RESUMEN
OBJECTIVES: Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride synthesis. DGAT1 is expressed in human enterocytes and is essential for fat absorption. Homozygous DGAT1 deficiency often presents with severe diarrhea and protein-losing enteropathy (PLE) in the 1st weeks of life. Because severe restriction of fat intake controls diarrhea and decreases PLE, total parenteral nutrition (TPN) was the initial standard therapy in infants and children. We present tertiary center experience managing infants and children with DGAT1 deficiency resulting in the development of a nutritional approach that minimizes the use of TPN. METHODS: From 2014 to 2020, 12 infants with DGAT1 deficiency were treated. Stool output, growth, and development, as well as essential fatty acid status, were monitored. This retrospective experience formed the basis for treatment recommendations, which include an ultralow fat formula with intermittent peripheral intravenous lipid infusions during the 1st year of life. RESULTS: All patients with prolonged intestinal fat exposure had PLE, which resolved when treated with the nutrition protocol. Essential fatty acid status as measured by triene:tetraene ratios normalized in all treated patients. Over time, early genetic diagnosis and prompt initiation of an ultralow fat diet with peripheral intravenous lipid infusions replaced the need for TPN. CONCLUSIONS: Children with DGAT1 deficiency respond to dietary restriction of lipids. Management with a novel nutritional approach provides effective treatment for infants with DGAT1 deficiency, treats diarrhea and PLE, promotes growth and development, avoids TPN dependency, and decreases the potential for essential fatty acid deficiency.
Asunto(s)
Diacilglicerol O-Acetiltransferasa , Diarrea , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/deficiencia , Diarrea/dietoterapia , Diarrea/genética , Grasas de la Dieta/administración & dosificación , Homocigoto , Mutación , Nutrición Parenteral Total/métodos , Estudios RetrospectivosRESUMEN
Deficiency in diacylglycerol acyltransferase (DGAT1) is a rare cause of neonatal diarrhea, without a known mechanism or in vitro model. A patient presenting at our institution at 7 weeks of life with failure to thrive and diarrhea was found by whole-exome sequencing to have a homozygous DGAT1 truncation mutation. Duodenal biopsies showed loss of DGAT1 and deficits in apical membrane transporters and junctional proteins in enterocytes. When placed on a very low-fat diet, the patient's diarrhea resolved with normalization of brush border transporter localization in endoscopic biopsies. DGAT1 knockdown in Caco2-BBe cells modeled the deficits in apical trafficking, with loss of apical DPPIV and junctional occludin. Elevation in cellular lipid levels, including diacylglycerol (DAG) and phospholipid metabolites of DAG, was documented by lipid analysis in DGAT1 knockdown cells. Culture of the DGAT1 knockdown cells in lipid-depleted media led to re-establishment of occludin and return of apical DPPIV. DGAT1 loss appears to elicit global changes in enterocyte polarized trafficking that could account for deficits in absorption seen in the patient. The in vitro modeling of this disease should allow for investigation of possible therapeutic targets.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/genética , Diarrea Infantil/genética , Enfermedades del Sistema Digestivo/genética , Células CACO-2 , Preescolar , Diacilglicerol O-Acetiltransferasa/deficiencia , Diacilglicerol O-Acetiltransferasa/metabolismo , Diarrea Infantil/patología , Enfermedades del Sistema Digestivo/patología , Humanos , Lactante , Absorción Intestinal , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patología , Masculino , Transporte de ProteínasRESUMEN
BACKGROUND: Early-onset chronic diarrhoea often indicates a congenital disorder. Mutation in diacylglycerol o-acyltransferase 1 (DGAT1) has recently been linked to early-onset chronic diarrhoea. To date, only a few cases of DGAT1 deficiency have been reported. Diarrhoea in those cases was severe and developed in the neonatal period or within 2 months after birth. CASE PRESENTATION: Here, we report a female patient with DGAT1 mutations with delayed-onset chronic diarrhoea. The patient had vomiting, hypoalbuminemia, hypertriglyceridemia, and failure to thrive at early infancy. Her intractable chronic diarrhoea occurred until she was 8 months of age. A compound heterozygous DGAT1 mutation was found in the patient, which was first found in the Chinese population. Her symptoms and nutrition status improved after nutritional therapy, including a fat restriction diet. CONCLUSIONS: This case expanded our knowledge of the clinical features of patients with DGAT1 mutations. Intractable diarrhoea with delayed onset could also be a congenital disorder.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/genética , Diarrea/genética , Insuficiencia de Crecimiento/genética , Hipertrigliceridemia/genética , Hipoalbuminemia/genética , Mutación , Vómitos/genética , Edad de Inicio , Secuencia de Bases , Diacilglicerol O-Acetiltransferasa/deficiencia , Diarrea/dietoterapia , Diarrea/metabolismo , Diarrea/fisiopatología , Dieta con Restricción de Grasas , Insuficiencia de Crecimiento/dietoterapia , Insuficiencia de Crecimiento/metabolismo , Insuficiencia de Crecimiento/fisiopatología , Femenino , Expresión Génica , Heterocigoto , Humanos , Hipertrigliceridemia/dietoterapia , Hipertrigliceridemia/metabolismo , Hipertrigliceridemia/fisiopatología , Hipoalbuminemia/dietoterapia , Hipoalbuminemia/metabolismo , Hipoalbuminemia/fisiopatología , Lactante , Índice de Severidad de la Enfermedad , Vómitos/dietoterapia , Vómitos/metabolismo , Vómitos/fisiopatologíaRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is characterized by excess lipid accumulation in hepatocytes and represents a huge public health problem owing to its propensity to progress to nonalcoholic steatohepatitis, fibrosis, and liver failure. The lipids stored in hepatic steatosis (HS) are primarily triglycerides (TGs) synthesized by two acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Either DGAT1 or DGAT2 catalyzes this reaction, and these enzymes have been suggested to differentially utilize exogenous or endogenously synthesized fatty acids, respectively. DGAT2 has been linked to storage of fatty acids from de novo lipogenesis, a process increased in NAFLD. However, whether DGAT2 is more responsible for lipid accumulation in NAFLD and progression to fibrosis is currently unknown. Also, it is unresolved whether DGAT2 can be safely inhibited as a therapy for NAFLD. Here, we induced NAFLD-like disease in mice by feeding a diet rich in fructose, saturated fat, and cholesterol and found that hepatocyte-specific Dgat2 deficiency reduced expression of de novo lipogenesis genes and lowered liver TGs by ~70%. Importantly, the reduction in steatosis was not accompanied by increased inflammation or fibrosis, and insulin and glucose metabolism were unchanged. Conclusion: This study suggests that hepatic DGAT2 deficiency successfully reduces diet-induced HS and supports development of DGAT2 inhibitors as a therapeutic strategy for treating NAFLD and preventing downstream consequences.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/fisiología , Hepatitis/etiología , Hepatocitos/enzimología , Cirrosis Hepática Experimental/etiología , Enfermedad del Hígado Graso no Alcohólico/prevención & control , Animales , Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Diacilglicerol O-Acetiltransferasa/deficiencia , Grasas de la Dieta/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Triglicéridos/metabolismoRESUMEN
We investigated whether, in view of its activity being expressed on both aspects of the endoplasmic reticulum (ER; dual membrane topology), diacylglycerol acyltransferase 1 (DGAT1) plays a distinctive role in determining the triglyceride (TAG) content of VLDL particles secreted by the liver. Mice in which the DGAT1 gene was specifically ablated in hepatocytes (DGAT1-LKO mice) had the same number of VLDL particles (apoB concentration) in the plasma 1 h after Triton 1339 treatment, but these particles were approximately half the size of VLDL particles secreted by control mice and had a proportionately decreased content of TAG, with normal cholesterol and cholesteryl ester contents. Analyses of purified microsomal fractions prepared from 16 h fasted control and DAGT1-LKO mice showed that the TAG/protein ratio in the ER was significantly lower in the latter. Electron micrographs of these livers showed that those from DGAT1-LKO mice did not show the increased lipid content of the smooth ER shown by control livers. The effects of DGAT1- and DGAT2-specific inhibitors on apoB secretion by HepG2 cells showed that DGAT1 is not indispensable for apoB secretion and demonstrated redundancy in the ability of the two enzymes to support apoB secretion. Therefore, our findings show that DGAT1 is essential for the complete lipidation and maturation of VLDL particles within the lumen of the ER, consistent with its dual topology within the ER membrane. In the mouse, DGAT2 can support apoB secretion (particle number) even when TAG availability for full VLDL lipidation is restricted in the absence of DGAT1.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/metabolismo , Lipoproteínas VLDL/química , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Tamaño de la Partícula , Animales , Apolipoproteínas B/metabolismo , Diacilglicerol O-Acetiltransferasa/deficiencia , Diacilglicerol O-Acetiltransferasa/genética , Retículo Endoplásmico/metabolismo , Regulación Enzimológica de la Expresión Génica , Técnicas de Inactivación de Genes , Células Hep G2 , Hepatocitos/metabolismo , Humanos , Lipogénesis , Hígado/citología , Ratones , ARN Mensajero/genéticaRESUMEN
Dietary lipids are taken up as FAs by the intestinal epithelium and converted by diacylglycerol acyltransferase (DGAT) enzymes into triglycerides, which are packaged in chylomicrons or stored in cytoplasmic lipid droplets (LDs). DGAT1-deficient patients suffer from vomiting, diarrhea, and protein losing enteropathy, illustrating the importance of this process to intestinal homeostasis. Previously, we have shown that DGAT1 deficiency causes decreased LD formation and resistance to unsaturated FA lipotoxicity in patient-derived intestinal organoids. However, LD formation was not completely abolished in patient-derived organoids, suggesting the presence of an alternative mechanism for LD formation. Here, we show an unexpected role for DGAT2 in lipid metabolism, as DGAT2 partially compensates for LD formation and lipotoxicity in DGAT1-deficient intestinal stem cells. Furthermore, we show that (un)saturated FA-induced lipotoxicity is mediated by ER stress. More importantly, we demonstrate that overexpression of DGAT2 fully compensates for the loss of DGAT1 in organoids, indicating that induced DGAT2 expression in patient cells may serve as a therapeutic target in the future.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/deficiencia , Diacilglicerol O-Acetiltransferasa/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Intestinos/citología , Lípidos/efectos adversos , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Preescolar , Femenino , Humanos , Gotas Lipídicas/efectos de los fármacos , Gotas Lipídicas/metabolismo , MasculinoRESUMEN
BACKGROUND & AIMS: Congenital diarrheal disorders are rare inherited intestinal disorders characterized by intractable, sometimes life-threatening, diarrhea and nutrient malabsorption; some have been associated with mutations in diacylglycerol-acyltransferase 1 (DGAT1), which catalyzes formation of triacylglycerol from diacylglycerol and acyl-CoA. We investigated the mechanisms by which DGAT1 deficiency contributes to intestinal failure using patient-derived organoids. METHODS: We collected blood samples from 10 patients, from 6 unrelated pedigrees, who presented with early-onset severe diarrhea and/or vomiting, hypoalbuminemia, and/or (fatal) protein-losing enteropathy with intestinal failure; we performed next-generation sequencing analysis of DNA from 8 patients. Organoids were generated from duodenal biopsies from 3 patients and 3 healthy individuals (controls). Caco-2 cells and patient-derived dermal fibroblasts were transfected or transduced with vectors that express full-length or mutant forms of DGAT1 or full-length DGAT2. We performed CRISPR/Cas9-guided disruption of DGAT1 in control intestinal organoids. Cells and organoids were analyzed by immunoblot, immunofluorescence, flow cytometry, chromatography, quantitative real-time polymerase chain reaction, and for the activity of caspases 3 and 7. RESULTS: In the 10 patients, we identified 5 bi-allelic loss-of-function mutations in DGAT1. In patient-derived fibroblasts and organoids, the mutations reduced expression of DGAT1 protein and altered triacylglycerol metabolism, resulting in decreased lipid droplet formation after oleic acid addition. Expression of full-length DGAT2 in patient-derived fibroblasts restored formation of lipid droplets. Organoids derived from patients with DGAT1 mutations were more susceptible to lipid-induced cell death than control organoids. CONCLUSIONS: We identified a large cohort of patients with congenital diarrheal disorders with mutations in DGAT1 that reduced expression of its product; dermal fibroblasts and intestinal organoids derived from these patients had altered lipid metabolism and were susceptible to lipid-induced cell death. Expression of full-length wildtype DGAT1 or DGAT2 restored normal lipid metabolism in these cells. These findings indicate the importance of DGAT1 in fat metabolism and lipotoxicity in the intestinal epithelium. A fat-free diet might serve as the first line of therapy for patients with reduced DGAT1 expression. It is important to identify genetic variants associated with congenital diarrheal disorders for proper diagnosis and selection of treatment strategies.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/genética , Duodeno/metabolismo , Fibroblastos/metabolismo , Hipoalbuminemia/genética , Trastornos del Metabolismo de los Lípidos/genética , Organoides/metabolismo , Enteropatías Perdedoras de Proteínas/genética , Células CACO-2 , Estudios de Casos y Controles , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Niño , Preescolar , Consanguinidad , Dermis/citología , Diacilglicerol O-Acetiltransferasa/deficiencia , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Países Bajos , Forboles , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ADN , TurquíaRESUMEN
Diacylglycerol acyltransferase (DGAT) is expressed abundantly in intestine, liver, and adipose tissues. DGAT1 is the crucial and rate-limiting enzyme that mediates the final step in triacylglycerol (TAG) resynthesis during dietary fat absorption. However, too much triacylglycerol (TAG) reserve will lead to genetic obesity (Hubert et al., 2000). DGAT1 knockout mice could survive and displayed a reduction in the postprandial rise of plasma TG, and increased sensitivity of insulin and leptin. Here we report the discovery and characterization of a novel selective DGAT1 inhibitor 29 to potentially treat obesity. Compound 29 showed lipid lowering effect in mouse lipid tolerance test (LTT) and also reduced body weight in DIO mice without observable liver damage.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Grasas de la Dieta/efectos adversos , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacología , Ácidos Grasos Insaturados/farmacología , Obesidad/tratamiento farmacológico , Administración Oral , Aminoácidos Aromáticos , Animales , Disponibilidad Biológica , Peso Corporal/efectos de los fármacos , Diacilglicerol O-Acetiltransferasa/deficiencia , Diacilglicerol O-Acetiltransferasa/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Ácidos Grasos Insaturados/administración & dosificación , Ácidos Grasos Insaturados/química , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Estructura Molecular , Obesidad/metabolismo , Relación Estructura-ActividadRESUMEN
Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is a key enzyme in triacylglycerol (TG) biosynthesis. Here we show that genetic deficiency and pharmacological inhibition of DGAT1 in mice alters cholesterol metabolism. Cholesterol absorption, as assessed by acute cholesterol uptake, was significantly decreased in the small intestine and liver upon DGAT1 deficiency/inhibition. Ablation of DGAT1 in the intestine (I-DGAT1(-/-)) alone is sufficient to cause these effects. Consequences of I-DGAT1 deficiency phenocopy findings in whole-body DGAT1(-/-) and DGAT1 inhibitor-treated mice. We show that deficiency/inhibition of DGAT1 affects cholesterol metabolism via reduced chylomicron size and increased trans-intestinal cholesterol excretion. These effects are independent of cholesterol uptake at the apical surface of enterocytes but mediated through altered dietary fatty acid metabolism. Our findings provide insight into a novel role of DGAT1 and identify a pathway by which intestinal DGAT1 deficiency affects whole-body cholesterol homeostasis in mice. Targeting intestinal DGAT1 may represent a novel approach for treating hypercholesterolemia.
Asunto(s)
Colesterol/metabolismo , Diacilglicerol O-Acetiltransferasa/genética , Hipercolesterolemia/tratamiento farmacológico , Metabolismo de los Lípidos/genética , Triglicéridos/metabolismo , Animales , Diacilglicerol O-Acetiltransferasa/deficiencia , Diacilglicerol O-Acetiltransferasa/metabolismo , Grasas de la Dieta , Ácidos Grasos/metabolismo , Hipercolesterolemia/metabolismo , Absorción Intestinal/genética , Lipogénesis/genética , Hígado/metabolismo , RatonesRESUMEN
OBJECTIVE: States of insulin resistance, hyperinsulinemia, and hepatic steatosis are associated with increased secretion of triglycerides (TG) and apolipoprotein B (apoB), even though insulin targets apoB for degradation. We used hepatic-specific "phosphatase and tensin homologue deleted on chromosome 10" (Pten) knockout (hPten-ko) mice, with increased hepatic insulin signaling, to determine the relative roles of insulin signaling and hepatic TG in regulating apoB secretion. METHODS AND RESULTS: TG and apoB secretion was elevated in hPten-ko mice. When hepatic TG was reduced by inhibition of diacylglycerol acyltransferase 1/diacylglycerol acyltransferase 2 or sterol regulatory element-binding protein-1c, both TG secretion and apoB secretion fell without changes in hepatic insulin signaling. Acute reconstitution of hPten reduced hepatic TG content, and both TG and apoB secretion fell within 4 days despite decreased hepatic insulin signaling. Acute depletion of hepatic Pten by adenoviral introduction of Cre into Pten floxed mice caused steatosis within 4 days, and secretion of both TG and apoB increased despite increased hepatic insulin signaling. Even when steatosis after acute Pten depletion was prevented by pretreatment with SREBP-1c antisense oligonucleotides, apoB secretion was not reduced after 4 days. Ex vivo results were in primary hepatocytes were similar. CONCLUSIONS: Either hepatic TG is the dominant regulator of apoB secretion or any inhibitory effects of hepatic insulin signaling on apoB secretion is very short-lived.
Asunto(s)
Apolipoproteínas B/metabolismo , Insulina/metabolismo , Hígado/metabolismo , Transducción de Señal/fisiología , Triglicéridos/metabolismo , Animales , Diacilglicerol O-Acetiltransferasa/deficiencia , Diacilglicerol O-Acetiltransferasa/genética , Hígado Graso/metabolismo , Lipogénesis/fisiología , Masculino , Ratones , Ratones Noqueados , Modelos Animales , Fosfohidrolasa PTEN/deficiencia , Fosfohidrolasa PTEN/genéticaAsunto(s)
Diacilglicerol O-Acetiltransferasa/genética , Diarrea/etiología , Enfermedades Genéticas Congénitas/diagnóstico , Enteropatías Perdedoras de Proteínas/etiología , Raquitismo/etiología , Desequilibrio Hidroelectrolítico/etiología , Diacilglicerol O-Acetiltransferasa/deficiencia , Diarrea/congénito , Femenino , Enfermedades Genéticas Congénitas/complicaciones , Marcadores Genéticos , Humanos , LactanteRESUMEN
Microsomal triglyceride transfer protein (Mtp) inhibitors represent a novel therapeutic approach to lower circulating LDL cholesterol, although therapeutic development has been hindered by the observed increase in hepatic triglycerides and liver steatosis following treatment. Here, we used small interfering RNAs (siRNA) targeting Mtp to achieve target-specific silencing to study this phenomenon and to determine to what extent liver steatosis is induced by changes in Mtp expression. We observed that Mtp silencing led to a decrease in many genes involved in hepatic triglyceride synthesis. Given the role of diacylglycerol O-acyltransferase 2 (Dgat2) in regulating hepatic triglyceride synthesis, we then evaluated whether target-specific silencing of both Dgat2 and Mtp were sufficient to attenuate Mtp silencing-induced liver steatosis. We showed that the simultaneous inhibition of Dgat2 and Mtp led to a decrease in plasma cholesterol and a reduction in the accumulation of hepatic triglycerides caused by the inhibition of Mtp. Collectively, these findings provide a proof-of-principle for a triglyceride synthesis/Mtp inhibitor combination and represent a potentially novel approach for therapeutic development in which targeting multiple pathways can achieve the desired response.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diacilglicerol O-Acetiltransferasa/deficiencia , Diacilglicerol O-Acetiltransferasa/genética , Hígado Graso/genética , Silenciador del Gen , ARN Interferente Pequeño/genética , Animales , Apolipoproteínas B/deficiencia , Apolipoproteínas B/genética , Colesterol/sangre , Hígado Graso/sangre , Hígado Graso/enzimología , Hígado Graso/metabolismo , Hígado/metabolismo , Masculino , Ratones , Triglicéridos/metabolismoRESUMEN
Acyl CoA:diacylglycerol acyltransferase (DGAT) 1 catalyzes the final step of triglyceride (TG) synthesis. We show that acute administration of a DGAT1 inhibitor (DGAT1i) by oral gavage or genetic deletion of intestinal Dgat1 (intestine-Dgat1(-/-)) markedly reduced postprandial plasma TG and retinyl ester excursions by inhibiting chylomicron secretion in mice. Loss of DGAT1 activity did not affect the efficiency of retinol esterification, but it did reduce TG and retinoid accumulation in the small intestine. In contrast, inhibition of microsomal triglyceride transfer protein (MTP) reduced chylomicron secretion after oral fat/retinol loads, but with accumulation of dietary TG and retinoids in the small intestine. Lack of intestinal accumulation of TG and retinoids in DGAT1i-treated or intestine-Dgat1(-/-) mice resulted, in part, from delayed gastric emptying associated with increased plasma levels of glucagon-like peptide (GLP)-1. However, neither bypassing the stomach through duodenal oil injection nor inhibiting the receptor for GLP-1 normalized postprandial TG or retinyl esters excursions in the absence of DGAT1 activity. In summary, intestinal DGAT1 inhibition or deficiency acutely delayed gastric emptying and inhibited chylomicron secretion; however, the latter occurred when gastric emptying was normal or when lipid was administered directly into the small intestine. Long-term hepatic retinoid metabolism was not impacted by DGAT1 inhibition.
Asunto(s)
Quilomicrones/metabolismo , Diacilglicerol O-Acetiltransferasa/deficiencia , Diacilglicerol O-Acetiltransferasa/metabolismo , Vaciamiento Gástrico/genética , Periodo Posprandial/fisiología , Triglicéridos/metabolismo , Animales , Carbamatos/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/metabolismo , Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Diacilglicerol O-Acetiltransferasa/genética , Vaciamiento Gástrico/efectos de los fármacos , Péptido 1 Similar al Glucagón/sangre , Receptor del Péptido 1 Similar al Glucagón , Indoles/farmacología , Masculino , Ratones , Ratones Mutantes , Fragmentos de Péptidos/farmacología , Periodo Posprandial/genética , Receptores de Glucagón/antagonistas & inhibidores , Retinoides/metabolismo , Triglicéridos/sangreRESUMEN
Triacylglycerols (TG) are the major storage molecules of metabolic energy and fatty acids in several tissues. The final step in TG biosynthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. Lack of whole body DGAT1 is associated with reduced lipid-induced inflammation. Since one major component of atherosclerosis is chronic inflammation we hypothesized that DGAT1 deficiency might ameliorate atherosclerotic lesion development. We therefore crossbred Apolipoprotein E-deficient (ApoE(-/-)) mice with Dgat1(-/-) mice. ApoE(-/-) and ApoE(-/-)Dgat1(-/-) mice were fed Western-type diet (WTD) for 9weeks and thereafter examined for plaque formation. The mean atherosclerotic lesion area was substantially reduced in ApoE(-/-)Dgat1(-/-) compared with ApoE(-/-) mice in en face and aortic valve section analyses. The reduced lesion size was associated with decreased cholesterol uptake and absorption by the intestine, reduced plasma TG and cholesterol concentrations and increased cholesterol efflux from macrophages. The expression of adhesion molecules was reduced in aortas of ApoE(-/-)Dgat1(-/-) mice, which might be the reason for less migration capacities of monocytes and macrophages and the observed decreased amount of macrophages within the plaques. From our results we conclude that the lack of DGAT1 is atheroprotective, implicating an additional application of DGAT1 inhibitors with regard to maintaining cholesterol homeostasis and attenuating atherosclerosis.
Asunto(s)
Aorta/metabolismo , Apolipoproteínas E/deficiencia , Aterosclerosis , Colesterol/sangre , Diacilglicerol O-Acetiltransferasa/deficiencia , Placa Aterosclerótica/sangre , Triglicéridos/sangre , Acilcoenzima A/sangre , Animales , Aorta/patología , Apolipoproteínas E/genética , Aterosclerosis/sangre , Aterosclerosis/enzimología , Aterosclerosis/genética , Movimiento Celular/genética , Células Cultivadas , Cruzamientos Genéticos , Diacilglicerol O-Acetiltransferasa/genética , Modelos Animales de Enfermedad , Femenino , Humanos , Inmunohistoquímica , Absorción Intestinal/genética , Mucosa Intestinal/metabolismo , Metabolismo de los Lípidos/genética , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Placa Aterosclerótica/patologíaRESUMEN
Diacylglycerol (DAG) acyl transferase 1 (Dgat1) knockout ((-/-)) mice are resistant to high-fat-induced obesity and insulin resistance, but the reasons are unclear. Dgat1(-/-) mice had reduced mRNA levels of all three Ppar genes and genes involved in fatty acid oxidation in the myocardium of Dgat1(-/-) mice. Although DGAT1 converts DAG to triglyceride (TG), tissue levels of DAG were not increased in Dgat1(-/-) mice. Hearts of chow-diet Dgat1(-/-) mice were larger than those of wild-type (WT) mice, but cardiac function was normal. Skeletal muscles from Dgat1(-/-) mice were also larger. Muscle hypertrophy factors phospho-AKT and phospho-mTOR were increased in Dgat1(-/-) cardiac and skeletal muscle. In contrast to muscle, liver from Dgat1(-/-) mice had no reduction in mRNA levels of genes mediating fatty acid oxidation. Glucose uptake was increased in cardiac and skeletal muscle in Dgat1(-/-) mice. Treatment with an inhibitor specific for DGAT1 led to similarly striking reductions in mRNA levels of genes mediating fatty acid oxidation in cardiac and skeletal muscle. These changes were reproduced in cultured myocytes with the DGAT1 inhibitor, which also blocked the increase in mRNA levels of Ppar genes and their targets induced by palmitic acid. Thus, loss of DGAT1 activity in muscles decreases mRNA levels of genes involved in lipid uptake and oxidation.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/metabolismo , Músculo Esquelético/metabolismo , Miocardio/metabolismo , Receptores Activados del Proliferador del Peroxisoma/genética , Absorciometría de Fotón , Animales , Western Blotting , Línea Celular , Ceramidas/metabolismo , Diacilglicerol O-Acetiltransferasa/deficiencia , Diacilglicerol O-Acetiltransferasa/genética , Diglicéridos/metabolismo , Ecocardiografía , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Glucosa/metabolismo , Lipoproteínas VLDL/metabolismo , Masculino , Ratones , Ratones Noqueados , Mioblastos/efectos de los fármacos , Mioblastos/metabolismo , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Tamaño de los Órganos/genética , Ácido Palmítico/farmacología , Reacción en Cadena de la PolimerasaRESUMEN
Deletion of the acyltransferases responsible for triglyceride and steryl ester synthesis in Saccharomyces cerevisiae serves as a genetic model of diseases where lipid overload is a component. The yeast mutants lack detectable neutral lipids and cytoplasmic lipid droplets and are strikingly sensitive to unsaturated fatty acids. Expression of human diacylglycerol acyltransferase 2 in the yeast mutants was sufficient to reverse these phenotypes. Similar to mammalian cells, fatty acid-mediated death in yeast is apoptotic and presaged by transcriptional induction of stress-response pathways, elevated oxidative stress, and activation of the unfolded protein response. To identify pathways that protect cells from lipid excess, we performed genetic interaction and transcriptional profiling screens with the yeast acyltransferase mutants. We thus identified diacylglycerol kinase-mediated phosphatidic acid biosynthesis and production of phosphatidylcholine via methylation of phosphatidylethanolamine as modifiers of lipotoxicity. Accordingly, the combined ablation of phospholipid and triglyceride biosynthesis increased sensitivity to saturated fatty acids. Similarly, normal sphingolipid biosynthesis and vesicular transport were required for optimal growth upon denudation of triglyceride biosynthesis and also mediated resistance to exogenous fatty acids. In metazoans, many of these processes are implicated in insulin secretion thus linking lipotoxicity with early aspects of pancreatic beta-cell dysfunction, diabetes, and the metabolic syndrome.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/deficiencia , Ácidos Grasos/toxicidad , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología , Esteroles/metabolismo , Muerte Celular/efectos de los fármacos , Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Ácidos Grasos/metabolismo , Regulación Fúngica de la Expresión Génica , Humanos , Viabilidad Microbiana , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Increased fat deposition in skeletal muscle is associated with insulin resistance. However, exercise increases both intramyocellular fat stores and insulin sensitivity, a phenomenon referred to as "the athlete's paradox". In this study, we provide evidence that augmenting triglyceride synthesis in skeletal muscle is intrinsically connected with increased insulin sensitivity. Exercise increased diacylglycerol (DAG) acyltransferase (DGAT) activity in skeletal muscle. Channeling fatty acid substrates into TG resulted in decreased DAG and ceramide levels. Transgenic overexpression of DGAT1 in mouse skeletal muscle replicated these findings and protected mice against high-fat diet-induced insulin resistance. Moreover, in isolated muscle, DGAT1 deficiency exacerbated insulin resistance caused by fatty acids, whereas DGAT1 overexpression mitigated the detrimental effect of fatty acids. The heightened insulin sensitivity in the transgenic mice was associated with attenuated fat-induced activation of DAG-responsive PKCs and the stress mediator JNK1. Consistent with these changes, serine phosphorylation of insulin receptor substrate 1 was reduced, and Akt activation and glucose 4 membrane translocation were increased. In conclusion, upregulation of DGAT1 in skeletal muscle is sufficient to recreate the athlete's paradox and illustrates a mechanism of exercise-induced enhancement of muscle insulin sensitivity. Thus, increasing muscle DGAT activity may offer a new approach to prevent and treat insulin resistance and type 2 diabetes mellitus.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/genética , Diacilglicerol O-Acetiltransferasa/metabolismo , Resistencia a la Insulina/fisiología , Músculo Esquelético/metabolismo , Triglicéridos/biosíntesis , Animales , Secuencia de Bases , Cartilla de ADN/genética , Diabetes Mellitus Tipo 2/prevención & control , Diacilglicerol O-Acetiltransferasa/deficiencia , Grasas de la Dieta/administración & dosificación , Humanos , Resistencia a la Insulina/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Esfuerzo Físico/fisiología , Regulación hacia ArribaRESUMEN
UNLABELLED: Nonalcoholic fatty liver disease, characterized by the accumulation of triacylglycerols (TGs) and other lipids in the liver, often accompanies obesity and is a risk factor for nonalcoholic steatohepatitis and fibrosis. To treat or prevent fatty liver, a thorough understanding of hepatic fatty acid and TG metabolism is crucial. To investigate the role of acyl CoA:diacylglycerol acyltransferase 1 (DGAT1), a key enzyme of TG synthesis, in fatty liver development, we studied mice with global and liver-specific knockout of Dgat1. DGAT1 was required for hepatic steatosis induced by a high-fat diet and prolonged fasting, which are both characterized by delivery of exogenous fatty acids to the liver. Studies in primary hepatocytes showed that DGAT1 deficiency protected against hepatic steatosis by reducing synthesis and increasing the oxidation of fatty acids. In contrast, lipodystrophy (aP2-SREBP-1c436) and liver X receptor activation (T0901317), which increase de novo fatty acid synthesis in liver, caused steatosis independently of DGAT1. Pharmacologic inhibition of Dgat1 with antisense oligonucleotides protected against fatty liver induced by a high-fat diet. CONCLUSION: Our findings identify a specific role for hepatic DGAT1 in esterification of exogenous fatty acids and indicate that DGAT1 contributes to hepatic steatosis induced by this mechanism.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/deficiencia , Ácidos Grasos/efectos adversos , Hígado Graso/enzimología , Animales , Grasas de la Dieta/efectos adversos , Ayuno/efectos adversos , Ácidos Grasos/administración & dosificación , Ácidos Grasos/biosíntesis , Hígado Graso/etiología , Hígado Graso/prevención & control , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Oligonucleótidos Antisentido/farmacologíaRESUMEN
Brown adipocytes store metabolic energy as triglycerides (TGs) in lipid droplets (LDs). Fatty acids released from brown adipocyte LDs by lipolysis are thought to activate and fuel UCP1-mediated thermogenesis. Here, we test this hypothesis by preventing fatty acid storage in murine brown adipocytes through brown adipose tissue (BAT)-specific deletions of the TG synthesis enzymes DGAT1 and DGAT2 (BA-DGAT KO). Despite the absence of TGs in brown adipocytes, BAT is functional, and BA-DGAT-KO mice maintain euthermia during acute or chronic cold exposure. As apparent adaptations to the lack of TG, brown adipocytes of BA-DGAT-KO mice appear to use circulating glucose and fatty acids, and stored glycogen, to fuel thermogenesis. Moreover, BA-DGAT-KO mice are resistant to diet-induced glucose intolerance, likely because of increased glucose disposal by BAT. We conclude that TGs in BAT are dispensable for its contribution to cold-induced thermogenesis, at least when other fuel sources are available.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Frío , Gotas Lipídicas/metabolismo , Termogénesis , Adaptación Fisiológica , Adipocitos Beige/metabolismo , Adipocitos Marrones/metabolismo , Animales , Diacilglicerol O-Acetiltransferasa/deficiencia , Diacilglicerol O-Acetiltransferasa/metabolismo , Dieta Alta en Grasa , Intolerancia a la Glucosa/patología , Ratones Noqueados , Triglicéridos/metabolismoRESUMEN
Acyl-CoA:diacylglycerol acyltransferases (DGATs) are enzymes that catalyze the formation of triglyceride (TG) from acyl-CoA and diacylglycerol. Two DGATs have been identified which belong to two distinct gene families and both are ubiquitously expressed. DGAT2 knockout mice are lipopenic and die shortly after birth. In the current study, wild type mice were treated with increasing doses (25-60 mg/kg twice weekly) of a DGAT2 gene-specific antisense oligonucleotide (ASO). Treatment resulted in a dose dependent decrease in hepatic DGAT2 gene expression (up to 80%) which was associated with a 40% decrease in hepatic DGAT2 activity and a 45% decrease in hepatic TG. Decreased levels of DGAT2 resulted in a significant dose dependent decrease in VLDL TG secretion (up to 52%) and reduced plasma TG, total cholesterol, and ApoB. Similar results were obtained when DGAT1 KO mice were treated with the DGAT2 ASO. Treatment of ob/ob mice with the DGAT2 ASO resulted in significant decreases in weight gain (10%), adipose weight (25%) and hepatic TG content (80%). Our findings indicate that the majority of TG destined for secretion by liver is synthesized by DGAT2 and suggests that DGAT2 may be a therapeutic target for treatment of hypertriglyceridemia, hepatic steatosis and obesity.