Cellular energy sensing and signaling by AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an energy sensing/signaling protein that, when activated, increases ATP production by stimulating glucose uptake and fatty acid oxidation while at the same time inhibiting ATP=consuming processes such as protein synthesis. Chronic activation of AMPK inhibits expression of lipogenic enzymes in the liver and enhances expression of mitochondrial oxidative enzymes in skeletal muscle. Deficiency of muscle LKB1, the upstream kinase of AMPK, results in greater fluctuation in energy charge during muscle contraction and decreased capacity for exercise at higher work rates. Because AMPK enhances both glucose uptake and fatty acid oxidation in skeletal muscle, it has become a target for prevention and treatment of type 2 diabetes and obesity.     

Regulation of Na+-coupled glucose carrier SGLT1 by AMP-activated protein kinase

Abstract

AMP-activated protein kinase (AMPK), a serine/threonine kinase activated upon energy depletion, stimulates energy production and limits energy utilization. It has previously been shown to enhance cellular glucose uptake through the GLUT family of facilitative glucose transporters. The present study explored the possibility that AMPK may regulate Na⁺-coupled glucose transport through SGLT1 (SLC5A1). To this end, SGLT1 was expressed in Xenopus oocytes with and without AMPK and electrogenic glucose transport determined by dual electrode voltage clamping experiments. In SGLT1-expressing oocytes but not in oocytes injected with water or expressing constitutively active ^γR70QAMPK (α1β1γ1(R70Q)) alone, the addition of glucose to the extracellular bath generated a current (I_g), which was half maximal (K_M) at ≈ 650 μM glucose concentration. Coexpression of ^γR70QAMPK did not affect K_M but significantly enhanced the maximal current (≈ 1.7 fold). Coexpression of wild type AMPK or the kinase dead ^αK45RAMPK mutant (α1(K45R)β1γ1) did not appreciably affect I_g. According to confocal microscopy and Western Blotting, AICAR (1 mM), phenformin (1 mM) and A-769662 (10 μM) enhanced the SGLT1 protein abundance in the cell membrane of Caco2 cells suggesting that AMPK activity may increase membrane translocation of SGLT1. These observations support a role for AMPK in the regulation of Na⁺-coupled glucose transport.     

Opinion: alternative views of AMP-activated protein kinase

Genes most closely related to adenosine monophosphate (AMP)-activated protein kinase, including SAD kinases and Par-1 regulate cell polarity, although AMP-activated protein kinase (AMPK) modulates cellular energy status. LKB1 (Par-4) is required for normal activation of AMPK in the liver and also regulates cell polarity. AMPK is proposed to inhibit energy consuming activity while initiating energy producing activity during energy limitation. Demonstration that metformin, a common drug for Type 2 diabetes, requires LKB1 for full therapeutic benefit has increased interest in AMPK signaling. Despite the potential importance of AMPK signaling for diabetes, metabolic syndrome and even cancer, the developmental processes regulated by AMPK in genetically mutant animals require further elucidation. Mouse conditional null mutants for AMPK activity will allow genetic elucidation of AMPK function in vivo. This perspective focuses on sequence and structural moieties of AMPK and genetic analysis of AMPK mutations. Interestingly, the predicted protein structure of the carboxy-terminus of AMPKα resembles the carboxy-terminal KA-1 domain of MARK3, a Par-1 orthologue.     

Isodihydrocapsiate stimulates plasma glucose uptake by activation of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme that is implicated as a key factor in controlling whole body homeostasis, including fatty acid oxidation and glucose uptake. We report that a synthetic structural isomer of dihydrocapsiate, isodihydrocapsiate (8-methylnonanoic acid 3-hydroxy-4-methoxy benzyl ester) improves type 2 diabetes by activating AMPK through the LKB1 pathway. In L6 myotube cells, phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) and glucose uptake were significantly increased, whereas these effects were attenuated by an AMPK inhibitor, compound C. In addition, increased phosphorylation of AMPK and ACC by isodihydrocapsiate was significantly reduced by radicicol, an LKB1 destabilizer, suggesting that increased glucose uptake in L6 cells with isodihydrocapsiate treatment is predominantly accomplished by a LKB1-mediated AMPK activation pathway. Oral administration of isodihydrocapsiate to diabetic (db/db) mice reduced blood glucose levels by 40% after a 4-week treatment period. Our results support the development of isodihydrocapsiate as a potential therapeutic agent to target AMPK in type 2 diabetes.     

LKB1, an upstream AMPK kinase, regulates glucose and lipid metabolism in cultured liver and muscle cells

LKB1 is a 50 kDa serine/threonine kinase that phosphorylates and activates the catalytic subunit of AMPK at its T-loop residue Thr 172. We prepared adenoviruses expressing the constitutive active (wild-type) form (CA) or dominant negative (kinase inactive, D194A mutant) form (DN) of LKB1 and overexpressed these proteins in cultured myotubes (C2C12 cells) and rat hepatoma cells (FAO cells). When analyzed by immunoblotting with the antibody against Thr172-phosphorylated AMPK, the phosphorylation of AMPK was increased (2.5-fold) and decreased (0.4-fold) in cells expressing CA and DN LKB1, respectively, as compared with Lac-Z expressing control cells. Immunoprecipitation experiments, using isoform-specific antibody, revealed these alterations of AMPK phosphorylation to be attributable to altered phosphorylation of AMPK alpha2, but not alpha1 catalytic subunits, strongly suggesting the alpha2 catalytic subunit to be the major substrate for LKB1 in mammalian cells. In addition, adiponectin or AICAR-stimulated AMPK phosphorylation was inhibited by overexpression of DN LKB1, while phenformin-stimulated phosphorylation was unaffected. These results may explain the difference in AMPK activation mechanisms between AMP and phenformin, and also indicate that AMPK phosphorylation by LKB1 is involved in AMP-stimulated AMPK activation. As a downstream target for AMPK, AICAR-induced glucose uptake and ACCbeta phosphorylation were found to be significantly reduced in DN LKB1 expressing C2C12 cells. The expression of key enzymes for gluconeogenesis, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, was also dependent on LKB1 activities in FAO cells. These results demonstrate that LKB1 is a crucial regulator of AMPK activation in muscle and liver cells and, therefore, that LKB1 activity is potentially of importance to our understanding of glucose and lipid metabolism.     

Molecular mechanisms of tumor suppression by LKB1

The LKB1 tumor suppressor gene is frequently mutated in sporadic lung adenocarcinomas and cervical cancers and germline mutations are causative for Peutz-Jeghers syndrome characterized by gastrointestinal polyposis. The intracellular LKB1 kinase is implicated in regulating polarity, metabolism, cell differentiation, and proliferation – all functions potentially contributing to tumor suppression. LKB1 acts as an activating kinase of at least 14 kinases mediating LKB1 functions in a complex signaling network with partial overlaps. Regulation of the LKB1 signaling network is highly context dependent, and spatially organized in various cellular compartments. Also the mechanisms by which LKB1 activity suppresses tumorigenesis is context dependent, where recent observations are providing hints on the molecular mechanisms involved.     

Concurrent regulation of AMP-activated protein kinase and SIRT1 in mammalian cells

We examined in HepG2 cells whether glucose-induced changes in AMP-activated protein kinase (AMPK) activity could be mediated by SIRT1, an NAD⁺-dependent histone/protein deacetylase that has been linked to the increase in longevity caused by caloric restriction. Incubation with 25 vs. 5 mM glucose for 6 h concurrently diminished the phosphorylation of AMPK (Thr 172) and ACC (Ser 79), increased lactate release, and decreased the abundance and activity of SIRT1. In contrast, incubation with pyruvate (0.1 and 1 mM) for 2 h increased AMPK phosphorylation and SIRT1 abundance and activity. The putative SIRT1 activators resveratrol and quercetin also increased AMPK phosphorylation. None of the tested compounds (low or high glucose, pyruvate, and resveratrol) significantly altered the AMP/ATP ratio. Collectively, these findings raise the possibility that glucose-induced changes in AMPK are linked to alterations in SIRT1 abundance and activity and possibly cellular redox state.     

Diet-induced obese rats exhibit impaired LKB1-AMPK signaling in hypothalamus and adipose tissue

AMPK not only acts as a sensor of cellular energy status but also plays a critical role in the energy balance of the body. In this study, LKB1-AMPK signaling was investigated in diet-induced obese (DIO) and diet resistant (DR) rats. In hypothalamus, DIO rats had lower level of LKB1, AMPKα and pAMPKα than chow-fed or DR rats. Both orexigenic peptide NPY and anorexigenic peptide POMC expression were reduced in hypothalamus of DIO rats. i.c.v. injection of AICAR, an activator of AMPK, increased NPY expression but did not alter POMC expression in DIO rats. In periphery, LKB1 protein content and pAMPKα level were lower in the adipose tissue of DIO rats compared to chow-fed and DR rats. Moreover, pAMPKα and LKB1 protein levels obtained from epididymal fat pad were inversely correlated with epididymal fat mass. LKB1 protein content and pAMPKα in skeletal muscle of DIO rats were not different from those in the muscles of chow-fed and DR rats. In summary, DIO rats, but not DR rats, have impaired LKB1-AMPK signaling in hypothalamus and adipose tissue, suggesting the disturbed energy balance observed in DIO rats is related with abnormalities of AMPK signaling in a tissue specific manner.     

14-3-3 protein directly interacts with the kinase domain of calcium/calmodulin-dependent protein kinase kinase (CaMKK2)

Background

Calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a member of the Ca²⁺/calmodulin-dependent kinase (CaMK) family involved in adiposity regulation, glucose homeostasis and cancer. This upstream activator of CaMKI, CaMKIV and AMP-activated protein kinase is inhibited by phosphorylation, which also triggers an association with the scaffolding protein 14-3-3. However, the role of 14-3-3 in the regulation of CaMKK2 remains unknown.

Lopinavir impairs protein synthesis and induces eEF2 phosphorylation via the activation of AMP-activated protein kinase

HIV anti-retroviral drugs decrease protein synthesis, although the underlying regulatory mechanisms of this process are not fully established. Therefore, we investigated the effects of the HIV protease inhibitor lopinavir (LPV) on protein metabolism. We also characterized the mechanisms that mediate the effects of this drug on elongation factor-2 (eEF2), a key component of the translational machinery. Treatment of C2C12 myocytes with LPV produced a dose-dependent inhibitory effect on protein synthesis. This effect was observed at 15 min and was maintained for at least 4 h. Mechanistically, LPV increased the phosphorylation of eEF2 and thereby decreased the activity of this protein. Increased phosphorylation of eEF2 was associated with increased activity of its upstream regulators AMP-activated protein kinase (AMPK) and eEF2 kinase (eEF2K). Both AMPK and eEF2K directly phosphorylated eEF2 in an in vitro kinase assay suggesting two distinct paths lead to eEF2 phosphorylation. To verify this connection, myocytes were treated with the AMPK inhibitor compound C. Compound C blocked eEF2K and eEF2 phosphorylation, demonstrating that LPV affects eEF2 activity via an AMPK-eEF2K dependent pathway. In contrast, incubation of myocytes with rottlerin suppressed eEF2K, but not eEF2 phosphorylation, suggesting that eEF2 can be regulated independent of eEF2K. Finally, LPV did not affect PP2A activity when either eEF2 or peptide was used as the substrate. Collectively, these results indicate that LPV decreases protein synthesis, at least in part, via inhibition of eEF2. This appears regulated by AMPK which can act directly on eEF2 or indirectly via the action of eEF2K.     

Inhibition of Kir2.1 (KCNJ2) by the AMP-activated protein kinase

The inward rectifier K⁺ channel Kir2.1 participates in the maintenance of the cell membrane potential in a variety of cells including neurons and cardiac myocytes. Mutations of KCNJ2 encoding Kir2.1 underlie the Andersen–Tawil syndrome, a rare disorder clinically characterized by periodic paralysis, cardiac arrhythmia and skeletal abnormalities. The maintenance of the cardiac cell membrane potential is decreased in ischaemia, which is known to stimulate the AMP-activated serine/threonine protein kinase (AMPK). This energy-sensing kinase stimulates energy production and limits energy utilization. The present study explored whether AMPK regulates Kir2.1. To this end, cRNA encoding Kir2.1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), of the kinase dead mutant ^αK45RAMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. Kir2.1 activity was determined in two-electrode voltage-clamp experiments. Moreover, Kir2.1 protein abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced Kir2.1-mediated currents and Kir2.1 protein abundance in the cell membrane. Expression of wild type Nedd4-2 or of Nedd4-2^S795A lacking an AMPK phosphorylation consensus sequence downregulated Kir2.1 currents. The effect of wild type Nedd4-2 but not of Nedd4-2^S795A was significantly augmented by additional coexpression of AMPK. In conclusion, AMPK is a potent regulator of Kir2.1. AMPK is at least partially effective through phosphorylation of the ubiquitin ligase Nedd4-2.     

Activation of lipoprotein lipase by glucose-dependent insulinotropic polypeptide in adipocytes. A role for a protein kinase B, LKB1, and AMP-activated protein kinase cascade

Glucose-dependent insulinotropic polypeptide (GIP) has been mainly studied because of its glucose-dependent insulinotropic action and its ability to regulate beta-cell proliferation and survival. Considerably less is known about the effects of GIP on fat metabolism, and the present study was directed at identifying the mechanisms underlying its stimulatory action on lipoprotein lipase (LPL). In differentiated 3T3-L1 adipocytes, GIP, in the presence of insulin, increased LPL activity and triglyceride accumulation through a pathway involving increased phosphorylation of protein kinase B (PKB) and reductions in phosphorylated LKB1 and AMP-activated protein kinase (AMPK). Knockdown of AMPK using RNA interference and application of the AMPK inhibitor, Compound C, supported this conclusion. In contrast, the other major incretin hormone, glucagon-like peptide-1, exhibited no significant effects on LPL activity or PKB, LKB1, or AMPK phosphorylation. Cultured subcutaneous human adipocytes showed similar responses to GIP but with greater sensitivity. Chronic elevation of circulating GIP levels in the Vancouver diabetic fatty Zucker rat in vivo resulted in increased LPL activity and elevated triglyceride accumulation in epididymal fat tissue, combined with a modulation of PKB, LKB1, and AMPK phosphorylation similar to that observed in vitro. This appears to be the first demonstration of a GIP-stimulated signal transduction pathway involved in increasing fat storage in adipocytes.     

Role of deleted in breast cancer 1 (DBC1) protein in SIRT1 deacetylase activation induced by protein kinase A and AMP-activated protein kinase

The NAD(+)-dependent deacetylase SIRT1 is a key regulator of several aspects of metabolism and aging. SIRT1 activation is beneficial for several human diseases, including metabolic syndrome, diabetes, obesity, liver steatosis, and Alzheimer disease. We have recently shown that the protein deleted in breast cancer 1 (DBC1) is a key regulator of SIRT1 activity in vivo. Furthermore, SIRT1 and DBC1 form a dynamic complex that is regulated by the energetic state of the organism. Understanding how the interaction between SIRT1 and DBC1 is regulated is therefore essential to design strategies aimed to activate SIRT1. Here, we investigated which pathways can lead to the dissociation of SIRT1 and DBC1 and consequently to SIRT1 activation. We observed that PKA activation leads to a fast and transient activation of SIRT1 that is DBC1-dependent. In fact, an increase in cAMP/PKA activity resulted in the dissociation of SIRT1 and DBC1 in an AMP-activated protein kinase (AMPK)-dependent manner. Pharmacological AMPK activation led to SIRT1 activation by a DBC1-dependent mechanism. Indeed, we found that AMPK activators promote SIRT1-DBC1 dissociation in cells, resulting in an increase in SIRT1 activity. In addition, we observed that the SIRT1 activation promoted by PKA and AMPK occurs without changes in the intracellular levels of NAD(+). We propose that PKA and AMPK can acutely activate SIRT1 by inducing dissociation of SIRT1 from its endogenous inhibitor DBC1. Our experiments provide new insight on the in vivo mechanism of SIRT1 regulation and a new avenue for the development of pharmacological SIRT1 activators targeted at the dissociation of the SIRT1-DBC1 complex.     

AMP-activated protein kinase positively regulates FGF-2-stimulated VEGF synthesis in osteoblasts

AMP-activated protein kinase (AMPK) is recognized as a regulator of energy homeostasis. We have previously reported that basic fibroblast growth factor (FGF-2) stimulates vascular endothelial growth factor (VEGF) release through the activation of p44/p42 mitogen-activated protein (MAP) kinase and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) in osteoblast-like MC3T3-E1 cells. In the present study, we investigated the involvement of AMPK in FGF-2-stimulated VEGF release in these cells. FGF-2 time-dependently induced the phosphorylation of AMPK α-subunit (Thr-172). Compound C, an AMPK inhibitor, which suppressed the FGF-2-induced phosphorylation of AMPK, significantly inhibited the VEGF release stimulated by FGF-2. The AMPK inhibitor also reduced the mRNA expression of VEGF induced by FGF-2. The FGF-2-induced phosphorylation of both p44/p42 MAP kinase and SAPK/JNK was attenuated by compound C. These results strongly suggest that AMPK positively regulates the FGF-2-stimulated VEGF synthesis via p44/p42 MAP kinase and SAPK/JNK in osteoblasts.     

LKB1 and AMPK instruct cone nuclear position to modify visual function

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Loss of AMP-activated protein kinase in X-linked adrenoleukodystrophy patient-derived fibroblasts and lymphocytes

X-Adrenoleukodystrophy (X-ALD) is a peroxisomal disorder characterized by accumulation of very-long-chain (VLC) fatty acids, which induces inflammatory disease and alterations in cellular redox, both of which are reported to play a role in the pathogenesis of the severe form of the disease (childhood cerebral ALD). While the mutation defect in ABCD1 gene is common to all forms of X-ALD it fails to account for the spectrum of phenotypic variability seen in X-ALD patients, strongly suggesting a role for as yet unidentified modifier gene(s). Here we report, for the first time, loss of AMP-activated protein kinase alpha1 (AMPKα1) in patient-derived fibroblasts and lymphocytes of the severe cerebral form of X-ALD (ALD), and not in the milder adrenomyeloneuropathy (AMN) form. Decrease in AMPK was observed at both protein and mRNA levels. AMPK loss in ALD patient-derived fibroblasts was associated with increased ubiquitination. Using the Seahorse Bioscience XF^e96 Flux Analyzer for measuring the mitochondrial oxygen consumption and extracellular acidification rate we show that ALD patient-derived fibroblasts have a significantly lower “metabolic state” than AMN fibroblasts. Unstimulated ALD patient-derived lymphocytes had significantly higher proinflammatory gene expression. Selective AMPK loss represents a novel physiopathogenic factor in X-ALD disease mechanism. Strategies aimed at upregulating/recovering AMPK levels might have beneficial therapeutic effects in X-ALD.     

Activation of protein kinase C zeta by peroxynitrite regulates LKB1-dependent AMP-activated protein kinase in cultured endothelial cells

We previously reported the phosphoinositide 3-kinase-dependent activation of the 5'-AMP-activated kinase (AMPK) by peroxynitrite (ONOO-) and hypoxia-reoxygenation in cultured endothelial cells. Here we show the molecular mechanism of activation of this pathway. Exposure of bovine aortic endothelial cells to ONOO- significantly increased the phosphorylation of both Thr172 of AMPK and Ser1179 of endothelial nitric-oxide synthase, a known downstream enzyme of AMPK. In addition, activation of AMPK by ONOO- was accompanied by increased phosphorylation of protein kinase Czeta (PKCzeta) (Thr410/403) and translocation of cytosolic PKCzeta into the membrane. Further, inhibition of PKCzeta abrogated ONOO- -induced AMPK-Thr172 phosphorylation as that of endothelial nitric-oxide synthase. Furthermore, overexpression of a constitutively active PKCzeta mutant enhanced the phosphorylation of AMPK-Thr172, suggesting that PKCzeta is upstream of AMPK activation. In contrast, ONOO- activated PKCzeta in LKB1-deficient HeLa-S3 but affected neither AMPK-Thr172 nor AMPK activity. These data suggest that LKB1 is required for PKCzeta-enhanced AMPK activation. In vitro, recombinant PKCzeta phosphorylated LKB1 at Ser428, resulting in phosphorylation of AMPK at Thr172. Further, direct mutation of Ser428 of LKB1 into alanine, like the kinase-inactive LKB1 mutant, abolished ONOO- -induced AMPK activation. In several cell types originating from human, rat, and mouse, inhibition of PKCzeta significantly attenuated the phosphorylation of both LKB1-Ser428 and AMPK-Thr172 that were enhanced by ONOO-. Taken together, we conclude that PKCzeta can regulate AMPK activity by increasing the Ser428 phosphorylation of LKB1, resulting in association of LKB1 with AMPK and consequent AMPK Thr172 phosphorylation by LKB1.     

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca²⁺-dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.     

Inhibition of the heterotetrameric K+ channel KCNQ1/KCNE1 by the AMP-activated protein kinase

Abstract

The heterotetrameric K⁺-channel KCNQ1/KCNE1 is expressed in heart, skeletal muscle, liver and several epithelia including the renal proximal tubule. In the heart, it contributes to the repolarization of cardiomyocytes. The repolarization is impaired in ischemia. Ischemia stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase, sensing energy depletion and stimulating several cellular mechanisms to enhance energy production and to limit energy utilization. AMPK has previously been shown to downregulate the epithelial Na⁺ channel ENaC, an effect mediated by the ubiquitin ligase Nedd4-2. The present study explored whether AMPK regulates KCNQ1/KCNE1. To this end, cRNA encoding KCNQ1/KCNE1 was injected into Xenopus oocytes with and without additional injection of wild type AMPK (AMPKα1 + AMPKβ1 + AMPKγ1), of the constitutively active ^γR70QAMPK (α1β1γ1(R70Q)), of the kinase dead mutant ^αK45RAMPK (α1(K45R)β1γ1), or of the ubiquitin ligase Nedd4-2. KCNQ1/KCNE1 activity was determined in two electrode voltage clamp experiments. Moreover, KCNQ1 abundance in the cell membrane was determined by immunostaining and subsequent confocal imaging. As a result, wild type and constitutively active AMPK significantly reduced KCNQ1/KCNE1-mediated currents and reduced KCNQ1 abundance in the cell membrane. Similarly, Nedd4-2 decreased KCNQ1/KCNE1-mediated currents and KCNQ1 protein abundance in the cell membrane. Activation of AMPK in isolated perfused proximal renal tubules by AICAR (10 mM) was followed by significant depolarization. In conclusion, AMPK is a potent regulator of KCNQ1/KCNE1.