Flux Analysis and Metabolomics for Rat Hepatocyte Gluconeogenesis

Increased gluconeogenesis is an early sign of liver insulin resistance in pre-diabetic patients; this metabolic pathway is targeted by the anti-diabetic agent metformin. We explored the metabolic variations responsible for the flux distribution of glucose production in liver. We applied stable isotope tracer labeling using deuterated water (2H2O) and other precursors to elucidate detailed metabolic fluxes of the gluconeogenic pathway in rat hepatocytes and H4IIEC3 hepatomas models of glucose production. Medium glucose is measured enzymatically and further analyzed for isotopic enrichment using Gas Chromatography/Mass Spectrometry (GC/MS) of isotopomer spectra of multiple glucose fragments. Data are analyzed using Matlab-based package Metran for flux estimation. Hepatocytes treated with metformin produced lower amounts of glucose while the ones treated with glipizide, a sulfonylurea, had higher glucose production. Metformin treatment countered the effects of glipizide on the cells. These methods have the potential to become clinical assays for the measurement of liver glucose output, thus allowing a better understanding of the pathophysiology of Type-2 Diabetes.