Reminiscence of 40-year research on nitrogen metabolism.
Bottom Line: These amino acids are not efficiently metabolized in the liver, so I had to consider the physiology of extrahepatic tissues as well.Finally, I came across a huge protease complex, the proteasome.Whether these players, small amino acid metabolizing enzymes and the huge protease complex, danced well in harmony on my playground or not, I still do not know.
Affiliation: Tokushima University, Japan.
This article summarizes my research over 40 years. The main theme of my work is nitrogen metabolism of amino acids, though later I focused on protein turnover in the cell. In the first years of my research work, I was busy dissecting the pathways involved in the metabolism of certain amino acids and their related enzymes. Then I became interested in the physiology and regulation of matabolism of these amino acids. For that, I used primary cultured hepatocytes, which contain many liver-specific enzymes. However, this play field was very rough around 1970 and hence I had to smooth them (differentiated) first. We discovered a specific growth factor (hepatocyte growth factor, HGF) in rat platelets. Exceptionally, I also worked on branched chain amino acids (valine, leucine and isoleucine). These amino acids are not efficiently metabolized in the liver, so I had to consider the physiology of extrahepatic tissues as well. Finally, I came across a huge protease complex, the proteasome. Whether these players, small amino acid metabolizing enzymes and the huge protease complex, danced well in harmony on my playground or not, I still do not know.
Mentions: Our main interest in hepatocyte research, however, was whether these cells could proliferate in vitro in the presence of growth factor. It has been known for long time that the partially hepatectomized rat liver could proliferate actively and recover the original volume. We showed that there was indeed DNA synthesis of primary cultured hepatocytes in the presence of epidermal growth factor (EGF). Then we searched for a growth factor specific for hepatocytes. We checked for such factors in various rat tissues and found that circulating platelets carry such factor. We isolated platelets from over 5,000 rats and purified the factor. Further analysis showed it was more than 80,000 Da in size and consisted of a heterodimer. We named it HGF. Around the same time, Dr. Toshikazu Nakamura who was one of the most active researchers in my laboratory was appointed as a Professor in Science at Kyushu University. Later, he identified the unique structure of HGF.21) He also found that HGF was not only a liver growth factor, but also played important roles in diverse cellular functions such as angiogenesis, morphogenesis or anti-liver cirrhosis activity. Dr. Nakamura then moved to Osaka University and is still currently in the same University, where he reported that one domain of HGF (NK4), which functions as a specific inhibitor of HGF, is a potent anti-tumor drug.22) After he left Tokushima, I became interested in the relation between hepatocyte function and morphology of the cells in primary cultures. It is common to use monolayer cultures to examine the cell activities, but this is quite an artificial setup and it should be more natural to mimic the in vivo state. Several studies have described more natural culture conditions such as coating dishes with extracellular substratum, addition of various hormones or nutrients.23) I found an interesting report using positively charged culture dishes, in which cells aggregate to become round and detach from dishes to float in the medium.24) The cultured spheroid-shaped rat hepatocytes have similar morphological features (Fig. 1). This technique yielded some remarkably interesting results. Glucagon, insulin and glucocorticoids regulate the expression of liver-specific enzymes in spheroid cultures more natural than in monolayer cultures. For example, glucokinase is upregulated while hexokinase is downregulated in spheroid cultures25) (Fig. 2), as if hepatocytes are mature cells at resting condition. Interestingly, the transfer of the spheroid cultured cells to monolayer cultures resulted in the gradual reversal of the expression; i.e., downregulation of glucokinase and upregulation of hexokinase. The latter pattern is similar to that seen in hepatoma cells. Of course, the cultured hepatocytes were not cancerous cells in both the monolayer and spheroid cultures, but they seem to transform into the Gl state of the cell cycle from a resting state. In spheroid cultures, cells do not proliferate even in the presence of HGF. It is possible that the intracellular structure of the cells in spheroid cultures would generate more natural signals to regulate gene expressions, although this could not have been studied since gene structure analysis was not available. Thus, we considered that the intracellular environment seems more important for signal transduction to gene activation. However, my knowledge of intracellular structure such as integrin and cytoskeleton was limited to allow me investigate this area. This challenge was also close to my retirement from the University.