N13195

Glucose absorption was assessed using the protocol previously outlined in the literature [7 (link),15 (link)]. Briefly, well-differentiated C2C12 myoblasts underwent a 16 h starvation phase in low-glucose DMEM (11885084, Gibco BRL, Middlesex, UK) to deplete essential nutrients. Subsequently, these cells were exposed to various QG concentrations for 6 h, followed by a 30 m treatment with 50 nM insulin in a no-glucose medium (11966025, Gibco BRL, Middlesex, UK) to stimulate glucose uptake. After the treatment, the cells were washed twice with 1 × PBS (LB 001-01, Welgene, Gyeongsan-si, Republic of Korea) and then incubated with a 100 μM solution of the fluorescent glucose analog, 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose (2-NBDG; N13195, Thermo Fisher Scientific, Waltham, MA, USA), for 90 min. The level of glucose uptake by the cells was measured by detecting the fluorescence at an excitation/emission wavelength of 465/540 nm using a Victor^TM X4 plate reader.

Isolated primary hepatocytes were blocked with rat IgG antibody (1 μg/ml, MAB005, R&D systems) in phosphate buffered saline (PBS) containing 1% (w/v) BSA for 15 minutes at room temperature. Next, cells were washed three times and incubated with the rat anti-GLUT2-allophycocyanin (APC) antibody (1 μg/ml, FAB1440A, R&D systems), mouse anti-asialoglycoprotein receptor 1 (ASGR1) (1 μg/ml, AF2755, R&D systems) with Alexa Flour 488 conjugated anti-mouse IgG antibody in PBS containing 1% (w/v) BSA. For lipids staining or glucose uptake assay, primary hepatocytes and Hep3B were incubated with BODIPY (20 μM, D-3922, Invitrogen) or 2-NBDG (500 μM, N13195, Invitrogen), respectively. After incubation, cells were washed three times and promptly analyzed on a FACSCalibur (BD Immunocytometry System).

The fluorescent analogue of glucose (Glc), 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) Amino)-2-Deoxyglucose (2NBDG) was used for the evaluation of glucose uptake. Briefly, 5x10⁵ T cells were incubated for 30 minutes in Glc free media (12633-012, Invitrogen) to normalize the Glc uptake rate across different samples, then incubated with 100 μM of 2NBDG (N13195, Invitrogen) resuspended in 100 μl of Glc free media for 30 minutes at 37°C with the addition or not of 2-DG. Upon extensive washes with PBS, the cells were analyzed with flow cytometry for green fluorescence (FL-1 channel). Data are presented as mean fluorescence intensity (MFI). Alternatively, the Glc uptake luminescence-based assay was used as per the manufacturer’s instructions (J1341, Promega).

HepG2 cells (4 × 10⁵) were seeded onto 6-well plates and cultured in the supplemented medium (EMEM) until 70–80% confluency was reached. Subsequently, the cells were grown in galactose medium for 16 h and in low-glucose medium supplemented with 20 mM 2-DG for 24 h with KR. On the next day, the cells remaining in galactose medium were treated for 6 h with 20, 40, and 80 µM KR. The cells were then washed with PBS, added to culture media (low glucose when possible) supplemented with 100 µM 2-NBDG that shows excitation and emission maxima at 465/540 nm (a fluorescent glucose analog used to monitor glucose uptake in living cells as an indicator of cell viability; Life Technologies #N13195), and incubated for 1 h at 37 °C with 5% CO₂. Subsequently, the cells were detached with trypsin (Thermo Fisher Scientific) and washed twice with 1 ml DPBS (Thermo Fisher Scientific). Stained cells were immediately analyzed with excitation at 488 nm by Accuri C6 flow cytometer (Becton Dickinson).

2-[N-(7-Nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG) is taken up into cells through glucose transporters and phosphorylated by hexokinase. sEND.1 cells were seeded in a 24 well plate. After overnight culture, cells were treated with or without IFNγ (100 ng/mL, 315-05, Peprotech) and AZD5363 (10 μM, HY-15431, MCE) for 12 h. The culture medium was discarded and cells were washed with PBS (pH 7.4). This was followed by the addition of low-glucose culture media supplemented with 2-NBDG (100 µM, N13195, Life Technologies) and an incubation of 45 min at 37 °C. Next, cells were washed with PBS and trypsin was added, and they were harvested, centrifuged at 1,500 rpm for 5 min at 4 °C, washed twice with ice-cold PBS, and kept on ice. A control sample lacking 2-NBDG was used to set the blank in the flow cytometer and gate parameters for 2-NBDG detection.