Maintaining a stable blood glucose concentration through periods of nutrient availability or fasting is an essential physiological equilibrium [1]. Much of this balance is orchestrated by hepatocytes in the liver, which control several metabolic pathways to regulate glucose blood levels, such as glycogenesis, glycogenolysis, glycolysis, and gluconeogenesis [2]. During periods when a dietary glucose supply is not available, hepatocytes can secrete large quantities of glucose into the blood. Glucose is produced and released either via breakdown of stored glycogen (glycogenolysis and glycophagy) [3,4] or de novo synthesis using carbon substrates such as pyruvate, lactate, and amino acids (gluconeogenesis) [5].

Lipid metabolism plays a crucial role in maintaining energy homeostasis and is deeply interlinked with glucose metabolism [6]. Gluconeogenesis is an energy-demanding process, which is primarily derived from the beta(β)-oxidation of fatty acids (FAs), stored in hepatocytes as triglycerides (TGs) or delivered by the adipose tissue via the blood circulation during periods of starvation [7]. The breakdown of fatty acids produces molecules such as acetyl-CoA and NADH, which can enter the citric acid cycle and the electron transport chain, respectively, to generate ATP that is necessary for the production of glucose [7]. While the link between fatty acid β-oxidation and gluconeogenesis is widely accepted and characterized, the metabolic connection with other lipids, such as phospholipids and sphingolipids is complex and partially unknown.

Unsurprisingly, impaired or dysregulated glucose production and lipid metabolism, often associated with pathologies such as diabetes, obesity and inborn errors of metabolism, can lead to severe and life-threatening medical conditions [[8], [15]]. Studying the process of glucose and lipid metabolism during periods of nutrient deprivation is crucial to understand how metabolic disorders lead to potentially life-threatening scenarios. Consequently, in vitro glucose production assays, in which hepatocytes are stimulated to secrete glucose under controlled conditions are central biological readouts that hold immense importance in the field. These assays rely on metabolically competent human hepatocyte models, experimental conditions that simulate the physiological nutrient availability oscillations found in vivo and robust assay to quantify molecular targets.

Primary human hepatocytes (PHH) are still considered as the gold standard in metabolic disease research but present several limitations, such as their limited availability, donor-to-donor variation, rapid de-differentiation and high costs. Hence, researchers have been exploring the possibility to use alternative hepatocyte sources by comparing their metabolic competence to primary cells. Direct comparisons are needed for specific assays in order to evaluate the biological response of non-primary hepatocytes exposed to a metabolic challenge.

Glucose production, for example, has been measured for primary hepatocytes, iPSC-derived hepatocytes (iPSC-Hep) and hepatoma cell lines and typically involve a prolonged period of starvation (3–24 h) in which the cells are cultured in absence (or reduced levels) of glucose but in presence of gluconeogenic substrates, such as glycerol, lactate or pyruvate [[16], [17], [18], [19], [20]].

Recently, expandable primary hepatocytes (Upcyte-Hep) have been generated by inducing low expression of the human papilloma virus [21,22] but their competence for energy metabolism studies remains unclear [23]. Furthermore, hepatic organoids (Orgs) emerged as an exciting possibility to generate patient-derived expandable cells that can differentiate into hepatocyte-like cells and recent progress started to determine their exact application landscape to study liver metabolism and disease [24,25]. Yet, more research is needed in order to identify specific applications of alternatives to primary hepatocytes in metabolism studies.

With this study, we aimed to characterize and compare the metabolic capabilities of various in vitro models of human hepatocytes such as iPSC-Hep, HepG2, Upcyte-Hep and organoids with PHH. Specifically, we challenged the models with a glucose production assay by measuring secreted glucose levels and induction of gluconeogenesis-related genes. Finally, we used a highly sensitive targeted lipidomics method to study the lipid profile of these hepatocyte models to monitor the modulation of intracellular lipid composition as a result of the glucose production challenge. This study aimed to further characterize and evaluate alternatives to primary hepatocyte models for energy metabolism studies.