PROKARYOTIC EXPRESSION, ANTIBODY PREPARATION, AND REGULATION BY DIETARY STARCH LEVELS OF G6PC AND GCK IN MICROPTERUS SALMOIDES

The dysregulation of glucose-G6P (glucose-6-phosphate) interconversion is thought to be an important reason for the low glucose tolerance of carnivorous fish. However, it remains unclear if this phenomenon applies to largemouth bass (Micropterus salmoides, LMB). To investigate the regulatory mechanism of glucose homeostasis influenced by nutritional factors in LMB, we cloned and constructed recombinant plasmid vectors containing truncated sequences of glucose-6-phosphatase catalytic subunit (g6pc) and glucokinase (gck). These recombinant plasmid vectors were transformed into Escherichia coli Rosetta receptor cells and successfully expressed using 1 mmol/L IPTG (isopropyl-β-D-thiogalactopyranoside) induction overnight at 16℃. Following the lysis receptor cells, truncated G6pc, and Gck recombinant proteins were purified by GST-tag affinity chromatography from the supernatants. The purified recombinant proteins were emulsified with Freund's adjuvant to create immunogens, and immunized to KM mice for five times. G6pc and Gck polyclonal antibodies with high specificity were successfully prepared with titers exceeding 1﹕3000 and 1﹕10000, respectively. Western blot analysis showed that both G6pc and Gck in LMB were mainly distributed in the liver. Immunofluorescent staining indicated that the G6pc positive signals were localized around the nucleus, whereas Gck positive signals spread throughout the hepatocytes. After an 8-weeks feeding trial, the results showed that Gck level and the expression of genes involved in glycolysis and glycogenesis in the liver of LMB increased gradually with dietary starch levels rising from 8% to 20% in 6% increments. Conversely, G6pc levels and the expression of genes involved in gluconeogenesis and glycogenolysis were only down-regulated at 14% starch. These results suggested that a high starch diet (20%) could induce a futile cycle of glucose-G6P interconversion in the liver of LMB. In summary, specific polyclonal antibodies of G6pc and Gck were successfully prepared in LMB, and the regulation of their expression by dietary starch levels was evaluated in this study. This research lays the foundation for further elucidating the roles of G6pc and Gck in the glucose homeostasis of LMB.