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1.
Nat Commun ; 11(1): 5352, 2020 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-33097693

RESUMO

Prime editing is a recent genome editing technology using fusion proteins of Cas9-nickase and reverse transcriptase, that holds promise to correct the vast majority of genetic defects. Here, we develop prime editing for primary adult stem cells grown in organoid culture models. First, we generate precise in-frame deletions in the gene encoding ß-catenin (CTNNB1) that result in proliferation independent of Wnt-stimuli, mimicking a mechanism of the development of liver cancer. Moreover, prime editing functionally recovers disease-causing mutations in intestinal organoids from patients with DGAT1-deficiency and liver organoids from a patient with Wilson disease (ATP7B). Prime editing is as efficient in 3D grown organoids as in 2D grown cell lines and offers greater precision than Cas9-mediated homology directed repair (HDR). Base editing remains more reliable than prime editing but is restricted to a subgroup of pathogenic mutations. Whole-genome sequencing of four prime-edited clonal organoid lines reveals absence of genome-wide off-target effects underscoring therapeutic potential of this versatile and precise gene editing strategy.


Assuntos
Edição de Genes/métodos , Organoides/metabolismo , beta Catenina/genética , Sistemas CRISPR-Cas , Linhagem Celular , Proliferação de Células , ATPases Transportadoras de Cobre/genética , Desoxirribonuclease I/metabolismo , Diacilglicerol O-Aciltransferase/genética , Células HEK293 , Degeneração Hepatolenticular/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Mutação , Reparo de DNA por Recombinação , Células-Tronco , Reparo Gênico Alvo-Dirigido/métodos
2.
J Steroid Biochem Mol Biol ; 202: 105721, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32565248

RESUMO

Accumulating evidence shows that granulosa cells within both mammalian and avian ovaries have the ability to synthesize fatty acids through de novo lipogenesis and to accumulate triglycerides essential for oocyte and ovarian development. However, very little is known about the exact roles of key genes involved in the lipid metabolic pathway in granulosa cells. The goal of this study was to investigate the differential actions of diacylglycerol acyltransferase (DGAT) 1 and 2, which are recognized as the rate-limiting enzymes catalyzing the last step of triglyceride biosynthesis, in regulating lipid metabolism and steroidogenesis in granulosa cells of goose follicles at different developmental stages. It was observed that the mRNAs encoding DGAT1 and DGAT2 were ubiquitous in all examined granulosa cell layers but exhibited distinct expression profiles during follicle development. Notably, the mRNA levels of DGAT1, DGAT2, FSHR, LHR, STAR, CYP11A1, and 3ßHSD remained almost constant in all except for 1-2 follicles within the 8-10 mm cohort, followed by an acute increase/decrease in the F5 follicles. At the cellular level, siRNA-mediated downregulation of DGAT1 or DGAT2 did not change the amount of lipids accumulated in both undifferentiated- and differentiated granulosa cells, while overexpression of DGAT2 promoted lipid accumulation and expression of lipogenic-related genes in these cells. Meanwhile, we found that interfering DGAT2 had no effect but interfering DGAT1 or overexpressing DGAT2 stimulated progesterone secretion in undifferentiated granulosa cells; in contrast, interference or overexpression of DGAT1/2 failed to change progesterone levels in differentiated granulosa cells but differently modulated expression of steroidogenic-related genes. Therefore, it could be concluded that DGAT1 is less efficient than DGAT2 in promoting lipid accumulation in both undifferentiated- and differentiated granulosa cells and that DGAT1 negatively while DGAT2 positively regulates progesterone production in undifferentiated granulosa cells.


Assuntos
Diacilglicerol O-Aciltransferase/genética , Células da Granulosa/metabolismo , Metabolismo dos Lipídeos , Progesterona/metabolismo , Animais , Proteínas Aviárias/genética , Diferenciação Celular , Células Cultivadas , Feminino , Gansos
3.
Nature ; 581(7808): 329-332, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32433610

RESUMO

Diacylglycerol O-acyltransferase 1 (DGAT1) synthesizes triacylglycerides and is required for dietary fat absorption and fat storage in humans1. DGAT1 belongs to the membrane-bound O-acyltransferase (MBOAT) superfamily, members of which are found in all kingdoms of life and are involved in the acylation of lipids and proteins2,3. How human DGAT1 and other mammalian members of the MBOAT family recognize their substrates and catalyse their reactions is unknown. The absence of three-dimensional structures also hampers rational targeting of DGAT1 for therapeutic purposes. Here we present the cryo-electron microscopy structure of human DGAT1 in complex with an oleoyl-CoA substrate. Each DGAT1 protomer has nine transmembrane helices, eight of which form a conserved structural fold that we name the MBOAT fold. The MBOAT fold in DGAT1 forms a hollow chamber in the membrane that encloses highly conserved catalytic residues. The chamber has separate entrances for each of the two substrates, fatty acyl-CoA and diacylglycerol. DGAT1 can exist as either a homodimer or a homotetramer and the two forms have similar enzymatic activity. The N terminus of DGAT1 interacts with the neighbouring protomer and these interactions are required for enzymatic activity.


Assuntos
Microscopia Crioeletrônica , Diacilglicerol O-Aciltransferase/química , Diacilglicerol O-Aciltransferase/metabolismo , Acil Coenzima A/química , Acil Coenzima A/metabolismo , Sítios de Ligação , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/ultraestrutura , Diglicerídeos/metabolismo , Humanos , Modelos Moleculares , Multimerização Proteica , Relação Estrutura-Atividade , Triglicerídeos/metabolismo
4.
Immunity ; 52(4): 620-634.e6, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32268121

RESUMO

Innate lymphoid cells (ILCs) play an important role in the control and maintenance of barrier immunity. However, chronic activation of ILCs results in immune-mediated pathology. Here, we show that tissue-resident type 2 ILCs (ILC2s) display a distinct metabolic signature upon chronic activation. In the context of allergen-driven airway inflammation, ILC2s increase their uptake of both external lipids and glucose. Externally acquired fatty acids are transiently stored in lipid droplets and converted into phospholipids to promote the proliferation of ILC2s. This metabolic program is imprinted by interleukin-33 (IL-33) and regulated by the genes Pparg and Dgat1, which are both controlled by glucose availability and mTOR signaling. Restricting dietary glucose by feeding mice a ketogenic diet largely ablated ILC2-mediated airway inflammation by impairing fatty acid metabolism and the formation of lipid droplets. Together, these results reveal that pathogenic ILC2 responses require lipid metabolism and identify ketogenic diet as a potent intervention strategy to treat airway inflammation.


Assuntos
Alérgenos/administração & dosagem , Asma/dietoterapia , Diacilglicerol O-Aciltransferase/imunologia , Dieta Cetogênica/métodos , Interleucina-33/imunologia , Gotículas Lipídicas/metabolismo , Subpopulações de Linfócitos T/imunologia , Alternaria/química , Animais , Asma/induzido quimicamente , Asma/imunologia , Asma/patologia , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Citocinas/administração & dosagem , Diacilglicerol O-Aciltransferase/genética , Modelos Animais de Doenças , Ácidos Graxos/imunologia , Ácidos Graxos/metabolismo , Regulação da Expressão Gênica , Glucose/imunologia , Glucose/metabolismo , Imunidade Inata , Interleucina-33/administração & dosagem , Interleucina-33/genética , Interleucinas/administração & dosagem , Gotículas Lipídicas/imunologia , Pulmão/efeitos dos fármacos , Pulmão/imunologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , PPAR gama/genética , PPAR gama/imunologia , Papaína/administração & dosagem , Fosfolipídeos/imunologia , Fosfolipídeos/metabolismo , Cultura Primária de Células , Subpopulações de Linfócitos T/classificação , Subpopulações de Linfócitos T/efeitos dos fármacos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologia
5.
Proc Natl Acad Sci U S A ; 117(11): 6216-6222, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32123083

RESUMO

Cyanobacteria are unicellular prokaryotic algae that perform oxygenic photosynthesis, similar to plants. The cells harbor thylakoid membranes composed of lipids related to those of chloroplasts in plants to accommodate the complexes of photosynthesis. The occurrence of storage lipids, including triacylglycerol or wax esters, which are found in plants, animals, and some bacteria, nevertheless remained unclear in cyanobacteria. We show here that the cyanobacterium Synechocystis sp. PCC6803 accumulates both triacylglycerol and wax esters (fatty acid phytyl esters). Phytyl esters accumulate in higher levels under abiotic stress conditions. The analysis of an insertional mutant revealed that the acyltransferase slr2103, with sequence similarity to plant esterase/lipase/thioesterase (ELT) proteins, is essential for triacylglycerol and phytyl ester synthesis in Synechocystis The recombinant slr2103 enzyme showed acyltransferase activity with phytol and diacylglycerol, thus producing phytyl esters and triacylglycerol. Acyl-CoA thioesters were the preferred acyl donors, while acyl-ACP (acyl carrier protein), free fatty acids, or galactolipid-bound fatty acids were poor substrates. The slr2103 protein sequence is unrelated to acyltransferases from bacteria (AtfA) or plants (DGAT1, DGAT2, PDAT), and therefore establishes an independent group of bacterial acyltransferases involved in triacylglycerol and wax ester synthesis. The identification of the gene slr2103 responsible for triacylglycerol synthesis in cyanobacteria opens the possibility of using prokaryotic photosynthetic cells in biotechnological applications.


Assuntos
Proteínas de Bactérias/metabolismo , Diacilglicerol O-Aciltransferase/metabolismo , Ésteres/metabolismo , Synechocystis/enzimologia , Triglicerídeos/biossíntese , Proteínas de Bactérias/genética , Diacilglicerol O-Aciltransferase/genética , Técnicas de Inativação de Genes , Fitol/metabolismo , Synechocystis/genética , Ceras/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-32058036

RESUMO

Besides energy storage and membrane biogenesis, lipids are known for their numerous biological functions. The two essential lipids, diacylglycerol (DG) and phosphatidic acid (PA), are shown to be associated with cell signalling processes. In this study, we examined whether triglyceride-deficient yeast mutants (tgΔ), dga1Δ and dga1Δlro1Δ, may play an important role in mevalonate (MEV) pathway regulation. Our metabolite analyses revealed that tgΔ cells showed high levels of squalene (SQ) and ergosterol (ERG), which are key indicators of MEV pathway activity. In addition, gene expression studies indicated that the MEV pathway genes in tgΔ cells were significantly upregulated. Interestingly, tgΔ cells exhibited high diacylglycerol kinase1 (DGK1) expression. Furthermore, DGK1 overexpression in WT and tgΔ phenotypes causes a substantial elevation in SQ and ERG levels, and we also found a significant increase in transcript levels of MEV pathway genes, confirming the new role of DGK1 in MEV pathway regulation. This suggests that high DG phosphorylation activity increases the PA pool that may induce the upregulation of MEV pathway in tgΔ cells. The induced MEV pathway is one of the key strategies in the field of synthetic biology for improved production of terpenoids in yeast. Thus, to examine whether increased endogenous MEV pathway flux can be redirected to triterpenoid, ß-Amyrin synthase gene was heterologously expressed in DGK1 overexpressing tgΔ cells that led to significant production of ß-Amyrin, a natural triterpenoid. In conclusion, our findings provide a novel strategy to increase MEV pathway precursors by modulating endogenous signal lipids for improved production of terpenoids.


Assuntos
Regulação Fúngica da Expressão Gênica , Ácido Mevalônico/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Triglicerídeos/deficiência , Aciltransferases/genética , Diacilglicerol O-Aciltransferase/genética , Ergosterol/biossíntese , Técnicas de Inativação de Genes , Redes e Vias Metabólicas/genética , Ácido Oleanólico/análogos & derivados , Ácido Oleanólico/biossíntese , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Esqualeno/metabolismo , Biologia Sintética/métodos , Ativação Transcricional , Regulação para Cima
7.
J Dairy Sci ; 103(3): 2514-2522, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31882213

RESUMO

It has been shown that milk infrared (IR) spectroscopy can be used to predict detailed milk fat composition. In addition, polymorphisms with substantial effects on milk fat composition have been identified. In this study, we investigated the combined use of milk IR spectroscopy and genotypes of dairy cows on the accuracy of predicting milk fat composition. Milk fat composition data based on gas chromatography and milk IR spectra were available for 1,456 Dutch Holstein Friesian cows. In addition, genotypes for the diacylglycerol acyltransferase 1 (DGAT1) K232A and stearoyl-CoA desaturase 1 (SCD1) A293V polymorphisms and a SNP located in an intron of the fatty acid synthase (FASN) gene were available. Adding SCD1 genotypes to the milk IR spectra resulted in a considerable improvement of the prediction accuracy for the unsaturated fatty acids C10:1, C12:1, C14:1 cis-9, and C16:1 cis-9 and their corresponding unsaturation indices. Adding DGAT1 genotypes to the milk IR spectra resulted in an improvement of the prediction accuracy for C16:1 cis-9 and C16 index. Adding genotypes of the FASN SNP to the IR spectra did not improve prediction of milk fat composition. This study demonstrated the potential of combining milk IR spectra with genotypic information from 3 polymorphisms to predict milk fat composition. We hypothesize that prediction accuracy of milk fat composition can be further improved by combining milk IR spectra with genomic breeding values.


Assuntos
Bovinos , Gorduras/análise , Genótipo , Leite/química , Espectrofotometria Infravermelho/veterinária , Alelos , Animais , Cruzamento , Bovinos/genética , Diacilglicerol O-Aciltransferase/genética , Gorduras na Dieta/análise , Ácidos Graxos Insaturados/análise , Feminino , Polimorfismo Genético
8.
J Dairy Sci ; 102(12): 11124-11141, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31563305

RESUMO

In genome-wide association studies (GWAS), sample size is the most important factor affecting statistical power that is under control of the investigator, posing a major challenge in understanding the genetics underlying difficult-to-measure traits. Combining data sets available from different populations for joint or meta-analysis is a promising alternative to increasing sample sizes available for GWAS. Simulation studies indicate statistical advantages from combining raw data or GWAS summaries in enhancing quantitative trait loci (QTL) detection power. However, the complexity of genetics underlying most quantitative traits, which itself is not fully understood, is difficult to fully capture in simulated data sets. In this study, population-specific and combined-population GWAS as well as a meta-analysis of the population-specific GWAS summaries were carried out with the objective of assessing the advantages and challenges of different data-combining strategies in enhancing detection power of GWAS using milk fatty acid (FA) traits as examples. Gas chromatography (GC) quantified milk FA samples and high-density (HD) genotypes were available from 1,566 Dutch, 614 Danish, and 700 Chinese Holstein Friesian cows. Using the joint GWAS, 28 additional genomic regions were detected, with significant associations to at least 1 FA, compared with the population-specific analyses. Some of these additional regions were also detected using the implemented meta-analysis. Furthermore, using the frequently reported variants of the diacylglycerol acyltransferase 1 (DGAT1) and stearoyl-CoA desaturase (SCD1) genes, we show that significant associations were established with more FA traits in the joint GWAS than the remaining scenarios. However, there were few regions detected in the population-specific analyses that were not detected using the joint GWAS or the meta-analyses. Our results show that combining multi-population data set can be a powerful tool to enhance detection power in GWAS for seldom-recorded traits. Detection of a higher number of regions using the meta-analysis, compared with any of the population-specific analyses also emphasizes the utility of these methods in the absence of raw multi-population data sets to undertake joint GWAS.


Assuntos
Conjuntos de Dados como Assunto , Estudo de Associação Genômica Ampla/veterinária , Glicolipídeos/análise , Glicoproteínas/análise , Metanálise como Assunto , Leite/química , Animais , Bovinos , Cromatografia Gasosa , Diacilglicerol O-Aciltransferase/genética , Ácidos Graxos/análise , Feminino , Estudo de Associação Genômica Ampla/métodos , Genótipo , Locos de Características Quantitativas , Estearoil-CoA Dessaturase/genética
9.
J Plant Physiol ; 242: 153019, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31437808

RESUMO

Acyl-CoA:diacylglycerol acyltransferase (DGAT) is a key enzyme in the Kennedy pathway of triacylglycerol (TAG) synthesis. It catalyzes the acyl-CoA-dependent acylation of sn-1, 2-diacylglycerol to form TAG. DGATs in soybean (Glycine max) have been reported, but their functions are largely unclear. Here we cloned three members of DGAT1 and four members of DGAT2 family from soybean, named GmDGAT1A to GmDGAT1C, and GmDGAT2A to GmDGAT2D, respectively. GmDGAT1A and GmDGAT1C were expressed at a high level in immature seeds, GmDGAT2B in mature seeds, and GmDGAT2C in older leaves. The seven genes were transformed into the H1246 quadruple mutant yeast strain, in which GmDGAT1A, GmDGAT1B, GmDGAT1C, GmDGAT2A, and GmDGAT2B had the ability to produce TAG. Six genes were transformed into Arabidopsis respectively, and constitutive expression of GmDGAT1A and GmDGAT1B resulted in an increase in oil content at the cost of reduced protein content in seeds. Overexpression of GmDGAT1A produced heavier weight of individual seed, but did not affect the weight of total seeds from a plant. Our results reveal the functions of soybean DGATs in seed oil synthesis using transgenic Arabidopsis. The implications for the biotechnological modification of the oil contents in soybeans by altering DGAT expression are discussed.


Assuntos
Arabidopsis/metabolismo , Diacilglicerol O-Aciltransferase/genética , Óleos Vegetais/metabolismo , Soja/enzimologia , Triglicerídeos/biossíntese , Arabidopsis/genética , Diacilglicerol O-Aciltransferase/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Genômica , Filogenia , Plantas Geneticamente Modificadas/metabolismo , Sementes/genética , Sementes/metabolismo , Soja/metabolismo , Triglicerídeos/metabolismo
10.
Genes Genomics ; 41(11): 1265-1271, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31388977

RESUMO

BACKGROUND: Diacylglycerol O-acyltransferase 1 (DGAT1) plays a key role in the synthesis of triglycerides. Recent studies have shown that a transition mutation resulting in substitutions of guanine by adenine in the DGAT1 gene in cattle has considerable effects on milk yield and composition. Currently, there is no systematic research reporting on the utilization of this gene segment in Iranian buffalo (Bubalus bubalis). OBJECTIVE: In this study, the genetic differentiation of three indigenous Iranian buffalo populations was investigated in the region spanning exon 3 to exon 17 of the DGAT1 gene. METHODS: A total of 200 buffaloes were genotyped, all the samples were sequenced directly in both directions with forward and reverse sequencing primers. RESULTS: Sequence analysis showed novel SNPs compared to the reference GenBank sequence (DQ886485) at nucleotide positions g.6097A>G, g.7036C>T, g.7338G>A, g.7710C>T, g.8087C>T, g.8259G>A, g.8275G>A, g.8367C>T, and g.8426C>T. No polymorphisms were found within exon 8. Therefore, the K232A position was thought to be a conserved and fixed region for high milk fat content (K allele) in Bos indicus and all buffalo breeds. Comparison with Indian buffalo revealed three exonic SNPs, one of which was nonsynonymous. A unique 22 bp insertion was observed in intron 10 of DGAT1. Linkage disequilibrium analysis allowed the identification of nine haplotypes among the sampled animals. To our knowledge, this is the first report of sequencing analysis of the DGAT1 gene in Iranian buffalo. CONCLUSION: Our results suggest that genetic diversity exists and could be useful in examining the association between the DGAT1 gene and milk production traits in buffalo.


Assuntos
Búfalos/genética , Diacilglicerol O-Aciltransferase/genética , Haplótipos , Polimorfismo de Nucleotídeo Único , Animais , Diacilglicerol O-Aciltransferase/química , Conformação Proteica
11.
Am J Physiol Regul Integr Comp Physiol ; 317(4): R552-R562, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31411897

RESUMO

The main goal of this study was to compare the impact of total body leptin deficiency with neuronal-specific leptin receptor (LR) deletion on metabolic and cardiovascular regulation. Liver fat, diacylglycerol acyltransferase-2 (DGTA2), and CD36 protein content were measured in wild-type (WT), nervous system LR-deficient (LR/Nestin-Cre), and leptin deficient (ob/ob) mice. Blood pressure (BP) and heart rate (HR) were recorded by telemetry, and motor activity (MA) and oxygen consumption (V̇o2) were monitored at 24 wk of age. Female and male LR/Nestin-Cre and ob/ob mice were heavier than WT mice (62 ± 5 and 61 ± 3 vs. 31 ± 1 g) and hyperphagic (6.2 ± 0.5 and 6.1 ± 0.7 vs. 3.5 ± 1.0 g/day), with reduced V̇o2 (27 ± 1 and 33 ± 1 vs 49 ± 3 ml·kg-1·min-1) and decreased MA (3 ± 1 and 7 ± 2 vs 676 ± 105 cm/h). They were also hyperinsulinemic and hyperglycemic compared with WT mice. LR/Nestin-Cre mice had high levels of plasma leptin, while ob/ob mice had undetectable leptin levels. Despite comparable obesity, LR/Nestin-Cre mice had lower liver fat content, DGTA2, and CD36 protein levels than ob/ob mice. Male WT, LR/Nestin-Cre, and ob/ob mice exhibited similar BP (111 ± 3, 110 ± 1 and 109 ± 2 mmHg). Female LR/Nestin-Cre and ob/ob mice, however, had higher BP than WT females despite similar metabolic phenotypes compared with male LR/Nestin-Cre and ob/ob mice. These results indicate that although nervous system LRs play a crucial role in regulating body weight and glucose homeostasis, peripheral LRs regulate liver fat deposition. In addition, our results suggest potential sex differences in the impact of obesity on BP regulation.


Assuntos
Diacilglicerol O-Aciltransferase/metabolismo , Leptina/genética , Receptores para Leptina/metabolismo , Tecido Adiposo/metabolismo , Antagonistas Adrenérgicos , Aldosterona/sangue , Antagonistas de Receptores de Angiotensina/farmacologia , Animais , Glicemia , Pressão Sanguínea , Antígenos CD36/genética , Antígenos CD36/metabolismo , Diacilglicerol O-Aciltransferase/genética , Feminino , Regulação da Expressão Gênica , Frequência Cardíaca/fisiologia , Leptina/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Obesos , Antagonistas de Receptores de Mineralocorticoides/farmacologia , Receptores Adrenérgicos/metabolismo , Receptores para Leptina/genética , Estresse Fisiológico
12.
Anim Reprod Sci ; 208: 106104, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31405453

RESUMO

The peri-calving period is characterized by a negative energy balance, which leads to lipid mobilization. Thus, during this period, the liver has important functions related to optimizing milk yield, preventing metabolic and infectious diseases, and improving fertility. To clarify the relationship between liver fatty acid metabolism and reproductive performance, the present study was conducted to assess the abundance of specific hepatic proteins related to lipid metabolism in both plasma and follicular fluid in dairy cattle with different days to conception (DC). Sixteen animals were grouped according to DC, as more and fewer DC (MDC and FDC, respectively). Blood and liver biopsies were sampled 14 days before the expected calving date and 4, 14 and 28 days after calving. The plasma beta-hydroxybutyric acid (BHBA) concentrations and the liver triacylglycerol (TAG) content were greater in the MDC group (P <  0.05), whereas the protein abundance of carnitine palmitoyl transferase 1 was greater in the FDC group (P < 0.05). Additionally, total bilirubin (TBil) concentration was less in the FDC than MDC group on day 28 (P < 0.05). These results indicate lipid mobilization and liver fatty acid oxidation capacity in dairy cows could contribute to the adaptations and reproductive performance.


Assuntos
Bovinos/fisiologia , Ácidos Graxos/metabolismo , Fígado/metabolismo , Reprodução/fisiologia , Acil-CoA Oxidase/genética , Acil-CoA Oxidase/metabolismo , Ração Animal , Criação de Animais Domésticos , Animais , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Bovinos/sangue , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Feminino , Regulação da Expressão Gênica , Metabolismo dos Lipídeos , PPAR alfa/genética , PPAR alfa/metabolismo , Período Periparto/sangue , Período Periparto/fisiologia , Gravidez , Triglicerídeos/metabolismo
13.
Genes (Basel) ; 10(8)2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31398921

RESUMO

A dose of proanthocyanidins with satiating properties proved to be able to limit body weight increase several weeks after administration under exposure to a cafeteria diet. Here we describe some of the molecular targets and the duration of the effects. We treated rats with 500 mg grape seed proanthocyanidin extract (GSPE)/kg BW for ten days. Seven or seventeen weeks after the last GSPE dose, while animals were on a cafeteria diet, we used reverse transcriptase-polymerase chain reaction (RT-PCR) to measure the mRNA of the key energy metabolism enzymes from the liver, adipose depots and muscle. We found that a reduction in the expression of adipose Lpl might explain the lower amount of adipose tissue in rats seven weeks after the last GSPE dose. The liver showed increased expression of Cpt1a and Hmgs2 together with a reduction in Fasn and Dgat2. In addition, muscle showed a higher fatty oxidation (Oxct1 and Cpt1b mRNA). However, after seventeen weeks, there was a completely different gene expression pattern. At the conclusion of the study, seven weeks after the last GSPE administration there was a limitation in adipose accrual that might be mediated by an inhibition of the gene expression of the adipose tissue Lpl. Concomitantly there was an increase in fatty acid oxidation in liver and muscle.


Assuntos
Adiposidade/efeitos dos fármacos , Depressores do Apetite/farmacologia , Dieta da Carga de Carboidratos/efeitos adversos , Dieta Ocidental/efeitos adversos , Sobrepeso/prevenção & controle , Proantocianidinas/farmacologia , Tecido Adiposo/metabolismo , Animais , Depressores do Apetite/uso terapêutico , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Coenzima A-Transferases/genética , Coenzima A-Transferases/metabolismo , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Feminino , Leptina/genética , Leptina/metabolismo , Fígado/metabolismo , Músculo Esquelético/metabolismo , Sobrepeso/tratamento farmacológico , Proantocianidinas/uso terapêutico , Ratos , Vitis/química
14.
J Dairy Sci ; 102(9): 8159-8174, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31301836

RESUMO

We performed genome-wide association analyses for milk, fat, and protein yields and somatic cell score based on lactation stages in the first 3 parities of Canadian Ayrshire, Holstein, and Jersey cattle. The genome-wide association analyses were performed considering 3 different lactation stages for each trait and parity: from 5 to 95, from 96 to 215, and from 216 to 305 d in milk. Effects of single nucleotide polymorphisms (SNP) for each lactation stage, trait, parity, and breed were estimated by back-solving the direct breeding values estimated using the genomic best linear unbiased predictor and single-trait random regression test-day models containing only the fixed population average curve and the random genomic curves. To identify important genomic regions related to the analyzed lactation stages, traits, parities and breeds, moving windows (SNP-by-SNP) of 20 adjacent SNP explaining more than 0.30% of total genetic variance were selected for further analyses of candidate genes. A lower number of genomic windows with a relatively higher proportion of the explained genetic variance was found in the Holstein breed compared with the Ayrshire and Jersey breeds. Genomic regions associated with the analyzed traits were located on 12, 8, and 15 chromosomes for the Ayrshire, Holstein, and Jersey breeds, respectively. Especially for the Holstein breed, many of the identified candidate genes supported previous reports in the literature. However, well-known genes with major effects on milk production traits (e.g., diacylglycerol O-acyltransferase 1) showed contrasting results among lactation stages, traits, and parities of different breeds. Therefore, our results suggest evidence of differential sets of candidate genes underlying the phenotypic expression of the analyzed traits across breeds, parities, and lactation stages. Further functional studies are needed to validate our findings in independent populations.


Assuntos
Bovinos/genética , Estudo de Associação Genômica Ampla/veterinária , Genoma/genética , Lactação/genética , Leite/metabolismo , Polimorfismo de Nucleotídeo Único/genética , Animais , Cruzamento , Bovinos/fisiologia , Diacilglicerol O-Aciltransferase/genética , Feminino , Paridade , Fenótipo , Gravidez
15.
Protein Eng Des Sel ; 32(1): 25-32, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31251342

RESUMO

Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/química , Diacilglicerol O-Aciltransferase/química , Evolução Molecular Direcionada , Actinobacteria/genética , Proteínas de Bactérias/genética , Diacilglicerol O-Aciltransferase/genética
16.
J Dairy Sci ; 102(8): 6842-6852, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31178185

RESUMO

In the present study, we aimed to investigate the changes in triacylglycerol (TAG) composition as affected by alterations in the cows' diet due to seasonal variations and genetic factors. For this study, 50 milk fat samples in winter and 50 in summer were used from 25 cows with the DGAT1 KK genotype and 25 cows with the DGAT1 AA genotype. The samples were analyzed for milk fat content (%), fat composition, and TAG composition. We found that the content of TAG species CN54 was higher and that of CN34 and CN36 lower in summer than in winter. This seasonal variation in TAG profile was related to seasonal changes in the fatty acids C14:0, C16:0, C18:0, C18:1 cis-9, total unsaturated fatty acids, and total long-chain fatty acids, most likely resulting from dietary differences between seasons. Furthermore, we quantified the effect of DGAT1 K232A polymorphism on TAG profile and detected a significant effect on TAG species CN36, with higher values for the DGAT1 KK genotype. When adjusting for differences in fat content, we found no significant effects of the DGAT1 K232A polymorphism on TAG profile. We detected a significant interaction between DGAT1 K232A polymorphism and season for TAG species CN42 and CN52; in summer, the KK genotype was associated with higher levels for CN42 than the AA genotype, whereas in winter, the difference between the genotypes was small. For CN52, in summer the AA genotype was associated with higher levels than the KK genotype. In winter, the difference between the genotypes was also small. We show that, regardless of preference for DGAT1 genotype (AA or KK) and depending on the availability of FA according to season, UFA (C18:1 cis-9), short-chain FA (C6:0 and C10:0), and medium-chain FA might be esterified on the glycerol backbone of the TAG, keeping the structure characteristics of each TAG species. To our knowledge, this is the first report on the interaction effect of DGAT1 K232A polymorphism and season on the TAG composition in milk fat.


Assuntos
Bovinos/genética , Diacilglicerol O-Aciltransferase/genética , Dieta/veterinária , Genótipo , Leite/química , Triglicerídeos/análise , Animais , Bovinos/fisiologia , Ácidos Graxos/análise , Ácidos Graxos Insaturados/análise , Feminino , Polimorfismo Genético/genética , Estações do Ano
17.
J Dairy Sci ; 102(8): 7536-7547, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31178189

RESUMO

High blood concentrations of nonesterified fatty acids (NEFA) and altered lipid metabolism are key characteristics of fatty liver in dairy cows. In nonruminants, the mitochondrial membrane protein mitofusin 2 (MFN2) plays important roles in regulating mitochondrial function and intrahepatic lipid metabolism. Whether MFN2 is associated with hepatic lipid metabolism in dairy cows with moderate fatty liver is unknown. Therefore, to investigate changes in MFN2 expression and lipid metabolic status in dairy cows with moderate fatty liver, blood and liver samples were collected from healthy dairy cows (n = 10) and cows with moderate fatty liver (n = 10). To determine the effects of MFN2 on lipid metabolism in vitro, hepatocytes isolated from healthy calves were used for small interfering RNA-mediated silencing of MFN2 or adenovirus-mediated overexpression of MFN2 for 48 h, or treated with 0, 0.6, 1.2, or 2.4 mM NEFA for 12 h. Milk production and plasma glucose concentrations in dairy cows with moderate fatty liver were lower, but concentrations of NEFA and ß-hydroxybutyrate (BHB) were greater in dairy cows with moderate fatty liver. Dairy cows with moderate fatty liver displayed hepatic lipid accumulation and lower abundance of hepatic MFN2, peroxisome proliferator-activated receptor-α (PPARα), and carnitine palmitoyltransferase 1A (CPT1A). However, sterol regulatory element-binding protein 1c (SREBP-1c), acetyl CoA carboxylase 1 (ACACA), fatty acid synthase (FASN), and diacylglycerol acyltransferase 1 (DGAT1) were more abundant in the livers of dairy cows with moderate fatty liver. In vitro, exogenous NEFA treatment upregulated abundance of SREBP-1c, ACACA, FASN, and DGAT1, and downregulated the abundance of PPARα and CPT1A. These changes were associated with greater lipid accumulation in calf hepatocytes, and MFN2 silencing aggravated this effect. In contrast, overexpression of MFN2-ameliorated exogenous NEFA-induced lipid accumulation by downregulating the abundance of SREBP-1c, ACACA, FASN, and DGAT1, and upregulating the abundance of PPARα and CPT1A in calf hepatocytes. Overall, these data suggest that one cause for the negative effect of excessive NEFA on hepatic lipid accumulation is the inhibition of MFN2. As such, these mechanisms partly explain the development of hepatic steatosis in dairy cows.


Assuntos
Doenças dos Bovinos/metabolismo , Bovinos/metabolismo , Fígado Gorduroso/veterinária , GTP Fosfo-Hidrolases/metabolismo , Metabolismo dos Lipídeos , Fígado/metabolismo , Ácido 3-Hidroxibutírico/metabolismo , Animais , Bovinos/genética , Doenças dos Bovinos/enzimologia , Doenças dos Bovinos/genética , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Fígado Gorduroso/enzimologia , Fígado Gorduroso/genética , Fígado Gorduroso/metabolismo , Feminino , GTP Fosfo-Hidrolases/genética , Hepatócitos/enzimologia , Hepatócitos/metabolismo , Mitocôndrias/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo
18.
Proteomics ; 19(13): e1900160, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31099964

RESUMO

Lipids are emerging as key regulators of apoptosis. Specific lipid species are associated with apoptosis with important functional roles, but the understanding of the regulation of these lipid species is still limited. It has been previously shown by our laboratory that polyunsaturated triacylglycerols accumulate and get stored within lipid droplets during apoptosis via activated glycerolipid biosynthesis. In this work, the biochemical mechanisms that result in the activation of glycerolipid biosynthesis and, consequently, triacylglycerol and lipid droplet accumulation during apoptosis are investigated. The transcriptomes of control and apoptotic HCT-116 cells are compared and gene enrichment analysis revealed the upregulation of p38 mitogen-activated protein kinase (MAPK). It is shown that p38 MAPK regulates triacylglycerol biosynthesis through diacylglycerol acyltransferase1 during apoptosis. Perilipin 2 and cytosolic phospholipase A2delta are also shown to be involved in lipid droplet and polyunsaturated triacylglycerol accumulation in this process. Overall, the results provide new insights into the upregulation of glycerolipid synthesis during apoptosis.


Assuntos
Apoptose/fisiologia , Diacilglicerol O-Aciltransferase/metabolismo , Triglicerídeos/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Diacilglicerol O-Aciltransferase/genética , Fosfolipases A2 do Grupo IV/metabolismo , Células HCT116 , Humanos , Gotículas Lipídicas/metabolismo , Perilipina-2/metabolismo , Análise de Sequência de RNA , Ativação Transcricional/genética , Transcriptoma/genética
19.
J Agric Food Chem ; 67(21): 6007-6018, 2019 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-31060359

RESUMO

4EBP1 is a chief downstream factor of mTORC1, and PPARγ is a key lipogenesis-related transcription factor. mTORC1 and PPARγ are associated with lipid metabolism. However, it is unknown which effector protein connects mTORC1 and PPARγ. This study investigated the interaction between 4EBP1 with PPARγ as part of the underlying mechanism by which insulin-induced lipid synthesis and secretion are regulated by mTORC1 in primary bovine mammary epithelial cells (pBMECs). Rapamycin, a specific inhibitor of mTORC1, downregulated 4EBP1 phosphorylation and the expression of PPARγ and the following lipogenic genes: lipin 1, DGAT1, ACC, and FAS. Rapamycin also decreased the levels of intracellular triacylglycerol (TAG); 10 types of fatty acid; and the accumulation of TAG, palmitic acid (PA), and stearic acid (SA) in the cell culture medium. Inactivation of mTORC1 by shRaptor or shRheb attenuated the synthesis and secretion of TAG and PA. In contrast, activation of mTORC1 by Rheb overexpression promoted 4EBP1 phosphorylation and PPARγ expression and upregulated the mRNA and protein levels of lipin 1, DGAT1, ACC, and FAS, whereas the levels of intracellular and extracellular TAG, PA, and SA also rose. Further, 4EBP1 interacted directly with PPARγ. Inactivation of mTORC1 by shRaptor prevented the nuclear location of PPARγ. These results demonstrate that mTORC1 regulates lipid synthesis and secretion by inducing the expression of lipin 1, DGAT1, ACC, and FAS, which is likely mediated by the 4EBP1/PPARγ axis. This finding constitutes a novel mechanism by which lipid synthesis and secretion are regulated in pBMECs.


Assuntos
Células Epiteliais/metabolismo , Fatores de Iniciação em Eucariotos/metabolismo , Regulação da Expressão Gênica , Insulina/metabolismo , Lipogênese , Glândulas Mamárias Animais/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , PPAR gama/metabolismo , Animais , Carbono-Carbono Liases/genética , Carbono-Carbono Liases/metabolismo , Bovinos , Diacilglicerol O-Aciltransferase/genética , Diacilglicerol O-Aciltransferase/metabolismo , Fatores de Iniciação em Eucariotos/genética , Feminino , Glândulas Mamárias Animais/citologia , Glândulas Mamárias Animais/enzimologia , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , PPAR gama/genética , Triglicerídeos/metabolismo
20.
BMC Genomics ; 20(1): 402, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31117950

RESUMO

BACKGROUND: Cotton (Gossypium spp.) is the most important natural fiber crop worldwide, and cottonseed oil is its most important byproduct. Phospholipid: diacylglycerol acyltransferase (PDAT) is important in TAG biosynthesis, as it catalyzes the transfer of a fatty acyl moiety from the sn-2 position of a phospholipid to the sn-3 position of sn-1, 2-diacylglyerol to form triacylglycerol (TAG) and a lysophospholipid. However, little is known about the genes encoding PDATs involved in cottonseed oil biosynthesis. RESULTS: A comprehensive genome-wide analysis of G. hirsutum, G. barbadense, G. arboreum, and G. raimondii herein identified 12, 11, 6 and 6 PDATs, respectively. These genes were divided into 3 subfamilies, and a PDAT-like subfamily was identified in comparison with dicotyledonous Arabidopsis. All GhPDATs contained one or two LCAT domains at the C-terminus, while most GhPDATs contained a preserved single transmembrane region at the N-terminus. A chromosomal distribution analysis showed that the 12 GhPDAT genes in G. hirsutum were distributed in 10 chromosomes. However, none of the GhPDATs was co-localized with quantitative trait loci (QTL) for cottonseed oil content, suggesting that their sequence variations are not genetically associated with the natural variation in cottonseed oil content. Most GhPDATs were expressed during the cottonseed oil accumulation stage. Ectopic expression of GhPDAT1d increased Arabidopsis seed oil content. CONCLUSIONS: Our comprehensive genome-wide analysis of the cotton PDAT gene family provides a foundation for further studies into the use of PDAT genes to increase cottonseed oil content through biotechnology.


Assuntos
Diacilglicerol O-Aciltransferase/genética , Regulação da Expressão Gênica de Plantas , Gossypium/genética , Família Multigênica , Fosfolipídeos/análise , Óleos Vegetais/análise , Proteínas de Plantas/genética , Diacilglicerol O-Aciltransferase/metabolismo , Evolução Molecular , Gossypium/crescimento & desenvolvimento , Gossypium/metabolismo , Filogenia , Proteínas de Plantas/metabolismo , Sementes/química
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