Activation of AMP-activated protein kinase reduces cAMP-mediated epithelial chloride secretion

2003 ◽  
Vol 285 (5) ◽  
pp. G850-G860 ◽  
Author(s):  
John Walker ◽  
Humberto B. Jijon ◽  
Thomas Churchill ◽  
Marianne Kulka ◽  
Karen L. Madsen
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Protein Kinase ◽  
Chronic Inflammation ◽  
Chloride Secretion ◽  
Wild Type ◽  
Colonic Tissue ◽  
T84 Cells ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is activated in response to fluctuations in cellular energy status caused by oxidative stress. One of its targets is the cystic fibrosis transmembrane conductance regulator (CFTR), which is the predominant Cl-secretory channel in colonic tissue. The aim of this study was to determine the role of AMPK in the modulation of colonic chloride secretion under conditions of oxidative stress and chronic inflammation. Chloride secretion and AMPK activity were examined in colonic tissue from adult IL-10-deficient and wild-type 129 Sv/Ev mice in the presence and absence of pharmacological AMPK inhibitors and activators, respectively. Apical levels of CFTR were measured in brush-border membrane vesicles. Cell culture studies in human colonic T84 monolayers examined the effect of hydrogen peroxide and pharmacological activation of AMPK on forskolin-stimulated chloride secretion. Inflamed colons from IL-10-deficient mice exhibited hyporesponsiveness to forskolin stimulation in association with reductions in surface CFTR expression and increased AMPK activity. Inhibition of AMPK restored tissue responsiveness to forskolin, whereas stimulation of AMPK with 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) induced tissue hyporesponsivness in wild-type mice. T84 cells exposed to hydrogen peroxide demonstrated a time-dependent increase in AMPK activity and reduction of forskolin-stimulated chloride secretion. Inhibition of AMPK prevented the reduction in chloride secretion. Treatment of cells with the AMPK activator, AICAR, resulted in a decreased chloride secretion. In conclusion, AMPK activation is linked with reductions in cAMP-mediated epithelial chloride flux and may be a contributing factor to the hyporesponsiveness seen under conditions of chronic inflammation.

Physiological modulation of CFTR activity by AMP-activated protein kinase in polarized T84 cells

2003 ◽  
Vol 284 (5) ◽  
pp. C1297-C1308 ◽  
Author(s):  
Kenneth R. Hallows ◽  
Gary P. Kobinger ◽  
James M. Wilson ◽  
Lee A. Witters ◽  
J. Kevin Foskett
Keyword(s):  
Protein Kinase ◽  
Short Circuit ◽  
Basolateral Membrane ◽  
Physiological Role ◽  
Dominant Negative ◽  
T84 Cells ◽  
Physiological Relevance ◽  
Polarized Epithelia ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-gated Cl− channel and cellular conductance regulator, but the detailed mechanisms of CFTR regulation and its regulation of other transport proteins remain obscure. We previously identified the metabolic sensor AMP-activated protein kinase (AMPK) as a novel protein interacting with CFTR and found that AMPK phosphorylated CFTR and inhibited CFTR-dependent whole cell conductances when coexpressed with CFTR in Xenopus oocytes. To address the physiological relevance of the CFTR-AMPK interaction, we have now studied polarized epithelia and have evaluated the localization of endogenous AMPK and CFTR and measured CFTR activity with modulation of AMPK activity. By immunofluorescent imaging, AMPK and CFTR share an overlapping apical distribution in several rat epithelial tissues, including nasopharynx, submandibular gland, pancreas, and ileum. CFTR-dependent short-circuit currents ( Isc ) were measured in polarized T84 cells grown on permeable supports, and several independent methods were used to modulate endogenous AMPK activity. Activation of endogenous AMPK with the cell-permeant adenosine analog 5-amino-4-imidazolecarboxamide-1-β-d-ribofuranoside (AICAR) inhibited forskolin-stimulated CFTR-dependent I sc in nonpermeabilized monolayers and monolayers with nystatin permeabilization of the basolateral membrane. Raising intracellular AMP concentration in monolayers with basolateral membranes permeabilized with α-toxin also inhibited CFTR, an effect that was unrelated to adenosine receptors. Finally, overexpression of a kinase-dead mutant AMPK-α1 subunit (α1-K45R) enhanced forskolin-stimulated I sc in polarized T84 monolayers, consistent with a dominant-negative reduction in the inhibition of CFTR by endogenous AMPK. These results indicate that AMPK plays a physiological role in modulating CFTR activity in polarized epithelia and suggest a novel paradigm for the coupling of ion transport to cellular metabolism.

scholarly journals The regulation of AMP-activated protein kinase by phosphorylation

2000 ◽  
Vol 345 (3) ◽  
pp. 437-443 ◽  
Author(s):  
Silvie C. STEIN ◽  
Angela WOODS ◽  
Neil A. JONES ◽  
Matthew D. DAVISON ◽  
David CARLING
Keyword(s):  
Protein Kinase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Aspartic Acid Residue ◽  
Α Subunit ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase ◽  
Mutant Complex

The AMP-activated protein kinase (AMPK) cascade is activated by an increase in the AMP/ATP ratio within the cell. AMPK is regulated allosterically by AMP and by reversible phosphorylation. Threonine-172 within the catalytic subunit (α) of AMPK (Thr172) was identified as the major site phosphorylated by the AMP-activated protein kinase kinase (AMPKK) in vitro. We have used site-directed mutagenesis to study the role of phosphorylation of Thr172 on AMPK activity. Mutation of Thr172 to an aspartic acid residue (T172D) in either α1 or α2 resulted in a kinase complex with approx. 50% the activity of the corresponding wild-type complex. The activity of wild-type AMPK decreased by greater than 90% following treatment with protein phosphatases, whereas the activity of the T172D mutant complex fell by only 10-15%. Mutation of Thr172 to an alanine residue (T172A) almost completely abolished kinase activity. These results indicate that phosphorylation of Thr172 accounts for most of the activation by AMPKK, but that other sites are involved. In support of this we have shown that AMPKK phosphorylates at least two other sites on the α subunit and one site on the β subunit. Furthermore, we provide evidence that phosphorylation of Thr172 may be involved in the sensitivity of the AMPK complex to AMP.

scholarly journals AMP-Activated Protein Kinase α1 but Not α2 Catalytic Subunit Potentiates Myogenin Expression and Myogenesis

2013 ◽  
Vol 33 (22) ◽  
pp. 4517-4525 ◽  
Author(s):  
Xing Fu ◽  
Jun-Xing Zhao ◽  
Mei-Jun Zhu ◽  
Marc Foretz ◽  
Benoit Viollet ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Fluorescent Protein ◽  
Tibialis Anterior Muscle ◽  
Muscle Fibers ◽  
Specific Effect ◽  
C2c12 Cells ◽  
Wild Type ◽  
Ampk Activity ◽  
Green Fluorescent ◽  
Amp Activated Protein Kinase

The link between AMP-activated protein kinase (AMPK) and myogenesis remains poorly defined. AMPK has two catalytic α subunits, α1 and α2. We postulated that AMPK promotes myogenesis in an isoform-specific manner. Primary myoblasts were prepared from AMPK knockout (KO) mice and AMPK conditional KO mice, and knockout of the α1 but not the α2 subunit resulted in downregulation of myogenin and reduced myogenesis. Myogenin expression and myogenesis were nearly abolished in the absence of both AMPKα1 and AMPKα2, while enhanced AMPK activity promoted myogenesis and myotube formation. The AMPKα1-specific effect on myogenesis was likely due to the dominant expression of α1 in myoblasts. These results were confirmed in C2C12 cells. To further evaluate the necessity of the AMPKα1 subunit for myogenesisin vivo, we prepared both DsRed AMPKα1 knockout myoblasts and enhanced green fluorescent protein (EGFP) wild-type myoblasts, which were cotransplanted into tibialis anterior muscle. A number of green fluorescent muscle fibers were observed, showing the fusion of engrafted wild-type myoblasts with muscle fibers; on the other hand, very few or no red muscle fibers were observed, indicating the absence of myogenic capacity of AMPKα1 knockout myoblasts. In summary, these results indicate that AMPK activity promotes myogenesis through a mechanism mediated by AMPKα1.

scholarly journals Escherichia coli MutM Suppresses Illegitimate Recombination Induced by Oxidative Stress

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 439-446 ◽  
Author(s):  
Masaaki Onda ◽  
Katsuhiro Hanada ◽  
Hirokazu Kawachi ◽  
Hideo Ikeda
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Double Strand Breaks ◽  
Illegitimate Recombination ◽  
Recombination Analysis ◽  
Wild Type ◽  
Strand Breaks ◽  
In The Wild

Abstract DNA damage by oxidative stress is one of the causes of mutagenesis. However, whether or not DNA damage induces illegitimate recombination has not been determined. To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of λbio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination. To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process. The frequency of λbio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type. Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination. Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total λbio transducing phages in the wild type or in the mutM mutant, respectively. The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA.

scholarly journals Glucose regulates AMP-activated protein kinase activity and gene expression in clonal, hypothalamic neurons expressing proopiomelanocortin: additive effects of leptin or insulin

2007 ◽  
Vol 192 (3) ◽  
pp. 605-614 ◽  
Author(s):  
Fang Cai ◽  
Armen V Gyulkhandanyan ◽  
Michael B Wheeler ◽  
Denise D Belsham
Keyword(s):  
Protein Kinase ◽  
Energy Homeostasis ◽  
Cell Model ◽  
Neuronal Cell ◽  
Cell Types ◽  
Glucose Sensing ◽  
Protein Kinase Activity ◽  
Energy Status ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The mammalian hypothalamus comprises an array of phenotypically distinct cell types that interpret peripheral signals of energy status and, in turn, elicits an appropriate response to maintain energy homeostasis. We used a clonal representative hypothalamic cell model expressing proopiomelanocortin (POMC; N-43/5) to study changes in AMP-activated protein kinase (AMPK) activity and glucose responsiveness. We have demonstrated the presence of cellular machinery responsible for glucose sensing in the cell line, including glucokinase, glucose transporters, and appropriate ion channels. ATP-sensitive potassium channels were functional and responded to glucose. The N-43/5 POMC neurons may therefore be an appropriate cell model to study glucose-sensing mechanisms in the hypothalamus. In N-43/5 POMC neurons, increasing glucose concentrations decreased phospho-AMPK activity. As a relevant downstream effect, we found that POMC transcription increased with 2.8 and 16.7 mM glucose. Upon addition of leptin, with either no glucose or with 5 mM glucose, we found that leptin decreased AMPK activity in N-43/5 POMC neurons, but had no significant effect at 25 mM glucose, whereas insulin decreased AMPK activity at only 5 mM glucose. These results demonstrate that individual hypothalamic neuronal cell types, such as the POMC neuron, can have distinct responses to peripheral signals that relay energy status to the brain, and will therefore be activated uniquely to control neuroendocrine function.

scholarly journals Adrenaline is a critical mediator of acute exercise-induced AMP-activated protein kinase activation in adipocytes

2007 ◽  
Vol 403 (3) ◽  
pp. 473-481 ◽  
Author(s):  
Ho-Jin Koh ◽  
Michael F. Hirshman ◽  
Huamei He ◽  
Yangfeng Li ◽  
Yasuko Manabe ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Protein Kinase Activity ◽  
Chronic Exercise ◽  
Rat Adipocytes ◽  
Compound C ◽  
Isolated Adipocytes ◽  
Ampk Activity ◽  
Exercise Induced ◽  
Amp Activated Protein Kinase

Exercise increases AMPK (AMP-activated protein kinase) activity in human and rat adipocytes, but the underlying molecular mechanisms and functional consequences of this activation are not known. Since adrenaline (epinephrine) concentrations increase with exercise, in the present study we hypothesized that adrenaline activates AMPK in adipocytes. We show that a single bout of exercise increases AMPKα1 and α2 activities and ACC (acetyl-CoA carboxylase) Ser79 phosphorylation in rat adipocytes. Similarly to exercise, adrenaline treatment in vivo increased AMPK activities and ACC phosphorylation. Pre-treatment of rats with the β-blocker propranolol fully blocked exercise-induced AMPK activation. Increased AMPK activity with exercise and adrenaline treatment in vivo was accompanied by an increased AMP/ATP ratio. Adrenaline incubation of isolated adipocytes also increased the AMP/ATP ratio and AMPK activities, an effect blocked by propranolol. Adrenaline incubation increased lipolysis in isolated adipocytes, and Compound C, an AMPK inhibitor, attenuated this effect. Finally, a potential role for AMPK in the decreased adiposity associated with chronic exercise was suggested by marked increases in AMPKα1 and α2 activities in adipocytes from rats trained for 6 weeks. In conclusion, both acute and chronic exercise are significant regulators of AMPK activity in rat adipocytes. Our findings suggest that adrenaline plays a critical role in exercise-stimulated AMPKα1 and α2 activities in adipocytes, and that AMPK can function in the regulation of lipolysis.

Abstract 337: Activation of Myocardial AMP-activated Protein Kinase by Metformin Prevents Progression of Heart Failure in Dogs; Involvement of eNOS Activation

Circulation ◽  
2008 ◽  
Vol 118 (suppl_18) ◽  
Author(s):  
Hideyuki Sasaki ◽  
Hiroshi Asanuma ◽  
Masashi Fujita ◽  
Hiroyuki Takahama ◽  
Masanori Asakura ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Nitric Oxide ◽  
Heart Failure ◽  
Cell Death ◽  
Protein Kinase ◽  
Left Ventricular ◽  
Vehicle Group ◽  
Mrna Levels ◽  
Compound C ◽  
Amp Activated Protein Kinase

Background; Several studies have shown that metformin activates AMP-activated protein kinase (AMPK), which mediates potent cardioprotection against ischemia-reperfusion injury. AMPK is also activated in experimental failing myocardium, suggesting that activation of AMPK is beneficial for the pathophysiology of heart failure. We investigated whether metformin prevents oxidative stress-induced cell death in rat cardiomyocytes and attenuates the progression of heart failure in dogs. Methods and Results; The treatment with metformin (10 μmol/L) protected the rat cultured cardiomyocytes against cell death due to H 2 O 2 exposure (50 μmol/L) as indicated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), TUNEL staining, and flow cytometry. These effects were blunted by an AMPK inhibitor, compound-C (20 μmol/L), suggesting that the activation of AMPK decreased the extent of apoptosis-induced cell death due to H 2 O 2 exposure. Continuous rapid ventricular pacing (230/min for 4 weeks) in dogs caused heart failure and the treatment with metformin (100 mg/kg/day PO, n=8) decreased left ventricular (LV) end-diastolic dimension (32.8±0.4 vs. 36.5±1.0 mm, p< 0.01) and pressure (11.8±1.1 vs. 22±0.9 mmHg, p< 0.01), and increased LV fractional shortening (18.6±1.8 vs. 9.6±0.7 %, p< 0.01) along with enhanced phosphorylation of AMPK and the decreased the number of TUNEL-positive cells of the LV myocardium compared with the vehicle group (n=8). Interestingly, metformin increased the protein and mRNA levels of endothelial nitric oxide synthase of the LV myocardium and plasma nitric oxide levels. Metformin improved the plasma insulin resistance without increased myocardial GLUT-4 translocation. Furthermore, the subcutaneous administration of AICAR (50 mg/kg/every other day), another AMPK activator mediated the equivalent effects to metformin, strengthening the pivotal role of AMPK in reduction of apoptosis and prevention of heart failure. Conclusions; Activation of myocardial AMPK attenuated the oxidative stress-induced cardiomyocyte apoptosis and prevented the progression of heart failure in dogs, along with eNOS activation. Thus, metformin or AICAR may be applicable as a novel therapy for heart failure.

AMP kinase is not required for the GLUT4 response to exercise and denervation in skeletal muscle

2004 ◽  
Vol 287 (4) ◽  
pp. E739-E743 ◽  
Author(s):  
Burton F. Holmes ◽  
David B. Lang ◽  
Morris J. Birnbaum ◽  
James Mu ◽  
G. Lynis Dohm
Keyword(s):  
Skeletal Muscle ◽  
Dominant Negative ◽  
Treadmill Running ◽  
Wild Type ◽  
Α Subunit ◽  
Ampk Activity ◽  
Denervated Muscles ◽  
Amp Activated Protein Kinase ◽  
Response To Exercise

An acute bout of exercise increases muscle GLUT4 mRNA in mice, and denervation decreases GLUT4 mRNA. AMP-activated protein kinase (AMPK) activity in skeletal muscle is also increased by exercise, and GLUT4 mRNA is increased in mouse skeletal muscle after treatment with AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside(AICAR). These findings suggest that AMPK activation might be responsible for the increase in GLUT4 mRNA expression in response to exercise. To investigate the role of AMPK in GLUT4 regulation in response to exercise and denervation, transgenic mice with a mutated AMPK α-subunit (dominant negative; AMPK-DN) were studied. GLUT4 did not increase in AMPK-DN mice that were treated with AICAR, demonstrating that muscle AMPK is inactive. Exercise (two 3-h bouts of treadmill running separated by 1 h of rest) increased GLUT4 mRNA in both wild-type and AMPK-DN mice. Likewise, denervation decreased GLUT4 mRNA in both wild-type and AMPK-DN mice. GLUT4 mRNA was also increased by AICAR treatment in both the innervated and denervated muscles. These data demonstrate that AMPK is not required for the response of GLUT4 mRNA to exercise and denervation.

scholarly journals Novel Antioxidant Properties of Doxycycline

2018 ◽  
Vol 19 (12) ◽  
pp. 4078 ◽  
Author(s):  
Dahn Clemens ◽  
Michael Duryee ◽  
Cleofes Sarmiento ◽  
Andrew Chiou ◽  
Jacob McGowan ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Reactive Oxygen Species ◽  
Hydrogen Peroxide ◽  
Chronic Inflammation ◽  
Antioxidant Properties ◽  
Antibacterial Properties ◽  
Reactive Oxygen ◽  
Protein Adducts ◽  
Oxygen Species ◽  
Free System

Doxycycline (DOX), a derivative of tetracycline, is a broad-spectrum antibiotic that exhibits a number of therapeutic activities in addition to its antibacterial properties. For example, DOX has been used in the management of a number of diseases characterized by chronic inflammation. One potential mechanism by which DOX inhibits the progression of these diseases is by reducing oxidative stress, thereby inhibiting subsequent lipid peroxidation and inflammatory responses. Herein, we tested the hypothesis that DOX directly scavenges reactive oxygen species (ROS) and inhibits the formation of redox-mediated malondialdehyde-acetaldehyde (MAA) protein adducts. Using a cell-free system, we demonstrated that DOX scavenged reactive oxygen species (ROS) produced during the formation of MAA-adducts and inhibits the formation of MAA-protein adducts. To determine whether DOX scavenges specific ROS, we examined the ability of DOX to directly scavenge superoxide and hydrogen peroxide. Using electron paramagnetic resonance (EPR) spectroscopy, we found that DOX directly scavenged superoxide, but not hydrogen peroxide. Additionally, we found that DOX inhibits MAA-induced activation of Nrf2, a redox-sensitive transcription factor. Together, these findings demonstrate the under-recognized direct antioxidant property of DOX that may help to explain its therapeutic potential in the treatment of conditions characterized by chronic inflammation and increased oxidative stress.

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