After 24 h of FA treatment exposure, media was harvested from each plate, pooled within triplicate, and stored at −80 °C for subsequent quantification of glucose (Autokit Glucose, Wako Diagnostics, Richmond, VA), TG (L-Type Triglyceride M, Wako Diagnostics, Richmond, VA), and BHB (commercial kit, CataChemWell-T, Awareness Technologies, Westport, CT).
After removal of media, cells were harvested for subsequent quantification of cellular TG and DNA as previously described16 (link). Total cellular TG was normalized to corresponding total DNA within each culture plate prior to averaging within triplicates. Total glucose, BHB, and TG in the pooled media sample were normalized to total DNA averaged across the triplicate.
To characterize the temporal pattern of glucose export in this culture system, a parallel set of isolated hepatocytes were cultured for the serial sampling of media for glucose analysis. After 4 h of incubation following cell seeding, media was refreshed with base media without glucose, Met, and choline chloride and cells were randomly assigned in triplicate to media containing either 0 or 1 mM FA. Cells were cultured for the next 20 h before cell media was refreshed with the same treatments. Over the culture period from 8 to 48 h, 25 µL of media from each triplicate was serially collected and pooled across triplicates every 4 h for subsequent analysis of glucose.
After removal of media, cells were harvested for subsequent quantification of cellular TG and DNA as previously described16 (link). Total cellular TG was normalized to corresponding total DNA within each culture plate prior to averaging within triplicates. Total glucose, BHB, and TG in the pooled media sample were normalized to total DNA averaged across the triplicate.
To characterize the temporal pattern of glucose export in this culture system, a parallel set of isolated hepatocytes were cultured for the serial sampling of media for glucose analysis. After 4 h of incubation following cell seeding, media was refreshed with base media without glucose, Met, and choline chloride and cells were randomly assigned in triplicate to media containing either 0 or 1 mM FA. Cells were cultured for the next 20 h before cell media was refreshed with the same treatments. Over the culture period from 8 to 48 h, 25 µL of media from each triplicate was serially collected and pooled across triplicates every 4 h for subsequent analysis of glucose.
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