Metabolic engineering analysis of post-burn hepatic hypermetabolism

Abstract

Metabolic engineering refers to the directed improvement of product formation or cellular properties through the modification of specific biochemical reactions or introduction of new ones with the use of recombinant DNA technology. It has been used to investigate and modify intermediary metabolism in a variety of microbial organisms of biotechnological interest. An emerging area of application for metabolic engineering is medicine, in particular the study of metabolic disorders, where analysis and manipulation of metabolic pathways have obvious relevance. Central to metabolic engineering is the notion that metabolism results from the concerted and coordinated activities of biochemical pathways connected through shared intermediates in the form of common reactants, products, and catalysts. According to this "metabolic network" concept, an enhanced understanding of metabolism and cellular function is obtained by considering the component biochemical reactions together, rather than individually. In this light, this thesis work was motivated by the idea that the application of metabolic engineering analysis to biological systems relevant to human disease has the potential to provide valuable insight into the biochemical underpinnings behind metabolic disorders. In the present dissertation, this idea was explored by investigating a metabolic disorder known clinically as hypermetabolism that is associated with the systemic inflammatory response to severe injury. At the whole body level, hypermetabolism is characterized by elevated resting energy expenditure and increased turnover of proteins, fatty acids, and carbohydrates.

(cont.) If this state persists over a period of days to weeks, the patient is predisposed to muscle wasting, progressive organ dysfunction, multiple organ failure, and ultimately death. Unfortunately, existing nutritional therapies are inadequate for preventing the onset of persistent hypermetabolism, because many of the mechanistic details of this process are poorly understood. An important player in the hypermetabolic response to injury is the liver, which responsible for synthesizing healing factors from muscle protein derived amino acids, converting carbohydrate and lipid fuel resources to useful energy substrates, and eliminating waste products generated by these processes. In order to better understand the biochemical underpinnings behind injury derived hypermetabolism in the liver, the following specific aims were addressed: 1) to develop and validate tissue and organ models of injury for the liver; 2) to delineate activity changes in the major metabolic pathways in the liver during the developmental period of hypermetabolism; and 3) to build diagnostic tools for detecting and grading the injury derived metabolic abnormalities in the liver. A particularly useful metabolic engineering tool is metabolic flux analysis (MFA), which refers to a methodology whereby intracellular reaction fluxes are estimated using a stoichiometric model for the major intracellular reactions and applying mass balances around intracellular metabolites. A powerful feature of this methodology is its ability to consider cellular biochemistry in terms of a network of reactions ...