Etude du Lou/C, un rat résistant à l"obésité (contrôle de la prise alimentaire par l'AMPK hypothalamique, augmentation du métabolisme hépatique du glycérol, phénocopie partielle du Lou/C par un traitement chronique à la diiodothyronine)

Le travail décrit dans ce manuscrit s'intéresse à la résistance à l'obésité et a eu pour objectif d'étudier les particularités métaboliques du rat Lou/C, une souche résistante à l'obésité. Dans un premier temps, notre travail a consisté à déterminer si l'hypophagie spontanée du Lou/C était liée à une modification de la voie de contrôle de la prise alimentaire dépendante de l'AMPK hypothalamique. Nos résultats montrent que le jeûne n'active pas l'AMPK hypothalamique chez le Lou/C, alors qu'il le fait chez le rat témoin. Cette observation pourrait expliquer, au moins en partie, la faible prise alimentaire du Lou/C. La deuxième partie de ce travail porte sur le métabolisme énergétique hépatique. En comparaison au rat témoin, nous avons montré que les hépatocytes de Lou/C avaient une plus grande capacité à métaboliser le glycérol, reflétant une gestion des potentiels d'oxydo-réduction différente. En effet, une augmentation de l'activité de la G3PdH mitochondriale permet au Lou/C de maintenir une cellule hépatique plus oxydée, et d'oxyder plus d'acides gras. Dans la troisième partie, nous présentons les résultats concernant les effets d'un traitement chronique à la diiodothyronine sur le phénotype et le métabolisme énergétique hépatique de rats témoins. Ce traitement induit des changements qui miment le phénotype du rat Lou/C : une stabilisation de la masse corporelle, un cytosol et une mitochondrie plus oxydés associés à une augmentation de l'activité et de l'expression de la G3PdH mitochondriale, une augmentation des capacités oxydatives mitochondriales. L'ensemble de ces données suggère que la T2 pourrait être impliquée dans le phénotype du rat résistant à l'obésité, Lou/e.The work reported here aimed at studying the phenotypical and metabolic characteristics of the Lou/C rat, a strain resistant to obesity. ln the first part, we studied whether the spontaneous hypophagia of Lou/C rats could be related to alterations of the hypothalamic AMPK pathway that controls food intake. Our results showed that starvation did not activate hypothalamic AMPK in Lou/C rats whereas it did in control rats. This observation could explain, at least partly, the lower food intake of Lou/C. The second part of our work deals with hepatic energy metabolism. Compared with control rats, the Ii vers of Lou/C rats were found to have a greater capacity to use glycerol, which reflected a different redox management. This could result from a higher content and activity of the mitochondrial G3PdH, which allow Lou/C rats to keep a more oxidized state and to enhance the rate offatty acid oxidation. The last part ofthis manuscript presents our results concerning the effects of a chronic 3,5 diiodothyronine (T2) treatment on the phenotype and hepatic energy metabolism of control rats. This treatment induced, in control rats, metabolic changes that mimicked to sorne extent the phenotype of Lou/C rats: their body weight was stable, their cytosol and mitochondria redox state were more oxidized and corresponded to a higher content and activity of the mitochondrial G3PdH, their mitochondria had enhanced oxidative capacities. Taken together our results suggest that T2 might be implicated in the peculiar phenotype of the resistant to obesity Lou/C rats.GRENOBLE1-BU Sciences (384212103) / SudocSudocFranceF

Régulation du métabolisme énergétique par l’AMPK

L’ensemble du monde vivant a développé les mécanismes nécessaires à son adaptation métabolique en réponse à des contraintes externes variables, condition essentielle de survie. Que ce soit au niveau de l’organisme entier, où toute une série de mécanismes hormonaux et neuronaux peuvent agir, ou au niveau cellulaire, siège d’une régulation métabolique fine, il est impératif de répondre de manière adéquate aux modifications de l’environnement qui visent à modifier l’équilibre énergétique. Les stress énergétiques sont variés et incluent des déficits en apport énergétique (déficit en glucose, en acides aminés, en oxygène…) et/ou des augmentations en demande énergétique (croissance, exercice sportif…). L’organisme dispose de nombreux moyens pour répondre à ces changements parmi lesquels figure la protéine kinase activée par l’AMP (AMPK, AMP-activated protein kinase), caractérisée depuis peu comme un senseur métabolique. Enzyme ubiquitaire, l’AMPK participe à la régulation coordonnée du métabolisme énergétique, de la prise alimentaire et de la sensibilité des tissus en réponse à de nombreux signaux métaboliques et hormonaux. Ces propriétés lui confèrent donc un rôle de cible pharmacologique potentielle à visée métabolique (diabète, insulinorésistance, obésité) et cardiologique (ischémie cardiaque, complications liées au diabète)

Beyond AICA riboside: In search of new specific AMP-activated protein kinase activators.

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to activate the AMP-activated protein kinase (AMPK), a metabolic sensor involved in both cellular and whole body energy homeostasis. However, it has been recently highlighted that AICA riboside also exerts AMPK-independent effects, mainly on AMP-regulated enzymes and mitochondrial oxidative phosphorylation (OXPHOS), leading to the conclusion that new compounds with reduced off target effects are needed to specifically activate AMPK. Here, we review recent findings on newly discovered AMPK activators, notably on A-769662, a nonnucleoside compound from the thienopyridone family. We also report that A-769662 is able to activate AMPK and stimulate glucose uptake in both L6 cells and primary myotubes derived from human satellite cells. In addition, A-769662 increases AMPK activity and phosphorylation of its main downstream targets in primary cultured rat hepatocytes but, by contrast with AICA riboside, does neither affect mitochondrial OXPHOS nor change cellular AMP:ATP ratio. We conclude that A-769662 could be one of the new promising chemical agents to activate AMPK with limited AMPK-independent side effects. (c) 2008 IUBMB IUBMB Life, 2008

Régulation du métabolisme énergétique par l'AMPK : une nouvelle voie thérapeutique pour le traitement des maladies métaboliques et cardiaques

[Regulation of energy metabolism by AMPK: a novel therapeutic approach for the treatment of metabolic and cardiovascular diseases] The 5' AMP-activated protein kinase (AMPK) is a sensor of cellular energy homeostasis well conserved in all eukaryotic cells. AMPK is activated by rising AMP and falling ATP, either by inhibiting ATP production or by accelerating ATP consumption, by a complex mechanism that results in an ultrasensitive response. AMPK is a heterotrimeric enzyme complex consisting of a catalytic subunit a and two regulatory subunits beta and gamma. AMP activates the system by binding to the gamma subunit that triggers phosphorylation of the catalytic alpha subunit by the upstream kinases LKB1 and CaMKK beta. Once activated, it switches on catabolic pathways (such as fatty acid oxidation and glycolysis) and switches off ATP-consuming pathways (such as lipogenesis) both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression. Dominant mutations in the regulatory gamma subunit isoforms cause hypertrophy of cardiac and skeletal muscle providing a link in human diseases caused by defects in energy metabolism. As well as acting at the level of the individual cell, the system also regulates food intake and energy expenditure at the whole body level, in particular by mediating the effects of adipokines such as leptin and adiponectin. Moreover, the AMPK system is one of the probable target for the anti-diabetic drug metformin and rosiglitazone. The relationship between AMPK activation and beneficial metabolic effects provides the rationale for the development of new therapeutic strategies. Thus, pharmacological AMPK activation may, through signaling, metabolic and gene expression effects, reduce the risk of Type 2 diabetes, metabolic syndrome and cardiac diseases

Effects of a high-fat diet on energy metabolism and ROS production in rat liver.

International audienceBACKGROUND & AIMS: A high-fat diet affects liver metabolism, leading to steatosis, a complex disorder related to insulin resistance and mitochondrial alterations. Steatosis is still poorly understood since diverse effects have been reported, depending on the different experimental models used. METHODS: We hereby report the effects of an 8 week high-fat diet on liver energy metabolism in a rat model, investigated in both isolated mitochondria and hepatocytes. RESULTS: Liver mass was unchanged but lipid content and composition were markedly affected. State-3 mitochondrial oxidative phosphorylation was inhibited, contrasting with unaffected cytochrome content. Oxidative phosphorylation stoichiometry was unaffected, as were ATPase and adenine nucleotide translocator proteins and mRNAs. Mitochondrial acylcarnitine-related H(2)O(2) production was substantially higher and the mitochondrial quinone pool was smaller and more reduced. Cellular consequences of these mitochondrial alterations were investigated in perifused, freshly isolated hepatocytes. Ketogenesis and fatty acid-dependent respiration were lower, indicating a lower β-oxidation rate contrasting with higher RNA contents of CD36, FABP, CPT-1, and AcylCoA dehydrogenases. Concomitantly, the cellular redox state was more reduced in the mitochondrial matrix but more oxidized in the cytosol: these opposing changes are in agreement with a significantly higher in situ mitochondrial proton motive force. CONCLUSIONS: A high-fat diet results in both a decrease in mitochondrial quinone pool and a profound modification in mitochondrial lipid composition. These changes appear to play a key role in the resulting inhibition of fatty acid oxidation and of mitochondrial oxidative-phosphorylation associated with an increased mitochondrial ROS production. Mitochondrial quinone pool could have prospects as a crucial event, potentially leading to interesting therapeutic perspectives

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside and metformin inhibit hepatic glucose phosphorylation by an AMP-activated protein kinase-independent effect on glucokinase translocation.

AMP-activated protein kinase (AMPK) controls glucose uptake and glycolysis in muscle. Little is known about its role in liver glucose uptake, which is controlled by glucokinase. We report here that 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR), metformin, and oligomycin activated AMPK and inhibited glucose phosphorylation and glycolysis in rat hepatocytes. In vitro experiments demonstrated that this inhibition was not due to direct phosphorylation of glucokinase or its regulatory protein by AMPK. By contrast, AMPK phosphorylated liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase without affecting activity. Inhibitors of the endothelial nitric oxide synthase, stress kinases, and phosphatidylinositol 3-kinase pathways did not counteract the effects of AICAR, metformin, or oligomycin, suggesting that these signaling pathways were not involved. Interestingly, the inhibitory effect on glucose phosphorylation of these well-known AMPK activators persisted in primary cultured hepatocytes from newly engineered mice lacking both liver alpha1 and alpha2 AMPK catalytic subunits, demonstrating that this effect was clearly not mediated by AMPK. Finally, AICAR, metformin, and oligomycin were found to inhibit the glucose-induced translocation of glucokinase from the nucleus to the cytosol by a mechanism that could be related to the decrease in intracellular ATP concentrations observed in these conditions

High expression of thyroid hormone receptors and mitochondrial glycerol-3-phosphate dehydrogenase in the liver is linked to enhanced fatty acid oxidation in Lou/C, a rat strain resistant to obesity.

International audienceBesides its well recognized role in lipid and carbohydrate metabolisms, glycerol is involved in the regulation of cellular energy homeostasis via glycerol-3-phosphate, a key metabolite in the translocation of reducing power across the mitochondrial inner membrane with mitochondrial glycerol-3-phosphate dehydrogenase. Here, we report a high rate of gluconeogenesis from glycerol and fatty acid oxidation in hepatocytes from Lou/C, a peculiar rat strain derived from Wistar, which is resistant to age- and diet-related obesity. This feature, associated with elevated cellular respiration and cytosolic ATP/ADP and NAD(+)/NADH ratios, was linked to a high expression and activity of mitochondrial glycerol-3-phosphate dehydrogenase. Interestingly, this strain exhibited high expression and protein content of thyroid hormone receptor, whereas circulating thyroid hormone levels were slightly decreased and hepatic thyroid hormone carrier MCT-8 mRNA levels were not modified. We propose that an enhanced liver thyroid hormone receptor in Lou/C may explain its unique resistance to obesity by increasing fatty acid oxidation and lowering liver oxidative phosphorylation stoichiometry at the translocation of reducing power into mitochondria

The flavonoid silibinin decreases glucose-6-phosphate hydrolysis in perfused rat hepatocytes by an inhibitory effect on glucose-6-phosphatase.

BACKGROUND/AIMS: The flavonoid silibinin has been reported to be beneficial in several hepatic disorders. Recent evidence also suggests that silibinin could be beneficial in the treatment of type 2 diabetes, owing to its anti-hyperglycemic properties. However, the mechanism(s) underlying these metabolic effects remains unknown. METHODS: The effects of silibinin on liver gluconeogenesis were studied by titrating hepatocytes from starved rats with sub-saturating concentrations of various exogenous substrates in a perifusion system. Hepatocytes from fed rats were also used to investigate glycogenolysis from endogenous glycogen. The effect of silibinin on glucose-6-phosphatase kinetics was determined in intact and permeabilized rat liver microsomes. RESULTS: Silibinin induced a dose-dependent inhibition of gluconeogenesis associated with a potent decrease in glucose-6-phosphate hydrolysis. This effect was demonstrated whatever the gluconeogenic substrates used, i.e. dihydroxyacetone, lactate/pyruvate, glycerol and fructose. In addition, silibinin decreased the glucagon-induced stimulation of both gluconeogenesis and glycogenolysis, this being associated with a reduction of glucose-6-phosphate hydrolysis. Silibinin inhibits glucose-6-phosphatase in rat liver microsomes in a concentration-dependent manner that could explain the decrease in glucose-6-phosphate hydrolysis seen in intact cells. CONCLUSION: The inhibitory effect of silibinin on both hepatic glucose-6-phosphatase and gluconeogenesis suggests that its use may be interesting in treatment of type 2 diabetes