Application of (13)C-filtered (1)H NMR to evaluate drug action on gluconeogenesis and glycogenolysis simultaneously in isolated rat hepatocytes.

The effects of two inhibitors of hepatic glucose production, AICAR (5-aminoimidazole-4-carboxamide riboside) and metformin, whose precise mechanisms of action are a matter of some controversy, have been investigated in isolated rat hepatocytes by application of a novel NMR-based method whereby effects on metabolic flow from the two glucose-producing pathways, glycogenolysis and gluconeogenesis, and also lactate production, can be studied simultaneously. Hepatocytes were pre-incubated for 24 h with 15 mM 1-(13)C-glucose to load the cells with labeled glycogen, which under subsequent glycogenolytic conditions would yield predominantly 1-(13)C glucose and 3-(13)C-lactate, followed (after washing) by incubation in media with 2-(13)C-glycerol, which under subsequent gluconeogenic conditions would yield 2,5-(13)C-glucose, or if metabolized to lactate, 2-(13)C-lactate. Glucose production was then stimulated by glucagon for 3 h in the absence or presence of the inhibitors and then incubation media were analyzed by (13)C-HSQC (heteronuclear single quantum coherence)-filtered (1)H NMR spectra. The results show that metformin only inhibits glucose production by inhibition of gluconeogenesis, but also that it increases lactate production from both glycogenolysis and from glycerol, whereas, and contrary to expectations, AICAR inhibits glucose production by inhibiting both gluconeogenesis and glycogenolysis, and also increases lactate production from glycerol. The data show that application of this methodology can be used to answer important questions about drug action on hepatic metabolism that are not readily accessible by alternative means.