Contribution of gene expression to metabolic fluxes in hypermetabolic livers induced through burn injury and cecal ligation and puncture in rats

Abstract

Severe injury activates many stress-related and inflammatory pathways that can lead to a systemic hypermetabolic state. Prior studies using perfused hypermetabolic rat livers have identified intrinsic metabolic flux changes that were not dependent upon the continual presence of elevated stress hormones and substrate loads. We investigated the hypothesis that such changes may be due to persistent alterations in gene expression. A systemic hypermetabolic response was induced in rats by applying a moderate burn injury followed 2 days later by cecum ligation and puncture (CLP) to produce sepsis. Control animals received a sham-burn followed by CLP, or a sham-burn followed by sham-CLP. Two days after CLP, livers were analyzed for gene expression changes using DNA microarrays and for metabolism alterations by ex vivo perfusion coupled with Metabolic Flux Analysis. Burn injury prior to CLP increased fluxes while decreases in gene expression levels were observed. Conversely, CLP alone significantly increased metabolic gene expression, but decreased many of the corresponding metabolic fluxes. Burn injury combined with CLP led to the most dramatic changes, where concurrent changes in fluxes and gene expression levels occurred in about 1/3 of the reactions. The data are consistent with the notion that in this model, burn injury prior to CLP increased fluxes through post-translational mechanisms with little contribution of gene expression, while CLP treatment up-regulated the metabolic machinery by transcriptional mechanisms. Overall, these data show that mRNA changes measured at a single time point by DNA microarray analysis do not reliably predict metabolic flux changes in perfused livers.

Metabolic flux determination in perfused livers by mass balance analysis: Effect of fasting

“…The big challenge is that flux measurements of these reactions or pathways require more complex techniques, such as isotope-labeling, which are experimentally difficult and expensive. Therefore, most of the liver perfusion systems were performed by using fasted rats so that glycolysis, glycogenesis, and fatty acid synthesis were assumed to be inactive (Banta et al, 2007;Lee et al, 2000Lee et al, , 2003. However, it is not well documented whether fasting truly eliminates the glycolytic pathway or some other reactions related to lipid metabolism, in disease states.…”

“…A range has to be assigned for O 2 and CO 2 external fluxes; otherwise physiologically irrelevant flux distribution could be obtained. Based on previous perfused liver studies (Arai et al, 2001;Banta et al, 2005Banta et al, , 2007Lee et al, 2000Lee et al, , 2003, a wide range (between 50-350 mmol/ g liver/h) is attributed to oxygen uptake and carbon dioxide output for both states.…”

“…The liver metabolic network used in this work was originally developed for perfused livers and hepatocyte cultures (Banta et al, 2007;Chan et al, 2003;Lee et al, 2000Lee et al, , 2003 and was modified to simultaneously include both glycolytic and gluconeogenic pathways, fatty acid synthesis and oxidation, as well as glycogenesis and glycogenolysis (Table SI in ''Supplementary Materials''). Given the physiological properties of liver and the perfusate composition, the network involves all possible major liver-specific pathways such as gluconeogenesis, glycolysis, urea cycle, fatty acid metabolism, pentose phosphate pathway, TCA cycle, glycogen metabolism, and amino acid metabolism.…”

“…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 (Arai et al, 2001;Banta et al, 2005Banta et al, , 2007Lee et al, 2000Lee et al, , 2003Yokoyama et al, 2005). A critical component of these investigations is to evaluate the impact of these insults on glucose and nitrogen metabolism since one of the major functions of the liver is to maintain physiological circulating glucose and ammonia levels.…”

“…A caveat of this approach is that fasting induces metabolic changes that superpose to disease-related changes. In some instances, a different metabolic model was used for the fed state, and both the fed and fasted models were fitted to the data; the best fitting model was then used to derive the fluxes (Arai et al, 2001;Banta et al, 2005Banta et al, , 2007Lee et al, 2000Lee et al, , 2003Yokoyama et al, 2005). This implied that the glycolytic and gluconegenic states are mutually exclusive.…”