Effet de l'AICAriboside sur le métabolisme des lipides

Fatty acid mobilization from adipose tissue is regulated by hormone-sensitive lipase (HSL). This enzyme catalyzes the initial, rate-limiting step in the hydrolysis of stored triacylglycerols. HSL is also regulated by phosphorylation/dephosphorylation. Phosphorylation by cyclic-AMP-dependent protein kinase (PKA) in response to lipolytic agents such as epinephrine activates the enzyme. This initiates lipolysis, resulting in the release of free fatty acids which can be oxidized in other tissues to provide energy. The main anti-lipolytic agent, insulin, reduces the level of phosphorylation of HSL, thereby inactivating the enzyme. HSL is also phosphorylated in vitro by AMP-PK.
AMP-PK is a recently recognized multisubstrate protein kinase with appears to play a central role in the regulation of lipid metabolism. AMP-PK phosphorylates ACC at serine residues 79, 1200 and 1215. Phosphorylation of the first of these sites causes inactivation of the enzyme by both decreasing Vmax and increasing Ka for citrate. HMG-CoA reductase is phosphorylated by AMP-PK at serine 871 and this phosphorylation profoundly decreases Vmax. HSL is phosphorylated by AMP-PK at serine 565. This has no direct effect on enzyme activity, but completely prevents phosphorylation at serine 563, and consequently abolished activation by PKA. As its name suggest, the activity of AMP-PK is increased by AMP, and this occurs by a dual mechanism: a direct up to 5-fold allosteric stimulation, and an AMP-promoted phosphorylation by an upstream AMP-PK kinase, which causes a 10-fold activation. These two effects are probably due to binding to a single site on AMP-PK itself. AMP-PK kinase is also stimulated by palmitoyl-CoA. Dephosphorylation of AMP-PK is catalyzed by protein phosphatase 2C. It has been proposed that the function of the AMP-PK kinase – AMP-PK cascade is to exert a protective role when the cell is under stress, resulting in a decrease in ATP and increase in AMP. The elevation of AMP, by switching off the pathways of lipid synthesis and perhaps other ATP-consuming processes, should allow preservation of ATP for highly vital functions. AMP-PK may be part of an ancient and highly conserved regulatory system. Indeed, a structurally analogous protein kinase, with several identical functional criteria, is found in yeasts and plants.
AICAriboside (5-amino-4-imidazolecarboxamide riboside) is the nucleoside corresponding to AICAribotide (AICAR or ZMP), an intermediate of te de novo pathway of purine nucleotide biosynthesis. It is taken up and metabolized by various mammalian tissues, including rat hepatocytes. The initial step in the metabolism of AICAriboside has been reported to exert various effects in eukaryotic cells. These include inhibition of glycolysis and gluconeogenesis in isolated rat hepatocytes.
The inhibitory action of AICAriboside on glycolysis, with supplies acetyl-Coa, the common substrate if the synthesis of fatty acids and cholesterol, was the starting point of this work.
Chapter 2, described the effects of AICAriboside on fatty acid and cholesterol synthesis in isolated rat hepatocytes. We show that AICAriboside dose-dependently inhibits, up to near-complete arrest at 500 µM, the synthesis of fatty acids, measured by incorporation of tritiated water in the saponifiable lipid fraction. This inhibition was recorded in the presence of not only glucose, but also endogenous substrates and non-glycolytic precursors such as lactate/pyruvate, amino acids and acetate. AICAriboside also inhibits the incorporation of tritiated water in the non-saponifiable lipid fraction, which is composed mainly of cholesterol. This inhibition was similarly observed in the presence of the various precursors. We have verified that cholesterol synthesis was specifically inhibited by the nucleoside. Two types of experiments (cell washing after a preincubation with AICAriboside to eliminate the nucleoside, and incubation of cells with 5’-iodotubercidin, an inhibitor of adenosine kinase) have shown that the inhibitory effects of AICAriboside on lipid synthesis require its metabolization. Metabolites of AICAriboside are mainly ZMP, which accumulated to approximately 5 µmol/g of cells after 20 min, and ZTP, which can reach approximatively 1,5 µmol/g of cells after one jour of incubation with 500 µM of the nucleoside. Since accumulation of AMP, with which ZMP is structurally ralted, also inhibits lipid synthesis, we have concluded hat ZMP might be responsible for the actions of AICAriboside in cells.
Chapter 3 related the effects of the addition of AICAriboside to isolated hepatocytes on the activity states of ACC and HMG-CoA reductase. Both enzymes were progressively and dose-dependently inactivated in the presence of AICAriboside. Near-complete inactivations were recorded after 5 to 10 min with 200 – 500 µM AICAriboside. These inactivations were correlated with the intracellular accumulation of ZMP. Measurement of the total activity of ACC showed that it was not modified. The absence of effect of ZMP on the activity of purified fatty acid synthase (the enzymatic complex catalyzing fatty acid synthesis after the formation of malonyl-CoA by ACC), and on the incorporation of tritiated mevalonolactone (the product of the reaction of HMG-CoA reductase) into cholesterol, accords with the conclusion that inhibitions are exerted at the level of, respectively, ACC ad HMG-CoA reductase.
Chapter 4 describes a kinetic study of the effects of ZMP, compared with those of AMP, on purified AMP-PK from rat liver. The activity of AMP-PK was assayed by measuring the phosphorylation of a peptide, termed SAMS, baserd in the sequence around serine 79, the site of ACC which is specifically phosphorylated by AMP-PK. The study has shown that ZMP can stimulate allosterically AMP-PK to the same extent as AMP, although markedly higher concentrations are required. Nevertheless, the stimulatory concentrations are the same as those measured after incubation of cells with AICAriboside. Indeed, half-maximal stimulation, measured in the presence of physiological concentrations of AMP and ATP, was obtained at about 5 mM ZMP. These findings can thus explain that inactivations of ACC and HMG-CoA reductase, both substrates of AMP-PK. AMP and ZMP have the same effects on the substrates saturation curves for the SAMS-peptide and ATP. Studies of the interaction between ZMP and AMP indicated competition at the same binding site. A study of the specificity of this site performed with other nucleoside monophosphates has shown that the N6 amino group of the purine cycle seems required for binding to AMP-PK.
Chapter 5 reports a study of the effect of AICAriboside on the phosphorylation state of AMP-PK in isolated hepatocytes. The measurements of the activity of AMP-PK were performed at saturating concentrations of the allosteric activator AMP in extracts of hepatocytes that had been incubated with the nucleoside. Under these conditions changes in activity reflect covalent modifications of AMP-PK. Incubation with AICAriboside induced a dose-dependent activation of AMP-PK, correlated with the accumulation of ZMP. We conclude that ZMP, similarly to AMP, also has the ability to stimulate AMP-PK kinase thereby inducing a phosphorylation which results in activation of AMP-PK. This demonstrates that the accumulation f ZMP, which follows the addition of AICAriboside to suspensions of isolated hepatocytes, increases the activity of AMP-PK, both by allosteric stimulation and by activation.
Chapter 6 is a study of the effect of AICAriboside on lipolysis in adipocytes isolated from rat epididymal tissue. Our results show that ZMP accumulates in these cells (reaching a concentration similar to that of ATP) and that preincubation with AICARiboside prevents in a dose-dependent manner the release of glycerol induced by epinephrine. The anti-lipolytic action of AICAriboside is similar to that of insulin. 5-iodotubercidin prevents the effect of AICAriboside. These results suggest that ZMP stimulated AMP-PK in adipocytes leading to phosphorylation of HSL, thereby preventing phosphorylation and activation by PKA

In this thesis we report a study of the effects of AICAribosis (5-amino 4-imidazolecarboxamide riboside) on the synthesis of fatty acids and cholesterol in isolated rat hepatocytes. The influence of AICAriboside on lipolysis in rat adipose tissue has also been briefly investigated.
Chapter 1 is devoted to a short description of the three pathways mentioned, and to a survey of the recent literature concerning the regulation of their rate-limiting enzymes. The metabolism and metabolic effects of AICAriboside are also reviewed.
Lipids are essential components of mammalian cell membranes, play a crucial role in energy storage, and provide critical messengers for cellular regulation. Both fatty acid and cholesterol synthesis are most active in the liver. The rate-limiting enzymes of both pathways are acetyl-CoA carboxylase and 3-hydroxy-3-methylglutaryl-CoA reductase, respectively.
Acetyl-Coa carboxylase (ACC) catalyzes the ATP-dependent carboxylation of acetyl-CoA into malonyl-CoA. It is subjected to three regulatory mechanisms. An allosteric regulation is accomplished by the fatty acid precursor, citrate, which stimulates the enzyme, and by the end-products of fatty acid synthesis, fatty acylc-CoA, which are feed-back inhibitors. Covalent modifications, catalyzed by protein kinases and phosphatises, convert ACC from an active dephosphorylated form, into an inactive phosphorylated form and vice-versa. The purified enzyme can be phosphorylated in vitro on several serine residues by a variety of protein kinases, including cAMP- and calcium/calmodulin-dependent protein kinases, protein kinase C, casein kinase 1 and 2, and AMP-activated protein kinase (AMP-PK). In vivo, however, phosphorylation of ACC seems to be mainly performed by AMP-PK. Dephosphorylation of ACC is predominantly accomplished by protein phosphatise 2A. The allosteric and covalent regulations of ACC are linked. Indeed, dephospharylation of the enzyme transforms it into a form which is capable of functioning in the presence of physiological concentrations of this relevant cellular metabolites. The third regulatory mechanism of ACC is a long term control of its activity. It depends on nutritional status and involves changes in the levels of the enzyme’s mRNA, and of the rates of synthesis and degradation of the enzyme protein.
3-hydroxy-3-methylglutaryl-CoA reductase (HGM-CoA reductase) catalyzes the reduction of HGM-CoA into mevalonate by two equivalents pf NADPH. The expression of the enzyme is tightly controlled by several feedback-regulation mechanisms involving repression of transcription of the HMG-CoA gene, inhibition of translation of its mRNA and stimulation of degradation of the enzyme protein by mevalonate-derived products or hormones. In addition, similarly to ACC, HGM-CoA reductase is regulated by interconversion of an active, dephosphorylated form, into an inactive, phosphorylated form. Three protein kinases can phosphorylate HMG-CoA reductase on a single serine residue in vitro, but in intact cells this phosphorylation seems to be performed only by AMP-PK. Protein phosphatase 2A is apparently responsible for dephoshorylation of HGM-CoA reductase.

L’AICAriboside (amino imidazole carboxamide riboside) est la forme déphosphorylée de l’AICAribotide ou AICAR. E dernier, plus souvent appelé ZMP à l’heure actuelle, est un intermédiaire de la voie de synthèse des nucléotides puriques.
Nous avons montré que l’AICAriboside inhibe la synthèse des acides gras et celle du cholestérol dans des hépatocytes isolés de rat, et qu’il freine la libération des acides gras sous l’action de l’adrénaline dans des adipocytes isolés à partir de tissu graisseux épididymaire de rat.
Nous proposons que le mécanisme de ces actions se fasse selon la séquence suivante : le métabolite monophosphorylé de l’AICAriboside, le ZMP, s’accumule dans les hépatocytes et les adipocytes et y induit une augmentation de l’activité de la protéine kinase dépendante de l’AMP (AMP-PK) en se comportant comme l’AMP, dont il est un analogue structurel, c’est-à-dire en stimulant allostrériquement l’enzyme et en provoquant sa phosphorylation par une AMP-PK kinase. L’AMP-PK activée phosphoryle alors les enzymes limitantes des voies métaboliques mentionnées ci-dessus, à savoir l’acétyl coenzyme A carboxylase (ACC) pour la synthèse des acides gras, l’3-hydroxy-3-méthylglutayl coenzyme A réductase (HGM-CoA) pour la synthèse du cholestérol et l’hormone)sensitive lipase (HSL) pour la lipolyse. La phosphorylation des deux premières aboutit à leur inactivation et à l’inhibition des deux voies de synthèse. La phosphorylation de l’HSL par l’AMP-PK empêche la phospharylation ainsi que l’activation de la même enzyme par la protéine kinase dépendante de l’AMP cyclique sous l’action des agents β-adrénergiques.
Les éléments-clé nous permettant de tirer ces conclusions sont les suivants : (1) Après addition d’AICA-riboside à la suspension cellulaire, nous avons mesuré des concentrations de ZMP de l’ordre du millimolaire, tant dans les hépatocytes que dans les adipocytes. (2) Une étude cinétique réalisée sur une préparation purifiée d’AMP-PK nous a montré que le ZMP stimulait effectivement l’enzyme avec la même intensité que l’AMP en se liant au même site allostérique pour lequel les deux effecteurs entrent en compétition, mais avec une affinité environ 15 fois supérieure pour l’AMP que pour le ZMP. Toutefois, en présence d’AICAriboside, les concentrations intracellulaires de ZMP sont équivalentes à celles nécessaires pour stimuler l’enzyme. (3) L’AMP-PK est activée dans les hépatocytes isolés incubés en présence d’AICAriboside et l’intensité de cet effet est corrélée avec la concentration de ZMP mesurée dans les cellules. (4) L’activité de l’ACC et celle de l’HMG-CoA réductase dans les hépatocytes sont abaissées en présence des concentrations d’AICAriboside qui provoquent l’activation de l’AMP-PJ. (5) Bien que n’ayant pas réalisé de mesure directe de l’activité de l’HSL dans les adipocytes, l’observation qui nous avons faite d’une inhibition de l’action lipolytique de l’adrénaline due à la présence d’AICAriboside dans le milieux d’incubation est en accord avec la propriété connue de cette enzyme d’être phosphorylée par l’AMP-PK et de devenir ainsi insensible à une activation par le PKA.
Ces résultats ouvrent des perspectives thérapeutiques : l’AICAriboside est le sel composé connu qui inhibe de façon parallèle la synthèse des acides gras et celle du cholestérol et pourrait donc être utilisée dans le traitement de l’hypercholestérolémie et de l’hypertriglycéridémie associées