(645e) High Resolution Flux Estimation of Gluconeogenesis Using A Novel [U-13C,2H8]Glycerol Tracer in Mice

The liver plays a key role in glucose and energy homeostasis by regulating rates of glucose synthesis and storage in response to nutritional and hormonal status. During fasting, hepatic gluconeogenesis (GNG) and glycogenolysis are the main pathways contributing to endogenous glucose production. To determine metabolic pathway fluxes in vivo investigators rely heavily on the use of stable isotope methods. It has been challenging, however, to develop an accurate method for determining fluxes of hepatic glucose production in mice. To address limitations of current methods, we have developed a novel method for comprehensive analysis of metabolic fluxes in the gluconeogenesis pathway in living mice. This method makes use of novel [U-¹³C,²H₈]glycerol tracer and deuterated water ²H₂O; GC/MS analysis of glucose, and model-based interpretation of glucose mass isotopomer distributions (MID) to quantify a detailed map of hepatic metabolism consisting of 11 independent fluxes. The integrated analysis of MIDs of glucose from incorporation of ¹³C and ²H-isotopes is facilitated by the recently developed elementary metabolite unit (EMU) framework that efficiently simulates labeling patterns from multiple isotopic tracers, advances in computational techniques for large-scale nonlinear regression, and statistical analysis of confidence intervals of estimated fluxes. The comprehensive snap shot of hepatic metabolism obtained with our method makes it highly suitable for studies of diseases such as diabetes and for evaluating the effects of potential therapeutic agents on hepatic function.

References

Antoniewicz, M.R., Kelleher, J.K., and Stephanopoulos, G., 2007. Elementary Metabolite Units (EMU): a novel framework for modeling isotopic distributions. Metab Eng, 9, 68-86.

Antoniewicz, M.R., Kelleher, J.K., and Stephanopoulos, G., 2006. Determination of confidence intervals of metabolic fluxes estimated from stable isotope measurements. Metab Eng, 8, 324-337.