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Animals were individually housed in a temperature-controlled (24°C to 27°C) facility with a 12-h light/dark cycle (lights on 22:00) and free access to food and water. After a minimum of 2 weeks acclimation to the facility, the mice were randomized according to their body weight, so...
Cells were then stimulated with agonists for 30 min in serum-free DMEM with 0.1 % BSA. cAMP was assayed using the Cisbio cAMP Dynamic 2 HTRF kit. 3-parameter logistic fitting was performed with fitting to the global maximum for the assay. Ratio of the cAMP response for Dicretin ...
Specialised or high-cost pharmaceutical drugs used for the treatment of viral hepatitis, multiple sclerosis, HIV and diabetes are distributed free of charge by the Brazilian government. The glucagon peptide was included in this group of high-cost biopharmaceuticals in 2008. Although its main ...
(B) Gluconeogenic gene expression of WT hepatocytes after 13 hrs in glucose-free DMEM in the absence or presence of LPA (2.5 μM), glucagon (10 nM) and/or insulin (100 nM); n = 3. (C) Representative Western blot and quantification of PEPCK expression in WT primary ...
(Life Technologies) according to the manufacturer’s protocol. Reverse transcription products were diluted 1:3 in nuclease-free water, and 1 μl was used for qPCR with the Kapa Sybr Fast qPCR kit (Kapa Biosystems) in combination with the CFX96 Real Time PCR thermocycler (Biorad). The ...
Free fatty acids were measured by a commercially available enzymatic NEFA kit (Wako Diagnostics, Richmond, USA) with intra-assay CV of 4.2% and inter-assay CV of 8.5%. RNA was extracted from muscle biopsies as described in Ref. [26]. Muscle ANGPTL4 mRNA content in the biopsies was ...
HEK-293 cells expressing GCGR-WT or desired GCGR mutant were stimulated with glucagon after starvation for 1 h in serum-free media. Following stimulation cells were solubilized in ice-cold lysis buffer containing 50 mM Hepes (pH 7.5), 2 mM EDTA (pH 8.0), 250 mM NaCl, 10% (v/v) glycer...
This procedure consisted of three iterative steps: (a) random conformational change of a dihedral angle according to the biased probability Monte Carlo method (35); (b) local minimization of all free dihedral angles; and (c) acceptance or rejection of the new conformation based on the ...
Cells were then treated with various concentrations (0, 10, 50, and 100 µg/ml) of l-PGDS for 2 h in 1 mM glucose; glucagon secretion was then measured using a Millipore Milliplex kit. High concentrations of l-PGDS were found to reduce the amount of glucagon secretion into the media...