The rate of gluconeogenesis from various precursors in the perfused rat liver

1. The rates of gluconeogenesis from many precursors have been measured in the perfused rat liver and, for comparison, in rat liver slices. All livers were from rats starved for 48hr. Under optimum conditions the rates in perfused liver were three to five times those found under optimum conditions in slices. 2. Rapid gluconeogenesis (rates of above 0·5μmole/g./min.) were found with lactate, pyruvate, alanine, serine, proline, fructose, dihydroxyacetone, sorbitol, xylitol. Unexpectedly other amino acids, notably glutamate and aspartate, and the intermediates of the tricarboxylic acid cycle (with the exception of oxaloacetate), reacted very slowly and were not readily removed from the perfusion medium, presumably because of permeability barriers which prevent the passage of highly charged negative ions. Glutamine and asparagine formed glucose more readily than the corresponding amino acids. 3. Glucagon increased the rate of gluconeogenesis from lactate and pyruvate but not from any other precursor tested. This occurred when the liver was virtually completely depleted of glycogen. Two sites of action of glucagon must therefore be postulated: one concerned with mobilization of liver glycogen, the other with the promotion of gluconeogenesis. Sliced liver did not respond to glucagon. 4. Pyruvate and oxaloacetate formed substantial quantities of lactate on perfusion, which indicates that the reducing power provided in the cytoplasm was in excess of the needs of gluconeogenesis. 5. Values for the content of intermediary metabolites of gluconeogenesis in the perfused liver are reported. The values for most intermediates rose on addition of lactate. 6. The rates of gluconeogenesis from lactate and pyruvate were not affected by wide variations of the lactate/pyruvate ratio in the perfusion medium.     

The influence of ammonium and calcium lons on gluconeogenesis in isolated rat hepatocytes and their response to glucagon and epinephrine

The use of n-butylmalonate as an inhibitor of malate transport from mitochondria and of aminooxyacetate as an inhibitor of glutamate-aspartate transaminase indicated that rat liver hepatocytes employ the aspartate shuttle for gluconeogenesis from lactate which supplies reducing equivalents to the cytosolic NAD system. In contrast, malate is transported from mitochondria to cytosol for gluconeogenesis from pyruvate. This conclusion is corroborated by the finding that the addition of ammonium ions enhances gluconeogenesis from lactate but inhibits glucose formation from pyruvate. In hepatocytes, glucagon and epinephrine have relatively little effect on glucose synthesis from lactate. Ammonium ions permit both of these hormones to exert their usual stimulation of gluconeogenesis from lactate.Calcium ions (1.3 mm) enhance gluconeogenesis from lactate and from lactatepyruvate mixtures (10:1). The stimulatory effects of Ca²⁺ and NH₄⁺ are additive and, when lactate is the substrate, the rates of gluconeogenesis achieved are so high as to preclude further stimulation by glucagon.     

Metabolism of propionate by sheep liver: Pathway of propionate metabolism in aged homogenate and mitochondria

Experiments were conducted with aged nuclear-free homogenate of sheep liver and aged mitochondria in an attempt to measure both the extent of oxidation of propionate and the distribution of label from [2-¹⁴C]propionate in the products. With nuclear-free homogenate, propionate was 44% oxidized with the accumulation of succinate, fumarate, malate and some citrate. Recovery of ¹⁴C in these intermediates and respiratory carbon dioxide was only 33%, but additional label was detected in endogenous glutamate and aspartate. With washed mitochondria 30% oxidation of metabolized propionate occurred, and proportionately more citrate and malate accumulated. Recovery of ¹⁴C in dicarboxylic acids, citrate, α-oxoglutarate, glutamate, aspartate and respiratory carbon dioxide was 91%. The specific activities of the products and the distribution of label in the carbon atoms of the dicarboxylic acids were consistent with the operation solely of the methylmalonate pathway together with limited oxidation of the succinate formed by the tricarboxylic acid cycle via pyruvate. In a final experiment with mitochondria the label consumed from [2-¹⁴C]propionate was entirely recovered in the intermediates of the tricarboxylic acid cycle, glutamate, aspartate, methylmalonate and respiratory carbon dioxide.     

The effect of propionate on the metabolism of pyruvate and lactate in the perfused rat liver

1. Rates of gluconeogenesis in the perfused rat liver from propionate, l-lactate, pyruvate and the combination of propionate with either lactate or pyruvate were measured. Less than additive rates were obtained with either propionate plus lactate or propionate plus pyruvate. 2. The uptake of pyruvate plus lactate from the perfusion medium was decreased more seriously when propionate was present with lactate than with pyruvate. 3. The use of [2-(14)C]pyruvate in the presence of propionate showed that the decreased disappearance of pyruvate plus lactate did not result in their formation from propionate. 4. The addition of sodium butyrate to the perfusion medium caused an inhibition of gluconeogenesis from propionate and stimulated gluconeogenesis and uptake of pyruvate and lactate. 5. The observations are consistent with there being a sparing effect of propionate on lactate and pyruvate metabolism.     

The mechanism of Ca2(+)-related control of gluconeogenesis in perfused liver.

A kinetic expression for rat-liver mitochondrial aspartate formation in situ was developed in order to determine whether hormonally induced decreases in 2-oxoglutarate levels can regulate hepatic gluconeogenesis from lactate via control of aspartate formation. Previous studies from this laboratory showed that 2-oxoglutarate can inhibit aspartate production by isolated mitochondria. These present studies were designed to probe the physiological significance of the decrease in 2-oxoglutarate levels observed when Ca2(+)-mobilizing gluconeogenic hormones are administered to isolate perfused rat livers. First, estimates were made of the kinetic constants which determine the rate of aspartate formation in isolated mitochondria. The concentrations of the substrates and products of this process were then measured in perfused livers. From these values, it was possible to estimate aspartate efflux from mitochondria in situ. The calculated rates of aspartate production were increased by decreases in 2-oxoglutarate levels which occurred when glucagon or phenylephrine was added to the perfused livers. Glucagon also effected an inhibition of pyruvate kinase, evidenced by the fact that the calculated rate of aspartate efflux equalled the rate of gluconeogenesis (the difference between the two is equivalent to the pyruvate-kinase flux). By contrast, in control livers and with phenylephrine stimulation, aspartate formation was higher than gluconeogenesis suggesting significant pyruvate-kinase flux in this condition. The calculations also show a correlating increase in flux through pyruvate carboxylase (30% with phenylephrine, 15% with glucagon, compared with approximately 50% increases in gluconeogenic flux). The mechanism of this increase is discussed.     

Studies on the relationship between glycolysis, lipogenesis, gluconeogenesis, and pyruvate kinase activity of rat and chicken hepatocytes.

Chicken hepatocytes synthesize glucose and fatty acids at rates which are faster than rat hepatocytes. The former also consume exogenous lactate and pyruvate at a much faster rate and, in contrast to rat hepatocytes, do not accumulate large quantities of lactate and pyruvate by aerobic glycolysis. α-Cyano-4-hydroxycinnamate, an inhibitor of pyruvate transport, causes lactate and pyruvate accumulation by chicken hepatocytes. Glucagon and N⁶,O²′-dibutyryl adenosine 3′,5′-monophosphate (dibutyryl cyclic AMP) convert pyruvate kinase (EC 2.7.1.40) of rat hepatocytes to a less active form. This effect explains, in part, inhibition of glycolysis, inhibition of lipogenesis, stimulation of gluconeogenesis, and inhibition of the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment by these compounds. In contrast, pyruvate kinase of chicken hepatocytes is refractory to inhibition by glucagon or dibutyryl cyclic AMP. Rat liver is known to have predominantly the type L isozyme of pyruvate kinase and chicken liver predominantly the type K. Thus, only the type L isozyme appears subject to interconversion between active and inactive forms by a cyclic AMP-dependent, phosphorylation-dephos-phorylation mechanism. This explains why the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasmic compartment of chicken hepatocytes is insensitive to cyclic AMP. However, glucagon and dibutyryl cyclic AMP inhibit net glucose utilization, inhibit fatty acid synthesis, inhibit lactate and pyruvate accumulation in the presence of α-cyano-4-hydroxycinnamate, and stimulate gluconeogenesis from lactate and dihydroxyacetone by chicken hepatocytes. Thus, a site of action of cyclic AMP distinct from pyruvate kinase must exist in the glycolytic-gluconeogenic pathway of chicken liver.     

Accumulation and disposal of tricarboxylic acid cycle intermediates during propionate oxidation in the isolated perfused rat heart

The role of the metabolite disposal mechanisms in the regulation of the tricarboxylic acid cycle pool size was studied in isolated perfused rat hearts oxidizing 2 mM propionate. Malate and succinate accumulated during the propionate metabolism. A further 118% increase in the malate concentration and 600% increase in the succinate concentration and a slight inhibition of the propionate uptake were observed during a subsequent KCl-induced arrest of the heart metabolizing propionate. When the mechanical activity of the heart was restored, the malate and succinate concentrations returned to the same levels as before the arrest of the heart, but the propionate uptake did not rise significantly. The mean disposal rates of the tricarboxylic acid cycle metabolites during the cardiac arrest and subsequent restoration of the activity were 1.4 and 2.4 μmol/min per g dry weight, respectively. During cardiac arrest the malate carbon disposed was almost totally recovered as C₃ compounds, whereas after the increase in the ATP-consumption most of it was oxidized. The results show that propionate is oxidized by heart muscle at an appreciable rate but the disposal rate of the tricarboxylic acid cycle intermediates is not tightly regulated by the cellular energy state. Although the metabolite pool size of the tricarboxylic acid cycle responds to change in the ATP consumption, the energy state appears to have a greater effect on the fate of the C₃ compounds formed than on the actual rate of C₄ compound disposition.     

Phenethylbiguanide and the inhibition of hepatic gluconeogenesis in the guinea pig

1. Phenethylbiguanide inhibits the synthesis of phosphoenolpyruvate from malate or 2-oxoglutarate by isolated guinea-pig liver mitochondria. This inhibition is time- and concentration-dependent, with the maximum decrease in the rate of phosphoenolpyruvate synthesis (80%) evident after 10min of incubation with 1mm-phenethylbiguanide. 2. The phosphorylation of ADP by these mitochondria is also inhibited at increasing concentrations of phenethylbiguanide and there is a progressive increase in AMP formation. Guinea-pig liver mitochondria are more sensitive to this inhibition in oxidative phosphorylation caused by phenethylbiguanide than are rat liver mitochondria. 3. Simultaneous measurements of O(2) consumption and ADP phosphorylation with guinea-pig liver mitochondria oxidizing malate plus glutamate in State 3 indicated that phenethylbiguanide at low concentrations (0.1mm) inhibits respiration at Site 1. At higher phenethylbiguanide concentrations Site 2 is also inhibited. 4. Gluconeogenesis from lactate, pyruvate, alanine and glycerol by isolated perfused guinea-pig liver is inhibited to various degrees by phenethylbiguanide. Alanine is the most sensitive to inhibition (60% inhibition of the maximum rate by 0.1mm-phenethylbiguanide), whereas glycerol is relatively insensitive (25% inhibition at 4mm). 5. Gluconeogenesis from lactate and pyruvate by perfused rat liver was also inhibited by phenethylbiguanide, but only at high concentrations (8mm). Unlike guinea-pig liver, the inhibitory effect of phenethylbiguanide on rat liver was reversible after the termination of phenethylbiguanide infusion. 6. The time-course of inhibition of gluconeogenesis from the various substrates used in this study indicated a time-dependency which was related in part to the concentration of infused phenethylbiguanidine. This time-course closely paralleled that noted for the inhibition by phenethylbiguanide of phosphoenolpyruvate synthesis in isolated guinea-pig liver mitochondria.     

The redistribution of carbon label by the reactions involved in glycolysis, gluconeogenesis and the tricarboxylic acid cycle in rat liver

A scheme is presented that shows how the reactions involved in gluconeogenesis, glycolysis and the tricarboxylic acid cycle are linked in rat liver. Equations are developed that show how label is redistributed in aspartate, glutamate and phosphopyruvate when it is introduced as specifically labelled pyruvate or glucose either at a constant rate (steady-state theory) or at a variable rate (non-steady-state theory). For steady-state theory the fractions of label introduced as specifically labelled pyruvate that are incorporated into glucose and carbon dioxide are also given, and for both theories the specific radioactivities of aspartate and glutamate relative to the specific radioactivity of the substrate. The theories allow for entry of label into the tricarboxylic acid cycle via both oxaloacetate and acetyl-CoA, for (14)CO(2) fixation and for loss of label from the tricarboxylic acid cycle in glutamate, but not for losses in citrate. They also allow for incomplete symmetrization of label in oxaloacetate due to incomplete equilibration with fumarate both in the extramitochondrial part of the cell and in the mitochondrion on entry of oxaloacetate into the tricarboxylic acid cycle. In the latter case failure both of oxaloacetate to equilibrate with malate and of malate to equilibrate with fumarate are considered.     

Effect of glucagon on metabolite compartmentation in isolated rat liver cells during gluconeogenesis from lactate

1. The subcellular distribution of adenine nucleotides, acetyl-CoA, CoA, glutamate, 2-oxoglutarate, malate, oxaloacetate, pyruvate, phosphoenolpyruvate, 3-phosphoglycerate, glucose 6-phosphate, aspartate and citrate was studied in isolated hepatocytes in the absence and presence of glucagon by using a modified digitonin procedure for cell fractionation. 2. In the absence of glucagon, the cytosol contains about two-thirds of cellular ATP, some 40-50% of ADP, acetyl-CoA, citrate and phosphoenolpyruvate, more than 75% of total 2-oxoglutarate, glutamate, malate, oxaloacetate, pyruvate, 3-phosphoglycerate and aspartate, and all of glucose 6-phosphate. 3. In the presence of glucagon the cytosolic space shows an increase in the content of malate, phosphoenolpyruvate and 3-phosphoglycerate by more than 60%, and those of aspartate and glucose 6-phosphate rise by about 25%. Other metabolites remain unchanged. After glucagon treatment, cytosolic pyruvate is decreased by 37%, whereas glutamate and 2-oxoglutarate decrease by 70%. The [NAD(+)]/[NADH] ratios calculated from the cytosolic concentrations of the reactants of lactate dehydrogenase and malate dehydrogenase were the same. Glucagon shifts this ratio and also that of the [NADP(+)]/[NADPH] couple towards a more reduced state. 4. In the mitochondrial space glucagon causes an increase in the acetyl-CoA and ATP contents by 25%, and an increase in [phosphoenolpyruvate] by 50%. Other metabolites are not changed by glucagon. Oxaloacetate in the matrix is only slightly decreased after glucagon, yet glutamate and 2-oxoglutarate fall to about 25% of the respective control values. The [NAD(+)]/[NADH] ratios as calculated from the [3-hydroxybutyrate]/[acetoacetate] ratio and from the matrix [malate]/[oxaloacetate] couple are lowered by glucagon, yet in the latter case the values are about tenfold higher than in the former. 5. Glucagon and oleate stimulate gluconeogenesis from lactate to nearly the same extent. Oleate, however, does not produce the changes in cellular 2-oxoglutarate and glutamate as observed with glucagon. 6. The changes of the subcellular metabolite distribution after glucagon are compatible with the proposal that the stimulation of gluconeogenesis results from as yet unknown action(s) of the hormone at the mitochondrial level in concert with its established effects on proteolysis and lipolysis.     

Formation and dissimilation of oxalacetate and pyruvate Pseudomonas citronellolis grown on noncarbohydrate substrates.

Metabolism of lactate as a carbon source by Pseudomonas citronellolis occurred via a nicotinamide adenine dinucleotide (NAD)-independent L-lactate dehydrogenase, which was present in cells grown on DL-lactate but was not present in cells grown on acetate, aspartate, citrate, glucose, glutamate, or malate. The cells also possessed a constitutive, NAD-independent malate dehydrogenase instead of the conventional NAD-dependent malate dehydrogenase instead of the conventional NAD-dependent enzyme in the tricarboxylic acid cycle. Both enzymes were particulate and used dichlorophenolindo-phenol or oxygen as an electron acceptor. In acetate-grown cells, the activity of pyruvate dehydrogenase and NAD phosphate-linked malate enzyme decreased, cells grown on glucose or lactate. This was consistent with the need to maintain a supply of oxalacetate for metabolism of acetate via the tricarboxylic acid cycle. Changes in enzyme activities suggest that gluconeogenesis from noncarbohydrate carbon sources occurs via the malate enzyme (when oxalacetate decarboxylase is inhibited) or a combination of the NAD-independent malate dehydrogenase and oxalacetate decarboxylase.     

Effects of added adenine nucleotides on renal carbohydrate metabolism

1. The regulatory effects that adenine nucleotides are known to exert on enzymes of glycolysis and gluconeogenesis were demonstrated to operate in kidney-cortex slices and in the isolated perfused rat kidney by the addition of exogenous ATP, ADP and AMP to the incubation or perfusion media. 2. Both preparations rapidly converted added ATP into ADP and AMP, and ADP into AMP; added AMP was rapidly dephosphorylated. AMP formed from ATP was dephosphorylated at a lower rate than was added AMP, especially when the initial ATP concentration was high (10mm). Deamination of added AMP occurred more slowly than dephosphorylation of AMP. 3. Gluconeogenesis from lactate or propionate by rat kidney-cortex slices, and from lactate by the isolated perfused rat kidney, was inhibited by the addition of adenine nucleotides to the incubation or perfusion media. In contrast, oxygen consumption and the utilization of propionate or lactate by slices were not significantly affected by added ATP or AMP. 4. The extent and rapidity of onset of the inhibition of renal gluconeogenesis were proportional to the AMP concentration in the medium and the tissue, and were not due to the production of acid or P(i) or the formation of complexes with Mg(2+) ions. 5. Glucose uptake by kidney-cortex slices was stimulated 30-50% by added ATP, but the extra glucose removed was not oxidized to carbon dioxide and did not all appear as lactate. Glucose uptake, but not lactate production, by the isolated perfused kidney was also stimulated by the addition of ATP or AMP. 6. In the presence of either glucose or lactate, ATP and AMP greatly increased the concentrations of C(3) phosphorylated intermediates and fructose 1,6-diphosphate in the kidney. There was a simultaneous rise in the concentration of malate and fall in the concentration of alpha-oxoglutarate. 7. The effects of added adenine nucleotides on renal carbohydrate metabolism seem to be mainly due to an increased concentration of intracellular AMP, which inhibits fructose diphosphatase and deinhibits phosphofructokinase. This conclusion is supported by the accumulation of intermediates of the glycolytic pathway between fructose diphosphate and pyruvate. 8. ATP or ADP (10mm) added to the medium perfusing an isolated rat kidney temporarily increased the renal vascular resistance, greatly diminishing the flow rate of perfusion medium for a period of several minutes.     

Gluconeogenesis in isolated intact lamb liver cells. Effects of glucagon and butyrate.

1. Isolated lamb liver cells were prepared from 24-h-starved animals by venous perfusion of the excised caudate lobe with buffer containing collagenase. On the basis of Trypan-Blue exclusion, rate of O2 uptake, adenine nucleotide content and retention of constitutive enzymes, these cells were judged to be intact. 2. Isolated caudate-lobe liver cells showed rates of gluconeogenesis from 10 mM-propionate and 10 mM-lactate that compared favourably with rates determined in isolated median-lobe cells and with rates determined with the isolated perfused lamb liver. 3. The gluconeogenic potential of substrates tested depended on the lamb's age. Cells prepared from suckling lambs (up to 20 days of age and essentially non-ruminant) showed highest rates from galactose, serine and alanine; those prepared from post-weaned lambs (older than 30 days of age and ruminant) showed highest rates from propionate, lactate and fructose. 4. Gluconeogenic rates from endogeneous precursors, 10 mM-propionate and 10mM-galactose, were linear for 1 h and were both stimulated by 1 muM-glucagon. Provided the endogenous rate of gluconeogenesis remained unchanged after substrate addition, glucagon caused a net stimulation of gluconeogenesis from each of these substrates. 5. Gluconeogenic capacity and glucagon sensitivity were examined in cells maintained in substrate-free oxygenated buffer at 37 degrees, 22 degrees and * degrees C. Even under the best of the three conditions of storage that were tested (i.e. at 22 degrees C in gelatin-containing buffer) deterioration of the lamb cells proceeded rapidly, and loss of glucagon responsiveness preceeded the loss of ability to convert precursor into glucose. 6. n-Butyric acid, 2-methylpropanoic acid and 3-methylbutanoic acid at concentrations comparable with those found in lamb portal-vein blood each stimulated gluconeogenesis from 10mM-galactose or 10mM-propionate; gluconeogenesis from galactose was stimulated to the greater extent. 7. The regulatory effects of glucagon and sodium butyrate on lamb liver-cell gluconeogenesis and glycogenolysis were compared. Glucagon (1 muM) and 2mM-butyrate accelerated the rate of glucose formation of liver cells of 24h-starved animals from lactate+pyruvate or fructose. Insulin (20nM) decreased both gluconeogenesis and the efficacy of 1 muM-glucagon. For lactate+pyruvate as substrate, the stimulatory effect of butyrate was additive to that of 1muM-glucagon and for both lactate+pyruvate and fructose the stimulatory effect of butyrate was not influenced by 20nM-insulin. In contrast with glucagon, which stimulated the rate of glycogenolysis in cells prepared from fed lambs, butyrate (0.1-20mM) had no effect. 8. It is concluded that glucagon and butyrate stimulate lamb liver-cell gluconeogenesis by different mechanisms.     

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.     

Interrelations between ureogenesis and gluconeogenesis in isolated hepatocytes. The role of antion transport and the competition for energy.

1. Glucose synthesis from lactate plus pyruvate and from lactate plus alanine was measured in the presence or absence of 1mM-oleate or 2mM-octanoate at low (2mM) or high (8mM) concentrations of NH4Cl. 2. Both fatty acids alone or with 2mM-NH4Cl doubled glucose production from lactate plus pyruvate. Glucose synthesis from lactate plus alanine, in the presence of oleate, was decreased 16% by 2mM-NH4Cl. 3. In the presence of fatty acids, 8mM-NH4Cl decreased gluconeogenesis by 60-65% from both lactate plus pyruvate and lactate plus alanine. This inhibition was correlated with a high accumulation of aspartate and a drastic decrease in 2-oxoglutarate and malate in the cells. 4. In the presence of 2mM- or 8 mM-NH4Cl, oleate and glucogenic precursors, the addition of 2.5mM-ornithine stimulated urea synthesis. 5. This was paralleled by a decrease of 16% in glucose synthesis from lactate plus pyruvate in the presence of 2mM-NH4Cl and had no effect at 8mM-NH4Cl. In the system producing glucose from lactate plus alanine, ornithine completely reversed the inhibition caused by 2mM-NH4Cl and only partly that by 8mM-NH4Cl. 6. Gluconeogenesis from pyruvate was also inhibited by 2mM-NH4Cl in the presence of oleate or ethanol. This way due to the decrease of malate, which is the C4 precursor of glucose in this system. 7. The limitation of gluconeogenesis by 2-oxoglutarate and malate concentrations in the liver cell and the competition for energy between glucose and urea synthesis is discussed.     

Hormonal control of [14C]glucose synthesis from [U-14C]dihydroxyacetone and glycerol in isolated rat hepatocytes.

The hormonal control of [14C]glucose synthesis from [U-14C-A1dihydroxyacetone was studied in hepatocytes from fed and starved rats. In cells from fed rats, glucagon lowered the concentration of substrate giving half-half-maximal rates of incorporation while it had little or no effect on the maximal rate. Inhibitors of gluconeogenesis from pyruvate had no effect on the ability of the hormone to stimulate the synthesis of [14C]glucose from dihydroxyacetone. The concentrations of glucagon and epinephrine giving half-maximal stimulation from dihydroxacetone were 0.3 to 0.4 mM and 0.3 to 0.5 muM, respectively. The meaximal catecholamine stimulation was much less than the maximal stimulation by glucagon and was mediated largely by the alpha receptor. Insulin had no effect on the basal rate of [14C]clucose synthesis but inhibited the effect of submaximal concentration of glucagon or of any concentration of catecholamine. Glucagon had no effect on the uptake of dihydroxyacetone but suppressed its conversion to lactate and pyruvate. This suppression accounted for most of the increase in glucose synthesis. In cells from gasted rats, where lactate production is greatly reduced and the rate of glucose synthesis is elevated, glucagon did not stimulate gluconeogenesis from dihydroxyacetone. Findings with glycerol as substrate were similar to those with dihyroxyacetone. Ethanol also stimulated glucose production from dihydroxyacetone while reducing proportionately the production of lactate. Ethanol is known to generate reducing equivalents fro clyceraldehyde-3-phosphate dehydrogenase and presumably thereby inhibits carbon flux to lactate at this site. Its effect was additive with that of glucagon. Estimates of the steady state levels of intermediary metabolites and flux rates suggested that glucagon activated conversion of fructose diphosphate to fructose 6-phosphate and suppressed conversion of phosphoenolpyruvate to pyruvate. More direct evidence for an inhibition of pyruvate kinase was the observation that brief exposure of cells to glucagon caused up to 70% inhibition of the enzyme activity in homogenates of these cells. The inhibition was not seen when the enzyme was assayed with 20 muM fructose diphosphate. The effect of glucagon to lower fructose diphosphate levels in intact cells may promote the inhibition of pyruvate kinase. The inhibition of pyruvate kinase may reduce recycling in the pathway of gluconeogenesis from major physiological substrates and probably accounts fromsome but not all the stimulatory effect of glucagon.     

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

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

Gluconeogenesis in the kidney cortex. Effects of d-malate and amino-oxyacetate

1. Rat kidney-cortex slices incubated with d-malate alone formed very little glucose. d-Malate, however, augmented gluconeogenesis from l-lactate and inhibited gluconeogenesis from pyruvate and l-malate. 2. d-Malate had little effect on the rate of the tricarboxylic acid cycle with or without other substrates added. 3. d-Malate inhibited the activity of the l-malate dehydrogenase in a high-speed-supernatant fraction from kidney cortex. 4. It was concluded that d-malate inhibited either the operation of the cytoplasmic l-malate dehydrogenase or malate outflow from the mitochondria in the intact kidney-cortex cell. This supports the hypothesis of Lardy, Paetkau & Walter (1965) and Krebs, Gascoyne & Notton (1967) on the role of malate as carrier for carbon and reducing equivalents in gluconeogenesis. 5. Gluconeogenesis from l-lactate in kidney-cortex slices was strongly inhibited by a low concentration (0.1mm) of amino-oxyacetate, whereas glucose formation from pyruvate, malate, aspartate and several other compounds was only slightly affected. 6. High concentrations of l-aspartate largely reversed the inhibition of gluconeogenesis from l-lactate caused by amino-oxyacetate. 7. Amino-oxyacetate inhibited strongly the glutamate-oxaloacetate transaminase in the 30000g supernatant fraction of a kidney-cortex homogenate. The presence of l-aspartate decreased the inhibition of the transaminase by amino-oxyacetate. 8. Detritiation of l-[2-(3)H]aspartate was inhibited by 90% during an incubation of kidney-cortex slices with l-lactate and amino-oxyacetate. 9. Low concentrations (10mum) of artificial electron acceptors such as Methylene Blue and phenazine methosulphate abolished most of the inhibition of gluconeogenesis from l-lactate by amino-oxyacetate. This is interpreted as an activation of net malate outflow from the mitochondria by-passing the inhibited transfer of oxaloacetate. 10. These findings support the concept that transamination to aspartate is involved in the transfer of oxaloacetate from mitochondria to cytosol required in gluconeogenesis from l-lactate.     

Mechanism of 3-mercaptopicolinic acid inhibition of hepatic phosphoenolpyruvate carboxykinase (GTP).

The hypoglycemic agent 3-mercaptopicolinic acid inhibits gluconeogenesis from lactate by isolated, perfused livers from fasted rats and guinea pigs. A 3-mercaptopicolinate concentration of 50 muM caused a sharp decrease in glucose synthesis, with virtually complete inhibition at 100 muM. This inhibitory effect was reversed completely when 3-mercaptopicolinate was removed and the rate of glucose synthesis returned to normal values within 2 min. Oxygen consumption was not altered, even at the highest concentration of inhibitor. Gluconeogenesis from glycerol by guinea pig liver was blocked completely by 100 muM 3-mercaptopicolinate but was inhibited only partially in rat liver. After removal of the inhibitor glucose synthesis returned to levels higher than noted before the addition of this compound. The formation of P-enolpyruvate bu isolated guinea pig liver mitochondria metabolizing alpha-ketoglutarate (State 3) was inhibited markedly by 3-mercaptopicolinate, but malate conversion to P-enolpyruvate was considerably less sensitive. Kinetic studies with purified P-enolpyruvate carboxykinase from rat liver cytosol indicate that 3-mercaptopicolinate is a noncompetitive inhibitor with respect to both oxalacetate and MnGTP2-, and that simulataeous saturation with both substrates does not diminish this inhibition. The inhibitory effects of 3-mercaptopicolinate occur primarily by decreasing the rate of product formation while having relatively minor effects on the apparent Michaelis constants for substrates. Inhibition constants for slope and intercept effects ranged from 3 to 9 muM 3-mercaptopicolinate, and the inhibition patterns were dependent on the concentration of free Mn2+ present. Comparison of the inhibition constants with the observed inhibition of gluconeogenesis in livers perfused with 3-mercaptopicolinate supports the contention that P-enolpyruvate carboxykinase is the site of action of this inhibitor. The possibility that 3-mercaptopicolinate inhibition occurs by binding either free or bound manganese was eliminated by determination of the dissociation constant of 0.51 mM for the manganese-3-mercaptopicolinate complex. In addition, no tightly bound, slowly exchanging metal was bound to purified enzyme protein. These results suggest that 3-mercaptopicolinate inhibits by the removal of a tightly bound, rapidly exchanging metal ion other than Mn2+.     

Differential effect of glucagon on gluconeogenesis in periportal and pericentral regions of the liver lobule.

The effect of glucagon on gluconeogenesis was measured in periportal and pericentral regions of the liver lobule by monitoring changes in rates of O2 uptake on the surface of the perfused liver with miniature O2 electrodes after infusion of lactate. When lactate (2 mM) was infused into livers from starved rats perfused in the anterograde direction, O2 uptake was increased 2.5-fold more in periportal than in pericentral regions, reflecting increased energy demands for glucose synthesis. Under these conditions, glucagon infusion in the presence of lactate increased O2 uptake exclusively in periportal regions of the liver lobule. Thus, when perfusion is in the physiological anterograde direction, the metabolic actions of glucagon predominate in periportal regions of the liver lobule under gluconeogenic conditions in the starved state. When livers were perfused in the retrograde direction, however, glucagon stimulated O2 uptake exclusively in pericentral regions. Thus glucagon only stimulates gluconeogenesis in 'upstream' regions of the liver lobule irrespective of the direction of flow.