Mob00

Standard i.p. glucose tolerance testing was performed using a 20% glucose solution in a weight-adjusted dosage (2 mg/g body weight). The procedure started 7–8 hours after food removal and initiating of the anesthesia. Whole tail blood sampling was performed at 0, 30, 60, 90 and 120 minutes. Glucose levels were measured using a standard measurement devise (Contour XT, Bayer, Germany). Insulin and leptin measurements were performed using commercially available ELISA kits (10-1247-01, Mercodia, Sweden, Cat.Nr. MOB00, R&D Systems, USA). For further information, see supplementary material. The intracerebroventricular insulin injection was performed using a 33-gauge blunt-tip cannula fixed on a 10 μl Hamilton syringe (WorldPrecision Instruments, FL, USA). The third ventricle was targeted using the stereotactic coordinates AP: −2.3, ML: 0.0, DV: −8.5 mm. 2 μl of 4mU/l insulin or saline was injected at a pace of 1 μl/min via a precision syringe pump (Micro4™, World Precision Instruments, FL, USA).

Pre-adipocytes were plated at a density of 200,000 cells/12-well. The next day, cells were exposed to CRF peptides plus/minus LPS at different time points and cell culture supernatants were collected and stored at −80 C until used for the determination of adipokine and interleukin concentration by ELISA.
Parallel experiments were performed in fully differentiated adipocytes. In brief, pre-adipocytes were cultured to 12-well plates at a density of 20,000 cells per well and forced to differentiate as mentioned above. Then, fully differentiated adipocytes were exposed to CRF peptides plus/minus LPS at different time points and cell culture supernatants were collected and stored at −80 C. Cell culture supernatants were also collected from adipocytes exposed to CRF peptides plus/minus LPS during the differentiation process.
Adiponectin was measured by MRP300 and Duoset (DY1119), while Leptin was measured by MOB00 and Duoset (DY498) purchased from R&D (R&D, NE). ELISA assays for IL-6 (DY406), CXCL1/KC (DY453), TNF-α (DY410), IL-1b (DY401) and IL-10 (DY417) were all purchased from R&D. For normalization of the measurements, cells were harvested and sonicated for quantification of total cellular proteins as previously described [14] (link).

The Ad and leptin concentrations were measured according to the manufacturer’s instructions (Ad: MRP300, leptin: MOB00, R&D Systems, Minneapolis, MN, USA).

Adiponectin and leptin were assessed in pgAT homogenates using ELISA kits (MOB00 and MRP300, R&D Systems, Minneapolis, MN, USA). Samples of adipose tissue homogenates (4.5 µg and 75 µg of protein lysate for adiponectin and leptin, respectively) were assayed in triplicate in 96 well-plates that were read in a BioRad iMark microplate spectrophotometer. The optical density was determined by subtracting the value obtained at 570 nm of wavelength from the value obtained at 450 nm according to the manufacturer’s instructions.

The Ad and leptin plasmatic concentration were measured by using the mouse Ad/Acrp30 Quantikine ELISA Kit and the mouse Leptin Quantikine ELISA Kit, according to the manufacturer’s instructions (Ad: MRP300; Leptin: MOB00, R&D Systems, Minneapolis, MN, United States).