Journal: Journal of Neuroinflammation

Article Title: Microglia-derived TNF-α contributes to RVLM neuronal mitochondrial dysfunction via blocking the AMPK–Sirt3 pathway in stress-induced hypertension

doi: 10.1186/s12974-023-02818-6

Figure Lengend Snippet: TNF-α led to neuronal mitochondrial dysfunction via downregulating the AMPK-Sirt3 pathway. A Representative immunofluorescence images and quantitative analysis showed colocalization of Sirt3 and tyrosine hydroxylase (TH)-positive neurons in the RVLM of control and SIH groups. Scale bar = 500 or 20 μm. B Representative immunoblot bands and quantitative analysis showed the expression of p-AMPK and Sirt3 in the RVLM of control and SIH rats. C Representative immunoblot bands and quantitative analysis of p-AMPK and Sirt3 expression in N2a cells treated with or without TNF-α. D Representative images and quantitative analysis showed mitochondrial membrane potential (MMP) in control, TNF-α, and TNF-α + A769662 groups. Scale bar = 50 μm. E Representative immunoblot bands and quantitative analysis showed the expression of mitochondrial respiratory chain complexes (complexes II-SDHB, III-UQCRC2, and V-ATP5A) in N2a cells with different treatments. F Representative fluorescence images of reactive oxygen species (ROS) detection and statistical data of ROS production in N2a cells with or without TNF-α treatment. Scale bar = 50 μm. G The levels of superoxide dismutase (SOD) and catalase (CAT) were quantified by using commercial kits in each group. Data were shown as mean ± SEM. Statistical significance was determined using two-tailed unpaired Student’s t -test ( A – C ) and one-way ANOVA followed by post hoc Bonferroni test ( D – G ). n = 12 slices from 6 rats, two slices per rat ( A ). n = 3 rats per group ( B ). n = 3 of independent cell culture preparations ( C , E ). n = 6 of independent cell culture preparations ( G ). n = 12 slices from 6 of independent cell culture preparations, two slices per independent cell culture preparation ( D , F ). *p < 0.05 vs. SIH group. # p < 0.05, ## p < 0.01 vs. TNF-α group

Article Snippet: The AMPK activator A769662 was purchased from MedChemExpress (Cat.NO.HY-50662, USA).

Techniques: Immunofluorescence, Control, Western Blot, Expressing, Membrane, Fluorescence, Two Tailed Test, Cell Culture

Journal: Journal of Neuroinflammation

Article Title: Microglia-derived TNF-α contributes to RVLM neuronal mitochondrial dysfunction via blocking the AMPK–Sirt3 pathway in stress-induced hypertension

doi: 10.1186/s12974-023-02818-6

Figure Lengend Snippet: Schematic diagram illustrating the mechanisms of the pressor effect of microglia-derived TNF-α via impairing neuronal mitochondrial function in the RVLM. A769662: AMPK activator; CAT: catalase; ETC: electron transport chain; MMP: mitochondrial membrane potential; R7050: TNF-α receptor antagonist; ROS: reactive oxygen species; SOD: superoxide dismutase; TNF-α: tumor necrosis factor-α

Article Snippet: The AMPK activator A769662 was purchased from MedChemExpress (Cat.NO.HY-50662, USA).

Techniques: Derivative Assay, Membrane

Journal: The Journal of biological chemistry

Article Title: Glucose-6-phosphate dehydrogenase exerts antistress effects independently of its enzymatic activity.

doi: 10.1016/j.jbc.2022.102587

Figure Lengend Snippet: Figure 3. G6PD facilitates AMPK activity independently of its dehydrogenase activity. A, Western blotting analysis of G6PD, pACC1-Ser79, ACC1, pAMPKα-Thr172, and AMPK in HeLa/WT and HeLa/G6PD-KO cells treated with 500 μM AICAR for 8 h or 24 h. B, cell survival of HeLa/G6PD-KO cells treated with 1 μM antimycin A for 24 h, in the presence or absence of 500 μM AICAR (pretreatment for 12 h). C, Western blotting analysis of G6PD, pACC1-Ser79, ACC1, pAMPKα-Thr172, and AMPKα in HeLa cells with different states of G6PD, as indicated, treated with 50 μM or 100 μM A769662 for 8 h. D, cell survival of HeLa/G6PD-KO cells treated with or without PMS (1 μM), H2O2 (100 μM), antimycin A (1 μM) for 24 h, in the presence or absence of 100 μM A769662 (pretreatment for 16 h). E and F, Flag-AMPKα1 immunoprecipitates from HeLa/G6PD-KO cells were aliquoted to incubate with recombinant G6PD-Wt or R198P protein in the presence or absence of ATP in an in vitro kinase assay. pACC1-Ser79, ACC1, pAMPKα-The172, and AMPKα were detected using Western blot. **p < 0.01 (t test). AICAR, 5-amino-4-imidazolecarboxamide ribonucleoside; G6PD, glucose-6-phosphate dehydrogenase.

Article Snippet: A769662 was obtained from Selleck.

Techniques: Activity Assay, Western Blot, Recombinant, In Vitro, Kinase Assay

Journal: eLife

Article Title: Continuous sensing of nutrients and growth factors by the mTORC1-TFEB axis

doi: 10.7554/eLife.74903

Figure Lengend Snippet:

Article Snippet: A769662 , DMSO , Tocris , 3336.

Techniques: Solvent

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of CPT2 exacerbates cardiac dysfunction and inflammation in experimental endotoxaemia

doi: 10.1111/jcmm.15809

Figure Lengend Snippet: Changes in body temperature and blood glucose concentration 4 h after LPS administration

Article Snippet: C75 and A769662 were purchased from Tocris Bioscience and Carbosynth Ltd., respectively.

Techniques: Concentration Assay, Control, Saline

Journal: Journal of Cellular and Molecular Medicine

Article Title: Inhibition of CPT2 exacerbates cardiac dysfunction and inflammation in experimental endotoxaemia

doi: 10.1111/jcmm.15809

Figure Lengend Snippet: LPS‐induced changes in mitochondrial energy metabolism pattern in cardiac tissues. Respiration rate (A) and flux control factors (B) in permeabilized cardiac fibres. LPS administration decreased fatty acid oxidation pathway‐dependent oxidative phosphorylation (A,B) and induced pyruvate metabolism stimulation without affecting other pathways (B). Treatment with both C75 and A769662 restored the fatty acid oxidation pathway‐linked OXPHOS coupling efficiency and subsequently decreased pyruvate metabolism (B). Alongside after aminocarnitine administration, the fatty acid oxidation pathway‐linked OXPHOS coupling efficiency remained decreased, and pyruvate metabolism was inhibited (B). Each value represents the mean ± SEM of 5 animals. *Significant difference between saline control and LPS control groups (Student's t test, P < .05). # Significantly different from the LPS control group (ANOVA followed by Dunnett's test, P < .05). FAO, fatty acid oxidation; F(N), fatty acid oxidation‐dependent pathway (FADH 2 and NADH); N, NADH‐pathway; LEAK, substrate metabolism‐dependent state; OXPHOS, oxidative phosphorylation‐dependent state; D, ADP; P, pyruvate; PC, palmitoylcarnitine; Rot, rotenone; S, succinate

Article Snippet: C75 and A769662 were purchased from Tocris Bioscience and Carbosynth Ltd., respectively.

Techniques: Control, Phospho-proteomics, Saline

Journal:

Article Title: AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H + -ATPase accumulation in epididymal clear cells

doi: 10.1152/ajpcell.00004.2009

Figure Lengend Snippet: AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) inhibits the pH-mediated V-ATPase accumulation at the apical membrane of clear cells. A and B: confocal images of double immunofluorescence labeling for V-ATPase distribution (green) and for the endocytic marker horseradish peroxidase (HRP, red) in clear cells perfused for 60 min with phosphate-buffered saline (PBS) pH 7.8 (A) or PBS pH 7.8 + AICAR (2 mM) (B). Arrows demarcate the base of the microvilli in clear cells. Treatment of cells with the AMPK activator AICAR prevented the elongation of apical microvilli observed upon exposure to pH 7.8 and prevented accumulation of the V-ATPase at the apical pole. C and D: level of V-ATPase accumulation in microvilli in the cells from A and B was quantified by measuring the area occupied by V-ATPase-labeled microvilli (enclosed in the white line), normalized for the width of the cells at the apical pole (blue line) for each cell. E: quantification of the surface occupied by V-ATPase-labeled microvilli of clear cells normalized by the apical width of each cell under the conditions shown in A and B. Data shown are means ± SE from three tissues and at least 30 cells per condition (*P < 0.05). Scale bar = 5 μm.

Article Snippet: To further confirm that these findings resulted from AMPK activation in this epididymis/VD in vivo perfusion system, we repeated the above experiments using a second direct and specific AMPK activator, Abbott compound A-769662.

Techniques: Membrane, Immunofluorescence, Labeling, Marker, Saline

Journal:

Article Title: AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H + -ATPase accumulation in epididymal clear cells

doi: 10.1152/ajpcell.00004.2009

Figure Lengend Snippet: The AMPK activator A-769662 inhibits the pH-mediated V-ATPase accumulation at the apical membrane of clear cells. A and B: confocal images of double immunofluroscence labeling of the V-ATPase distribution (green) and the endocytic marker HRP (red) in clear cells perfused for 60 min with PBS pH 7.8 (A) or PBS pH 7.8 containing AMPK activator Abbott compound A-769662 (200 μM) (B). Arrows demarcate the base of the microvilli in clear cells. Treatment of cells A-769662 prevented the elongation of apical microvilli observed upon exposure to pH 7.8 and prevented accumulation of the V-ATPase at the apical pole. C: quantification of the surface occupied by V-ATPase-labeled microvilli of clear cells normalized by the apical width of each cell under the conditions shown in A and B. Data shown are means ± SE from three tissues and at least 30 cells per condition (*P < 0.05). Scale bar = 5 μm.

Article Snippet: To further confirm that these findings resulted from AMPK activation in this epididymis/VD in vivo perfusion system, we repeated the above experiments using a second direct and specific AMPK activator, Abbott compound A-769662.

Techniques: Membrane, Labeling, Marker

Journal:

Article Title: AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H + -ATPase accumulation in epididymal clear cells

doi: 10.1152/ajpcell.00004.2009

Figure Lengend Snippet: AMPK activator AICAR inhibits the PKA-mediated V-ATPase accumulation at the apical membrane of clear cells. A, B, and C: confocal images of the distribution of V-ATPase (green) and of the endocytic marker HRP (red) by immunofluorescence labeling in clear cells perfused with PBS pH 6.5 for 75 min (A); PBS pH 6.5 for 45 min, followed by PBS pH 6.5 containing PKA activator N6-monobutyryl-cAMP (6-MB-cAMP, 100 μM) for an additional 30 min (B); or PBS (pH 6.5) containing AICAR (2 mM) for 45 min, followed by PBS (pH 6.5) plus AICAR plus 6-MB-cAMP for an additional 30 min (C). Arrows demarcate the bases of the microvilli. Treatment of cells with AMPK activator AICAR prevented the elongation of apical microvilli observed upon exposure to a specific PKA activator 6-MB-cAMP and prevented accumulation of the V-ATPase at the apical pole. D: quantification of V-ATPase accumulation in apical microvilli for at least three independent perfusions and at least 30 cells per condition. Data shown are the means ± SE (*P < 0.05, relative to PBS, pH 6.5; #P < 0.05, relative to PBS, pH 6.5; **P < 0.05, relative to + 6-MB-cAMP). Scale bar = 7.5 μm.

Article Snippet: To further confirm that these findings resulted from AMPK activation in this epididymis/VD in vivo perfusion system, we repeated the above experiments using a second direct and specific AMPK activator, Abbott compound A-769662.

Techniques: Membrane, Marker, Immunofluorescence, Labeling

Journal:

Article Title: AMP-activated protein kinase inhibits alkaline pH- and PKA-induced apical vacuolar H + -ATPase accumulation in epididymal clear cells

doi: 10.1152/ajpcell.00004.2009

Figure Lengend Snippet: PKA- and AMPK-dependent in vivo phosphorylation of the V-ATPase A subunit in HEK-293 cells. FLAG-tagged A subunit was transfected into HEK-293 cells expressing an irrelevant mammalian shRNA (Fig. 7, “CON” cells) or in AMPK KD cells expressing an shRNA for AMPK-α1. Cells were then incubated with [32P]orthophosphate for 2 h in the presence of a PKA activator (6-MB-cAMP, last 20 min of labeling period) or in the presence of PKA inhibitor myristoylated protein kinase inhibitor (mPKI) (for entire labeling period), followed by lysis of the cells, immunoprecipitation using an anti-FLAG antibody, SDS-PAGE, and immunoblotting using an anti-FLAG antibody. A: typical phospho-screen image (top) revealing the signal of phosphorylated A subunit under the indicated conditions in CON or AMPK KD cells. The Western blot (bottom) confirms similar protein expression and loading of the gel for the different conditions. B: quantification of V-ATPase A subunit phosphorylation signal normalized for protein expression in vivo (*P = 1.6 × 10−5; **P = 1.3 × 10−4; #P = 3.7 × 10−4; ##P = 3.2 × 10−6; §P = 4.9 × 10−6, and §§P = 0.016) indicates that the differences between the indicated conditions were statistically significant by analysis of variance; (n = 4 experiments). C: relative PKA-dependent A subunit phosphorylation in CON and AMPK KD cells as calculated by the difference in phosphorylation signal between 6-MB-cAMP- and mPKI-treated cells over the total phosphorylation signal with 6-MB-cAMP treatment (comparing lanes 2 and 3 for CON and lanes 5 and 6 for AMPK KD). *P < 0.05, unpaired t-test.

Article Snippet: To further confirm that these findings resulted from AMPK activation in this epididymis/VD in vivo perfusion system, we repeated the above experiments using a second direct and specific AMPK activator, Abbott compound A-769662.

Techniques: In Vivo, Phospho-proteomics, Transfection, Expressing, shRNA, Incubation, Labeling, Lysis, Immunoprecipitation, SDS Page, Western Blot