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1.
Sci Rep ; 14(1): 729, 2024 01 06.
Article in English | MEDLINE | ID: mdl-38184689

ABSTRACT

To investigate the role and potential mechanism of serine/threonine kinase 36 (STK36) in docetaxel resistance-prostate cancer (PCa). The expression of STK36 in PCa and the correlation with clinicopathological characteristics of PCa patients were analyzed using the data from different databases and tissue microarrays. To investigate the role of STK36 on cell proliferation, invasion, and migration, STK36 was overexpressed and silenced in DU-145 and PC-3 cell lines. Cell counting kit-8 (CCK8) was used to test cell proliferation. Cell invasion and migration were detected by cell wound scratch assay and trans well, respectively. The expression profile of STK36, E-Cadherin, and Vimentin was analyzed by Western blot. Cell apoptosis was detected by the TUNEL assay. STK36 expression was upregulated in PCa tissue compared with adjacent benign PCa tissue; it was higher in patients with advanced stages compared with lower stages and was significantly correlated with decreased overall survival. Up-regulation of STK36 significantly promoted the proliferation, invasion, and migration of DU-145 and PC-3 cells and compensated for the suppression caused by docetaxel treatment in vitro. A striking apoptosis inhibition could be observed when dealing with docetaxel, although the apoptosis of DU-145 and PC-3 cells was not affected by the STK36 exclusive overexpression. Besides, E-Cadherin expression was restrained while the expression levels of vimentin were all enhanced. The knockdown of STK36 reversed the above process. STK36 up-regulation could accelerate the biological behavior and docetaxel resistance of PCa by epithelial-mesenchymal transition (EMT) activation. STK36 may be potentially used as a target in PCa resolvent with docetaxel.


Subject(s)
Prostatic Neoplasms , Male , Humans , Docetaxel/pharmacology , Vimentin/genetics , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/genetics , Cadherins/genetics , Epithelial-Mesenchymal Transition/genetics , Serine , Protein Serine-Threonine Kinases/genetics
2.
Langmuir ; 39(49): 18101-18112, 2023 12 12.
Article in English | MEDLINE | ID: mdl-38038444

ABSTRACT

CD47 on the surface of tumor cells has become a research hot spot in immunotherapy and anticancer therapy, as it can bind to SIRPα protein on the surface of macrophages, which ultimately leads to immune escape of tumor cells. In the present study, molecular interactions between CD47 and human SIRPα proteins (including variant 1, V1 and variant 2, V2) were analyzed through molecular dynamics (MD) simulation and the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. Hydrophobic interactions were found as the main driving force for the binding of CD47 on SIRPα. The residues including pyroglutamate acid (Z)1, L2, E35, Y37, E97, L101, and T102 of CD47 were identified with a significant favorable contribution to the binding of CD47 on SIRPα (both V1 and V2). Based on this, a peptide inhibitor library with the sequence ZLXRTLXEXY was designed (X represents the arbitrary residue of 20 standard amino acids) and then screened using molecular docking, MD simulations, and experimental validation. Finally, a peptide ZLIRTLHEWY was determined with high affinity with SIRPα from 8000 candidates, containing 6/10 residues favorable for the binding on SIRPα V1 and 8/10 residues favorable for the binding on SIRPα V2, which was thus considered to have potential anticancer function.


Subject(s)
CD47 Antigen , Neoplasms , Humans , CD47 Antigen/genetics , CD47 Antigen/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/metabolism , Molecular Docking Simulation , Biomimetics , Antigens, Differentiation/chemistry , Antigens, Differentiation/metabolism , Peptides/pharmacology , Peptide Library , Phagocytosis
3.
Appl Microbiol Biotechnol ; 107(23): 7089-7104, 2023 Dec.
Article in English | MEDLINE | ID: mdl-37733049

ABSTRACT

Nitriles are of significant interest in the flavor and fragrance industries with potential application in cosmetics due to their higher stability than analogous aldehydes. However, the traditional methods to prepare nitriles need toxic reagents and hash conditions. This work aimed to develop a chemoenzymatic strategy to synthesize nitriles from natural aldehydes with aldoxime as the intermediate. A non-classical aldoxime dehydratase (Oxd) was discovered from the fungus Aspergillus ibericus (OxdAsp) to catalyze the dehydration of aldoximes to corresponding nitriles under mild conditions. The amino acid sequence of OxdAsp exhibits an approximately 20% identity with bacterial Oxds. OxdAsp contains a heme prosthetic group bound with the axial H287 in the catalytic pocket. The structure models of OxdAsp with substrates suggest that its catalytic triad is Y138-R141-E192, which is different from the classically bacterial Oxds of His-Arg-Ser/Thr. The catalytic mechanism of OxdAsp was proposed based on the mutagenesis of key residues. The hydroxyl group of the substrate is fixed by E192 to increase its basicity. Y138 acts as a general acid-based catalyst, and its phenolic proton is polarized by the adjacent R141. The protonated Y138 would donate a proton to the hydroxyl group of the substrate and eliminate a water molecule from aldoxime to produce nitrile. The recombinant OxdAsp can efficiently dehydrate citronellal oxime and cinnamaldoxime to citronellyl nitrile and cinnamonitrile in aqueous media, which are applied as fragrance ingredients in the food and cosmetic fields. KEY POINTS: • A novel aldoxime dehydratase from the Aspergillus genus was first characterized as a heme-binding protein. • The catalytic mechanism was predicted based on the molecular interactions of the catalytic pocket with the substrate. • A chemoenzymatic strategy was developed to synthesize nitriles from natural aldehydes with aldoxime as the intermediate.


Subject(s)
Bacteria , Protons , Bacteria/metabolism , Hydro-Lyases/metabolism , Nitriles/metabolism , Aldehydes
4.
Stat Appl Genet Mol Biol ; 22(1)2023 01 01.
Article in English | MEDLINE | ID: mdl-36724206

ABSTRACT

Many human disease conditions need to be measured by ordinal phenotypes, so analysis of ordinal phenotypes is valuable in genome-wide association studies (GWAS). However, existing association methods for dichotomous or quantitative phenotypes are not appropriate to ordinal phenotypes. Therefore, based on an aggregated Cauchy association test, we propose a fast and efficient association method to test the association between genetic variants and an ordinal phenotype. To enrich association signals of rare variants, we first use the burden method to aggregate rare variants. Then we respectively test the significance of the aggregated rare variants and other common variants. Finally, the combination of transformed variant-level P values is taken as test statistic, that approximately follows Cauchy distribution under the null hypothesis. Extensive simulation studies and analysis of GAW19 show that our proposed method is powerful and computationally fast as a gene-based method. Especially, in the presence of an extremely low proportion of causal variants in a gene, our method has better performance.


Subject(s)
Genetic Variation , Genome-Wide Association Study , Humans , Phenotype , Models, Genetic , Computer Simulation
5.
Langmuir ; 38(23): 7114-7120, 2022 06 14.
Article in English | MEDLINE | ID: mdl-35623058

ABSTRACT

The development of antithrombotic peptides targeting collagen was proven effective, and an effective antithrombotic peptide LEKNSTY was obtained in part I. However, the plasma stability of LEKNSTY was found to be not good enough. In this part, the LEKNSTY was further optimized for improvement in plasma stability by substitution using d-amino acid residues. Two novel antithrombotic peptides LekNStY and lEKnsTy were designed, where lowercase letters represent d-amino acid residues. Improvements in plasma stability of both LekNStY and lEKnsTy were experimentally confirmed. Moreover, good binding of these antithrombotic peptides on the collagen surface was confirmed by molecular dynamics simulation and experimental validation. For example, a Kd of only 0.75 ± 0.10 µM was observed for lEKnsTy. Moreover, LekNStY and lEKnsTy were found to inhibit platelet adhesion on the collagen surface more effectively than LEKNSTY, and the IC50 of lEKnsTy was only 2/5 of that of LEKNSTY. These results confirmed the successful design of LekNStY and lEKnsTy that had good plasma stability and could effectively inhibit arterial thrombosis, which would be helpful for the research into interfaces involved in thrombus formation and the development of antithrombotic nanomedicine.


Subject(s)
Fibrinolytic Agents , Thrombosis , Amino Acids , Collagen/metabolism , Fibrinolytic Agents/chemistry , Fibrinolytic Agents/pharmacology , Humans , Peptides/pharmacology , Platelet Adhesiveness , Thrombosis/drug therapy , Thrombosis/prevention & control
6.
JCI Insight ; 6(1)2021 01 11.
Article in English | MEDLINE | ID: mdl-33320834

ABSTRACT

Subjects with obesity frequently have elevated serum vasopressin levels, noted by measuring the stable analog, copeptin. Vasopressin acts primarily to reabsorb water via urinary concentration. However, fat is also a source of metabolic water, raising the possibility that vasopressin might have a role in fat accumulation. Fructose has also been reported to stimulate vasopressin. Here, we tested the hypothesis that fructose-induced metabolic syndrome is mediated by vasopressin. Orally administered fructose, glucose, or high-fructose corn syrup increased vasopressin (copeptin) concentrations and was mediated by fructokinase, an enzyme specific for fructose metabolism. Suppressing vasopressin with hydration both prevented and ameliorated fructose-induced metabolic syndrome. The vasopressin effects were mediated by the vasopressin 1b receptor (V1bR), as V1bR-KO mice were completely protected, whereas V1a-KO mice paradoxically showed worse metabolic syndrome. The mechanism is likely mediated in part by de novo expression of V1bR in the liver that amplifies fructokinase expression in response to fructose. Thus, our studies document a role for vasopressin in water conservation via the accumulation of fat as a source of metabolic water. Clinically, they also suggest that increased water intake may be a beneficial way to both prevent or treat metabolic syndrome.


Subject(s)
Fructose/metabolism , Metabolic Syndrome/metabolism , Receptors, Vasopressin/metabolism , Vasopressins/metabolism , Animals , Disease Models, Animal , Drinking/physiology , Fructokinases/metabolism , Fructose/administration & dosage , Hep G2 Cells , Humans , Liver/metabolism , Male , Metabolic Syndrome/chemically induced , Mice , Mice, Inbred C57BL , Mice, Knockout , Receptors, Vasopressin/deficiency , Receptors, Vasopressin/genetics , Vasopressins/antagonists & inhibitors , Vasopressins/biosynthesis
8.
J Biol Chem ; 294(11): 4272-4281, 2019 03 15.
Article in English | MEDLINE | ID: mdl-30651350

ABSTRACT

Dietary, fructose-containing sugars have been strongly associated with the development of nonalcoholic fatty liver disease (NAFLD). Recent studies suggest that fructose also can be produced via the polyol pathway in the liver, where it may induce hepatic fat accumulation. Moreover, fructose metabolism yields uric acid, which is highly associated with NAFLD. Here, using biochemical assays, reporter gene expression, and confocal fluorescence microscopy, we investigated whether uric acid regulates aldose reductase, a key enzyme in the polyol pathway. We evaluated whether soluble uric acid regulates aldose reductase expression both in cultured hepatocytes (HepG2 cells) and in the liver of hyperuricemic rats and whether this stimulation is associated with endogenous fructose production and fat accumulation. Uric acid dose-dependently stimulated aldose reductase expression in the HepG2 cells, and this stimulation was associated with endogenous fructose production and triglyceride accumulation. This stimulatory mechanism was mediated by uric acid-induced oxidative stress and stimulation of the transcription factor nuclear factor of activated T cells 5 (NFAT5). Uric acid also amplified the effects of elevated glucose levels to stimulate hepatocyte triglyceride accumulation. Hyperuricemic rats exhibited elevated hepatic aldose reductase expression, endogenous fructose accumulation, and fat buildup that was significantly reduced by co-administration of the xanthine oxidase inhibitor allopurinol. These results suggest that uric acid generated during fructose metabolism may act as a positive feedback mechanism that stimulates endogenous fructose production by stimulating aldose reductase in the polyol pathway. Our findings suggest an amplifying mechanism whereby soft drinks rich in glucose and fructose can induce NAFLD.


Subject(s)
Adipose Tissue/metabolism , Aldehyde Reductase/metabolism , Fructose/biosynthesis , Non-alcoholic Fatty Liver Disease/metabolism , Polymers/metabolism , Uric Acid/pharmacology , Animals , Dose-Response Relationship, Drug , Enzyme Activation/drug effects , Fructose/metabolism , Hep G2 Cells , Humans , Male , Non-alcoholic Fatty Liver Disease/chemically induced , Non-alcoholic Fatty Liver Disease/pathology , Oxidative Stress/drug effects , Polymers/analysis , Rats , Rats, Wistar , Tumor Cells, Cultured , Uric Acid/metabolism
9.
Proc Natl Acad Sci U S A ; 115(12): 3138-3143, 2018 03 20.
Article in English | MEDLINE | ID: mdl-29507217

ABSTRACT

Dietary guidelines for obesity typically focus on three food groups (carbohydrates, fat, and protein) and caloric restriction. Intake of noncaloric nutrients, such as salt, are rarely discussed. However, recently high salt intake has been reported to predict the development of obesity and insulin resistance. The mechanism for this effect is unknown. Here we show that high intake of salt activates the aldose reductase-fructokinase pathway in the liver and hypothalamus, leading to endogenous fructose production with the development of leptin resistance and hyperphagia that cause obesity, insulin resistance, and fatty liver. A high-salt diet was also found to predict the development of diabetes and nonalcoholic fatty liver disease in a healthy population. These studies provide insights into the pathogenesis of obesity and diabetes and raise the potential for reduction in salt intake as an additional interventional approach for reducing the risk for developing obesity and metabolic syndrome.


Subject(s)
Fructose/metabolism , Leptin/blood , Non-alcoholic Fatty Liver Disease/chemically induced , Obesity/chemically induced , Sodium Chloride, Dietary/adverse effects , Adult , Aged , Aged, 80 and over , Animals , Diabetes Mellitus/chemically induced , Fructokinases/genetics , Humans , Leptin/genetics , Metabolic Syndrome/chemically induced , Metabolic Syndrome/genetics , Mice, Inbred C57BL , Mice, Knockout , Middle Aged , Obesity/metabolism , Sucrose/adverse effects , Sucrose/analogs & derivatives , Transcription Factors/genetics , Transcription Factors/metabolism
10.
J Clin Invest ; 128(6): 2226-2238, 2018 06 01.
Article in English | MEDLINE | ID: mdl-29533924

ABSTRACT

Increasing evidence suggests a role for excessive intake of fructose in the Western diet as a contributor to the current epidemics of metabolic syndrome and obesity. Hereditary fructose intolerance (HFI) is a difficult and potentially lethal orphan disease associated with impaired fructose metabolism. In HFI, the deficiency of aldolase B results in the accumulation of intracellular phosphorylated fructose, leading to phosphate sequestration and depletion, increased adenosine triphosphate (ATP) turnover, and a plethora of conditions that lead to clinical manifestations such as fatty liver, hyperuricemia, Fanconi syndrome, and severe hypoglycemia. Unfortunately, there is currently no treatment for HFI, and avoiding sugar and fructose has become challenging in our society. In this report, through use of genetically modified mice and pharmacological inhibitors, we demonstrate that the absence or inhibition of ketohexokinase (Khk), an enzyme upstream of aldolase B, is sufficient to prevent hypoglycemia and liver and intestinal injury associated with HFI. Herein we provide evidence for the first time to our knowledge of a potential therapeutic approach for HFI. Mechanistically, our studies suggest that it is the inhibition of the Khk C isoform, not the A isoform, that protects animals from HFI.


Subject(s)
Fructokinases/antagonists & inhibitors , Fructokinases/metabolism , Fructose Intolerance/enzymology , Animals , Fructokinases/genetics , Fructose/genetics , Fructose/metabolism , Fructose Intolerance/drug therapy , Fructose Intolerance/genetics , Fructose-Bisphosphate Aldolase/antagonists & inhibitors , Fructose-Bisphosphate Aldolase/genetics , Fructose-Bisphosphate Aldolase/metabolism , Isoenzymes/antagonists & inhibitors , Isoenzymes/genetics , Mice , Mice, Knockout
11.
Dig Dis Sci ; 63(4): 1025-1034, 2018 Apr.
Article in English | MEDLINE | ID: mdl-29417326

ABSTRACT

INTRODUCTION: More than 50% of patients with esophageal cancer already have inoperable disease at the time of diagnosis. Controversy surrounds the outcomes of patients with advanced esophageal cancer who receive palliative care by either stent alone or stent plus an additional modality. We set out to perform a systematic review and meta-analysis of studies assessing the use of metal stents as treatment options for symptomatic improvement, survival, and adverse events. METHODS: We searched Embase, MEDLINE, and the Cochrane Central Register of Controlled Trials (CENTRAL) from inception until January 14, 2016, as well as other databases for randomized controlled trials (RCTs) comparing esophageal stent versus either esophageal stent plus brachytherapy, radiotherapy, or chemotherapy. For quality assurance purposes throughout the systematic review, multiple independent extractions were performed, and the process was executed as per the standards of the Cochrane collaboration. Primary outcomes were mean change in dysphagia score, overall survival, and quality of life. Secondary outcomes were adverse events including fever, severe pain, aspiration, fistula, stent migration, perforation, and restenosis. RESULTS: Eight RCTs enrolling 732 patients were included with three distinct comparisons: stents combination therapy vs stents alone (5 studies, n = 417), stents alone versus brachytherapy alone (2 studies, n = 274), and stents + brachytherapy vs brachytherapy alone (1 study, n = 41). Stents combination therapy was defined as stents plus radiotherapy, chemotherapy, or both. Mean change in dysphagia scores favored stents combination therapy versus stents alone, and the effect was seen in patients surviving longer than 3 months. Stents combination therapy was also associated with a more favorable overall survival. The risks of stent migration, aspiration pneumonia, and restenosis were lower in the stents combination group compared to stents alone, while the risks of severe pain, hemorrhage, and fistula formation were higher. Changes in dysphagia scores and overall survival did not differ significantly in the brachytherapy-alone vs stents-alone comparison. The risk of fistula formation and hemorrhage were higher in the stents-alone group, while the risk of perforation was lower, compared to brachytherapy alone. Quality of life improvements were seen in all treatment groups, but were not pooled in analysis due to differing methods of measurement. DISCUSSION: While there appears to be no immediate short-term differences, those who live longer than 3 months experience a significant improvement in dysphagia score using a stents combination therapy approach vs stents alone. The combination therapy significantly improves the overall survival as well as showed improvements in quality of life scores. Larger randomized controlled trials are needed to assess improvements in dysphagia score, overall survival, quality of life, and adverse events.


Subject(s)
Carcinoma/therapy , Esophageal Neoplasms/therapy , Self Expandable Metallic Stents , Carcinoma/mortality , Carcinoma/pathology , Combined Modality Therapy , Deglutition Disorders/etiology , Deglutition Disorders/therapy , Esophageal Neoplasms/mortality , Esophageal Neoplasms/pathology , Humans , Quality of Life
12.
Nat Commun ; 8: 14181, 2017 02 13.
Article in English | MEDLINE | ID: mdl-28194018

ABSTRACT

Acute kidney injury is associated with high mortality, especially in intensive care unit patients. The polyol pathway is a metabolic route able to convert glucose into fructose. Here we show the detrimental role of endogenous fructose production by the polyol pathway and its metabolism through fructokinase in the pathogenesis of ischaemic acute kidney injury (iAKI). Consistent with elevated urinary fructose in AKI patients, mice undergoing iAKI show significant polyol pathway activation in the kidney cortex characterized by high levels of aldose reductase, sorbitol and endogenous fructose. Wild type but not fructokinase knockout animals demonstrate severe kidney injury associated with ATP depletion, elevated uric acid, oxidative stress and inflammation. Interestingly, both the renal injury and dysfunction in wild-type mice undergoing iAKI is significantly ameliorated when exposed to luteolin, a recently discovered fructokinase inhibitor. This study demonstrates a role for fructokinase and endogenous fructose as mediators of acute renal disease.


Subject(s)
Acute Kidney Injury/prevention & control , Fructokinases/antagonists & inhibitors , Kidney/drug effects , Luteolin/pharmacology , Acute Kidney Injury/etiology , Acute Kidney Injury/metabolism , Aldehyde Reductase/metabolism , Animals , Cell Line , Fructokinases/genetics , Fructokinases/metabolism , Fructose/metabolism , Fructose/urine , Humans , Ischemia/complications , Kidney/metabolism , Kidney/pathology , Mice, Inbred C57BL , Mice, Knockout , Oxidative Stress/drug effects , Protective Agents/pharmacology , Uric Acid/metabolism
13.
PLoS One ; 10(4): e0123509, 2015.
Article in English | MEDLINE | ID: mdl-25856396

ABSTRACT

Hibernating animals develop fatty liver when active in summertime and undergo a switch to a fat oxidation state in the winter. We hypothesized that this switch might be determined by AMP and the dominance of opposing effects: metabolism through AMP deaminase (AMPD2) (summer) and activation of AMP-activated protein kinase (AMPK) (winter). Liver samples were obtained from 13-lined ground squirrels at different times during the year, including summer and multiples stages of winter hibernation, and fat synthesis and ß-fatty acid oxidation were evaluated. Changes in fat metabolism were correlated with changes in AMPD2 activity and intrahepatic uric acid (downstream product of AMPD2), as well as changes in AMPK and intrahepatic ß-hydroxybutyrate (a marker of fat oxidation). Hepatic fat accumulation occurred during the summer with relatively increased enzymes associated with fat synthesis (FAS, ACL and ACC) and decreased enoyl CoA hydratase (ECH1) and carnitine palmitoyltransferase 1A (CPT1A), rate limiting enzymes of fat oxidation. In summer, AMPD2 activity and intrahepatic uric acid levels were high and hepatic AMPK activity was low. In contrast, the active phosphorylated form of AMPK and ß-hydroxybutyrate both increased during winter hibernation. Therefore, changes in AMPD2 and AMPK activity were paralleled with changes in fat synthesis and fat oxidation rates during the summer-winter cycle. These data illuminate the opposing forces of metabolism of AMP by AMPD2 and its availability to activate AMPK as a switch that governs fat metabolism in the liver of hibernating ground squirrel.


Subject(s)
AMP Deaminase/metabolism , AMP-Activated Protein Kinases/metabolism , Adipose Tissue/metabolism , Hibernation/physiology , Animals , Fatty Acids/metabolism , Glucose/metabolism , Hibernation/genetics , Lipid Metabolism/genetics , Liver/metabolism , Liver/physiology , Oxidation-Reduction , Sciuridae/metabolism , Sciuridae/physiology , Seasons
14.
J Am Soc Nephrol ; 25(11): 2526-38, 2014 Nov.
Article in English | MEDLINE | ID: mdl-24876114

ABSTRACT

Diabetes is associated with activation of the polyol pathway, in which glucose is converted to sorbitol by aldose reductase. Previous studies focused on the role of sorbitol in mediating diabetic complications. However, in the proximal tubule, sorbitol can be converted to fructose, which is then metabolized largely by fructokinase, also known as ketohexokinase, leading to ATP depletion, proinflammatory cytokine expression, and oxidative stress. We and others recently identified a potential deleterious role of dietary fructose in the generation of tubulointerstitial injury and the acceleration of CKD. In this study, we investigated the potential role of endogenous fructose production, as opposed to dietary fructose, and its metabolism through fructokinase in the development of diabetic nephropathy. Wild-type mice with streptozotocin-induced diabetes developed proteinuria, reduced GFR, and renal glomerular and proximal tubular injury. Increased renal expression of aldose reductase; elevated levels of renal sorbitol, fructose, and uric acid; and low levels of ATP confirmed activation of the fructokinase pathway. Furthermore, renal expression of inflammatory cytokines with macrophage infiltration was prominent. In contrast, diabetic fructokinase-deficient mice demonstrated significantly less proteinuria, renal dysfunction, renal injury, and inflammation. These studies identify fructokinase as a novel mediator of diabetic nephropathy and document a novel role for endogenous fructose production, or fructoneogenesis, in driving renal disease.


Subject(s)
Diabetes Mellitus, Experimental/metabolism , Diabetic Nephropathies/metabolism , Fructokinases/metabolism , Fructose/biosynthesis , Fructose/metabolism , Kidney Tubules, Proximal/enzymology , Animals , Blood Glucose/metabolism , Body Weight , Cell Line, Transformed , Chemokines/metabolism , Cytokines/metabolism , Diabetes Mellitus, Experimental/pathology , Diabetic Nephropathies/pathology , Humans , Kidney Cortex/enzymology , Kidney Cortex/pathology , Kidney Glomerulus/cytology , Kidney Glomerulus/pathology , Kidney Tubules, Proximal/pathology , Macrophages/metabolism , Macrophages/pathology , Mice, Inbred C57BL , Mice, Knockout , Polymers/metabolism
15.
FASEB J ; 28(8): 3339-50, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24755741

ABSTRACT

Reduced AMP kinase (AMPK) activity has been shown to play a key deleterious role in increased hepatic gluconeogenesis in diabetes, but the mechanism whereby this occurs remains unclear. In this article, we document that another AMP-dependent enzyme, AMP deaminase (AMPD) is activated in the liver of diabetic mice, which parallels with a significant reduction in AMPK activity and a significant increase in intracellular glucose accumulation in human HepG2 cells. AMPD activation is induced by a reduction in intracellular phosphate levels, which is characteristic of insulin resistance and diabetic states. Increased gluconeogenesis is mediated by reduced TORC2 phosphorylation at Ser171 by AMPK in these cells, as well as by the up-regulation of the rate-limiting enzymes PEPCK and G6Pc. The mechanism whereby AMPD controls AMPK activation depends on the production of a specific AMP downstream metabolite through AMPD, uric acid. In this regard, humans have higher uric acid levels than most mammals due to a mutation in uricase, the enzyme involved in uric acid degradation in most mammals, that developed during a period of famine in Europe 1.5 × 10(7) yr ago. Here, working with resurrected ancestral uricases obtained from early hominids, we show that their expression on HepG2 cells is enough to blunt gluconeogenesis in parallel with an up-regulation of AMPK activity. These studies identify a key role AMPD and uric acid in mediating hepatic gluconeogenesis in the diabetic state, via a mechanism involving AMPK down-regulation and overexpression of PEPCK and G6Pc. The uricase mutation in the Miocene likely provided a survival advantage to help maintain glucose levels under conditions of near starvation, but today likely has a role in the pathogenesis of diabetes.


Subject(s)
AMP Deaminase/physiology , Gluconeogenesis/physiology , Liver/metabolism , Starvation/physiopathology , Uric Acid/metabolism , AMP Deaminase/antagonists & inhibitors , AMP Deaminase/genetics , AMP-Activated Protein Kinases/physiology , Animals , Diabetes Mellitus, Experimental/metabolism , Europe , Gene Expression Regulation, Enzymologic , Gluconeogenesis/drug effects , Glucose-6-Phosphatase/biosynthesis , Hep G2 Cells , History, Ancient , Hominidae/physiology , Humans , Insulin/metabolism , Insulin Resistance , Insulin Secretion , Liver/enzymology , Male , Mechanistic Target of Rapamycin Complex 2 , Mice , Mice, Inbred C57BL , Models, Biological , Multiprotein Complexes/physiology , Phosphates/metabolism , Phosphates/pharmacology , Phosphoenolpyruvate Carboxykinase (ATP)/biosynthesis , Recombinant Fusion Proteins/metabolism , Selection, Genetic , Specific Pathogen-Free Organisms , Starvation/history , TOR Serine-Threonine Kinases/physiology , Transduction, Genetic , Urate Oxidase/genetics , Urate Oxidase/history , Urate Oxidase/metabolism , Uric Acid/pharmacology
16.
Nat Commun ; 4: 2434, 2013.
Article in English | MEDLINE | ID: mdl-24022321

ABSTRACT

Carbohydrates with high glycaemic index are proposed to promote the development of obesity, insulin resistance and fatty liver, but the mechanism by which this occurs remains unknown. High serum glucose concentrations are known to induce the polyol pathway and increase fructose generation in the liver. Here we show that this hepatic, endogenously produced fructose causes systemic metabolic changes. We demonstrate that mice unable to metabolize fructose are protected from an increase in energy intake and body weight, visceral obesity, fatty liver, elevated insulin levels and hyperleptinaemia after exposure to 10% glucose for 14 weeks. In normal mice, glucose consumption is accompanied by aldose reductase and polyol pathway activation in steatotic areas. In this regard, we show that aldose reductase-deficient mice are protected against glucose-induced fatty liver. We conclude that endogenous fructose generation and metabolism in the liver represents an important mechanism by which glucose promotes the development of metabolic syndrome.


Subject(s)
Fructose/biosynthesis , Fructose/metabolism , Liver/metabolism , Liver/pathology , Metabolic Syndrome/metabolism , Metabolic Syndrome/pathology , Aldehyde Reductase/metabolism , Animals , Energy Metabolism , Fatty Liver/metabolism , Feeding Behavior , Fructokinases/deficiency , Fructokinases/metabolism , Glucose/metabolism , Hep G2 Cells , Humans , Liver/enzymology , Liver/physiopathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Polymers/metabolism
17.
PLoS One ; 7(10): e47948, 2012.
Article in English | MEDLINE | ID: mdl-23112875

ABSTRACT

Excessive dietary fructose intake may have an important role in the current epidemics of fatty liver, obesity and diabetes as its intake parallels the development of these syndromes and because it can induce features of metabolic syndrome. The effects of fructose to induce fatty liver, hypertriglyceridemia and insulin resistance, however, vary dramatically among individuals. The first step in fructose metabolism is mediated by fructokinase (KHK), which phosphorylates fructose to fructose-1-phosphate; intracellular uric acid is also generated as a consequence of the transient ATP depletion that occurs during this reaction. Here we show in human hepatocytes that uric acid up-regulates KHK expression thus leading to the amplification of the lipogenic effects of fructose. Inhibition of uric acid production markedly blocked fructose-induced triglyceride accumulation in hepatocytes in vitro and in vivo. The mechanism whereby uric acid stimulates KHK expression involves the activation of the transcription factor ChREBP, which, in turn, results in the transcriptional activation of KHK by binding to a specific sequence within its promoter. Since subjects sensitive to fructose often develop phenotypes associated with hyperuricemia, uric acid may be an underlying factor in sensitizing hepatocytes to fructose metabolism during the development of fatty liver.


Subject(s)
Fatty Liver/metabolism , Fructokinases/metabolism , Fructose/metabolism , Hepatocytes/metabolism , Uric Acid/metabolism , Allopurinol/pharmacology , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Enzyme Inhibitors/pharmacology , Fatty Liver/genetics , Fatty Liver/pathology , Fructokinases/genetics , Hep G2 Cells , Hepatocytes/drug effects , Hepatocytes/pathology , Humans , Liver/metabolism , Liver/pathology , Male , Rats , Rats, Sprague-Dawley , Transcriptional Activation , Up-Regulation/drug effects , Uric Acid/antagonists & inhibitors
18.
PLoS One ; 7(11): e48801, 2012.
Article in English | MEDLINE | ID: mdl-23152807

ABSTRACT

Fatty liver (hepatic steatosis) is associated with nucleotide turnover, loss of ATP and generation of adenosine monophosphate (AMP). It is well known that in fatty liver, activity of the AMP-activated kinase (AMPK) is reduced and that its stimulation can prevent hepatic steatosis by both enhancing fat oxidation and reducing lipogenesis. Here we show that another AMP dependent enzyme, AMPD2, has opposing effects on fatty acid oxidation when compared to AMPK. In human hepatocytres, AMPD2 activation -either by overexpression or by lowering intracellular phosphate levels with fructose- is associated with a significant reduction in AMPK activity. Likewise, silencing of AMPK spontaneously increases AMPD activity, demonstrating that these enzymes counter-regulate each other. Furthermore, we show that a downstream product of AMP metabolism through AMPD2, uric acid, can inhibit AMPK activity in human hepatocytes. Finally, we show that fructose-induced fat accumulation in hepatocytes is due to a dominant stimulation of AMPD2 despite stimulating AMPK. In this regard, AMPD2-deficient hepatocytes demonstrate a further activation of AMPK after fructose exposure in association with increased fatty acid oxidation, and conversely silencing AMPK enhances AMPD-dependent fat accumulation. In vivo, we show that sucrose fed rats also develop fatty liver that is blocked by metformin in association with both a reduction in AMPD activity and an increase in AMPK activity. In summary, AMPD and AMPK are both important in hepatic fat accumulation and counter-regulate each other. We present the novel finding that uric acid inhibits AMPK kinase activity in fructose-fed hepatocytes thus providing new insights into the pathogenesis of fatty liver.


Subject(s)
AMP Deaminase/metabolism , Adenylate Kinase/metabolism , Fatty Liver/metabolism , Adenylate Kinase/genetics , Animals , Carbon-Carbon Double Bond Isomerases/metabolism , Enzyme Activation/drug effects , Fats/metabolism , Fatty Liver/enzymology , Fructose/metabolism , Fructose/pharmacology , Gene Expression Regulation , Hep G2 Cells , Hepatocytes/drug effects , Hepatocytes/metabolism , Humans , Isoenzymes , Male , Metformin/pharmacology , Oxidation-Reduction , Rats , Uric Acid/metabolism
19.
J Biol Chem ; 287(48): 40732-44, 2012 Nov 23.
Article in English | MEDLINE | ID: mdl-23035112

ABSTRACT

BACKGROUND: Uric acid is an independent risk factor in fructose-induced fatty liver, but whether it is a marker or a cause remains unknown. RESULTS: Hepatocytes exposed to uric acid developed mitochondrial dysfunction and increased de novo lipogenesis, and its blockade prevented fructose-induced lipogenesis. CONCLUSION: Rather than a consequence, uric acid induces fatty liver SIGNIFICANCE: Hyperuricemic people are more prone to develop fructose-induced fatty liver. Metabolic syndrome represents a collection of abnormalities that includes fatty liver, and it currently affects one-third of the United States population and has become a major health concern worldwide. Fructose intake, primarily from added sugars in soft drinks, can induce fatty liver in animals and is epidemiologically associated with nonalcoholic fatty liver disease in humans. Fructose is considered lipogenic due to its ability to generate triglycerides as a direct consequence of the metabolism of the fructose molecule. Here, we show that fructose also stimulates triglyceride synthesis via a purine-degrading pathway that is triggered from the rapid phosphorylation of fructose by fructokinase. Generated AMP enters into the purine degradation pathway through the activation of AMP deaminase resulting in uric acid production and the generation of mitochondrial oxidants. Mitochondrial oxidative stress results in the inhibition of aconitase in the Krebs cycle, resulting in the accumulation of citrate and the stimulation of ATP citrate lyase and fatty-acid synthase leading to de novo lipogeneis. These studies provide new insights into the pathogenesis of hepatic fat accumulation under normal and diseased states.


Subject(s)
Fatty Liver/metabolism , Mitochondria/metabolism , Oxidative Stress , Uric Acid/metabolism , Fructose/metabolism , Hep G2 Cells , Humans , Lipogenesis , Triglycerides/metabolism , Uric Acid/adverse effects
20.
Metabolism ; 60(9): 1259-70, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21489572

ABSTRACT

Fructose induces metabolic syndrome in rats; but studies have been criticized for using high concentrations of fructose that are not physiologic, for using only pure fructose, and for not controlling for energy intake. We tested the hypothesis that a 40% sucrose diet (containing 20% fructose) might induce features of metabolic syndrome in male breeder rats independent of excess energy intake. Male Sprague-Dawley breeder rats were pair fed 40% sucrose or isocaloric starch diet for 4 months and evaluated for metabolic syndrome and diabetes. In vitro studies were performed in rat insulinoma cells (RIN-m5F) exposed to uric acid, and markers of inflammation were assessed. Rats fed a 40% sucrose diet developed accelerated features of metabolic syndrome with up-regulation of fructose-dependent transporter Glut5 and fructokinase. Fatty liver and low-grade pancreatic inflammation also occurred. Uric acid was found to stimulate inflammatory mediators and oxidative stress in islet cells in vitro. Sucrose, at concentrations ingested by a subset of Americans, can accelerate metabolic syndrome, fatty liver, and type 2 diabetes mellitus in male breeder rats; and the effects are independent of excess energy intake.


Subject(s)
Energy Intake , Fatty Liver/etiology , Pancreatitis/etiology , Sucrose/toxicity , Animals , Anion Transport Proteins/genetics , Cell Line, Tumor , Fructose/metabolism , Male , Metabolic Syndrome/etiology , Rats , Rats, Sprague-Dawley , Starch/administration & dosage , Sucrose/administration & dosage
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