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1.
J Endocrinol ; 243(2): 111-123, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31454789

RESUMO

Obesity and type 2 diabetes (T2D) are both complicated endocrine disorders resulting from an interaction between multiple predisposing genes and environmental triggers, while diet and exercise have key influence on metabolic disorders. Previous reports demonstrated that 2-aminoadipic acid (2-AAA), an intermediate metabolite of lysine metabolism, could modulate insulin secretion and predict T2D, suggesting the role of 2-AAA in glycolipid metabolism. Here, we showed that treatment of diet-induced obesity (DIO) mice with 2-AAA significantly reduced body weight, decreased fat accumulation and lowered fasting glucose. Furthermore, Dhtkd1-/- mice, in which the substrate of DHTKD1 2-AAA increased to a significant high level, were resistant to DIO and obesity-related insulin resistance. Further study showed that 2-AAA induced higher energy expenditure due to increased adipocyte thermogenesis via upregulating PGC1α and UCP1 mediated by ß3AR activation, and stimulated lipolysis depending on enhanced expression of hormone-sensitive lipase (HSL) through activating ß3AR signaling. Moreover, 2-AAA could alleviate the diabetic symptoms of db/db mice. Our data showed that 2-AAA played an important role in regulating glycolipid metabolism independent of diet and exercise, implying that improving the level of 2-AAA in vivo could be developed as a strategy in the treatment of obesity or diabetes.


Assuntos
Ácido 2-Aminoadípico/farmacologia , Peso Corporal/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Obesidade/metabolismo , Ácido 2-Aminoadípico/metabolismo , Células 3T3-L1 , Tecido Adiposo/citologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Glicemia/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Dieta Hiperlipídica/efeitos adversos , Cetona Oxirredutases/genética , Cetona Oxirredutases/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/etiologia , Obesidade/fisiopatologia , Substâncias Protetoras/farmacologia , Receptores Adrenérgicos beta 3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Termogênese/efeitos dos fármacos
2.
Biol Pharm Bull ; 42(7): 1140-1145, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31257290

RESUMO

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is characterized by disabling fatigue of at least 6 months, in addition to symptoms such as muscle pain and muscle weakness. There is no treatment provides long-term benefits to most patients. Recently, clinical research suggested the involvement of pyruvate dehydrogenase (PDH) in ME/CFS. PDH is a crucial enzyme in the mitochondria matrix that links glycolysis to the tricarboxylic acid cycle and oxidative phosphorylation. However, it is little known whether PDH could be a therapeutic target. The purpose of this study was to establish ME/CFS in mice and to investigate the involvement of PDH in ME/CFS. To induce the chronic fatigue in mice, a repeated forced swimming test was conducted. To evaluate fatigue, we measured immobility time in forced swimming test and starting time of grooming. An open field test was conducted on day 8. After 25 d of the forced swimming test, the mitochondrial fraction in gastrocnemius muscle was isolated and PDH activity was measured. Moreover, we evaluated the effect of PDH activation by administering sodium dichloroacetate (DCA). In ME/CFS mice group, the immobility time and starting time of grooming increased time-dependently. In addition, the moved distance was decreased in ME/CFS mice. PDH activity was decreased in the mitochondrial fraction of the gastrocnemius muscle of the forced swimming group. DCA treatment may be beneficial in preventing fatigue-like behavior in ME/CFS. These findings indicate that ME/CFS model was established in mice and that a decrease in mitochondrial PDH activity is involved with the symptom of ME/CFS.


Assuntos
Modelos Animais de Doenças , Síndrome de Fadiga Crônica/enzimologia , Síndrome de Fadiga Crônica/fisiopatologia , Cetona Oxirredutases/fisiologia , Natação , Animais , Comportamento Animal , Ácido Dicloroacético/farmacologia , Ácido Dicloroacético/uso terapêutico , Síndrome de Fadiga Crônica/tratamento farmacológico , Masculino , Camundongos Endogâmicos ICR , Mitocôndrias Musculares/fisiologia , Músculo Esquelético/fisiologia
3.
World J Gastroenterol ; 25(23): 2947-2960, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31249452

RESUMO

BACKGROUND: Changes in N-linked glycosylation have been observed in the circulation of individuals with hepatocellular carcinoma. In particular, an elevation in the level of core fucosylation has been observed. However, the mechanisms through which core fucose is increased are not well understood. We hypothesized that a review of the literature and related bioinformatic review regarding six genes known to be involved in the attachment of core fucosylation, the synthesis of the fucosylation substrate guanosine diphosphate (GDP)-fucose, or the transport of the substrate into the Golgi might offer mechanistic insight into the regulation of core fucose levels. AIM: To survey the literature to capture the involvement of genes regulating core N-linked fucosylation in hepatocellular carcinoma. METHODS: The PubMed biomedical literature database was searched for the association of hepatocellular carcinoma and each of the core fucose-related genes and their protein products. We also queried The Cancer Genome Atlas Liver hepatocellular carcinoma (LIHC) dataset for genetic, epigenetic and gene expression changes for the set of six genes using the tools at cBioportal. RESULTS: A total of 27 citations involving one or more of the core fucosylation-related genes (FPGT, FUK, FUT8, GMDS, SLC35C1, TSTA3) and hepatocellular carcinoma were identified. The same set of gene symbols was used to query the 371 patients with liver cancer in the LIHC dataset to identify the frequency of mRNA over or under expression, as well as non-synonymous mutations, copy number variation and methylation level. Although all six genes trended to more samples displaying over expression relative to under-expression, it was noted that a number of tumor samples had undergone amplification of the genes of the de novo synthesis pathway, GMDS (27 samples) and TSTA3 (78 samples). In contrast, the other four genes had undergone amplification in 2 or fewer samples. CONCLUSION: Amplification of genes involved in the de novo pathway for generation of GDP-fucose, GMDS and TSTA3, likely contributes to the elevated core fucose observed in hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Redes e Vias Metabólicas/genética , Carboidratos Epimerases/metabolismo , Carcinoma Hepatocelular/patologia , Variações do Número de Cópias de DNA , Metilação de DNA , Glicoproteínas/metabolismo , Glicosilação , Guanosina Difosfato Fucose/metabolismo , Humanos , Hidroliases/metabolismo , Cetona Oxirredutases/metabolismo , Neoplasias Hepáticas/patologia , Mutação
4.
PLoS One ; 14(6): e0218126, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31188872

RESUMO

Obesity is associated with increased incidence and worse prognosis of more than one dozen tumor types; however, the molecular mechanisms for this association remain under debate. We hypothesized that insulin, which is elevated in obesity-driven insulin resistance, would increase tumor glucose oxidation in obesity-associated tumors. To test this hypothesis, we applied and validated a stable isotope method to measure the ratio of pyruvate dehydrogenase flux to citrate synthase flux (VPDH/VCS, i.e. the percent of total mitochondrial oxidation fueled by glucose) in tumor cells. Using this method, we found that three tumor cell lines associated with obesity (colon cancer [MC38], breast cancer [4T1], and prostate cancer [TRAMP-C3] cells) increase VPDH/VCS in response to physiologic concentrations of insulin. In contrast, three tumor cell lines that are not associated with obesity (melanoma [YUMM1.7], B cell lymphoma [BCL1 clone 5B1b], and small cell lung cancer [NCI-H69] cells) exhibited no oxidative response to insulin. The observed increase in glucose oxidation in response to insulin correlated with a dose-dependent increase in cell division in obesity-associated tumor cell lines when grown in insulin, whereas no alteration in cell division was seen in tumor types not associated with obesity. These data reveal that a shift in substrate preference in the setting of physiologic insulin may comprise a metabolic signature of obesity-associated tumors that differs from that of those not associated with obesity.


Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Insulina/farmacologia , Mitocôndrias/efeitos dos fármacos , Alanina/metabolismo , Neoplasias da Mama/complicações , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Citrato (si)-Sintase/genética , Citrato (si)-Sintase/metabolismo , Neoplasias do Colo/complicações , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Feminino , Ácido Glutâmico/metabolismo , Humanos , Insulina/metabolismo , Marcação por Isótopo , Cetona Oxirredutases/genética , Cetona Oxirredutases/metabolismo , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Masculino , Melanoma/genética , Melanoma/metabolismo , Melanoma/patologia , Mitocôndrias/metabolismo , Obesidade/complicações , Obesidade/genética , Obesidade/metabolismo , Especificidade de Órgãos , Oxirredução , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/complicações , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de Sinais , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/metabolismo , Carcinoma de Pequenas Células do Pulmão/patologia
5.
ACS Synth Biol ; 8(5): 1153-1167, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30973696

RESUMO

O-acetylhomoserine (OAH) is a promising platform chemical for the production of l-methionine and other valuable compounds. However, the relative low titer and yield of OAH greatly limit its industrial production and cost-effective application. In this study, we successfully constructed an efficient OAH-producing strain with high titer and yield by combining protein and metabolic engineering strategies in E. coli. Initially, an OAH-producing strain was created by reconstruction of biosynthetic pathway and deletion of degradation and competitive pathways, which accumulated 1.68 g/L of OAH. Subsequently, several metabolic engineering strategies were implemented to improve the production of OAH. The pathway flux of OAH was enhanced by eliminating byproduct accumulation, increasing oxaloacetate supply and promoting the biosynthesis of precursor homoserine, resulting in a 1.79-fold increase in OAH production. Moreover, protein engineering was applied to improve the properties of the rate-limiting enzyme homoserine acetyltransferase (MetXlm) based on evolutionary conservation analysis and structure-guided engineering. The resulting triple F147L-M182I-M240A mutant of MetXlm exhibited a 12.15-fold increase in specific activity, and the optimized expression of the MetXlm mutant led to a 57.14% improvement in OAH production. Furthermore, the precursor acetyl-CoA supply and NADPH generation were also enhanced to facilitate the biosynthesis of OAH by promoting CoA biosynthesis, overexpressing heterogeneous acetyl-CoA synthetase (ACS), and introducing NADP-dependent pyruvate dehydrogenase (PDH). Finally, the engineered strain OAH-7 produced 62.7 g/L of OAH with yield and productivity values of 0.45 g/g glucose and 1.08 g/L/h, respectively, in a 7.5 L fed-batch fermenter, which was the highest OAH production ever reported.


Assuntos
Acetiltransferases/metabolismo , Coenzima A Ligases/metabolismo , Escherichia coli/metabolismo , Homosserina/metabolismo , Engenharia Metabólica/métodos , Acetilcoenzima A/metabolismo , Acetiltransferases/genética , Biomassa , Coenzima A Ligases/genética , Homosserina/química , Cetona Oxirredutases/genética , Cetona Oxirredutases/metabolismo , Cinética , Mutagênese Sítio-Dirigida , NADP/metabolismo
6.
Mol Med Rep ; 19(5): 4484-4490, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30896807

RESUMO

Charcot­Marie­Tooth (CMT) disease is a group of motor and sensory neuropathies with a high degree of pathological and genetic heterogenicity. The present study described 2 patients with CMT in a Chinese Han pedigree. The proband exhibited the classic manifestation of CMT with slowly progressing muscular atrophy and weakness. Electrophysiological examination highlighted axonal and demyelinating features. His mother did not have any symptoms, but did exhibit abnormal electrophysiological results. Next­generation sequencing technology was employed to screen mutations in the genes associated with inherited motor never diseases. A novel mutation, c.528_530delAGT, in the gap junction protein beta 1 (GJB1) gene for CMTX, and a rare variation, c.2369C>T, in the dehydrogenase E1 and transketolase domain containing 1 (DHTKD1) gene for CMT disease type 2Q (CMT2Q), were identified in the proband and his mother. The results were verified by Sanger sequencing. Although the in silico analysis predicted no change in the 3­dimensional structure, the clinical and electrophysiological presentation in the pedigree and the high evolutionary conservation of the affected amino acid supported the hypothesis that the c.528_530delAGT mutation in the GJB1 gene may be pathogenic in this pedigree. In silico analysis and high evolutionary conservation suggested the pathogenicity of the c.2369C>T mutation in the DHTKD1 gene; however, the clinical and electrophysiological performances of the proband and his mother did not conform to those of CMT2Q caused by the DHTKD1 gene. The present study provided additional information concerning the range of mutations of the GJB1 gene, which facilitated the understanding of the genotype­phenotype association of CMT.


Assuntos
Grupo com Ancestrais do Continente Asiático/genética , Doença de Charcot-Marie-Tooth/patologia , Conexinas/genética , Cetona Oxirredutases/genética , Adulto , Doença de Charcot-Marie-Tooth/genética , China , Conexinas/química , Análise Mutacional de DNA , Eletromiografia , Deleção de Genes , Humanos , Cetona Oxirredutases/química , Masculino , Pessoa de Meia-Idade , Linhagem , Polimorfismo de Nucleotídeo Único , Estrutura Terciária de Proteína
7.
Mol Genet Metab ; 126(4): 388-396, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30709776

RESUMO

Inbred mouse strains are a cornerstone of translational research but paradoxically many strains carry mild inborn errors of metabolism. For example, α-aminoadipic acidemia and branched-chain ketoacid dehydrogenase deficiency are known in C57BL/6J mice. Using RNA sequencing, we now reveal the causal variants in Dhtkd1 and Bckdhb, and the molecular mechanism underlying these metabolic defects. C57BL/6J mice have decreased Dhtkd1 mRNA expression due to a solitary long terminal repeat (LTR) in intron 4 of Dhtkd1. This LTR harbors an alternate splice donor site leading to a partial splicing defect and as a consequence decreased total and functional Dhtkd1 mRNA, decreased DHTKD1 protein and α-aminoadipic acidemia. Similarly, C57BL/6J mice have decreased Bckdhb mRNA expression due to an LTR retrotransposon in intron 1 of Bckdhb. This transposable element encodes an alternative exon 1 causing aberrant splicing, decreased total and functional Bckdhb mRNA and decreased BCKDHB protein. Using a targeted metabolomics screen, we also reveal elevated plasma C5-carnitine in 129 substrains. This biochemical phenotype resembles isovaleric acidemia and is caused by an exonic splice mutation in Ivd leading to partial skipping of exon 10 and IVD protein deficiency. In summary, this study identifies three causal variants underlying mild inborn errors of metabolism in commonly used inbred mouse strains.


Assuntos
Erros Inatos do Metabolismo/genética , Camundongos Endogâmicos/genética , Animais , Elementos de DNA Transponíveis/genética , Cetona Oxirredutases/genética , Masculino , Erros Inatos do Metabolismo/diagnóstico , Metabolômica , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Fenótipo , Análise de Sequência de RNA
8.
Biochem Genet ; 57(3): 443-454, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30644007

RESUMO

3-Methyl-1-butanol (3MB) is a promising biofuel that can be produced from 2-ketoisocaproate via the common L-leucine biosynthesis pathway. Corynebacterium glutamicum was chosen as a host bacterium because of its strong resistance to isobutanol. In the current study, several strategies were designed to overproduce 3MB in C. glutamicum through a non-fermentation pathway. The engineered C. glutamicum mutant was obtained by silencing the pyruvate dehydrogenase gene complex (aceE) and deleting the lactic dehydrogenase gene (ldh), followed by mutagenesis with diethyl sulfate (DES) and selection with Fmoc-3-4-thiazolyl-L-alanine (FTA). The mutant could produce 659 mg/L of 3MB after 12 h of incubation. To facilitate carbon flux to 3MB biosynthesis, the engineered recombinant was also constructed without branched-chain acid aminotransferase (ilvE) activity by deleting the ilvE gene. This recombinant could produce 697 mg/L of 3MB after 12 h of incubation.


Assuntos
Corynebacterium glutamicum/genética , Corynebacterium glutamicum/metabolismo , Engenharia Genética , Mutação , Pentanóis/metabolismo , Cromossomos Bacterianos , Genes Bacterianos , Cetona Oxirredutases/genética
9.
J Biol Chem ; 294(13): 5137-5145, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696768

RESUMO

NADPH: 2-ketopropyl-coenzyme M oxidoreductase/carboxylase (2-KPCC) is a bacterial disulfide oxidoreductase (DSOR) that, uniquely in this family, catalyzes CO2 fixation. 2-KPCC differs from other DSORs by having a phenylalanine that replaces a conserved histidine, which in typical DSORs is essential for stabilizing the reduced, reactive form of the active site. Here, using site-directed mutagenesis and stopped-flow kinetics, we examined the reactive form of 2-KPCC and its single turnover reactions with a suicide substrate and CO2 The reductive half-reaction of 2-KPCC was kinetically and spectroscopically similar to that of a typical DSOR, GSH reductase, in which the active-site histidine had been replaced with an alanine. However, the reduced, reactive form of 2-KPCC was distinct from those typical DSORs. In the absence of the histidine, the flavin and disulfide moieties were no longer coupled via a covalent or charge transfer interaction as in typical DSORs. Similar to thioredoxins, the pKa between 7.5 and 8.1 that controls reactivity appeared to be due to a single proton shared between the cysteines of the dithiol, which effectively stabilizes the attacking cysteine sulfide and renders it capable of breaking the strong C-S bond of the substrate. The lack of a histidine protected 2-KPCC's reactive intermediate from unwanted protonation; however, without its input as a catalytic acid-base, the oxidative half-reaction where carboxylation takes place was remarkably slow, limiting the overall reaction rate. We conclude that stringent regulation of protons in the DSOR active site supports C-S bond cleavage and selectivity for CO2 fixation.


Assuntos
Dióxido de Carbono/metabolismo , Cetona Oxirredutases/metabolismo , Xanthobacter/enzimologia , Domínio Catalítico , Cetona Oxirredutases/química , Cinética , Modelos Moleculares , NADP/metabolismo , Oxirredução , Especificidade por Substrato , Xanthobacter/química , Xanthobacter/metabolismo
10.
Mol Med Rep ; 19(2): 1065-1073, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30569177

RESUMO

Postmenopausal osteoporosis (PMOP) is a major public health concern worldwide. The present study aimed to provide evidence to assist in the development of specific novel biomarkers for PMOP. Differentially expressed genes (DEGs) were identified between PMOP and normal controls by integrated microarray analyses of the Gene Expression Omnibus (GEO) database, and the optimal diagnostic gene biomarkers for PMOP were identified with LASSO and Boruta algorithms. Classification models, including support vector machine (SVM), decision tree and random forests models, were established to test the diagnostic value of identified gene biomarkers for PMOP. Functional annotations and protein­protein interaction (PPI) network constructions were also conducted. Integrated microarray analyses (GSE56815, GSE13850 and GSE7429) of the GEO database were employed, and 1,320 DEGs were identified between PMOP and normal controls. An 11­gene combination was also identified as an optimal biomarker for PMOP by feature selection and classification methods using SVM, decision tree and random forest models. This combination was comprised of the following genes: Dehydrogenase E1 and transketolase domain containing 1 (DHTKD1), osteoclast stimulating factor 1 (OSTF1), G protein­coupled receptor 116 (GPR116), BCL2 interacting killer, adrenoceptor ß1 (ADRB1), neogenin 1 (NEO1), RB binding protein 4 (RBBP4), GPR87, cylicin 2, EF­hand calcium binding domain 1 and DEAH­box helicase 35. RBBP4 (degree=12) was revealed to be the hub gene of this PMOP­specific PPI network. Among these 11 genes, three genes (OSTF1, ADRB1 and NEO1) were speculated to serve roles in PMOP by regulating the balance between bone formation and bone resorption, while two genes (GPR87 and GPR116) may be involved in PMOP by regulating the nuclear factor­κB signaling pathway. Furthermore, DHTKD1 and RBBP4 may be involved in PMOP by regulating mitochondrial dysfunction and interacting with ESR1, respectively. In conclusion, the findings of the current study provided an insight for exploring the mechanism and developing novel biomarkers for PMOP. Further studies are required to test the diagnostic value for PMOP prior to use in a clinical setting.


Assuntos
Redes Reguladoras de Genes , Cetona Oxirredutases/genética , Osteoporose Pós-Menopausa/genética , Proteínas/genética , Receptores Adrenérgicos beta 1/genética , Receptores Acoplados a Proteínas-G/genética , Idoso , Biomarcadores/metabolismo , Estudos de Casos e Controles , Biologia Computacional/métodos , Bases de Dados Genéticas , Árvores de Decisões , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Cetona Oxirredutases/metabolismo , Análise em Microsséries , Pessoa de Meia-Idade , Anotação de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Osteoporose Pós-Menopausa/metabolismo , Osteoporose Pós-Menopausa/patologia , Mapeamento de Interação de Proteínas , Proteínas/metabolismo , Receptores Adrenérgicos beta 1/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Proteína 4 de Ligação ao Retinoblastoma/genética , Proteína 4 de Ligação ao Retinoblastoma/metabolismo , Transdução de Sinais , Máquina de Vetores de Suporte
11.
J Clin Endocrinol Metab ; 104(5): 1841-1854, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30544235

RESUMO

CONTEXT: Skeletal muscle molecular mechanisms underlying insulin resistance in women with polycystic ovary syndrome (PCOS) are poorly understood. OBJECTIVE: To provide insight into mechanisms regulating skeletal muscle insulin resistance in women who are lean with PCOS. PARTICIPANTS AND METHODS: A hyperinsulinemic-euglycemic clamp with skeletal muscle biopsies was performed. Thirteen women who are lean who have hyperandrogenism and PCOS and seven age- and body mass index-matched healthy control subjects were enrolled. Skeletal muscle protein expression and phosphorylation were analyzed by Western blotting and intramuscular lipid content was measured by thin-layer chromatography. RESULTS: Women with PCOS had 25% lower whole-body insulin sensitivity and 40% lower plasma adiponectin concentration than in control subjects. Intramuscular triacylglycerol, sn-1.3 diacylglycerol, and ceramide contents in skeletal muscle were higher (40%, 50%, and 300%, respectively) in women with PCOS than in control subjects. Activation of insulin signaling did not differ between groups. In women with PCOS, the insulin-stimulated glucose oxidation was reduced and insulin-stimulated dephosphorylation of pyruvate dehydrogenase (PDH) Ser293 was absent. AMP-activated protein kinase (AMPK) α2 protein expression and basal Thr172 phosphorylation were 45% and 50% lower in women with PCOS than in control subjects, respectively. CONCLUSIONS: Whole-body insulin resistance in women who are lean who have hyperandrogenism and PCOS was not related to changes in the proximal part of the insulin signaling cascade in skeletal muscle despite lipid accumulation. Rather, reduced insulin sensitivity was potentially related to plasma adiponectin levels playing a modulating role in human skeletal muscle via AMPK. Furthermore, abnormal PDH regulation may contribute to reduced whole-body metabolic flexibility and thereby insulin resistance.


Assuntos
Hiperandrogenismo/fisiopatologia , Resistência à Insulina , Insulina/metabolismo , Músculo Esquelético/fisiopatologia , Síndrome do Ovário Policístico/fisiopatologia , Magreza/fisiopatologia , Proteínas Quinases Ativadas por AMP/metabolismo , Adiponectina/metabolismo , Adulto , Biomarcadores/metabolismo , Índice de Massa Corporal , Estudos de Casos e Controles , Feminino , Seguimentos , Técnica Clamp de Glucose , Humanos , Cetona Oxirredutases/metabolismo , Masculino , Fosforilação , Prognóstico
12.
Chem Commun (Camb) ; 54(79): 11208-11211, 2018 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-30230493

RESUMO

In the past decade it has become clear that many microbes harbor enzymes that employ an unusual flavin cofactor, the F420 deazaflavin cofactor. Herein we show that F420-dependent reductases (FDRs) can successfully perform enantio-, regio- and chemoselective ene-reductions. For the first time, we have demonstrated that F420H2-driven reductases can be used as biocatalysts for the reduction of α,ß-unsaturated ketones and aldehydes with good conversions (>99%) and excellent regioselectivities and enantiomeric excesses (>99% ee). Noteworthily, FDRs typically display an opposite enantioselectivity when compared to the well established FMN-dependent Old Yellow Enzymes (OYEs).


Assuntos
Aldeído Oxirredutases/química , Proteínas de Bactérias/química , Cetona Oxirredutases/química , Riboflavina/análogos & derivados , Aldeídos/química , Catálise , Cetonas/química , Mycobacterium/enzimologia , Oxirredução , Rhodococcus/enzimologia , Riboflavina/química , Estereoisomerismo
13.
J Biol Chem ; 293(45): 17402-17417, 2018 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-30232153

RESUMO

In vertebrate cells, mitochondrial Ca2+ uptake by the mitochondrial calcium uniporter (MCU) leads to Ca2+-mediated stimulation of an intramitochondrial pyruvate dehydrogenase phosphatase (PDP). This enzyme dephosphorylates serine residues in the E1α subunit of pyruvate dehydrogenase (PDH), thereby activating PDH and resulting in increased ATP production. Although a phosphorylation/dephosphorylation cycle for the E1α subunit of PDH from nonvertebrate organisms has been described, the Ca2+-mediated PDP activation has not been studied. In this work, we investigated the Ca2+ sensitivity of two recombinant PDPs from the protozoan human parasites Trypanosoma cruzi (TcPDP) and T. brucei (TbPDP) and generated a TcPDP-KO cell line to establish TcPDP's role in cell bioenergetics and survival. Moreover, the mitochondrial localization of the TcPDP was studied by CRISPR/Cas9-mediated endogenous tagging. Our results indicate that TcPDP and TbPDP both are Ca2+-sensitive phosphatases. Of note, TcPDP-KO epimastigotes exhibited increased levels of phosphorylated TcPDH, slower growth and lower oxygen consumption rates than control cells, an increased AMP/ATP ratio and autophagy under starvation conditions, and reduced differentiation into infective metacyclic forms. Furthermore, TcPDP-KO trypomastigotes were impaired in infecting cultured host cells. We conclude that TcPDP is a Ca2+-stimulated mitochondrial phosphatase that dephosphorylates TcPDH and is required for normal growth, differentiation, infectivity, and energy metabolism in T. cruzi Our results support the view that one of the main roles of the MCU is linked to the regulation of intramitochondrial dehydrogenases.


Assuntos
Doença de Chagas/enzimologia , Metabolismo Energético , Cetona Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/enzimologia , Linhagem Celular , Doença de Chagas/genética , Doença de Chagas/patologia , Técnicas de Silenciamento de Genes , Humanos , Cetona Oxirredutases/genética , Fosforilação/genética , Proteínas de Protozoários/genética , Trypanosoma cruzi/genética
14.
Sci Rep ; 8(1): 11485, 2018 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-30065264

RESUMO

In a GM-CSF driven myeloid cell deficient mouse model (Csf2-/-) that has preserved insulin sensitivity despite increased adiposity, we used unbiased three-dimensional integration of proteome profiles, metabolic profiles, and gene regulatory networks to understand adipose tissue proteome-wide changes and their metabolic implications. Multi-dimensional liquid chromatography mass spectrometry and extended multiplex mass labeling was used to analyze proteomes of epididymal adipose tissues isolated from Csf2+/+ and Csf2-/- mice that were fed low fat, high fat, or high fat plus cholesterol diets for 8 weeks. The metabolic health (as measured by body weight, adiposity, plasma fasting glucose, insulin, triglycerides, phospholipids, total cholesterol levels, and glucose and insulin tolerance tests) deteriorated with diet for both genotypes, while mice lacking Csf2 were protected from insulin resistance. Regardless of diet, 30 mostly mitochondrial, branch chain amino acids (BCAA), and lysine metabolism proteins were altered between Csf2-/- and Csf2+/+ mice (FDR < 0.05). Lack of GM-CSF driven myeloid cells lead to reduced adipose tissue 2-oxoglutarate dehydrogenase complex (DHTKD1) levels and subsequent increase in plasma 2-aminoadipate (2-AA) levels, both of which are reported to correlate with insulin resistance. Tissue DHTKD1 levels were >4-fold upregulated and plasma 2-AA levels were >2 fold reduced in Csf2-/- mice (p < 0.05). GM-CSF driven myeloid cells link peripheral insulin sensitivity to adiposity via lysine metabolism involving DHTKD1/2-AA axis in a diet independent manner.


Assuntos
Tecido Adiposo/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Resistência à Insulina/fisiologia , Células Mieloides/metabolismo , Ganho de Peso/fisiologia , Adiposidade/fisiologia , Animais , Peso Corporal/fisiologia , Colesterol/metabolismo , Dieta Hiperlipídica , Gorduras na Dieta , Metabolismo Energético/fisiologia , Glucose/metabolismo , Insulina/metabolismo , Cetona Oxirredutases/metabolismo , Metabolismo dos Lipídeos/fisiologia , Masculino , Camundongos , Triglicerídeos/metabolismo
15.
Proc Natl Acad Sci U S A ; 115(30): E7063-E7072, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29987032

RESUMO

The lack of attachment of lipoic acid to its cognate enzyme proteins results in devastating human metabolic disorders. These mitochondrial disorders are evident soon after birth and generally result in early death. The mutations causing specific defects in lipoyl assembly map in three genes, LIAS, LIPT1, and LIPT2 Although physiological roles have been proposed for the encoded proteins, only the LIPT1 protein had been studied at the enzyme level. LIPT1 was reported to catalyze only the second partial reaction of the classical lipoate ligase mechanism. We report that the physiologically relevant LIPT1 enzyme activity is transfer of lipoyl moieties from the H protein of the glycine cleavage system to the E2 subunits of the 2-oxoacid dehydrogenases required for respiration (e.g., pyruvate dehydrogenase) and amino acid degradation. We also report that LIPT2 encodes an octanoyl transferase that initiates lipoyl group assembly. The human pathway is now biochemically defined.


Assuntos
Aciltransferases/metabolismo , Ácido Tióctico/metabolismo , Aciltransferases/genética , Biocatálise , Humanos , Cetona Oxirredutases/metabolismo , Ácido Tióctico/genética
16.
Technol Cancer Res Treat ; 17: 1533033818781405, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29950151

RESUMO

Esophageal squamous cell carcinoma is the sixth most lethal cancer worldwide and the fourth most lethal cancer in China. Tissue-specific transplantation antigen P35B codifies the enzyme GDP-d-mannose-4,6-dehydratase, which participates in the biosynthesis of GDP-l-fucose. GDP-l-fucose is an important substrate involved in the biosynthesis of many glycoproteins. Cancer cells are often accompanied by the changes in glycoprotein structure, which affects the adhesion, invasion, and metastasis of cells. It is not clear whether tissue-specific transplantation antigen P35B has any effect on the development of esophageal squamous cell carcinoma. We used an immunohistochemical method to assess the expression of tissue-specific transplantation antigen P35B in 104 esophageal squamous cell carcinoma samples. The results showed tissue-specific transplantation antigen P35B expression was associated with some clinical features in patients, such as age ( P = .017), clinical stage ( P = .010), and lymph node metastasis ( P = .043). Kaplan-Meier analysis and log-rank test showed that patients with esophageal squamous cell carcinoma having high tissue-specific transplantation antigen P35B expression had a worse prognosis compared to the patients with low expression ( P = .048). Multivariate Cox proportional hazards regression model showed that high expression of tissue-specific transplantation antigen P35B could predict poor prognosis for patients with esophageal squamous cell carcinoma independently. In conclusion, abnormal fucosylation might participate in the progress of esophageal squamous cell carcinoma and tissue-specific transplantation antigen P35B may serve as a novel biomarker for prognosis of patients with esophageal squamous cell carcinoma.


Assuntos
Biomarcadores Tumorais/análise , Carboidratos Epimerases/biossíntese , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/patologia , Cetona Oxirredutases/biossíntese , Adulto , Idoso , Área Sob a Curva , Neoplasias Esofágicas/mortalidade , Carcinoma de Células Escamosas do Esôfago/mortalidade , Feminino , Humanos , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Prognóstico , Modelos de Riscos Proporcionais , Curva ROC , Sensibilidade e Especificidade
17.
Epilepsy Res ; 145: 77-81, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29913407

RESUMO

A recent report has found that glucose oxidation and the activity of pyruvate dehydrogenase (PDH) are reduced in the chronic stage of the pilocarpine mouse epilepsy model. This is likely caused by increased phosphorylation by PDH kinase of the E1α subunit of PDH, downregulating its activity. Inhibition of this phosphorylation has not yet been explored as a possible approach to treat epilepsy. Chronic dichloroacetate (DCA, 50 and 100 mg/kg/day) treatment was tested in acute seizure and the chronic pilocarpine models. We also determined the effects on phosphorylation state, activity and protein levels of PDH in the chronic stage of the pilocarpine model. DCA treatment did not increase latencies to seizures in the acute flurothyl seizure test and was slightly proconvulsant in the 6 Hz test. The latencies to seizures in a second-hit flurothyl test were decreased in SE vs. No SE mice in the chronic stage, but were not restored by DCA. In mice that had experienced pilocarpine-induced SE and were in the chronic "epileptic" stage of the model, PDH activity was reduced by 65% compared to "healthy" No SE mice. This was partially alleviated with DCA treatment. Also, PDH protein levels were decreased by 37% and phosphorylation at Ser300 of PDH was increased by 52% in SE mice, but were not significantly changed with DCA. Moreover DCA treatment decreased the amounts of total PDH by 23% in No SE mice, which may explain the proconvulsant effects in the 6 Hz test. The reduction in PDH protein levels during the chronic epileptic stage suggests increased degradation of the protein, which may contribute to the deficient glucose oxidation found in epilepsy. Taken together, DCA did not have any anti-convulsant effects in the tested models. Future studies utilising other PDH kinase inhibitors are required to determine whether this treatment approach is viable.


Assuntos
Anticonvulsivantes/uso terapêutico , Ácido Dicloroacético/uso terapêutico , Epilepsia/tratamento farmacológico , Análise de Variância , Animais , Convulsivantes/toxicidade , Modelos Animais de Doenças , Estimulação Elétrica , Epilepsia/induzido quimicamente , Flurotila/toxicidade , Cetona Oxirredutases/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Pilocarpina/toxicidade
18.
Clin Cancer Res ; 24(17): 4215-4224, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-29798908

RESUMO

Purpose: Virotherapies are maturing in the clinical setting. Adenoviruses (Ad) are excellent vectors for the manipulability and tolerance of transgenes. Poor tumor selectivity, off-target sequestration, and immune inactivation hamper clinical efficacy. We sought to completely redesign Ad5 into a refined, tumor-selective virotherapy targeted to αvß6 integrin, which is expressed in a range of aggressively transformed epithelial cancers but nondetectable in healthy tissues.Experimental Design: Ad5NULL-A20 harbors mutations in each major capsid protein to preclude uptake via all native pathways. Tumor-tropism via αvß6 targeting was achieved by genetic insertion of A20 peptide (NAVPNLRGDLQVLAQKVART) within the fiber knob protein. The vector's selectivity in vitro and in vivo was assessed.Results: The tropism-ablating triple mutation completely blocked all native cell entry pathways of Ad5NULL-A20 via coxsackie and adenovirus receptor (CAR), αvß3/5 integrins, and coagulation factor 10 (FX). Ad5NULL-A20 efficiently and selectively transduced αvß6+ cell lines and primary clinical ascites-derived EOC ex vivo, including in the presence of preexisting anti-Ad5 immunity. In vivo biodistribution of Ad5NULL-A20 following systemic delivery in non-tumor-bearing mice was significantly reduced in all off-target organs, including a remarkable 107-fold reduced genome accumulation in the liver compared with Ad5. Tumor uptake, transgene expression, and efficacy were confirmed in a peritoneal SKOV3 xenograft model of human EOC, where oncolytic Ad5NULL-A20-treated animals demonstrated significantly improved survival compared with those treated with oncolytic Ad5.Conclusions: Oncolytic Ad5NULL-A20 virotherapies represent an excellent vector for local and systemic targeting of αvß6-overexpressing cancers and exciting platforms for tumor-selective overexpression of therapeutic anticancer modalities, including immune checkpoint inhibitors. Clin Cancer Res; 24(17); 4215-24. ©2018 AACR.


Assuntos
Antígenos de Neoplasias/genética , Carcinoma Epitelial do Ovário/genética , Carcinoma Epitelial do Ovário/terapia , Integrinas/genética , Terapia Viral Oncolítica , Adenoviridae/genética , Animais , Carboidratos Epimerases/genética , Carcinoma Epitelial do Ovário/patologia , Carcinoma Epitelial do Ovário/virologia , Linhagem Celular Tumoral , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus/genética , Feminino , Genes cdc/genética , Vetores Genéticos/genética , Vetores Genéticos/farmacologia , Humanos , Cetona Oxirredutases/genética , Camundongos , Vírus Oncolíticos/genética , Distribuição Tecidual , Transdução Genética , Tropismo/genética
19.
JCI Insight ; 3(8)2018 04 19.
Artigo em Inglês | MEDLINE | ID: mdl-29669943

RESUMO

Eosinophilic esophagitis (EoE) is an allergic inflammatory esophageal disorder with a complex underlying genetic etiology often associated with other comorbidities. Using whole-exome sequencing (WES) of 63 patients with EoE and 60 unaffected family members and family-based trio analysis, we sought to uncover rare coding variants. WES analysis identified 5 rare, damaging variants in dehydrogenase E1 and transketolase domain-containing 1 (DHTKD1). Rare variant burden analysis revealed an overabundance of putative, potentially damaging DHTKD1 mutations in EoE (P = 0.01). Interestingly, we also identified 7 variants in the DHTKD1 homolog oxoglutarate dehydrogenase-like (OGDHL). Using shRNA-transduced esophageal epithelial cells and/or patient fibroblasts, we further showed that disruption of normal DHTKD1 or OGDHL expression blunts mitochondrial function. Finally, we demonstrated that the loss of DHTKD1 expression increased ROS production and induced the expression of viperin, a gene previously shown to be involved in production of Th2 cytokines in T cells. Viperin had increased expression in esophageal biopsies of EoE patients compared with control individuals and was upregulated by IL-13 in esophageal epithelial cells. These data identify a series of rare genetic variants implicating DHTKD1 and OGDHL in the genetic etiology of EoE and underscore a potential pathogenic role for mitochondrial dysfunction in EoE.


Assuntos
Esofagite Eosinofílica/congênito , Esofagite Eosinofílica/imunologia , Complexo Cetoglutarato Desidrogenase/metabolismo , Mitocôndrias/metabolismo , Oxirredutases/genética , Adulto , Criança , Citocinas/metabolismo , Esofagite Eosinofílica/etiologia , Esofagite Eosinofílica/patologia , Células Epiteliais/metabolismo , Feminino , Fibroblastos/metabolismo , Humanos , Interleucina-13/metabolismo , Cetona Oxirredutases , Masculino , Mitocôndrias/fisiologia , Mutação , Oxirredutases/metabolismo , Proteínas , RNA Interferente Pequeno/genética , Linfócitos T/metabolismo , Regulação para Cima/genética , Sequenciamento Completo do Exoma/métodos
20.
Cell Physiol Biochem ; 46(2): 727-739, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29621749

RESUMO

BACKGROUND/AIMS: Recent interest in the use of cannabinoids as therapeutic agents has revealed the involvement of the endogenous cannabinoid system (ECS) in the regulation of the cardiovascular system in hypertension. Abnormalities in glucose metabolism and insulin action are commonly detected in hypertensive animals. Thus, potential antihypertensive drugs should be investigated with respect to modulation of glucose homeostasis. Therefore, the aim of the present study was to evaluate the effects of the ECS activation after chronic fatty acid amide hydrolase inhibitor (URB597) administration on plasma glucose and insulin concentrations as well as parameters of myocardial glucose metabolism in the deoxycorticosterone acetate (DOCA)-salt hypertensive rats, an animal model of secondary hypertension. METHODS: Hypertension was induced by DOCA (25mg/kg) injections and addition of 1% NaCl in the drinking water for six weeks. Chronic activation of the ECS was performed by URB597 (1mg/kg) injections for two weeks. We examined fasting plasma levels of insulin (ELISA), glucose and intramyocardial glycogen (colorimetric method). Expressions of glucose transporters (GLUT1, 4) and selected proteins engaged in GLUT translocation as well as glucose metabolism were determined using Western blotting. RESULTS: Hypertension induced hypoinsulinemia with concomitant lack of significant changes in glycemia, reduced intramyocardial glycogen content and increased pyruvate dehydrogenase (PDH) expression in the cardiac muscle. Importantly, chronic URB597 administration in the hypertensive rats increased insulin concentration, elevated plasmalemmal GLUT1 and GLUT4 expression and concomitantly improved myocardial glycogen storage. CONCLUSION: Chronic administration of fatty acid amide hydrolase (FAAH) inhibitor has potential protective properties on myocardial glucose metabolism in hypertension.


Assuntos
Benzamidas/uso terapêutico , Carbamatos/uso terapêutico , Glucose/metabolismo , Hipertensão/patologia , Miocárdio/metabolismo , Animais , Benzamidas/farmacologia , Glicemia/análise , Carbamatos/farmacologia , Acetato de Desoxicorticosterona/toxicidade , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glicogênio/análise , Hipertensão/induzido quimicamente , Hipertensão/tratamento farmacológico , Insulina/sangue , Cetona Oxirredutases/metabolismo , Masculino , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/patologia , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Wistar
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