gst-pparγ protein  (Boston Biochem)

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Glutamine activates peroxisome proliferator-activated receptor-? in intestinal epithelial cells via 15-S-HETE and 13-OXO-ODE: a novel mechanism

doi: 10.1152/ajpgi.00174.2011

Figure Lengend Snippet: Peroxisome proliferator-activated receptor-γ (PPAR-γ) DNA binding activity is increased by glutamine (Gln) stimulation in small bowel epithelial cells. Intestinal epithelial cells, IEC-6, were incubated in serum-free media overnight and then treated with the indicated concentrations of Gln for 24 h. A: PCR was performed for evaluation of PPAR-γ mRNA expression and showed no change in mRNA expression with increasing Gln concentration. B: Western blotting was performed for measurement of protein expression and similarly showed no change in protein expression with increasing Gln concentration. C: DNA binding activity was measured in nuclear extracts and demonstrated a dose-dependent increase in PPAR-γ DNA binding activity. The EMSA and corresponding densitometry data are shown. D: DNA binding activity also demonstrated a time-dependent increase in PPAR-γ DNA binding activity. At 24 h, activity increased 3.1-fold. The EMSA and corresponding densitometry data are shown.

Article Snippet: Three micrograms of purified PPAR-γ (GST-PPAR-γ fusion protein from Thermo Fisher Scientific), 10 nM [ 3 H]BRL49653 (rosiglitazone) (50 Ci/mmol) (American Radiolabeled Chemicals), and 5 μM or 50 μM of glutamine were incubated with 5 μM of rosiglitazone or vehicle in a buffer containing 10 mM Tris (pH 8.0), 50 mM KCl, and 10 mM DTT at 4°C for 3 h ( 27 ).

Techniques: Binding Assay, Activity Assay, Incubation, Expressing, Concentration Assay, Western Blot

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Glutamine activates peroxisome proliferator-activated receptor-? in intestinal epithelial cells via 15-S-HETE and 13-OXO-ODE: a novel mechanism

doi: 10.1152/ajpgi.00174.2011

Figure Lengend Snippet: PPAR-γ transcriptional activity is increased by Gln, but inhibited by GW9662 (GW) in a dose-dependent fashion. A: confluent IEC-6 cells were transiently transfected with PPAR-γ response element (PPRE)-luciferase and then incubated in Gln (2–20 mM) for 24 h. Whole cell lysates were assayed for luciferase activity and normalized to Renilla luciferase expression. PPAR-γ transcriptional activity was increased by Gln in a dose-dependent fashion, reaching maximal activity after 10 mM Gln. B: confluent IEC-6 cells were transiently transfected with PPRE-luc and then preincubated with GW (0–20 μM), followed by 10 mM Gln. Whole cell lysates were assayed for luciferase activity and normalized to Renilla luciferase expression. There was an inverse correlation between PPAR-γ transcriptional activity and GW concentration, reaching maximal inhibition after 10 μM GW. C: cells were preincubated in 10 μM GW, followed by either 2 mM or 10 mM Gln, and nuclear DNA binding activity was determined. A representative EMSA is shown. D: densitometric analysis revealed that Gln increased DNA binding activity, which was significantly inhibited by GW. Changes in activity followed changes in PPAR-γ DNA binding activity. All results represent the means ± SE of triplicate determinations. aP < 0.05 and bP < 0.01.

Article Snippet: Three micrograms of purified PPAR-γ (GST-PPAR-γ fusion protein from Thermo Fisher Scientific), 10 nM [ 3 H]BRL49653 (rosiglitazone) (50 Ci/mmol) (American Radiolabeled Chemicals), and 5 μM or 50 μM of glutamine were incubated with 5 μM of rosiglitazone or vehicle in a buffer containing 10 mM Tris (pH 8.0), 50 mM KCl, and 10 mM DTT at 4°C for 3 h ( 27 ).

Techniques: Activity Assay, Transfection, Luciferase, Incubation, Expressing, Concentration Assay, Inhibition, Binding Assay

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Glutamine activates peroxisome proliferator-activated receptor-? in intestinal epithelial cells via 15-S-HETE and 13-OXO-ODE: a novel mechanism

doi: 10.1152/ajpgi.00174.2011

Figure Lengend Snippet: Gln is not a direct PPAR-γ ligand. A ligand binding assay was performed using purified PPAR-γ. [3H]rosiglitazone (Ros; PPAR-γ synthetic ligand) was incubated with either 5 μM or 50 μM of Gln, 5 μM of Ros, or vehicle. The amount of [3H]Ros bound to PPAR-γ in the presence of vehicle was assigned as 100% binding. There was no significant displacement of bound Ros by an equimolar concentration or even a 10-fold concentration of Gln, indicating that Gln is not a PPAR-γ ligand. Results represent the means ± SE of triplicate determinations.

Article Snippet: Three micrograms of purified PPAR-γ (GST-PPAR-γ fusion protein from Thermo Fisher Scientific), 10 nM [ 3 H]BRL49653 (rosiglitazone) (50 Ci/mmol) (American Radiolabeled Chemicals), and 5 μM or 50 μM of glutamine were incubated with 5 μM of rosiglitazone or vehicle in a buffer containing 10 mM Tris (pH 8.0), 50 mM KCl, and 10 mM DTT at 4°C for 3 h ( 27 ).

Techniques: Ligand Binding Assay, Purification, Incubation, Binding Assay, Concentration Assay

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Glutamine activates peroxisome proliferator-activated receptor-? in intestinal epithelial cells via 15-S-HETE and 13-OXO-ODE: a novel mechanism

doi: 10.1152/ajpgi.00174.2011

Figure Lengend Snippet: Potential endogenous ligands of PPAR-γ in small bowel intestinal epithelial cells. IEC-6 cells were treated with the indicated concentrations of Gln for 24 h. A: 15-deoxy-d-Δ12,14-PGJ2 (15d-PGJ2) concentrations were measured, and expression decreased in a dose-dependent fashion. B: to examine lipoxygenase metabolites after exposure to Gln, cells were processed for liquid chromatography/tandem mass spectrometry. Detection was validated using deuterated standards of potential ligands. All values were normalized to cell number. Data shown are from pooled lysates from three separate experiments. There was an increase in both 15-hydroxyeicosatetraenoic acid (15-HETE) and dehydrogenated 13-hydroxyoctaolecadienoic acid (13-OXO-ODE) after increasing concentrations of Gln. 13-HODE, 13-hydroxyoctaolecadienoic acid. a P < 0.05 and b P < 0.01.

Article Snippet: Three micrograms of purified PPAR-γ (GST-PPAR-γ fusion protein from Thermo Fisher Scientific), 10 nM [ 3 H]BRL49653 (rosiglitazone) (50 Ci/mmol) (American Radiolabeled Chemicals), and 5 μM or 50 μM of glutamine were incubated with 5 μM of rosiglitazone or vehicle in a buffer containing 10 mM Tris (pH 8.0), 50 mM KCl, and 10 mM DTT at 4°C for 3 h ( 27 ).

Techniques: Expressing, Liquid Chromatography, Mass Spectrometry

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Glutamine activates peroxisome proliferator-activated receptor-? in intestinal epithelial cells via 15-S-HETE and 13-OXO-ODE: a novel mechanism

doi: 10.1152/ajpgi.00174.2011

Figure Lengend Snippet: Proposed pathway by which Gln activates PPAR-γ in small bowel intestinal epithelial cells. PPAR-γ can act as a nuclear receptor for both linoleic acid and arachidonic acid. Arachidonic acid can either be metabolized by the cyclooxygenase pathway to 15-PGJ2 or the lipoxygenase pathway. Gln stimulation resulted in a decrease in 15-PGJ2. Alternatively, arachidonic acid can be metabolized to 15-S-hydroperoxyeicosatetraenoic acid (15-S-HPETE) and then 15-S-HETE. Linoleic acid can be metabolized to 13-S-hydroperoxyoctadecadienoic acid (13-S-HPODE) and then to 13-S-HODE. 13-HODE dehydrogenase then converts 13-S-HODE into 13-OXO-ODE. The common link between 15-S-HETE and 13-OXO-ODE is glutathione (GSH), which is involved in the production of these metabolic by-products, and itself is a metabolite of Gln via glutamate. In response to Gln stimulation, 15-S-HETE and 13-OXO-ODE then enter the nucleus, bind to the ligand binding domain (LBD), and activate PPAR-γ. Once activated, PPAR-γ heterodimerizes with the retinoid X receptor (RXR), and then this complex binds to a specific DNA element, PPRE, in the promoter of target genes where it can activate transcription (8). DBD, DNA binding domain.

Article Snippet: Three micrograms of purified PPAR-γ (GST-PPAR-γ fusion protein from Thermo Fisher Scientific), 10 nM [ 3 H]BRL49653 (rosiglitazone) (50 Ci/mmol) (American Radiolabeled Chemicals), and 5 μM or 50 μM of glutamine were incubated with 5 μM of rosiglitazone or vehicle in a buffer containing 10 mM Tris (pH 8.0), 50 mM KCl, and 10 mM DTT at 4°C for 3 h ( 27 ).

Techniques: Ligand Binding Assay, Binding Assay

Journal: American Journal of Physiology - Gastrointestinal and Liver Physiology

Article Title: Glutamine activates peroxisome proliferator-activated receptor-? in intestinal epithelial cells via 15-S-HETE and 13-OXO-ODE: a novel mechanism

doi: 10.1152/ajpgi.00174.2011

Figure Lengend Snippet: Glutamate increases PPAR-γ DNA binding activity in small bowel intestinal epithelial cells. IEC-6 cells were incubated in serum-free media overnight and then treated with either 10 mM Gln or the indicated concentrations of glutamate for 24 h. DNA binding activity was measured in nuclear extracts and demonstrated a dose-dependent increase in PPAR-γ DNA binding activity by glutamate.

Article Snippet: Three micrograms of purified PPAR-γ (GST-PPAR-γ fusion protein from Thermo Fisher Scientific), 10 nM [ 3 H]BRL49653 (rosiglitazone) (50 Ci/mmol) (American Radiolabeled Chemicals), and 5 μM or 50 μM of glutamine were incubated with 5 μM of rosiglitazone or vehicle in a buffer containing 10 mM Tris (pH 8.0), 50 mM KCl, and 10 mM DTT at 4°C for 3 h ( 27 ).

Techniques: Binding Assay, Activity Assay, Incubation