Stable isotope resolved metabolomics of primary human hepatocytes reveals a stressed phenotype

The development of techniques allowing the culturing of primary mammalian hepatocytes has provided great insights into liver physiology. For most applications, it is desirable for hepatocytes in culture to mimic hepatocytes in vivo. We used stable isotope resolved metabolomics (SIRM) to assess glucose and glutamine utilization in primary rat and human hepatocytes maintained in standard culture media. Primary rat hepatocytes readily metabolized 13 C-glucose and 13 C-glutamine made evident by 13 C incorporation into glycogen, glycolytic end products, and Krebs cycle intermediates and responded to insulin and glucagon appropriately. In contrast, no glucose or glutamine consumption was detected in primary human hepatocytes over 4 h of exposure to high media concentrations of 13 C-glucose or 13 C-glutamine even in the presence of insulin. Nonetheless, cultured human hepatocytes were metabolically active and viable, as demonstrated by incorporation of media 13 C-octanoic acid into Krebs cycle intermediates and ketone bodies. The failure to utilize glucose was not due to inhibition of glucokinase (hexokinase IV) since the human hepatocytes could readily incorporate 13 C-glucose into glucuronic acid, as demonstrated by the production of 13 C-glucuronide conjugates after addition of acetaminophen to the media. These novel observations support inhibition of phosphofructokinase-1, the other regulatory enzyme in glycolysis. Parts of this phenotype could be reproduced in the rat hepatocytes by replacing insulin with glucagon to the media. We conclude that under standard culture conditions human hepatocytes are in an extreme starved state. We believe this may result from prolonged fasting in the human liver donors combined with exposure to stress hormones such as, epinephrine, glucagon, and cortisol. Efforts should now be exerted to find culture conditions that will reverse this state to achieve more metabolically relevant cultures of human hepatocytes.