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Glycerol can be metabolized to glucose via gluconeogenesis or lactate via glycolysis. It is unknown if glycerol is metabolized similarly in the portal and systemic circulations in humans. Eight metabolically healthy overnight-fasted individuals received equimolar amounts of 13C3-glycerol orally and intravenously on two separate occasions with serial blood draws over four hours. Serum samples underwent liquid chromatography–mass spectrometry analysis. Oral 13C3-glycerol administration led to higher average serum glucose enrichment than intravenous administration (5.02 ± 1.43 versus 4.07 ± 0.79%, p = 0.009). In contrast, intravenous 13C3-glycerol administration yielded higher average serum lactate enrichment than oral administration (5.67 ± 0.80 versus 4.85 ± 1.30%, p = 0.032). Peak serum glucose enrichment was also higher with oral administration (9.37 ± 2.93 versus 7.12 ± 1.28%, p = 0.010). Glycerol metabolism across the portal and systemic circulations is not congruent. Orally administered labeled glycerol led to greater labeled glucose production, while intravenously administration yielded greater lactate production. These data support direct glycerol to lactate conversion in humans.

The liver is among the principal organs for glucose homeostasis and metabolism. Studies of liver metabolism are limited by the inability to expand primary hepatocytes in vitro while maintaining their metabolic functions. Human hepatic three-dimensional (3D) organoids have been established using defined factors, yet hepatic organoids from adult donors showed impaired expansion. We examined conditions to facilitate the expansion of adult donor-derived hepatic organoids (HepAOs) and HepG2 cells in organoid cultures (HepGOs) using combinations of growth factors and small molecules. The expansion dynamics, gluconeogenic and HNF4α expression, and albumin secretion are assessed. The conditions tested allow the generation of HepAOs and HepGOs in 3D cultures. Nevertheless, gluconeogenic gene expression varies greatly between conditions. The organoid expansion rates are limited when including the TGFβ inhibitor A8301, while are relatively higher with Forskolin (FSK) and Oncostatin M (OSM). Notably, expanded HepGOs grown in the optimized condition maintain detectable gluconeogenic expression in a spatiotemporal distribution at 8 weeks. We present optimized conditions by limiting A8301 and incorporating FSK and OSM to allow the expansion of HepAOs from adult donors and HepGOs with gluconeogenic competence. These models increase the repertoire of human hepatic cellular tools available for use in liver metabolic assays.

Metabolic flux analysis (MFA) is an increasingly important tool to study metabolism quantitatively. Unlike the concentrations of metabolites, the fluxes, which are the rates at which intracellular metabolites interconvert, are not directly measurable. MFA uses stable isotope labeled tracers to reveal information related to the fluxes. The conceptual idea of MFA is that in tracer experiments the isotope labeling patterns of intracellular metabolites are determined by the fluxes, therefore by measuring the labeling patterns we can infer the fluxes in the network. In this review, we will discuss the basic concept of MFA using a simplified upper glycolysis network as an example. We will show how the fluxes are reflected in the isotope labeling patterns. The central idea we wish to deliver is that under metabolic and isotopic steady-state the labeling pattern of a metabolite is the flux-weighted average of the substrates’ labeling patterns. As a result, MFA can tell the relative contributions of converging metabolic pathways only when these pathways make substrates in different labeling patterns for the shared product. This is the fundamental principle guiding the design of isotope labeling experiment for MFA including tracer selection. In addition, we will also discuss the basic biochemical assumptions of MFA, and we will show the flux-solving procedure and result evaluation. Finally, we will highlight the link between isotopically stationary and nonstationary flux analysis.