Sugar alcohol degradation in Archaea: uptake and degradation of mannitol and sorbitol in Haloarcula hispanica.

The halophilic archaeon Haloarcula hispanica utilizes the sugar alcohols mannitol and sorbitol as carbon and energy sources. Genes, enzymes, and transcriptional regulators involved in uptake and degradation of these sugar alcohols were identified by growth experiments with deletion mutants and enzyme characterization. It is shown that both mannitol and sorbitol are taken up via a single ABC transporter of the CUT1 transporter family. Then, mannitol and sorbitol are oxidized to fructose by two distinct dehydrogenases. Fructose is further phosphorylated to fructose-1-phosphate by a haloarchaeal ketohexokinase, providing the first evidence for a physiological function of ketohexokinase in prokaryotes. Finally, fructose-1-phosphate is phosphorylated via fructose-1-phosphate kinase to fructose-1,6-bisphosphate, which is cleaved to triosephosphates by a Class I fructose-1,6-bisphosphate aldolase. Two distinct transcriptional regulators, acting as activators, have been identified: an IclR-like regulator involved in activating genes for sugar alcohol uptake and oxidation to fructose, and a GfcR-like regulator that likely activates genes involved in the degradation of fructose to pyruvate. This is the first comprehensive analysis of a sugar alcohol degradation pathway in Archaea.

Exploring Marine-Derived Compounds: In Silico Discovery of Selective Ketohexokinase (KHK) Inhibitors for Metabolic Disease Therapy.

The increasing prevalence of metabolic diseases, including nonalcoholic fatty liver disease (NAFLD), obesity, and type 2 diabetes, poses significant global health challenges. Ketohexokinase (KHK), an enzyme crucial in fructose metabolism, is a potential therapeutic target due to its role in these conditions. This study focused on the discovery of selective KHK inhibitors using in silico methods. We employed structure-based drug design (SBDD) and ligand-based drug design (LBDD) approaches, beginning with molecular docking to identify promising compounds, followed by induced-fit docking (IFD), molecular mechanics generalized Born and surface area continuum solvation (MM-GBSA), and molecular dynamics (MD) simulations to validate binding affinities. Additionally, shape-based screening was conducted to assess structural similarities. The findings highlight several potential inhibitors with favorable ADMET profiles, offering promising candidates for further development in the treatment of fructose-related metabolic disorders.

Residues in the fructose-binding pocket are required for ketohexokinase-A activity.

Excessive fructose consumption is a primary contributor to the global surges in obesity, cancer, and metabolic syndrome. Fructolysis is not robustly regulated and is initiated by ketohexokinase (KHK). In this study, we determined the crystal structure of KHK-A, one of two human isozymes of KHK, in the apo-state at 1.85 Å resolution, and we investigated the roles of residues in the fructose-binding pocket by mutational analysis. Introducing alanine at D15, N42, or N45 inactivated KHK-A, whereas mutating R141 or K174 reduced activity and thermodynamic stability. Kinetic studies revealed that the R141A and K174A mutations reduced fructose affinity by 2- to 4-fold compared to WT KHK-A, without affecting ATP affinity. Molecular dynamics simulations provided mechanistic insights into the potential roles of the mutated residues in ligand coordination and the maintenance of an open state in one monomer and a closed state in the other. Protein-protein interactome analysis indicated distinct expression patterns and downregulation of partner proteins in different tumor tissues, warranting a reevaluation of KHK's role in cancer development and progression. The connections between different cancer genes and the KHK signaling pathway suggest that KHK is a potential target for preventing cancer metastasis. This study enhances our understanding of KHK-A's structure and function and offers valuable insights into potential targets for developing treatments for obesity, cancer, and metabolic syndrome.

Michaelis-like complex of mouse ketohexokinase isoform C.

Over the past forty years there has been a drastic increase in fructose-related diseases, including obesity, heart disease and diabetes. Ketohexokinase (KHK), the first enzyme in the liver fructolysis pathway, catalyzes the ATP-dependent phosphorylation of fructose to fructose 1-phosphate. Understanding the role of KHK in disease-related processes is crucial for the management and prevention of this growing epidemic. Molecular insight into the structure-function relationship in ligand binding and catalysis by KHK is needed for the design of therapeutic inhibitory ligands. Ketohexokinase has two isoforms: ketohexokinase A (KHK-A) is produced ubiquitously at low levels, whereas ketohexokinase C (KHK-C) is found at much higher levels, specifically in the liver, kidneys and intestines. Structures of the unliganded and liganded human isoforms KHK-A and KHK-C are known, as well as structures of unliganded and inhibitor-bound mouse KHK-C (mKHK-C), which shares 90% sequence identity with human KHK-C. Here, a high-resolution X-ray crystal structure of mKHK-C refined to 1.79 Šresolution is presented. The structure was determined in a complex with both the substrate fructose and the product of catalysis, ADP, providing a view of the Michaelis-like complex of the mouse ortholog. Comparison to unliganded structures suggests that KHK undergoes a conformational change upon binding of substrates that places the enzyme in a catalytically competent form in which the ß-sheet domain from one subunit rotates by 16.2°, acting as a lid for the opposing active site. Similar kinetic parameters were calculated for the mouse and human enzymes and indicate that mice may be a suitable animal model for the study of fructose-related diseases. Knowledge of the similarity between the mouse and human enzymes is important for understanding preclinical efforts towards targeting this enzyme, and this ground-state, Michaelis-like complex suggests that a conformational change plays a role in the catalytic function of KHK-C.

Fructose-induced metabolic reprogramming of cancer cells.

Excess dietary fructose consumption has been long proposed as a culprit for the world-wide increase of incidence in metabolic disorders and cancer within the past decades. Understanding that cancer cells can gradually accumulate metabolic mutations in the tumor microenvironment, where glucose is often depleted, this raises the possibility that fructose can be utilized by cancer cells as an alternative source of carbon. Indeed, recent research has increasingly identified various mechanisms that show how cancer cells can metabolize fructose to support their proliferating and migrating needs. In light of this growing interest, this review will summarize the recent advances in understanding how fructose can metabolically reprogram different types of cancer cells, as well as how these metabolic adaptations can positively support cancer cells development and malignancy.

Identification and characterization of a novel type of ketohexokinase from the haloarchaeon Haloferax volcanii.

Ketohexokinase (KHK) catalyzes the ATP-dependent phosphorylation of fructose, forming fructose-1-phosphate and ADP. The enzyme is well studied in Eukarya, in particular in humans and other vertebrates, but homologs have not been identified in Bacteria and Archaea. Here we report the identification of a novel type of KHK from the haloarchaeon Haloferax volcanii (HvKHK). The encoding gene khk was identified as HVO_1812. The gene was expressed as a 90-kDa homodimeric protein, catalyzing the phosphorylation of fructose with a Vmax value of 59 U/mg and apparent KM values for ATP and fructose of 0.47 and 1.29 mM, respectively. Homologs of HvKHK were only identified in a few haloarchaea and halophilic Bacteria. The protein showed low sequence identity to characterized KHKs from Eukarya and phylogenetic analyses indicate that haloarchaeal KHKs are largely separated from eukaryal KHKs. This is the first report of the identification of KHKs in prokaryotes that form a novel cluster of sugar kinases within the ribokinase/pfkB superfamily.

Ketohexokinase-A deficiency attenuates the proliferation via reducing ß-catenin in gastric cancer cells.

Overconsumption of fructose is closely related to cancer. Ketohexokinase (KHK) catalyzes the conversion from fructose to fructose-1-phosphate (F1P), which is the first and committed step of fructose metabolism. Recently, aberrant KHK activation has been identified in multiple malignancies. However, the roles of KHK in gastric cancer (GC) cells are largely unclear. Herein, we reveal that the expression of ketohexokinase-A (KHK-A), one alternatively spliced KHK isoform that possesses low affinity for fructose, was markedly increased in GC cells. Depletion of endogenous KHK-A expression using lentiviruses encoding short hairpin RNAs (shRNAs) or pharmaceutical disruption of KHK-A activity using KHK-IN-1 hydrochloride in GC NCI-N87 and HGC-27 cells inhibited the proliferation in vitro and in vivo. Additionally, the mitochondrial respiration in the GC cells with KHK-A deficiency compared with the control cells was significantly impaired. One commercially-available antibody array was used to explore the effects of KHK-A knockdown on signaling pathways, showing that ß-catenin was remarkably reduced in the KHK-A deficient GC cells compared with the control ones. Pharmaceutical reduction in ß-catenin levels slowed down the proliferation of GC cells. These data uncover that KHK-A promotes the proliferation in GC cells, indicating that this enzyme might be a promising therapeutical target for GC treatment.

Crystal structures of human and mouse ketohexokinase provide a structural basis for species- and isoform-selective inhibitor design.

A molecular understanding of the proteins involved in fructose metabolism is essential for controlling the current spread of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism starts with the phosphorylation of D-fructose to fructose 1-phosphate by ketohexokinase (KHK). KHK exists in two alternatively spliced isoforms: the hepatic and intestinal isoform KHK-C and the peripheral isoform KHK-A. Here, the structure of apo murine KHK (mKHK), which differs from structures of human KHK in overall conformation, is reported. An isoform-selective ligand, which offers a 50-fold higher potency on mKHK and human KHK-A compared with KHK-C, is further characterized. In mKHK, large-scale conformational changes are observed upon ligand binding. The structures suggest a combined strategy for the design of species- and isoform-selective KHK inhibitors.

Fructose induced KHK-C can increase ER stress independent of its effect on lipogenesis to drive liver disease in diet-induced and genetic models of NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a liver manifestation of metabolic syndrome, and is estimated to affect one billion individuals worldwide. An increased intake of a high-fat diet (HFD) and sugar-sweetened beverages are risk-factors for NAFLD development, but how their combined intake promotes progression to a more severe form of liver injury is unknown. Here we show that fructose metabolism via ketohexokinase (KHK) C isoform leads to unresolved endoplasmic reticulum (ER) stress when coupled with a HFD intake. Conversely, a liver-specific knockdown of KHK in mice consuming fructose on a HFD is adequate to improve the NAFLD activity score and exert a profound effect on the hepatic transcriptome. Overexpression of KHK-C in cultured hepatocytes is sufficient to induce ER stress in fructose free media. Upregulation of KHK-C is also observed in mice with genetically induced obesity or metabolic dysfunction, whereas KHK knockdown in these mice improves metabolic function. Additionally, in over 100 inbred strains of male or female mice hepatic KHK expression correlates positively with adiposity, insulin resistance, and liver triglycerides. Similarly, in 241 human subjects and their controls, hepatic Khk expression is upregulated in early, but not late stages of NAFLD. In summary, we describe a novel role of KHK-C in triggering ER stress, which offers a mechanistic understanding of how the combined intake of fructose and a HFD propagates the development of metabolic complications.

PFKFB3 Inhibits Fructose Metabolism in Pulmonary Microvascular Endothelial Cells.

Pulmonary microvascular endothelial cells contribute to the integrity of the lung gas exchange interface, and they are highly glycolytic. Although glucose and fructose represent discrete substrates available for glycolysis, pulmonary microvascular endothelial cells prefer glucose over fructose, and the mechanisms involved in this selection are unknown. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is an important glycolytic enzyme that drives glycolytic flux against negative feedback and links glycolytic and fructolytic pathways. We hypothesized that PFKFB3 inhibits fructose metabolism in pulmonary microvascular endothelial cells. We found that PFKFB3 knockout cells survive better than wild-type cells in fructose-rich medium under hypoxia. Seahorse assays, lactate and glucose measurements, and stable isotope tracing showed that PFKFB3 inhibits fructose-hexokinase-mediated glycolysis and oxidative phosphorylation. Microarray analysis revealed that fructose upregulates PFKFB3, and PFKFB3 knockout cells increase fructose-specific GLUT5 (glucose transporter 5) expression. Using conditional endothelial-specific PFKFB3 knockout mice, we demonstrated that endothelial PFKFB3 knockout increases lung tissue lactate production after fructose gavage. Last, we showed that pneumonia increases fructose in BAL fluid in mechanically ventilated ICU patients. Thus, PFKFB3 knockout increases GLUT5 expression and the hexokinase-mediated fructose use in pulmonary microvascular endothelial cells that promotes their survival. Our findings indicate that PFKFB3 is a molecular switch that controls glucose versus fructose use in glycolysis and help better understand lung endothelial cell metabolism during respiratory failure.

Recent Progress on Fructose Metabolism-Chrebp, Fructolysis, and Polyol Pathway.

Excess fructose intake is associated with obesity, fatty liver, tooth decay, cancer, and cardiovascular diseases. Even after the ingestion of fructose, fructose concentration in the portal blood is never high; fructose is further metabolized in the liver, and the blood fructose concentration is 1/100th of the glucose concentration. It was previously thought that fructose was metabolized in the liver and not in the small intestine, but it has been reported that metabolism in the small intestine also plays an important role in fructose metabolism. Glut5 knockout mice exhibit poor fructose absorption. In addition, endogenous fructose production via the polyol pathway has also received attention; gene deletion of aldose reductase (Ar), ketohexokinase (Khk), and triokinase (Tkfc) has been found to prevent the development of fructose-induced liver lipidosis. Carbohydrate response element-binding protein (Chrebp) regulates the expression of Glut5, Khk, aldolase b, and Tkfc. We review fructose metabolism with a focus on the roles of the glucose-activating transcription factor Chrebp, fructolysis, and the polyol pathway.

Ketohexokinase-C regulates global protein acetylation to decrease carnitine palmitoyltransferase 1a-mediated fatty acid oxidation.

BACKGROUND & AIMS: The consumption of sugar and a high-fat diet (HFD) promotes the development of obesity and metabolic dysfunction. Despite their well-known synergy, the mechanisms by which sugar worsens the outcomes associated with a HFD are largely elusive. METHODS: Six-week-old, male, C57Bl/6 J mice were fed either chow or a HFD and were provided with regular, fructose- or glucose-sweetened water. Moreover, cultured AML12 hepatocytes were engineered to overexpress ketohexokinase-C (KHK-C) using a lentivirus vector, while CRISPR-Cas9 was used to knockdown CPT1α. The cell culture experiments were complemented with in vivo studies using mice with hepatic overexpression of KHK-C and in mice with liver-specific CPT1α knockout. We used comprehensive metabolomics, electron microscopy, mitochondrial substrate phenotyping, proteomics and acetylome analysis to investigate underlying mechanisms. RESULTS: Fructose supplementation in mice fed normal chow and fructose or glucose supplementation in mice fed a HFD increase KHK-C, an enzyme that catalyzes the first step of fructolysis. Elevated KHK-C is associated with an increase in lipogenic proteins, such as ACLY, without affecting their mRNA expression. An increase in KHK-C also correlates with acetylation of CPT1α at K508, and lower CPT1α protein in vivo. In vitro, KHK-C overexpression lowers CPT1α and increases triglyceride accumulation. The effects of KHK-C are, in part, replicated by a knockdown of CPT1α. An increase in KHK-C correlates negatively with CPT1α protein levels in mice fed sugar and a HFD, but also in genetically obese db/db and lipodystrophic FIRKO mice. Mechanistically, overexpression of KHK-C in vitro increases global protein acetylation and decreases levels of the major cytoplasmic deacetylase, SIRT2. CONCLUSIONS: KHK-C-induced acetylation is a novel mechanism by which dietary fructose augments lipogenesis and decreases fatty acid oxidation to promote the development of metabolic complications. IMPACT AND IMPLICATIONS: Fructose is a highly lipogenic nutrient whose negative consequences have been largely attributed to increased de novo lipogenesis. Herein, we show that fructose upregulates ketohexokinase, which in turn modifies global protein acetylation, including acetylation of CPT1a, to decrease fatty acid oxidation. Our findings broaden the impact of dietary sugar beyond its lipogenic role and have implications on drug development aimed at reducing the harmful effects attributed to sugar metabolism.

Dietary Fructose and Fructose-Induced Pathologies.

The consumption of fructose as sugar and high-fructose corn syrup has markedly increased during the past several decades. This trend coincides with the exponential rise of metabolic diseases, including obesity, nonalcoholic fatty liver disease, cardiovascular disease, and diabetes. While the biochemical pathways of fructose metabolism were elucidated in the early 1990s, organismal-level fructose metabolism and its whole-body pathophysiological impacts have been only recently investigated. In this review, we discuss the history of fructose consumption, biochemical and molecular pathways involved in fructose metabolism in different organs and gut microbiota, the role of fructose in the pathogenesis of metabolic diseases, and the remaining questions to treat such diseases.

GLUT5 (SLC2A5) enables fructose-mediated proliferation independent of ketohexokinase.

BACKGROUND: Fructose is an abundant source of carbon and energy for cells to use for metabolism, but only certain cell types use fructose to proliferate. Tumor cells that acquire the ability to metabolize fructose have a fitness advantage over their neighboring cells, but the proteins that mediate fructose metabolism in this context are unknown. Here, we investigated the determinants of fructose-mediated cell proliferation. METHODS: Live cell imaging and crystal violet assays were used to characterize the ability of several cell lines (RKO, H508, HepG2, Huh7, HEK293T (293T), A172, U118-MG, U87, MCF-7, MDA-MB-468, PC3, DLD1 HCT116, and 22RV1) to proliferate in fructose (i.e., the fructolytic ability). Fructose metabolism gene expression was determined by RT-qPCR and western blot for each cell line. A positive selection approach was used to "train" non-fructolytic PC3 cells to utilize fructose for proliferation. RNA-seq was performed on parental and trained PC3 cells to find key transcripts associated with fructolytic ability. A CRISPR-cas9 plasmid containing KHK-specific sgRNA was transfected in 293T cells to generate KHK-/- cells. Lentiviral transduction was used to overexpress empty vector, KHK, or GLUT5 in cells. Metabolic profiling was done with seahorse metabolic flux analysis as well as LC/MS metabolomics. Cell Titer Glo was used to determine cell sensitivity to 2-deoxyglucose in media containing either fructose or glucose. RESULTS: We found that neither the tissue of origin nor expression level of any single gene related to fructose catabolism determine the fructolytic ability. However, cells cultured chronically in fructose can develop fructolytic ability. SLC2A5, encoding the fructose transporter, GLUT5, was specifically upregulated in these cells. Overexpression of GLUT5 in non-fructolytic cells enabled growth in fructose-containing media across cells of different origins. GLUT5 permitted fructose to flux through glycolysis using hexokinase (HK) and not ketohexokinase (KHK). CONCLUSIONS: We show that GLUT5 is a robust and generalizable driver of fructose-dependent cell proliferation. This indicates that fructose uptake is the limiting factor for fructose-mediated cell proliferation. We further demonstrate that cellular proliferation with fructose is independent of KHK.

Ketohexokinase inhibition improves NASH by reducing fructose-induced steatosis and fibrogenesis.

BACKGROUND & AIMS: Increasing evidence highlights dietary fructose as a major driver of non-alcoholic fatty liver disease (NAFLD) pathogenesis, the majority of which is cleared on first pass through the hepatic circulation by enzymatic phosphorylation to fructose-1-phosphate via the ketohexokinase (KHK) enzyme. Without a current approved therapy, disease management emphasises lifestyle interventions, but few patients adhere to such strategies. New targeted therapies are urgently required. METHODS: We have used a unique combination of human liver specimens, a murine dietary model of NAFLD and human multicellular co-culture systems to understand the hepatocellular consequences of fructose administration. We have also performed a detailed nuclear magnetic resonance-based metabolic tracing of the fate of isotopically labelled fructose upon administration to the human liver. RESULTS: Expression of KHK isoforms is found in multiple human hepatic cell types, although hepatocyte expression predominates. KHK knockout mice show a reduction in serum transaminase, reduced steatosis and altered fibrogenic response on an Amylin diet. Human co-cultures exposed to fructose exhibit steatosis and activation of lipogenic and fibrogenic gene expression, which were reduced by pharmacological inhibition of KHK activity. Analysis of human livers exposed to 13C-labelled fructose confirmed that steatosis, and associated effects, resulted from the accumulation of lipogenic precursors (such as glycerol) and enhanced glycolytic activity. All of these were dose-dependently reduced by administration of a KHK inhibitor. CONCLUSIONS: We have provided preclinical evidence using human livers to support the use of KHK inhibition to improve steatosis, fibrosis, and inflammation in the context of NAFLD. LAY SUMMARY: We have used a mouse model, human cells, and liver tissue to test how exposure to fructose can cause the liver to store excess fat and become damaged and scarred. We have then inhibited a key enzyme within the liver that is responsible for fructose metabolism. Our findings show that inhibition of fructose metabolism reduces liver injury and fibrosis in mouse and human livers and thus this may represent a potential route for treating patients with fatty liver disease in the future.

Inhibition of ketohexokinase in adults with NAFLD reduces liver fat and inflammatory markers: A randomized phase 2 trial.

BACKGROUND: Increased consumption of the lipogenic sugar fructose promotes the current epidemic of metabolic disease. Ketohexokinase (KHK) catalyzes the first committed step in fructose metabolism. In animal models, KHK inhibition decreases hepatic de novo lipogenesis and steatosis and corrects many metabolic abnormalities associated with insulin resistance. The consequences of inhibiting fructose metabolism in humans have not been tested. This randomized, double-blind, placebo-controlled, phase 2a study (NCT03256526) assessed the effect of the reversible KHK inhibitor PF-06835919 on metabolic parameters in participants with non-alcoholic fatty liver disease (NAFLD). METHODS: Adults with NAFLD (>6% whole liver fat [WLF] by magnetic resonance imaging-proton density fat fraction) received once-daily oral placebo or PF-06835919 75 mg or 300 mg for 6 weeks. Randomization (1:1:1) was via computer-generated randomization code with random permuted blocks. Endpoints included WLF (primary endpoint), safety/tolerability, and metabolic parameters. FINDINGS: Overall, 158 participants were screened and 53 randomized; 48 completed the trial (placebo, n = 17; PF-06835919 75 mg, n = 17; PF-06835919 300 mg, n = 14). Compared with placebo, significant reductions in WLF were observed in participants receiving PF-06835919 300 mg (difference of -18.73%; p = 0.04), but not with 75 mg. In addition, inhibition of KHK resulted in improvement in inflammatory markers. The incidence of treatment-emergent adverse events (AEs) was low and similar across treatment groups (26.3%, 23.5%, and 29.4% of participants in the placebo and PF-06835919 75 mg and 300 mg groups, respectively). No serious AEs were reported. CONCLUSIONS: Data suggest that KHK inhibition may be clinically beneficial in the treatment of adults with NAFLD and insulin resistance. FUNDING: This study was sponsored by Pfizer Inc.

KHK-A promotes the proliferation of oesophageal squamous cell carcinoma through the up-regulation of PRPS1.

BACKGROUND AND STUDY AIMS: The metabolism of dietary fructose by ketohexokinase (KHK) is an important step in glucose metabolism in various tumour types. However, the expression, function and underlying mechanisms of KHK in oesophageal squamous cell carcinoma (ESCC) remain largely unclear. The objective of this study was to investigate the effects of KHK-A, a peripheral isoform of KHK, on the proliferation of ESCC cell lines. MATERIAL AND METHODS: The function and mechanism of KHK-A in ESCC cells were investigated by constructing stable KHK-A-knockdown and -overexpressing ESCC cell lines (KYSE410 and KYSE150, respectively). The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry and colony formation assays were used to analyse the effects of KHK-A on cell proliferation, cell cycle and colony formation, respectively. KHK-A and phosphoribosyl pyrophosphate synthetase isoform 1 (PRPS1) mRNA and protein expressions in several ESCC cell lines were determined using routine reverse transcription-polymerase chain reaction and immunoblotting, respectively. KHK and PRPS1 expressions in ESCC tumour tissues and corresponding adjacent non-tumour tissues were evaluated according to the gene expression omnibus (GEO) database (GSE20347). RESULTS: In vitro experiments showed that KHK-A significantly promoted cell proliferation by modulating the G1/S phase transition in the cell cycle, which was probably regulated by PRPS1 expression. GEO database-based analysis showed that KHK levels were significantly higher in the ESCC tissues than in the corresponding adjacent non-tumour tissues. Pearson's correlation coefficient analysis showed that KHK expression in ESCC cell lines and tissues was significantly positively associated with the up-regulation of PRPS1, suggesting that KHK-A levels regulate PRPS1 expression in ESCC. CONCLUSION: KHK-A may serve as a driving gene in ESCC for the activation of PRPS1, resulting in the up-regulation of PRPS1. This could lead to enhanced nucleic acid synthesis for tumourigenesis. Our study showed that KHK-A is a potential target for ESCC diagnosis and therapy.

Organismal Fructose Metabolism in Health and Non-Alcoholic Fatty Liver Disease.

NAFLD has alarmingly increased, yet FDA-approved drugs are still lacking. An excessive intake of fructose, especially in liquid form, is a dietary risk factor of NAFLD. While fructose metabolism has been studied for decades, it is still controversial how fructose intake can cause NAFLD. It has long been believed that fructose metabolism solely happens in the liver and accordingly, numerous studies have investigated liver fructose metabolism using primary hepatocytes or liver cell lines in culture. While cultured cells are useful for studying detailed signaling pathways and metabolism in a cell-autonomous manner, it is equally important to understand fructose metabolism at the whole-body level in live organisms. In this regard, recent in vivo studies using genetically modified mice and stable isotope tracing have tremendously expanded our understanding of the complex interaction between fructose-catabolizing organs and gut microbiota. Here, we discuss how the aberrant distribution of fructose metabolism between organs and gut microbiota can contribute to NAFLD. We also address potential therapeutic interventions of fructose-elicited NAFLD.

Peroxisome-Deficiency and HIF-2α Signaling Are Negative Regulators of Ketohexokinase Expression.

Ketohexokinase (KHK) is the first and rate-limiting enzyme of fructose metabolism. Expression of the two alternatively spliced KHK isoforms, KHK-A and KHK-C, is tissue-specific and KHK-C is predominantly expressed in liver, kidney and intestine and responsible for the fructose-catabolizing function. While KHK isoform choice has been linked to the development of disorders such as obesity, diabetes, cardiovascular disease and cancer, little is known about the regulation of total KHK expression. In the present study, we investigated how hypoxic signaling influences fructose metabolism in the liver. Hypoxia or von Hippel-Lindau (VHL) tumor suppressor loss leads to the stabilization of hypoxia-inducible factors alpha (HIF-1α and HIF-2α) and the activation of their signaling to mediate adaptive responses. By studying liver-specific Vhl, Vhl/Hif1a, and Vhl/Epas1 knockout mice, we found that KHK expression is suppressed by HIF-2α (encoded by Epas1) but not by HIF-1α signaling on mRNA and protein levels. Reduced KHK levels were accompanied by downregulation of aldolase B (ALDOB) in the livers of Vhl and Vhl/Hif1a knockout mice, further indicating inhibited fructose metabolism. HIF-1α and HIF-2α have both overlapping and distinct target genes but are differentially regulated depending on the cell type and physiologic or pathologic conditions. HIF-2α activation augments peroxisome degradation in mammalian cells by pexophagy and thereby changes lipid composition reminiscent of peroxisomal disorders. We further demonstrated that fructose metabolism is negatively regulated by peroxisome-deficiency in a Pex2 knockout Zellweger mouse model, which lacks functional peroxisomes and is characterized by widespread metabolic dysfunction. Repression of fructolytic genes in Pex2 knockout mice appeared to be independent of PPARα signaling and nutritional status. Interestingly, our results demonstrate that both HIF-2α and peroxisome-deficiency result in downregulation of Khk independent of splicing as both isoforms, Khka as well as Khkc, are significantly downregulated. Hence, our study offers new and unexpected insights into the general regulation of KHK, and therefore fructolysis. We revealed a novel regulatory function of HIF-2α, suggesting that HIF-1α and HIF-2α have tissue-specific opposing roles in the regulation of Khk expression, isoform choice and fructolysis. In addition, we discovered a previously unknown function of peroxisomes in the regulation of fructose metabolism.

Bone Growth is Influenced by Fructose in Adolescent Male Mice Lacking Ketohexokinase (KHK).

Fructose is metabolized in the cytoplasm by the enzyme ketohexokinase (KHK), and excessive consumption may affect bone health. Previous work in calcium-restricted, growing mice demonstrated that fructose disrupted intestinal calcium transport. Thus, we hypothesized that the observed effects on bone were dependent on fructose metabolism and took advantage of a KHK knockout (KO) model to assess direct effects of high plasma fructose on the long bones of growing mice. Four groups (n = 12) of 4-week-old, male, C57Bl/6 background, congenic mice with intact KHK (wild-type, WT) or global knockout of both isoforms of KHK-A/C (KHK-KO), were fed 20% glucose (control diet) or fructose for 8 weeks. Dietary fructose increased by 40-fold plasma fructose in KHK-KO compared to the other three groups (p < 0.05). Obesity (no differences in epididymal fat or body weight) or altered insulin was not observed in either genotype. The femurs of KHK-KO mice with the highest levels of plasma fructose were shorter (2%). Surprisingly, despite the long-term blockade of KHK, fructose feeding resulted in greater bone mineral density, percent volume, and number of trabeculae as measured by µCT in the distal femur of KHK-KO. Moreover, higher plasma fructose concentrations correlated with greater trabecular bone volume, greater work-to-fracture in three-point bending of the femur mid-shaft, and greater plasma sclerostin. Since the metabolism of fructose is severely inhibited in the KHK-KO condition, our data suggest mechanism(s) that alter bone growth may be related to the plasma concentration of fructose.