5 3h d glucose

ECs were incubated in fully supplemented EGM2 medium with 100 μM unlabeled palmitate and 50 μM carnitine. Cells were incubated for 2 hr in growth medium containing 2 μCi/mL [9,10-³H]-palmitate Thereafter, the supernatant was transferred into glass vials sealed with rubber stoppers. ³H₂O was captured in hanging wells containing a piece of Whatman paper soaked with H₂O over a period of 48 hr at 37°C to reach saturation (Schoors et al., 2015 (link)). Radioactivity was determined by liquid scintillation counting. Glycolysis was measured analogously to fatty acid oxidation (cf supra) using 80 mCi/mmol [5-³H]-D-glucose (Perkin Elmer) (De Bock et al., 2013 (link)).

INS-1 832/13 and αTC1-6 cells were seeded in 24-well tissue-culture plates and cultured overnight. Prior to assay, cells were washed in PBS and pre-incubated as described for hormone secretion. Then, the buffer was removed and 500 μl of HBSS containing D-[5-³H] glucose (specific activity 19.63 Ci/mmol; Perkin Elmer Life Science) and glucose added to reach the same concentrations as were used for determination of hormone secretion. Subsequently, cells were incubated for 30 min at 37°C, followed by addition of 100 μl of 10% trichloracetic acid to prevent further metabolism of D-[5-³H] glucose. The cells were harvested from the wells and 500 μl lysate transferred to 1.5 ml eppendorf tubes. The tubes were placed inside sealed scintillation vials, containing 500 μl of water and incubated at 56°C overnight to permit ³H₂O formed by the cells to evaporate and equilibrate with water in the vials. The vials were cooled to room temperature and ³H content in the water measured by liquid scintillation spectrometry [41 (link)]. Glucose utilization was calculated as previously described in detail [42 (link)].

Glycolysis was analyzed as described (Ashcroft, et al. 1972 (link); Tamarit-Rodriguez, et al. 1998 (link); Wang and Iynedjian 1997 (link)). Briefly, aliquots of ~100 WT and germline G6pc2 KO islets were incubated for 2 hrs at 37°C in Krebs bicarbonate buffer containing 5.6 mM glucose spiked with 1 μl D-[5-³H]glucose (Perkin Elmer, Waltham, MA; 10Ci/mmol; 1 mCi/ml) in a volume of 100 μl. Following the incubation, islets were pelleted by centrifugation. The supernatant was retained for analysis of ³H₂O generation whereas DNA content of the cell pellet was measured using the Hoechst reagent (Sigma, St. Louis, MO) as described (Ashcroft et al. 1972 (link)).