Glucose colorimetric detection kit

After VEM treatment, cells were collected and resuspended in Biolog IF-M1 medium (catalog# 72301, Biolog, Inc.) supplemented with penicillin/streptomycin, 5% FBS, and 0.3 mM glutamine. When indicated, 4 mM sodium lactate and/or 4 mM glucose were added to the cell suspension. The cell density was adjusted to 4 × 10⁵ cells per mL. Then, 500 µL of the cell suspension was transferred to each well of a 24-well plate and incubated at 37 °C. At desired time points (t = 24 h and 48 h), the cell suspension was removed and centrifuged at 900 rpm for 5 min and the supernatant was transferred to a 1.5 mL microcentrifuge tube. The glucose and lactate concentrations in samples and standard solutions were measured using a glucose colorimetric detection kit (catalog# EIAGLUC, Thermo Fisher Scientific) and a lactate assay kit (catalog# MAK064-1KT, Sigma Aldrich) following the vendor’s protocol. For the lactate assay, the supernatant was deproteinized with a 10 kDa MWCO spin filter. This additional step was needed as the presence of lactate dehydrogenase can degrade lactate and interfere with the readings. Standard curves were used to calculate the amount of glucose or lactic acid consumed by the cells daily. The data were then normalized by the number of cells.

Cryopreserved human primary hepatocytes (Lonza) were plated overnight on collagen-coated 12-well plates at 1 × 10⁵ cells per well in MM (Lonza). 24 h after plating, cells were serum-starved in DMEM base medium (Sigma) supplemented with 1 g/L glucose (Sigma), 3.7 g/L sodium bicarbonate (Sigma), and 4 mM L-glutamine (Corning) overnight, followed by 24 h incubation in 0.3 ml glucose-production medium: DMEM base with 2 mM glutamine, 3.7 g/L sodium bicarbonate, 15 mM HEPES (ThermoFisher), 20 mM lactate (Sigma), 2 mM pyruvate (Fisher) and 0.1 mM pCPT-cAMP (Sigma). After 24 h, 50 μL of medium was removed for glucose detection with Invitrogen glucose Colorimetric Detection kit (#EIAGLUC), according to manufacturer’s protocol, and read on a plate reader (Multiskan GO, Thermo-Scientific). Because hepatocytes were extensively washed prior to cell incubation in glucose-free media for this assay, the only potential source of glucose in the media is hepatic production. The prolonged culture of cells in low glucose media prior to the assay depletes hepatocytes of glycogen stores. The media used during this assay contains high concentrations of gluconeogenic substrates, primarily lactate, favoring gluconeogenesis^{67 (link),68 (link)}.