Separation of enzymatically distinct populations of trout hepatocytes

Abstract

Two different approaches were chosen to investigate the heterogeneity of rainbow trout (Oncorhynchus mykiss) hepatocytes. (i) Hepatocytes were isolated following selective liver infusion with a short pulse of digitonin from either the portal or the hepatic vein. Cell isolates, presumably enriched in cells from either "periportal" or "perivenous" liver regions, were then compared with cells obtained from the nondigitonized part of the same liver (control). No significant differences between hepatocytes were found in the activities of six key metabolic enzymes or in the rate of pyruvate oxidation. All cell isolates were responsive to glucagon, glucagon-like peptide, and insulin (all at 10 nM). If the liver was digitonized through the hepatic vein, resulting cell isolates revealed higher rates of gluconeogenesis from pyruvate, and gluconeogenic flux was more responsive to glucagon (at 10 nM) than controls. The experimental evidence for metabolic zonation in trout liver is weak. (ii) Enzymatically different subpopulations of hepatocytes were successfully separated by Percoll gradient centrifugation after isolation of cells by the conventional collagenase perfusion technique. Two prominent subpopulations of hepatocytes differed in the activities of glycogen phosphorylase, pyruvate kinase, lactate dehydrogenase, citrate synthase, glutamate dehydrogenase, phosphoenolpyruvate carboxykinase, and alanine and aspartate aminotransferases, but not in their content of glycogen. However, the relative distribution of enzymes did not follow the mammalian pattern of perivenous or periportal hepatocytes. These data support cellular heterogeneity in the trout liver, with gluconeogenesis and oxidative metabolism dominating in some hepatocytes, whereas others display a relatively larger scope for glycogen breakdown, glycolysis, and amino acid metabolism.