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1.
PLoS One ; 15(4): e0231506, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298312

RESUMO

Hypercholesterolaemia is considered an important cause of atherosclerotic cardiovascular disease. In a previous investigation, we demonstrated that cultured hepatoma cells treated with hypercholesterolaemic sera compared with cells treated with normocholesterolaemic sera show overexpression of mRNAs related to mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS2). In the present work, using an NMR metabolomic analysis, we demonstrate that the hypercholesterolaemic blood sera previously used to treat cultured hepatoma cells are characterized by a metabolomic profile that is significantly different from the normocholesterolaemic sera. Acetate, acetone, 2-hydroxybutyrate, cysteine, valine, and glutamine are the metabolites distinguishing the two groups. Abnormalities in the concentrations of these metabolites reflect alterations in energy-related pathways, such as pantothenate and CoA biosynthesis, pyruvate, glycolysis/gluconeogenesis, the citrate cycle, and ketone bodies. Regarding ketone bodies, the pathway is regulated by HMGCS2; therefore, serum samples previously found to be able to increase HMGCS2 mRNA levels in cultured cells also contain higher amounts of the metabolites of its encoded enzyme protein product.


Assuntos
Hipercolesterolemia/sangue , Metabolômica , Adulto , Carcinoma Hepatocelular/metabolismo , Estudos de Casos e Controles , Linhagem Celular Tumoral , Expressão Gênica/fisiologia , Humanos , Hidroximetilglutaril-CoA Sintase/metabolismo , Hipercolesterolemia/metabolismo , Neoplasias Hepáticas/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Redes e Vias Metabólicas , Pessoa de Meia-Idade
2.
PLoS One ; 15(1): e0227411, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31910233

RESUMO

Disorders/differences of sex development (DSD) cause profound psychological and reproductive consequences for the affected individuals, however, most are still unexplained at the molecular level. Here, we present a novel gene, 3-hydroxy-3-methylglutaryl coenzyme A synthase 2 (HMGCS2), encoding a metabolic enzyme in the liver important for energy production from fatty acids, that shows an unusual expression pattern in developing fetal mouse gonads. Shortly after gonadal sex determination it is up-regulated in the developing testes following a very similar spatial and temporal pattern as the male-determining gene Sry in Sertoli cells before switching to ovarian enriched expression. To test if Hmgcs2 is important for gonad development in mammals, we pursued two lines of investigations. Firstly, we generated Hmgcs2-null mice using CRISPR/Cas9 and found that these mice had gonads that developed normally even on a sensitized background. Secondly, we screened 46,XY DSD patients with gonadal dysgenesis and identified two unrelated patients with a deletion and a deleterious missense variant in HMGCS2 respectively. However, both variants were heterozygous, suggesting that HMGCS2 might not be the causative gene. Analysis of a larger number of patients in the future might shed more light into the possible association of HMGCS2 with human gonadal development.


Assuntos
Transtornos do Desenvolvimento Sexual/genética , Disgenesia Gonadal/genética , Gônadas/crescimento & desenvolvimento , Hidroximetilglutaril-CoA Sintase/genética , Adolescente , Animais , Transtornos do Desenvolvimento Sexual/patologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Disgenesia Gonadal/patologia , Gônadas/patologia , Heterozigoto , Humanos , Masculino , Camundongos , Mutação de Sentido Incorreto/genética , Ovário/crescimento & desenvolvimento , Ovário/patologia , Células de Sertoli/metabolismo , Proteína da Região Y Determinante do Sexo/genética , Testículo/crescimento & desenvolvimento , Testículo/patologia
3.
Chin J Nat Med ; 17(8): 575-584, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31472894

RESUMO

3-Hydroxy-3-methylglutaryl-CoA synthase (HMGS) is the first committed enzyme in the MVA pathway and involved in the biosynthesis of terpenes in Tripterygium wilfordii. The full-length cDNA and a 515 bp RNAi target fragment of TwHMGS were ligated into the pH7WG2D and pK7GWIWG2D vectors to respectively overexpress and silence, TwHMGS was overexpressed and silenced in T. wilfordii suspension cells using biolistic-gun mediated transformation, which resulted in 2-fold increase and a drop to 70% in the expression level compared to cells with empty vector controls. During TwHMGS overexpression, the expression of TwHMGR, TwDXR and TwTPS7v2 was significantly upregulated to the control. In the RNAi group, the expression of TwHMGR, TwDXS, TwDXR and TwMCT visibly displayed downregulation to the control. The cells with TwHMGS overexpressed produced twice higher than the control value. These results proved that differential expression of TwHMGS determined the production of triptolide in T. wilfordii and laterally caused different trends of relative gene expression in the terpene biosynthetic pathway. Finally, the substrate acetyl-CoA was docked into the active site of TwHMGS, suggesting the key residues including His247, Lys256 and Arg296 undergo electrostatic or H-bond interactions with acetyl-CoA.


Assuntos
Diterpenos/metabolismo , Regulação da Expressão Gênica de Plantas , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Fenantrenos/metabolismo , Proteínas de Plantas/metabolismo , Tripterygium/metabolismo , Acetilcoenzima A/metabolismo , Sequência de Aminoácidos , Vias Biossintéticas , Domínio Catalítico , Compostos de Epóxi/metabolismo , Hidroximetilglutaril-CoA Sintase/química , Modelos Moleculares , Proteínas de Plantas/química , Proteínas de Plantas/genética , Interferência de RNA , Terpenos/metabolismo , Tripterygium/enzimologia , Tripterygium/genética , Triterpenos/metabolismo
4.
Cell ; 178(5): 1115-1131.e15, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31442404

RESUMO

Little is known about how metabolites couple tissue-specific stem cell function with physiology. Here we show that, in the mammalian small intestine, the expression of Hmgcs2 (3-hydroxy-3-methylglutaryl-CoA synthetase 2), the gene encoding the rate-limiting enzyme in the production of ketone bodies, including beta-hydroxybutyrate (ßOHB), distinguishes self-renewing Lgr5+ stem cells (ISCs) from differentiated cell types. Hmgcs2 loss depletes ßOHB levels in Lgr5+ ISCs and skews their differentiation toward secretory cell fates, which can be rescued by exogenous ßOHB and class I histone deacetylase (HDAC) inhibitor treatment. Mechanistically, ßOHB acts by inhibiting HDACs to reinforce Notch signaling, instructing ISC self-renewal and lineage decisions. Notably, although a high-fat ketogenic diet elevates ISC function and post-injury regeneration through ßOHB-mediated Notch signaling, a glucose-supplemented diet has the opposite effects. These findings reveal how control of ßOHB-activated signaling in ISCs by diet helps to fine-tune stem cell adaptation in homeostasis and injury.


Assuntos
Dieta Hiperlipídica , Corpos Cetônicos/metabolismo , Células-Tronco/metabolismo , Ácido 3-Hidroxibutírico/sangue , Ácido 3-Hidroxibutírico/farmacologia , Idoso de 80 Anos ou mais , Animais , Diferenciação Celular/efeitos dos fármacos , Autorrenovação Celular , Feminino , Inibidores de Histona Desacetilases/farmacologia , Humanos , Hidroximetilglutaril-CoA Sintase/deficiência , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Intestinos/citologia , Intestinos/patologia , Masculino , Camundongos , Camundongos Knockout , Receptores Acoplados a Proteínas-G/metabolismo , Receptores Notch/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/citologia , Adulto Jovem
5.
Environ Microbiol ; 21(11): 4270-4282, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31435987

RESUMO

Acyltransferase (AT)-less type I polyketide synthases (PKSs) produce complex natural products due to the presence of many unique tailoring enzymes. The 3-hydroxy-3-methylglutaryl coenzyme A synthases (HCSs) are responsible for ß-alkylation of the growing polyketide intermediates in AT-less type I PKSs. In this study, we discovered a large group of HCSs, closely associated with the characterized and orphan AT-less type I PKSs through in silico genome mining, sequence and genome neighbourhood network analyses. Using HCS-based probes, the survey of 1207 in-house strains and 18 soil samples from different geographic locations revealed the vast diversity of HCS-containing AT-less type I PKSs. The presence of HCSs in many AT-less type I PKSs suggests their co-evolutionary relationship. This study provides a new probe to study the abundance and diversity of AT-less type I PKSs in the environment and microbial strain collections. Our study should inspire future efforts to discover new polyketide natural products from AT-less type I PKSs.


Assuntos
Hidroximetilglutaril-CoA Sintase/análise , Policetídeo Sintases/análise , Solo/química , Bactérias/genética , Produtos Biológicos/análise , Genoma Bacteriano , Microbiologia do Solo
6.
Biotechnol Lett ; 41(8-9): 1077-1091, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31236789

RESUMO

OBJECTIVES: 3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase (HMGS) is an important enzyme in mevalonate (MVA) pathway of isoprenoid biosynthesis, which regulates the rubber biosynthetic pathway in rubber tree (Hevea brasiliensis) in coordination with HMG-CoA reductase (HMGR). However, little information is available about the regulation of HMGS gene expression. To understand the mechanism controlling the HbHMGS1 gene expression, we characterized the HbHMGS1 promoter sequence in transgenic plants with the ß-glucuronidase (GUS) reporter gene. RESULTS: GUS activity analysis of the transgenic plants showed that the HbHMGS1 promoter is active in all organs of the transgenic Arabidopsis plants during various developmental stages (from 6 to 45-day-old). Deletion of different portions of the upstream HbHMGS1 promoter identified sequences responsible for either positive or negative regulation of the GUS expression. Particularly, the - 454 bp HbHMGS1 promoter resulted in a 2.19-fold increase in promoter activity compared with the CaMV 35S promoter, suggesting that the - 454 bp HbHMGS1 promoter is a super-strong near-constitutive promoter. In addition, a number of promoter regions important for the responsiveness to ethylene, methyl jasmonate (MeJA) and gibberellic acid (GA) were identified. CONCLUSION: The - 454 bp HbHMGS1 promoter has great application potential in plant transformation studies as an alternative to the CaMV 35S promoter. The HbHMGS1 promoter may play important roles in regulating ethylene-, MeJA- and GA-mediated gene expression. The functional complexity of cis-elements revealed by this study remains to be elucidated.


Assuntos
Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Hevea/enzimologia , Hidroximetilglutaril-CoA Sintase/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Sequências Reguladoras de Ácido Nucleico , Análise Mutacional de DNA , Perfilação da Expressão Gênica , Hevea/genética , Hidroximetilglutaril-CoA Sintase/biossíntese , Proteínas de Plantas/biossíntese , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Deleção de Sequência
7.
Pathol Res Pract ; 215(8): 152464, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31176575

RESUMO

BACKGROUND: Accumulating studies reported that 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) may function as either an oncogene or a tumor suppressor in various human cancers. However, its involvement in prostate cancer (PCa) remains unknown. Therefore, the aim of this study was to investigate the clinical significance of HMGCS2 expression and its functions in PCa. METHODS: Expression levels of HMGCS2 mRNA and protein were detected by quantitative Polymerase Chain Reaction (qPCR), Western blot and immunohistochemistry, respectively. Associations of HMGCS2 expression with various clinicopathological features and patients' prognosis of PCa were statistically evaluated. Roles of HMGCS2 dysregulation in cell proliferation, invasion and migration of PCa cell lines were also determined. RESULTS: HMGCS2 protein expression was significantly reduced in PCa tissues compared to adjacent benign prostate tissues at protein levels (P < 0.05). Clinically, low HMGCS2 mRNA expression was dramatically associated with high Gleason score (GS) and pathological grade, as well as the presence of distant metastasis of PCa patients. In addition, PCa patients with low HMGCS2 mRNA expression more frequently had shorter disease-free survival and biochemical recurrence-free survival (all P < 0.05). HMGCS2 expression was identified as an independent factor to predict both disease-free and biochemical recurrence-free survivals of PCa patients. Moreover, loss-of-function experiments demonstrated that HMGCS2 knockdown-expression promotes cell proliferation, colony formation, invasion and migration of PCa cells in vitro and lower the apoptotic rate of PCa cells in vitro. CONCLUSIONS: Our data indicate that HMGCS2 may be capable of predicting the risk of biochemical recurrence in PCa patients after radical prostatectomy and functions as a tumor suppressor in PCa cancer, implying its related pathway potential as a drug candidate in anti-PCa therapy.


Assuntos
Regulação Neoplásica da Expressão Gênica/genética , Hidroximetilglutaril-CoA Sintase/genética , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Idoso , Biomarcadores Tumorais/metabolismo , Progressão da Doença , Intervalo Livre de Doença , Genes Supressores de Tumor/fisiologia , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores/métodos , Próstata/patologia , Neoplasias da Próstata/diagnóstico
8.
Cells ; 8(5)2019 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-31058869

RESUMO

Polyploidization is important for the speciation and subsequent evolution of many plant species. Analyses of the duplicated genes produced via polyploidization events may clarify the origin and evolution of gene families. During terpene biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A synthase (HMGS) functions as a key enzyme in the mevalonate pathway. In this study, we first identified a total of 53 HMGS genes in 23 land plant species, while no HMGS genes were detected in three green algae species. The phylogenetic analysis suggested that plant HMGS genes may have originated from a common ancestral gene before clustering in different branches during the divergence of plant lineages. Then, we detected six HMGS genes in the allotetraploid cotton species (Gossypium hirsutum), which was twice that of the two diploid cotton species (Gossypium raimondii and Gossypium arboreum). The comparison of gene structures and phylogenetic analysis of HMGS genes revealed conserved evolution during polyploidization in Gossypium. Moreover, the expression patterns indicated that six GhHMGS genes were expressed in all tested tissues, with most genes considerably expressed in the roots, and they were responsive to various phytohormone treatments and abiotic stresses. The sequence and expression divergence of duplicated genes in G. hirsutum implied the sub-functionalization of GhHMGS1A and GhHMGS1D as well as GhHMGS3A and GhHMGS3D, whereas it implied the pseudogenization of GhHMGS2A and GhHMGS2D. Collectively, our study unraveled the evolutionary history of HMGS genes in green plants and from diploid to allotetraploid in cotton and illustrated the different evolutionary fates of duplicated HMGS genes resulting from polyploidization.


Assuntos
Sequência Conservada/genética , Evolução Molecular , Genes de Plantas , Variação Genética , Gossypium/enzimologia , Gossypium/genética , Hidroximetilglutaril-CoA Sintase/genética , Poliploidia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Filogenia , Reguladores de Crescimento de Planta/farmacologia , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética
9.
Acta Histochem ; 121(5): 584-594, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31079945

RESUMO

The Cholesterol-synthesizing proteins (HMGCS1 and HMGCS2) are mitochondrial enzymes that believed to catalyze the first reaction of ketogenesis, the process by which energy is provided from fats in the absence of carbohydrates. Typically, astrocytes developed from its progenitor cells in the embryonic optic nerve and enriched with HMGCS1 and 2. However, the detailed histomorphology of camel HMGCS1 and 2 remains to be clearly defined. Here, we investigated the changes that associate with astrocytes differentiation within the developing camel optic nerve. Firstly, we isolated cDNAs encoding HMGCS1 and 2 from the optic nerve. Then, we found that HMGCS1 shared high similarity to human, while HMGCS2 showed a lower similarity and was more diverse. Immunohistochemical studies revealed that distinct correlation of astrocytes differentiation with HMGCS1 and 2 expressions in the developing camel optic nerve. Both encoded proteins were localized throughout the cytoplasm, as well as the nuclei of the astrocytes. In addition, semi-quantitative PCR analysis and western analysis confirmed that both HMGCS1 and 2 were highly expressed in camel optic nerve as well as other tissue, but they were lower in both skeletal and heart muscles. Moreover, various stains such as Sudan black and florescence filipin stains were used to visualize the free cholesterol in the astrocytes, indicating the enzymatic activity of HMGCS1 and 2. Together, our study reported the first comprehensive investigation of the molecular cloning and cellular expression of HMGCS1 and 2 in the optic nerve of dromedary camel.


Assuntos
Camelus/embriologia , Colesterol/biossíntese , Hidroximetilglutaril-CoA Sintase/metabolismo , Nervo Óptico/embriologia , Sequência de Aminoácidos , Animais , Camelus/anatomia & histologia , Camelus/genética , Camelus/metabolismo , Clonagem Molecular , Desenvolvimento Embrionário , Hidroximetilglutaril-CoA Sintase/química , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/imunologia , Nervo Óptico/anatomia & histologia , Nervo Óptico/metabolismo , Alinhamento de Sequência , Transcriptoma
10.
Blood Adv ; 3(8): 1244-1254, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30987969

RESUMO

Erythropoiesis is the process by which new red blood cells (RBCs) are formed and defects in this process can lead to anemia or thalassemia. The GATA1 transcription factor is an established mediator of RBC development. However, the upstream mechanisms that regulate the expression of GATA1 are not completely characterized. Cholesterol is 1 potential upstream mediator of GATA1 expression because previously published studies suggest that defects in cholesterol synthesis disrupt RBC differentiation. Here we characterize RBC development in a zebrafish harboring a single missense mutation in the hmgcs1 gene (Vu57 allele). hmgcs1 encodes the first enzyme in the cholesterol synthesis pathway and mutation of hmgcs1 inhibits cholesterol synthesis. We analyzed the number of RBCs in hmgcs1 mutants and their wild-type siblings. Mutation of hmgcs1 resulted in a decrease in the number of mature RBCs, which coincides with reduced gata1a expression. We combined these experiments with pharmacological inhibition and confirmed that cholesterol and isoprenoid synthesis are essential for RBC differentiation, but that gata1a expression is isoprenoid dependent. Collectively, our results reveal 2 novel upstream regulators of RBC development and suggest that appropriate cholesterol homeostasis is critical for primitive erythropoiesis.


Assuntos
Diferenciação Celular/genética , Eritrócitos/enzimologia , Eritropoese/genética , Hidroximetilglutaril-CoA Sintase , Mutação de Sentido Incorreto , Terpenos/metabolismo , Peixe-Zebra , Substituição de Aminoácidos , Animais , Colesterol/biossíntese , Colesterol/genética , Fator de Transcrição GATA1/biossíntese , Fator de Transcrição GATA1/genética , Regulação da Expressão Gênica , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/biossíntese , Proteínas de Peixe-Zebra/genética
11.
In Vitro Cell Dev Biol Anim ; 55(5): 368-375, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31025252

RESUMO

Ketosis is a condition where ketone bodies are produced as an alternative energy source, due to insufficient glucose for energy production so that the body switches from carbohydrate metabolism to mostly fat metabolism. In this study, we examined the anti-ketosis effects of silibinin, a major active component of silymarin. We induced ketosis in FL83B mouse hepatocytes in vitro by culturing in low glucose media and compared results to hepatocytes maintained in high-glucose conditions. We quantified ß-hydroxybutyrate (BHB) levels with a colorimetric assay. In low-glucose conditions, silibinin reduced the amount of BHB produced, compared to high-glucose conditions; thus, silibinin exhibited an anti-ketotic effect. Ketone body formation during beta oxidation is mediated by 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2). The nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) regulates the transcription of HMGCS2, and plays a vital role in BHB levels. We showed that silibinin inhibited the expression of HMGCS2 and NF-kB at transcriptional and translational levels. Silibinin also inhibited the nuclear translocation of NF-kB and its DNA binding activity. To elucidate the relationship between HMGCS2 and NF-kB, we tested inhibited and over-expressed NF-kB. We found that NF-kB acted as a positive regulator for HMGCS2 under ketosis treatment conditions.


Assuntos
Hidroximetilglutaril-CoA Sintase/genética , Cetose/tratamento farmacológico , NF-kappa B/genética , Silibina/farmacologia , Ácido 3-Hidroxibutírico/metabolismo , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Colorimetria , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Hepatócitos/efeitos dos fármacos , Humanos , Corpos Cetônicos/biossíntese , Corpos Cetônicos/metabolismo , Cetose/genética , Cetose/metabolismo , Cetose/patologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Transdução de Sinais/efeitos dos fármacos , Silimarina/química , Silimarina/farmacologia
12.
Mar Drugs ; 17(2)2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30791608

RESUMO

Five new (fusarisolins A⁻E, 1 to 5) and three known (6 to 8) polyketides were isolated from the marine-derived fungus Fusarium solani H918, along with six known phenolics (9 to 14). Their structures were established by comprehensive spectroscopic data analyses, methoxyphenylacetic acid (MPA) method, chemical conversion, and by comparison with data reported in the literature. Compounds 1 and 2 are the first two naturally occurring 21 carbons polyketides featuring a rare ß- and γ-lactone unit, respectively. All isolates (1 to 14) were evaluated for their inhibitory effects against tea pathogenic fungus Pestalotiopsis theae and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase gene expression. Compound 8 showed potent antifungal activity with an ED50 value of 55 µM, while 1, 8, 13, and 14 significantly inhibited HMG-CoA synthase gene expression.


Assuntos
Fusarium/química , Policetídeos/química , Policetídeos/farmacologia , Antifúngicos/farmacologia , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Produtos Biológicos/farmacologia , Fusarium/metabolismo , Hidroximetilglutaril-CoA Sintase/metabolismo , Conformação Molecular , Policetídeos/isolamento & purificação , Policetídeos/metabolismo
13.
Am J Physiol Gastrointest Liver Physiol ; 316(5): G623-G631, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30767679

RESUMO

Ketosis is a metabolic adaptation to fasting, nonalcoholic fatty liver disease (NAFLD), and prolonged exercise. ß-OH butyrate acts as a transcriptional regulator and at G protein-coupled receptors to modulate cellular signaling pathways in a hormone-like manner. While physiological ketosis is often adaptive, chronic hyperketonemia may contribute to the metabolic dysfunction of NAFLD. To understand how ß-OH butyrate signaling affects hepatic metabolism, we compared the hepatic fasting response in control and 3-hydroxy-3-methylglutaryl-CoA synthase II (HMGCS2) knockdown mice that are unable to elevate ß-OH butyrate production. To establish that rescue of ketone metabolic/endocrine signaling would restore the normal hepatic fasting response, we gave intraperitoneal injections of ß-OH butyrate (5.7 mmol/kg) to HMGCS2 knockdown and control mice every 2 h for the final 9 h of a 16-h fast. In hypoketonemic, HMGCS2 knockdown mice, fasting more robustly increased mRNA expression of uncoupling protein 2 (UCP2), a protein critical for supporting fatty acid oxidation and ketogenesis. In turn, exogenous ß-OH butyrate administration to HMGCS2 knockdown mice decreased fasting UCP2 mRNA expression to that observed in control mice. Also supporting feedback at the transcriptional level, ß-OH butyrate lowered the fasting-induced expression of HMGCS2 mRNA in control mice. ß-OH butyrate also regulates the glycemic response to fasting. The fast-induced fall in serum glucose was absent in HMGCS2 knockdown mice but was restored by ß-OH butyrate administration. These data propose that endogenous ß-OH butyrate signaling transcriptionally regulates hepatic fatty acid oxidation and ketogenesis, while modulating glucose tolerance. NEW & NOTEWORTHY Ketogenesis regulates whole body glucose metabolism and ß-OH butyrate produced by the liver feeds back to inhibit hepatic ß-oxidation and ketogenesis during fasting.


Assuntos
Jejum/fisiologia , Ácidos Graxos/metabolismo , Corpos Cetônicos/biossíntese , Cetonas/metabolismo , Fígado/metabolismo , Adaptação Fisiológica , Animais , Glicemia/metabolismo , Butiratos/metabolismo , Regulação da Expressão Gênica , Hidroximetilglutaril-CoA Sintase/metabolismo , Cetose/metabolismo , Camundongos , Camundongos Knockout , Oxirredução , Transdução de Sinais , Proteína Desacopladora 2/metabolismo
14.
Theranostics ; 9(3): 900-919, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30809316

RESUMO

Circular RNAs (circRNAs), a novel class of endogenous RNAs, have been recently shown to participate in cellular development and several pathophysiological processes. The identification of dysregulated circRNAs and their function in cancer have attracted considerable attention. Nevertheless, the expression profile and role of circRNAs in human hepatoblastoma (HB) remain to be studied. In this report, we analyzed the expression prolife of circRNAs in HB tissues and identified circHMGCS1 (3-hydroxy-3-methylglutaryl-CoA synthase 1; hsa_circ_0072391) as a remarkably upregulated circRNA. Methods: The expression prolife of circRNAs in HB tissues were investigated through circRNA sequencing analyses. ISH and qRT-PCR assays were performed to measure the expression level of circHMGCS1. The effect of knocking down circHMGCS1 in HB cells in vitro and in vivo were evaluated by colony formation assay, flow cytometry, xenograft tumors assay and untargeted metabolomics assay. MRE analysis and dual luciferase assay were performed to explore the underlying molecular mechanisms. Results: HB patients with high circHMGCS1 expression have shorted overall survival. Knockdown of circHMGCS1 inhibits HB cells proliferation and induces apoptosis. CircHMGCS1 regulates IGF2 and IGF1R expression via sponging miR-503-5p, and affects the downstream PI3K-Akt signaling pathway to regulate HB cell proliferation and glutaminolysis. Conclusions: The circHMGCS1/miR-503-5p/IGF-PI3K-Akt axis regulates the proliferation, apoptosis and glutaminolysis of HB cells, implying that circHMGCS1 is a promising therapeutic target and prognostic marker for HB patients.


Assuntos
Proliferação de Células , Glutamina/metabolismo , Hepatoblastoma/patologia , Hidroximetilglutaril-CoA Sintase/genética , RNA Circular/metabolismo , Transdução de Sinais , Somatomedinas/metabolismo , Linhagem Celular Tumoral , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hepatoblastoma/mortalidade , Hepatócitos/patologia , Humanos , RNA Circular/genética , Análise de Sobrevida
15.
Lipids Health Dis ; 18(1): 11, 2019 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-30621686

RESUMO

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is closely linked to obesity, type 2 diabetes and other metabolic disorders worldwide. Crocin is a carotenoid compound possessing various pharmacological activities. In the present study, we aimed to investigate the effect on fatty liver under diabetic and obese condition and to examine the possible role of AMP-activated protein kinase (AMPK) signaling. METHODS: db/db mice were administrated with crocin and injected with LV-shAMPK or its negative control lentivirus. Metabolic dysfunction, lipogenesis and fatty acid-oxidation in liver were evaluated. RESULTS: In db/db mice, we found that oral administration of crocin significantly upregulated the phosphorylation of AMPK and downregulated the phosphorylation of mTOR in liver. Crocin reduced liver weight, serum levels of alanine aminotransferase, alanine aminotransferase, and liver triglyceride content, and attenuated morphological injury of liver in db/db mice. Crocin inhibited the mRNA expression of lipogenesis-associated genes, including sterol regulatory element binding protein-1c, peroxisome proliferator-activated receptor γ, fatty acid synthase, stearoyl-CoA desaturase 1, and diacylglycerol acyltransferase 1, and increased the mRNA expression of genes involved in the regulation of ß-oxidation of fatty acids, including PPARα, acyl-CoA oxidase 1, carnitine palmitoyltransferase 1, and 3-hydroxy-3-methylglutaryl-CoA synthase 2. Moreover, treatment of crocin resulted in a amelioration of general metabolic disorder, as evidenced by decreased fasting blood glucose, reduced serum levels of insulin, triglyceride, total cholesterol, and non-esterified fatty acid, and improved glucose intolerance. Crocin-induced protective effects against fatty liver and metabolic disorder were significantly blocked by lentivirus-mediated downregulation of AMPK. CONCLUSIONS: The results suggest that crocin can inhibit lipogenesis and promote ß-oxidation of fatty acids through activation of AMPK, leading to improvement of fatty liver and metabolic dysfunction. Therefore, crocin may be a potential promising option for the clinical treatment for NAFLD and associated metabolic diseases.


Assuntos
Proteínas Quinases Ativadas por AMP/genética , Fármacos Antiobesidade/farmacologia , Carotenoides/farmacologia , Diabetes Mellitus Tipo 2/tratamento farmacológico , Hipoglicemiantes/farmacologia , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Proteínas Quinases Ativadas por AMP/metabolismo , Acil-CoA Oxidase/genética , Acil-CoA Oxidase/metabolismo , Alanina Transaminase/sangue , Alanina Transaminase/genética , Animais , Aspartato Aminotransferases/sangue , Aspartato Aminotransferases/genética , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Diacilglicerol O-Aciltransferase/antagonistas & inibidores , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Modelos Animais de Doenças , Ácido Graxo Sintases/antagonistas & inibidores , Ácido Graxo Sintases/genética , Ácido Graxo Sintases/metabolismo , Regulação da Expressão Gênica , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Lipogênese/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , PPAR alfa/agonistas , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gama/antagonistas & inibidores , PPAR gama/genética , PPAR gama/metabolismo , Transdução de Sinais , Estearoil-CoA Dessaturase/antagonistas & inibidores , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/antagonistas & inibidores , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Resultado do Tratamento , Triglicerídeos/sangue
16.
Endocrine ; 63(3): 615-631, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30460485

RESUMO

PURPOSE: Diabetic mellitus-induced erectile dysfunction (DMED) represents a significant complication associated with diabetes mellitus (DM) that greatly affects human life quality. Various reports have highlighted the involvement of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) in the regulation of mitochondrial fatty acid oxidation, which has also been linked with DM. Through bioinformatics analysis, HMGCS2 was determined to be a novel target among DM patients suffering from erectile dysfunction (ED), and enriched in the Ras/ERK/PPAR signaling axis. Owing to the fact that the key mechanism HMGCS2 involved in DM remains largely unknown, we set out to investigate the role of the Ras/MAPK/PPARγ signaling axis and HMGCS2 in the corpus cavernosal endothelial cells (CCECs) of rats with DMED. METHODS: Firstly, bioinformatics analysis was used to screen out differentially expressed genes in DMED. Then, to investigate the influence of the Ras/MAPK/PPARγ signaling axis and HMGCS2 on DMED, a rat model of DMED was established and injected with Simvastatin and si-Hmgcs2. The individual expression patterns of Ras, MAPK, PPARγ and HMGCS2 were determined by RT-qPCR, immunohistochemistry and western blot analysis methods. Afterwards, to investigate the mechanism of Ras/MAPK/PPARγ signaling axis and HMGCS2, CCECs were isolated from DMED rats and transfected with agonists and inhibitors of the Ras/MAPK/PPARγ signaling axis and siRNA of HMGCS2, with their respective functions in apoptosis and impairment of CCECs evaluated using TUNEL staining and flow cytometry. RESULTS: Microarray analysis and KEGG pathway enrichment analysis revealed that Ras/ERK/PPAR signaling axis mediated HMGCS2 in DMED. Among the DMED rats, the Ras/MAPK/PPAR signaling axis was also activated while the expression of HMGCS2 was upregulated. The activation of Ras was determined to be capable of upregulating ERK expression which resulted in the inhibition of the transcription of PPARγ and subsequent upregulation of HMGCS2 expression. The inhibited activation of the Ras/ERK/PPAR signaling axis and silencing HMGCS2 were observed to provide an alleviatory effect on the injury of DMED while acting to inhibit the apoptosis of CCECs. CONCLUSION: Collectively, the key findings suggested that suppression of the Ras/MAPK/PPARγ signaling axis could downregulate expression of HMGCS2, so as to alleviate DMED. This study defines the potential treatment for DMED through inhibition of the Ras/MAPK/PPARγ signaling axis and silencing HMGCS2.


Assuntos
Diabetes Mellitus Experimental/complicações , Disfunção Erétil/tratamento farmacológico , Hidroximetilglutaril-CoA Sintase/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases , Sinvastatina/uso terapêutico , Animais , Ensaios de Seleção de Medicamentos Antitumorais , Células Endoteliais/metabolismo , Disfunção Erétil/enzimologia , Disfunção Erétil/etiologia , Hidroximetilglutaril-CoA Sintase/metabolismo , Masculino , PPAR gama/metabolismo , Pênis/metabolismo , RNA Interferente Pequeno/uso terapêutico , Ratos Sprague-Dawley , Sinvastatina/farmacologia , Proteínas ras/metabolismo
17.
Neuropharmacology ; 148: 377-393, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-28987936

RESUMO

The brain is a high energy-consuming organ that typically utilizes glucose as the main energy source for cerebral activity. When glucose becomes scarce under conditions of stress, ketone bodies, such as ß-hydroxybutyrate, acetoacetate and acetone, become extremely important. Alterations in brain energy metabolism have been observed in psychostimulant abusers; however, the mode of brain metabolic programming in cocaine dependence remains largely unknown. Here, we profiled the metabolites and metabolic enzymes from brain nucleus accumbens (NAc) of mice exposed to cocaine. We found that cocaine modified energy metabolism and markedly activated ketogenesis pathway in the NAc. The expression of HMG-CoA synthase 2 (HMGCS2), a critical rate-limiting ketogenesis enzyme, was markedly up-regulated. After switching metabolic pathways from ketogenesis to glycolysis through activation of glucokinase, cocaine-evoked metabolic reprogramming regained homeostasis, and the cocaine effect was attenuated. Importantly, both the pharmacological and genetic inhibition of HMGCS2 significantly suppressed cocaine-induced ketogenesis and behavior. In conclusion, cocaine induces a remarkable energy reprogramming in the NAc, which is characterized by HMGCS2-driven ketogenesis. Such effect may facilitate adaptations to cocaine-induced energy stress in the brain. Our findings establish an important link between drug-induced energy reprogramming and cocaine effect, and may have implication in the treatment of cocaine addiction.


Assuntos
Cocaína/farmacologia , Metabolismo Energético/efeitos dos fármacos , Hidroximetilglutaril-CoA Sintase/biossíntese , Corpos Cetônicos/metabolismo , Animais , Homeostase , Hidroximetilglutaril-CoA Sintase/antagonistas & inibidores , Masculino , Camundongos , Núcleo Accumbens/metabolismo , Regulação para Cima/efeitos dos fármacos
18.
Int J Mol Sci ; 19(12)2018 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-30567295

RESUMO

BACKGROUND: Lipid expression is increased in the atrial myocytes of mitral regurgitation (MR) patients. This study aimed to investigate key regulatory genes and mechanisms of atrial lipotoxic myopathy in MR. METHODS: The HL-1 atrial myocytes were subjected to uniaxial cyclic stretching for eight hours. Fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism were analyzed by PCR assay (168 genes). RESULTS: The stretched myocytes had significantly larger cell size and higher lipid expression than non-stretched myocytes (all p < 0.001). Fatty acid metabolism, lipoprotein signaling, and cholesterol metabolism in the myocytes were analyzed by PCR assay (168 genes). In comparison with their counterparts in non-stretched myocytes, seven genes in stretched monocytes (Idi1, Olr1, Nr1h4, Fabp2, Prkag3, Slc27a5, Fabp6) revealed differential upregulation with an altered fold change >1.5. Nine genes in stretched monocytes (Apoa4, Hmgcs2, Apol8, Srebf1, Acsm4, Fabp1, Acox2, Acsl6, Gk) revealed differential downregulation with an altered fold change <0.67. Canonical pathway analysis, using Ingenuity Pathway Analysis software, revealed that the only genes in the "superpathway of cholesterol biosynthesis" were Idi1 (upregulated) and Hmgcs2 (downregulated). The fraction of stretched myocytes expressing Nile red was significantly decreased by RNA interference of Idi1 (p < 0.05) and was significantly decreased by plasmid transfection of Hmgcs2 (p = 0.004). CONCLUSIONS: The Idi1 and Hmgcs2 genes have regulatory roles in atrial lipotoxic myopathy associated with atrial enlargement.


Assuntos
Isomerases de Ligação Dupla Carbono-Carbono/genética , Hidroximetilglutaril-CoA Sintase/genética , Metabolismo dos Lipídeos/genética , Insuficiência da Valva Mitral/genética , Linhagem Celular , Colesterol/genética , Colesterol/metabolismo , Citometria de Fluxo , Regulação da Expressão Gênica/genética , Átrios do Coração/metabolismo , Átrios do Coração/fisiopatologia , Hemiterpenos , Humanos , Lipídeos/genética , Lipoproteínas/genética , Lipoproteínas/metabolismo , Insuficiência da Valva Mitral/metabolismo , Insuficiência da Valva Mitral/fisiopatologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Transdução de Sinais
19.
Breast Cancer Res ; 20(1): 150, 2018 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-30537987

RESUMO

BACKGROUND: Prevention of triple-negative breast cancer (TNBC) is hampered by lack of knowledge about the drivers of tumorigenesis. METHODS: To identify molecular markers and their downstream networks that can potentially be targeted for TNBC prevention, we analyzed small RNA and RNA sequencing of a cell line model that represent early stages of TNBC development. We have identified direct gene targets of isomiRNA-140-3p and by using cell-based and in vivo model systems we have demonstrated the utility of targeting downstream pathways for prevention of TNBC. RESULTS: These analyses showed that 5'isomiRNA of miR-140-3p (miR-140-3p-1) and its novel direct gene targets, HMG-CoA reductase (HMGCR) and HMG-CoA synthase 1(HMGCS1), key enzymes in the cholesterol biosynthesis pathway, were deregulated in the normal-to-preneoplastic transition. Upregulation in the cholesterol pathway creates metabolic vulnerability that can be targeted. Consistent with this hypothesis, we found direct targeting of miR-140-3p-1 and its downstream pathway by fluvastatin to inhibit growth of these preneoplastic MCF10.AT1 cells. However, although, fluvastatin inhibited the growth of MCF10.AT1-derived xenografts, histological progression remained unchanged. The cholesterol pathway is highly regulated, and HMGCR enzymatic activity inhibition is known to trigger a feedback response leading to restoration of the pathway. Indeed, we found fluvastatin-induced HMGCR transcript levels to be directly correlated with the degree of histological progression of lesions, indicating that the extent of cholesterol pathway suppression directly correlates with abrogation of the tumorigenic process. To block the HMGCR feedback response to statins, we treated resistant preneoplastic cells with an activator of AMP-activated protein kinase (AMPK), a brake in the cholesterol feedback pathway. AMPK activation by aspirin and metformin effectively abrogated the statin-induced aberrant upregulation of HMGCR and sensitized these resistant cells to fluvastatin. CONCLUSIONS: These results suggest the potential use of combined treatment with statin and aspirin for prevention of TNBC.


Assuntos
Biomarcadores Tumorais/genética , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Sintase/genética , Ácido Mevalônico/metabolismo , MicroRNAs/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Animais , Aspirina/farmacologia , Aspirina/uso terapêutico , Vias Biossintéticas/efeitos dos fármacos , Vias Biossintéticas/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica/genética , Colesterol/biossíntese , Retroalimentação Fisiológica/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Hidroximetilglutaril-CoA Redutases/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Inibidores de Hidroximetilglutaril-CoA Redutases/uso terapêutico , Hidroximetilglutaril-CoA Sintase/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , Análise de Sequência de RNA , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/prevenção & controle , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
20.
Food Funct ; 9(11): 5936-5949, 2018 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-30378628

RESUMO

In this study, the protective effects of a carboxymethyl polysaccharide CMP33 from Poria cocos against inflammatory bowel disease (IBD) were investigated using TNBS-induced colitis in mice. The results showed that CMP33 markedly ameliorated the severity of colitis, including a 2-fold decrease in the mortality rate, a 50% decrease in disease activity index, and a 36%-44% decrease in macro- or microscopic histopathological score, compared with TNBS administration. Moreover, CMP33 decreased the levels of pro-inflammatory cytokines and increased the levels of anti-inflammatory cytokines in the colon tissue and serum of colitic mice. Using iTRAQ-coupled- nano-HPLC-MS/MS-based proteomics, the protein profiles after TNBS, high- or low-dose CMP33 and salazosulfapyridine (SASP) treatments were compared and many differentially expressed proteins were identified. Among them, 7 proteins (Hmgcs2, Fabp2, Hp, B4galnt2, B3gnt6, Sap and Ca1) were proposed to be the common targeting protein group (TPG) of CMP33 and drug SASP. Particularly, some targeting proteins were CMP33-dose-specific: high-dose-specific TPG (Mtco3, Gal-6, Mptx, S100 g and Hpx) and low-dose-specific TPG (Zg16, Hexb, Insl5, Cept1, Hspb6 and Ifi27l2b), suggesting the complex acting mechanism of CMP33. GC-TOF-MS-based metabolomics revealed that oleic acid and dihydrotestosterone could be the common targets of CMP33 and SASP. By integrative analysis of proteomics and metabolomics, key protein-metabolite pathways (PMP) were identified, PMP for high-dose: 2-hydroxybutyric acid - (GPT, GGH) - glutathione - ALB - testosterone - TTR - dihydrotestosterone; PMP for low-dose: (PYY, FABP2, HMGCS2) - oleic acid - TTR - dihydrotestosterone. In total, these results demonstrated the protective effects of CMP33 against IBD in mice through the potential TPG and PMP.


Assuntos
Doenças Inflamatórias Intestinais/tratamento farmacológico , Polissacarídeos/farmacologia , Wolfiporia/química , Animais , Cromatografia Líquida de Alta Pressão , Colo/efeitos dos fármacos , Colo/metabolismo , Citocinas/sangue , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Haptoglobinas/genética , Haptoglobinas/metabolismo , Hidroximetilglutaril-CoA Sintase/genética , Hidroximetilglutaril-CoA Sintase/metabolismo , Doenças Inflamatórias Intestinais/induzido quimicamente , Masculino , Malondialdeído/sangue , Metabolômica , Camundongos , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Peroxidase/sangue , Proteômica , Sulfassalazina , Espectrometria de Massas em Tandem , Ácido Trinitrobenzenossulfônico
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