Excessive fructose consumption in Western diets correlates with increases in obesity, insulin resistance, kidney disease, and non-alcoholic fatty liver disease (NAFLD), collectively part of metabolic syndrome (MBS). Liver and kidneys metabolize 50-70% of ingested fructose, but the fate of remaining fructose remains poorly understood. Moreover, the correlation of fructose ingestion with MBS highlights the need for better understanding of whole-body fructose metabolism, in both health and disease. To that end, valid rodent models for fructose metabolism must reflect the same metabolism in humans. A serious autosomal recessive defect in fructose metabolism, called hereditary fructose intolerance (HFI), is caused by mutations in the aldolase B gene (ALDOB, human; Aldo2, mouse). With low levels of fructose exposure, HFI patients develop NAFLD and liver fibrosis, sharing pathologies with MBS. Targeting Aldo2 for deletion in mice (Aldo2-/-) provides a major step in validating that fructose metabolism in mice mimics that in humans. Like HFI patients, Aldo2-/- mice exposed to chronic, low-level dietary fructose show failure to thrive, liver dysfunction, and potential mortality. The fructose-induced symptoms of HFI and MBS result from flux through the ketohexokinase (KHK)-mediated pathway, and the metabolite Fru 1-P. Bioinformatic analysis reveals gene expression for this pathway is highest in liver, as expected; surprisingly, brain is predicted to have expression levels similar to kidney. This predicted gene expression is validated via RNA in situ hybridization, quantification of enzyme activities, presence of transport proteins, and measuring fructose oxidation rates in adult mice brains. Within the brain, regions of the cerebellum, hippocampus, cortex, and olfactory bulb show the highest population of cells expressing Fru-1-P pathway genes. In these regions, enzyme activities for both KHK and aldolase, and rates of fructolytic flux, are many times that seen in liver slices. Additionally, brains of mice on a high fructose diet show a three-fold increase in KHK activity. This suggests that not only are these regions of the brain capable of metabolizing fructose, but that they are also capable of responding to increases in dietary fructose. This work provides a foundation for research of long-term consequences of excessive fructose consumption in multiple organs. / 2017-06-21T00:00:00Z

Over the past forty years, there has been an increase in obesity, diabetes, and heart disease, collectively known as metabolic syndrome (MetS), in which fructose has been implicated. In addition to MetS, hereditary fructose intolerance (HFI) has no known treatment aside from the difficult removal of fructose from the diet. Ketohexokinase (KHK) is the first enzyme in the fructose metabolic pathway and catalyzes an ATP-dependent reaction that phosphorylates fructose to fructose 1-phosphate. For effective inhibitor development, it is key to understand the KHK-catalytic mechanism. To that end, the research described in this thesis focuses on two goals: 1) understanding how KHK functions in its role as a metabolic enzyme, using structure-function analysis to inform the development of KHK inhibitors, and 2) investigating how these findings can be used to make KHK a prime therapeutic target for alleviating diseases such as HFI and MetS. The X-ray crystal structure of the mouse-liver isozyme, KHK-C (mKHK-C), was determined at a resolution of 1.79 Å. The mKHK-C structure is in complex with the substrate fructose and the product of catalysis, ADP, forming a ground-state complex. The mKHK-C structure has nearly identical secondary structure to its human homolog and has similar steady-state kinetic parameters validating the use of mouse models for exploring the pre-clinical efficacy of KHK-C inhibitors. Furthermore, six structures of human KHK-C in complex with inhibitors and ligands are presented. These structures support the kinetic analyses showing these inhibitors are all competitive with ATP and reveal the shape and polarity of the ATP-binding pocket to achieve inhibition constants (Ki) as low as 50 nM. Lastly, comparison of all KHK structures demonstrate that the β-sheet domain of KHK is capable of 30.3° rotation of the β-sheet domain towards the active site of the opposing dimer subunit. Kinetic experiments using site-directed mutants of human KHK-C and various viscogens confirmed that a conformational change is linked to KHK’s catalytic function. This research provides a foundation for further development of more specific KHK inhibitors aimed at HFI and MetS therapies. / 2022-10-30T00:00:00Z