A reexamination of the role of the cytosolic alanine aminotransferase in hepatic gluconeogenesis

The relative importance of the mitochondrial and cytosolic alanine aminotransferase isozymes for providing pyruvate from alanine for further metabolism in the mitochondrial compartment was examined in the isolated perfused rat liver. The experimental rationale employed depends upon the supposition that gluconeogenesis from alanine and the decarboxylation of infused [1-14C]alanine should be diminished by pyruvate transport inhibitors (e.g., alpha-cyanocinnamate) in proportion to the contribution of the cytosolic alanine aminotransferase for generating pyruvate. alpha-Cyanocinnamate inhibited the endogenous rate of glucose production in perfused livers derived from 24-h-fasted rats. The rate of [1-14C]alanine decarboxylation at low (1 mM) and high (10 mM) perfusate alanine concentrations was inhibited by 9.5 and 42%, respectively, in the presence of alpha-cyanocinnamate. In livers from fasted animals perfused with either 1 or 10 mM alanine, alpha-cyanocinnamate caused a substantial increase in the rates of both lactate and pyruvate production. Elevating the hepatic ketogenic rate during infusion of acetate in livers, perfused with alanine, stimulated both the rates of alanine decarboxylation and glucose production; the extent of stimulation of these two metabolic parameters was determined to be a function of the alanine concentration in the perfusate. The stimulation of the rate of alanine decarboxylation during acetate-induced ketogenesis was reversed by co-infusion of alpha-cyanocinnamate with simultaneous increases in the rates of lactate and pyruvate production. The results indicate that during rapid ketogenesis, cytosolic transamination of alanine contributes at least 19% (at 1 mM alanine) and 55% (at 10 mM alanine) of the pyruvate for gluconeogenesis.     

Role of fructose 2,6-bisphosphate in the control by glucagon of gluconeogenesis from various precursors in isolated rat hepatocytes.

Hepatocytes from overnight-starved rats were incubated with 1-20 mM-fructose, -dihydroxyacetone, -glycerol, -alanine or -lactate and -pyruvate with or without 0.1 microM-glucagon. The production of glucose and lactate was measured, as was the content of fructose 2,6-bisphosphate. The concentrations of fructose (below 5 mM) and dihydroxyacetone (above 1 mM) that gave rise to an increase in fructose 2,6-bisphosphate were those at which a glucagon effect on the production of glucose and lactate could be observed. Glycerol had no effect on fructose 2,6-bisphosphate content or on production of lactate, and glucagon did not stimulate the production of glucose from this precursor. With alanine or lactate/pyruvate as substrates, glucagon stimulated glucose production whether the concentration of fructose 2,6-bisphosphate was increased or not. The extent of inactivation of pyruvate kinase by glucagon was not affected by the presence of the various gluconeogenic precursors. The role of fructose 2,6-bisphosphate in the effect of glucagon on gluconeogenesis from precursors entering the pathway at the level of triose phosphates or pyruvate is discussed.     

The regulation of hepatic gluconeogenesis using alpha-ketoisovalerate and propionate as precursors

The regulation of gluconeogenesis from alpha-ketoisovalerate and propionate was investigated in perfused livers from fasted rats. With alpha-ketoisovalerate as the gluconeogenic precursor, infusion of beta-hydroxybutyrate and acetate stimulated the rate of alpha-ketoisovalerate decarboxylation, but inhibited the rate of glucose production. Oleate, on the other hand, inhibited both alpha-ketoisovalerate decarboxylation and glucose production. When propionate was the primary gluconeogenic substrate, oleate, beta-hydroxybutyrate, and acetate infusion did not significantly alter hepatic glucose production. The present studies suggest that gluconeogenesis from alpha-ketoisovalerate is regulated at the level of various dehydrogenases prior to formation of propionyl-CoA, but subsequent to the branched-chain alpha-keto acid dehydrogenase reaction.     

alpha-Ketobutyrate metabolism in perfused rat liver: regulation of alpha-ketobutyrate decarboxylation and effects of alpha-ketobutyrate on pyruvate dehydrogenase

The oxidative decarboxylation and subsequent production of glucose from alpha-ketobutyrate were studied using perfused livers from fasted rats. The production of 14CO2 from alpha-keto-[1-14C]butyrate increased monotonically while the production of glucose from alpha-ketobutyrate was biphasic as the perfusate concentration of alpha-ketobutyrate was increased. The biphasic gluconeogenic response using alpha-ketobutyrate as the gluconeogenic precursor was similar to that observed with propionate. The decarboxylation of alpha-ketobutyrate was found to be exquisitely sensitive to the effects of the monocarboxylate transport inhibitor, alpha-cyanocinnamate. Infusion of beta-hydroxybutyrate caused a substantial inhibition of alpha-ketobutyrate decarboxylation while dichloroacetate, a pyruvate dehydrogenase kinase inhibitor, did not stimulate the metabolism of alpha-ketobutyrate but was inhibitory. The effects of alpha-ketobutyrate infusion on pyruvate decarboxylation were tested and it was found that at low perfusate pyruvate concentrations (ca. 0.25 mM) increasing alpha-ketobutyrate led to increasing inhibition of pyruvate decarboxylation, while at high perfusate pyruvate concentrations (ca. 2.5 mM) an initial inhibition was apparent which did not increase substantially with increasing alpha-ketobutyrate concentrations. The results obtained indicate that the regulation of alpha-ketobutyrate metabolism by oxidative decarboxylation differs significantly from that of pyruvate. In addition, while the rate of gluconeogenesis using alpha-ketobutyrate as a precursor was remarkably similar to that using propionate as a gluconeogenic precursor, the effects of alpha-ketobutyrate on the oxidative decarboxylation of pyruvate were qualitatively different from the effects of propionate on pyruvate metabolism.     

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.     

Interaction of oxamate with the gluconeogenic pathway in rat liver

Oxamate, a structural analog of pyruvate, known as a potent inhibitor of lactic dehydrogenase, lactic dehydrogenase, produces an inhibition of gluconeogenic flux in isolated perfused rat liver or hepatocyte suspensions from low concentrations of pyruvate (less than 0.5 mM) or substrates yielding pyruvate. The following observations indicate that oxamate inhibits flux through pyruvate carboxylase: accumulation of substrates and decreased concentration of all metabolic intermediates beyond pyruvate; decreased levels of aspartate, glutamate, and alanine; and enhanced ketone body production, which is a sensitive indicator of decreased mitochondrial free oxaloacetate levels. The decreased pyruvate carboxylase flux does not seem to be the result of a direct inhibitory action of oxamate on this enzyme but is secondary to a decreased rate of pyruvate entry into the mitochondria. This assumption is based on the following observations: Above 0.4 mM pyruvate, no significant inhibitory effect of oxamate on gluconeogenesis was observed. The competitive nature of oxamate inhibition is in conflict with its effect on isolated pyruvate carboxylase which is noncompetitive for pyruvate. Fatty acid oxidation was effective in stimulating gluconeogenesis in the presence of oxamate only at concentrations of pyruvate above 0.4 mM. Since only at low pyruvate concentrations its entry into the mitochondria occurs via the monocarboxylate translocator, from these observations it follows that pyruvate transport across the mitochondrial membrane, and not its carboxylation, is the first nonequilibrium step in the gluconeogenic pathway. In the presence of oxamate, fatty acid oxidation inhibited gluconeogenesis from lactate, alanine, and low pyruvate concentrations (less than 0.5 mM), and the rate of transfer of reducing equivalents to the cytosol was significantly decreased. Whether fatty acids stimulate or inhibit gluconeogenesis appears to correlate with the rate of flux through pyruvate carboxylase which ultimately seems to rely on pyruvate availability. Unless adequate rates of oxaloacetate formation are maintained, the shift of the mitochondrial NAD couple to a more reduced state during fatty acid oxidation seems to decrease mitochondrial oxaloacetate resulting in a decreased rate of transfer of carbon and reducing power to the cytosol.     

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.     

Characterization of gluconeogenesis in hepatocytes isolated from the American eel,Anguilla rostrata LeSueur,

Summary Isolated hepatocyte preparations from fed immature American eels,Anguilla rostrata Le Sueur, were used to study gluconeogenic, lipogenic, glycogenic and oxidative rates of radioactively labelled lactate, glycerol, alanine and aspartate. Eel hepatocytes maintain membrane integrity and energy charge during a 2 h incubation period and are considered a viable preparation for studying fish liver metabolism.Incubating eel hepatocytes with 10 mM substrates, the following results were obtained: glycerol, alanine and lactate, in that order, were effective gluconeogenic substrates; these three substrates reduced glucose release from glycogen stores, while aspartate had no such effect; lactate, alanine and aspartate led to high rates of glycerol production, with subsequent incorporation into lipid; incorporation into glycogen was low from all substrates; and, alanine oxidation was seven times higher than that observed with other substrates.When eel hepatocytes were incubated with low or physiological substrate concentrations gluconeogenic rates from lactate were twice those from alanine; rates from aspartate were very low. Glucagon stimulated lactate gluconeogenesis, but not amino acid gluconeogenesis, and had no significant effect on glycogenolysis. Cortisol increased gluconeogenic rates from 1 mM lactate.Thus, in the presence of adequate substrate, eel liver gluconeogenesis is preferentially stimulated relative to glycogenolysis to produce plasma glucose. These data support three important roles for gluconeogenesis: the recycling of muscle lactate, the synthesis of glucose from dietary amino acids to supplement glucose levels, and the production of glycerol for lipogenesis.This work was supported from operating grants to TWM from the National Research Council of Canada (A6944)     

Gluconeogenic and lipogenic properties of isolated kidney tubules from the domestic fowl (Gallus domesticus)

Isolated kidney tubules synthesize glucose actively from fructose, lactate, glycerol and pyruvate and, to a lesser extent, from a variety of amino acids. Ethanol stimulated gluconeogenesis from pyruvate and inhibited it from lactate. The aminotransferase inhibitor, aminooxyacetate, greatly reduced synthesis from lactate but not from pyruvate. Quinolinate inhibited gluconeogenesis from both precursors, indicating an active role for cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in the gluconeogenic pathway. Incorporation of lactate or glucose into triglycerides was relatively low, and since no fatty acid synthase (FAS) activity could be detected, probably represented chain elongation or reesterification.     

The effect of acetate on the regulation of the branched chain α-keto acid and the pyruvate dehydrogenase complexes in the perfused rat liver

The regulatory consequences of acetate infusion on the pyruvate and the branched chain α-keto acid dehydrogenase reactions in the isolated, perfused rat liver were investigated. Metabolic flux through these two decarboxylation reactions was monitored by measuring the rate of ¹⁴CO₂ production from infused 1-¹⁴C-labeled substrates. When acetate was presented to the liver as the sole substrate the rate of ketogenesis which resulted was maximal at concentrations of acetate in excess of 10 mm. The increase in hepatic ketogenesis during acetate infusion was not accompanied by an alteration of the mitochondrial oxidation-reduction state as measured by the ratio of β-hydroxybutyrate/ acetoacetate in the effluent perfusate. While acetate infusion did not affect the rate of α-keto[1-¹⁴C]isocaproate decarboxylation, the rate of α-keto[1-¹⁴C]isovalerate decarboxylation was stimulated appreciably upon acetate addition. No change was observed in the amount of extractable branched chain α-keto acid dehydrogenase during acetate infusion. The rate of [1-¹⁴C]pyruvate decarboxylation was stimulated in the presence of acetate at low (<1 mm) but not at high (>1 mm) perfusate pyruvate concentrations. The stimulation of the metabolic flux through the pyruvate dehydrogenase reaction upon acetate infusion was accompanied by an increase in the activation state of the pyruvate dehydrogenase complex from 25.7 to 35.6% in the active form. In a liver perfused in the presence of the pyruvate dehydrogenase kinase inhibitor, dichloroacetate, at a low concentration of pyruvate (0.05 mm) the infusion of acetate did not affect the rate of pyruvate decarboxylation. As the rate of mitochondrial acetoacetate efflux is increased during acetate infusion the stimulation of pyruvate and α-ketoisovalerate decarboxylation is attributed to an accelerated rate of exchange of mitochondrial acetoacetate for cytosolic pyruvate or α-ketoisovalerate on the monocarboxylate transporter.     

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.     

The effects of cyclopropane carboxylate on hepatic pyruvate metabolism

The effects of cyclopropane carboxylate on gluconeogenesis and pyruvate decarboxylation from [1-14C]-labeled pyruvate and lactate were investigated in perfused livers from fasted rats. With high concentrations of pyruvate (greater than or equal to 0.5 mM) in the perfusion medium, infusion of cyclopropane carboxylate inhibited pyruvate decarboxylation and gluconeogenesis by 30 and 40%, respectively. With low, more physiological concentrations of pyruvate (50 microM) or with lactate (1 mM), cyclopropane carboxylate, at a concentration which elicits maximal inhibition of pyruvate decarboxylation from pyruvate (greater than or equal to 0.5 mM), did not affect either pyruvate decarboxylation or gluconeogenesis. Evidence is presented for the rapid formation of the coenzyme-A ester of cyclopropane carboxylate in perfused livers. Infusion of l-(-)carnitine (20 mM) prevented the inhibitory effects of cyclopropane carboxylate on pyruvate decarboxylation and gluconeogenesis from pyruvate (greater than or equal to 0.5 mM). Interestingly, no decrease in the tissue level of cyclopropanecarboxyl-CoA occurs under these conditions. The present study suggests that cyclopropane carboxylate, through a presently ill-defined mediator, inhibits pyruvate decarboxylation and gluconeogenesis by interfering with the pyruvate----oxalacetate----phosphoenolpyruvate----pyruvate cycle when pyruvate (greater than or equal to 0.5mM) supports gluconeogenesis.     

Stimulation by alpha-adrenergic agonists of Ca2+ fluxes, mitochondrial oxidation and gluconeogenesis in perfused rat liver.

Glucose output from perfused livers of 48 h-starved rats was stimulated by phenylephrine (2 microM) when lactate, pyruvate, alanine, glycerol, sorbitol, dihydroxyacetone or fructose were used as gluconeogenic precursors. Phenylephrine-induced increases in glucose output were immediately preceded by a transient efflux of Ca2+ and a sustained increase in oxygen uptake. Phenylephrine decreased the perfusate [lactate]/[pyruvate] ratio when sorbitol or glycerol was present, but increased the ratio when alanine, dihydroxyacetone or fructose was present. Phenylephrine induced a rapid increase in the perfusate [beta-hydroxybutyrate]/[acetoacetate] ratio and increased total ketone-body output by 40-50% with all substrates. The oxidation of [1-14C]octanoate or 2-oxo[1-14C]glutarate to 14CO2 was increased by up to 200% by phenylephrine. All responses to phenylephrine infusion were diminished after depletion of the hepatic alpha-agonist-sensitive pool of Ca2+ and returned toward maximal responses after Ca2+ re-addition. Phenylephrine-induced increases in glucose output from lactate, sorbitol and glycerol were inhibited by the transaminase inhibitor amino-oxyacetate by 95%, 75% and 66% respectively. Data presented suggest that the mobilization of an intracellular pool of Ca2+ is involved in the activation of gluconeogenesis by alpha-adrenergic agonists in perfused rat liver. alpha-Adrenergic activation of gluconeogenesis is apparently accompanied by increases in fatty acid oxidation and tricarboxylic acid-cycle flux. An enhanced transfer of reducing equivalents from the cytoplasmic to the mitochondrial compartment may also be involved in the stimulation of glucose output from the relatively reduced substrates glycerol and sorbitol and may arise principally from an increased flux through the malate-aspartate shuttle.     

Inhibitory effect of tumor necrosis factor α on gluconeogenesis in perfused rat liver

Tumor necrosis factor α (TNFα) is a cytokine involved in many metabolic responses in both normal and pathological states. Considering that the effects of TNFα on hepatic gluconeogenesis are inconclusive, we investigated the influence of this cytokine in gluconeogenesis from various glucose precursors. TNFα (10 μg/kg) was intravenously injected in rats; 6 h later, gluconeogenesis from alanine, lactate, glutamine, glycerol, and several related metabolic parameters were evaluated in situ perfused liver. TNFα reduced the hepatic glucose production (p < 0.001), increased the pyruvate production (p < 0.01), and had no effect on the lactate and urea production from alanine. TNFα also reduced the glucose production (p < 0.01), but had no effect on the pyruvate production from lactate. In addition, TNFα did not alter the hepatic glucose production from glutamine nor from glycerol. It can be concluded that the TNFα inhibited hepatic gluconeogenesis from alanine and lactate, which enter in gluconeogenic pathway before the pyruvate carboxylase step, but not from glutamine and glycerol, which enter in this pathway after the pyruvate carboxylase step, suggesting an important role of this metabolic step in the changes mediated by TNFα.     

Iron deficiency decreases gluconeogenesis in isolated rat hepatocytes

Dietary iron deficiency in rats results in increased blood glucose turnover and recycling. We measured the rates of glucose production in isolated hepatocytes from iron-sufficient (Fe+) and iron-deficient (Fe-) rats to assess the intrinsic capacity of the Fe- liver to carry out gluconeogenesis. Low-iron and control diets were given to 21-day-old female rats. After 4-5 wk, hemoglobin concentrations averaged 4.1 g/dl in the Fe- and 14.3 g/dl in the Fe+ animals. In the hepatocytes from Fe- rats, there was a 35% decrease in the rate of glucose production from 1 mM pyruvate + 10 mM lactate, a 48% decrease from 0.1 mM pyruvate + 1 mM lactate, a 39% decrease from 1 mM alanine, and a 48% decrease from 1 mM glycerol. The addition of 5 microM norepinephrine or 0.5 microM glucagon to the incubation media produced stimulatory effects on hepatocytes from both Fe- and Fe+ rats, resulting in the maintenance of an average difference of 38% in the rates of gluconeogenesis between the two groups. Studies on isolated liver mitochondria and cytosol revealed alpha-glycerophosphate-cytochrome c reductase and phospho(enol)pyruvate carboxykinase activities to be decreased by 27% in Fe- rats. We conclude that because severe dietary iron deficiency decreases gluconeogenesis in isolated rat hepatocytes, the increased gluconeogenesis demonstrated by Fe- rats in vivo is attributable to increased availability of gluconeogenic substrates and upregulation of the pathway.     

Rapid effects of insulin and glucose on the hepatic incorporation of gluconeogenic substrates into glyceride glycerol and glycogen

1. The hepatic utilization of gluconeogenic substrates was investigated shortly after portal infusion of either insulin or glucose in fasted rats. 2. After 20 min of insulin infusion blood glucose concentration decreased. However, neither glucose generation from precursors such as alanine or pyruvate nor their incorporation into fatty acids was modified. Under these conditions, insulin rapidly increased the incorporation of gluconeogenic substrates into the hepatic glyceride glycerol fraction. Insulin treatment led to a decrease in substrate incorporation into liver glycogen. 3. After 20 min of portal glucose infusion both plasma insulin and glucose concentrations increased and the incorporation of pyruvate into hepatic glyceride glycerol and into glycogen was also stimulated. 4. A close relationship was observed between blood glucose concentrations and the level of incorporation of gluconeogenic substrates into liver glycogen. 5. In conclusion, during fasting insulin stimulates the incorporation of gluconeogenic substrates into the glycerol moiety of hepatic glycerides, which may be the preferential mechanism through which fatty acid esterification is accomplished during refeeding. This effect of insulin is rapid and detected even before other classical modifications induced by the hormone such as gluconeogenesis inhibition or lipogenesis activation. Furthermore, the effect is not related to insulin-induced hypoglycemia since glucose infusion mimics insulin action on glyceride glycerol synthesis.     

Development of gluconeogenesis from different substrates in newborn rabbit hepatocytes

The rates of glucose production from various substrates entering gluconeogenesis at different steps were investigated in hepatocytes isolated from term-fetus and newborn rabbits fasted during the first 2 days of life. The data were compared to the rate of glucose production measured in hepatocytes from young rabbits (50-60 days) starved for 48 h. The net production of glucose from substrates (lactate, pyruvate, propionate, alanine) entering gluconeogenesis below phosphoenolpyruvate was very low at birth and increased during the first day of life, in relation with an increased cytosolic phosphoenolpyruvate carboxykinase activity. The net production of glucose from precursors entering gluconeogenesis at the level of triose phosphates (dihydroxyacetone, fructose) was low at birth but a maximal capacity for gluconeogenesis was reached within 6 h after birth. This enhanced gluconeogenic capacity was associated with a fall in hepatic fructose 2,6-bisphosphate concentration and a reduced glycolytic flux. In contrast, a high glucose production from galactose was already present at birth and did not rise at 24 or 48 h after delivery. These results suggest that the development of gluconeogenic capacity in hepatocytes isolated from newborn rabbit is dependent upon two factors, a decrease in the F2,6-P2 concentration which reduces the glycolytic flux and an increase in the activity of cytosolic phosphoenolpyruvate carboxykinase.     

The identification of the major excreted protein (MEP) from a transformed mouse fibroblast cell line as a catalytically active precursor form of cathepsin L.

Incubation of hepatocytes from 24 h-starved rats in the presence of 0.5 mM-adenosine decreased gluconeogenesis from lactate, but not from alanine. The inhibition of gluconeogenesis was associated with a stimulation of ketone-body production and an inhibition of pyruvate oxidation. These metabolic changes were suppressed in the presence of iodotubercidin (an inhibitor of adenosine kinase), but were reinforced in the presence of deoxycoformycin (an inhibitor of adenosine deaminase); 2-chloroadenosine induced no change in gluconeogenesis from lactate. These data indicate that the inhibition of gluconeogenesis by adenosine probably results from its conversion into adenine nucleotides. In the presence of lactate or pyruvate, but not with alanine or asparagine, this conversion resulted in a decrease in the [ATP]/[ADP] ratio in both mitochondrial and cytosolic compartments. Adenosine decreased the Pi concentration with all gluconeogenic substrates.     

Gluconeogenesis in chick embryo isolated hepatocytes

1. The effectiveness of gluconeogenic precursors in hepatocytes isolated from 18 day old chick embryos is:Lactate much much greater than pyruvate greater than alanine = glutamine greater than glycerol and other amino acids. This result is qualitatively and quantitatively similar to hepatocytes isolated after hatching. 2. In the presence of endogenous glycogenolysis, conversion of [U-14C]lactate to glucose was used to estimate gluconeogenic flux and its control by hormones. 3. Glucagon failed to stimulate lactate gluconeogenesis although simultaneously increasing glycogenolysis. Insulin had no effects on gluconeogenesis.     

Nonhepatic glucose production in humans

Extrahepatic glucose release was evaluated during the anhepatic phase of liver transplantation in 14 recipients for localized hepatocarcinoma with mild or absent cirrhosis, who received a bolus of [6,6-2H2]glucose and l-[3-13C]alanine or l-[1,2-13C2]glutamine to measure glucose kinetics and to prove whether gluconeogenesis occurred from alanine and glutamine. Twelve were studied again 7 mo thereafter along with seven healthy subjects. At the beginning of the anhepatic phase, plasma glucose was increased and then declined by 15%/h. The right kidney released glucose, with an arteriovenous gradient of -3.7 mg/dl. Arterial and portal glucose concentrations were similar. The glucose clearance was 25% reduced, but glucose uptake was similar to that of the control groups. Glucose production was 9.5 +/- 0.9 micromol.kg-1. min-1, 30% less than in controls. Glucose became enriched with 13C from alanine and especially glutamine, proving the extrahepatic gluconeogenesis. The gluconeogenic precursors alanine, glutamine, lactate, pyruvate, and glycerol, insulin, and the counterregulatory hormones epinephrine, cortisol, growth hormone, and glucagon were increased severalfold. Extrahepatic organs synthesize glucose at a rate similar to that of postabsorptive healthy subjects when hepatic production is absent, and gluconeogenic precursors and counterregulatory hormones are markedly increased. The kidney is the main, but possibly not the unique, source of extrahepatic glucose production.