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.     

Rat kidney mitochondrial carbonic anhydrase

Mitochondrial carbonic anhydrase has previously been quantitated in liver mitochondria; it was not detected in guinea pig kidney cortical mitochondria. Evidence of this enzyme in rat kidney cortical mitochondria is reported. Electron microscopy showed that intact mitochondria were free of other intracellular organelles. When intact kidney mitochondria were added to isotonic 3'-(N'-morpholino) propanesulfonic acid buffer with 25 mM KHCO3 (1% labeled with 18O) the rate of disappearance of C18O16O was biphasic; this indicates that there is carbonic anhydrase within the inner mitochondrial membrane. Intact rat kidney mitochondria were assayed for carbonic anhydrase activity at 4 degrees C by the changing pH technique. The rate of CO2 hydration in the presence and absence of intact mitochondria was identical; this rate increased when Triton X-100 was added which indicates that all carbonic anhydrase is inside the inner mitochondrial membrane. Carbonic anhydrase activity was quantitated as kenz (units, ml.s-1 mg-1 mitochondrial protein) at 37 degrees C, pH 7.4, in 25 mM NaHCO3 (1% labeled with 18O) by following the rate of disappearance of C18O16O from solutions before and after addition of disrupted mitochondria. Values of Kenz for liver and kidney mitochondria from rats given free access to normal rat chow and water at neutral pH were 0.06 and 0.08 (respectively). Values of kenz for liver and kidney mitochondria from rats fed as above and with free access to water adjusted to pH 2.5 with HCl were 0.04 and 0.16, respectively. Values of kenz for rats starved for 48 h were 0.06 and 0.12 (respectively). The values of kenz remained 0.11-0.14 in liver mitochondria from guinea pigs fed normally, given dilute acid, or starved and the value was always at zero in guinea pig kidney mitochondria. Values of Kenz were measured with disrupted mitochondria by the 18O technique as a function of pH at 25 degrees C, 25 to 75 mM NaHCO3, ionic strength 0.3. From pH 7.0 to 8.0 kenz increased threefold for mitochondria from rat liver, fed rat kidney, and acid rat kidney, and increased eightfold for mitochondria from guinea pig liver. kenz was decreased similarly by increasing HCO3- in mitochondria from rat liver, fed kidney, and acid kidney; it is concluded that carbonic anhydrase in rat liver mitochondria is probably the same isozyme as in rat kidney mitochondria. The published observation that rat kidney cortices are up to 10 times as gluconeogenic from pyruvate as guinea pig kidney cortices can be explained by the presence of mitochondrial carbonic anhydrase in rat but not guinea pig mitochondria.     

Carbonic anhydrase in guinea pig skeletal muscle mitochondria

The presence of carbonic anhydrase activity was demonstrated in guinea pig skeletal muscle mitochondria purified by Percoll gradient centrifugation such that contamination by sarcoplasmic reticulum vesicles was less than 5%. Assay of purified heavy sarcoplasmic reticulum vesicles for carbonic anhydrase activity showed these to have somewhat less activity than the mitochondria, so that any contribution by sarcoplasmic reticulum vesicles to mitochondrial activity would be negligible. In agreement with this observation, rabbit skeletal muscle mitochondria prepared by the Percoll method had no detectable activity. Assay of the guinea pig muscle mitochondrial enzyme activity in the presence of Triton X-100 showed a sixfold greater activity than in its absence, indicating a matrix location for the carbonic anhydrase. The enzyme is highly sensitive to the sulfonamide inhibitor ethoxzolamide, with Ki = 8.7 nM. The activation energy obtained from the rate constant for CO2 hydration, kenz with units (mg/ml)-1 s-1, over the range 4 to 37 degrees C was 12.8 kcal/mol. These properties are those expected for a carbonic anhydrase of the CA II class of isozymes, rather than for CA I, CA III, and the liver mitochondrial enzyme CA V.     

Effect of long-chain fatty acyl-CoA on mitochondrial and cytosolic ATP/ADP ratios in the intact liver cell.

The effect of long-chain acyl-CoA on subcellular adenine nucleotide systems was studied in the intact liver cell. Long-chain acyl-CoA content was varied by varying the nutritional state (fed and starved states) or by addition of oleate. Starvation led to an increase in the mitochondrial and a decrease in the cytosolic ATP/ADP ratio in liver both in vivo and in the isolated perfused organ as compared with the fed state. The changes were reversed on re-feeding glucose in liver in vivo or on infusion of substrates (glucose, glycerol) in the perfused liver, respectively. Similar changes in mitochondrial and cytosolic ATP/ADP ratios occurred on addition of oleate, but, importantly, not with a short-chain fatty acid such as octanoate. It is concluded that long-chain acyl-CoA exerts an inhibitory effect on mitochondrial adenine nucleotide translocation in the intact cell, as was previously postulated in the literature from data obtained with isolated mitochondria. The physiological relevance with respect to pyruvate metabolism, i.e. regulation of pyruvate carboxylase and pyruvate dehydrogenase by the mitochondrial ATP/ADP ratio, is discussed.     

Regulation of flux through pyruvate dehydrogenase and pyruvate carboxylase in rat hepatocytes. Effects of fatty acids and glucagon

The regulation of flux through pyruvate dehydrogenase (PDH) and pyruvate carboxylase (PC) by fatty acids and glucagon was studied in situ, in intact hepatocyte suspensions. The rate of pyruvate metabolized by carboxylation plus decarboxylation was determined from the incorporation of [1-14C]pyruvate into 14CO2 plus [14C]glucose. The flux through PDH was determined from the rate of formation of 14CO2 from [1-14C]pyruvate corrected for other decarboxylation reactions (citrate cycle, phosphoenolpyruvate carboxykinase and malic enzyme), and the flux through PC was determined by subtracting the flux through PDH from the total pyruvate metabolized. With 0.5 mM pyruvate as substrate the ratio of flux through PDH/PC was 1.9 in hepatocytes from fed rats and 1.4 in hepatocytes from 24 h-starved rats. In hepatocytes from fed rats, octanoate (0.8 mM) and palmitate (0.5 mM) increased the flux through PDH (59-76%) and PC (80-83%) without altering the PDH/PC flux ratios. Glucagon did not affect the flux through PDH but it increased the flux through PC twofold, thereby decreasing the PDH/PC flux ratio to the value of hepatocytes from starved rats. In hepatocytes from starved rats, fatty acids had similar effects on pyruvate metabolism as in hepatocytes from fed rats, however glucagon did not increase the flux through PC. 2[5(4-Chlorophenyl)pentyl]oxirane-2-carboxylate (100 microM) an inhibitor of carnitine palmitoyl transferase I, reversed the palmitate-stimulated but not the octanoate-stimulated flux through PDH, in cells from fed rats, indicating that the effects of fatty acids on PDH are secondary to the beta-oxidation of fatty acids. This inhibitor also reversed the stimulatory effect of palmitate on PC and partially inhibited the flux through PC in the presence of octanoate suggesting an effect of POCA independent of fatty acid oxidation. It is concluded that the effects of fatty acids on pyruvate metabolism are probably secondary to increased pyruvate uptake by mitochondria in exchange for acetoacetate. Glucagon favours the partitioning of pyruvate towards carboxylation, by increasing the flux through pyruvate carboxylase, without directly inhibiting the flux through PDH.     

Interactions between alpha-ketoisovalerate metabolism and the pathways of gluconeogenesis and urea synthesis in isolated hepatocytes

The mechanism of inhibition of pyruvate carboxylase, pyruvate dehydrogenase, and carbamyl phosphate synthetase induced by alpha-ketoisovalerate metabolism has been investigated in isolated rat hepatocytes incubated with lactate, pyruvate, ammonia, and ornithine as substrates. Half-maximum inhibitions of flux through each of these enzyme steps were obtained with 0.3 mM alpha-ketoisovalerate. The inhibition of pyruvate carboxylase flux by alpha-ketoisovalerate was largely reversed by oleate addition, but pyruvate dehydrogenase flux was inhibited further. Inhibition of flux through pyruvate carboxylase could be attributed mainly to the fall of its allosteric activator, acetyl-CoA, with some additional effect due to inhibition by methylmalonyl-CoA. Tissue acetyl-CoA levels decrease as a result of an inhibition of the active form of pyruvate dehydrogenase. Kinetic studies with the purified pig heart pyruvate dehydrogenase complex showed that methyl-malonyl-CoA, propionyl-CoA, and isobutyryl-CoA were inhibitory, the latter noncompetitive with CoASH with an apparent Ki of 90 microM. The observed inhibition of pyruvate dehydrogenase flux correlated with increases of the acetyl-CoA/CoASH and propionyl-CoA/CoASH ratios and isobutyryl-CoA levels, while increases of the mitochondrial NADH/NAD+ ratio explained differences between the effects of alpha-ketoisovalerate and propionate. Carbamyl phosphate synthetase I purified from rat liver was shown to be inhibited directly by methylmalonyl-CoA (apparent Ki of 5 mM). Inhibition of flux through carbamyl phosphate synthetase during alpha-ketoisovalerate metabolism could be attributed both to a direct inhibitory effect of methyl-malonyl-CoA and to a diminished activation by N-acetylglutamate. Direct effects of various acyl-CoA metabolites on these key enzymes may explain symptoms of hypoglycemia and hyperammonemia observed in patients with inherited disorders of organic acid metabolism.     

Carbonic anhydrase in human platelets.

The carbonic anhydrase activity of human platelets was investigated by measuring the kinetics of CO2 hydration in supernatants of platelet lysates by using a pH stopped-flow apparatus. An average carbonic anhydrase concentration of 2.1 microM was determined for pellets of human platelets. Analysis of the kinetic properties of this carbonic anhydrase yielded a Km value of 1.0 mM, a catalytic-centre activity kcat. of 130000 s-1 and an inhibition constant Ki towards ethoxzolamide of 0.3 nM. From these values, CO2 hydration inside platelets is estimated to be accelerated by a factor of 2500. When platelet lysates were subjected to affinity chromatography, only the high-activity carbonic anhydrase II could be eluted from the affinity column, whereas the carbonic anhydrase isoenzyme I, which is known to occur in high concentrations in human erythrocytes, appeared to be absent.     

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.     

The relationship between mitochondrial heterogeneity and gluconeogenesis in liver mitochondria of the rat, pigeon and guinea pig.

Isolated mitochondria of pigeon and guinea pig liver were subjected to zonal centrifugation. With pigeon liver mitochondria there was uniform distribution of pyruvate carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, aspartate aminotransferase and glutamate dehydrogenase activities. Guinea pig liver mitochondria demonstrated two pyruvate carboxylase and phosphoenolpyruvate carboxykinase maxima but only one maximum with aspartate aminotransferase, malate dehydrogenase and glutamate dehydrogenase. Mitochondrial enzyme levels in rat, pigeon and guinea pig indicate different roles of certain gluconeogenic enzymes in the transport of carbon and hydrogen in and out of mitochondria.     

The kinetics of transport of lactate and pyruvate into isolated cardiac myocytes from guinea pig. Kinetic evidence for the presence of a carrier distinct from that in erythrocytes and hepatocytes.

1. Time courses for the uptake of L-lactate, D-lactate and pyruvate into isolated cardiac ventricular myocytes from guinea pig were determined at 11 degrees C or 0 degrees C (for pyruvate) in a citrate-based buffer by using a silicone-oil-filtration technique. These conditions enabled initial rates of transport to be measured without interference from metabolism of the substrates. 2. At a concentration of 0.5 mM, transport of all these substrates was inhibited by approx. 90% by 5 mM-alpha-cyano-4-hydroxycinnamate; at 10 mM-L-lactate a considerable portion of transport could not be inhibited. 3. Initial rates of L-lactate and pyruvate uptake in the presence of 5 mM-alpha-cyano-4-hydroxycinnamate were linearly related to the concentration of the monocarboxylate and probably represented diffusion of the free acid. The inhibitor-sensitive component of uptake obeyed Michaelis-Menten kinetics, with Km values for L-lactate and pyruvate of 2.3 and 0.066 mM respectively. 4. Pyruvate and D-lactate inhibited the transport of L-lactate, with Ki values (competitive) of 0.077 and 6.6 mM respectively; the Ki for pyruvate was very similar to its Km for transport. The Ki for alpha-cyano-4-hydroxycinnamate as a non-competitive inhibitor was 0.042 mM. 5. These results indicate that L-lactate, D-lactate and pyruvate share a common carrier in guinea-pig cardiac myocytes; the low stereoselectivity for L-lactate over D-lactate and the high affinity for pyruvate distinguish it from the carrier in erythrocytes and hepatocytes. The metabolic roles for this novel carrier in heart are discussed.     

Characterization of oxalate as a catabolite of dichloroacetate responsible for the inhibition of gluconeogenesis and pyruvate carboxylation in rat liver cells

In isolated hepatocytes, dichloroacetate decreased glucose synthesis from lactate, pyruvate and alanine, but not from substrates which bypass pyruvate carboxylase (propionate, glycerol). It was also found to inhibit pyruvate carboxylation in isolated mitochondria, but only after a preincubation period, and had no effect on partially purified pyruvate carboxylase. Hepatocytes and liver mitochondria metabolized [¹⁴C] dichloroacetate to oxalate which inhibits pyruvate carboxylase and mimics, without preincubation, the effects of dichloroacetate in mitochondrial pyruvate carboxylation. Thus, oxalate appears to be responsible for the inhibition of gluconeogenesis by dichloroacetate at the level of pyruvate carboxylation.     

Illumination increases the affinity of phosphoenolpyruvate carboxylase to bicarbonate in leaves of a C4 plant, Amaranthus hypochondriacus

Illumination increased markedly the affinity to bicarbonate of phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) in leaves of Amaranthus hypochondriacus L., a C4 plant. When leaves were illuminated, the apparent Km for (HCO3-) of PEPC decreased by about 50% concurrent with a 2- to 5-fold increase in Vmax and 3- to 4-fold increase in Ki for malate. The inclusion of ethoxyzolamide, an inhibitor of carbonic anhydrase, during the assay had no effect on kinetic and regulatory properties of PEPC indicating that carbonic anhydrase was not involved during light-induced sensitization of PEPC to HCO3-. Pretreatment of leaf discs with cycloheximide (CHX), a cytosolic protein synthesis inhibitor, suppressed significantly the light-enhanced decrease in apparent Km (HCO3-). Further, in vitro phosphorylation of purified dark-form PEPC by protein kinase A (PKA) decreased the apparent Km (HCO3-) of the enzyme, in addition increasing Ki (malate) as expected. Such changes, due to in vitro phosphorylation of purified PEPC by PKA, occurred only with wild-type PEPC, but not in the mutant form of maize (S15D) which is already a mimic of the phosphorylated enzyme. These results suggest that phosphorylation of the enzyme is important during the sensitization of PEPC to HCO3- by illumination in C4 leaves. Since illumination is expected to increase the cytosolic pH and the availability of dissolved HCO3- in mesophyll cells, the sensitization by light of PEPC to HCO3- could be physiologically quite significant.     

CO2 fixation kinetics of Halothiobacillus neapolitanus mutant carboxysomes lacking carbonic anhydrase suggest the shell acts as a diffusional barrier for CO2

The widely accepted models for the role of carboxysomes in the carbon-concentrating mechanism of autotrophic bacteria predict the carboxysomal carbonic anhydrase to be a crucial component. The enzyme is thought to dehydrate abundant cytosolic bicarbonate and provide ribulose 1.5-bisphosphate carboxylase/oxygenase (RubisCO) sequestered within the carboxysome with sufficiently high concentrations of its substrate, CO(2), to permit its efficient fixation onto ribulose 1,5-bisphosphate. In this study, structure and function of carboxysomes purified from wild type Halothiobacillus neapolitanus and from a high CO(2)-requiring mutant that is devoid of carboxysomal carbonic anhydrase were compared. The kinetic constants for the carbon fixation reaction confirmed the importance of a functional carboxysomal carbonic anhydrase for efficient catalysis by RubisCO. Furthermore, comparisons of the reaction in intact and broken microcompartments and by purified carboxysomal RubisCO implicated the protein shell of the microcompartment as impeding diffusion of CO(2) into and out of the carboxysome interior.     

The effect of adenosine and chloroadenosine on sex differences in the energy metabolism of rat hepatocytes

The intensity and regulation of metabolic pathways are different depending on the sex of the source animal for hepatocytes isolated from mature rats. In cells from fed animals incubated without exogenous substrate, ATP level and ketone body production are higher in males (+25% and +100%) and lactate production is higher (+64%) in females; oleate enhances mitochondrial pyruvate oxidation in hepatocytes from fed male rats but not from fed females; in cells from starved animals oleate increases gluconeogenesis in both sexes at saturating levels of gluconeogenic substrates. However, at physiological levels (1 mM lactate and 0.1 mM pyruvate), this activation can only be detected in cells from males. In both sexes, oleate activation is abolished by adenosine which reduces in parallel the mitochondrial oxidation of pyruvate; chloroadenosine, an A2-receptor agonist, increases glycogenolysis strongly in hepatocytes from male animals (+80%) but only very slightly in female cells (+12%).     

Stimulation by glucose of gluconeogenesis in hepatocytes isolated from starved rats.

Control properties of the gluconeogenic pathway in hepatocytes isolated from starved rats were studied in the presence of glucose. The following observations were made. (1) Glucose stimulated the rate of glucose production from 20 mM-glycerol, from a mixture of 20 mM-lactate and 2 mM-pyruvate, or from pyruvate alone; no stimulation was observed with 20 mM-alanine or 20 mM-dihydroxyacetone. Maximal stimulation was obtained between 2 and 5 mM-glucose, depending on the conditions. At concentrations above 6 mM, gluconeogenesis declined again, so that at 10 mM-glucose the glucose production rate became equal to that in its absence. (2) With glycerol, stimulation of gluconeogenesis by glucose was accompanied by oxidation of cytosolic NADH and reduction of mitochondrial NAD+ and was insensitive to the transaminase inhibitor amino-oxyacetate; this indicated that glucose accelerated the rate of transport of cytosolic reducing equivalents to the mitochondria via the glycerol 1-phosphate shuttle. (3) With lactate plus pyruvate (10:1) as substrates, stimulation of gluconeogenesis by glucose was almost additive to that obtained with glucagon. From an analysis of the effect of glucose on the curves relating gluconeogenic flux and the steady-state intracellular concentrations of gluconeogenic intermediates under various conditions, in the absence and presence of glucagon, it was concluded that addition of glucose stimulated both phosphoenolpyruvate carboxykinase and pyruvate carboxylase activity.     

Comparison of 18O exchange and pH stop-flow assays for carbonic anhydrase

The hydration velocity of CO2 (0.002 M) catalyzed by bovine carbonic anhydrase (BCA) was measured at 25 degrees C and pH 7.4 by three different techniques: two initial-rate (steady-state) stop-flow methods, one using a glass pH electrode (in Hannover, method 1) and one using spectrophotometric measurements of a pH indicator (in Philadelphia, method 2), and an exchange method in which the disappearance of C18O16O from a bicarbonate solution was determined at equilibrium (in Philadelphia, method 3). The Michaelis-Menten constant (Km) and the inhibition constants for chloride (Ki,Cl) and ethoxzolamide (Ki,ez) were the same for methods 1, 2, and 3. The turnover numbers were 270,000, 400,000, and 555,000 s-1 by methods 1, 2, and 3, respectively. Values for CO2 hydration velocity measured by methods 2 and 3 on the same solution of BCA at the same time were the same. Km, maximal reaction velocity (Vmax), Ki,ez, and Ki,Cl obtained from normal human hemolysate at 37 degrees C and pH 7.2 by methods 2 and 3 were the same. Km and Vmax of the carbonic anhydrase isozyme CA III of homogenate from rabbit soleus were also identical by methods 1 and 3. According to Michaelis-Menten theory, the values of Km and Vmax obtained by method 3 should have been significantly smaller than those obtained by methods 1 and 2. We conclude that the catalytic step itself is apparently not rate limiting under physiological conditions and that method 3 can be used to obtain Michaelis-Menten characteristics of carbonic anhydrase.     

Regulation of mitochondrial pyruvate carboxylation in isolated hepatocytes by acute insulin treatment.

The effect of acute insulin treatment of hepatocytes on pyruvate carboxylation in both isolated mitochondria and cells rendered permeable by filipin was examined. Challenging the cells with insulin alone had no effect on either the basal rate of pyruvate carboxylation or gluconeogenesis, although it did suppress the responses to both glucagon and catecholamines. Insulin treatment was unable to antagonize the enhanced rate of pyruvate carboxylation caused by stimulation of the cells with either angiotensin or vasopressin. Neither insulin nor the gluconeogenic hormones altered the total extractable pyruvate carboxylase activity in the isolated mitochondria, suggesting that the effect of hormones at the level of the isolated intact organelle was mediated via alterations in the intramitochondrial concentrations of effector molecules, notably ATP and the [ATP]/[ADP] ratio and substrate availability. The alterations in pyruvate carboxylation correlate well with glucose synthesis in terms of sensitivity to effector molecules, putative second messengers and time of onset of the response, indicating that alterations in the flux through this enzyme are compatible with it being an important site in the control of gluconeogenesis from C3 precursors.     

Fatty acid effects on gluconeogenesis in goat, calf and guinea pig hepatocytes

1. Regulation of hepatic gluconeogenesis by fatty acid was studied in goat, calf and guinea pig hepatocytes. 2. Fatty acid effects on gluconeogenesis were dependent upon species; fatty acid and gluconeogenic substrate. 3. Oleate and octanoate inhibited gluconeogenesis from propionate in guinea pig hepatocytes and stimulated it in goat hepatocytes. 4. Oleate and octanoate markedly inhibited gluconeogenesis from lactate in guinea pig hepatocytes whereas octanoate, but not oleate, decreased glucose production from lactate in goat hepatocytes. 5. Effects of fatty acids on gluconeogenesis in calf hepatocytes were similar to goat hepatocytes suggesting control of gluconeogenesis is similar among ruminant species but differs from guinea pigs.     

Aminooxyacetate inhibits gluconeogenesis by isolated chicken hepatocytes

Although the pathway for glucose synthesis from lactate in avian liver is not thought to involve transamination steps, inhibitors of transamination (aminooxyacetate and L-2-amino-4-methoxy-trans-3-butenoic acid) block lactate gluconeogenesis by isolated chicken hepatocytes. Inhibition of glucose synthesis from lactate by aminooxyacetate is accompanied by a large increase in the lactate-to-pyruvate ratio. Oleate largely relieves inhibition by aminooxyacetate and lowers the lactate-to-pyruvate ratio. In parallel studies with rat hepatocytes, oleate did not overcome aminooxyacetate inhibition of glucose synthesis. The ratios of lactate used to glucose formed were greater than 2 with both rat and chicken hepatocytes, were increased by aminooxyacetate, and were restored toward 2 by oleate. Thus, in the absence of oleate, lactate is oxidized to provide the energy needed to meet the metabolic demand of chicken hepatocytes. Excess cytosolic reducing equivalents generated by the oxidation of lactate to pyruvate are transferred from the cytosol to the mitosol by the malate-aspartate shuttle. Aminooxyacetate inhibits the shuttle and, consequently, glucose synthesis for want of pyruvate.     

Regulation of hepatic gluconeogenesis in the guinea pig by fatty acids and ammonia.

Octanoate and L-palmitylcarnitine inhibited the synthesis of P-enolpyruvate from alpha-ketoglutarate and malate by isolated guinea pig liver mitochondria. A 50% reduction in P-enolpyruvate formation was obtained with 0.1 to 0.2 mM octanoate or with 0.06 to 0.10 mM L-palmitylcarnitine. At these concentrations, oxidative phosphorylation remained intact and only much higher concentrations of fatty acids altered this process. The addition of NH4Cl in the presence of malate and increasing concentrations of alpha-ketoglutarate (or vice versa) enhanced the formation of glutamate, aspartate, and P-enolpyruvate. The addition of increasing concentrations of NH4Cl in the presence of fixed amounts of malate and alpha-ketoglutarate had a similar effect. Furthermore, the inhibition of P-enolpyruvate synthesis by fatty acids and the reduction of the acetoacetate to beta-hydroxybutyrate ratio were reversed by the addition of NH4Cl. Cycloheximide, which blocks energy transfer at site 1 of the respiratory chain, decreased P-enolpyruvate formation. When cycloheximide and either octanoate or L-palmitylcarnitine were added together, there was an even greater reduction in P-enolpyruvate synthesis from either malate or alpha-ketoglutarate than was noted with either fatty acid alone. Since cycloheximide lowers the rate of ATP synthesis this may in turn reduce P-enolpyruvate formation by a mechanism independent of changes in the mitochondrial NAD+/NADH ratio caused by fatty acids. In the isolated perfused liver metabolizing lactate, the inhibitory effect of octanoate on gluconeogenesis was partially relieved by the addition of 1 mM NH4Cl, but remained unchanged in the presence of 2 mM NH4Cl, despite a highly oxidized NAD+/NADH ratio in the mitochondria. In contrast to glucose synthesis, urea formation was markedly increased during the infusion of 1 mM as well as 2 mM NH4Cl. After cessation of NH4Cl infusion, there was an increase in glucose production, to a rate as high as that observed in the absence of octanoate. This increase was accompanied by the disappearance of alanine, aspartate, and glutamate which had been stored in the liver during NH4Cl infusion. Urea synthesis also decreased progressively. These results indicate that gluconeogenesis in guinea pig liver is regulated, in part, by alterations in the mitochondrial oxidation-reduction state. However, the modulation of this effect by changing the concentrations of intermediates of the aspartate aminotransferase reaction indicates competition for oxalacetate between the aminotransferase reaction and P-enolpyruvate carboxykinase.