Aglycemic growth enhances carbohydrate metabolism and induces sensitivity to menadione in cultured tumor-derived cells

Abstract

Background Hepatocellular carcinoma (HCC) is the most prevalent form of liver malignancy and carries poor prognoses due to late presentation of symptoms. Treatment of late-stage HCC relies heavily on chemotherapeutics, many of which target cellular energy metabolism. A key platform for testing candidate chemotherapeutic compounds is the intrahepatic orthotopic xenograft (IOX) model in rodents. Translational efficacy from the IOX model to clinical use is limited (in part) by variation in the metabolic phenotypes of the tumor-derived cells that can be induced by selective adaptation to subculture conditions. Methods In this study, a detailed multilevel systems approach combining microscopy, respirometry, potentiometry, and extracellular flux analysis (EFA) was utilized to examine metabolic adaptations that occur under aglycemic growth media conditions in HCC-derived (HEPG2) cells. We hypothesized that aglycemic growth would result in adaptive “aerobic poise” characterized by enhanced capacity for oxidative phosphorylation over a range of physiological energetic demand states. Results Aglycemic growth did not invoke adaptive changes in mitochondrial content, network complexity, or intrinsic functional capacity/efficiency. In intact cells, aglycemic growth markedly enhanced fermentative glycolytic substrate-level phosphorylation during glucose refeeding and enhanced responsiveness of both fermentation and oxidative phosphorylation to stimulated energy demand. Additionally, aglycemic growth induced sensitivity of HEPG2 cells to the provitamin menadione at a 25-fold lower dose compared to control cells. Conclusions These findings indicate that growth media conditions have substantial effects on the energy metabolism of subcultured tumor-derived cells, which may have significant implications for chemotherapeutic sensitivity during incorporation in IOX testing panels. Additionally, the metabolic phenotyping approach used in this study provides a practical workflow that can be incorporated with IOX screening practices to aid in deciphering the metabolic underpinnings of chemotherapeutic drug sensitivity.

From OCR and ECAR to energy: Perspectives on the design and interpretation of bioenergetics studies

“…For example, measurement of the electrochemical potential across the inner mitochondrial membrane (IMM) is proportional to the energetic state of the ETS and can be measured potentiometrically or with fluorescent organic cations (e.g. tetramethyl rhodamine methyl ester) (31,32). The redox potential of the reduced pyridine nucleotide pool (NAD(P)H) within specific subcellular compartments can be proportional to the J o u r n a l P r e -p r o o f energetic state of specific pathways and can be monitored via autofluorescence (ideally fluorescence lifetime) (33,34).…”

“…It is directly proportional to the power transferred through the phosphate potential (Box 1), which if measured over the duration of a cellular process is proportional to the total work required by the process itself (75). Directly measuring JATP is complex and limited by technical challenges ( 76), but estimating JATP from EFA data is straightforward and has been recently described in detail (9,10,31). Importantly, there is a major caveat to this approach: i.e.…”

“…ECAR units can be made to be more stoichiometrically useful by conversion to a proton efflux rate (JH + ; a.k.a proton production rate-PPR) by empirically determining the buffer capacity of the media via titration of known amounts of strong acid (31,115). This J o u r n a l P r e -p r o o f conversion is useful because JH + can be linked to the substrate level ATP phosphorylation rate (APRGlyc) via the following expression(10).…”

“…Abandoning the simplified view that cellular energy transducing pathways are independent is a recommended start. Instead, research should focus on metabolic system integration and adaptation under experimental conditions conducted in the most physiologically relevant micronutrient environment that can be reasonably achieved (10,31,127). Non-physiological metabolic states may inform the limitations of the assay, aid in standardizing among replicates, or aid in interpolating a model of rate limiting steps in the pathway (45).…”

“…To reiterate, the IM/PC-based assays described in previous sections are intended to provide additional resolution on observations made in intact cells, with the ultimate goal of returning to the intact cells to test the new hypotheses (Figure 5). To a limited degree substrate oxidation patterns can be isolated in intact cells using strategic substrate deprivation/refeeding and appropriate inhibitors (31,187). Under conditions in which specific pathways can be reasonably isolated, JATP can be estimated using theoretical ATP/O ratios, which can then be compared to JATP calculated from empirically derived ATP/O ratios.…”