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Finely-tuned regulation of AMP-activated protein kinase is crucial for human adult erythropoiesis.

ABSTRACT: AMP-activated protein kinase (AMPK) is a heterotrimeric complex containing ?, ?, and ? subunits involved in maintaining integrity and survival of murine red blood cells. Indeed, Ampk ?1-/- , Ampk ?1-/- and Ampk ?1-/- mice develop hemolytic anemia and the plasma membrane of their red blood cells shows elasticity defects. The membrane composition evolves continuously along erythropoiesis and during red blood cell maturation; defects due to the absence of Ampk could be initiated during erythropoiesis. We, therefore, studied the role of AMPK during human erythropoiesis. Our data show that AMPK activation had two distinct phases in primary erythroblasts. The phosphorylation of AMPK (Thr172) and its target acetyl CoA carboxylase (Ser79) was elevated in immature erythroblasts (glycophorin Alow), then decreased conjointly with erythroid differentiation. In erythroblasts, knockdown of the ?1 catalytic subunit by short hairpin RNA led to a decrease in cell proliferation and alterations in the expression of membrane proteins (band 3 and glycophorin A) associated with an increase in phosphorylation of adducin (Ser726). AMPK activation in mature erythroblasts (glycophorin Ahigh), achieved through the use of direct activators (GSK621 and compound 991), induced cell cycle arrest in the S phase, the induction of autophagy and caspase-dependent apoptosis, whereas no such effects were observed in similarly treated immature erythroblasts. Thus, our work suggests that AMPK activation during the final stages of erythropoiesis is deleterious. As the use of direct AMPK activators is being considered as a treatment in several pathologies (diabetes, acute myeloid leukemia), this observation is pivotal. Our data highlighted the importance of the finely-tuned regulation of AMPK during human erythropoiesis.

SUBMITTER: Ladli M

PROVIDER: S-EPMC6518903 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Finely-tuned regulation of AMP-activated protein kinase is crucial for human adult erythropoiesis.

Ladli Meriem M Richard Cyrielle C Aguilar Lilia Cantero LC Ducamp Sarah S Bondu Sabrina S Sujobert Pierre P Tamburini Jérôme J Lacombe Catherine C Azar Nabih N Foretz Marc M Zermati Yael Y Mayeux Patrick P Viollet Benoit B Verdier Frédérique F

Haematologica 20181011 5

AMP-activated protein kinase (AMPK) is a heterotrimeric complex containing α, β, and γ subunits involved in maintaining integrity and survival of murine red blood cells. Indeed, Ampk α1-/- , Ampk β1-/- and Ampk γ1-/- mice develop hemolytic anemia and the plasma membrane of their red blood cells shows elasticity defects. The membrane composition evolves continuously along erythropoiesis and during red blood cell maturation; defects due ...[more]

PMID: 30309849

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Project description:The metabolic sensor AMP-activated protein kinase (AMPK) regulates several transport proteins, potentially coupling transport activity to cellular stress and energy levels. The creatine transporter (CRT; SLC6A8) mediates creatine uptake into several cell types, including kidney epithelial cells, where it has been proposed that CRT is important for reclamation of filtered creatine, a process critical for total body creatine homeostasis. Creatine and phosphocreatine provide an intracellular, high-energy phosphate-buffering system essential for maintaining ATP supply in tissues with high energy demands. To test our hypothesis that CRT is regulated by AMPK in the kidney, we examined CRT and AMPK distribution in the kidney and the regulation of CRT by AMPK in cells. By immunofluorescence staining, we detected CRT at the apical pole in a polarized mouse S3 proximal tubule cell line and in native rat kidney proximal tubules, a distribution overlapping with AMPK. Two-electrode voltage-clamp (TEV) measurements of Na(+)-dependent creatine uptake into CRT-expressing Xenopus laevis oocytes demonstrated that AMPK inhibited CRT via a reduction in its Michaelis-Menten V(max) parameter. [(14)C]creatine uptake and apical surface biotinylation measurements in polarized S3 cells demonstrated parallel reductions in creatine influx and CRT apical membrane expression after AMPK activation with the AMP-mimetic compound 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside. In oocyte TEV experiments, rapamycin and the AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranosyl 5'-monophosphate (ZMP) inhibited CRT currents, but there was no additive inhibition of CRT by ZMP, suggesting that AMPK may inhibit CRT indirectly via the mammalian target of rapamycin pathway. We conclude that AMPK inhibits apical membrane CRT expression in kidney proximal tubule cells, which could be important in reducing cellular energy expenditure and unnecessary creatine reabsorption under conditions of local and whole body metabolic stress.

Project description:Background and purposeRegulating P2X7 receptor-mediated activation of NLRP3 inflammasomes could be a therapeutic strategy to treat alcoholic hepatosteatosis. We investigated whether this process was modulated by gentiopicroside, the main active secoiridoid glycoside from Gentiana manshurica Kitagawa.Experimental approachIn vivo models of acute and chronic alcoholic hepatosteatosis were established by intragastrically administered ethanol or using chronic plus binge ethanol feeding of Lieber-DeCarli liquid diet to male C57BL/6 mice. In vitro, HepG2 cells were treated with ethanol. RAW 264.7 macrophages and murine bone marrow-derived macrophages (BMDMs) were stimulated with LPS and ATP.Key resultsIn both the acute and chronic alcohol-induced mouse hepatosteatosis models, gentiopicroside decreased serum aminotransferases and triglyceride accumulation. Up-regulated SREBP1, down-regulated PPARα and phosphorylated acetyl-CoA carboxylase caused by acute and chronic alcohol feeding were modulated by gentiopicroside, through the elevation of LKB1 and AMPK. Suppression of P2X7 receptor-NLRP3 activation by gentiopicroside inhibited IL-1β production. In ethanol-exposed HepG2 cells, gentiopicroside reduced lipogenesis and promoted lipid oxidation via activation of P2X7 receptor-NLRP3 inflammasomes. Genetic or pharmacological blockade of P2X7 receptors enhanced AMPK activity and reduced SREBP1 expression in ethanol-treated HepG2 cells. Gentiopicroside down-regulated P2X7 receptor-mediated inflammatory responses in LPS/ATP-stimulated RAW 264.7 macrophages and BMDMs. IL-1β from macrophages accelerated lipid accumulation in hepatocytes. Depleting macrophages by clodronate liposomes ameliorated alcoholic hepatosteatosis, and it was further alleviated by gentiopicroside.Conclusions and implicationsActivation of LKB1/AMPK signalling by gentiopicroside was mediated by the P2X7 receptor-NLRP3 inflammasome, suggesting the therapeutic value of blocking P2X7 receptors in the treatment of alcoholic hepatosteatosis.

Project description:Thioredoxin-interacting protein (TXNIP) regulates critical biological processes including inflammation, stress and apoptosis. TXNIP is upregulated by glucose and is a critical mediator of hyperglycemia-induced beta-cell apoptosis in diabetes. In contrast, the saturated long-chain fatty acid palmitate, although toxic to the beta-cell, inhibits TXNIP expression. The mechanisms involved in the opposing effects of glucose and fatty acids on TXNIP expression are unknown. We found that both palmitate and oleate inhibited TXNIP in a rat beta-cell line and islets. Palmitate inhibition of TXNIP was independent of fatty acid beta-oxidation or esterification. AMP-activated protein kinase (AMPK) has an important role in cellular energy sensing and control of metabolic homeostasis; therefore we investigated its involvement in nutrient regulation of TXNIP. As expected, glucose inhibited whereas palmitate stimulated AMPK. Pharmacologic activators of AMPK mimicked fatty acids by inhibiting TXNIP. AMPK knockdown increased TXNIP expression in presence of high glucose with and without palmitate, indicating that nutrient (glucose and fatty acids) effects on TXNIP are mediated in part via modulation of AMPK activity. TXNIP is transcriptionally regulated by carbohydrate response element-binding protein (ChREBP). Palmitate inhibited glucose-stimulated ChREBP nuclear entry and recruitment to the Txnip promoter, thereby inhibiting Txnip transcription. We conclude that AMPK is an important regulator of Txnip transcription via modulation of ChREBP activity. The divergent effects of glucose and fatty acids on TXNIP expression result in part from their opposing effects on AMPK activity. In light of the important role of TXNIP in beta-cell apoptosis, its inhibition by fatty acids can be regarded as an adaptive/protective response to glucolipotoxicity. The finding that AMPK mediates nutrient regulation of TXNIP may have important implications for the pathophysiology and treatment of diabetes.

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Finely-tuned regulation of AMP-activated protein kinase is crucial for human adult erythropoiesis.

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Finely-tuned regulation of AMP-activated protein kinase is crucial for human adult erythropoiesis.

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