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Stimulation of GLP-1 Receptor Inhibits Methylglyoxal-Induced Mitochondrial Dysfunctions in H9c2 Cardiomyoblasts: Potential Role of Epac/PI3K/Akt Pathway.

ABSTRACT: Accumulation of methylglyoxal (MG) contributes to oxidative stress, apoptosis, and mitochondrial dysfunction, leading to the development of type 2 diabetes and cardiovascular diseases. Inhibition of mitochondrial abnormalities induced by MG in the heart may improve and delay the progression of heart failure. Although glucagon-like peptide-1 receptor (GLP-1R) agonists have been used as anti-diabetic drugs and GLP-1R has been detected in the heart, the cardioprotective effects of GLP-1R agonists on the inhibition of MG-induced oxidative stress and mitochondrial abnormalities have not been elucidated. Stimulation of GLP-1Rs leads to cAMP elevation and subsequently activates PKA- and/or Epac-dependent signaling pathway. However, the signaling pathway involved in the prevention of MG-induced mitochondrial dysfunctions in the heart has not been clarified so far. In the present study, we demonstrated that stimulation of GLP-1Rs with exendin-4 inhibited MG-induced intracellular and mitochondrial reactive oxygen species (ROS) production and apoptosis in H9c2 cardiomyoblasts. GLP-1R stimulation also improved the alterations of mitochondrial membrane potential (MMP) and expressions of genes related to mitochondrial functions and dynamics induced by MG. In addition, stimulation of GLP-1R exhibits antioxidant and antiapoptotic effects as well as the improvement of mitochondrial functions through cAMP/Epac/PI3K/Akt signaling pathway in H9c2 cells. Our study is the first work demonstrating a novel signaling pathway for cardioprotective effects of GLP-1R agonist on inhibition of oxidative stress and prevention of mitochondrial dysfunction. Thus, GLP-1R agonist represents a potential therapeutic target for inhibition of oxidative stress and modulation of mitochondrial functions in the heart.

SUBMITTER: Nuamnaichati N

PROVIDER: S-EPMC7274035 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Stimulation of GLP-1 Receptor Inhibits Methylglyoxal-Induced Mitochondrial Dysfunctions in H9c2 Cardiomyoblasts: Potential Role of Epac/PI3K/Akt Pathway.

Nuamnaichati Narawat N Mangmool Supachoke S Chattipakorn Nipon N Parichatikanond Warisara W

Frontiers in pharmacology 20200529

Accumulation of methylglyoxal (MG) contributes to oxidative stress, apoptosis, and mitochondrial dysfunction, leading to the development of type 2 diabetes and cardiovascular diseases. Inhibition of mitochondrial abnormalities induced by MG in the heart may improve and delay the progression of heart failure. Although glucagon-like peptide-1 receptor (GLP-1R) agonists have been used as anti-diabetic drugs and GLP-1R has been detected in the heart, the cardioprotective effects of GLP-1R agonists o ...[more]

PMID: 32547400

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Project description:Background and aimsActivation of the cAMP/Epac signalling stabilises endothelial barrier function. Moreover, its activation is accompanied by an activation of PI3K/Akt and MEK/ERK signalling in diverse cell types but their impact on endothelial barrier function is largely unknown. Here the role of PI3K/Akt and MEK/ERK signalling in cAMP/Epac-mediated endothelial barrier stabilisation was analysed.MethodsEndothelial barrier function was analysed in cultured human umbilical vein endothelial cells (HUVECs) by measuring flux of albumin. A modified cAMP analogue 8-pCPT-2'-O-Me-cAMP (Epac agonist) was used to specifically activate cAMP/Epac signalling.ResultsEpac agonist reduces the basal and attenuates thrombin-induced endothelial hyperpermeability accompanied by an activation of PI3K/Akt and MEK/ERK signalling. The qPCR data demonstrate HUVECs express PI3Kα, PI3Kβ, and PI3Kγ but not PI3Kδ isoforms. The western blot data demonstrate Epac agonist activates PI3Kα and PI3Kβ isoforms. Inhibition of MEK/ERK but not PI3K/Akt pathway potentiates the endothelial barrier protective effects of cAMP/Epac signalling. Inhibition of MEK/ERK signalling in the presence of Epac agonist induces a reorganisation of actin cytoskeleton to the cell periphery, enhanced VE-cadherin localisation at cell-cell junctions, and dephosphorylation of myosin light chains (MLC) but not inhibition of RhoA/Rock signalling. Moreover, Epac agonist promotes endothelial cell (EC) survival via reduction in activities of pro-apoptotic caspases in a PI3K/Akt and MEK/ERK signalling-dependent manner.ConclusionOur data demonstrate that the Epac agonist simultaneously activates diverse signalling pathways in ECs, which may have differential effects on endothelial barrier function. It activates PI3K/Akt and MEK/ERK signalling which mainly govern its pro-survival effects on ECs. Inhibition of MEK/ERK but not PI3K/Akt signalling enhances barrier stabilising and barrier protective effects of cAMP/Epac activation.Chemical compounds used in this study8-pCPT-2'-O-Me-cAMP (PubChem CID: 9913268); Akt inhibitor VIII (PubChem CID: 10196499); AS-252424 (PubChem CID: 11630874); IC-87114 (PubChem CID: 9908783); PD 98059 (PubChem CID: 4713); PIK-75 (PubChem CID: 10275789); TGX-221 (PubChem CID: 9907093); Thrombin (PubChem CID: 90470996); U0126 (PubChem CID: 3006531); Wortmannin (PubChem CID: 312145).

Project description:Mitochondria are susceptible to damage resulting from their activity as energy providers. Damaged mitochondria can cause harm to the cell and thus mitochondria are subjected to elaborate quality-control mechanisms including elimination via lysosomal degradation in a process termed mitophagy. Basal mitophagy is a house-keeping mechanism fine-tuning the number of mitochondria according to the metabolic state of the cell. However, the molecular mechanisms underlying basal mitophagy remain largely elusive. In this study, we visualized and assessed the level of mitophagy in H9c2 cardiomyoblasts at basal conditions and after OXPHOS induction by galactose adaptation. We used cells with a stable expression of a pH-sensitive fluorescent mitochondrial reporter and applied state-of-the-art imaging techniques and image analysis. Our data showed a significant increase in acidic mitochondria after galactose adaptation. Using a machine-learning approach we also demonstrated increased mitochondrial fragmentation by OXPHOS induction. Furthermore, super-resolution microscopy of live cells enabled capturing of mitochondrial fragments within lysosomes as well as dynamic transfer of mitochondrial contents to lysosomes. Applying correlative light and electron microscopy we revealed the ultrastructure of the acidic mitochondria confirming their proximity to the mitochondrial network, ER and lysosomes. Finally, exploiting siRNA knockdown strategy combined with flux perturbation with lysosomal inhibitors, we demonstrated the importance of both canonical as well as non-canonical autophagy mediators in lysosomal degradation of mitochondria after OXPHOS induction. Taken together, our high-resolution imaging approaches applied on H9c2 cells provide novel insights on mitophagy during physiologically relevant conditions. The implication of redundant underlying mechanisms highlights the fundamental importance of mitophagy.Abbreviations: ATG: autophagy related; ATG7: autophagy related 7; ATP: adenosine triphosphate; BafA1: bafilomycin A1; CLEM: correlative light and electron microscopy; EGFP: enhanced green fluorescent protein; MAP1LC3B: microtubule associated protein 1 light chain 3 beta; OXPHOS: oxidative phosphorylation; PepA: pepstatin A; PLA: proximity ligation assay; PRKN: parkin RBR E3 ubiquitin protein ligase; RAB5A: RAB5A, member RAS oncogene family; RAB7A: RAB7A, member RAS oncogene family; RAB9A: RAB9A, member RAS oncogene family; ROS: reactive oxygen species; SIM: structured illumination microscopy; siRNA: short interfering RNA; SYNJ2BP: synaptojanin 2 binding protein; TEM: transmission electron microscopy; TOMM20: translocase of outer mitochondrial membrane 20; ULK1: unc-51 like kinase 1.

Dataset Information

Stimulation of GLP-1 Receptor Inhibits Methylglyoxal-Induced Mitochondrial Dysfunctions in H9c2 Cardiomyoblasts: Potential Role of Epac/PI3K/Akt Pathway.

Publications

Stimulation of GLP-1 Receptor Inhibits Methylglyoxal-Induced Mitochondrial Dysfunctions in H9c2 Cardiomyoblasts: Potential Role of Epac/PI3K/Akt Pathway.

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