| 1. |
- Ducommun, Serge, et al.
(författare)
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Chemical genetic screen identifies Gapex-5/GAPVD1 and STBD1 as novel AMPK substrates
- 2019
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Ingår i: Cellular Signalling. - : Elsevier BV. - 0898-6568 .- 1873-3913. ; 57, s. 45-57
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Tidskriftsartikel (refereegranskat)abstract
- AMP-activated protein kinase (AMPK) is a key regulator of cellular energy homeostasis, acting as a sensor of energy and nutrient status. As such, AMPK is considered a promising drug target for treatment of medical conditions particularly associated with metabolic dysfunctions. To better understand the downstream effectors and physiological consequences of AMPK activation, we have employed a chemical genetic screen in mouse primary hepatocytes in an attempt to identify novel AMPK targets. Treatment of hepatocytes with a potent and specific AMPK activator 991 resulted in identification of 65 proteins phosphorylated upon AMPK activation, which are involved in a variety of cellular processes such as lipid/glycogen metabolism, vesicle trafficking, and cytoskeleton organisation. Further characterisation and validation using mass spectrometry followed by immunoblotting analysis with phosphorylation site-specific antibodies identified AMPK-dependent phosphorylation of Gapex-5 (also known as GTPase-activating protein and VPS9 domain-containing protein 1 (GAPVD1)) on Ser902 in hepatocytes and starch-binding domain 1 (STBD1) on Ser175 in multiple cells/tissues. As new promising roles of AMPK as a key metabolic regulator continue to emerge, the substrates we identified could provide new mechanistic and therapeutic insights into AMPK-activating drugs in the liver.
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| 2. |
- Göransson, Olga, et al.
(författare)
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Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase
- 2007
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 282:45, s. 32549-32560
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Tidskriftsartikel (refereegranskat)abstract
- 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. We found that it has no effect on the isolated alpha subunit kinase domain, with or without the associated autoinhibitory domain, or on interaction of glycogen with the beta subunit glycogen- binding domain. Although it mimics actions of AMP, it has no effect on binding of AMP to the isolated Bateman domains of the gamma subunit. The addition of A- 769662 to mouse embryonic fibroblasts or primary mouse hepatocytes stimulates phosphorylation of acetyl- CoA carboxylase ( ACC), effects that are completely abolished in AMPK- alpha 1(-/-) alpha 2(-/-) cells but not in TAK1(-/-) mouse embryonic fibroblasts. Phosphorylation of AMPK and ACC in response to A- 769662 is also abolished in isolated mouse skeletal muscle lacking LKB1, a major upstream kinase for AMPK in this tissue. However, in HeLa cells, which lack LKB1 but express the alternate upstream kinase calmodulin- dependent protein kinase kinase-beta, 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. We propose that this direct and specific AMPK activator will be a valuable experimental tool to understand the physiological roles of AMPK.
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| 3. |
- Mogilenko, Denis A., et al.
(författare)
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Metabolic and Innate Immune Cues Merge into a Specific Inflammatory Response via the UPR
- 2019
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Ingår i: Cell. - : CELL PRESS. - 0092-8674 .- 1097-4172. ; 177:5, s. 1201-
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Tidskriftsartikel (refereegranskat)abstract
- Innate immune responses are intricately linked with intracellular metabolism of myeloid cells. Toll-like receptor (TLR) stimulation shifts intracellular metabolism toward glycolysis, while anti-inflammatory signals depend on enhanced mitochondrial respiration. How exogenous metabolic signals affect the immune response is unknown. We demonstrate that TLR-dependent responses of dendritic cells (DCs) are exacerbated by a high-fatty-acid (FA) metabolic environment. FAs suppress the TLR-induced hexokinase activity and perturb tricarboxylic acid cycle metabolism. These metabolic changes enhance mitochondria! reactive oxygen species (mtROS) production and, in turn, the unfolded protein response (UPR), leading to a distinct transcriptomic signature with IL-23 as hallmark. Interestingly, chemical or genetic suppression of glycolysis was sufficient to induce this specific immune response. Conversely, reducing mtROS production or DC-specific deficiency in XBP1 attenuated IL-23 expression and skin inflammation in an IL-23-dependent model of psoriasis. Thus, fine-tuning of innate immunity depends on optimization of metabolic demands and minimization of mtROS-induced UPR.
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| 4. |
- Mounier, Remi, et al.
(författare)
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AMPK alpha 1 Regulates Macrophage Skewing at the Time of Resolution of Inflammation during Skeletal Muscle Regeneration
- 2013
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Ingår i: Cell Metabolism. - : Elsevier BV. - 1550-4131. ; 18:2, s. 251-264
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Tidskriftsartikel (refereegranskat)abstract
- Macrophages control the resolution of inflammation through the transition from a proinflammatory (M1) to an anti-inflammatory (M2) phenotype. Here, we present evidence for a role of AMPK alpha 1, a master regulator of energy homeostasis, in macrophage skewing that occurs during skeletal muscle regeneration. Muscle regeneration was impaired in AMPK alpha 1(-/-) mice. In vivo loss-of-function (LysM-Cre;AMPK alpha 1(fl/fl) mouse) and rescue (bone marrow transplantation) experiments showed that macrophagic AMPK alpha 1 was required for muscle regeneration. Cell-based experiments revealed that AMPK alpha 1(-/-) macrophages did not fully acquire the phenotype or the functions of M2 cells. In vivo, AMPK alpha 1(-/-) leukocytes did not acquire the expression of M2 markers during muscle regeneration. Skewing from M1 toward M2 phenotype upon phagocytosis of necrotic and apoptotic cells was impaired in AMPK alpha 1(-/-) macrophages and when AMPK activation was prevented by the inhibition of its upstream activator, CaMKK beta. In conclusion, AMPK alpha 1 is crucial for phagocytosis-induced macrophage skewing from a pro-to anti-inflammatory phenotype at the time of resolution of inflammation.
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| 5. |
- Patel, Kashyap, et al.
(författare)
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The LKB1-salt-inducible kinase pathway functions as a key gluconeogenic suppressor in the liver.
- 2014
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 5:Aug 4
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Tidskriftsartikel (refereegranskat)abstract
- LKB1 is a master kinase that regulates metabolism and growth through adenosine monophosphate-activated protein kinase (AMPK) and 12 other closely related kinases. Liver-specific ablation of LKB1 causes increased glucose production in hepatocytes in vitro and hyperglycaemia in fasting mice in vivo. Here we report that the salt-inducible kinases (SIK1, 2 and 3), members of the AMPK-related kinase family, play a key role as gluconeogenic suppressors downstream of LKB1 in the liver. The selective SIK inhibitor HG-9-91-01 promotes dephosphorylation of transcriptional co-activators CRTC2/3 resulting in enhanced gluconeogenic gene expression and glucose production in hepatocytes, an effect that is abolished when an HG-9-91-01-insensitive mutant SIK is introduced or LKB1 is ablated. Although SIK2 was proposed as a key regulator of insulin-mediated suppression of gluconeogenesis, we provide genetic evidence that liver-specific ablation of SIK2 alone has no effect on gluconeogenesis and insulin does not modulate SIK2 phosphorylation or activity. Collectively, we demonstrate that the LKB1-SIK pathway functions as a key gluconeogenic gatekeeper in the liver.
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| 6. |
- Treebak, Jonas T, et al.
(författare)
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AMPK-mediated AS160 phosphorylation in skeletal muscle is dependent on AMPK catalytic and regulatory subunits.
- 2006
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Ingår i: Diabetes. - : American Diabetes Association. - 0012-1797 .- 1939-327X. ; 55:7, s. 2051-2058
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Tidskriftsartikel (refereegranskat)abstract
- AMP-activated protein kinase (AMPK) is a heterotrimeric protein that regulates glucose transport mediated by cellular stress or pharmacological agonists such as 5-aminoimidazole-4-carboxamide 1 beta-D-ribonucleoside (AICAR). AS160, a Rab GTPase-activating protein, provides a mechanism linking AMPK signaling to glucose uptake. We show that AICAR increases AMPK, acetyl-CoA carboxylase, and AS160 phosphorylation by insulin-independent mechanisms in isolated skeletal muscle. Recombinant AMPK heterotrimeric complexes (alpha 1 beta 1 gamma l and alpha 2 beta 2 gamma 1) phosphorylate AS160 in a cell-free assay. In mice deficient in AMPK signaling (alpha 2 AMPK knockout [KO], alpha 2 AMPK kinase dead [KD], and gamma 3 AMPK KO), AICAR effects on AS160 phosphorylation were severely blunted, highlighting that complexes containing alpha 2 and gamma 3 are necessary for AICAR-stimulated AS160 phosphorylation in intact skeletal muscle. Contraction-mediated AS160 phosphorylation was also impaired in alpha 2 AMPK KO and KD but not gamma 3 AMPK KO mice. Our results implicate AS160 as a downstream target of AMPK.
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| 7. |
- Zibrova, Darya, et al.
(författare)
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GFAT1 phosphorylation by AMPK promotes VEGF-induced angiogenesis
- 2017
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Ingår i: Biochemical Journal. - 0264-6021. ; 474:6, s. 983-1001
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Tidskriftsartikel (refereegranskat)abstract
- Activation of AMP-activated protein kinase (AMPK) in endothelial cells regulates energy homeostasis, stress protection and angiogenesis, but the underlying mechanisms are incompletely understood. Using a label-free phosphoproteomic analysis, we identified glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1) as an AMPK substrate. GFAT1 is the rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP) and as such controls the modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc). In the present study, we tested the hypothesis that AMPK controls O-GlcNAc levels and function of endothelial cells via GFAT1 phosphorylation using biochemical, pharmacological, genetic and in vitro angiogenesis approaches. Activation of AMPK in primary human endothelial cells by 5-aminoimidazole-4-carboxamide riboside (AICAR) or by vascular endothelial growth factor (VEGF) led to GFAT1 phosphorylation at serine 243. This effect was not seen when AMPK was down-regulated by siRNA. Upon AMPK activation, diminished GFAT activity and reduced O-GlcNAc levels were observed in endothelial cells containing wild-type (WT)-GFAT1 but not in cells expressing non-phosphorylatable S243A-GFAT1. Pharmacological inhibition or siRNA-mediated down-regulation of GFAT1 potentiated VEGF-induced sprouting, indicating that GFAT1 acts as a negative regulator of angiogenesis. In cells expressing S243A-GFAT1, VEGF-induced sprouting was reduced, suggesting that VEGF relieves the inhibitory action of GFAT1/HBP on angiogenesis via AMPK-mediated GFAT1 phosphorylation. Activation of GFAT1/HBP by high glucose led to impairment of vascular sprouting, whereas GFAT1 inhibition improved sprouting even if glucose level was high. Our findings provide novel mechanistic insights into the role of HBP in angiogenesis. They suggest that targeting AMPK in endothelium might help to ameliorate hyperglycaemia-induced vascular dysfunction associated with metabolic disorders.
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