scholarly journals Enhanced activation of cellular AMPK by dual-small molecule treatment: AICAR and A769662

2014 ◽  
Vol 306 (6) ◽  
pp. E688-E696 ◽  
Author(s):  
Serge Ducommun ◽  
Rebecca J. Ford ◽  
Laurent Bultot ◽  
Maria Deak ◽  
Luc Bertrand ◽  
...  
Keyword(s):  
Synergistic Effect ◽  
Metabolic Diseases ◽  
Cellular Tissue ◽  
Intact Cells ◽  
Downstream Signaling ◽  
Upstream Kinase ◽  
A Cell ◽  
Single Compound ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a key cellular energy sensor and regulator of metabolic homeostasis. Activation of AMPK provides beneficial outcomes in fighting against metabolic disorders such as insulin resistance and type 2 diabetes. Currently, there is no allosteric AMPK activator available for the treatment of metabolic diseases, and limited compounds are available to robustly stimulate cellular/tissue AMPK in a specific manner. Here we investigated whether simultaneous administration of two different pharmacological AMPK activators, which bind and act on different sites, would result in an additive or synergistic effect on AMPK and its downstream signaling and physiological events in intact cells. We observed that cotreating primary hepatocytes with the AMP mimetic 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) and a low dose (1 μM) of the allosteric activator A769662 produced a synergistic effect on AMPK Thr172 phosphorylation and catalytic activity, which was associated with a more profound increase/decrease in phosphorylation of downstream AMPK targets and inhibition of hepatic lipogenesis compared with single-compound treatment. Mechanistically, we found that cotreatment does not stimulate LKB1, upstream kinase for AMPK, but it protects against dephosphorylation of Thr172 phosphorylation by protein phosphatase PP2Cα in an additive manner in a cell-free assay. Collectively, we demonstrate that AICAR sensitizes the effect of A769662 and promotes AMPK activity and its downstream events. The study demonstrates the feasibility of promoting AMPK activity by using two activators with distinct modes of action in order to achieve a greater activation of AMPK and downstream signaling.

Hypoxia and AMP independently regulate AMP-activated protein kinase activity in heart

2005 ◽  
Vol 288 (5) ◽  
pp. H2412-H2421 ◽  
Author(s):  
Markus Frederich ◽  
Li Zhang ◽  
James A. Balschi
Keyword(s):  
Protein Kinase ◽  
Metabolic Inhibitors ◽  
Protein Kinase Activity ◽  
Α Subunit ◽  
Rat Hearts ◽  
Upstream Kinase ◽  
Ampk Activity ◽  
Amp Activated Protein Kinase

The hypothesis was tested that hypoxia increases AMP-activated protein kinase (AMPK) activity independently of AMP concentration ([AMP]) in heart. In isolated perfused rat hearts, cytosolic [AMP] was changed from 0.2 to 16 μM using metabolic inhibitors during both normal oxygenation (95% O2-5% CO2, normoxia) and limited oxygenation (95% N2-5% CO2, hypoxia). Total AMPK activity measured in vitro ranged from 2 to 40 pmol·min−1·mg protein−1 in normoxic hearts and from 5 to 55 pmol·min−1·mg protein−1 in hypoxic hearts. The dependence of the in vitro total AMPK activity on the in vivo cytosolic [AMP] was determined by fitting the measurements from individual hearts to a hyperbolic equation. The [AMP] resulting in half-maximal total AMPK activity ( A0.5) was 3 ± 1 μM for hypoxic hearts and 28 ± 13 μM for normoxic hearts. The A0.5 for α2-isoform AMPK activity was 2 ± 1 μM for hypoxic hearts and 13 ± 8 μM for normoxic hearts. Total AMPK activity correlated with the phosphorylation of the Thr172 residue of the AMPK α-subunit. In potassium-arrested hearts perfused with variable O2 content, α-subunit Thr172 phosphorylation increased at O2 ≤ 21% even though [AMP] was <0.3 μM. Thus hypoxia or O2 ≤ 21% increased AMPK phosphorylation and activity independently of cytosolic [AMP]. The hypoxic increase in AMPK activity may result from either direct phosphorylation of Thr172 by an upstream kinase or reduction in the A0.5 for [AMP].

scholarly journals Calmodulin-dependent protein kinase kinase-β activates AMPK without forming a stable complex: synergistic effects of Ca2+ and AMP

2010 ◽  
Vol 426 (1) ◽  
pp. 109-118 ◽  
Author(s):  
Sarah Fogarty ◽  
Simon A. Hawley ◽  
Kevin A. Green ◽  
Nazan Saner ◽  
Kirsty J. Mustard ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Synergistic Effects ◽  
Stable Complex ◽  
Dependent Protein Kinase ◽  
Intact Cells ◽  
Ampk Phosphorylation ◽  
Upstream Kinase ◽  
Dependent Protein ◽  
Amp Activated Protein Kinase ◽  
Protein Kinase Kinase

Activation of AMPK (AMP-activated protein kinase) by phosphorylation at Thr172 is catalysed by at least two distinct upstream kinases, i.e. the tumour suppressor LKB1, and CaMKKβ (Ca2+/calmodulin-dependent protein kinase kinase-β). The sequence around Thr172 is highly conserved between the two catalytic subunit isoforms of AMPK and the 12 AMPK-related kinases, and LKB1 has been shown to act upstream of all of them. In the present paper we report that none of the AMPK-related kinases tested could be phosphorylated or activated in intact cells or cell-free assays by CaMKKβ, although we did observe a slow phosphorylation and activation of BRSK1 (brain-specific kinase 1) by CaMKKα. Despite recent reports, we could not find any evidence that the α and/or β subunits of AMPK formed a stable complex with CaMKKβ. We also showed that increasing AMP concentrations in HeLa cells (which lack LKB1) had no effect on basal AMPK phosphorylation, but enhanced the ability of agents that increase intracellular Ca2+ to activate AMPK. This is consistent with the effect of AMP on phosphorylation of Thr172 being due to inhibition of dephosphorylation, and confirms that the effect of AMP is independent of the upstream kinase utilized.

scholarly journals AMP-activated protein kinase is activated by low glucose in cell lines derived from pancreatic β cells, and may regulate insulin release

1998 ◽  
Vol 335 (3) ◽  
pp. 533-539 ◽  
Author(s):  
Ian P. SALT ◽  
Gabriele JOHNSON ◽  
Stephen J. H. ASHCROFT ◽  
D. Grahame HARDIE
Keyword(s):  
Insulin Secretion ◽  
Protein Kinase ◽  
Insulin Release ◽  
Cell Lines ◽  
Cell Types ◽  
Β Cell ◽  
Upstream Kinase ◽  
Ampk Activity ◽  
Two Parameters ◽  
Amp Activated Protein Kinase

The role of the AMP-activated protein kinase (AMPK) cascade in the glucose-sensitive pancreatic β cell lines HIT-T15 and INS-1 was addressed. In both cell types, removal of glucose leads to a > 5-fold activation of AMPK activity. Activation of AMPK was due to phosphorylation, since the effect was reversed by protein phosphatase treatment of the extracts, and was restored by re-addition of MgATP and the purified upstream kinase. When the effects of different concentrations of medium glucose were examined, insulin secretion and AMPK activity were inversely related, and varied over the same concentration range. The activation in response to glucose removal appeared to be due to changes in the concentration of the known regulators of the cascade, i.e. AMP and ATP, since AMPK activation was associated with a large increase in the cellular AMP/ATP ratio, and the two parameters varied over the same range of glucose concentrations. In late-passage HIT-T15 cells that had lost the glucose-dependent insulin secretion response, both AMPK activity and the AMP/ATP ratio also became insensitive to the extracellular glucose concentration. Treatment of INS-1 cells, but not HIT-T15 cells, with AICA riboside (5-aminoimidazole-4-carboxamide riboside) results in accumulation of the ribotide, ZMP (AICA riboside monophosphate), and activation of AMPK. AICA riboside treatment of INS-1 cells, and of isolated rat islets, had both inhibitory and stimulatory effects on insulin secretion. These results show that in β cell lines the AMP-activated protein kinase, like its yeast homologue the SNF1 complex, can respond to the level of glucose in the medium, and may be involved in regulating insulin release.

scholarly journals Spatial regulation of AMPK signaling revealed by a sensitive kinase activity reporter

2021 ◽  
Author(s):  
Danielle L Schmitt ◽  
Stephanie D Curtis ◽  
Allen Leung ◽  
Jin-fan Zhang ◽  
Mingyuan Chen ◽  
...  
Keyword(s):  
Dynamic Range ◽  
High Sensitivity ◽  
Dynamic Activity ◽  
Ampk Signaling ◽  
Upstream Kinase ◽  
Cellular Energetics ◽  
Liver Kinase B1 ◽  
Ampk Activity ◽  
Nuclear Shuttling ◽  
Amp Activated Protein Kinase

AMP-activated protein kinase (AMPK) is a master regulator of cellular energetics which coordinates metabolism by phosphorylating a plethora of substrates throughout the cell. But whether AMPK activity is regulated at different subcellular locations to provide precise spatial and temporal control over metabolism is unclear. Genetically encoded AMPK activity reporters (AMPKAR) have provided a window into spatial AMPK activity, but the limited dynamic range of current AMPKARs hinders detailed study. To monitor the dynamic activity of AMPK with high sensitivity, we developed a single-fluorophore AMPK activity reporter (ExRai AMPKAR) that exhibits an excitation ratiometric fluorescence change upon phosphorylation by AMPK, with over 3-fold greater response compared to previous AMPKARs. Using subcellularly localized ExRai AMPKAR, we found that the activity of AMPK at the lysosome and mitochondria are differentially regulated. While different activating conditions, irrespective of their effects on ATP, robustly yet gradually increase mitochondrial AMPK activity, lysosomal AMPK activity accumulates with much faster kinetics. Genetic deletion of the canonical upstream kinase liver kinase B1 (LKB1) resulted in slower AMPK activity at lysosomes but did not affect the response amplitude at either location, in sharp contrast to the necessity of LKB1 for maximal cytoplasmic AMPK activity. We further discovered AMPK activity in the nucleus, which resulted from LKB1-mediated cytoplasmic activation of AMPK followed by nuclear shuttling. Thus, a new, sensitive reporter for AMPK activity, ExRai AMPKAR, in complement with mathematical and biophysical methods, captured subcellular AMPK activity dynamics in living cells and unveiled complex regulation of AMPK signaling within subcellular compartments.

scholarly journals Shedding light on the intricate puzzle of ghrelin's effects on appetite regulation

2009 ◽  
Vol 202 (2) ◽  
pp. 191-198 ◽  
Author(s):  
Blerina Kola ◽  
Márta Korbonits
Keyword(s):  
Uncoupling Protein ◽  
Metabolic Effects ◽  
Metabolic Enzyme ◽  
Appetite Regulation ◽  
Calmodulin Kinase ◽  
Wide Range ◽  
Upstream Kinase ◽  
Ampk Activity ◽  
Acid Pathway ◽  
Amp Activated Protein Kinase

Ghrelin, a hormone primarily produced by the stomach, has a wide range of metabolic and non-metabolic effects. It also stimulates food intake through activation of various hypothalamic and brain stem neurons. A series of recent studies have explored the intracellular mechanisms of the appetite-inducing effect of ghrelin in the hypothalamus, shedding light on the intricate mechanisms of appetite regulation. AMP-activated protein kinase (AMPK) is a key metabolic enzyme involved in appetite regulation. Calmodulin kinase kinase 2 (CaMKK2) has been identified as an upstream kinase of AMPK and a key mediator in the effect of ghrelin on AMPK activity. The fatty acid pathway, hypothalamic mitochondrial respiration, and uncoupling protein 2 have been outlined as downstream targets of AMPK and mediators of ghrelin's appetite stimulating effect. This short overview summarises the present data in this field.

Glycans in scaffold design in tissue reconstruction

2021 ◽  
pp. 088391152199784
Author(s):  
Nipun Jain ◽  
Shashi Singh
Keyword(s):  
Cell Attachment ◽  
Low Cost ◽  
Growth Conditions ◽  
Downstream Signaling ◽  
Cell Carrier ◽  
Wide Range ◽  
Proliferation And Differentiation ◽  
Different Types ◽  
Tissue Reconstruction ◽  
A Cell

Development of an artificial tissue by tissue engineering is witnessed to be one of the long lasting clarified solutions for the damaged tissue function restoration. To accomplish this, a scaffold is designed as a cell carrier in which the extracellular matrix (ECM) performs a prominent task of controlling the inoculated cell’s destiny. ECM composition, topography and mechanical properties lead to different types of interactions between cells and ECM components that trigger an assortment of cellular reactions via diverse sensing mechanisms and downstream signaling pathways. The polysaccharides in the form of proteoglycans and glycoproteins yield better outcomes when included in the designed matrices. Glycosaminoglycan (GAG) chains present on proteoglycans show a wide range of operations such as sequestering of critical effector morphogens which encourage proficient nutrient contribution toward the growing stem cells for their development and endurance. In this review we discuss how the glycosylation aspects are of considerable importance in everyday housekeeping functions of a cell especially when placed in a controlled environment under ideal growth conditions. Hydrogels made from these GAG chains have been used extensively as a resorbable material that mimics the natural ECM functions for an efficient control over cell attachment, permeability, viability, proliferation, and differentiation processes. Also the incorporation of non-mammalian polysaccharides can elicit specific receptor responses which authorize the creation of numerous vigorous frameworks while prolonging the low cost and immunogenicity of the substance.

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.

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.

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