Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Oleoylethanolamide modulates glucagon-like peptide-1 receptor agonist signaling and enhances exendin-4-mediated weight loss in obese mice

doi: 10.1152/ajpregu.00459.2017

Figure Lengend Snippet: Oleoylethanolamide (OEA) enhances glucagon-like peptide-1 (GLP-1)-mediated cAMP production and β-arrestin recruitment to the GLP-1 receptor (GLP-1R) without affecting PKA activity. A–G: cAMP production (A–C and G) was measured in cAMP Hunter CHO-K1 GLP-1R Gs (CHOK1-GLP-1R) cells, and β-arrestin recruitment to the GLP-1R (D–F) was measured in CHOK1-GLP-1R-β-arrestin cells after stimulation with various concentrations of GLP-1 or exendin-4 (Ex4) in the absence and presence of OEA (9.2 µM). Dashed line in G is the concentration at which OEA was used for all cell experiments (9.2 µM). Data points reflect the mean ± SE of 3 experimental replicates containing 4 technical replicates each. *GLP-1R agonist (GLP-1RA) vs. GLP-1RA+OEA, #Ex4 vs. GLP-1 (P < 0.05). The x-axis values represent log [Peptide]. H: phosphorylation of PKA substrates was assessed in CHOK1-GLP-1R cells after stimulation with GLP-1 (20 pM), OEA (10 µM), or GLP-1+OEA. Bars reflect means ± SE of 3 experimental replicates consisting of 2 technical replicates each. †vs. vehicle (P < 0.05). Representative immunoblot is shown. Images obtained from different locations on the original immunoblot are separated by white spaces. I: PathScan (Akt Signaling Antibody Array Kit, no. 9700; Cell Signaling Technology) analysis was used to assess phosphorylation of phospho-ERK1/2 (Thr421/Tyr204) in CHOK1-GLP-1R cells after stimulation with vehicle, GLP-1 (20 pM), OEA (10 µM), or GLP-1+OEA for 5, 15, or 60 min. Bars reflect means ± SE of 4 experimental replicates consisting of 2 technical replicates (P < 0.05).

Article Snippet: GLP-1-(7–36) amide (no. 2082; Tocris), Ex4 (ab120214; Abcam), Lira (H-6724; Bachem), and OEA (O0383; Sigma) were used for cell experiments.

Techniques: Activity Assay, Concentration Assay, Western Blot, Ab Array

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Oleoylethanolamide modulates glucagon-like peptide-1 receptor agonist signaling and enhances exendin-4-mediated weight loss in obese mice

doi: 10.1152/ajpregu.00459.2017

Figure Lengend Snippet: Oleoylethanolamide (OEA) does not enhance nutrient-sensing pathways that mediate the hypophagic action of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists. cAMP Hunter CHO-K1 GLP-1R Gs cells were incubated for 5, 15, or 60 min with either GLP-1 (20 pM; A–C) or exendin-4 (Ex4, 20 pM; D–F) in the absence or presence of OEA (10 µM). PathScan (Akt Signaling Antibody Array Kit, no. 9700; Cell Signaling Technology) analysis was then performed to measure the phosphorylated state of targets within AMPK, Akt, and mTOR signaling pathways. Bars reflect means ± SE of 3 experimental replicates consisting of 2 technical replicates each. 4E-BP1, eukaryotic translation initiation factor 4E-binding protein 1; p70S6K, 70-kDa ribosomal protein S6 kinase; PRAS40, 40-kDa proline-rich Akt substrate. *vs. vehicle (P < 0.05).

Article Snippet: GLP-1-(7–36) amide (no. 2082; Tocris), Ex4 (ab120214; Abcam), Lira (H-6724; Bachem), and OEA (O0383; Sigma) were used for cell experiments.

Techniques: Incubation, Ab Array, Binding Assay

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Oleoylethanolamide modulates glucagon-like peptide-1 receptor agonist signaling and enhances exendin-4-mediated weight loss in obese mice

doi: 10.1152/ajpregu.00459.2017

Figure Lengend Snippet: Oleoylethanolamide (OEA) enhances glycolysis in the presence of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) agonists (GLP-1RA) in a GLP-1R-dependent manner. Extracellular acidification rate (ECAR; A and B) and oxygen consumption rate (OCR; C and D) were measured in cAMP Hunter CHO-K1 GLP-1R Gs cells with a Seahorse XF96 analyzer to evaluate glycolysis and mitochondrial respiration, respectively, in the presence of GLP-1 (20 pM), exendin-4 (Ex4, 20 pM), OEA (9.2 µM), or GLP-1RA+OEA. For analysis of GLP-1R-mediated effects, cells were exposed to exendin-9 (Ex9, 500 nM) for 80 min before addition of treatments to inhibit GLP-1R signaling. Data points represent the means ± SE of 3 experimental replicates containing 3–5 technical replicates each. Data were normalized to baseline ECAR or OCR and are represented as fold change from vehicle. *GLP-1RA vs. GLP-1RA+OEA. †GLP-1RA+OEA vs. Ex9-GLP-1RA+OEA (P < 0.05).

Article Snippet: GLP-1-(7–36) amide (no. 2082; Tocris), Ex4 (ab120214; Abcam), Lira (H-6724; Bachem), and OEA (O0383; Sigma) were used for cell experiments.

Techniques:

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Oleoylethanolamide modulates glucagon-like peptide-1 receptor agonist signaling and enhances exendin-4-mediated weight loss in obese mice

doi: 10.1152/ajpregu.00459.2017

Figure Lengend Snippet: Oleoylethanolamide (OEA) does not alter the hypophagic action of glucagon-like peptide-1 (GLP-1) or exendin-4 (Ex4). A–E: cumulative, 18-h food intake was measured using a PromethION metabolic cage system in lean C57BL/6J mice after intraperitoneal injection of vehicle, OEA (8 mg/kg), GLP-1 receptor agonist (GLP-1RA, 3 µg/kg), or GLP-1RA+OEA at the onset of the dark cycle and after 5 h of food deprivation. Data points reflect the means ± SE (n = 12–17/group) *vs. vehicle at same time point (P < 0.05). F–L: chow-fed C57BL/6J mice received an intracerebroventricular injection of artificial cerebrospinal fluid (ACSF, 2 µl) or 2-deoxyglucose (2-DG, 5 mM, 2 µl) followed 15 min later with intraperitoneal injection of vehicle, OEA (8 mg/kg), Ex4 (3 µg/kg), or Ex4+OEA. Data points reflect the means ± SE (n = 7–8/group). *vs. ACSF-vehicle at same time point (P < 0.05). Black and white bars represent dark and light periods, respectively.

Article Snippet: GLP-1-(7–36) amide (no. 2082; Tocris), Ex4 (ab120214; Abcam), Lira (H-6724; Bachem), and OEA (O0383; Sigma) were used for cell experiments.

Techniques: Injection

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Oleoylethanolamide modulates glucagon-like peptide-1 receptor agonist signaling and enhances exendin-4-mediated weight loss in obese mice

doi: 10.1152/ajpregu.00459.2017

Figure Lengend Snippet: Exendin-4 (Ex4) + oleoylethanolamide (OEA) promotes greater weight loss than Ex4 or OEA alone in diet-induced obese (DIO) mice. A–C: body weights (expressed as %, raw data and delta) of male, C57BL/6J, DIO mice were measured daily during chronic treatment (7 days, intraperitoneal injection, twice a day) with vehicle, OEA (5 mg/kg), Ex4 (100 µg/kg), or Ex4+OEA. D and E: fat (D) and lean mass (E) were measured with NMR before treatments began and after the 7-day treatment course. Values are means ± SE (n = 9–12). d, Days; Pre, preceding treatment; Veh, vehicle. *vs. vehicle; †vs. OEA; #vs. Ex4 (n = 9–12/group, P < 0.05).

Article Snippet: GLP-1-(7–36) amide (no. 2082; Tocris), Ex4 (ab120214; Abcam), Lira (H-6724; Bachem), and OEA (O0383; Sigma) were used for cell experiments.

Techniques: Injection

Journal: American Journal of Physiology - Regulatory, Integrative and Comparative Physiology

Article Title: Oleoylethanolamide modulates glucagon-like peptide-1 receptor agonist signaling and enhances exendin-4-mediated weight loss in obese mice

doi: 10.1152/ajpregu.00459.2017

Figure Lengend Snippet: Exendin-4 (Ex4) and Ex4 + oleoylethanolamide (OEA) transiently reduce 24-h food intake and increase energy expenditure (EE)/resting metabolic rate (RMR). Components of energy balance [food intake, EE, locomotor activity, and respiratory quotient (RQ)] were measured in male, diet-induced obese, C57BL/6J mice with a PromethION metabolic cage system preceding (Pre) and during chronic treatment (7 days, intraperitoneal injection, twice a day) with vehicle, OEA (5 mg/kg), Ex4 (100 µg/kg), or Ex4+OEA. A and B: 24-h averages of food intake (kcal; A) and EE (kcal; B) as well as 7-day averages (Insets) were measured. *vs. vehicle within day (P < 0.05). C: comparison of raw EE in the first 24 h of treatment (day 1) and the final 24 h of treatment (day 7). †Day 1 vs. day 7 within treatment group (P < 0.05). D: analysis of covariance (ANCOVA)-adjusted (covariate: lean mass) EE during the final 24 h of treatment [day 7: light (12 h) and dark (12 h) periods]. #vs. vehicle (P < 0.05). E: ANCOVA-adjusted (covariate: lean mass) RMR during the final 24 h (day 7) of treatment. RMR was calculated as the mean EE (kcal/h) during the 30 min of lowest activity over a 24-h period. #vs. vehicle (P < 0.05). F and G: 24-h averages of RQ (F) and locomotor activity (G) were collected by CO2-to-O2 ratio and beam break, respectively. *vs. vehicle within day (P < 0.05). All values reflect the means ± SE (n = 9–12). d, Days; Veh, vehicle.

Article Snippet: GLP-1-(7–36) amide (no. 2082; Tocris), Ex4 (ab120214; Abcam), Lira (H-6724; Bachem), and OEA (O0383; Sigma) were used for cell experiments.

Techniques: Activity Assay, Injection