(504g) Metabolic Flux Determination in Perfused Livers by Mass Balance Analysis: Effect of Fasting

Isolated perfused liver systems have been extensively used to characterize the detailed metabolic changes in various abnormal metabolic conditions, for example systemic burns and trauma, infection, and other insults [1,2,3,4,5,6]. However, most of these studies were carried out in fasted animals to facilitate implementation of mass balance analysis methods to calculate intracellular flux distributions from extracellular metabolite measurements. Under these conditions, certain simplifying assumptions can be used, such as lack of glycogen storage and inhibition of all glycolytic enzymes. Although this is fairly true in normal individuals, it is not necessarily the case in many diseases states where the regulation between fed and fasted states may be abnormal. Thus, there is a need to develop a ?unified? metabolic flux analysis approach that is not limited to the fasted state, but rather that could be similarly applied to both fed and fasted states.

The aim of this work was to compare the differences in hepatic metabolism between fasted and fed states using data from perfused livers isolated from fed and fasted rats. Net metabolic fluxes for amino acids, glucose, urea and other metabolites entering and exiting the liver were used as the primary measurements for both groups of animals, and applied to the same metabolic flux analysis model. A number of possible flux distributions within the ranges obtained from the flux spectrum approach [7] were determined by Monte Carlo sampling analysis [8]. Singular Value Decomposition analysis was then used to analyze the solution space and find the estimated flux behaviors that capture the system variability.

Considering both experimental observations and mathematical analysis, the results show that 24 h fasting results in up-regulation of TCA cycle, urea cycle and gluconeogenic pathway. Glutamate metabolism, electron transport chain reaction, and fatty acid oxidation are also slightly up-regulated whereas aspartate metabolism is down-regulated after fasting. Limited glucose production from glycogen is observed at the fasted state. Moreover, SVD analysis, providing a better comparison for two states, clearly shows that glycogen breakdown has a great influence on glycolysis and electron transport chain reaction which should have been observed in the samples. This behavior is less dominant among the samples in the fasted state. On the contrary, in fasted state, glycogen breakdown mostly affects the PPP and TCA cycle. In conclusion, this analysis provides a detailed description of the metabolic changes in two different states, considering the entire steady state flux space and dominant flux patterns that capture the system variations within the flux space.

References

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