Nellie Taleux

Metformin activates AMP-activated protein kinase in primary human hepatocytes by decreasing cellular energy status.

Aim/hypothesisThe glucose-lowering drug metformin has been shown to activate hepatic AMP-activated protein kinase (AMPK), a master kinase regulating cellular energy homeostasis. However, the underlying mechanisms remain controversial and have never been investigated in primary human hepatocytes.MethodsHepatocytes isolated from rat, mouse and human livers were treated with various concentrations of metformin. Isoform-specific AMPKα abundance and activity, as well as intracellular adenine nucleotide levels and mitochondrial oxygen consumption rates were determined at different time points.ResultsMetformin dose- and time-dependently increased AMPK activity in rat and human hepatocytes, an effect associated with a significant rise in cellular AMP:ATP ratio. Surprisingly, we found that AMPKα2 activity was undetectable in human compared with rat hepatocytes, while AMPKα1 activities were comparable. Accordingly, metformin only increased AMPKα1 activity in human hepatocytes, although both AMPKα isoforms were activated in rat hepatocytes. Analysis of mRNA expression and protein levels confirmed that only AMPKα1 is present in human hepatocytes; it also showed that the distribution of β and γ regulatory subunits differed between species. Finally, we demonstrated that the increase in AMP:ATP ratio in hepatocytes from liver-specific Ampkα1/2 (also known as Prkaa1/2) knockout mice and humans is due to a similar and specific inhibition of the mitochondrial respiratory-chain complex 1 by metformin.Conclusions/interpretationActivation of hepatic AMPK by metformin results from a decrease in cellular energy status owing to metformin’s AMPK-independent inhibition of the mitochondrial respiratory-chain complex 1. The unique profile of AMPK subunits found in human hepatocytes should be considered when developing new pharmacological agents to target the kinase.

AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside

AICA riboside (5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside) has been extensively used in cells to activate the AMPK (AMP-activated protein kinase), a metabolic sensor involved in cell energy homoeostasis. In the present study, we investigated the effects of AICA riboside on mitochondrial oxidative; phosphorylation. AICA riboside was found to dose-dependently inhibit the oligomycin-sensitive JO2 (oxygen consumption rate) of isolated rat hepatocytes. A decrease in P(i) (inorganic phosphate), ATP, AMP and total adenine nucleotide contents was also observed with AICA riboside concentrations >0.1 mM. Interestingly, in hepatocytes from mice lacking both alpha1 and alpha2 AMPK catalytic subunits, basal JO2 and expression of several mitochondrial proteins were significantly reduced compared with wild-type mice, suggesting that mitochondrial biogenesis was perturbed. However, inhibition of JO2 by AICA riboside was still present in the mutant mice and thus was clearly not mediated by AMPK. In permeabilized hepatocytes, this inhibition was no longer evident, suggesting that it could be due to intracellular accumulation of Z nucleotides and/or loss of adenine nucleotides and P(i). ZMP did indeed inhibit respiration in isolated rat mitochondria through a direct effect on the respiratory-chain complex I. In addition, inhibition of JO2 by AICA riboside was also potentiated in cells incubated with fructose to deplete adenine nucleotides and P(i). We conclude that AICA riboside inhibits cellular respiration by an AMPK-independent mechanism that likely results from the combined intracellular P(i) depletion and ZMP accumulation. Our data also demonstrate that the cellular effects of AICA riboside are not necessarily caused by AMPK activation and that their interpretation should be taken with caution.

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

5‐Aminoimidazole‐4‐carboxamide‐1‐β‐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.

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.

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

Besides 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.

Lack of starvation-induced activation of AMP-activated protein kinase in the hypothalamus of the Lou/C rats resistant to obesity

Objective:The AMP-activated protein kinase (AMPK) is involved in the control of food intake by the hypothalamus. The aim of this work was to investigate if modification of hypothalamic AMPK regulation could be related to the spontaneous food restriction of Lou/C rats, a strain resistant to obesity exhibiting a 40% reduction in caloric intake compared with their lean Wistar counterparts.Design:Three-month-old male Lou/C rats were compared with age-matched male Wistar rats in both fed ad libitum and 24-h food deprivation state.Measurements and results:We first confirmed that starvation activated both isoforms of AMPK catalytic α subunits and enhanced the phosphorylation state of its downstream targets acetyl-CoA carboxylase and elongation factor 2 in the hypothalamus of Wistar rats. These changes were not observed in the hypothalamus of Lou/C rats. Interestingly, the starvation-induced changes in hypothalamic mRNA levels of the main orexigenic and anorexigenic neuropeptides were also blunted in the Lou/C rats. Analysis of the concentrations of circulating substrates and hormones known to regulate hypothalamic AMPK indicated that the starvation-induced changes in ghrelin, adiponectin and leptin were not observed in Lou/C rats. Furthermore, an increased phosphorylation state of signal transducer and activator of transcription 3 (STAT3), which admittedly mediates leptin signaling, was evidenced in the hypothalamus of the starved Lou/C rats, as well as modifications of expression of the leptin-sensitive genes suppressor of cytokine signaling-3 and stearoyl-coenzyme A desaturase 1. In addition, despite reduced leptin level in fed Lou/C rats, the phosphorylation state of hypothalamic STAT3 remained similar to that found in fed Wistar rats, an adaptation that could be explained by the concomitant increase in ObRb leptin receptor mRNA expression.Conclusion:Activation of hypothalamic AMPK by starvation, which stimulates food intake through changes in (an)orexigenic neuropeptides in the normal rats, was not observed in the spontaneously hypophagic Lou/C rats.

Liver mitochondrial properties from the obesity-resistant Lou/C rat

Objective:The first objective was to evaluate the influence of caloric intake on liver mitochondrial properties. The second objective was aimed at determining the impact of increasing fat intake on these properties.Design:Lou/C rats, displaying an inborn low caloric intake and resistant to diet-induced obesity, were compared to Wistar rats fed either ad libitum or pair-fed. An additional group of Lou/C rats were allowed to increase their fat intake by adjusting their diet from a standard high carbohydrate low-fat diet to a high-fat carbohydrate-free diet.Measurements:Hydrogen peroxide (H2O2) generation, oxygen consumption rate (J O2), membrane potential (ΔΨ), activity of respiratory chain complexes, cytochrome contents, oxidative phosphorylation efficiency (OPE) and uncoupling protein 2 (UCP2) expression were determined in liver mitochondria.Results:H2O2 production was higher in Lou/C than Wistar rats with glutamate/malate and/or succinate, octanoyl-carnitine, as substrates. These mitochondrial features cannot be mimicked by pair-feeding Wistar rats and remained unaltered by increasing fat intake. Enhanced H2O2 production by mitochondria from Lou/C rats is due to an increased reverse electron flow through the respiratory-chain complex I and a higher medium-chain acyl-CoA dehydrogenase activity. While J O2 was similar over a large range of ΔΨ in both strains, Lou/C rats were able to sustain higher membrane potential and respiratory rate. In addition, mitochondria from Lou/C rats displayed a decrease in OPE that cannot be explained by increased expression of UCP2 but rather to a slip in proton pumping by cytochrome oxidase.Conclusions:Liver mitochondria from Lou/C rats display higher reactive oxygen species (ROS) generation but to deplete upstream electron-rich intermediates responsible for ROS generation, these animals increased intrinsic uncoupling of cytochrome oxidase. It is likely that liver mitochondrial properties allowed this strain of rat to display higher insulin sensitivity and resist diet-induced obesity.

O32 Le mécanisme d’activation de l’AMPK hépatique par la metformine est lié à une modification de l’état énergétique cellulaire

Introduction La metformine est aujourd’hui un des principaux antidiabetiques oraux utilises dans le traitement du diabete de type 2. Son mecanisme d’action cellulaire exact reste toutefois a elucider. La decouverte de l’effet activateur de la metformine sur la proteine kinase activee par l’AMP (AMPK), une enzyme jouant un role de senseur energetique cellulaire, pourrait expliquer certaines de ses proprietes therapeutiques. Cependant, la voie de signalisation impliquee dans cette activation, notamment au niveau hepatique, reste controversee. En effet, malgre le fait qu’il ait ete montre que la metformine inhibe le complexe 1 de la chaine respiratoire mitochondriale, des travaux initiaux ont plutot conclus que celle-ci activait l’AMPK sans modifier l’etat energetique cellulaire. Cependant, cette assertion est aujourd’hui de plus en plus souvent remise en question. Le but de notre etude etait de tester l’hypothese selon laquelle l’activation de l’AMPK hepatique par la metformine est bien associee a une modification de l’energetique cellulaire. Materiels et methodes Des hepatocytes isoles de foies de rat ou humains ont ete mis en culture et traites avec differentes concentrations de metformine. L’expression et l’activite de l’AMPK ainsi que les concentrations intracellulaires des nucleotides de l’adenine ont ete determinees. La consommation d’oxygene d’hepatocytes isoles de foies de souris controle ou genetiquement inactivees pour les deux sous-unites catalytiques de l’AMPK (AMPKα1α2-/-LS) a egalement ete mesuree apres traitement a la metformine. Resultats Des concentrations therapeutiques de metformine (>100 μm) activent l’AMPK, aussi bien dans les hepatocytes murins qu’humains, cet effet etant clairement associe a une augmentation du rapport AMP/ATP. Contrairement aux hepatocytes de rats, ceux provenant de foies humains n’expriment quasiment pas d’AMPKα2 et la metformine n’active donc que l’isoforme α1 chez cette espece. De maniere interessante, l’inhibition du complexe 1 de la chaine respiratoire par la metformine persiste dans les hepatocytes de souris AMPKα1α2-/-LS demontrant que ses effets mitochondriaux sont egalement independants de l’AMPK. Conclusion Nos travaux confirment que la mitochondrie est la cible premiere de la metformine et que l’activation de l’AMPK hepatique est associee a une modification des nucleotides de l’adenine (baisse de l’ATP, augmentation du rapport AMP/ ATP), consequence de son inhibition du complexe 1 de la chaine respiratoire.