Hepatic urea synthesis and pH regulation. Role of CO2, HCO3-, pH and the activity of carbonic anhydrase

In isolated perfused rat liver, urea synthesis from ammonium ions was dependent on extracellular HCO3- and CO2 concentrations when the HCO3-/CO2 ratio in the influent perfusate was constant (pH 7.4). Urea synthesis was half-maximal at HCO3- = 4 mM, CO2 = 0.19 mM and was maximal at HCO3- and CO2 concentrations above 20 mM and 0.96 mM, respectively. At physiological HCO3- (25 mM) and CO2 (1.2 mM) concentrations in the influent perfusate, acetazolamide, the inhibitor of carbonic anhydrase, inhibited urea synthesis from ammonium ions (1 mM) by 50-60% and led to a 70% decrease in citrulline tissue levels. Acetazolamide concentrations required for maximal inhibition of urea synthesis were 0.01-0.1 mM. At subphysiological HCO3- and CO2 concentrations, inhibition of urea synthesis by acetazolamide was increased up to 90%. Inhibition of urea synthesis by acetazolamide was fully overcome in the presence of unphysiologically high HCO3- and CO2 concentrations, indicating that the inhibitory effect of acetazolamide is due to an inhibition of carbonic-anhydrase-catalyzed HCO3- supply for carbamoyl-phosphate synthetase, which can be bypassed when the uncatalyzed intramitochondrial HCO3- formation from portal CO2 is stimulated in the presence of high portal CO2 concentrations. With respect to HCO3- supply of mitochondrial carbamoyl-phosphate synthetase, urea synthesis can be separated into a carbonic-anhydrase-dependent (sensitive to acetazolamide at 0.5 mM) and a carbonic-anhydrase-independent (insensitive to acetazolamide) portion. Carbonic-anhydrase-independent urea synthesis linearly increased with the portal 'total CO2 addition' (which was experimentally determined to be CO2 addition plus 0.036 HCO3- addition) and was independent of the perfusate pH. At a constant 'total CO2 addition', carbonic-anhydrase-dependent urea synthesis was strongly affected by perfusate pH and increased about threefold when the perfusate pH was raised from 6.9 to 7.8. It is concluded that the pH dependent regulation of urea synthesis is predominantly due to mitochondrial carbonic anhydrase-catalyzed HCO3- supply for carbamoyl phosphate synthesis, whereas there is no control of urea synthesis by pH at the level of the five enzymes of the urea cycle. Because HCO3- provision for carbamoyl phosphate synthetase increases with increasing portal CO2 concentrations even in the absence of carbonic anhydrase activity, susceptibility of ureogenesis to pH decreases with increasing portal CO2 concentrations. This may explain the different response of urea synthesis to chronic metabolic and chronic respiratory acidosis in vivo.     

Hepatocyte heterogeneity in glutamate uptake by isolated perfused rat liver

Glutamate is simultaneously taken up and released by perfused rat liver, as shown by 14CO2 production from [1-14C]glutamate in the presence of a net glutamate release by the liver, turning to a net glutamate uptake at portal glutamate concentrations above 0.3 mM. 14CO2 production from portal [1-14C]glutamate is decreased by about 60% in the presence of ammonium ions. This effect is not observed during inhibition of glutamine synthetase by methionine sulfoximine. 14CO2 production from [1-14C]glutamate is not influenced by glutamine. Also, when glutamate accumulates intracellularly during the metabolism of glutamine (added at high concentrations, 5 mM), 14CO2 production from [1-14C]glutamate is not affected. If labeled glutamate is generated intracellularly from added [U-14C]proline, stimulation of glutamine synthesis by ammonium ions did not affect 14CO2 production from [U-14C]proline. After induction of a perivenous liver cell necrosis by CCL4, i.e. conditions associated with an almost complete loss of perivenous glutamine synthesis but no effect on periportal urea synthesis, 14CO2 production from [1-14C]glutamate is decreased by about 70%. The results are explained by hepatocyte heterogeneity in glutamate metabolism and indicate a predominant uptake of glutamate (that reaches the liver by the vena portae) by the small perivenous population of glutamine-synthesizing hepatocytes, whereas glutamate production from glutamine or proline is predominantly periportal. In view of the size of the glutamine synthetase-containing hepatocyte pool [Gebhardt, R. and Mecke, D. (1983) EMBO J. 2, 567-570], glutamate transport capacity of these hepatocytes would be about 20-fold higher as compared to other hepatocytes.     

Inhibition of CA V decreases glucose synthesis from pyruvate

The carbonic anhydrase inhibitor acetazolamide reduces citrulline synthesis by intact guinea pig liver mitochondria and also inhibits mitochondrial carbonic anhydrase (CA V) and the more lipophilic carbonic anhydrase inhibitor ethoxzolamide reduces urea synthesis by intact guinea pig hepatocytes in parallel with its inhibition of total hepatocytic carbonic anhydrase activity. Intact hepatocytes from 48-h starved male guinea pig livers were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 5 mM pyruvate, 5 mM lactate, 3 mM ornithine, 10 mM NH4Cl, 1 mM oleate; with these inclusions both urea and glucose synthesis start with HCO3- -requiring enzymes, carbamyl phosphate synthetase I and pyruvate carboxylase, respectively. Urea and glucose synthesis were inhibited in parallel by increasing concentrations of ethoxzolamide, estimated Ki for each approximately 0.1 mM. In other experiments hepatocytes were incubated at 37 degrees C in Krebs-Henseleit with 95% O2/5% CO2 at pH 7.1 with 10 mM glutamine, 1 mM oleate; with these inclusions glucose synthesis no longer starts with a HCO3- -requiring enzyme. Urea synthesis was inhibited by ethoxzolamide with an estimated Ki of 0.1 mM, but glucose synthesis was unaffected. Intact mitochondria were prepared from 48-h starved male guinea pig livers. Pyruvate carboxylase activity of intact mitochondria was determined in isotonic KCl-Hepes buffer, pH 7.4, 25 degrees C, with 7.5 mM pyruvate, 3 mM ATP, and 10 mM NaHCO3. Inclusion of ethoxzolamide resulted in reduction in the rate of pyruvate carboxylation in intact mitochondria, but not in disrupted mitochondria. It is concluded that carbonic anhydrase is functionally important for gluconeogenesis in the male guinea pig liver when there is a requirement for bicarbonate as substrate.     

Participation of endogenous fatty acids in the secretory activity of the pancreatic B-cell.

The pancreatic B-cell may represent a fuel-sensor organ, the release of insulin evoked by nutrient secretagogues being attributable to an increased oxidation of exogenous and/or endogenous substrates. The participation of endogenous fatty acids in the secretory response of isolated rat pancreatic islets was investigated. Methyl palmoxirate (McN-3716, 0.1 mM), an inhibitor of long-chain-fatty-acid oxidation, suppressed the oxidation of exogenous [U-14C]palmitate and inhibited 14CO2 output from islets prelabelled with [U-14C]palmitate. Methyl palmoxirate failed to affect the oxidation of exogenous D-[U-14C]glucose or L-[U-14C]glutamine, the production of NH4+ and the output of 14CO2 from islets prelabelled with L-[U-14C]glutamine. In the absence of exogenous nutrient and after a lag period of about 60 min, methyl palmoxirate decreased O2 uptake to 69% of the control value. Methyl palmoxirate inhibited insulin release evoked by D-glucose, D-glyceraldehyde, 2-oxoisohexanoate, L-leucine, 2-aminobicyclo[2.2.1]heptane-2-carboxylate or 3-phenylpyruvate. However, methyl palmoxirate failed to affect insulin release when the oxidation of endogenous fatty acids was already suppressed, e.g. in the presence of pyruvate or L-glutamine. These findings support the view that insulin release evoked by nutrient secretagogues tightly depends on the overall rate of nutrient oxidation, including that of endogenous fatty acids.     

Hepatocyte heterogeneity in glutamate metabolism and bidirectional transport in perfused rat liver

1. The metabolic fate of infused [1-14C]glutamate was studied in perfused rat liver. The 14C label taken up by the liver was recovered to 85 +/- 2% as 14CO2 and [14C]glutamine. Whereas 14CO2 production accounted for about 70% of the [1-14C]glutamate taken up under conditions of low endogenous rates of glutamine synthesis, stepwise stimulation of glutamine synthesis by NH4Cl increased 14C incorporation into glutamine at the expense of 14CO2 production. Extrapolation to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of vascular glutamate taken up by the liver for glutamine synthesis. This was observed in both, antegrade and retrograde perfusions and suggests an almost exclusive uptake of glutamate into perivenous glutamine-synthetase-containing hepatocytes. 2. Glutamate was simultaneously taken up and released from perfused rat liver. At a near-physiological influent glutamate concentration (0.1 mM), the rates of unidirectional glutamate influx and efflux were similar (about 100 and 120 nmol g-1 min-1, respectively). 3. During infusion of [1-14C]oxoglutarate (50 microM), addition of glutamate (2 mM) did not affect hepatic uptake of [1-14C]oxoglutarate. However, it increased labeled glutamate release from the liver about 10-fold (from 9 +/- 2 to 86 +/- 20 nmol g-1 min-1; n = 4), whereas 14CO2 production from labeled oxoglutarate decreased by about 40%. This suggests not only different mechanisms of oxoglutarate and glutamate transport across the plasma membrane, but also points to a glutamate/glutamate exchange. 4. Oxoglutarate was recently shown to be taken up almost exclusively by perivenous glutamine-synthetase-containing hepatocytes [Stoll, B & H?ussinger, D. (1989) Eur. J. Biochem. 181, 709-716] and [1-14C]oxoglutarate (9 microM) was used to label selectively the intracellular glutamate pool in this perivenous cell population. The specific radioactivity of this intracellular (perivenous) glutamate pool was assessed by measuring the specific radioactivity of newly synthesized glutamine which is continuously released from these cells into the perfusate. Comparison of the specific radioactivities of glutamine and glutamate released from perivenous cells indicates that about 60% of total glutamate release from the liver is derived from the perivenous glutamine-synthetase-containing cell population. Following addition of unlabeled glutamate (0.1 mM), unidirectional glutamate efflux from perivenous cells increased from about 30 to 80 nmol g-1 min-1, whereas glutamate efflux from non-perivenous (presumably periportal) hepatocytes remained largely unaltered (i.e. 20-30 nmol g-1 min-1). 5. It is concluded that, in the intact liver, vascular glutamate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase.     

Possible role of carbonic anhydrase in rat pancreatic islets: enzymatic, secretory, metabolic, ionic, and electrical aspects

The presence of carbonic anhydrase (type V) was recently documented in rat and mouse pancreatic islet beta-cells by immunostaining and Western blotting. In the present study, the activity of carbonic anhydrase was measured in rat islet homogenates and shown to be about four times lower than in rat parotid cells. The pattern for the inhibitory action of acetazolamide on carbonic anhydrase activity also differed in islet and parotid cell homogenates, suggesting the presence of different isoenzymes. NaN3 inhibited carbonic anhydrase activity in islet homogenates and both D-[U-14C]glucose oxidation and glucose-stimulated insulin secretion. Acetazolamide (0.3-10.0 mM) also decreased glucose-induced insulin output but failed to affect adversely D-[U-14C]glucose oxidation, although it inhibited the conversion of D-[5-3H]glucose to [3H]OH and that of D-[U-14C]glucose to acidic metabolites. Hydrochlorothiazide (3.0-10.0 mM), which also caused a concentration-related inhibition of the secretory response, like acetazolamide (5.0-10.0 mM), decreased H(14)CO3- production from D-[U-14C]glucose (16.7 mM). Acetazolamide (5.0 mM) did not affect the activity of volume-sensitive anion channels in beta-cells but lowered intracellular pH and adversely affected both the bioelectrical response to d-glucose and its effect on the cytosolic concentration of Ca2+ in these cells. The lowering of cellular pH by acetazolamide, which could well be due to inhibition of carbonic anhydrase, might in turn account for inhibition of glycolysis. The perturbation of stimulus-secretion coupling in the beta-cells exposed to acetazolamide may thus involve impaired circulation in the pyruvate-malate shuttle, altered mitochondrial Ca2+ accumulation, and perturbation of Cl- fluxes, resulting in both decreased bioelectrical activity and insulin release.     

Functional hepatocyte heterogeneity. Vascular 2-oxoglutarate is almost exclusively taken up by perivenous, glutamine-synthetase-containing hepatocytes

1. In isolated perfused rat liver maximal rates of 2-[1-14C]oxoglutarate uptake were about 0.4 mumol.g-1 .min-1; half-maximal rates of 2-[14C]oxoglutarate uptake were observed with influent concentrations of about 100 microM. 2-[14C]Oxoglutarate uptake by the liver was not affected by the direction of perfusion, but was decreased by about 80-90% when Na+ in the perfusion fluid was substituted by choline+, suggesting a Na+-dependence of hepatic 2-oxoglutarate uptake. In the absence of added ammonia, [14C]oxoglutarate uptake by the liver was about twice the net oxoglutarate uptake, indicating a simultaneous release of unlabeled oxoglutarate from perfused rat liver. 2. 14C-Labeled metabolites derived from [1-14C]oxoglutarate and recovered in the effluent perfusate were 14CO2 and 14C-labeled glutamate and glutamine; they accounted for 85-100% of the radiolabel taken up by the liver. 14CO2 was the major product (more than 70%) from [1-14C]oxoglutarate taken up the liver, provided glutamine synthesis was either inhibited by methionine sulfoximine or the endogenous rate of glutamine production was below 40 nmol.g-1.min-1. 3. Stimulation of glutamine synthesis by ammonia did not affect [14C]oxoglutarate uptake by the liver, but considerably increased net hepatic oxoglutarate uptake, indicating a decreased release of unlabeled oxoglutarate from the liver. Stepwise stimulation of hepatic glutamine synthesis led to a gradual decrease of 14CO2 production and radiolabel was recovered increasingly as [14C]glutamine in the effluent. At high rates of glutamine formation (i.e. about 0.6 mumol.g-1.min-1), about 60% of the [1-14C]oxoglutarate taken up by the liver was recovered in the effluent as [14C]glutamine. 14CO2 and [14C]glutamine production from added [1-14C]oxoglutarate were dependent on the rate of hepatic glutamine synthesis but not on the direction of perfusion. Extrapolation of 14C incorporation into glutamine to maximal rates of hepatic glutamine synthesis yielded an about 100% utilization of the [14C]oxoglutarate taken up by the liver for glutamine synthesis. This was again true for both the antegrade and the retrograde perfusion directions. On the other hand, addition of ammonia did not affect 14CO2 production from labeled oxoglutarate, when glutamine synthetase was inhibited by methionine sulfoximine. 4. The data suggest that vascular oxoglutarate is almost exclusively taken up by the small perivenous hepatocyte population containing glutamine synthetase, i.e. a cell population comprising only 6-7% of all hepatocytes. Thus, the findings demonstrate the existence of a, to date, uniquely zonally distributed oxoglutarate transport system which is probably Na+-dependent in the plasma membrane.(ABSTRACT TRUNCATED AT 400 WORDS)     

Alteration in Neuronal-Glial Metabolism of Glutamate by the Neurotoxin Kainic Acid

The effect of the excitotoxin kainic acid on glutamate and glutamine metabolism was studied in cerebellar slices incubated with D-[2-14C]glucose, [U-14C]gamma-aminobutyric acid, [3H]acetate, [U-14C]glutamate, and [U-14C]glutamine as precursors. Kainic acid (1 mM) strongly inhibited the labeling of glutamine relative to that of glutamate from all precursors except [2-14C]glucose and [U-14C]glutamine. Kainic acid did not inhibit glutamine synthetase directly. The data indicate that in the cerebellum kainic acid inhibits the synthesis of glutamine from the small pool of glutamate that is thought to be associated with glial cells. Kainic acid also markedly stimulated the efflux of glutamate from cerebellar slices and this release was not sensitive to tetrodotoxin. Kainic acid stimulated efflux of both glucose- and acetate-labeled glutamate. In contrast, veratridine released glucose-labeled glutamate preferentially via a tetrodotoxin-sensitive mechanism. Kainic acid did not release [U-14C]glutamate from synaptosomal fractions. These results suggest that the bulk of the glutamate released from cerebellar slices by kainic acid comes from nonsynaptic pools.     

Comparison between transport and degradation of leucine and glutamine by peripheral human lymphocytes exposed to concanavalin A

Transport and pathways of leucine and glutamine degradation were evaluated in resting human peripheral lymphocytes and compared with the changes induced by concanavalin A (ConA). Cells were incubated with [1-14C]leucine (0.15 mM), [U-14C]leucine (0.15 mM), or [U-14C]glutamine (0.4 mM) after culture with or without 2, 5, 7, or 10 micrograms/ml ConA for 2, 18, or 24 hours, respectively. Initial rates of transport of leucine and glutamine were augmented 2.7-fold and threefold by the mitogen. Leucine transamination, irreversible oxidation, and catabolism beyond isovaleryl-CoA were increased by 90%, 20%, and 60%, respectively. Glutamine utilization increased threefold; accumulation of glutamate, aspartate, and ammonia increased by 700%, 50%, and 100%, respectively, and 14CO2 production by about 400% in response to ConA. The results indicate that ConA stimulates to about the same extent transport of leucine and glutamine into lymphocytes. Glutamine is mainly channeled into catabolic pathways, while leucine remains largely preserved. It is suggested that these metabolic changes provide more leucine for incorporation into protein and more N- and C-atoms required for the synthesis of macromolecules and energy from glutamine.     

Effect of CO2 concentration on phosphatidylcholine and phosphatidylglycerol metabolism in surfactant and residual lung fractions.

An investigation of the effect of change of total CO(2) concentration from 7 to 43 mM at pH 7.35 in the medium perfusing isolated rat lungs on [U-(14)C]glucose incorporation into lung phospholipids has been carried out. The incorporation of [U-(14)C]glucose into phosphatidylcholine and phosphatidylglycerol of the surfactant fraction and of the remaining lung tissue (residual fraction) was observed. Increased CO(2) concentration increased [U-(14)C]glucose incorporation into phosphatidylcholine of the surfactant fraction and residual fraction by 43 and 50%, respectively, during a 2 hr perfusion. Likewise, incorporation of [U-(14)C]glucose into phosphatidylglycerol was increased 22 and 34% into the surfactant and residual fractions, respectively. The percentage of [U-(14)C]glucose incorporated into the fatty acid moieties of phosphatidylcholine of both fractions increased as a result of increased CO(2) concentration. The increase in the incorporation of [U-(14)C]glucose into the fatty acid moieties of phosphatidylcholine was confirmed by an average increase of 56 and 77% in the specific activity of palmitic acid isolated from phosphatidylcholine of the surfactant and residual fraction, respectively, as a result of increased CO(2) concentration. The results suggest that alteration in extracellular CO(2) concentration affects the de novo synthesis from glucose of phosphatidylcholine and phosphatidylglycerol of the surfactant-lipoprotein fraction of lung.     

Acute Effect of Ammonia on Branched-Chain Amino Acid Oxidation and Incorporation into Proteins in Astrocytes and in Neurons in Primary Cultures

14CO2 production and incorporation of label into proteins from the labeled branched-chain amino acids, leucine, valine, and isoleucine, were determined in primary cultures of neurons and of undifferentiated and differentiated astrocytes from mouse cerebral cortex in the absence and presence of 3 mM ammonium chloride. Production of 14CO2 from [1-14C]leucine and [1-14C]valine was larger than 14CO2 production from [U-14C]leucine and [U-14C]valine in both astrocytes and neurons. In most cases more 14CO2 was produced in astrocytes than in neurons. Incorporation of labeled branched-chain amino acids into proteins varied with the cell type and with the amino acid. Addition of 3 mM ammonium chloride greatly suppressed 14CO2 production from [1-14C]-labeled branched chain amino acids but had little effect on 14CO2 production from [U-14C]-labeled branched-chain amino acids in astrocytes. Ammonium ion, at this concentration, suppressed the incorporation of label from all three branched-chain amino acids into proteins of astrocytes. In contrast, ammonium ion had very little effect on the metabolism (oxidation and incorporation into proteins) of these amino acids in neurons. The possible implications of these findings are discussed, especially regarding whether they signify variations in metabolic fluxes and/or in magnitudes of precursor pools.     

Inhibition of mitochondrial carbonic anhydrase and ureagenesis: a discrepancy examined

The amount of urea synthesized in intact guinea pig hepatocytes in 60 min ([urea]t=60), was determined at 37 degrees C in Krebs-Henseleit buffer plus (in mM) 10 NH4Cl, 5 lactate, and 10 ornithine in 5% CO2-95% O2. The concentrations of sulfonamide carbonic anhydrase (CA) inhibitors required to reduce the rate of urea synthesis by 50% (I50) were (in mM): 0.07 ethoxzolamide, 0.5 methazolamide, 0.7 acetazolamide, and 5.0 p-aminomethylbenzenesulfonamide. At 37 degrees C acetazolamide and ethoxzolamide reduced citrulline synthesis by intact mitochondria in medium containing (in mM) 50 3-(N-morpholino)propanesulfonic acid, 35 KCl, 5 KH2PO4, 2 adenosine triphosphate, 10 ornithine, 10 NH4Cl, 1 [ethylene-bis(oxyethylenenitrile)]tetraacetic acid, 1 MgCl2, 20 pyruvate, and 25 KHCO3 (pH 7.4) in 5% CO2-95% O2; the inhibition by ethoxzolamide was not decreased greater than 50%; 25% inhibition was achieved by 0.65 microM ethoxzolamide. Inhibition constant (Ki) values for CA activity of disrupted mitochondria at 37 degrees C were 0.03 microM ethoxzolamide and 0.16 microM acetazolamide, and for disrupted hepatocytes were 150 microM ethoxzolamide and 50 microM acetazolamide. p-Aminomethylaminosulfonamide-affinity column purification yields one band of 29,000 mol wt for CA V purified from disrupted mitochondria; homogenized whole-liver supernatant yields an additional band of 20,000 mol wt (at greater than 100 times the concentration of CA V), which has some glutathione S-transferase activity. It is concluded that this 20,000-mol wt protein modifies the potency of ethoxzolamide in the liver cytosol.     

Glutamine metabolism in rat hepatocytes. Stimulation by a nonmetabolizable analog of leucine

The metabolic effects of beta-(+/-)-2-aminobicyclo-(2.2.1)-heptane-2-carboxylic acid (BCH), a nonmetabolizable analog of leucine and known activator of glutamate dehydrogenase, were studied in hepatocytes isolated from fed and fasted rats. With glutamine as substrate, BCH stimulated in a concentration-dependent manner urea synthesis in both physiological states and glucose formation in hepatocytes from fasted rats. Despite the much higher rates of ureagenesis in the fasted animals, the degree of stimulation by BCH, over 2-fold, was similar. The effect of the drug was specific for glutamine since the rates of urea synthesis from NH4Cl, alanine, and asparagine were essentially unaltered. The stimulation of glutamine catabolism by BCH led to a decrease in the content of intracellular glutamine. The redox states of the mitochondrial and cytosolic nicotinamide adenine dinucleotides remained unaltered. In hepatocytes isolated from fasted rats and incubated with 5 mM glutamine the BCH-induced increases in urea, ammonia, and the amino acids, glutamate, aspartate, and alanine, accounted fully for the 2.4-fold rise in glutamine utilization. The stimulatory effects of BCH and glucagon on the formation of glucose, urea, and 14CO2 from [U-14C]glutamine were additive. Aminooxyacetate, and inhibitor of transaminases, neither blocked glutamine catabolism (as measured by the sum of urea, ammonia, and glutamate) nor prevented its activation by BCH. It is suggested that, in isolated hepatocytes, BCH-induced stimulation of glucose and urea formation from glutamine results from activation of glutaminase by a mechanism which is distinct from that of glucagon.     

Relative role of the glutaminase, glutamate dehydrogenase, and AMP-deaminase pathways in hepatic ureagenesis: studies with 15N.

We have studied the relative roles of the glutaminase versus glutamate dehydrogenase (GLDH) and purine nucleotide cycle (PNC) pathways in furnishing ammonia for urea synthesis. Isolated rat hepatocytes were incubated at pH 7.4 and 37 degrees C in Krebs buffer supplemented with 0.1 mM L-ornithine and 1 mM [2-15N]glutamine, [5-15N]glutamine, [15N]aspartate, or [15N]glutamate as the sole labeled nitrogen source in the presence and absence of 1 mM amino-oxyacetate (AOA). A separate series of incubations was carried out in a medium containing either 15N-labeled precursor together with an additional 19 unlabeled amino acids at concentrations similar to those of rat plasma. GC-MS was utilized to determine the precursor product relationship and the flux of 15N-labeled substrate toward 15NH3, the 6-amino group of adenine nucleotides ([6-15NH2]adenine), 15N-amino acids, and [15N]urea. Following 40 min incubation with [15N]aspartate the isotopic enrichment of singly and doubly labeled urea was 70 and 20 atom % excess, respectively; with [15N]glutamate these values were approximately 65 and approximately 30 atom % excess for singly and doubly labeled urea, respectively. In experiments with [15N]aspartate as a sole substrate 15NH3 enrichment exceeded that in [6-NH2]adenine, indicating that [6-15NH2]adenine could not be a major precursor to 15NH3. Addition of AOA inhibited the formation of [15N]glutamate, 15NH3 and doubly labeled urea from [15N]aspartate. However, AOA had little effect on [6-15NH2]adenine production. In experiments with [15N]glutamate, AOA inhibited the formation of [15N]aspartate and doubly labeled urea, whereas 15NH3 formation was increased. In the presence of a physiologic amino acid mixture, [15N]glutamate contributed less than 5% to urea-N. In contrast, the amide and the amino nitrogen of glutamine contributed approximately 65% of total urea-N regardless of the incubation medium. The current data indicate that when glutamate is a sole substrate the flux through GLDH is more prominent in furnishing NH3 for urea synthesis than the flux through the PNC. However, in experiments with medium containing a mixture of amino acids utilized by the rat liver in vivo, the fraction of NH3 derived via GLDH or PNC was negligible compared with the amount of ammonia derived via the glutaminase pathway. Therefore, the current data suggest that ammonia derived from 5-N of glutamine via glutaminase is the major source of nitrogen for hepatic urea-genesis.     

Competition of glycerol with other oxidizable substrates in rat brain.

The rate of conversion of [1,3-14C]glycerol into 14CO2 was measured in the presence and absence of unlabelled alternative substrates in whole homogenates from the brains of young (4-6 and 18-20 days old) and adult rats. Unlabelled glucose decreased 14CO2 production from [1,3-14C]glycerol by about 40% at all ages studied. Unlabelled 3-hydroxybutyrate significantly decreased the 14CO2 production from both low (0.2 mM) and high (2.0 mM) concentrations of glycerol in 4-6- and 18-20-day-old rat pups. However, the addition of 3-hydroxybutyrate had no effect on the rate of 14CO2 production from 2.0 mM-glycerol in adult rats, suggesting that the interaction of 3-hydroxybutyrate with glycerol in adult rat brain is complex and may be related to the biphasic kinetics previously reported for glycerol oxidation. Unlabelled glutamine decreased the production of 14CO2 by brain homogenates from 18-20-day-old and adult rats, but not in 4-6-day-old rat pups. In the converse situation, the addition of unlabelled glycerol to whole brain homogenates had little effect on the rate of 14CO2 production from [6-14C]glucose, 3-hydroxy[3-14C]butyrate and [U-14C]glutamine, although some significant differences were noted. Collectively these results suggest that glycerol and these other substrates may be metabolized in separate subcellular compartments in brain such that the products of glucose, 3-hydroxybutyrate and glutamine metabolism can dilute the oxidation of glycerol, but the converse cannot occur. The data also demonstrate that there are complex age-related changes in the interaction of glycerol with 3-hydroxybutyrate and glutamine. The fact that glycerol oxidation was only partially suppressed by the addition of 1-5 mM-glucose, -3-hydroxybutyrate or -glutamine could also suggest that glycerol may be selectively utilized as an energy substrate in some discrete brain region.     

Glyceroneogenesis and the supply of glycerol-3-phosphate for glyceride-glycerol synthesis in liver slices of fasted and diabetic rats

The pathways of glycerol-3-phosphate (G3P) generation for glyceride synthesis were examined in precision-cut liver slices of fasted and diabetic rats. The incorporation of 5 mM [U-(14)C]glucose into glyceride-glycerol, used to evaluate G3P generation via glycolysis, was reduced by approximately 26-36% in liver slices of fasted and diabetic rats. The glycolytic flux was reduced by approximately 60% in both groups. The incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol (glyceroneogenesis) increased approximately 50% and approximately 36% in slices of fasted and diabetic rats, respectively, which also showed a two-fold increase in the activity phosphoenolpyruvate carboxykinase. The increased incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol by slices of fasted rats was not affected by the addition of 5 mM glucose to the incubation medium. The activity of glycerokinase and the incorporation of 1 mM [U-(14)C]glycerol into glyceride-glycerol, evaluators of G3P formation by direct glycerol phosphorylation, did not differ significantly from controls in slices of the two experimental groups. Rates of incorporation of 1 mM [2-(14)C]pyruvate and [U-(14)C]glycerol into glucose of incubation medium (gluconeogenesis) were approximately 140 and approximately 20% higher in fasted and diabetic slices than in control slices. It could be estimated that glyceroneogenesis by liver slices of fasted rats contributed with approximately 20% of G3P generated for glyceride-glycerol synthesis, the glycolytic pathway with approximately 5%, and direct phosphorylation of glycerol by glycerokinase with approximately 75%. Pyruvate contributed with 54% and glycerol with 46% of gluconeogenesis. The present data indicate that glyceroneogenesis has a significant participation in the generation of G3P needed for the increased glyceride-glycerol synthesis in liver during fasting and diabetes.     

Inhibition of lipogenesis by vasopressin and angiotensin II in glycogen-depleted hepatocytes

Vasopressin and angiotensin II inhibited lipogenesis (measured with 3H2O) in hepatocytes from fed rats. Inhibition was also observed with hepatocytes from fed rats which had been depleted of glycogen in vitro and incubated with lactate + pyruvate (5 mM + 0.5 mM) as substrates. The inhibitory actions of the hormones are therefore independent of hormone-mediated changes in glycogenolytic or glycolytic flux from glycogen, and thus the site(s) of hormone action must be subsequent to the formation of lactate. (-)Hydroxycitrate, a specific inhibitor of ATP-citrate lyase, decreased lipogenesis in hepatocytes from fed rats incubated with lactate + pyruvate by approx. 51% but had little effect on lipogenesis in glycogen-depleted hepatocytes similarly incubated. There was parallel inhibition of incorporation of 14C from [U-14C]lactate into fatty acid and lipogenesis as measured with 3H2O in each case. Thus depletion of glycogen, or conceivably the process of glycogen-depletion (incubation with dibutyryl cyclic AMP) causes a change in the rate-determining step(s) for lipogenesis from lactate. Vasopressin and angiotensin II also decreased lipogenesis and incorporation of 14C into fatty acids in glycogen-depleted hepatocytes provided with [U-14C]proline as opposed to [U-14C]-lactate. However, proline-stimulated lipogenesis was inhibited by (-)hydroxycitrate, and proline-stimulated lipogenesis and incorporation of 14C from [U-14C]-proline were not decreased in parallel by this inhibitor (inhibition of 52% and 85% respectively). It is inferred that lactate and proline stimulate lipogenesis by different mechanisms and incorporation of 14C from [U-14C]proline and [U-14C]lactate into fatty acid occurs via different routes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Ammonia and β-Methylene-dl-Aspartate on the Oxidation of Glucose and Pyruvate by Neurons and Astrocytes in Primary Culture

Both ammonia and beta-methylene-DL-aspartate (beta-MA), an irreversible inhibitor of aspartate aminotransferase activity and thus of the malate-aspartate shuttle, were found previously to decrease oxidative metabolism in cerebral cortex slices. In the present work, the possibility that ammonia and beta-MA affect energy metabolism by a common mechanism (i.e., via inhibition of the malate-aspartate shuttle) was investigated using primary cultures of neurons and astrocytes. Incubation of astrocytes for 30 min with 5 mM beta-MA resulted in a decreased production of 14CO2 from [U-14C]glucose, but did not affect 14CO2 production from [2-14C]pyruvate. Conversely, incubation of astrocytes with 3 mM ammonium chloride resulted in decreased 14CO2 production from [2-14C]pyruvate, but 14CO2 production from [U-14C]glucose was not significantly affected. Ammonium chloride had no significant effect on 14CO2 production from either [U-14C]glucose or [2-14]pyruvate by neurons. However, incubation of neurons with beta-MA or beta-MA plus ammonium chloride resulted in a approximately 45% decrease of 14CO2 production from both [U-14C]glucose and [2-14C]pyruvate. A 2-h incubation of astrocytes with beta-MA resulted in no change in ATP levels, but a 35% decrease in phosphocreatine. Similar treatment of neurons resulted in greater than 50% decrease in ATP, but had little effect on phosphocreatine. beta-MA also caused a decrease in glutamate and aspartate content of neurons, but not of astrocytes. The different metabolic responses of neurons and astrocytes towards beta-MA were probably not due to a differential inhibition of aspartate aminotransferase which was inhibited by approximately 45% in astrocytes and by approximately 55% in neurons.     

Competition among oxidizable substrates in brains of young and adult rats. Dissociated cells.

The rates of conversion of D-(-)-3-hydroxy[3-14C]butyrate, [3-14C]acetoacetate, [6-14C]glucose and [U-14C]glutamine into 14CO2 were measured in the presence and absence of alternative oxidizable substrates in intact dissociated cells from the brains of young and adult rats. When unlabelled glutamine was added to [6-14C]glucose or unlabelled glucose was added to [U-14C]glutamine, the rate of 14CO2 production was decreased in both young and adult rats. The rate of oxidation of 3-hydroxy[3-14C]butyrate was also decreased by the addition of unlabelled glutamine in both age groups, but in the reverse situation, i.e. unlabelled 3-hydroxybutyrate added to [U-14C]glutamine, only the brain cells from young rats were affected. No significant effects were seen when glutamine and acetoacetate were combined. The addition of either of the two ketone bodies to [6-14C]glucose markedly lowered the rate of 14CO2 production in young rats, but in the adult only 3-hydroxybutyrate was effective and the magnitude of decrease in the rate of [6-14C]glucose oxidation was much lower than in young animals. Unlabelled glucose decreased the rate of [3-14C]acetoacetate oxidation to a minor extent in brain cells from both age groups; when added to 3-hydroxy[3-14C]butyrate, glucose had no effect in young rats and greatly enhanced 14CO2 production in adult brain cells. Many of these patterns of substrate interaction in dissociated brain cells differ from those in whole homogenates; they may be a function of the plasma membranes and the role of a carrier-mediated transport system or a reflection of a difference in the population of cell types or subcellular organelles in these two preparations.     

Effect of CO 2 concentration on phospholipid metabolism in the isolated perfused rat lung

Studies have been carried out on the incorporation of [U-(14)C]glucose, [2-(14)C]pyruvate, [2-(14)C]acetate, and [1-(14)C]-palmitate into the phospholipids of the isolated perfused rat lung in the presence of either 6 or 45 mm total CO(2) concentration in the perfusion medium. Incorporation of [U-(14)C]glucose into total phospholipid and into the phosphatidylcholine fraction was increased 19-53% over the 2-hr perfusion period in lungs perfused with medium containing 45 as compared with 6 mm CO(2). The incorporation of [2-(14)C]acetate, [2-(14)C]-pyruvate, and [1-(14)C]palmitate was not affected by the change in medium CO(2) concentration. Increased incorporation of [U-(14)C]glucose combined with a shift toward greater incorporation into the fatty acids of the phosphatidylcholine fraction produced a maximum increase of 90% in [U-(14)C]glucose incorporation into the fatty acids of phosphatidylcholine after 2 hr of perfusion in the presence of medium containing 45 mm CO(2) as compared with 6 mm CO(2). The increase in medium CO(2) concentration produced as much as a 150% increase in [U-(14)C]glucose incorporation into palmitate derived from the phosphatidylcholine fraction. The results provide evidence that glucose functions as an important precursor of palmitate in the phosphatidylcholine fraction of lung phospholipids and that the CO(2) concentration of the perfusion medium affects the incorporation of glucose into palmitate.