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Klaus Klarskov - One of the best experts on this subject based on the ideXlab platform.
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identification of liver protein targets modified by tienilic acid metabolites using a two dimensional western blot mass spectrometry approach
International Journal of Mass Spectrometry, 2007Co-Authors: Ruth Menque Methogo, Patrick M. Dansette, Klaus KlarskovAbstract:A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, Ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.
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Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach
International Journal of Mass Spectrometry, 2007Co-Authors: Ruth Menque Methogo, Patrick M. Dansette, Klaus KlarskovAbstract:International audienceA combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, Ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase
Ronaldo P. Ferraris - One of the best experts on this subject based on the ideXlab platform.
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cell type specific Ketohexokinase dependent induction by fructose of lipogenic gene expression in mouse small intestine
Journal of Nutrition, 2020Co-Authors: Arwa Aljawadi, Kunihiro Kishida, Chirag Patel, Reilly J. Shiarella, Emmanuellie Romelus, Madelyn J. Auvinen, Joshua Guardia, Sarah C. Pearce, Nan Gao, Ronaldo P. FerrarisAbstract:BACKGROUND High intakes of fructose are associated with metabolic diseases, including hypertriglyceridemia and intestinal tumor growth. Although small intestinal epithelia consist of many different cell types, express lipogenic genes, and convert dietary fructose to fatty acids, there is no information on the identity of the cell type(s) mediating this conversion and on the effects of fructose on lipogenic gene expression. OBJECTIVES We hypothesized that fructose regulates the intestinal expression of genes involved in lipid and apolipoprotein synthesis, that regulation depends on the fructose transporter solute carrier family 2 member a5 [Slc2a5 (glucose transporter 5)] and on Ketohexokinase (Khk), and that regulation occurs only in enterocytes. METHODS We compared lipogenic gene expression among different organs from wild-type adult male C57BL mice consuming a standard vivarium nonpurified diet. We then gavaged twice daily for 2.5 d fructose or glucose solutions (15%, 0.3 mL per mouse) into wild-type, Slc2a5-knockout (KO), and Khk-KO mice with free access to the nonpurified diet and determined expression of representative lipogenic genes. Finally, from mice fed the nonpurified diet, we made organoids highly enriched in enterocyte, goblet, Paneth, or stem cells and then incubated them overnight in 10 mM fructose or glucose. RESULTS Most lipogenic genes were significantly expressed in the intestine relative to the kidney, liver, lung, and skeletal muscle. In vivo expression of Srebf1, Acaca, Fasn, Scd1, Dgat1, Gk, Apoa4, and Apob mRNA and of Scd1 protein increased (P < 0.05) by 3- to 20-fold in wild-type, but not in Slc2a5-KO and Khk-KO, mice gavaged with fructose. In vitro, Slc2a5- and Khk-dependent, fructose-induced increases, which ranged from 1.5- to 4-fold (P < 0.05), in mRNA concentrations of all these genes were observed only in organoids enriched in enterocytes. CONCLUSIONS Fructose specifically stimulates expression of mouse small intestinal genes for lipid and apolipoprotein synthesis. Secretory and stem cells seem incapable of transport- and metabolism-dependent lipogenesis, occurring only in absorptive enterocytes.
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Bone Growth is Influenced by Fructose in Adolescent Male Mice Lacking Ketohexokinase (KHK)
Calcified Tissue International, 2020Co-Authors: Edek A. J. Williams, Veronique Douard, Keiichiro Sugimoto, Hiroshi Inui, Fabienne Devime, Xufei Zhang, Kunihiro Kishida, Ronaldo P. Ferraris, J. Christopher FrittonAbstract: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
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bone growth is influenced by fructose in adolescent male mice lacking Ketohexokinase khk
Calcified Tissue International, 2020Co-Authors: Edek Williams, Veronique Douard, Keiichiro Sugimoto, Hiroshi Inui, Fabienne Devime, Xufei Zhang, Kunihiro Kishida, Ronaldo P. Ferraris, Christopher J FrittonAbstract: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.
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Role of metabolism and intracellular trafficking in fructose-induced GLUT5 regulation (822.4)
The FASEB Journal, 2014Co-Authors: Chirag Patel, Veronique Douard, Phuntila Tharabenjasin, E Topaktas, Ronaldo P. FerrarisAbstract:Marked increases in fructose consumption have been linked to various diseases. Fructose (F) upregulates its own absorption by increasing the expression & activity of the intestinal F transporter GLUT5, but the underlying mechanism is not well understood. Defects or limits in regulation can lead to adult-onset F intolerance or to F malabsorption in infants. I tested the hypothesis that F transport via GLUT5, metabolism via Ketohexokinase (KHK), and GLUT5 intracellular trafficking to the apical membrane via the GTPase Rab11a are required for GLUT5 upregulation. WAdult wildtype (wt), GLUT5-/-, and KHK-/-, and Rab11a-/- mice were receivedreceiving 30% glucose (G), fructose (F) or lysine (L) by gavage twice a d for 3 d at X.X ml/kgays. 17 d old wt and Rab11a-/- mice were gavaged with either 30% G or F. Afterwards, GLUT5 activity as well as mRNA and protein expression determined in the small intestine. Activity and expression of GLUT5 and of other enzymes involved in F metabolism increased in F-gavaged mice com...
David T. Bonthron - One of the best experts on this subject based on the ideXlab platform.
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Abstract 18170: Role of Ketohexokinase in Fructose-Mediated Insulin Resistance and Endothelial Dysfunction
Circulation, 2017Co-Authors: Asjad Visnagri, David T. Bonthron, Hema Viswambharan, Yilizila Abudushalamu, Luke Morris, Nadira Yuldasheva, Mark T. Kearney, Aruna AsipuAbstract:Introduction: Diet-related cardiovascular disease is a major cause of mortality. Over-consumption of fructose, widely used in soft drinks, results in adverse metabolic effects, including insulin re...
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Both isoforms of Ketohexokinase are dispensable for normal growth and development
Physiological genomics, 2010Co-Authors: Christine P. Diggle, Aruna Asipu, Bruce E. Hayward, Michael Shires, Ian M. Carr, Cassey Mcrae, Doreen M. Crellin, Julie Fisher, Alexander F. Markham, David T. BonthronAbstract:Dietary fructose intake has dramatically increased over recent decades and is implicated in the high rates of obesity, hypertension, and type 2 diabetes (metabolic syndrome) in Western societies. T...
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Ketohexokinase: expression and localization of the principal fructose-metabolizing enzyme.
The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, 2009Co-Authors: Christine P. Diggle, Aruna Asipu, Bruce E. Hayward, Michael Shires, Derek Leitch, David Brooke, Ian M. Carr, Alex F. Markham, David T. BonthronAbstract:Ketohexokinase (KHK, also known as fructokinase) initiates the pathway through which most dietary fructose is metabolized. Very little is known about the cellular localization of this enzyme. Alternatively spliced KHK-C and KHK-A mRNAs are known, but the existence of the KHK-A protein isoform has not been demonstrated in vivo. Using antibodies to KHK for immunohistochemistry and Western blotting of rodent tissues, including those from mouse knockouts, coupled with RT-PCR assays, we determined the distribution of the splice variants. The highly expressed KHK-C isoform localized to hepatocytes in the liver and to the straight segment of the proximal renal tubule. In both tissues, cytoplasmic and nuclear staining was observed. The KHK-A mRNA isoform was observed exclusively in a range of other tissues, and by Western blotting, the presence of endogenous immunoreactive KHK-A protein was shown for the first time, proving that the KHK-A mRNA is translated into KHK-A protein in vivo, and supporting the suggestion that this evolutionarily conserved isoform is physiologically functional. However, the low levels of KHK-A expression prevented its immunohistochemical localization within these tissues. Our results highlight that the use of in vivo biological controls (tissues from knockout animals) is required to distinguish genuine KHK immunoreactivity from experimental artifact.
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Structures of alternatively spliced isoforms of human Ketohexokinase.
Acta Crystallographica Section D Biological Crystallography, 2009Co-Authors: Chi H. Trinh, Aruna Asipu, David T. Bonthron, Simon E. V. PhillipsAbstract:A molecular understanding of the unique aspects of dietary fructose metabolism may be the key to understanding and controlling the current epidemic of fructose-related obesity, diabetes and related adverse metabolic states in Western populations. Fructose catabolism is initiated by its phosphorylation to fructose 1-phosphate, which is performed by Ketohexokinase (KHK). Here, the crystal structures of the two alternatively spliced isoforms of human Ketohexokinase, hepatic KHK-C and the peripheral isoform KHK-A, and of the ternary complex of KHK-A with the substrate fructose and AMP-PNP are reported. The structure of the KHK-A ternary complex revealed an active site with both the substrate fructose and the ATP analogue in positions ready for phosphorylation following a reaction mechanism similar to that of the pfkB family of carbohydrate kinases. Hepatic KHK deficiency causes the benign disorder essential fructosuria. The effects of the disease-causing mutations (Gly40Arg and Ala43Thr) have been modelled in the context of the KHK structure.
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Properties of normal and mutant recombinant human Ketohexokinases and implications for the pathogenesis of essential fructosuria.
Diabetes, 2003Co-Authors: Aruna Asipu, Bruce E. Hayward, John O'reilly, David T. BonthronAbstract:Alternative splicing of the Ketohexokinase (fructokinase) gene generates a "central" predominantly hepatic isoform (Ketohexokinase-C) and a more widely distributed Ketohexokinase-A. Only the abundant hepatic isoform is known to possess activity, and no function is defined for the lower levels of Ketohexokinase-A in peripheral tissues. Hepatic Ketohexokinase deficiency causes the benign disorder essential fructosuria. The molecular basis of this has been defined in one family (compound heterozygosity for mutations Gly40Arg and Ala43Thr). Here we show that both Ketohexokinase isoforms are indeed active. Ketohexokinase-A has much poorer substrate affinity than Ketohexokinase-C for fructose but is considerably more thermostable. The Gly40Arg mutation seems null, rendering both Ketohexokinase-A and Ketohexokinase-C inactive and largely insoluble. The Ala43Thr mutant retains activity, but this mutation decreases the thermal stability of both Ketohexokinase-A and Ketohexokinase-C. At physiologic temperature, this results in significant loss of Ketohexokinase-C activity but not of Ketohexokinase-A. Affected individuals who carry both mutations therefore probably have a selective deficiency of hepatic Ketohexokinase, with peripheral Ketohexokinase-A being preserved. These findings raise the possibility that Ketohexokinase-A serves an unknown physiologic function that remains intact in essential fructosuria. Further mutation analysis in this rare disorder could illuminate the question of whether Ketohexokinase-A activity is, unlike that of Ketohexokinase-C, physiologically indispensable.
Ruth Menque Methogo - One of the best experts on this subject based on the ideXlab platform.
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identification of liver protein targets modified by tienilic acid metabolites using a two dimensional western blot mass spectrometry approach
International Journal of Mass Spectrometry, 2007Co-Authors: Ruth Menque Methogo, Patrick M. Dansette, Klaus KlarskovAbstract:A combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, Ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase.
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Identification of liver protein targets modified by tienilic acid metabolites using a two-dimensional Western blot-mass spectrometry approach
International Journal of Mass Spectrometry, 2007Co-Authors: Ruth Menque Methogo, Patrick M. Dansette, Klaus KlarskovAbstract:International audienceA combined approach based on two-dimensional electrophoresis-immuno-blotting and nanoliquid chromatography coupled on-line with electrospray ionization mass spectrometry (nLC-MS/MS) was used to identify proteins modified by a reactive intermediate of tienilic acid (TA). Liver homogenates from rats exposed to TA were fractionated using ultra centrifugation; four fractions were obtained and subjected to 2D electrophoresis. Following transfer to PVDF membranes, modified proteins were visualized after India ink staining, using an anti-serum raised against TA and ECL detection. Immuno-reactive spots were localized on the PVDF membrane by superposition of the ECL image, protein spots of interest were excised, digested on the membrane with trypsin followed by nLC-MS/MS analysis and protein identification. A total of 15 proteins were identified as likely targets modified by a TA reactive metabolite. These include selenium binding protein 2, senescence marker protein SMP-30, adenosine kinase, Acy1 protein, adenosylhomocysteinase, capping protein (actin filament), protein disulfide isomerase, fumarylacetoacetase, arginase chain A, Ketohexokinase, proteasome endopeptidase complex, triosephosphate isomerase, superoxide dismutase, dna-type molecular chaperone hsc73 and malate dehydrogenase
Naotake Tsuboi - One of the best experts on this subject based on the ideXlab platform.
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Fructose increases the activity of sodium hydrogen exchanger in renal proximal tubules that is dependent on Ketohexokinase.
The Journal of nutritional biochemistry, 2019Co-Authors: Takahiro Hayasaki, Takuji Ishimoto, Tomohito Doke, Akiyoshi Hirayama, Tomoyoshi Soga, Kazuhiro Furuhashi, Noritoshi Kato, Tomoki Kosugi, Naotake Tsuboi, Miguel A. LanaspaAbstract:High fructose intake has been known to induce metabolic syndrome in laboratory animals and humans. Although fructose intake enhances sodium reabsorption and elevates blood pressure, role of fructose metabolism in this process has not been studied. Here we show that by Ketohexokinase - the primary enzyme of fructose - is involved in regulation of renal sodium reabsorption and blood pressure via activation of the sodium hydrogen exchanger in renal proximal tubular cells. First, wild-type and Ketohexokinase knockout mice (Male, C57BL/6) were fed fructose water or tap water with or without a high salt diet. Only wild type mice fed the combination of fructose water and high salt diet displayed increased systolic blood pressure and decreased urinary sodium excretion. In contrast, Ketohexokinase knockout mice were protected. Second, urinary sodium excretion after intraperitoneal saline administration was reduced with the decreased phosphorylation of sodium hydrogen exchanger 3 in fructose-fed WT; these changes were not observed in the Ketohexokinase knockout mice, however. Third, knockdown of Ketohexokinase attenuated fructose-mediated increases of NHE activity with decreased cAMP levels in porcine renal proximal tubular cells (LLC-PK1). In conclusion, fructose metabolism by Ketohexokinase increases sodium hydrogen exchanger activity in renal proximal tubular cells via decreased intracellular cAMP level, resulting in increased renal sodium reabsorption and blood pressure in mice.
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Lacking Ketohexokinase-A exacerbates renal injury in streptozotocin-induced diabetic mice.
Metabolism: clinical and experimental, 2018Co-Authors: Tomohito Doke, Takahiro Hayasaki, Takuji Ishimoto, Akiyoshi Hirayama, Tomoyoshi Soga, Noritoshi Kato, Tomoki Kosugi, Satsuki Ikeda, Masako Hasebe, Naotake TsuboiAbstract:Abstract Objective Ketohexokinase (KHK), a primary enzyme in fructose metabolism, has two isoforms, namely, KHK-A and KHK-C. Previously, we reported that renal injury was reduced in streptozotocin-induced diabetic mice which lacked both isoforms. Although both isoforms express in kidney, it has not been elucidated whether each isoform plays distinct roles in the development of diabetic kidney disease (DKD). The aim of the study is to elucidate the role of KHK-A for DKD progression. Materials and Methods Diabetes was induced by five consecutive daily intraperitoneal injections of streptozotocin (50 mg/kg) in C57BL/6J wild-type mice, mice lacking KHK-A alone (KHK-A KO), and mice lacking both KHK-A and KHK-C (KHK-A/C KO). At 35 weeks, renal injury, inflammation, hypoxia, and oxidative stress were examined. Metabolomic analysis including polyol pathway, fructose metabolism, glycolysis, TCA (tricarboxylic acid) cycle, and NAD (nicotinamide adenine dinucleotide) metabolism in kidney and urine was done. Results Diabetic KHK-A KO mice developed severe renal injury compared to diabetic wild-type mice, and this was associated with further increases of intrarenal fructose, dihydroxyacetone phosphate (DHAP), TCA cycle intermediate levels, and severe inflammation. In contrast, renal injury was prevented in diabetic KHK-A/C KO mice compared to both wild-type and KHK-A KO diabetic mice. Further, diabetic KHK-A KO mice contained decreased renal NAD+ level with the increase of renal hypoxia-inducible factor 1-alpha expression despite having increased renal nicotinamide (NAM) level. Conclusion These results suggest that KHK-C might play a deleterious role in DKD progression through endogenous fructose metabolism, and that KHK-A plays a unique protective role against the development of DKD.
