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A-769662 activates AMPK {beta}1-containing complexes but induces glucose uptake through a PI3 kinase-dependent pathway in mouse skeletal muscle
2009-01-01
5'AMP-activated protein kinase (AMPK) regulates several aspects of metabolism. Recently, A-769662 was shown to activate AMPK in skeletal muscle. However, no biological effects of AMPK activation by A-769662 in this tissue have been reported. We hypothesized that A-769662 would increase glucose uptake in skeletal muscle. We studied incubated soleus and extensor digitorum longus (EDL) muscles from 129S6/sv and C57BL/6 mice. Glucose uptake increased only in soleus from 129S6/sv when concentrations of A-769662 was 500 microM (~15%, p
MECHANISM OF ACTION OF A-769662, A VALUABLE TOOL FOR ACTIVATION OF AMP-ACTIVATED PROTEIN KINASE
2007-11-09
We have studied the mechanism of A-769662, a new activator of AMP-activated protein kinase (AMPK). Unlike other pharmacological activators it directly activates native rat AMPK by mimicking...Full Text Available
MECHANISM OF ACTION OF A-769662, A VALUABLE TOOL FOR ACTIVATION OF AMP-ACTIVATED PROTEIN KINASE
2007-11-09
Full Text Available.We have studied the mechanism of A-769662, a new activator of AMP-activated protein kinase (AMPK). Unlike other pharmacological activators it directly activates native rat AMPK by mimicking both effects of AMP, i.e. allosteric activation and inhibition of dephosphorylation. It has no effect on the isolated α subunit kinase domain with or without the associated auto-inhibitory domain, on interaction of glycogen with the β subunit glycogen-binding domain, or on binding of AMP to the isolated Bateman domains of the γ subunit. Addition of A-769662 to mouse embryo fibroblasts (MEFs) or primary mouse hepatocytes stimulates phosphorylation of acetyl-CoA carboxylase (ACC), effects that are completely abolished in AMPK-α1−/−α2−/− cells, but not in TAK1−/− MEFs. Phosphorylation of AMPK and ACC in response to A-769662 is also abolished in LKB1−/− mouse muscle. However in HeLa cells, which lack LKB1 but express the alternate upstream kinase CaMKKβ, phosphorylation of AMPK and ACC in response to A-769662 still occurs. These results show that in intact cells the effects of A-769662 are independent of the upstream kinase utilized.
Beyond AICA Riboside: In Search of New Specific AMP-activated Protein Kinase Activators
2009-01-01
Full Text Available.Summary5-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.
2009-04-01
Full Text Available.Acidic luminal pH and low [HCO3−] maintain sperm quiescent during maturation in the epididymis. The vacuolar H+-ATPase (V-ATPase) in clear cells is a major contributor to epididymal luminal acidification. We have shown previously that protein kinase A (PKA), acting downstream of soluble adenylyl cyclase stimulation by alkaline luminal pH or HCO3−, induces V-ATPase apical membrane accumulation in clear cells. Here we examined whether the metabolic sensor AMP-activated protein kinase (AMPK) regulates this PKA-induced V-ATPase apical membrane accumulation. Immunofluorescence labeling of rat and non-human primate epididymides revealed specific AMPK expression in epithelial cells. Immunofluorescence labeling of rat epididymis showed that perfusion in vivo with the AMPK activators 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) or A-769662 induced a redistribution of the V-ATPase into subapical vesicles, even in the presence of a luminal alkaline (pH 7.8) buffer compared with that of controls perfused without drug. Moreover, preperfusion with AICAR blocked the PKA-mediated V-ATPase translocation to clear cell apical membranes induced by N6-monobutyryl-cAMP (6-MB-cAMP). Purified PKA and AMPK both phosphorylated V-ATPase A subunit in vitro. In HEK-293 cells [32P]orthophosphate in vivo labeling of the A subunit increased following PKA stimulation and decreased following RNA interference-mediated knockdown of AMPK. Finally, the extent of PKA-dependent in vivo phosphorylation of the A subunit increased with AMPK knockdown. In summary, our findings suggest that AMPK inhibits PKA-mediated V-ATPase apical accumulation in epididymal clear cells, that both kinases directly phosphorylate the V-ATPase A subunit in vitro and in vivo, and that AMPK inhibits PKA-dependent phosphorylation of this subunit. V-ATPase activity may be coupled to the sensing of acid-base status via PKA and to metabolic status via AMPK.
Uncoupling of the LKB1-AMPKα Energy Sensor Pathway by Growth Factors and Oncogenic BRAFV600E
Full Text Available.BackgroundUnderstanding the biochemical mechanisms contributing to melanoma development and progression is critical for therapeutical intervention. LKB1 is a multi-task Ser/Thr kinase that phosphorylates AMPK controlling cell growth and apoptosis under metabolic stress conditions. Additionally, LKB1Ser428 becomes phosphorylated in a RAS-Erk1/2-p90RSK pathway dependent manner. However, the connection between the RAS pathway and LKB1 is mostly unknown.Methodology/Principal FindingsUsing the UV induced HGF transgenic mouse melanoma model to investigate the interplay among HGF signaling, RAS pathway and PI3K pathway in melanoma, we identified LKB1 as a protein directly modified by HGF induced signaling. A variety of molecular techniques and tissue culture revealed that LKB1Ser428 (Ser431 in the mouse) is constitutively phosphorylated in BRAFV600E mutant melanoma cell lines and spontaneous mouse tumors with high RAS pathway activity. Interestingly, BRAFV600E mutant melanoma cells showed a very limited response to metabolic stress mediated by the LKB1-AMPK-mTOR pathway. Here we show for the first time that RAS pathway activation including BRAFV600E mutation promotes the uncoupling of AMPK from LKB1 by a mechanism that appears to be independent of LKB1Ser428 phosphorylation. Notably, the inhibition of the RAS pathway in BRAFV600E mutant melanoma cells recovered the complex formation and rescued the LKB1-AMPKα metabolic stress-induced response, increasing apoptosis in cooperation with the pro-apoptotic proteins Bad and Bim, and the down-regulation of Mcl-1.Conclusions/SignificanceThese data demonstrate that growth factor treatment and in particular oncogenic BRAFV600E induces the uncoupling of LKB1-AMPKα complexes providing at the same time a possible mechanism in cell proliferation that engages cell growth and cell division in response to mitogenic stimuli and resistance to low energy conditions in tumor cells. Importantly, this mechanism reveals a new level for therapeutical intervention particularly relevant in tumors harboring a deregulated RAS-Erk1/2 pathway.
Uncoupling of the LKB1-AMPKα Energy Sensor Pathway by Growth Factors and Oncogenic BRAFV600E
BackgroundUnderstanding the biochemical mechanisms contributing to melanoma development and progression is critical for therapeutical intervention. LKB1 is a multi-task Ser/Thr kinase...Full Text Available
Targeting the AMPK pathway for the treatment of Type 2 diabetes
2009-01-01
Type 2 diabetes is one of the fastest growing public health problems worldwide resulting from both environmental and genetic factors. It is characterized by the abnormal glucose and lipid metabolism...Full Text Available
Targeting the AMPK pathway for the treatment of Type 2 diabetes
2009-01-01
Full Text Available.Type 2 diabetes is one of the fastest growing public health problems worldwide resulting from both environmental and genetic factors. It is characterized by the abnormal glucose and lipid metabolism due in part to resistance to the actions of insulin in skeletal muscle, liver and fat. This may result from inadequate adaptation to environmental changes (e.g., imbalance between energy intake and energy expenditure). AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, acts as an integrator of regulatory signals monitoring systemic and cellular energy status. The growing realization that AMPK regulates the coordination of anabolic (synthesis and storage of glucose and fatty acids) and catabolic (oxidation of glucose and fatty acids) metabolic processes represents an attractive therapeutic target for intervention in many conditions of disordered energy balance. Recent evidences that pharmacological activation of AMPK improves blood glucose homeostasis, lipid profile and blood pressure in insulin-resistant rodents, make this protein kinase a novel therapeutic target in the treatment of type 2 diabetes. Consistent with these results, physical exercise and two major classes of antidiabetic drugs (biguanides and thiazolidinediones) have recently been reported to activate AMPK. In the present review, we update these topics and discuss the concept of targeting AMPK pathway for the treatment of type 2 diabetes.
TAK1 activates AMPK-dependent cytoprotective autophagy in TRAIL-treated epithelial cells
2009-03-18
The capacity of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) to trigger apoptosis preferentially in cancer cells, although sparing normal cells, has motivated clinical development...Full Text Available
TAK1 activates AMPK-dependent cytoprotective autophagy in TRAIL-treated epithelial cells
2009-03-18
Full Text Available.The capacity of tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) to trigger apoptosis preferentially in cancer cells, although sparing normal cells, has motivated clinical development of TRAIL receptor agonists as anti-cancer therapeutics. The molecular mechanisms responsible for the differential TRAIL sensitivity of normal and cancer cells are, however, poorly understood. Here, we show a novel signalling pathway that activates cytoprotective autophagy in untransformed human epithelial cells treated with TRAIL. TRAIL-induced autophagy is mediated by the AMP-activated protein kinase (AMPK) that inhibits mammalian target of rapamycin complex 1, a potent inhibitor of autophagy. Interestingly, the TRAIL-induced AMPK activation is refractory to the depletion of the two known AMPK-activating kinases, LKB1 and Ca(2+)/calmodulin-dependent kinase kinase-β, but depends on transforming growth factor-β-activating kinase 1 (TAK1) and TAK1-binding subunit 2. As TAK1 and AMPK are ubiquitously expressed kinases activated by numerous cytokines and developmental cues, these data are most likely to have broad implications for our understanding of cellular control of energy homoeostasis as well as the resistance of untransformed cells against TRAIL-induced apoptosis.
LKB1 and AMPK and the regulation of skeletal muscle metabolism
2008-05-01
Purpose of reviewTo address the role of LKB1 and AMP-activated protein kinase (AMPK) in glucose transport, fatty acid oxidation, and metabolic adaptations in skeletal...Full Text Available
LKB1 and AMPK and the regulation of skeletal muscle metabolism
2008-05-01
Full Text Available.Purpose of reviewTo address the role of LKB1 and AMP-activated protein kinase (AMPK) in glucose transport, fatty acid oxidation, and metabolic adaptations in skeletal muscle.Recent findingsContraction-mediated skeletal muscle glucose transport is decreased in muscle-specific LKB1 knockout mice, but not in whole body AMPKα2 knockout mice or AMPKα2 inactive transgenic mice.Chronic activation of AMPK by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) and β-guanadinopropionic acid enhances mitochondrial function in skeletal muscle, but AICAR or exercise-induced increases in mitochondrial markers are preserved in skeletal muscles from whole body AMPKα2 or muscle-specific LKB1 knockout mice.Pharmacological activation of AMPK increases glucose transport and fatty acid oxidation in skeletal muscle. Therefore, chronic activation of AMPK may be beneficial in the treatment of obesity and type 2 diabetes.SummaryLKB1 and AMPK play important roles in regulating metabolism in resting and contracting skeletal muscle.
2008-04-04
Insulin increases glucose transport by stimulating the trafficking of intracellular GLUT4 to the cell surface, a process known as GLUT4 translocation. A key protein in signaling this process is AS160,...Full Text Available
2008-04-04
Full Text Available.Insulin increases glucose transport by stimulating the trafficking of intracellular GLUT4 to the cell surface, a process known as GLUT4 translocation. A key protein in signaling this process is AS160, a Rab GTPase-activating protein (GAP) whose activity appears to be suppressed by Akt phosphorylation. Tbc1d1 is a Rab GAP with a sequence highly similar to that of AS160 and with the same Rab specificity as that of AS160. The role of Tbc1d1 in regulating GLUT4 trafficking has been unclear. Our previous study showed that overexpressed Tbc1d1 inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, even though insulin caused phosphorylation on its single canonical Akt motif. In the present study, we show in 3T3-L1 adipocytes that Tbc1d1 is only 1/20 as abundant as AS160, that knockdown of Tbc1d1 has no effect on insulin-stimulated GLUT4 translocation, and that overexpressed Tbc1d1 also inhibits GLUT4 translocation elicited by activated Akt expression. These results indicate that endogenous Tbc1d1 does not participate in insulin-regulated GLUT4 translocation in adipocytes and suggest that the GAP activity of Tbc1d1 is not suppressed by Akt phosphorylation. In addition, we discovered that Tbc1d1 is much more highly expressed in skeletal muscle than fat and that the AMP-activated protein kinase (AMPK) activator 5′-aminoimidazole-4-carboxamide ribonucleoside partially reversed the inhibition of insulin-stimulated GLUT4 translocation by overexpressed Tbc1d1 in 3T3-L1 adipocytes. 5′-Aminoimidazole-4-carboxamide ribonucleoside activation of the kinase AMPK is known to cause GLUT4 translocation in muscle. The above findings strongly suggest that Tbc1d1 is a component in the signal transduction pathway leading to AMPK-stimulated GLUT4 translocation in muscle.
Full Text Available.AMP activated protein kinase (AMPK) is a key regulator of cellular metabolism. AMPK activity is modulated in part by binding of AMP to the γ-subunit of the kinase, which increases the activity of the catalytic α-subunit. Because increased AMPK activity in the liver and in skeletal muscle leads to increased fatty acid oxidation and decreased cholesterol and fatty acid biosynthesis, activators of AMPK are being sought for treatment of type-2 diabetes and other metabolic disorders. The unique mechanism of AMPK activation offers an opportunity to develop small molecules that directly upregulate AMPK activity, and there exists a need for simplified methods to identify and characterize small-molecules that show isoform-specific effects on AMPK. We have developed a suite of fluorescence-based assays to identify and characterize such compounds, and have used these to characterize and compare activity of recombinant AMPK α1β1γ1 and α2β1γ1 isoforms in response to small molecule activators and inhibitors.
AMP activated protein kinase (AMPK) is a key regulator of cellular metabolism. AMPK activity is modulated in part by binding of AMP to the γ-subunit of the kinase, which increases the activity...Full Text Available
Can Patients with Type 2 Diabetes Be Treated with AMPK-Activators?
2008-10-01
Full Text Available.AMP-activated protein kinase (AMPK) has been identified as a target for development of pharmaceuticals for prevention and treatment of type 2 diabetes. Chemical activation of AMPK in experimental animals using 5-aminoimidazole-4-carboxamide (AICAR) has previously been reported to activate AMPK, stimulate fat oxidation, stimulate glucose uptake into muscle, inhibit lipogenesis and cholesterogenesis, increase hexokinase and GLUT-4 in muscle, and increase insulin sensitivity. Animals with metabolic conditions similar to type 2 diabetes show improvement in glucoregulation with chronic AICAR injections. A more recent study of normal human subjects demonstrated increased uptake of a glucose analogue into skeletal muscle in response to AICAR infusion. The current study by Boon, et al in this issue of Diabetologia demonstrates reduction of glucose production and improvement in glucoregulation in patients with type 2 diabetes infused with AICAR at much lower doses than were previously used in rats and mice. This study provides additional evidence that AMPK-activating pharmaceuticals may be useful in prevention and treatment of type 2 diabetes.
Can Patients with Type 2 Diabetes Be Treated with AMPK-Activators?
2008-10-01
AMP-activated protein kinase (AMPK) has been identified as a target for development of pharmaceuticals for prevention and treatment of type 2 diabetes. Chemical activation of AMPK in experimental...Full Text Available
Beyond AICA Riboside: In Search of New Specific AMP-activated Protein Kinase Activators
2009-01-01
Summary5-Aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICA riboside) has been extensively used in vitro and in vivo to...Full Text Available
Akt-dependent and independent mechanisms of mTOR regulation in cancer
2009-05-01
The protein kinase mTOR (mammalian target of rapamycin) is a critical regulator of cellular metabolism, growth, and proliferation. These processes contribute to tumor formation, and many cancers...Full Text Available
Akt-dependent and independent mechanisms of mTOR regulation in cancer
2009-05-01
Full Text Available.The protein kinase mTOR (mammalian target of rapamycin) is a critical regulator of cellular metabolism, growth, and proliferation. These processes contribute to tumor formation, and many cancers are characterized by aberrant activation of mTOR. Although activating mutations in mTOR itself have not been identified, deregulation of upstream components that regulate mTOR are prevalent in cancer. The prototypic mechanism of mTOR regulation in cells is through activation of the PI3K/Akt pathway, but mTOR receives input from multiple signaling pathways. This review will discuss Akt-dependent and independent mechanisms of mTOR regulation in response to mitogenic signals, as well as its regulation in response to energy and nutrient-sensing pathways. Preclinical and clinical studies have demonstrated that tumors bearing genetic alterations that activate mTOR are sensitive to pharmacologic inhibition of mTOR. Elucidation of novel pathways that regulate mTOR may help identify predictive factors for sensitivity to mTOR inhibitors and could provide new therapeutic targets for inhibiting the mTOR pathway in cancer. This review will also highlight pharmacologic approaches that inhibit mTOR via activation of the AMP-activated protein kinase (AMPK), an important inhibitor of the mTOR pathway and an emerging target in cancer.
AMP-activated protein kinase pathway: a potential therapeutic target in cardiometabolic disease
2009-04-01
Full Text Available.AMPK (AMP-activated protein kinase) is a heterotrimetric enzyme that is expressed in many tissues, including the heart and vasculature, and plays a central role in the regulation of energy homoeostasis. It is activated in response to stresses that lead to an increase in the cellular AMP/ATP ratio caused either by inhibition of ATP production (i.e. anoxia or ischaemia) or by accelerating ATP consumption (i.e. muscle contraction or fasting). In the heart, AMPK activity increases during ischaemia and functions to sustain ATP, cardiac function and myocardial viability. There is increasing evidence that AMPK is implicated in the pathophysiology of cardiovascular and metabolic diseases. A principle mode of AMPK activation is phosphorylation by upstream kinases [e.g. LKB1 and CaMK (Ca2+/calmodulin-dependent protein kinase], which leads to direct effects on tissues and phosphorylation of various downstream kinases [e.g. eEF2 (eukaryotic elongation factor 2) kinase and p70 S6 kinase]. These upstream and downstream kinases of AMPK have fundamental roles in glucose metabolism, fatty acid oxidation, protein synthesis and tumour suppression; consequently, they have been implicated in cardiac ischaemia, arrhythmias and hypertrophy. Recent mechanistic studies have shown that AMPK has an important role in the mechanism of action of MF (metformin), TDZs (thiazolinediones) and statins. Increased understanding of the beneficial effects of AMPK activation provides the rationale for targeting AMPK in the development of new therapeutic strategies for cardiometabolic disease.
AMP-activated protein kinase pathway: a potential therapeutic target in cardiometabolic disease
2009-04-01
AMPK (AMP-activated protein kinase) is a heterotrimetric enzyme that is expressed in many tissues, including the heart and vasculature, and plays a central role in the regulation of energy homoeostasis....Full Text Available
2009-04-01
Acidic luminal pH and low [HCO3−] maintain sperm quiescent during maturation in the epididymis. The vacuolar H+-ATPase (V-ATPase) in...Full Text Available
AMPK: Lessons from transgenic and knockout animals
2009-01-01
Full Text Available.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: Lessons from transgenic and knockout animals
2009-01-01
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...Full Text Available
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