [U-(14)C]glucose into fatty acids and cholesterol. 10. The concentration of total plasma amino acids was unchanged by ethanol, but the concentrations of alanine and glycine were decreased and ([glutamate]+[glutamine]) was raised. 11. It is proposed that the observed effects of ethanol on carbohydrate metabolism are due to an increased conversion of lactate into glucose, possibly by inhibition of pyruvate dehydrogenase. The increase in gluconeogenesis is accompanied by diminished substrate cycling at glucose-glucose 6-phosphate and at fructose 6-phosphate-fructose 1,6-bisphosphate.