Substrate Oxidation Control of Respiratory Rates in Primary Hepatocytes

Abstract

The goal of our studies is a bioenergetics profiling of primary rat hepatocytes using the Seahorse-XF analyzer in order to assess adaptation in response to metabolic stress or disease. Cellular oxygen consumption rates (JO2) were compared in enriched medium (DMEM) vs a balanced salt solution (HBSS) without added substrates. Hepatocytes exhibited higher basal JO2 in DMEM compared to HBSS and showed a proportional increase in oligomycin-insensitive JO2. The fractional increase in JO2 by uncoupler was higher in DMEM than in HBSS, presumably due to substrate supply by amino acids present in DMEM. These data suggests that substrate oxidative pathways exert significant control over basal and uncoupled respiration rates in primary rat hepatocytes. To further test this hypothesis, we assessed JO2 under different substrate conditions, in DMEM or HBSS medium. Addition of mono-methylsuccinate (MMS), a mitochondrial Complex III substrate, resulted in a large concentration- dependent stimulation of basal JO2 of hepatocytes in HBSS but a more limited stimulation in DMEM, likely reflecting availability of alternate substrates. In DMEM, physiological glucose concentrations (11mM) had little stimulatory effect, while higher concentrations (25mM) inhibited O2 uptake, thus exhibiting a “Crabtree-like” effect, which was not overcome by uncoupler treatment. This inhibitory effect of high glucose was not evident in HBSS, where basal JO2 increased with higher concentrations of glucose. Oligomycin-insensitive JO2, as a fraction of basal O2 uptake remained similar under all substrate conditions in DMEM and HBSS, apart from a small decrease at the highest MMS concentration. These results suggest a significant control exerted by substrate oxidative pathways over basal and uncoupler-stimulated respiration rates in primary rat hepatocytes. Electron supply may limit the rate of uncoupled respiration in hepatocytes, underestimating the reserve capacity in the electron transport chain. Supported by NIH grants AA018873 and AA017261.