ampk inhibitor bml  (Santa Cruz Biotechnology)

Journal: Redox Biology

Article Title: Pioglitazone-induced AMPK-Glutaminase-1 prevents high glucose-induced pancreatic β-cell dysfunction by glutathione antioxidant system

doi: 10.1016/j.redox.2021.102029

Figure Lengend Snippet: Effect of pioglitazone in the absence of AMPK on high glucose induced mitochondrial dysfunction. (A–C) INS-1 cells were treated with high glucose (30 mM) with Pioglitazone (10 μM) for 36 h with or without BML-275 (10 μM). The cell extracts were harvested and tested for protein levels with indicated antibodies. Actin was used as the loading control. (A) (* P < 0.001 vs control; ** P < 0.001 vs HG; *** P < 0.01 vs PIO) (B) (* P < 0.001 vs control; ** P < 0.001 vs HG; *** P < 0.001 vs PIO) (C) * P < 0.05 vs control; ** P < 0.05 vs HG; *** P < 0.05 vs PIO) (D) Co-IP of GLS1 with TRAP1 after pioglitazone treatment with BML-275 (10 μM) in high glucose conditions. (E) Measurement of relative GSH/GSSG ratios in INS-1 cells after pioglitazone treatment with BML-275 (10 μM) in high glucose conditions after 36 h (*P < 0.001 vs. control; **P < 0.005 vs. HG; ***P < 0.005 PIO). (F, G) Cellular ROS production and mitochondrial membrane potential after BML-275 treatment. (H) Cytosolic cytochrome c , cleaved casapase-3, BCL-2 and BCL-XL protein levels were quantified by immunoblotting (* P < 0.01 vs control; ** P < 0.01 vs HG; *** P < 0.01 vs PIO). Data are represented as mean ± SEM of three independent experiments.

Article Snippet: GW9662 (PPARγ antagonist) was purchased from Cayman (USA) and AMPK inhibitor (BML-275) from Enzo Life Sciences (USA).

Techniques: Co-Immunoprecipitation Assay, Western Blot

Journal: Redox Biology

Article Title: Pioglitazone-induced AMPK-Glutaminase-1 prevents high glucose-induced pancreatic β-cell dysfunction by glutathione antioxidant system

doi: 10.1016/j.redox.2021.102029

Figure Lengend Snippet: Pioglitazone effect on AMPK-GLS1 activation in Human pancreatic beta 1.1b4 cells. (A) Human pancreatic 1.1b4 cells were incubated with Pioglitazone (10 μM) for 48 h with high glucose (30 mM). Pioglitazone-induced AMPKα (Thr172) and ACC (Ser79), TRAP1/HSP75, and GLS1 was analyzed with western blotting (* P < 0.001 vs control; ** P < 0.005 vs HG). (B) Human pancreatic 1.1b4 cells were treated with high glucose (30 mM) with Pioglitazone (10 μM) for 48 h with or without BML-275 (10 μM). The cell extracts were harvested and tested for protein levels with indicated antibodies (* P < 0.001 vs control; ** P < 0.001 vs HG; *** P < 0.001 vs PIO). (C, D) Measurement of relative GSH/GSSG ratios and cellular ROS production were measured (*P < 0.001 vs. control; **P < 0.005 vs. HG; ***P < 0.005 PIO). (E, F) Cleaved caspase-3 and cell viability were measured using the Cell Counting Kit-8 (* P < 0.01 vs control; ** P < 0.005 vs HG; *** P < 0.005 vs PIO). (G) Pioglitazone activates AMPK, which causes a TRAP1/HSP75-GLS1 interaction, which increases the GSH/GSSG ratio and blocks mTORC1-induced maladaptive ER stress, protecting cells from high glucose toxicity.

Article Snippet: GW9662 (PPARγ antagonist) was purchased from Cayman (USA) and AMPK inhibitor (BML-275) from Enzo Life Sciences (USA).

Techniques: Activation Assay, Incubation, Western Blot, Cell Counting

Journal: Nutrients

Article Title: Maternal High Fat Diet Programs Male Mice Offspring Hyperphagia and Obesity: Mechanism of Increased Appetite Neurons via Altered Neurogenic Factors and Nutrient Sensor AMPK

doi: 10.3390/nu12113326

Figure Lengend Snippet: Activation of AMPK in vitro upregulates bHLH factors, suppresses appetite and increases POMC neuropeptide expression. ( A ) Control NPCs were treated in vitro with AMPK activator (0.5, 1, 2 mM; □) or vehicle (■). NPC protein expression with representative immunoblots of pAMPK, bHLH factors (Mash1, Ngn3) and ( B ) neuropeptides (POMC, AgRP). Values are fold change of vehicle treated ( N = 5; mean ± SE). * p < 0.01 vs control. Abbreviations: AICAR (5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside); pAMPK (phosphorylated 5’ AMP-activated protein kinase); Mash1 (Achaete-scute complex homolog-1); Ngn3 (neurogenin 3); POMC (proopiomelanocortin); AgRP (agouti-related protein); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

Article Snippet: To further explore AMPK-mediated effects, NPCs from postnatal 1-day old (p1) Control newborn males were cultured in differentiating medium and treated in vitro with AMPK activator (AICAR; 0.25, 1, 2 mM; #A9978, Sigma-Aldrich, St. Louis, MO, USA), AMPK inhibitor (1, 5, 25 μM Dorsomorphin dihydrochloride, Compound C BML-275,#21207, Cayman Chemicals, Ann Arbor, MI, USA) or vehicle.

Techniques: Activation Assay, In Vitro, Expressing, Control, Western Blot

Journal: Nutrients

Article Title: Maternal High Fat Diet Programs Male Mice Offspring Hyperphagia and Obesity: Mechanism of Increased Appetite Neurons via Altered Neurogenic Factors and Nutrient Sensor AMPK

doi: 10.3390/nu12113326

Figure Lengend Snippet: Suppression of AMPK in vitro downregulates bHLH factors and increases appetite and reduces satiety neuropeptide expression. ( A ) Control NPCs were treated in vitro with AMPK inhibitor (Compound C 1, 5, 25 µm; □) or vehicle (■). NPC protein expression with representative immunoblots of pAMPK, bHLH factors (Mash1, Ngn3) and ( B ) neuropeptides (POMC, AgRP). Values are fold change of vehicle treated ( N = 5; mean ± SE). * p < 0.001 vs control. Abbreviations: Compound C (Dorsomorphin dihydrochloride); pAMPK (phosphorylated 5’ AMP-activated protein kinase); Mash1 (Achaete-scute complex homolog-1); Ngn3 (neurogenin 3); POMC (proopiomelanocortin); AgRP (agouti-related protein); GAPDH (Glyceraldehyde 3-phosphate dehydrogenase).

Article Snippet: To further explore AMPK-mediated effects, NPCs from postnatal 1-day old (p1) Control newborn males were cultured in differentiating medium and treated in vitro with AMPK activator (AICAR; 0.25, 1, 2 mM; #A9978, Sigma-Aldrich, St. Louis, MO, USA), AMPK inhibitor (1, 5, 25 μM Dorsomorphin dihydrochloride, Compound C BML-275,#21207, Cayman Chemicals, Ann Arbor, MI, USA) or vehicle.

Techniques: In Vitro, Expressing, Control, Western Blot