Benoit Viollet

Systemic Treatment with the Antidiabetic Drug Metformin Selectively Impairs p53-Deficient Tumor Cell Growth

The effect of the antidiabetic drug metformin on tumor growth was investigated using the paired isogenic colon cancer cell lines HCT116 p53(+/+) and HCT116 p53(-/-). Treatment with metformin selectively suppressed the tumor growth of HCT116 p53(-/-) xenografts. Following treatment with metformin, we detected increased apoptosis in p53(-/-) tumor sections and an enhanced susceptibility of p53(-/-) cells to undergo apoptosis in vitro when subject to nutrient deprivation. Metformin is proposed to function in diabetes treatment as an indirect activator of AMP-activated protein kinase (AMPK). Treatment with AICAR, another AMPK activator, also showed a selective ability to inhibit p53(-/-) tumor growth in vivo. In the presence of either of the two drugs, HCT116 p53(+/+) cells, but not HCT116 p53(-/-) cells, activated autophagy. A similar p53-dependent induction of autophagy was observed when nontransformed mouse embryo fibroblasts were treated. Treatment with either metformin or AICAR also led to enhanced fatty acid beta-oxidation in p53(+/+) MEFs, but not in p53(-/-) MEFs. However, the magnitude of induction was significantly lower in metformin-treated cells, as metformin treatment also suppressed mitochondrial electron transport. Metformin-treated cells compensated for this suppression of oxidative phosphorylation by increasing their rate of glycolysis in a p53-dependent manner. Together, these data suggest that metformin treatment forces a metabolic conversion that p53(-/-) cells are unable to execute. Thus, metformin is selectively toxic to p53-deficient cells and provides a potential mechanism for the reduced incidence of tumors observed in patients being treated with metformin.

Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders

It is now becoming evident that the liver has an important role in the control of whole body metabolism of energy nutrients. In this review, we focus on recent findings showing that AMP‐activated protein kinase (AMPK) plays a major role in the control of hepatic metabolism. AMPK integrates nutritional and hormonal signals to promote energy balance by switching on catabolic pathways and switching off ATP‐consuming pathways, both by short‐term effects on phosphorylation of regulatory proteins and by long‐term effects on gene expression. Activation of AMPK in the liver leads to the stimulation of fatty acid oxidation and inhibition of lipogenesis, glucose production and protein synthesis. Medical interest in the AMPK system has recently increased with the demonstration that AMPK could mediate some of the effects of the fat cell‐derived adiponectin and the antidiabetic drugs metformin and thiazolidinediones. These findings reinforce the idea that pharmacological activation of AMPK may provide, through signalling and metabolic and gene expression effects, a new strategy for the management of metabolic hepatic disorders linked to type 2 diabetes and obesity.

5′-AMP-Activated Protein Kinase (AMPK) Is Induced by Low-Oxygen and Glucose Deprivation Conditions Found in Solid-Tumor Microenvironments

ABSTRACT Low oxygen gradients (hypoxia and anoxia) are important determinants of pathological conditions under which the tissue blood supply is deficient or defective, such as in solid tumors. We have been investigating the relationship between the activation of hypoxia-inducible factor 1 (HIF-1), the primary transcriptional regulator of the mammalian response to hypoxia, and 5′-AMP-activated protein kinase (AMPK), another regulatory system important for controlling cellular energy metabolism. In the present study, we used mouse embryo fibroblasts nullizygous for HIF-1α or AMPK expression to show that AMPK is rapidly activated in vitro by both physiological and pathophysiological low-oxygen conditions, independently of HIF-1 activity. These findings imply that HIF-1 and AMPK are components of a concerted cellular response to maintain energy homeostasis in low-oxygen or ischemic-tissue microenvironments. Finally, we used transformed derivatives of wild-type and HIF-1α- or AMPKα-null mouse embryo fibroblasts to determine whether AMPK is activated in vivo. We obtained evidence that AMPK is activated in authentic hypoxic tumor microenvironments and that this activity overlaps with regions of hypoxia detected by a chemical probe. We also showed that AMPK is important for the growth of this tumor model.

AMPK : Lessons from transgenic and knockout animals.

AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, has been proposed to function as a fuel gauge to monitor cellular energy status in response to nutritional environmental variations. AMPK system is a regulator of energy balance that, once activated by low energy status, switches on ATP-producing catabolic pathways (such as fatty acid oxidation and glycolysis), and switches off ATP-consuming anabolic pathways (such as lipogenesis), both by short-term effect on phosphorylation of regulatory proteins and by long-term effect on gene expression. Numerous observations obtained with pharmacological activators and agents that deplete intracellular ATP have been supportive of AMPK playing a role in the control of energy metabolism but none of these studies have provided conclusive evidence. Relatively recent developments in our understanding of precisely how AMPK complexes might operate to control energy metabolism is due in part to the development of transgenic and knockout mouse models. Although there are inevitable caveats with genetic models, some important findings have emerged. In the present review, we discuss recent findings obtained from animal models with inhibition or activation of AMPK signaling pathway.

AMPK dysregulation promotes diabetes-related reduction of superoxide and mitochondrial function

Diabetic microvascular complications have been considered to be mediated by a glucose-driven increase in mitochondrial superoxide anion production. Here, we report that superoxide production was reduced in the kidneys of a steptozotocin-induced mouse model of type 1 diabetes, as assessed by in vivo real-time transcutaneous fluorescence, confocal microscopy, and electron paramagnetic resonance analysis. Reduction of mitochondrial biogenesis and phosphorylation of pyruvate dehydrogenase (PDH) were observed in kidneys from diabetic mice. These observations were consistent with an overall reduction of mitochondrial glucose oxidation. Activity of AMPK, the major energy-sensing enzyme, was reduced in kidneys from both diabetic mice and humans. Mitochondrial biogenesis, PDH activity, and mitochondrial complex activity were rescued by treatment with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR). AICAR treatment induced superoxide production and was linked with glomerular matrix and albuminuria reduction in the diabetic kidney. Furthermore, diabetic heterozygous superoxide dismutase 2 (Sod2(+/-)) mice had no evidence of increased renal disease, and Ampka2(-/-) mice had increased albuminuria that was not reduced with AICAR treatment. Reduction of mitochondrial superoxide production with rotenone was sufficient to reduce AMPK phosphorylation in mouse kidneys. Taken together, these results demonstrate that diabetic kidneys have reduced superoxide and mitochondrial biogenesis and activation of AMPK enhances superoxide production and mitochondrial function while reducing disease activity.

The Energy Sensor AMPK Regulates T Cell Metabolic Adaptation and Effector Responses In Vivo

Naive T cells undergo metabolic reprogramming to support the increased energetic and biosynthetic demands of effector T cell function. However, how nutrient availability influences T cell metabolism and function remains poorly understood. Here we report plasticity in effector T cell metabolism in response to changing nutrient availability. Activated T cells were found to possess a glucose-sensitive metabolic checkpoint controlled by the energy sensor AMP-activated protein kinase (AMPK) that regulated mRNA translation and glutamine-dependent mitochondrial metabolism to maintain T cell bioenergetics and viability. T cells lacking AMPKα1 displayed reduced mitochondrial bioenergetics and cellular ATP in response to glucose limitation in vitro or pathogenic challenge in vivo. Finally, we demonstrated that AMPKα1 is essential for T helper 1 (Th1) and Th17 cell development and primary T cell responses to viral and bacterial infections in vivo. Our data highlight AMPK-dependent regulation of metabolic homeostasis as a key regulator of T cell-mediated adaptive immunity.

Neuroprotective Effects of Adenosine Monophosphate-Activated Protein Kinase Inhibition and Gene Deletion in Stroke

Background and Purpose— 5′ adenosine monophosphate-dependent protein kinase (AMPK) acts as a metabolic sensor. AMPK is elevated under ischemic conditions, but the role of AMPK in ischemic brain remains controversial. In this study, we examined the effects of AMPK inhibition using both pharmacological and genetic approaches in an in vivo stroke model. Methods— Focal stroke was induced by reversible middle cerebral artery occlusion in male wild-type mice as well as mice deficient in one of the isoforms of the catalytic subunit of AMPK, AMPK α-1 or α-2. Results— AMPK inhibition was neuroprotective after focal stroke. Mice deficient in AMPK α-2 demonstrated significantly smaller infarct volumes compared with wild-type littermates, whereas deletion of AMPK α-1 had no effect. Phosphorylation of a major upstream regulator of AMPK, LKB1, was also induced in stroke brain. Conclusions— AMPK activation is detrimental in a model of focal stroke. The AMPK catalytic isoform α-2 contributes to the deleterious effects of AMPK activation. AMPK inhibition leads to neuroprotection even when these agents are administered poststroke.

Activation of 5′-AMP-activated Kinase with Diabetes Drug Metformin Induces Casein Kinase Iϵ (CKIϵ)-dependent Degradation of Clock Protein mPer2

Metformin is one of the most commonly used first line drugs for type II diabetes. Metformin lowers serum glucose levels by activating 5′-AMP-activated kinase (AMPK), which maintains energy homeostasis by directly sensing the AMP/ATP ratio. AMPK plays a central role in food intake and energy metabolism through its activities in central nervous system and peripheral tissues. Since food intake and energy metabolism is synchronized to the light-dark (LD) cycle of the environment, we investigated the possibility that AMPK may affect circadian rhythm. We discovered that the circadian period of Rat-1 fibroblasts treated with metformin was shortened by 1 h. One of the regulators of the period length is casein kinase Iϵ (CKIϵ), which by phosphorylating and inducing the degradation of the circadian clock component, mPer2, shortens the period length. AMPK phosphorylates Ser-389 of CKIϵ, resulting in increased CKIϵ activity and degradation of mPer2. In peripheral tissues, injection of metformin leads to mPer2 degradation and a phase advance in the circadian expression pattern of clock genes in wild-type mice but not in AMPK α2 knock-out mice. We conclude that metformin and AMPK have a previously unrecognized role in regulating the circadian rhythm.

AMP Activated Protein Kinase-α2 Deficiency Exacerbates Pressure-Overload–Induced Left Ventricular Hypertrophy and Dysfunction in Mice

AMP activated protein kinase (AMPK) plays an important role in regulating myocardial metabolism and protein synthesis. Activation of AMPK attenuates hypertrophy in cultured cardiac myocytes, but the role of AMPK in regulating the development of myocardial hypertrophy in response to chronic pressure overload is not known. To test the hypothesis that AMPKα2 protects the heart against systolic overload–induced ventricular hypertrophy and dysfunction, we studied the response of AMPKα2 gene deficient (knockout [KO]) mice and wild-type mice subjected to 3 weeks of transverse aortic constriction (TAC). Although AMPKα2 KO had no effect on ventricular structure or function under control conditions, AMPKα2 KO significantly increased TAC-induced ventricular hypertrophy (ventricular mass increased 46% in wild-type mice compared with 65% in KO mice) while decreased left ventricular ejection fraction (ejection fraction decreased 14% in wild-type mice compared with a 43% decrease in KO mice). AMPKα2 KO also significantly exacerbated the TAC-induced increases of atrial natriuretic peptide, myocardial fibrosis, and cardiac myocyte size. AMPKα2 KO had no effect on total S6 ribosomal protein (S6), p70 S6 kinase, eukaryotic initiation factor 4E, and 4E binding protein-1 or their phosphorylation under basal conditions but significantly augmented the TAC-induced increases of p-p70 S6 kinaseThr389, p-S6Ser235, and p-eukaryotic initiation factor 4ESer209. AMPKα2 KO also enhanced the TAC-induced increase of p-4E binding protein-1Thr46 to a small degree and augmented the TAC-induced increase of p-AktSer473. These data indicate that AMPKα2 exerts a cardiac protective effect against pressure-overload–induced ventricular hypertrophy and dysfunction.

Upregulation of Mitochondrial Uncoupling Protein-2 by the AMP-Activated Protein Kinase in Endothelial Cells Attenuates Oxidative Stress in Diabetes

OBJECTIVE—Recent evidence suggests that the AMP-activated protein kinase (AMPK) is an important therapeutic target for diabetes. The present study was conducted to determine how AMPK activation suppressed tyrosine nitration of prostacyclin synthase in diabetes. RESEARCH DESIGN AND METHODS—Confluent human umbilical vein endothelial cells (HUVECs) or mice were treated with 5-amino-4-imidazole carboxamide riboside (AICAR) for the detection of AMPK phosphorylation and the expression of mitochondrial uncoupling protein (UCP)-2. RESULTS—Exposure of HUVECs to high glucose (30 mmol/l) increased superoxide anions (O2·−) and prostacyclin synthase nitration. In addition, overexpression of constitutively active AMPK (Ad-CA-AMPK) or the addition of AICAR reduced both O2·− and prostacyclin synthase nitration caused by high glucose, whereas adenoviral overexpression of dominant-negative AMPK mutants (Ad-DN-AMPK) enhanced the latter effects of high glucose. Exposure of HUVECs to either AICAR or metformin caused AMPK-dependent upregulation of both UCP-2 mRNA and UCP-2 protein. Furthermore, overexpression of UCP-2 significantly ablated both O2·− and prostacyclin synthase nitration triggered by high glucose. Furthermore, overexpression of Ad-CA-AMPK increased, whereas overexpression of Ad-DN-AMPK inhibited AICAR-induced phosphorylation of p38 kinase at Thr180/Tyr182. Inhibition of p38 kinase with SB239063, which had no effect on AICAR-induced AMPK-Thr172 phosphorylation, dose dependently suppressed AICAR-induced upregulation of UCP-2, suggesting that AMPK lies upstream of p38 kinase. Finally, AICAR markedly increased UCP-2 expression and reduced both O2·− and prostacyclin synthase nitration in diabetic wild-type mice but not in their AMPKα2-deficient counterparts in vivo. CONCLUSIONS—We conclude that AMPK activation increases UCP-2, resulting in the inhibition of both O2·− and prostacyclin synthase nitration in diabetes.