RESUMEN
The roles of DGAT1 and DGAT2 in lipid metabolism and insulin responsiveness of human skeletal muscle were studied using cryosections and myotubes prepared from muscle biopsies from control, athlete, and impaired glucose regulation (IGR) cohorts of men. The previously observed increases in intramuscular triacylglycerol (IMTG) in athletes and IGR were shown to be related to an increase in lipid droplet (LD) area in type I fibers in athletes but, conversely, in type II fibers in IGR subjects. Specific inhibition of both diacylglycerol acyltransferase (DGAT) 1 and 2 decreased fatty acid (FA) uptake by myotubes, whereas only DGAT2 inhibition also decreased fatty acid oxidation. Fatty acid uptake in myotubes was negatively correlated with the lactate thresholds of the respective donors. DGAT2 inhibition lowered acetate uptake and oxidation in myotubes from all cohorts whereas DGAT1 inhibition had no effect. A positive correlation between acetate oxidation in myotubes and resting metabolic rate (RMR) from fatty acid oxidation in vivo was observed. Myotubes from athletes and IGR had higher rates of de novo lipogenesis from acetate that were normalized by DGAT2 inhibition. Moreover, DGAT2 inhibition in myotubes also resulted in increased insulin-induced Akt phosphorylation. The differential effects of DGAT1 and DGAT2 inhibition suggest that the specialized role of DGAT2 in esterifying nascent diacylglycerols and de novo synthesized FA is associated with synthesis of a pool of triacylglycerol, which upon hydrolysis results in effectors that promote mitochondrial fatty acid oxidation but decrease insulin signaling in skeletal muscle cells.
Asunto(s)
Diacilglicerol O-Acetiltransferasa , Fibras Musculares Esqueléticas , Masculino , Humanos , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Glucosa/metabolismo , Insulina , Acetatos , Triglicéridos/metabolismo , Ácidos Grasos/metabolismoRESUMEN
Discovery of the Asgard superphylum of archaea provides new evidence supporting the two-domain model of life: eukaryotes originated from an Asgard-related archaeon that engulfed a bacterial endosymbiont. However, how eukaryotes acquired bacterial-like membrane lipids with a sn-glycerol-3-phosphate (G3P) backbone instead of the archaeal-like sn-glycerol-1-phosphate (G1P) backbone remains unknown. In this study, we reconstituted archaeal lipid production in Saccharomyces cerevisiae by expressing unsaturated archaeol-synthesizing enzymes. Using Golden Gate cloning for pathway assembly, modular gene replacement was performed, revealing the potential biosynthesis of both G1P- and G3P-based unsaturated archaeol by uncultured Asgard archaea. Unexpectedly, hybrid neutral lipids containing both archaeal isoprenoids and eukaryotic fatty acids were observed in recombinant S. cerevisiae. The ability of yeast and archaeal diacylglycerol acyltransferases to synthesize such hybrid lipids was demonstrated.
Asunto(s)
Archaea , Saccharomyces cerevisiae , Archaea/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Glicerol/metabolismo , Lípidos de la Membrana/metabolismo , Bacterias/metabolismo , Fosfatos/metabolismoRESUMEN
KEY MESSAGE: Two OsDGAT1 genes showed the ability to restore TAG and LB synthesis in yeast H1246. Alterations in the N-terminal region of OsDGAT1-1 gene revealed its regulatory role in gene function. Accumulation of triacylglycerol (TAG) or oil in vegetative tissues has emerged as a promising approach to meet the global needs of food, feed, and fuel. Rice (Oryza sativa) has been recognized as an important cereal crop containing nutritional rice bran oil with high economic value for renewable energy production. To identify the key component involved in storage lipid biosynthesis, two type-1 diacylglycerol acyltransferases (DGAT1) from rice were characterized for its in vivo function in the H1246 (dga1, lro1, are1 and are2) yeast quadruple mutant. The ectopic expression of rice DGAT1 (designated as OsDGAT1-1 and OsDGAT1-2) genes restored the capability of TAG synthesis and lipid body (LB) formation in H1246. OsDGAT1-1 showed nearly equal substrate preferences to C16:0-CoA and 18:1-CoA whereas OsDGAT1-2 displayed substrate selectivity for C16:0-CoA over 18:1-CoA, indicating that these enzymes have contrasting substrate specificities. In parallel, we have identified the intrinsically disordered region (IDR) at the N-terminal domains of OsDGAT1 proteins. The regulatory role of the N-terminal domain was dissected. Single point mutations at the phosphorylation sites and truncations of the N-terminal region highlighted reduced lipid accumulation capabilities among different OsDGAT1-1 variants.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/genética , Oryza/enzimología , Oryza/genética , Saccharomyces cerevisiae/metabolismo , Semillas/enzimología , Semillas/genética , Triglicéridos/metabolismo , Secuencia de Aminoácidos , Diacilglicerol O-Acetiltransferasa/química , Diacilglicerol O-Acetiltransferasa/metabolismo , Diglicéridos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Gotas Lipídicas/metabolismo , Mutación/genética , Fosforilación , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Dominios Proteicos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por SustratoRESUMEN
KEY MESSAGE: EgMADS21 regulates PUFA accumulation in oil palm. Oil palm (Elaeis guineensis Jacq.) is the most productive world oil crop, accounting for 36% of world plant oil production. However, the molecular mechanism of the transcriptional regulation of fatty acid accumulation and lipid synthesis in the mesocarp of oil palm by up- or downregulating the expression of genes involved in related pathways remains largely unknown. Here, an oil palm MADS-box gene, EgMADS21, was screened in a yeast one-hybrid assay using the EgDGAT2 promoter sequence as bait. EgMADS21 is preferentially expressed in early mesocarp developmental stages in oil palm fruit and presents a negative correlation with EgDGAT2 expression. The direct binding of EgMADS21 to the EgDGAT2 promoter was confirmed by electrophoretic mobility shift assay. Subsequently, transient expression of EgMADS21 in oil palm protoplasts revealed that EgMADS21 not only binds to the EgDGAT2 promoter but also negatively regulates the expression of EgDGAT2. Furthermore, EgMADS21 was stably overexpressed in transgenic oil palm embryoids by Agrobacterium-mediated transformation. In three independent transgenic lines, EgDGAT2 expression was significantly suppressed by the expression of EgMADS21. The content of linoleic acid (C18:2) in the three transgenic embryoids was significantly decreased, while that of oleic acid (C18:1) was significantly increased. Combined with the substrate preference of EgDGAT2 identified in previous research, the results demonstrate the molecular mechanism by which EgMADS21 regulates EgDGAT2 expression and ultimately affects fatty acid accumulation in the mesocarp of oil palm.
Asunto(s)
Arecaceae/genética , Arecaceae/metabolismo , Ácidos Grasos Insaturados/metabolismo , Proteínas de Plantas/genética , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Ácidos Grasos Insaturados/genética , Regulación de la Expresión Génica de las Plantas , Proteínas de Dominio MADS/genética , Proteínas de Dominio MADS/metabolismo , Aceite de Palma/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Protoplastos/metabolismoRESUMEN
SCOPE: The effects of sulforaphane (SFN) on the maturation of lipid droplets (LDs)-the storage units for free fatty acids and sterols as triacylglycerides (TAG) and cholesterol esters (CE)-are far from being understood, despite the fact that SFN is known to be beneficial for ameliorating lipid metabolism disorders. METHODS AND RESULTS: High-fat-intake models are established in both HHL-5 hepatocytes and rodents. The numbers and sizes of LDs are decreased by SFN. The accumulation of lipid core components (TAG & CE) is reduced and the expression of their key synthetases, acyl-coenzyme A: diacylglycerol acyltransferases 2 (DGAT2) and acyl-coenzyme A: cholesterol acyltransferases 1 (ACAT1), is also inhibited. Moreover, SFN decreases LD-associated protein PLIN2 and PLIN5 expression, but not that of PLIN1 and PLIN3, both in vivo and in vitro. Furthermore, over-expression of peroxisome proliferator-activated receptor gamma (PPARγ) induces the accumulation of TAG and the up-regulation of PLIN2 and PLIN5, which are not reversed by SFN. These results suggest that PPARγ may be a target of SFN in lipid metabolism. CONCLUSION: SFN disturbs LD maturation by inhibiting the formation of the neutral lipid core and decreases PLIN2 and PLIN5 via down-regulation of PPARγ.
Asunto(s)
Isotiocianatos/farmacología , Gotas Lipídicas/efectos de los fármacos , PPAR gamma/antagonistas & inhibidores , Perilipina-2/antagonistas & inhibidores , Perilipina-5/antagonistas & inhibidores , Animales , Células Cultivadas , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Gotas Lipídicas/fisiología , Masculino , Perilipina-2/fisiología , Perilipina-5/fisiología , Ratas , Ratas Wistar , Sulfóxidos , Triglicéridos/metabolismo , Triglicéridos/fisiologíaRESUMEN
Cinnamon polyphenol extract (CPE) improves people with insulin resistance. The objective was to investigate CPE and insulin on diacylglycerol acyltransferase (DGAT) gene expression important for lipid biosynthesis and compared it to anti-inflammatory tristetraprolin/zinc finger protein 36 (TTP/ZFP36) gene expression known to be regulated by both agents. Mouse 3T3-L1 adipocytes and RAW264.7 macrophages were treated with insulin and CPE followed by qPCR evaluation of DGAT and TTP mRNA levels. Insulin decreased DGAT1 and DGAT2 mRNA levels in adipocytes but had no effect on DGAT1 and increased DGAT2 mRNA levels 3-fold in macrophages. Insulin increased TTP mRNA levels 3-fold in adipocytes but had no effect in macrophages. CPE effect on DGAT1 gene expression was minimal but increased DGAT2 mRNA levels 2-4 fold in adipocytes and macrophages. CPE increased TTP mRNA levels 2-7 fold in adipocytes and macrophages. We conclude that CPE and insulin exhibited overlapping and independent effects on DGAT and TTP gene expression and suggest that CPE and insulin have profound effects on fat biosynthesis and inflammatory responses.
Asunto(s)
Cinnamomum zeylanicum/química , Diacilglicerol O-Acetiltransferasa/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Extractos Vegetales/farmacología , Polifenoles/farmacología , Células 3T3-L1 , Adipocitos/efectos de los fármacos , Adipocitos/enzimología , Animales , Diacilglicerol O-Acetiltransferasa/genética , Humanos , Resistencia a la Insulina , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Ratones , Células RAW 264.7 , ARN Mensajero/genética , Tristetraprolina/metabolismoRESUMEN
BACKGROUND: Triacylglycerols (TAGs) are the major form of energy storage in eukaryotes. Diacylglycerol acyltransferases (DGATs) catalyze the final and rate-limiting step of TAG biosynthesis. Mammalian DGATs are classified into DGAT1 and DGAT2 subfamilies. It was unclear which DGAT was the major isoform expressed in animal cells. The objective was to identify the major DGAT mRNA expressed in cultured mouse adipocytes and macrophages and compared it to that expressed in tung tree seeds. METHODS: qPCR evaluated DGAT mRNA levels in mouse 3 T3-L1 adipocytes and RAW264.7 macrophages and tung tree seeds. RESULTS: TaqMan qPCR showed that DGAT2 mRNA levels were 10-30 fold higher than DGAT1 in adipocytes and macrophages, and DGAT mRNA levels in adipocytes were 50-100-fold higher than those in macrophages. In contrast, the anti-inflammatory tristetraprolin/zinc finger protein 36 (TTP/ZFP36) mRNA levels were 2-4-fold higher in macrophages than those in adipocytes and similar to DGAT1 in adipocytes but 100-fold higher than DGAT1 in macrophages. SYBR Green qPCR analyses confirmed TaqMan qPCR results. DGAT2 mRNA as the major DGAT mRNA in the mouse cells was similar to that in tung tree seeds where DGAT2 mRNA levels were 10-20-fold higher than DGAT1 or DGAT3. CONCLUSION: The results demonstrated that DGAT2 mRNA was the major form of DGAT mRNA expressed in mouse adipocytes and macrophages and tung tree seeds.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/genética , ARN Mensajero/metabolismo , Células 3T3-L1 , Adipocitos/citología , Adipocitos/efectos de los fármacos , Adipocitos/enzimología , Aleurites/enzimología , Animales , Diacilglicerol O-Acetiltransferasa/metabolismo , Dimetilsulfóxido/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/enzimología , Ratones , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Células RAW 264.7 , Estabilidad del ARN , Reacción en Cadena en Tiempo Real de la Polimerasa , Semillas/enzimologíaRESUMEN
OBJECTIVE: To determine the effect of maternal obesity, gestational diabetes mellitus (GDM) and adipokines on the expression of genes involved in fatty acid uptake, transport, synthesis and metabolism. MATERIALS/METHODS: Human subcutaneous and omental adipose tissues were obtained from lean, overweight and obese normal glucose tolerant (NGT) women and women with GDM. Quantitative RT-PCR (qRT-PCR) was performed to determine the level of expression. Adipose tissue explants were performed to determine the effect of the adipokines TNFα, IL-1ß and leptin on adipose tissue gene expression. RESULTS: Pre-existing maternal obesity and GDM are associated with decreased expression in genes involved in fatty acid uptake and intracellular transport (LPL, FATP2, FATP6, FABPpm and ASCL1), triacylglyceride (TAG) biosynthesis (MGAT1,7 MGAT2 and DGAT1), lipogenesis (FASN) and lipolysis (PNPLA2, HSL and MGLL). Decreased gene expression was also observed for the transcription factors involved in lipid metabolism (LXRα, PPARα, PPARδ, PPARγ, RXRα and SREBP1c). On the other hand, the gene expression of the adipokines TNFα, IL-1ß and or leptin was increased in adipose tissue from obese and GDM women. Functional in vitro studies revealed that these adipokines decreased the gene expression of LPL, FATP2, FATP6, ASCL1, PNPLA2, PPARδ, PPARγ and RXRα. CONCLUSIONS: Pregnancies complicated by pre-existing maternal obesity and GDM are associated with abnormal adipose tissue lipid metabolism, which may play a role in the pathogenesis of these diseases.
Asunto(s)
Adipoquinas/metabolismo , Tejido Adiposo/metabolismo , Diabetes Gestacional/metabolismo , Ácidos Grasos/metabolismo , Metabolismo de los Lípidos/genética , Madres , Obesidad/metabolismo , Efectos Tardíos de la Exposición Prenatal/metabolismo , Adulto , Índice de Masa Corporal , Regulación hacia Abajo , Femenino , Humanos , Lipogénesis/genética , Lipólisis/genética , Masculino , Obesidad/complicaciones , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Regulación hacia ArribaRESUMEN
The oil from flax (Linum usitatissimum L.) has high amounts of α-linolenic acid (ALA; 18:3(cis)(Δ9,12,15)) and is one of the richest sources of omega-3 polyunsaturated fatty acids (ω-3-PUFAs). To produce â¼57% ALA in triacylglycerol (TAG), it is likely that flax contains enzymes that can efficiently transfer ALA to TAG. To test this hypothesis, we conducted a systematic characterization of TAG-synthesizing enzymes from flax. We identified several genes encoding acyl-CoA:diacylglycerol acyltransferases (DGATs) and phospholipid:diacylglycerol acyltransferases (PDATs) from the flax genome database. Due to recent genome duplication, duplicated gene pairs have been identified for all genes except DGAT2-2. Analysis of gene expression indicated that two DGAT1, two DGAT2, and four PDAT genes were preferentially expressed in flax embryos. Yeast functional analysis showed that DGAT1, DGAT2, and two PDAT enzymes restored TAG synthesis when produced recombinantly in yeast H1246 strain. The activity of particular PDAT enzymes (LuPDAT1 and LuPDAT2) was stimulated by the presence of ALA. Further seed-specific expression of flax genes in Arabidopsis thaliana indicated that DGAT1, PDAT1, and PDAT2 had significant effects on seed oil phenotype. Overall, this study indicated the existence of unique PDAT enzymes from flax that are able to preferentially catalyze the synthesis of TAG containing ALA acyl moieties. The identified LuPDATs may have practical applications for increasing the accumulation of ALA and other polyunsaturated fatty acids in oilseeds for food and industrial applications.