RESUMEN
Biochemistry of carbon assimilation in aerobic methylotrophs growing on reduced C1 compounds has been intensively studied due to the vital role of these microorganisms in nature. The biochemical pathways of carbon assimilation in methylotrophs growing on multi-carbon substrates are insufficiently explored. Here we elucidated the metabolic route of mannitol assimilation in the alphaproteobacterial facultative methylotroph Methylobrevis pamukkalensis PK2. Two key enzymes of mannitol metabolism, mannitol-2-dehydrogenase (MTD) and fructokinase (FruK), were obtained as His-tagged proteins by cloning and expression of mtd and fruK genes in Escherichia coli and characterized. Genomic analysis revealed that further transformation of fructose-6-phosphate proceeds via the Entner-Doudoroff pathway. During growth on mannitol + methanol mixture, the strain PK2 consumed both substrates simultaneously demonstrating independence of C1 and C6 metabolic pathways. Genome screening showed that genes for mannitol utilization enzymes are present in other alphaproteobacterial methylotrophs predominantly capable of living in association with plants. The capability to utilize a variety of carbohydrates (sorbitol, glucose, fructose, arabinose and xylose) suggests a broad adaptability of the strain PK2 to live in environments where availability of carbon substrate dynamically changes.
Asunto(s)
Fructoquinasas , Manitol , Manitol/metabolismo , Fructoquinasas/metabolismo , Fructoquinasas/genética , Manitol Deshidrogenasas/metabolismo , Manitol Deshidrogenasas/genética , Fructosafosfatos/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Redes y Vías Metabólicas/genética , Metanol/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/crecimiento & desarrolloRESUMEN
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 1.79â Å resolution 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.
Asunto(s)
Fructoquinasas , Animales , Fructoquinasas/química , Fructoquinasas/metabolismo , Ratones , Cristalografía por Rayos X , Isoenzimas/química , Modelos Moleculares , Conformación Proteica , Humanos , Fructosa/metabolismo , Fructosa/químicaRESUMEN
In Escherichia coli, the master transcription regulator catabolite repressor activator (Cra) regulates >100 genes in central metabolism. Cra binding to DNA is allosterically regulated by binding to fructose-1-phosphate (F-1-P), but the only documented source of F-1-P is from the concurrent import and phosphorylation of exogenous fructose. Thus, many have proposed that fructose-1,6-bisphosphate (F-1,6-BP) is also a physiological regulatory ligand. However, the role of F-1,6-BP has been widely debated. Here, we report that the E. coli enzyme fructose-1-kinase (FruK) can carry out its "reverse" reaction under physiological substrate concentrations to generate F-1-P from F-1,6-BP. We further show that FruK directly binds Cra with nanomolar affinity and forms higher order, heterocomplexes. Growth assays with a ΔfruK strain and fruK complementation show that FruK has a broader role in metabolism than fructose catabolism. Since fruK itself is repressed by Cra, these newly-reported events add layers to the dynamic regulation of E. coli's central metabolism that occur in response to changing nutrients. These findings might have wide-spread relevance to other γ-proteobacteria, which conserve both Cra and FruK.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Fructoquinasas/metabolismo , Fructoquinasas/genética , Fructosa/metabolismo , Fructosadifosfatos/metabolismo , Fructosafosfatos/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
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.
Asunto(s)
Proliferación Celular , Fructoquinasas , Neoplasias Gástricas , beta Catenina , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patología , Neoplasias Gástricas/genética , Humanos , beta Catenina/metabolismo , beta Catenina/genética , Animales , Línea Celular Tumoral , Fructoquinasas/metabolismo , Fructoquinasas/genética , Ratones , Ratones Desnudos , Regulación Neoplásica de la Expresión Génica , Ratones Endogámicos BALB CRESUMEN
Diabetes might be associated with increased cancer risk, with several studies reporting hyperglycemia as a primary oncogenic stimulant. Since glucose metabolism is linked to numerous metabolic pathways, it is difficult to specify the mechanisms underlying hyperglycemia-induced cancer progression. Here, we focused on the polyol pathway, which is dramatically activated under hyperglycemia and causes diabetic complications. We investigated whether polyol pathway-derived fructose facilitates hyperglycemia-induced gastric cancer metastasis. We performed bioinformatics analysis of gastric cancer datasets and immunohistochemical analyses of gastric cancer specimens, followed by transcriptomic and proteomic analyses to evaluate phenotypic changes in gastric cancer cells. Consequently, we found a clinical association between the polyol pathway and gastric cancer progression. In gastric cancer cell lines, hyperglycemia enhanced cell migration and invasion, cytoskeletal rearrangement, and epithelial-mesenchymal transition (EMT). The hyperglycemia-induced acquisition of metastatic potential was mediated by increased fructose derived from the polyol pathway, which stimulated the nuclear ketohexokinase-A (KHK-A) signaling pathway, thereby inducing EMT by repressing the CDH1 gene. In two different xenograft models of cancer metastasis, gastric cancers overexpressing AKR1B1 were found to be highly metastatic in diabetic mice, but these effects of AKR1B1 were attenuated by KHK-A knockdown. In conclusion, hyperglycemia induces fructose formation through the polyol pathway, which in turn stimulates the KHK-A signaling pathway, driving gastric cancer metastasis by inducing EMT. Thus, the polyol and KHK-A signaling pathways could be potential therapeutic targets to decrease the metastatic risk in gastric cancer patients with diabetes.
Asunto(s)
Diabetes Mellitus Experimental , Hiperglucemia , Polímeros , Neoplasias Gástricas , Humanos , Animales , Ratones , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Proteómica , Transducción de Señal , Hiperglucemia/complicaciones , Fructoquinasas/genética , Fructoquinasas/metabolismo , Fructosa/metabolismo , Transición Epitelial-Mesenquimal/genética , Movimiento Celular/genética , Línea Celular Tumoral , Aldehído Reductasa/genética , Aldehído Reductasa/metabolismo , Aldehído Reductasa/farmacologíaRESUMEN
Dementia, as an advanced diabetes-associated cognitive dysfunction (DACD), has become the second leading cause of death among diabetes patients. Given that little guidance is currently available to address the DACD process, it is imperative to understand the underlying mechanisms and screen out specific therapeutic targets. The excessive endogenous fructose produced under high glucose conditions can lead to metabolic syndrome and peripheral organ damage. Although generated by the brain, the role of endogenous fructose in the exacerbation of cognitive dysfunction is still unclear. Here, we performed a comprehensive study on leptin receptor-deficient T2DM mice and their littermate m/m mice and revealed that 24-week-old db/db mice had cognitive dysfunction and excessive endogenous fructose metabolism in the hippocampus by multiomics analysis and further experimental validation. We found that the rate-limiting enzyme of fructose metabolism, ketohexokinase, is primarily localized in microglia. It is upregulated in the hippocampus of db/db mice, which enhances mitochondrial damage and reactive oxygen species production by promoting nicotinamide adenine dinucleotide phosphate oxidase 4 (NOX4) expression and mitochondrial translocation. Inhibiting fructose metabolism via ketohexokinase depletion reduces microglial activation, leading to the restoration of mitochondrial homeostasis, recovery of structural synaptic plasticity, improvement of CA1 pyramidal neuron electrophysiology and alleviation of cognitive dysfunction. Our findings demonstrated that enhanced endogenous fructose metabolism in microglia plays a dominant role in diabetes-associated cognitive dysfunction and could become a potential target for DACD.
Asunto(s)
Disfunción Cognitiva , Diabetes Mellitus , Humanos , Ratones , Animales , Microglía/metabolismo , Fructosa/metabolismo , Disfunción Cognitiva/etiología , Encéfalo/metabolismo , Fructoquinasas/genética , Fructoquinasas/metabolismoRESUMEN
The identification of clinical candidate LY3522348 (compound 23) is described. LY3522348 is a highly selective, oral dual inhibitor of human ketohexokinase isoforms C and A (hKHK-C, hKHK-A). Optimization began with highly efficient (S)-2-(2-methylazetidin-1-yl)-6-(1H-pyrazol-4-yl)-4-(trifluoromethyl)nicotinonitrile (3). Efforts focused on developing absorption, distribution, metabolism, potency, and in vitro safety profiles to support oral QD dosing in patients. Structure-based design leveraged vectors for substitution of the pyrazole ring, which provided an opportunity to interact with several different proximal amino acid residues in the protein. LY3522348 displayed a robust pharmacodynamic response in a mouse model of fructose metabolism and was advanced into clinical trials.
Asunto(s)
Fructoquinasas , Ratones , Animales , HumanosRESUMEN
Excessive fructose absorption and its subsequent metabolisms are implicated in nonalcoholic fatty liver disease, obesity, and insulin resistance in humans. Ketohexokinase (KHK) is a primary enzyme involved in fructose metabolism via the conversion of fructose to fructose-1-phosphate. KHK inhibition might be a potential approach for the treatment of metabolic disorders. Herein, a series of novel KHK inhibitors were designed, synthesized, and evaluated. Among them, compound 14 exhibited more potent activity than PF-06835919 based on the rat KHK inhibition assay in vivo, and higher drug distribution concentration in the liver. Its good absorption, distribution, metabolism, and excretion and pharmacokinetic properties make it a promising clinical candidate.
Asunto(s)
Resistencia a la Insulina , Enfermedades Metabólicas , Animales , Humanos , Ratas , Fructoquinasas/antagonistas & inhibidores , Fructosa , Hígado/metabolismo , Enfermedades Metabólicas/tratamiento farmacológicoRESUMEN
The presence of obesity and metabolic syndrome is strongly linked with chronic kidney disease (CKD), but the mechanisms responsible for the association are poorly understood. Here, we tested the hypothesis that mice with obesity and metabolic syndrome might have increased susceptibility to CKD from liquid high fructose corn syrup (HFCS) by favoring the absorption and utilization of fructose. We evaluated the pound mouse model of metabolic syndrome to determine if it showed baseline differences in fructose transport and metabolism and whether it was more susceptible to chronic kidney disease when administered HFCS. Pound mice have increased expression of fructose transporter (Glut5) and fructokinase (the limiting enzyme driving fructose metabolism) associated with enhanced fructose absorption. Pound mice receiving HFCS rapidly develop CKD with increased mortality rates associated with intrarenal mitochondria loss and oxidative stress. In pound mice lacking fructokinase, the effect of HFCS to cause CKD and early mortality was aborted, associated with reductions in oxidative stress and fewer mitochondria loss. Obesity and metabolic syndrome show increased susceptibility to fructose-containing sugars and increased risk for CKD and mortality. Lowering added sugar intake may be beneficial in reducing the risk for CKD in subjects with metabolic syndrome.
Asunto(s)
Jarabe de Maíz Alto en Fructosa , Enfermedades Renales , Síndrome Metabólico , Ratones , Animales , Síndrome Metabólico/complicaciones , Jarabe de Maíz Alto en Fructosa/efectos adversos , Ratones Obesos , Sacarosa en la Dieta/efectos adversos , Sacarosa en la Dieta/metabolismo , Obesidad/etiología , Fructosa/metabolismo , Enfermedades Renales/inducido químicamente , FructoquinasasRESUMEN
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.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Animales , Femenino , Humanos , Masculino , Ratones , Dieta Alta en Grasa/efectos adversos , Fructoquinasas/genética , Fructoquinasas/metabolismo , Fructosa/farmacología , Lipogénesis/fisiología , Hígado/metabolismo , Modelos Genéticos , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismoRESUMEN
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.
Asunto(s)
Carnitina O-Palmitoiltransferasa , Hígado , Masculino , Ratones , Animales , Carnitina O-Palmitoiltransferasa/genética , Carnitina O-Palmitoiltransferasa/metabolismo , Carnitina O-Palmitoiltransferasa/farmacología , Acetilación , Hígado/metabolismo , Obesidad/metabolismo , Glucosa/metabolismo , Dieta Alta en Grasa/efectos adversos , Ácidos Grasos/metabolismo , Fructosa/metabolismo , Fructoquinasas/genética , Fructoquinasas/metabolismoRESUMEN
Fructokinase (FRK) and fructokinase-like (FLN), belonging to the phosphofructokinase B type subfamily, share substantial sequence similarity, and are crucial in various plant physiological processes. However, there is limited information regarding what functionally differentiates plant FRKs from FLNs. Here, a total of three CsFRKs and two CsFLNs were identified from the cucumber genome. Their significant difference lay in the structure of their G/AXGD motif, which existed as GAGD in CsFRKs, but as G/ASGD in CsFLNs. Comparative phylogenetic analysis classified CsFRKs and CsFLNs into five sub-branches consistent with their quite different exon/intron organizations. Both transcriptome data and RT-qPCR analyses revealed that CsFRK3 was the most active gene, with the highest expression in the majority of tissues tested. Moreover, the expression levels of two putative plastidic genes, CsFRK1 and CsFLN2, were significantly positively associated with chlorophyll accumulation in the chlorophyll-reduced cucumber mutant. Briefly, both CsFRK and CsFLN genes were involved in the development of sink tissues, especially CsFRK3. CsFRK1 and CsFLN2 were recognized as candidates in the chlorophyll biosynthesis pathway of cucumber. These results would greatly assist in further investigation on functional characterization of FRKs and FLNs, especially in the development and chlorophyll biosynthesis of cucumber.
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Cucumis sativus , Cucumis sativus/genética , Cucumis sativus/metabolismo , Filogenia , Fructoquinasas/genética , Fructoquinasas/metabolismo , Intrones , Clorofila/metabolismoRESUMEN
The control of the duration of the dormancy phase is a significant challenge in the potato industry and for seed producers. However, the proteome landscape involved in the regulation of the length of the dormancy period over potato cultivars remains largely unexplored. In this study, we performed for the first time a comparative proteome profiling of potato cultivars with differential duration of tuber dormancy. More specifically, the proteome profiling of Agata, Kennebec and Agria commercial potato varieties with short, medium and medium-long dormancy, respectively, was assessed at the endodormancy stage using high-resolution two-dimensional electrophoresis (2-DE) coupled to reversed-phase liquid chromatography-tandem mass spectrometry (LC-TripleTOF MS/MS). A total of 11 proteins/isoforms with statistically significant differential abundance among cultivars were detected on 2-DE gels and confidently identified by LC-TripleTOF MS/MS. Identified proteins have known functions related to tuber development, sprouting and the oxylipins biosynthesis pathway. Fructokinase, a mitochondrial ADP/ATP carrier, catalase isozyme 2 and heat shock 70 kDa were the proteins with the strongest response to dormancy variations. To the best of our knowledge, this study reports the first candidate proteins underlying variable dormancy length in potato cultivars.
Asunto(s)
Solanum tuberosum , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Catalasa/metabolismo , Fructoquinasas/análisis , Fructoquinasas/metabolismo , Isoenzimas/metabolismo , Oxilipinas/metabolismo , Proteínas de Plantas/metabolismo , Tubérculos de la Planta/química , Proteoma/metabolismo , Proteómica/métodos , Solanum tuberosum/química , Espectrometría de Masas en TándemRESUMEN
Low temperature is one of the main abiotic stresses that inhibit wheat growth and development. To understand the physiological mechanism of salt priming induced low temperature tolerance and its transgenerational effects, the chlorophyl b-deficient mutant (ANK) and its wild type (WT) wheat were subjected to low temperature stress after parental salt priming. Salt priming significantly decreased the levels of superoxide anions, hydrogen peroxide and malondialdehyde in both parental and offspring plants under low temperature. The catalase activity in parental wheat and activities of dehydroascorbate reductase and glutathione reductase in the offspring were significantly increased by salt priming under low temperature. Meanwhile, salt priming contributed to mantaining the integrity of chloroplast structure and relatively higher net photosynthetic rate (Pn) in both generations under low temperature. Salt priming also improved the carbohydrate metabolism enzyme activities of parental and offspring plants, such as phosphoglucomutase, fructokinase and sucrose synthase. In addition, ANK plants had significantly higher carbohydrate metabolism enzyme activities than WT plants. The differential expressed proteins (DEP) in seeds of two genotypes under salt priming were mainly related to homeostasis, electron transfer activity, photosynthesis and carbohydrate metabolism. Correlation network analysis showed that the expression of DEP under salt priming was significantly correlated to sucrose concentration and cytoplasmic peroxidase (POX) activity in WT, while that was correlated to various carbohydrate metabolism enzyme activities in ANK plants. These results indicated that the parental salt priming induced modulations of seed proteome regulated the ROS metabolism, photosynthetic carbon assimilation and carbohydrate metabolism, hence enhancing the low temperature tolerance in offspring wheat.
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Germinación , Triticum , Antioxidantes/metabolismo , Carbono/metabolismo , Catalasa/metabolismo , Fructoquinasas/metabolismo , Glutatión Reductasa/metabolismo , Peróxido de Hidrógeno/metabolismo , Malondialdehído/metabolismo , Fosfoglucomutasa/metabolismo , Fosfoglucomutasa/farmacología , Proteoma/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Semillas/metabolismo , Cloruro de Sodio/farmacología , Estrés Fisiológico , Sacarosa/metabolismo , Superóxidos/metabolismo , Temperatura , Triticum/metabolismoRESUMEN
Roles of fructose in elongating ovine conceptuses are poorly understood, despite it being the major hexose sugar in fetal fluids and plasma throughout gestation. Therefore, we determined if elongating ovine conceptuses utilize fructose via metabolic pathways for survival and development. Immunohistochemical analyses revealed that trophectoderm and extra-embryonic endoderm express ketohexokinase and aldolase B during the peri-implantation period of pregnancy for conversion of fructose into fructose-1-phosphate for entry into glycolysis and related metabolic pathways. Conceptus homogenates were cultured with 14C-labeled glucose and/or fructose under oxygenated and hypoxic conditions to assess contributions of glucose and fructose to the pentose cycle (PC), tricarboxylic acid cycle, glycoproteins, and lipid synthesis. Results indicated that both glucose and fructose contributed carbons to each of these pathways, except for lipid synthesis, and metabolized to pyruvate and lactate, with lactate being the primary product of glycolysis under oxygenated and hypoxic conditions. We also found that (1) conceptuses preferentially oxidized glucose over fructose (P < 0.05); (2) incorporation of fructose and glucose at 4 mM each into the PC by Day 16 conceptus homogenates was similar in the presence or absence of glucose, but incorporation of glucose into the PC was enhanced by the presence of fructose (P < 0.05); (3) incorporation of fructose into the PC in the absence of glucose was greater under oxygenated conditions (P < 0.01); and (4) incorporation of glucose into the PC under oxygenated conditions was greater in the presence of fructose (P = 0.05). These results indicate that fructose is an important metabolic substrate for ovine conceptuses.
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Fructosa-Bifosfato Aldolasa , Fructosa , Animales , Femenino , Fructoquinasas , Glucosa , Lactatos , Lípidos , Pentosas , Embarazo , Piruvatos , Ovinos , Oveja DomésticaRESUMEN
Rhizobium sp. RM solubilized tri-calcium phosphate (TCP: 324-463 µg ml-1) and rock phosphate (RP: 36-46.58 µg ml-1) in the presence of common rhizospheric sugars-glucose, arabinose, xylose and their combinations. Fructose, though did not support RP solubilization individually, surprisingly solubilized significantly higher phosphate when combined with aldoses. The highest TCP (644 µg ml-1) and RP (75 µg ml-1) solubilization was achieved in fructose + glucose combination. Presence of gluconate, malate and oxalate in culture supernatant indicated functioning of periplasmic glucose oxidation, the non-phosphorylative arabinose dehydrogenase pathway and the tricarboxylate (TCA) cycle, respectively. Aldoses, when present together, were co-utilized (monoauxic growth) however, when added with fructose, prevented the uptake of fructose yielding a typical diauxic growth. This presented an unusual sequential utilization of aldoses over a ketose (fructose) in strain RM. The prevention of fructose uptake by aldoses was investigated through real-time expression of key genes coding fructose transport proteins and initial enzymes of sugar metabolism. Fructose was actively transported via fructose-specific ABC transporters as suggested by upregulation of frcB and frcC only in fructose and fructose growth phases of fructose + aldose combinations. The probable route of initial fructose metabolism involved either fructokinase and/or xylose isomerase, as confirmed by enzyme activities. The upregulation of hfq and hprK genes only in aldose phase of fructose + aldose combinations suggested their possible involvement in governing the preferential utilization. The novel aspects of this study are enhanced organic acid mediated P solubilization in fructose + aldose combinations and a rare hierarchy of aldoses over fructose which is possibly regulated at the level of fructose transport and fructokinase. KEY POINTS: ⢠Sugars when provided in different dual combinations, supported enhanced P solubilization from complex phosphate sources like TCP and RP in Rhizobium sp. RM. ⢠Transcriptional status of genes in cells of RM when grown in different individual sugars and their combinations suggested that fructose might be a less preferred carbon source and hence was utilized after aldoses with the possible regulation by Hfq and HPrK. ⢠First study to present a unique phenomenon of sequential utilization of aldoses (glucose, arabinose and xylose) over fructose in a concentration-independent manner in Rhizobium sp. RM. and to present the effect of dual combinations of sugars on organic acid mediated P solubilization trait of rhizobia.
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Rhizobium , Arabinosa/metabolismo , Fructoquinasas/metabolismo , Fructosa/metabolismo , Glucosa/metabolismo , Compuestos Orgánicos/metabolismo , Fosfatos/metabolismo , Rhizobium/genética , Xilosa/metabolismoRESUMEN
PF-06835919 is a first-in-class ketohexokinase inhibitor (KHKi), recently under development for the treatment of metabolic and fatty liver diseases, which inhibited organic anion transporting polypeptide (OATP)1B1 in vitro and presented drug-drug interaction (DDI) risk. This study aims to investigate the dose-dependent effect of KHKi on OATP1B in vivo activity. We performed an open-label study comparing pharmacokinetics of atorvastatin (OATP1B probe) dosed alone (20 mg single dose) and coadministered with two dose strengths of KHKi (50 and 280 mg once daily) in 12 healthy participants. Additionally, changes in exposure of coproporphyrin-I (CP-I), an endogenous biomarker for OATP1B, were assessed in the atorvastatin study (1.12-fold and 1.49-fold increase in area under the plasma concentration-time profile (AUC) with once-daily 50 and 280 mg, respectively), and a separate single oral dose study of KHKi alone (100-600 mg, n = 6 healthy participants; up to a 1.80-fold increase in AUC). Geometric mean ratios (90% confidence interval) of atorvastatin AUC following 50 and 280 mg KHKi were 1.14 (1.00-1.30) and 1.54 (1.37-1.74), respectively. Physiologically-based pharmacokinetic modeling of CP-I plasma exposure following a single dose of KHKi predicted in vivo OATP1B inhibition from about 13% to 70% over the 100 to 600 mg dose range, while using the in vitro inhibition potency (1.9 µM). Model-based analysis correctly predicted "no-effect" (AUC ratio < 1.25) at the low dose range and "weak" effect (AUC ratio < 2) on atorvastatin pharmacokinetics at the high dose range of KHKi. This study exemplified the utility of biomarker-informed model-based approach in discerning even small effects on OATP1B activity in vivo, and to project DDI risk at the clinically relevant doses.
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Fructoquinasas , Atorvastatina , Biomarcadores , Interacciones Farmacológicas , Fructoquinasas/metabolismo , Humanos , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Medición de RiesgoRESUMEN
Colorectal cancer (CRC) is a leading cause of cancer-related deaths worldwide. Abundant metabolic fuels have been implicated as potential drivers of CRC. However, it remains unclear whether fructose, an ample sugar in daily diets, is essential for CRC growth. In the present study, we found that glucose levels were always insufficient in human CRC tissues. Compensating for this, fructose was flexibly utilized by tumor cells as an alternative energy source to maintain proliferation and exert chemotherapy resistance in vitro by upregulating GLUT5, a major fructose transporter encoded by SLC2A5. Mechanistically, in glucose-deprived but fructose-rich environments, GLUT5 could interact with ketohexokinase and inhibit its autophagy-dependent degradation, thus trapping fructose into glycolysis and tricarboxylic acid cycle for the malignant growth of CRC cells. In addition, reducing dietary fructose or pharmacological blockade of fructose utilization significantly reduced CRC growth and sensitized CRC cells to chemotherapy in vivo. Taken together, our findings highlight the role of elevated fructose utilization mediated by the GLUT5-KHK axis in governing CRC growth and imply that efforts to refine fructose intake or inhibit fructose-mediated actions may serve as potential therapeutic strategies.
Asunto(s)
Neoplasias Colorrectales , Fructoquinasas , Fructosa , Transportador de Glucosa de Tipo 5 , Proliferación Celular , Neoplasias Colorrectales/tratamiento farmacológico , Fructoquinasas/metabolismo , Fructosa/metabolismo , Glucosa , Transportador de Glucosa de Tipo 5/metabolismo , HumanosRESUMEN
Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer with poor prognosis. This study designated to figure out the effects of Ubiquitin Specific Peptidase 36 (USP36) on NSCLC. Data of this study demonstrated that upregulation of USP36 was observed in NSCLC tissues and cell lines. Overexpression of USP36 promoted NSCLC cell proliferation and inhibited NSCLC cell apoptosis. Knockdown of USP36 decreased Ketohexokinase A (KHK-A) and increased KHK-C expression at both RNA and protein levels. Expression of c-MYC and hnRNPH1/H2 was positively correlated with the expression of USP36. Upregulation of c-MYC reversed the downregulation of hnRNPH1/H2 induced inhibition of USP36. Overexpression of hnRNPH1/H2 reversed the downregulation of KHK-A induced inhibition of USP36. Results of in vivo xenograft model were consistent with the findings of in vitro experiments. In summary, overexpression of USP36 in NSCLC accelerated tumor growth through upregulation of KHK-A, which was medicated by stabilizing c-MYC to increase hnRNPH1/H2 expression.