Differential effects of tryptophan on glucose synthesis in rats and guinea pigs.

1. Tryptophan inhibition of gluconeogenesis in isolated rat liver cells is characterized by a 20 min lag period before linear rates of glucose output are attained. 2. Half-maximal inhibition of gluconeogenesis in isolated rat hepatocytes is produced by approx. 0.1 mM-tryptophan. 3. Tryptophan inhibits gluconeogenesis from all substrates giving rise to oxaloacetate, but stimulates glycerol-fuelled glucose production. 4. Gluconeogenesis in guinea-pig hepatocytes is insensitive to tryptophan. 5. Changes in metabolite concentrations in rat liver cells are consistent with a locus of inhibition at the step catalysed by phosphoenolpyruvate carboxykinase. 6. Inhibition of gluconeogenesis persists in cells from rats pretreated with tryptophan in vivo. 7. Tryptophan has no effect on urea production from alanine, but decreases [1-14C]palmitate oxidation to 14CO2 and is associated with an increased [hydroxybutyrate]/[acetoacetate] ratio. 8. These results are discussed with reference to the control of gluconeogenesis in various species.     

Effect of tryptophan on gluconeogenesis in the perfused liver of irradiated rats

The liver isolated at different times after exposure to 7 Gy radiation responded in a different way to the effect of tryptophan (0.75 g/l) used as a gluconeogenesis inhibitor. While 24 h after irradiation the addition of tryptophan inhibited gluconeogenesis from circulating exogenous amino acids, in 3 days, on the contrary, gluconeogenesis in the liver of donors was enhanced. It is suggested that these effects of tryptophan are associated with different functional status of the liver during the postirradiation observation period.     

Periportal and perivenous hepatocytes retain their zonal characteristics in primary culture

Periportal and perivenous hepatocytes from rat liver were isolated by combined digitonin-collagenase perfusion, and gluconeogenesis, urea synthesis and fatty acid synthesis was measured both in freshly isolated cells and in primary culture. A periportal zonation of gluconeogenesis and urea synthesis of about 3 and 1.5 fold, respectively, was observed. This zonation persisted unchanged for 23 hours in culture under identical conditions of incubation for periportal and perivenous cells. Fatty acid synthesis was not zonated.     

Gluconeogenesis in guinea pig renal tubule fragments--effects of noradrenaline, 3':5' cyclic AMP and angiotensin II

1. Tubule fragments were isolated by collagenase treatment of guinea pig kidney cortex. 2. 3':5'-Cyclic AMP increased gluconeogenesis from lactate, pyruvate, malate and fructose. 3. Noradrenaline decreased gluconeogenesis from lactate, pyruvate, 2-oxoglutarate and fructose. 4. Angiotensin II slightly, but significantly, increased gluconeogenesis from lactate. 5. Gluconeogenesis from glycerol as sole substrate was negligible. Gluconeogenesis from combinations of glycerol together with either lactate, pyruvate, 2-oxoglutarate or malate was appreciably greater than the sum of the glucose output observed when these substrates were added singly.     

Effect of compound D-600 (methoxyverapamil) on gluconeogenesis and on acceleration of the process by alpha-adrenergic stimuli in rat kidney tubules.

1. Tubule fragments were isolated from renal cortex of fed rats and glucose formation was measured after incubation with 5 mM-sodium lactate. 20 Compound D-600 (10-100 microM) decreased gluconeogenesis from lactate. This inhibition of the process by compound D-600 increased with increasing extracellular Ca2+ concentration, was overridden by noradrenaline and diminished by starvation for 24 h. 3. Inhibition of lactate-supported gluconeogenesis by compound D-600 was not prevented by the alpha 1-adrenoceptor antagonist thymoxamine. 4. Compound D-600 had little effect on gluconeogenesis from 2-oxoglutarate and increased gluconeogenesis from succinate. 5. Compound D-600 opposed stimulation of gluconeogenesis by noradrenaline or oxymetazoline (a selective alpha-adrenoceptor agonist) in a manner suggesting that compound D-600 is an alpha-adrenoceptor blocker. Oxymetazoline was more sensitive than noradrenaline to blockade by both compound D-600 and by the conventional alpha-adrenoceptor antagonist phentolamine. Noradrenaline became more sensitive to blockade by compound D-600 when extracellular Ca2+ was decreased. 6. Compound D-600 did not block stimulation of gluconeogenesis by angiotensin or cyclic AMP.     

Studies on the lack of glucagon response to clycogenolysis and gluconeogensis and decrease in protein synthesis in isolated hepatocytes from hypophysectomized animals.

Effect of various concentrations of glucagon on gluconeogenesis and glycogenolysis was studied in isolated hepatocytes obtained from normal and hypophysectomized animals. Addition of glucagon (10^?10 to 10^?6M) stimulated glycogenolysis and gluconeogenesis by 2–3 fold in normal hepatocytes. However, this concentration of glucagon had only a slight effect in isolated hepatocytes obtained from hypophysectomized animals. This lack of glucagon response was not due to reduction in glycogen levels in isolated hepatocytes obtained from hypophysectomized animals. Studies on the incorporation of¹⁴C-alanine,¹⁴C-leucine and¹⁴C-valine showed a 3–5 fold decrease in the incorporation of these amino acids into protein in hypophysectomized animals compared to normal controls.     

Interrelationship between drug oxidation ureogenesis and gluconeogenesis in isolated hepatocytes

1. The effect of increased ureogenesis--provoked by NH4Cl and ornithine--on gluconeogenesis and aminopyrine oxidation was studied in isolated hepatocytes prepared from 24 hr starved mice; lactate or fructose was used as gluconeogenic precursor. 2. Increased ureogenesis caused about 40% inhibition both on aminopyrine oxidation and gluconeogenesis when lactate was added as gluconeogenic substrate. 3. On the other hand, only 10% inhibition of aminopyrine oxidation and about 15% inhibition of gluconeogenesis were observed when fructose was used as gluconeogenic precursor. 4. Aminopyrine has been reported to inhibit gluconeogenesis from fructose by 30% and from lactate by 85%. The inhibitory effect of the combined addition of aminopyrine, NH4Cl and ornithine on gluconeogenesis was also dependent on the applied gluconeogenic precursor. 5. The provoked ureogenesis by ammonia and ornithine was not inhibited by aminopyrine. N6, O2-dibutyryl cAMP known to cause an increase of gluconeogenesis a decrease of aminopyrine oxidation enhanced the inhibitory action of increased ureogenesis on aminopyrine oxidation and on gluconeogenesis further. 6. The role of NADPH in the regulation of drug oxidation and ureogenesis is underlined.     

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.     

Inhibition of gluconeogenesis and lactate formation from pyruvate by N6, O2'-dibutyryl adenosine 3':5'-monophosphate.

N6,O2-Dibutyryl adenosine 3':5'-monophosphate (Bt2cAMP) inhibits gluconeogenesis and lactate formation but increases ketogenesis by isolated liver cells incubated with high concentrations of pyruvate. The inhibitory effects can not be explained on the basis of an inhibition of the pyruvate dehydrogenase complex nor by a change in the NAD+ oxidation-reduction potential of the mitochondrial compartment. Both oleate and 3-hydroxybutyrate substantially increase the rates of gluconeogenesis and lactate formation from pyruvate but do not overcome the inhibition caused by Bt2cAMP. A decreased effectiveness of pyruvate kinase is proposed to account for the inhibition of both gluconeogenesis and lactate formation by Bt2cAMP. This enzyme catalyzes a step required in the transfer of reducing equivalents from the mitochondrial compartment to the cytoplasm and participates in the formation of glucose and lactate from pyruvate by the overall reaction: 2 pyruvate- + 2 NADHmito + 4 ATP4- + 4 H2O leads to 1/2 glucose + lactate- + 2 NAD+ mito + 4 ADP3- + 4 HPO4(2)- + H+. Inhibition of pyruvate kinase promotes gluconeogenesis with most substrates but inhibits gluconeogenesis from pyruvate for want of cytoplasmic reducing equivalents.     

Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, suppresses hepatic gluconeogenesis through 5'-AMP-activated protein kinase

Epigallocatechin-3-gallate (EGCG), a main catechin of green tea, has been suggested to inhibit hepatic gluconeogenesis. However, the exact role and related mechanism have not been established. In this study, we examined the role of EGCG in hepatic gluconeogenesis at concentrations that are reachable by ingestion of pure EGCG or green tea, and are not toxic to hepatocytes. Our results show in isolated hepatocytes that EGCG at relatively low concentrations (相似文献   

Gluconeogenesis in goat hepatocytes is affected by calcium, ammonia and other key metabolites but not primarily through cytosolic redox state

1. Gluconeogenesis from propionate and lactate was studied in caprine hepatocytes. 2. Reducing cytosol with additions of ETOH, ammonium, or lactate decreased [2-14C]propionate conversion to glucose. 3. Calcium oxidized the cytosol and increased gluconeogenesis from propionate by 198% and from lactate by 220%. 4. Cells isolated from lactating does and wethers differed quantitatively in propionate conversion to glucose and response to calcium. 5. Acetoacetate decreased and 3-OH-butyrate slightly increased glucose production from propionate. 6. Neither ketone body had any significant effect on gluconeogenesis from lactate. 7. Results reported herein suggest gluconeogenesis from propionate is not limited by lack of cytosolic reducing equivalents.     

Gluconeogenesis in the perfused rat liver

1. A modification of the methods of Miller and of Schimassek for the perfusion of the isolated rat liver, suitable for the study of gluconeogenesis, is described. 2. The main modifications concern the operative technique (reducing the period of anoxia during the operation to 3min.) and the use of aged (non-glycolysing) red cells in the semi-synthetic perfusion medium. 3. The performance of the perfused liver was tested by measuring the rate of gluconeogenesis, of urea synthesis and the stability of adenine nucleotides. Higher rates of gluconeogenesis (1mumole/min./g.) from excess of lactate and of urea synthesis from excess of ammonia (4mumoles/min./g. in the presence of ornithine) were observed than are likely to occur in vivo where rates are limited by the rate of supply of precursor. The concentrations of the three adenine nucleotides in the liver tissue were maintained within 15% over a perfusion period of 135min. 4. Ca(2+), Na(+), K(+), Mg(2+) and phosphate were found to be required at physiological concentrations for optimum gluconeogenesis but bicarbonate and carbon dioxide could be largely replaced by phosphate buffer without affecting the rate of gluconeogenesis. 5. Maximal gluconeogenesis did not decrease maximal urea synthesis in the presence of ornithine and ammonia and vice versa. This indicates that the energy requirements were not limiting the rates of gluconeogenesis or of urea synthesis. 6. Addition of lactate, and especially ammonium salts, increased the uptake of oxygen more than expected on the basis of the ATP requirements of the gluconeogenesis and urea synthesis.     

Inhibition of hepatic gluconeogenesis by the Rp-diastereomer of adenosine cyclic 3'',5''-phosphorothioate.

The specific intracellular cyclic AMP-dependent protein kinase antagonist, the Rp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Rp-cAMPS), inhibited both basal and cyclic AMP-agonist-induced rates of gluconeogenesis in hepatocytes isolated from fasted rats. Incubation of the cells in the presence of pyruvate and lactate and either the Sp-diastereomer of adenosine cyclic 3',5'-phosphorothioate (Sp-cAMPS) or glucagon produced a concentration-dependent increase in the rate of gluconeogenic glucose production which was shifted to higher concentrations of Sp-cAMPS or glucagon in the presence of Rp-cAMPS. Incubation of the cells with Rp-cAMPS in the absence of agonist produced no increase in the rate of glucose production and, in most cases, 100 microM-Rp-cAMPS resulted in 14-20% decrease in the substrate-stimulated rate of glucose production. Sp-cAMPS-induced gluconeogenesis was inhibited half-maximally at 1 microM-Rp-cAMPS and glucagon-induced gluconeogenesis was inhibited half-maximally at 12 microM-Rp-cAMPS. Approx. 10-15% of the inhibition of gluconeogenesis observed in the presence of Rp-cAMPS was due to conversion of glucose 6-phosphate to liver glycogen, consistent with Rp-cAMPS-induced reactivation of glycogen synthase. The remaining 85-90% inhibition of gluconeogenic glucose production resulted from the action of Rp-cAMPS on the cyclic AMP-sensitive enzymes controlling the rate of gluconeogenesis.     

Regulation of carbohydrate metabolism in mouse liver. Effect of glucagon on gluconeogenic/glycolytic flux in isolated perfused livers.

1. Glucagon stimulated gluconeogenesis from both [U-14C]lactate and [14C]xylitol in isolated perfused mouse liver. 2. Addition of cyclic AMP also stimulated gluconeogenesis from [U-14C]lactate. 3. Glucagon caused a rapid (2.5 min) 12-fold increase in hepatic cyclic AMP but not cyclic GMP concentration. 4. Glucagon caused a rapid and stable decrease in hepatic fructose 1,6-diphosphatase activity measured in vitro. 5. The results are interpreted to indicate that glucagon stimulates hepatic gluconeogenesis in mice via cyclic AMP by two different mechanisms: (a) increased substrate uptake (i.e. utilization) and (b) increased gluconeogenic efficiency (i.e. inhibition of alternate substrate fates).     

Starvation and the metabolism of hepatocytes isolated from the American eel,Anguilla rostrata LeSeur

Summary Gluconeogenic, lipogenic, glycogenic and oxidative rates were estimated from¹⁴C-lactate,¹⁴C-alanine and¹⁴C-aspartate using a hepatocyte preparation isolated from starved immature American eels,Anguilla rostrata. Lactate gluconeogenesis increased significantly during starvation at 5 and 15°C. Alanine gluconeogenesis generally decreased during starvation. At the 2nd month of the starvation at 5 and 15°C, and the 8th month of starvation at 15°C, however, alanine gluconeogenesis was significantly higher than in the fed control. These increases in alanine gluconeogenesis occurred during a period of high glucose demand. Aspartate gluconeogenesis was quantitatively minor when compared to the other two substrates. Glycerol synthesis and esterification from the three substrates increased until the 5th month at 5 and 15°C followed by a gradual decline thereafter. Significant increases in glycogen synthesis occurred between the 3rd and the 5th months at 15°C, but rates were small compared to glucose synthesis. Rates of substrate oxidation appeared sufficient to provide adequate ATP to sustain gluconeogenesis in both the fed and starved eel hepatocyte. Glucagon stimulated lactate gluconeogenesis, but not amino acid gluconeogenesis in late starved eel hepatyocytes. Major changes in metabolite concentrations that occurred during starvation were increases in plasma glucose and amino acids; a significant liver glycogen depletion at the 2nd month followed by a return to control values at the third month; and, a significant protein depletion in white skeletal muscle at the 3rd month. These data suggest that lactate glucogeogenesis, but not amino acid gluconeogenesis or glycogenolysis, is the major source of tissue carbohydrates during eel starvation.This work was supported from operating grants to TWM from the National Research Council of Canada (A6944)     

Studies on the inhibition of gluconeogenesis by oxalate

Oxalate was shown to enter isolated rat hepatocytes and to inhibit gluconeogenesis from lactate, pyruvate, and alanine, but not from glutamine, proline, propionate or dihydroxyacetone. Oxalate apparently acts by inhibiting pyruvate carboxylase (EC 6.4.1.1). It is known to inhibit the isolated enzyme, and inhibition of gluconeogenesis was much greater in a bicarbonate-deficient medium where pyruvate carboxylase activity limits the overall rate of the pathway. A slight inhibition of gluconeogenesis from asparagine was observed, suggesting that oxalate may also inhibit gluconeogenesis at another site. Chelation of extracellular Ca²⁺ does not contribute to the inhibition of gluconeogenesis. Compared to oxalate, other Ca²⁺ chelators have little effect upon gluconeogenesis. Also, oxalate inhibits gluconeogenesis effectively both in low Ca²⁺ medium and in medium containing 2.6 mM Ca²⁺. Chelation of intracellular Ca²⁺ also appears to be of little importance, since oxalate does not block the glycogenolytic effects of epinephrine, vasopressin, and angiotensin which are thought to act via Ca²⁺ as the second messenger. The inhibition of gluconeogenesis could conceivably contribute to the toxic actions of oxalate and to the hypoglycemic action of dichloroacetate, a compound that is metabolized to oxalate. However, oxalate did not cause hypoglycemia in the suckling rat, a model in vivo system very dependent upon gluconeogenesis for maintenance of normal blood glucose levels. Thus, inhibition of gluconeogenesis is probably of little importance in oxalate toxicity and the hypoglycemic effects of dichloroacetate.     

Studies on the inhibition of insulin release,glycogenolysis and gluconeogenesis by somatostatin in the rat islets of Langerhans and isolated hepatocytes

The effect of somatostatin on insulin release, glycogenolysis and gluconeogenesis was studied in isolated islets of Langerhans and hepatocytes. Addition of somatostatin (0.2 μg – 100 μg) to isolated islets of Langerhans inhibited insulin release from 30 to 90 percent. Studies with isolated hepatocytes showed that somatostatin inhibited both glucagon-stimulated glycogenolysis and gluconeogenesis by 40–50 percent, whereas it had no effect on epinephrine-stimulated glycogenolysis.     

The hormonal control of gluconeogenesis by regulation of mitochondrial pyruvate carboxylation in isolated rat liver cells.

The possibility that hormones control hepatic gluconeogenesis via the regulation of the rate of mitochondrial pyruvate carboxylation was investigated with the use of suspensions of liver cells isolated from fasted rats. The mitochondria prepared from liver cells were judged in good condition as they exhibited satisfactory phosphorus-oxygen and respiratory control ratios and transported Ca2+ and K+ ions in an energy-dependent manner. Addition of glucagon, epinephrine, or cyclic adenosine 3':5'-monophosphate to liver cells caused a 50 to 80% increase in the rate of glucose synthesis from lactate. When mitochondria were isolated from the cells after treatment with these agonists, they displayed 2- to 3-fold increases in the rate of pyruvate carboxylation, pyruvate decarboxylation, and pyruvate uptake. These mitochondrial changes are similar to those obtained in hepatic mitochondria prepared from intact, hormone-treated rats. The mitochondrial responses were specific for agents that stimulated gluconeogenesis; no response occurred with 5'-AMP or cyclic adenosine 2':3'-monophosphate. In the cell suspensions, the dose response curves for the activation of mitochondrial pyruvate metabolism and for increased glucose synthesis from L-lactate were coincident with four different agonists. The mitochondrial changes resulting from stimulation with glucagon developed in 1 to 2 min after the rise in cyclic adenosine 3':5'-monophosphate and occurred at least as early as the increase in the rate of gluconeogenesis. When the intracellular level of cyclic adenosine 3':5'-monophosphate returned to basal values, the rates of mitochondrial pyruvate carboxylation and glucose synthesis also declined to control levels. It is concluded that the rate of mitochondrial pyruvate metabolisms can be increased by hormones and cyclic nucleotides and that control of mitochondrial pyruvate carboxylation is an important regulatory site of hepatic gluconeogenesis.     

Metabolism of adenosine through adenosine kinase inhibits gluconeogenesis in isolated rat hepatocytes

In isolated hepatocytes from fasted rats, 0.5 mM adenosine inhibited gluconeogenesis from glutamine, lactate and pyruvate. This inhibition was due to adenosine conversion through adenosine kinase. An increase in ketone body release was only observed in the presence of lactate or pyruvate, and the two phenomena (i.e. inhibition of gluconeogenesis and increased ketone-body release) were linked. With alanine, dihydroxyacetone or serine as substrates, adenosine did not change gluconeogenesis; however, its conversion through adenosine kinase also inhibited gluconeogenesis. With asparagine as substrate, 0.5 mM adenosine increased gluconeogenesis; this increase was due to adenosine conversion through adenosine deaminase. However, adenosine conversion through adenosine kinase inhibited gluconeogenesis from asparagine. Thus, whatever the substrate used, adenosine conversion through adenosine kinase inhibited gluconeogenesis. The inhibitory effect of adenosine on gluconeogenesis cannot be related to the decrease in Pi concentration and to the increase in ATP pool. Beside its effect on gluconeogenesis, adenosine inhibited ketogenesis measured without added substrate; adenosine conversion through adenosine kinase was also involved in the inhibition of ketogenesis.     

Studies on the differential response to glucagon concentration on gluconeogenesis in isolated hepatocytes containing high and low glycogen

Rat liver parenchymal cells were isolated with (a) collagenase alone and (b) with both collagenase and hyaluronidase. Addition of hyaluronidase significantly decreased intracellular glycogen content of cells from fed rats. Effects of various concentrations of glucagon on gluconeogenesis were also studied in isolated hepatocytes from fed and fasted rats. Glucagon at the concentration of 10^?12M to 10^?10M stimulated gluconeogenesis in fed rats. Higher concentrations (10^?8M) had no further stimulating effect. In fasted rats, glucagon at the concentrationsof 10^?12M had no effect whereas at 10^?10M to 10^?8M concentrations, it stimulated gluconeogenesis by 2 fold. These studies suggest that glucagon functions in gluconeogenesis both in the fed and fasted state.