Ablation of SGK1 Impairs Endothelial Cell Migration and Tube Formation Leading to Decreased Neo-Angiogenesis Following Myocardial Infarction

Serum and glucocorticoid inducible kinase 1 (SGK1) plays a pivotal role in early angiogenesis during embryonic development. In this study, we sought to define the SGK1 downstream signalling pathways in the adult heart and to elucidate their role in cardiac neo-angiogenesis and wound healing after myocardial ischemia. To this end, we employed a viable SGK1 knockout mouse model generated in a 129/SvJ background. Ablation of SGK1 in these mice caused a significant decrease in phosphorylation of SGK1 target protein NDRG1, which correlated with alterations in NF-κB signalling and expression of its downstream target protein, VEGF-A. Disruption of these signalling pathways was accompanied by smaller heart and body size. Moreover, the lack of SGK1 led to defective endothelial cell (ECs) migration and tube formation in vitro, and increased scarring with decreased angiogenesis in vivo after myocardial infarct. This study underscores the importance of SGK1 signalling in cardiac neo-angiogenesis and wound healing after an ischemic insult in vivo

Metformin inhibits hepatic gluconeogenesis in mice independently of the LKB1/AMPK pathway via a decrease in hepatic energy state.

pages 1-15International audienceMetformin is widely used to treat hyperglycemia in individuals with type 2 diabetes. Recently the LKB1/AMP-activated protein kinase (LKB1/AMPK) pathway was proposed to mediate the action of metformin on hepatic gluconeogenesis. However, the molecular mechanism by which this pathway operates had remained elusive. Surprisingly, here we have found that in mice lacking AMPK in the liver, blood glucose levels were comparable to those in wild-type mice, and the hypoglycemic effect of metformin was maintained. Hepatocytes lacking AMPK displayed normal glucose production and gluconeogenic gene expression compared with wild-type hepatocytes. In contrast, gluconeogenesis was upregulated in LKB1-deficient hepatocytes. Metformin decreased expression of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase), while cytosolic phosphoenolpyruvate carboxykinase (Pepck) gene expression was unaffected in wild-type, AMPK-deficient, and LKB1-deficient hepatocytes. Surprisingly, metformin-induced inhibition of glucose production was amplified in both AMPK- and LKB1-deficient compared with wild-type hepatocytes. This inhibition correlated in a dose-dependent manner with a reduction in intracellular ATP content, which is crucial for glucose production. Moreover, metformin-induced inhibition of glucose production was preserved under forced expression of gluconeogenic genes through PPARgamma coactivator 1alpha (PGC-1alpha) overexpression, indicating that metformin suppresses gluconeogenesis via a transcription-independent process. In conclusion, we demonstrate that metformin inhibits hepatic gluconeogenesis in an LKB1- and AMPK-independent manner via a decrease in hepatic energy state

Role of LKB-1 and AMPK in heart metabolism and function : lessons from animal models

L’AMP-activated protéine kinase (AMPK) est un senseur énergétique cellulaire qui contrôle différentes voies métaboliques telles que le métabolisme du glucose et des acides gras, ainsi que la synthèse protéique. L’AMPK est majoritairement activée en réponse à un stress énergétique et permet de maintenir la balance énergétique en inhibant les réactions anaboliques consommatrices d’ATP et en favorisant les réactions cataboliques productrices d’ATP. L’AMPK joue un rôle prépondérant dans le tissu cardiaque qui est un organe consommant une quantité importante d’énergie nécessaire au maintien de sa capacité contractile. Il est communément admis que le rôle de l’AMPK devient particulièrement critique dans des situations pathologiques énergétiquement défavorables comme lors d’une ischémie. Notre étude a consisté à l’évaluation du rôle exact de l’AMPK dans la régulation du métabolisme et de la fonction cardiaque en situations physiologiques et pathophysiologiques comme l’ischémie et l’hypertrophie, en utilisant les modèles de souris génétiquement modifiées dans lesquelles le gène de la sous-unité catalytique α2 (α2 -/-) ou celui de la LKB-1 (LKB-1-/-) a été invalidé. Les résultats obtenus montrent que la sous unité catalytique α2 AMPK est activée exclusivement par la LKB-1 et participe à la régulation du métabolisme du glucose, des acides gras et des protéines autant en conditions physiologiques que pathologiques. L’ α2 AMPK régule clairement des voies métaboliques telles que le captage du glucose et son stockage sous forme de glycogène. Au niveau moléculaire, l’AMPK participe également à la régulation de l’état de phosphorylation de l’acetyl-CoA-carboxylase et de la p70 ribosomal S6 protein kinase impliquées, respectivement, dans le métabolisme des acides gras et dans la synthèse protéique. La modification de ces paramètres, induite par l’absence de l’ α2 AMPK et présente en conditions physiologiques, a pour conséquence l’apparition d’effets défavorables majeurs en conditions pathologiques. En effet, les animaux α2 -/- sont caractérisés par une souffrance énergétique et fonctionnelle supérieure au cours d’un épisode d’ischémie/reperfusion. Ces animaux sont également plus sensibles à l’apparition d’une hypertrophie myocardique. En conclusion, nos travaux démontrent que l’ α2 AMPK participe à la régulation du métabolisme et de la fonction cardiaque et joue un rôle protecteur évident au cours d’une ischémie/reperfusion et du développement d’une hypertrophieAMP-activated protein kinase (AMPK) is a serine/threonine protein kinase which acts as a cellular energy gauge controlling many cellular processes such as glucose and fatty acid metabolism, and protein synthesis. This enzyme is activated during cellular energy stress in an attempt to adapt cellular metabolism by reducing energy consumption and increasing energy production in order to re-establish cellular energy balance. The AMPK pathway is particularly relevant in the heart as this organ has a very high energy demand to maintain its contractile activity. It becomes especially more critical during periods of ischemia and reperfusion. In this study we evaluated the role of AMPK in heart metabolism and function under physiological and pathological conditions, such as ischemia and hypertrophy, by using different genetic models: á2 AMPK total knockout (á2-/-) and LKB-1 muscle specific knockout (LKB-1-/-). Our results showed that the á2 catalytic AMPK isoform participated in the regulation of glucose, fatty acid and protein metabolism under pathological conditions. The impact of AMPK modulation on glycogen level, glucose uptake and phosphorylation of acetyl-CoA carboxylase (ACC) and p70S6 kinase, revealed the importance of this enzyme in the regulation of cardiac metabolism under physiological conditions. Moreover, á2 AMPK deletion, by exacerbating the ischemia-induced negative consequences and the development of cardiac hypertrophy, demonstrated the importance of AMPK under pathological conditions. In conclusion, our results provide evidence that AMPK activity and presence are required for normal cardiac energy metabolism and function, and protect the heart from the development of cardiac hypertrophy.Thèse de doctorat en sciences biomédicales (SBIM 3) -- UCL, 200

MTOR hyperactivation by ablation of tuberous sclerosis complex 2 in the mouse heart induces cardiac dysfunction with the increased number of small mitochondria mediated through the down-regulation of autophagy

Mammalian target of rapamycin complex 1 (mTORC1) is a key regulator of cell growth, proliferation and metabolism. mTORC1 regulates protein synthesis positively and autophagy negatively. Autophagy is a major system to manage bulk degradation and recycling of cytoplasmic components and organelles. Tuberous sclerosis complex (TSC) 1 and 2 form a heterodimeric complex and inactivate Ras homolog enriched in brain, resulting in inhibition of mTORC1. Here, we investigated the effects of hyperactivation of mTORC1 on cardiac function and structure using cardiac-specific TSC2-deficient (TSC2-/-) mice. TSC2-/- mice were born normally at the expected Mendelian ratio. However, the median life span of TSC2-/- mice was approximately 10 months and significantly shorter than that of control mice. TSC2-/- mice showed cardiac dysfunction and cardiomyocyte hypertrophy without considerable fibrosis, cell infiltration or apoptotic cardiomyocyte death. Ultrastructural analysis of TSC2-/- hearts revealed misalignment, aggregation and a decrease in the size and an increase in the number of mitochondria, but the mitochondrial function was maintained. Autophagic flux was inhibited, while the phosphorylation level of S6 or eukaryotic initiation factor 4E -binding protein 1, downstream of mTORC1, was increased. The upregulation of autophagic flux by trehalose treatment attenuated the cardiac phenotypes such as cardiac dysfunction and structural abnormalities of mitochondria in TSC2-/- hearts. The results suggest that autophagy via the TSC2-mTORC1 signaling pathway plays an important role in maintenance of cardiac function and mitochondrial quantity and size in the heart and could be a therapeutic target to maintain mitochondrial homeostasis in failing hearts

Differential regulation of eEF2 and p70S6K by AMPKalpha2 in heart.

Eukaryotic elongation factor 2 (eEF-2) and mammalian target of rapamycin (mTOR)-p70 ribosomal protein S6 kinase (p70S6K) signaling pathways control protein synthesis and are inhibited during myocardial ischemia. Intracellular acidosis and AMP-activated protein kinase (AMPK) activation, both occurring during ischemia, have been proposed to participate in this inhibition. We evaluated the contribution of AMPKα2, the main cardiac AMPK catalytic subunit isoform, in eEF2 and mTOR-p70S6K regulation using AMPKα2 KO mice. Hearts were perfused ex vivo with or without insulin, and then submitted or not to ischemia. Insulin pre-incubation was necessary to activate mTOR-p70S6K and evaluate their subsequent inhibition by ischemia. Ischemia decreased insulin-induced mTOR-p70S6K phosphorylation in WT and AMPKα2 KO mice to a similar extent. This AMPKα2-independent p70S6K inhibition correlated well with the inhibition of PKB/Akt, located upstream of mTOR-p70S6K and can be mimicked in cardiomyocytes by decreasing pH. By contrast, ischemia-induced inhibitory phosphorylation of eEF-2 was drastically reduced in AMPKα2 KO mice. Interestingly, AMPKα2 also played a role under normoxia. Its deletion increased the insulin-induced p70S6K stimulation. This p70S6K over-stimulation was associated with a decrease in inhibitory phosphorylation of Raptor, an mTOR partner identified as an AMPK target. In conclusion, AMPKα2 controls cardiac p70S6K under normoxia and regulates eEF-2 but not the mTOR-p70S6K pathway during ischemia. This challenges the accepted notion that mTOR-p70S6K is inhibited by myocardial ischemia mainly via an AMPK-dependent mechanism

AMPKalpha2 counteracts the development of cardiac hypertrophy induced by isoproterenol.

As AMP-activated protein kinase (AMPK) controls protein translation, an anti-hypertrophic effect of AMPK has been suggested. However, there is no genetic evidence to confirm this hypothesis. We investigated the contribution of AMPKalpha2 in the control of cardiac hypertrophy by using AMPKalpha2-/- mice submitted to isoproterenol. The isoproterenol-induced cardiac hypertrophy, measured by left ventricular mass and histological examination, was significantly higher in AMPKalpha2-/- than in WT animals. Moreover, the intensification of cardiac hypertrophy found in AMPKalpha2-/- mice can be linked to the abnormal basal overstimulation of the p70 ribosomal S6 protein kinase, an enzyme known to regulate protein translation and cell growth. In conclusion, this work shows that AMPKalpha2 plays a role of brake pedal for the development of cardiac hypertrophy

Deficiency of LKB1 in heart prevents ischemia-mediated activation of AMPKalpha2 but not AMPKalpha

Recent studies indicate that the LKB1 is a key regulator of the AMP-activated protein kinase (AMPK), which plays a crucial role in protecting cardiac muscle from damage during ischemia. We have employed mice that lack LKB1 in cardiac and skeletal muscle and studied how this affected the activity of cardiac AMPKalpha1/alpha2 under normoxic, ischemic, and anoxic conditions. In the heart lacking cardiac muscle LKB1, the basal activity of AMPKalpha2 was vastly reduced and not increased by ischemia or anoxia. Phosphorylation of AMPKalpha2 at the site of LKB1 phosphorylation (Thr172) or phosphorylation of acetyl-CoA carboxylase-2, a downstream substrate of AMPK, was ablated in ischemic heart lacking cardiac LKB1. Ischemia was found to increase the ADP-to-ATP (ADP/ATP) and AMP-to-ATP ratios (AMP/ATP) to a greater extent in LKB1-deficient cardiac muscle than in LKB1-expressing muscle. In contrast to AMPKalpha2, significant basal activity of AMPKalpha1 was observed in the lysates from the hearts lacking cardiac muscle LKB1, as well as in cardiomyocytes that had been isolated from these hearts. In the heart lacking cardiac LKB1, ischemia or anoxia induced a marked activation and phosphorylation of AMPKalpha1, to a level that was only moderately lower than observed in LKB1-expressing heart. Echocardiographic and morphological analysis of the cardiac LKB1-deficient hearts indicated that these hearts were not overtly dysfunctional, despite possessing a reduced weight and enlarged atria. These findings indicate that LKB1 plays a crucial role in regulating AMPKalpha2 activation and acetyl-CoA carboxylase-2 phosphorylation and also regulating cellular energy levels in response to ischemia. They also provide genetic evidence that an alternative upstream kinase can activate AMPKalpha1 in cardiac muscle