Journal: PLoS ONE

Article Title: Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms

doi: 10.1371/journal.pone.0198016

Figure Lengend Snippet: INS-1E cells were cultured in the presence of 11 mM glucose and test substances as indicated and described under Materials and methods. (A,D,F) Changes of TXNIP mRNA levels (ΔΔCt) vs control set to 100% in A and D; vs max effect in F. (B,E,G) Representative western blots of 172 Thr-P-AMPKα and AMPKα1; Tubulin is used as loading control. (C) Quantitative analysis of western blots of three independent experiments of B. Results are given as mean ± SEM of n = 3–4 independent experiments. *p<0.05, **p<0.01, *** p<0.001 denotes significance to control, i.e. 11 mM Glc at 1h; ## p<0.01 to 11 mM Glc at 24 h. $ $p<0.01 significant effect of palmitate in the presence of MS. §§p<0.01 significant effect of palmitate in the presence of MS and BML. Abbreviations: Glc, glucose; Pal, palmitate; MS, MS-275 (HDAC1/2/3 inhibitor); BML, BML-275 (AMPK inhibitor); AICAR, AMPK activator.

Article Snippet: The AMPK activator AICAR (#BML-EI-330, Enzo Life Sciences, Farmingdale, NY, USA), the PI3K inhibitor LY294002 (#440202, Merck Millipore, Burlington, MA, USA), the JNK inhibitor SP600125 (#S5567, Sigma-Aldrich, Munich, Germany), the ERK1/2 inhibitor PD98059 (#51300, Merck Millipore, Burlington, MA, USA), the AMPK inhibitor BML-275 (#BML-EI-369, Enzo Life Sciences, Farmingdale, NY, USA), the PKCα/β inhibitor Gö6976 (#365250, Merck Millipore, Burlington, MA, USA), the HDAC1/2/3 inhibitor MS-275 (#EPS002, Sigma Aldrich, Munich, Germany) and Actinomycin D (#A1410, Sigma-Aldrich, Munich, Germany) were dissolved in DMSO.

Techniques: Cell Culture, Western Blot, Tandem Mass Spectroscopy

Journal: PLoS ONE

Article Title: Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms

doi: 10.1371/journal.pone.0198016

Figure Lengend Snippet: (A-C) INS-1E cells were cultured for 24 h in the presence of 11 mM glucose and test substances as indicated and described under Materials and methods. (A) Percentage of TUNEL positive cells, *p<0.05 significant vs control culture, (B) correlation between TUNEL positive nuclei and relative TXNIP mRNA levels (∆Ct), (C) representative western blot for cleaved caspase 3, GAPDH was used as loading control; (D) representative pictures of INS-1E cells stained with the ROS sensor CellROXGreen. INS-1E cells were cultured for 2 h in the presence of different glucose concentrations, palmitate (600 μM) and menadione (100 μM; ROS inducer) as indicated. CellROXGreen (5 μM) was applied for 1 h in culture. Note the increased green fluorescence (increased ROS levels) in the cells exposed to 30 mM Glc or menadione vs 2.8 mM glucose and 30 mM Glc+palmitate. Abbreviations: Pal, P600, palmitate; BML, BML-275 (AMPK inhibitor); CC-3, cleaved caspase-3.

Article Snippet: The AMPK activator AICAR (#BML-EI-330, Enzo Life Sciences, Farmingdale, NY, USA), the PI3K inhibitor LY294002 (#440202, Merck Millipore, Burlington, MA, USA), the JNK inhibitor SP600125 (#S5567, Sigma-Aldrich, Munich, Germany), the ERK1/2 inhibitor PD98059 (#51300, Merck Millipore, Burlington, MA, USA), the AMPK inhibitor BML-275 (#BML-EI-369, Enzo Life Sciences, Farmingdale, NY, USA), the PKCα/β inhibitor Gö6976 (#365250, Merck Millipore, Burlington, MA, USA), the HDAC1/2/3 inhibitor MS-275 (#EPS002, Sigma Aldrich, Munich, Germany) and Actinomycin D (#A1410, Sigma-Aldrich, Munich, Germany) were dissolved in DMSO.

Techniques: Cell Culture, TUNEL Assay, Western Blot, Staining, Fluorescence

Journal: PLoS ONE

Article Title: Palmitate and insulin counteract glucose-induced thioredoxin interacting protein (TXNIP) expression in insulin secreting cells via distinct mechanisms

doi: 10.1371/journal.pone.0198016

Figure Lengend Snippet: Transcription of TXNIP is under the control of a protein complex consisting of ChREBP (carbohydrate-responsive element-binding protein) and MondoA as well as the histone acetyltransferase P300. ChREBP is negatively regulated by AMPK. Consequently, stimulation of AMPK by AICAR inhibits ChREBP and TXNIP expression, while inhibition of AMPK by glucose and BML-275 activates ChREBP and increases TXNIP mRNA levels. Histone deacetylase 1 (HDAC1), counteracts P300-mediated histone acetylation, and is involved in insulin-mediated downregulation of TXNIP expression. Thus, inhibition of PI3K, AKT or HDAC1/3 increases TXNIP mRNA levels. Fatty acids-mediated stimulation of insulin secretion occurs via FFAR1/GPR40, a signalling pathway not involved in regulation of TXNIP expression. Fatty acids activate AMPK and have an additional effect on TXNIP mRNA levels. Fatty acids counteract glucose-induced TXNIP expression and ROS elevation, events which do not impede the ER strees-associated lipotoxic effect.

Article Snippet: The AMPK activator AICAR (#BML-EI-330, Enzo Life Sciences, Farmingdale, NY, USA), the PI3K inhibitor LY294002 (#440202, Merck Millipore, Burlington, MA, USA), the JNK inhibitor SP600125 (#S5567, Sigma-Aldrich, Munich, Germany), the ERK1/2 inhibitor PD98059 (#51300, Merck Millipore, Burlington, MA, USA), the AMPK inhibitor BML-275 (#BML-EI-369, Enzo Life Sciences, Farmingdale, NY, USA), the PKCα/β inhibitor Gö6976 (#365250, Merck Millipore, Burlington, MA, USA), the HDAC1/2/3 inhibitor MS-275 (#EPS002, Sigma Aldrich, Munich, Germany) and Actinomycin D (#A1410, Sigma-Aldrich, Munich, Germany) were dissolved in DMSO.

Techniques: Binding Assay, Expressing, Inhibition, Histone Deacetylase Assay

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

Journal: Cancer Science

Article Title: Augmented O ‐Glc NA cylation of AMP ‐activated kinase promotes the proliferation of LoVo cells, a colon cancer cell line

doi: 10.1111/cas.13412

Figure Lengend Snippet: AMP ‐activated kinase ( AMPK ) inhibition accelerated cell growth and inactivated the mTOR pathway in LoVo cells. (a) LoVo cells were cultured in serum‐free DMEM with the indicated concentrations of BML ‐275 for 24 h. Cells were then lysed and subjected to Western blot analysis using anti‐phospho‐ AMPK ( pAMPK ) and phospho‐ mTOR (pm TOR ) antibodies. The total expression levels of AMPK and mTOR were used as controls. Experiments were carried out several times and representative data are shown. Ratios of phosphorylated to total AMPK or mTOR were analyzed statistically with Dunnett's test. * P < 0.05. Molecular weight markers are shown. (b) LoVo cells were cultured with the indicated concentrations of BML ‐275, an AMPK inhibitor, for 24 h. Growth was measured using CCK ‐8. Absorbance rates of the cells with the indicated concentrations of BML ‐275 relative to that of ones without BML ‐275 were analyzed statistically with Steel's test, * P < 0.05.

Article Snippet: Both AICAR and BML‐275 were obtained from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Inhibition, Cell Culture, Western Blot, Expressing, Molecular Weight, CCK-8 Assay