Network Modeling of Liver Metabolism to Predict Plasma Metabolite Changes During Short-Term Fasting in the Laboratory Rat

The liver—a central metabolic organ that integrates whole-body metabolism to maintain glucose and fatty-acid regulation, and detoxify ammonia—is susceptible to injuries induced by drugs and toxic substances. Although plasma metabolite profiles are increasingly investigated for their potential to detect liver injury earlier than current clinical markers, their utility may be compromised because such profiles are affected by the nutritional state and the physiological state of the animal, and by contributions from extrahepatic sources. To tease apart the contributions of liver and non-liver sources to alterations in plasma metabolite profiles, here we sought to computationally isolate the plasma metabolite changes originating in the liver during short-term fasting. We used a constraint-based metabolic modeling approach to integrate central carbon fluxes measured in our study, and physiological flux boundary conditions gathered from the literature, into a genome-scale model of rat liver metabolism. We then measured plasma metabolite profiles in rats fasted for 5–7 or 10–13 h to test our model predictions. Our computational model accounted for two-thirds of the observed directions of change (an increase or decrease) in plasma metabolites, indicating their origin in the liver. Specifically, our work suggests that changes in plasma lipid metabolites, which are reliably predicted by our liver metabolism model, are key features of short-term fasting. Our approach provides a mechanistic model for identifying plasma metabolite changes originating in the liver

Experimental and steady-state analysis of the GAL regulatory system in Kluyveromyces lactis

The galactose uptake mechanism in yeast is a well-studied regulatory network. The regulatory players in the Galactose Regulatory Mechanism (GAL system) are conserved in Saccharomyces cerevisiae and Kluyveromyces lactis, but the molecular mechanisms that occur as a result of the molecular interactions between them are different. The key differences in the GAL system of K. lactis relative to that of S. cerevisiae are: (a) the autoregulation of KlGAL4; (b) the dual role of KlGal1p as a metabolizing enzyme as well as a galactose-sensing protein; (c) the shuttling of KlGal1p between nucleus and cytoplasm; and (d) the nuclear confinement of KlGal80p. A steady-state model was used to elucidate the roles of these molecular mechanisms in the transcriptional response of the GAL system. The steady-state results were validated experimentally using measurements of β-galactosidase to represent the expression for genes having two binding sites. The results showed that the autoregulation of the synthesis of activator KlGal4p is responsible for the leaky expression of GAL genes, even at high glucose concentrations. Furthermore, GAL gene expression in K. lactis shows low expression levels because of the limiting function of the bifunctional protein KlGal1p towards the induction process in order to cope with the need for the metabolism of lactose/galactose. The steady-state model of the GAL system of K. lactis provides an opportunity to compare with the design prevailing in S. cerevisiae. The comparison indicates that the existence of a protein, Gal3p, dedicated to the sensing of galactose in S. cerevisiae as a result of genome duplication has resulted in a system which metabolizes galactose efficiently

Toxicant-Induced Metabolic Alterations in Lipid and Amino Acid Pathways Are Predictive of Acute Liver Toxicity in Rats

Liver disease and disorders associated with aberrant hepatocyte metabolism can be initiated via drug and environmental toxicant exposures. In this study, we tested the hypothesis that gene and metabolic profiling can reveal commonalities in liver response to different toxicants and provide the capability to identify early signatures of acute liver toxicity. We used Sprague Dawley rats and three classical hepatotoxicants: acetaminophen (2 g/kg), bromobenzene (0.4 g/kg), and carbon tetrachloride (0.3 g/kg), to identify early perturbations in liver metabolism after a single acute exposure dose. We measured changes in liver genes and plasma metabolites at two time points (5 and 10 h) and used genome-scale metabolic models to identify commonalities in liver responses across the three toxicants. We found strong correlations for gene and metabolic profiles between the toxicants, indicative of similarities in the liver response to toxicity. We identified several injury-specific pathways in lipid and amino acid metabolism that changed similarly across the three toxicants. Our findings suggest that several plasma metabolites in lipid and amino acid metabolism are strongly associated with the progression of liver toxicity, and as such, could be targeted and clinically assessed for their potential as early predictors of acute liver toxicity

Dynamic analysis of the KlGAL regulatory system in Kluyveromyces lactis: a comparative study with Saccharomyces cerevisiae

The GAL regulatory system is highly conserved in yeast species of Saccharomyces cerevisiae and Kluyveromyces lactis. While the GAL system is a well studied system in S. cerevisiae, the dynamic behavior of the KlGAL system in K. lactis has not been characterized. Here, we have characterized the GAL system in yeast K. lactis by developing a dynamic model and comparing its performance to its not-so-distant cousin S. cerevisiae. The present analysis demonstrates the significance of the autoregulatory feedbacks due to KlGal4p, KlGal80p, KlGal1p and Lac12p on the dynamic performance of the KlGAL switch. The model predicts the experimentally observed absence of bistability in the wild type strain of K. lactis, unlike the short term memory of preculturing conditions observed in S. cerevisiae. The performance of the GAL switch is distinct for the two yeast species although they share similarities in the molecular components. The analysis suggests that the whole genome duplication of S. cerevisiae, which resulted in a dedicated inducer protein, Gal3p, may be responsible for the high sensitivity of the system to galactose concentrations. On the other hand, K. lactis uses a bifunctional protein as an inducer in addition to its galactokinase activity, which restricts its regulatory role and hence higher galactose levels in the medium are needed to trigger the GAL system