G Van den Berghe

Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase.

AMP‐activated protein kinase is a multisubstrate protein kinase that, in liver, inactivates both acetyl‐CoA carboxylase, the rate‐limiting enzyme of fatty acid synthesis, and 3‐hydroxy‐3‐methyl‐glutaryl‐CoA reductase, the rate‐limiting enzyme of cholesterol synthesis. AICAR (5‐amino 4‐imidazolecarboxamide ribotide, ZMP) was found to stimulate up to 10‐fold rat liver AMP‐activated protein kinase, with a half‐maximal effect at approximately 5 mM. In accordance with previous observations, addition to suspensions of isolated rat hepatocytes of 50–500 μM AICAriboside, the nucleoside corresponding to ZMP, resulted in the accumulation of millimolar concentrations of the latter. This was accompanied by a dose‐dependent inactivation of both acetyl‐CoA carboxylase and 3‐hydroxy‐3‐methylglutaryl‐CoA reductase. Addition of 50–500 μM AICAriboside to hepatocyte suspensions incubated in the presence of various substrates, including glucose and lactate/pyruvate, caused a parallel inhibition of both fatty acid and cholesterol synthesis. With lactate/pyruvate (10/1 mM), half‐maximal inhibition was obtained at approximately 100 μM, and near‐complete inhibition at 500 μM AI‐CAriboside. These findings open new perspectives for the simultaneous control of triglyceride and cholesterol synthesis by pharmacological stimulators of AMP‐activated protein kinase.—Henin, N., Vincent, M.‐F., Gruber, H. E., Van den Berghe, G. Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP‐activated protein kinase. FASEB J. 9, 541–546 (1995)

The role of the liver in metabolic homeostasis: implications for inborn errors of metabolism.

SummaryThe mechanisms by which the liver maintains a constant supply of oxidizable substrates, which provide energy to the body as a whole, are reviewed. During feeding, the liver builds up energy stores in the form of glycogen and triglyceride, the latter being exported to adipose tissue. During fasting, it releases glucose and ketone bodies. Glucose is formed by degradation of glycogen and by gluconeogenesis from gluconeogenic amino acids provided by muscle. Ketone bodies are produced from fatty acids, released by adipose tissue, and from ketogenic amino acids. The major signals which control the transition between the fed and the fasted state are glucose, insulin and glucagon. These influence directly or indirectly the enzymes which regulate liver carbohydrate and fatty acid metabolism and thereby orient metabolic fluxes towards either energy storage or substrate release. In the fed state, the liver utilizes the energy generated by glucose oxidation to synthesize triglycerides. In the fasted state it utilizes that produced byβ-oxidation of fatty acids to synthesize glucose. The mechanisms whereby a number of inborn errors of glycogen metabolism, of gluconeogenesis and of ketogenesis cause hypoglycaemia are also briefly overviewed.

Pathways of adenine nucleotide catabolism in erythrocytes.

The exact pathway whereby the initial catabolism of the adenine nucleotides proceeds from AMP and the possibility of a recycling of adenosine were investigated in human erythrocytes. Adenine nucleotide catabolism, reflected by the production of hypoxanthine, is very slow under physiologic conditions and can be greatly increased by suppression of glucose or alkalinization of the medium. Experiments with inhibitors of adenosine deaminase and adenosine kinase demonstrated that under physiologic conditions the initial catabolism of AMP proceeds by way of a deamination of AMP, followed by dephosphorylation of inosine monophosphate, and that no recycling occurs between AMP and adenosine. Under glucose deprivation, approximately 75% of the 20-fold increase of the catabolism of the adenine nucleotides proceeded by way of a dephosphorylation of AMP followed by deamination of adenosine, and a small recycling of this nucleoside could be evidenced. Inhibition of adenosine transport showed that the dephosphorylation of AMP occurred intracellularly. When the incubation medium was alkalinized in the presence of glucose, the 15-fold increase in the conversion of AMP to hypoxanthine proceeded exclusively by way of AMP deaminase but a small recycling of adenosine could also be evidenced. The threefold elevation of intraerythrocytic inorganic phosphate (Pi) during glucose deprivation and its 50% decrease during alkalinization as well as experiments in which extracellular Pi was modified, indicate that the dephosphorylation of red blood cell AMP is mainly responsive to variations of AMP, whereas its deamination is more sensitive to Pi.

Inborn errors of the purine nucleotide cycle: adenylosuccinase deficiency.

Adenylosuccinase catalyses two reactions in purine metabolism: the conversion of succinylaminoimidazole carboxamide ribotide (SAICAR) into amino-imidazole carboxamide ribotide (AICAR) along the de novo synthesis of purine nucleotides, and the conversion of adenylosuccinate (S-AMP) into AMP in the conversion of IMP into AMP. The hallmarks of adenylosuccinase deficiency are the presence of succinylaminoimidazole carboxamide riboside (SAICAriboside) and succinyladenosine (S-Ado) in body fluids. These normally undetectable succinyl-purines are the products of the dephosphorylation, by cytosolic 5′-nucleotidase, of the two substrates of adenylosuccinase. The clinical picture of the enzyme deficiency is markedly heterogeneous with, as a rule, a profound, but nevertheless variable degree of psychomotor delay, often convulsions and/or autistic features, sometimes growth retardation and muscular dystrophy. The diagnostic tests that can be used for diagnosis, the enzyme and gene defects that have been identified, and the hypotheses that have been put forward to explain the pathophysiology of the disorder are reviewed.

The purine nucleotide cycle and its molecular defects.

Three enzymes of purine metabolism, adenylosuccinate synthetase, adenylosuccinate lyase and AMP deaminase, have been proposed to form a functional unit, termed the purine nucleotide cycle. This cycle converts AMP into IMP and reconverts IMP into AMP via adenylosuccinate, thereby producing NH3 and forming fumarate from aspartate. In muscle, the purine nucleotide cycle has been shown to function during intense exercise; the metabolic flux through the cycle has been proposed to play a role in the regeneration of ATP by pulling the adenylate kinase reaction in the direction of formation of ATP, and by providing Krebs cycle intermediates. In kidney, the purine nucleotide cycle was shown to account for the release of NH3 under the normal acid-base status, but not under acidotic conditions. In brain, the purine nucleotide cycle might function under conditions that induce a loss of ATP, and thereby contribute to its recovery. There is no evidence that the purine nucleotide cycle operates in liver. Deficiency of muscle AMP deaminase is an apparently frequent disorder, which might affect approximately 2% of the general population. The observation that it can be found in clinically asymptomatic individuals suggests, paradoxically, that the ATP-regenerating function which has been attributed to the purine nucleotide cycle is not essential for muscle function. Further work should be aimed at identifying the conditions under which AMP deaminase deficiency becomes symptomatic. Adenylosuccinate lyase deficiency provokes psychomotor retardation, often accompanied by autistic features. Its clinical heterogeneity justifies systematic screening in patients with unexplained mental deficiency. Additional studies are required to determine the mechanisms whereby this enzyme defect results in psychomotor retardation.

The control of glycogen synthesis in the liver

Abstract In the liver, glycogen synthetase exists in two forms, one of them (a) active, the other (b) inactive in the ionic conditions which exist in the cell. These two forms are interconvertible, presumably by phosphorylation and desphosphorylation under the action of a specific kinase and phospharylation and respectively, It seems probable that these two enzymes operate simultaneously and that the level of the synthetase a in the liver results from the addition of their antagonistic effects. In normal mice, glycogen synthetase is predominantly in the b form. It is converted into a within 3 to 5 min after the intravenous administration of glucose or within 2 or 3 hr after the administration of glucocorticoids. The a enzyme can then be reconverted into b within 1 to 3 min after the administration of glucagon, epinephrine or cyclic AMP. The effect of these various effectors has also been demonstrated in vitro, mostly thanks to the use of liver extracts from which glucose had been removed by gel filtration through a Sephadex column. The complete activation of glycogen synthetase in vitro requires the presence of salts. In their absence, synthetase b is converted into a form which has the kinetic properties of synthetase a but which is less active. Glucose markedly enhances the activation, both in the presence and in the absence of salts while glycogen is an inhibitor in the presence of salts only. The affinity of the synthetase phosphatase for glucose is decreased when glycogen is present. A stimulation of the phosphorylase phosphatase by glucose has also been observed. The treatment of mice by glucocorticoids induces the appearance in the liver of a synthetase phosphatase that is less sensitive to glucose stimulation and to glycogen inhibition than normally. No effect of the treatment on the system that inactivates glycogen synthetase could be demonstrated. An effect of cyclic AMP on the synthetase kinase is easily demonstrable in a liver extract in which the synthetase has been previously activated either by glucose or by glucocorticoids; a half-maximal effect has been obtained with a concentration equal to 2 × 10−7 m cyclic AMP. The two main effectors that act antagonistically on the glycogen synthetase and glycogen phosphorylase appear to be glucose and cyclic AMP; thanks to their action, the synthesis of glycogen is inhibited while its degeneration is stimulated and vice versa.

Residual adenylosuccinase activities in fibroblasts of adenylosuccinase-deficient children: parallel deficiency with adenylosuccinate and succinyl-AICAR in profoundly retarded patients and non-parallel deficiency in a mildly retarded girl.

SummaryAdenylosuccinase (ASase) catalyses both the conversion of succinylaminoimidazole carboxamide ribotide (succinyl-AICAR) into AICAR and that of adenylosuccinate into AMP in the synthesis of purine nucleotides. Its deficiency results in the accumulation in body fluids of the nucleosides corresponding to both substrates, succinyl-AICAriboside and succinyladenosine. Two main subtypes of the defect are type I with severe mental retardation and succinyladenosine/succinyl-AICAriboside ratios around 1, and type II with slight mental delay and succinyladenosine/succinyl-AICAriboside ratios around 4. We report that in fibroblasts of type I patients, the activity of ASase with both adenylosuccinate and succinyl-AICAR is about 30% of normal. In contrast, in type II fibroblasts, the activity with adenylosuccinate is only 3% of normal, whereas that with succinyl-AICAR is also 30% of normal. If also present in other tissues, this non-parallel deficiency provides an explanation for the higher concentration of succinyladenosine in type II. In type I fibroblasts, ASase is further characterized mainly by a 3-fold to 4-fold increase inKm for succinyl-AICAR, and by retarded elution from an anion exchanger. In type II fibroblasts, ASase is characterized by a similar increase inKm for succinyl-AICAR but by a potent inhibition by KCl and nucleoside triphosphates, and by a normal elution profile. These results suggest a modification of the surface charge of ASase in type I, and the addition of one or more positively charged residues in the active site in type II.

Adenylosuccinase Deficiency - a Newly Recognized Variant

Adenylosuccinase deficiency (McKusick 103050) is a genetic defect of de novo purine and AMP synthesis, first reported in 1984 (Jaeken and Van den Berghe 1984) (Figure 1). It results in the accumulation in CSF, plasma and urine of two normally undetectable compounds: succinyladenosine and succinylaminoimidazole carboxamide riboside (SAICA riboside)

Existence and role of substrate cycling between AMP and adenosine in isolated rabbit cardiomyocytes under control conditions and in ATP depletion.

BackgroundAdenosine, a physiological coronary vasodilator, has been proposed to regulate coronary circulation according to myocardial oxygen demand. In the present study, we investigated the mechanisms of adenosine formation and utilization in isolated rabbit cardiomyocytes and, in particular, the existence and the role of substrate cycling between AMP and adenosine in the regulation of its concentration. Methods and ResultsRabbit cardiomyocytes were isolated by collagenase perfusion and incubated in HEPES-buffered Krebs-Henseleit solution at 37°C, pH 7.4, in control conditions and in ATP depletion achieved by inhibiting glycolysis with 5 mmol/L iodoacetate. Under control conditions, adenosine accumulated at a rate of 4 pmol · min−1.10−6cells. The 13-fold elevation of adenosine accumulation induced by iodotubercidin (ITu), an inhibitor of adenosine kinase, proves that adenosine is normally recycled into AMP. This recycling involves 95% of the adenosine formed. In ATP depletion, adenosine accumulated at the rate of 335 pmol · min−1 · 10−6 cells and was no longer rephosphorylated after 20 minutes, as shown by the absence of effect of ITu after this time interval. Moreover, adenosine was deaminated, as indicated by the twofold increase of its accumulation induced by deoxycoformycin (dCF), an inhibitor of adenosine deaminase. Both in control conditions and in ATP depletion, adenosine-dialdehyde, an inhibitor of S-adenosylhomocysteine (SAH) hydrolase, had no significant effect on adenosine formation, indicating that the transmethylation pathway is not an important source of adenosine in rabbit cardiomyocytes. ConclusionsThe results indicate that recycling of adenosine into AMP is essential for the maintenance of low, nonvasodilatory concentrations of the nucleoside under control conditions and that interruption of recycling plays an important role in elevating adenosine during ATP depletion.

D-xylulose-induced depletion of ATP and Pi in isolated rat hepatocytes.

Xylitol is known to cause hepatic ATP catabolism by inducing the trapping of Pi in the form of glycerol 3‐P as a consequence of an increase in the NADH:NAD+ ratio, resulting from the oxidation of xylitol to d‐xylulose. The question was therefore raised whether d‐xylulose also depletes hepatic ATP. In isolated rat hepatocytes, 5 mM d‐xylulose decreased ATP by 80% within 5 min compared to 40% with 5 mM xylitol. Intracellular Pi decreased by 70% within the same time interval with both compounds, but was restored threefold faster with d‐xylulose. The rate of utilization of d‐xylulose reached 5 μmol · min−1 · g−1 of cells, as compared with 1.5 for xylitol, indicating that reduction of xylitol into d‐xylulose is a rate‐limiting step in the metabolism of the polyol. d‐Xylulose barely modified the concentration of glycerol 3‐P but increased xylulose 5‐P from 0.02 to 0.5 μmol/g within 5 min. The main cause of the ATP‐ and Pi‐depleting effects of d‐xylulose was found to be an accumulation of sedoheptulose 7‐P from a basal value of 0.1 to 5 μmol/g of cells after 10 min. Ribose 5‐P increased from 0.03 to 0.5 μmol/g at 5 min. Ribose 1‐P also accumulated, albeit outside of the cells. This extracellular accumulation can be explained by the release of intracellular purine nucleoside phosphorylase from damaged hepatocytes acting on inosine that had diffused out of the cells. Smaller increases in the concentrations of sedoheptulose 7‐P and pentose phosphates were recorded after incubations of the cells with xylitol.—Vincent, M. F.; Van den Berghe, G.; Hers, H‐G. d‐Xyluloseinduced depletion of ATP and Pi in isolated rat hepatocytes. FASEB J. 3: 1855‐1861; 1989.