5 thio d glucose

Nine-week-old male Sprague-Dawley rats (300-350 g) were fed in individual cages and allowed ad libitum access to food and water. Animals were given 7 days to adapt before the experiments. Rats underwent duodenal cannulation as previously described [19 ] and infusion catheters were placed in the proximal duodenum 1.5–2 cm downstream of the pyloric sphincter. Several additional groups of rats were stereotaxically implanted with a bilateral steel guide cannula positioned 2 mm above the caudomedial nucleus of the solitary tract (NTS). Bilateral catheters were inserted into the dorsal vagal complex (DVC) targeting NTS for injection. Accurate cannula placement was histologically confirmed by consumption of more than 1.5 g of chow within 60 min following a parenchymal injection of 24 μg of 5-thio-D-glucose (Sigma) in 0.1 μl of artificial cerebrospinal fluid (aCSF) per side and adenovirus expressing green fluorescent (Ad-GFP) (Additional file 1: Figure S1) [20 (link)]. Indwelling catheters were placed into the internal jugular vein and carotid artery for infusion and blood sampling during the pancreatic-euglycemic clamp (PEC).

Radiolabeling has been published previously (Seidensticker et al. 2014 (link)). In brief, 1-thio-β-D-glucose and 5-thio-D-glucose were obtained from Sigma-Aldrich® (St.Louis, USA). The glucose derivatives (in mean 5.1 µmol) were labeled with a maximum of 370 MBq [^99mTc] pertechnetate. The product was analyzed by high-performance liquid chromatography (HLPC) and thin-layer chromatography (TLC) and formulated in 0.9% NaCl for intravenous administration. The required minimum radiochemical purity for 1-TG and 5-TG was defined as 95%. Detailed analytic methods on radiolabeling process and purity assessment can be found in supplementary data (Additional File 2: Fig. S2 and Additional File 6).