Exendin 9 39

Cucurbitacin B (CuB, 100 μM), glucose (10%), denatonium benzoate (10 mM), 2-APB (10 μM), U73122 (10 μM), U73343 (10 μM), rolipram (50 μM), STO-690 (100 μM), Dorsomorphin (50 μM), forskolin (5 μM), and AICAR (5-Aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, 1–5 mM) were purchased from Sigma-Aldrich (Sigma-Aldrich, MO, United States). Gallein (10 μM) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Lactisole (10 μM) was purchased from Endeavour Speciality Chemicals (Daventry, United Kingdom). Metformin, Diabex tab was purchased from Daewoong Pharmaceutical Co., Ltd. (Gyeonggi-do, South Korea). PBS was purchased from Corning (NY, United States) Bovine serum albumin (BSA) was purchased from RMBIO (MT, United States). Exendin 9-39 was purchased from Abcam (Cambridge, United Kingdom).

Rats received a systemic infusion of either saline or exendin-(9-39) (GLP-1 receptor antagonist, 30 nmol/kg/min, Abcam, Cambridge, United Kingdom) via the jugular vein catheter for 20 min (Figure 1A). In the last 10 min, rats also received 30 pmol/kg/min of either GLP-1-FAM (AnaSpec, Fremont, CA, United States) or exendin-4-FAM (AnaSpec, Fremont, CA, United States) together with Dextran-TRITC (Sigma-Aldrich, St. Louis, MO, United States) (70 kD, vascular space marker, 30 pmol/kg/min). The infusion doses were based on our prior studies in rats that GLP-1 at 30 pmol/kg/min potently increased muscle microvascular perfusion and glucose use (Chai et al., 2012 (link), 2014 (link); Dong et al., 2013 (link)). After collecting plasma samples, rats were sacrificed via anesthetic overdose and various brain regions (cerebellum, cortex, hippocampus, hypothalamus, and brain stem) collected, homogenized and lysed in lysis buffer. Fluorescence from TRITC and FAM were measured in both plasma and brain lysates. Protein concentrations were determined in the brain lysates. Brain uptake of GLP-1-FAM and exendin-4-FAM were calculated using the following formula: