Dmem base medium

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)}.

For respiratory analyses, a basic mitochondrial stress test measuring oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) was performed using a Seahorse XF96 Extracellular Flux Analyzer (Agilent (Santa Clara, CA, USA)). Approximately 60,000–120,000 neurons were seeded per well into a Matrigel-coated Seahorse cell plate 2–5 days prior to the experiment. For the measurement, the medium was replaced with DMEM base medium (without phenol red; Sigma-Aldrich (St. Louis, MO, USA), #D5030 1L) supplemented with 4.5 mg/mL D-Glucose, 0.22 mg/mL pyruvate, and 2 mM glutamine. After eight measurements of basal respiration, hDaNs were challenged with subsequent injections to give final concentrations of (a) 0.8 µM Oligomycin, (b) 2.7 µM CCCP, and (c) 0.8 µM Rotenone with 4 µM Antimycin A. Each injection was followed by three measurements. OCR and ECAR measurement were normalized to the number of hDaNs seeded for each experiment. Basal respiration was calculated by subtracting the mean of the OCR measurements after injection (c) from the OCR of the third measurement of baseline. Maximal respiration was calculated by subtracting the mean of the OCR measurements after injection (c) from the mean of the OCR measurements after injection (b). Spare respiratory capacity was calculated as the percentage of maximal respiration of basal respiration.