RESUMEN
Sterol regulatory element-binding proteins (SREBPs) activate genes involved in the synthesis and trafficking of cholesterol and other lipids and are critical for maintaining lipid homeostasis. Aberrant SREBP activity, however, can contribute to obesity, fatty liver disease, and insulin resistance, hallmarks of metabolic syndrome. Our studies identify a conserved regulatory circuit in which SREBP-1 controls genes in the one-carbon cycle, which produces the methyl donor S-adenosylmethionine (SAMe). Methylation is critical for the synthesis of phosphatidylcholine (PC), a major membrane component, and we find that blocking SAMe or PC synthesis in C. elegans, mouse liver, and human cells causes elevated SREBP-1-dependent transcription and lipid droplet accumulation. Distinct from negative regulation of SREBP-2 by cholesterol, our data suggest a feedback mechanism whereby maturation of nuclear, transcriptionally active SREBP-1 is controlled by levels of PC. Thus, nutritional or genetic conditions limiting SAMe or PC production may activate SREBP-1, contributing to human metabolic disorders.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular Tumoral , Retículo Endoplásmico/metabolismo , Humanos , Lipogénesis , Ratones , Modelos Animales , Fosfatidilcolinas/biosíntesis , Interferencia de ARN , S-Adenosilmetionina/biosíntesisRESUMEN
Phosphatidylethanolamine (PE) N-methyltransferase (PEMT) accounts for â¼30% of hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet are protected against diet-induced obesity (DIO) and insulin resistance (IR) but develop nonalcoholic fatty liver disease (NAFLD) associated with a decreased PC:PE ratio. We investigated whether the lack of hepatic PEMT or the lack of PEMT in other tissues (where it is expressed at low levels) is responsible for or contributes to the protection against DIO and IR in Pemt-/- mice. Furthermore, we investigated whether decreasing PEMT expression with antisense oligonucleotides (ASOs) would result in metabolic benefits in both lean and obese mice without negatively impacting liver health. We both restored hepatic PEMT in Pemt-/- mice via adeno-associated virus delivery and decreased hepatic PEMT with ASOs in wild-type and ob/ob mice. Weight gain, insulin sensitivity, and indices of liver function were determined. We report that the protection against DIO and IR and the development of NAFLD is dependent on hepatic PEMT activity. NAFLD, associated with a significant decrease in the hepatic PC:PE ratio, was exacerbated by PEMT deficiency in obese mice, suggesting that phospholipid insufficiency promotes NAFLD progression during obesity or overnutrition. Hepatic PEMT is critical for maintaining phospholipid balance, which is crucial for a healthy liver.-Wan, S., van der Veen, J. N., Bakala N'Goma, J.-C., Nelson, R. C., Vance, D. E., Jacobs, R. L. Hepatic PEMT activity mediates liver health, weight gain, and insulin resistance.
Asunto(s)
Resistencia a la Insulina/fisiología , Hígado/metabolismo , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Animales , Dieta Alta en Grasa , Ratones Transgénicos , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Obesidad/metabolismo , Fosfatidiletanolamina N-Metiltransferasa/genética , Fosfatidiletanolaminas/metabolismoRESUMEN
Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet are protected from obesity and whole-body insulin resistance. However, Pemt-/- mice develop severe nonalcoholic steatohepatitis (NASH). Because NASH is often associated with hepatic insulin resistance, we investigated whether the increased insulin sensitivity in Pemt-/- mice was restricted to nonhepatic tissues or whether the liver was also insulin sensitive. Strikingly, the livers of Pemt-/- mice compared with those of Pemt+/+ mice were not insulin resistant, despite elevated levels of hepatic triacylglycerols and diacylglycerols, as well as increased hepatic inflammation and fibrosis. Endogenous glucose production was lower in Pemt-/- mice under both basal and hyperinsulinemic conditions. Experiments in primary hepatocytes and hepatoma cells revealed improved insulin signaling in the absence of PEMT, which was not due to changes in diacylglycerols, ceramides, or gangliosides. On the other hand, the phospholipid composition in hepatocytes seems critically important for insulin signaling such that lowering the PC:phosphatidylethanolamine (PE) ratio improves insulin signaling. Thus, treatments to reduce the PC:PE ratio in liver may protect against the development of hepatic insulin resistance.-Van der Veen, J. N., Lingrell, S., McCloskey, N., LeBlond, N. D., Galleguillos, D., Zhao, Y. Y., Curtis, J. M., Sipione, S., Fullerton, M. D., Vance, D. E., Jacobs, R. L. A role for phosphatidylcholine and phosphatidylethanolamine in hepatic insulin signaling.
Asunto(s)
Insulina/metabolismo , Hígado/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Animales , Resistencia a la Insulina/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Transducción de Señal/fisiologíaRESUMEN
The pioneering work of Eugene Kennedy in the 1950s established the choline pathway for phosphatidylcholine (PC) biosynthesis. However, the regulation of PC biosynthesis was poorly understood at that time. When I started my lab at the University of British Columbia in the 1970s, this was the focus of my research. This article provides my reflections on these studies that began with enzymology and the use of cultured mammalian cells, and progressed to utilize the techniques of molecular biology and gene-targeted mice. The research in my lab and others demonstrated that the regulated and rate-limiting step in the choline pathway for PC biosynthesis was catalyzed by CTP:phosphocholine cytidylyltransferase. This enzyme is regulated by its movement from a soluble form (largely in the nucleus) to a membrane-associated form where the enzyme becomes activated. Gene targeting in mice subsequently demonstrated that this gene is essential for development of mouse embryos. The other mammalian pathway for PC biosynthesis is catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT) that converts phosphatidylethanolamine to PC. Understanding of the regulation and function of the integral membrane protein PEMT was improved when the enzyme was purified (a masochistic endeavor) in 1987, leading to the cloning of the Pemt cDNA. Generation of knock-out mice that lacked PEMT showed that they were protected from atherosclerosis, diet-induced obesity, and insulin resistance. The protection from atherosclerosis appears to be due to decreased secretion of lipoproteins from the liver. We continue to investigate the mechanism(s) by which Pemt-/- mice are protected from weight gain and insulin resistance.
Asunto(s)
Aterosclerosis , Citidililtransferasa de Colina-Fosfato , Resistencia a la Insulina , Obesidad , Fosfatidilcolinas , Fosfatidiletanolamina N-Metiltransferasa , Animales , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Citidililtransferasa de Colina-Fosfato/genética , Citidililtransferasa de Colina-Fosfato/metabolismo , Dieta/efectos adversos , Embrión de Mamíferos/enzimología , Embrión de Mamíferos/patología , Marcación de Gen , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Ratones , Ratones Noqueados , Obesidad/inducido químicamente , Obesidad/enzimología , Obesidad/genética , Obesidad/patología , Fosfatidilcolinas/biosíntesis , Fosfatidilcolinas/genética , Fosfatidiletanolamina N-Metiltransferasa/genética , Fosfatidiletanolamina N-Metiltransferasa/metabolismoRESUMEN
Mice lacking phosphatidylethanolamine N-methyltransferase (PEMT) are protected from high-fat diet (HFD)-induced obesity and insulin resistance. However, these mice develop severe nonalcoholic fatty liver disease (NAFLD) when fed the HFD, which is mainly due to inadequate secretion of VLDL particles. Our aim was to prevent NAFLD development in mice lacking PEMT. We treated Pemt-/- mice with either ezetimibe or fenofibrate to see if either could ameliorate liver disease in these mice. Ezetimibe treatment did not reduce fat accumulation in Pemt-/- livers, nor did it reduce markers for hepatic inflammation or fibrosis. Fenofibrate, conversely, completely prevented the development of NAFLD in Pemt-/- mice: hepatic lipid levels, as well as markers of endoplasmic reticulum stress, inflammation, and fibrosis, in fenofibrate-treated Pemt-/- mice were similar to those in Pemt+/+ mice. Importantly, Pemt-/- mice were still protected against HFD-induced obesity and insulin resistance. Moreover, fenofibrate partially reversed hepatic steatosis and fibrosis in Pemt-/- mice when treatment was initiated after NAFLD had already been established. Increasing hepatic fatty acid oxidation can compensate for the lower VLDL-triacylglycerol secretion rate and prevent/reverse fatty liver disease in mice lacking PEMT.
Asunto(s)
Fenofibrato/administración & dosificación , Enfermedad del Hígado Graso no Alcohólico/tratamiento farmacológico , Obesidad/tratamiento farmacológico , Fosfatidiletanolamina N-Metiltransferasa/genética , Animales , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Ezetimiba/administración & dosificación , Humanos , Resistencia a la Insulina/genética , Metabolismo de los Lípidos/efectos de los fármacos , Lipoproteínas VLDL/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Ratones , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/genética , Obesidad/metabolismo , Obesidad/patología , Oxidación-Reducción/efectos de los fármacos , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Triglicéridos/metabolismoRESUMEN
Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC) in the liver. Mice lacking PEMT are protected from high-fat diet-induced obesity and insulin resistance, and exhibit increased whole-body energy expenditure and oxygen consumption. Since skeletal muscle is a major site of fatty acid oxidation and energy utilization, we determined if rates of fatty acid oxidation/oxygen consumption in muscle are higher in Pemt(-/-) mice than in Pemt(+/+) mice. Although PEMT is abundant in the liver, PEMT protein and activity were undetectable in four types of skeletal muscle. Moreover, amounts of PC and PE in the skeletal muscle were not altered by PEMT deficiency. Thus, we concluded that any influence of PEMT deficiency on skeletal muscle would be an indirect consequence of lack of PEMT in liver. Neither the in vivo rate of fatty acid uptake by muscle nor the rate of fatty acid oxidation in muscle explants and cultured myocytes depended upon Pemt genotype. Nor did PEMT deficiency increase oxygen consumption or respiratory function in skeletal muscle mitochondria. Thus, the increased whole body oxygen consumption in Pemt(-/-) mice, and resistance of these mice to diet-induced weight gain, are not primarily due to increased capacity of skeletal muscle for utilization of fatty acids as an energy source.
Asunto(s)
Ácidos Grasos/metabolismo , Hígado/enzimología , Músculo Esquelético/enzimología , Obesidad/enzimología , Fosfatidilcolinas/metabolismo , Fosfatidiletanolamina N-Metiltransferasa/deficiencia , Fosfatidiletanolaminas/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Grasas de la Dieta/efectos adversos , Metabolismo Energético , Expresión Génica , Resistencia a la Insulina , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/enzimología , Células Musculares/citología , Células Musculares/enzimología , Obesidad/etiología , Obesidad/genética , Oxidación-Reducción , Consumo de Oxígeno , Fosfatidiletanolamina N-Metiltransferasa/genética , Cultivo Primario de CélulasRESUMEN
Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) are the most abundant phospholipids in all mammalian cell membranes. In the 1950s, Eugene Kennedy and co-workers performed groundbreaking research that established the general outline of many of the pathways of phospholipid biosynthesis. In recent years, the importance of phospholipid metabolism in regulating lipid, lipoprotein and whole-body energy metabolism has been demonstrated in numerous dietary studies and knockout animal models. The purpose of this review is to highlight the unappreciated impact of phospholipid metabolism on health and disease. Abnormally high, and abnormally low, cellular PC/PE molar ratios in various tissues can influence energy metabolism and have been linked to disease progression. For example, inhibition of hepatic PC synthesis impairs very low density lipoprotein secretion and changes in hepatic phospholipid composition have been linked to fatty liver disease and impaired liver regeneration after surgery. The relative abundance of PC and PE regulates the size and dynamics of lipid droplets. In mitochondria, changes in the PC/PE molar ratio affect energy production. We highlight data showing that changes in the PC and/or PE content of various tissues are implicated in metabolic disorders such as atherosclerosis, insulin resistance and obesity. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá.
Asunto(s)
Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Animales , Hígado Graso Alcohólico/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Lipoproteínas VLDL/metabolismo , Hígado/metabolismo , Regeneración Hepática , Enfermedades Metabólicas/metabolismo , Mitocondrias/metabolismo , Músculo Esquelético/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismoRESUMEN
BACKGROUND & AIMS: Endoplasmic reticulum (ER) stress is associated with development of steatohepatitis. Phosphatidylethanolamine N-methyltransferase (PEMT) is a hepatic enzyme located on the ER and mitochondria-associated membranes and catalyzes phosphatidylcholine (PC) synthesis via methylation of phosphatidylethanolamine (PE). We hypothesized that PEMT deficiency in mice alters ER phospholipid content, thereby inducing ER stress and sensitizing the mice to diet-induced steatohepatitis. METHODS: PC and PE mass were measured in hepatic ER fractions from chow-fed and high fat-fed Pemt(-/-) and Pemt(+/+) mice. Proteins implicated in ER stress and the unfolded protein response (UPR) were assessed in mouse livers and in McArdle-RH7777 hepatoma cells that expressed or lacked PEMT. The chemical chaperone 4-phenyl butyric acid was administered to cells and HF-fed Pemt(-/-) mice to alleviate ER stress. RESULTS: In chow-fed Pemt(-/-) mice, the hepatic PC/PE ratio in the ER was lower than in Pemt(+/+) mice, and levels of ER stress markers, CHOP and BIP, were higher without activation of the UPR. In livers of HF-fed Pemt(-/-) mice the ER had a lower PC/PE ratio, and exhibited more ER stress and UPR activation. Similarly, the UPR was repressed in McArdle cells expressing PEMT compared with those lacking PEMT, with concomitantly lower levels of CHOP and BIP. 4-Phenyl butyric acid attenuated activation of the UPR and ER stress in McArdle cells lacking PEMT, but not the hepatic ER stress in HF-fed Pemt(-/-) mice. CONCLUSION: PEMT deficiency reduces the PC/PE ratio in the ER and induces ER stress, which sensitizes the mice to HF-induced steatohepatitis.
RESUMEN
Mice lacking phosphatidylethanolamine N-methyltransferase (PEMT, Pemt(-/-) mice) are resistant to high-fat diet (HFD)-induced obesity (DIO) but develop non-alcoholic steatohepatitis. PEMT expression is strongly induced during differentiation of 3T3-L1 adipocytes. Hence, we hypothesized that white adipose tissue (WAT) might be a key player in the protection against DIO in Pemt(-/-) mice. We fed Pemt(-/-) and Pemt(+/+) mice the HFD for 2 weeks, after which we examined adipocyte differentiation, adipogenesis and lipolysis in WAT. Pemt(-/-) mice gained less body weight, had reduced WAT mass and had smaller adipocytes than Pemt(+/+) mice. The protein levels of adipose differentiation markers FABP4, PPARγ and C/EBPß were not altered by genotype, but acetyl-CoA carboxylase expression and activation was reduced in the Pemt(-/-) mice. The in vivo conversion of [¹4C]acetate to [¹4C]TG in WAT was also lower in Pemt(-/-) mice. The release of glycerol from WAT explants was comparable between Pemt(+/+) and Pemt(-/-) mice under basal condition and in the presence of isoproterenol, indicating unaffected lipolytic capacity. Furthermore, the amounts of leptin, cytokines and chemokines in WAT were not altered by genotype in mice fed the HFD for 2 weeks. However, after 10 weeks of HFD, WAT from Pemt(-/-) mice had dramatically lower leptin, inflammatory cytokines (IL-1 and TNF-α) and chemokines (MCP-1 and RANTES), and significantly higher anti-inflammatory cytokine IL-10 than Pemt(+/+) mice. Together, our data show that PEMT deficiency did not affect the capability for differentiation and lipolysis in WAT. Decreased lipogenesis in WAT may contribute to the resistance to DIO in Pemt(-/-) mice.
Asunto(s)
Tejido Adiposo Blanco/enzimología , Dieta Alta en Grasa , Lipogénesis , Obesidad/prevención & control , Fosfatidiletanolamina N-Metiltransferasa/deficiencia , Adipocitos Blancos/enzimología , Adipogénesis , Tejido Adiposo Blanco/fisiopatología , Adiposidad , Animales , Biomarcadores/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animales de Enfermedad , Regulación hacia Abajo , Genotipo , Lípidos/sangre , Lipólisis , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/sangre , Obesidad/enzimología , Obesidad/genética , Obesidad/fisiopatología , Fenotipo , Fosfatidiletanolamina N-Metiltransferasa/genética , Factores Protectores , Factores de Tiempo , Aumento de PesoRESUMEN
Phosphatidylethanolamine N-methyltransferase (PEMT) is an important enzyme in hepatic phosphatidylcholine (PC) biosynthesis. Pemt(-/-) mice are protected against high-fat diet (HFD)-induced obesity and insulin resistance; however, these mice develop nonalcoholic fatty liver disease (NAFLD). We hypothesized that peroxisomal proliferator-activated receptor-γ (PPARγ) activation by pioglitazone might stimulate adipocyte proliferation, thereby directing lipids from the liver toward white adipose tissue. Pioglitazone might also act directly on PPARγ in the liver to improve NAFLD. Pemt(+/+) and Pemt(-/-) mice were fed a HFD with or without pioglitazone (20 mg·kg(-1)·day(-1)) for 10 wk. Pemt(-/-) mice were protected from HFD-induced obesity but developed NAFLD. Treatment with pioglitazone caused an increase in body weight gain in Pemt(-/-) mice that was mainly due to increased adiposity. Moreover, pioglitazone improved NAFLD in Pemt(-/-) mice, as indicated by a 35% reduction in liver weight and a 57% decrease in plasma alanine transaminase levels. Livers from HFD-fed Pemt(-/-) mice were steatotic, inflamed, and fibrotic. Hepatic steatosis was still evident in pioglitazone-treated Pemt(-/-) mice; however, treatment with pioglitazone reduced hepatic fibrosis, as evidenced by reduced Sirius red staining and lowered mRNA levels of collagen type Iα1 (Col1a1), tissue inhibitor of metalloproteinases 1 (Timp1), α-smooth muscle actin (Acta2), and transforming growth factor-ß (Tgf-ß). Similarly, oxidative stress and inflammation were reduced in livers from Pemt(-/-) mice upon treatment with pioglitazone. Together, these data show that activation of PPARγ in HFD-fed Pemt(-/-) mice improved liver function, while these mice were still protected against diet-induced obesity and insulin resistance.
Asunto(s)
Antiinfecciosos/farmacología , Hepatitis/prevención & control , Cirrosis Hepática Experimental/prevención & control , Hígado/efectos de los fármacos , Enfermedad del Hígado Graso no Alcohólico/prevención & control , PPAR gamma/agonistas , Fosfatidiletanolamina N-Metiltransferasa/deficiencia , Tiazolidinedionas/farmacología , Actinas/genética , Actinas/metabolismo , Adipocitos Blancos/efectos de los fármacos , Adipocitos Blancos/enzimología , Adipocitos Blancos/patología , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/enzimología , Tejido Adiposo Blanco/patología , Adiposidad/efectos de los fármacos , Animales , Proliferación Celular/efectos de los fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Dieta Alta en Grasa , Predisposición Genética a la Enfermedad , Hepatitis/enzimología , Hepatitis/genética , Hepatitis/patología , Resistencia a la Insulina , Hígado/enzimología , Hígado/patología , Cirrosis Hepática Experimental/enzimología , Cirrosis Hepática Experimental/genética , Cirrosis Hepática Experimental/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Enfermedad del Hígado Graso no Alcohólico/enzimología , Enfermedad del Hígado Graso no Alcohólico/genética , Enfermedad del Hígado Graso no Alcohólico/patología , Obesidad/enzimología , Obesidad/genética , Obesidad/prevención & control , Estrés Oxidativo/efectos de los fármacos , PPAR gamma/metabolismo , Fenotipo , Fosfatidiletanolamina N-Metiltransferasa/genética , Pioglitazona , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Inhibidor Tisular de Metaloproteinasa-1/genética , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
Mice that lack phosphatidylethanolamine N-methyltransferase (Pemt(-/-) mice) are protected from high-fat (HF) diet-induced obesity. HF-fed Pemt(-/-) mice show higher oxygen consumption and heat production, indicating that more energy might be utilized for thermogenesis and might account for the resistance to diet-induced weight gain. To test this hypothesis, HF-fed Pemt(-/-) and Pemt(+/+) mice were challenged with acute cold exposure at 4°C. Unexpectedly, HF-fed Pemt(-/-) mice developed hypothermia within 3 h of cold exposure. In contrast, chow-fed Pemt(-/-) mice, possessing similar body mass, maintained body temperature. Lack of PEMT did not impair the capacity for thermogenesis in skeletal muscle or brown adipose tissue. Plasma catecholamines were not altered by Pemt genotype, and stimulation of lipolysis was intact in brown and white adipose tissue of Pemt(-/-) mice. HF-fed Pemt(-/-) mice also developed higher systolic blood pressure, accompanied by reduced cardiac output. Choline supplementation reversed the cold-induced hypothermia in HF-fed Pemt(-/-) mice with no effect on blood pressure. Plasma glucose levels were â¼50% lower in HF-fed Pemt(-/-) mice compared with Pemt(+/+) mice. Choline supplementation normalized plasma hypoglycemia and the expression of proteins involved in gluconeogenesis. We propose that cold-induced hypothermia in HF-fed Pemt(-/-) mice is linked to plasma hypoglycemia due to compromised hepatic glucose production.
Asunto(s)
Metabolismo Energético/genética , Hipotermia/genética , Obesidad/metabolismo , Fosfatidiletanolamina N-Metiltransferasa/genética , Animales , Frío , Dieta Alta en Grasa , Glucosa/metabolismo , Humanos , Hipotermia/metabolismo , Hipotermia/patología , Lipólisis/genética , Hígado/metabolismo , Hígado/patología , Ratones , Obesidad/genética , Obesidad/patología , Consumo de Oxígeno/genéticaRESUMEN
Besides bulk amounts of SM, mammalian cells produce small quantities of the SM analog ceramide phosphoethanolamine (CPE). Little is known about the biological role of CPE or enzymes responsible for CPE production. Heterologous expression studies revealed that SM synthase (SMS)2 is a bifunctional enzyme producing both SM and CPE, whereas SMS-related protein (SMSr) serves as monofunctional CPE synthase. Acute disruption of SMSr catalytic activity in cultured cells causes a rise in endoplasmic reticulum (ER) ceramides, fragmentation of ER exit sites, and induction of mitochondrial apoptosis. To address the relevance of CPE biosynthesis in vivo, we analyzed the tissue-specific distribution of CPE in mice and generated mouse lines lacking SMSr and SMS2 catalytic activity. We found that CPE levels were >300-fold lower than SM in all tissues examined. Unexpectedly, combined inactivation of SMSr and SMS2 significantly reduced, but did not eliminate, tissue-specific CPE pools and had no obvious impact on mouse development or fertility. While SMSr is widely expressed and serves as the principal CPE synthase in the brain, blocking its catalytic activity did not affect ceramide levels or secretory pathway integrity in the brain or any other tissue. Our data provide a first inventory of CPE species and CPE-biosynthetic enzymes in mammals.
Asunto(s)
Biocatálisis , Esfingomielinas/biosíntesis , Transferasas (Grupos de Otros Fosfatos Sustitutos)/metabolismo , Animales , Encéfalo/citología , Encéfalo/enzimología , Encéfalo/metabolismo , Dominio Catalítico , Supervivencia Celular , Activación Enzimática , Exones/genética , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica , Hígado/citología , Hígado/enzimología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Especificidad de Órganos , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Mutación Puntual , Transporte de Proteínas , Esfingomielinas/metabolismo , Transferasas (Grupos de Otros Fosfatos Sustitutos)/química , Transferasas (Grupos de Otros Fosfatos Sustitutos)/deficiencia , Transferasas (Grupos de Otros Fosfatos Sustitutos)/genéticaRESUMEN
DNA methylation and histone acetylation inhibitors are widely used to study the role of epigenetic marks in the regulation of gene expression. In addition, several of these molecules are being tested in clinical trials or already in use in the clinic. Antimetabolites, such as the DNA-hypomethylating agent 5-azacytidine (5-AzaC), have been shown to lower malignant progression to acute myeloid leukemia and to prolong survival in patients with myelodysplastic syndromes. Here we examined the effects of DNA methylation inhibitors on the expression of lipid biosynthetic and uptake genes. Our data demonstrate that, independently of DNA methylation, 5-AzaC selectively and very potently reduces expression of key genes involved in cholesterol and lipid metabolism (e.g. PCSK9, HMGCR, and FASN) in all tested cell lines and in vivo in mouse liver. Treatment with 5-AzaC disturbed subcellular cholesterol homeostasis, thereby impeding activation of sterol regulatory element-binding proteins (key regulators of lipid metabolism). Through inhibition of UMP synthase, 5-AzaC also strongly induced expression of 1-acylglycerol-3-phosphate O-acyltransferase 9 (AGPAT9) and promoted triacylglycerol synthesis and cytosolic lipid droplet formation. Remarkably, complete reversal was obtained by the co-addition of either UMP or cytidine. Therefore, this study provides the first evidence that inhibition of the de novo pyrimidine synthesis by 5-AzaC disturbs cholesterol and lipid homeostasis, probably through the glycerolipid biosynthesis pathway, which may contribute mechanistically to its beneficial cytostatic properties.
Asunto(s)
Azacitidina/farmacología , Colesterol/metabolismo , Epigénesis Genética/efectos de los fármacos , Animales , Línea Celular Tumoral , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cricetinae , Metilación de ADN/efectos de los fármacos , Homeostasis/efectos de los fármacos , Humanos , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Pirimidinas/biosíntesis , Proteína 2 de Unión a Elementos Reguladores de Esteroles/genéticaRESUMEN
Phosphatidylcholine is made in the liver via the CDP-choline pathway and via the conversion of phosphatidylethanolamine to phosphatidylcholine by 3 transmethylation reactions from AdoMet catalyzed by phosphatidylethanolamine N-methyltransferase (PEMT). PEMT is a 22.3kDa integral transmembrane protein of the endoplasmic reticulum and mitochondria-associated membranes. The only tissue with quantitatively significant PEMT activity is liver; however, low levels of PEMT in adipocytes have been implicated in lipid droplet formation. PEMT activity is regulated by the concentration of substrates (phosphatidylethanolamine and AdoMet) as well as the ratio of AdoMet to AdoHcy. Transcription of PEMT is enhanced by estrogen whereas the transcription factor Sp1 is a negative regulator of PEMT transcription. Studies with mice that lack PEMT have provided novel insights into the function of this enzyme. PEMT activity is required to maintain hepatic membrane integrity and for the formation of choline when dietary choline supply is limited. PEMT is required for normal secretion of very low-density lipoproteins. The lack of PEMT protects against diet-induced atherosclerosis in two mouse models. Most unexpectedly, mice that lack PEMT are protected from diet-induced obesity and insulin resistance. Moreover, mice lacking PEMT have increased susceptibility to diet-induced fatty liver and steatohepatitis. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.
Asunto(s)
Fenómenos Fisiológicos Celulares , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Fosfolípidos/química , Animales , Humanos , Metilación , Ratones , Fosfolípidos/metabolismoRESUMEN
There is a paucity of information about phosphatidylcholine (PC) biosynthesis in bone formation. Thus, we characterized PC metabolism in both primary human osteoblasts (HOB) and human osteosarcoma MG-63 cells. Our results show that the CDP-choline pathway is the only de novo route for PC biosynthesis in both HOB and MG-63 cells. Both CK activity and CKα expression in MG-63 cells were significantly higher than those in HOB cells. Silencing of CKα in MG-63 cells had no significant effect on PC concentration but decreased the amount of phosphocholine by approximately 80%. The silencing of CKα also reduced cell proliferation. Moreover, pharmacological inhibition of CK activity impaired the mineralization capacity of MG-63 cells. Our data suggest that CK and its product phosphocholine are required for the normal growth and mineralization of MG-63 cells.
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Calcificación Fisiológica/genética , Colina Quinasa/genética , Osteogénesis/genética , Fosfatidilcolinas/biosíntesis , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Colina Quinasa/antagonistas & inhibidores , Colina Quinasa/metabolismo , Hemicolinio 3/farmacología , Humanos , Metabolismo de los Lípidos/genética , Osteoblastos/enzimología , Fosfatidilcolinas/genética , Fosfatidilcolinas/metabolismo , ARN Interferente PequeñoRESUMEN
BACKGROUND: Choline kinase has three isoforms encoded by the genes Chka and Chkb. Inactivation of Chka in mice results in embryonic lethality, whereas Chkb(-/-) mice display neonatal forelimb bone deformations. METHODS: To understand the mechanisms underlying the bone deformations, we compared the biology and biochemistry of bone formation from embryonic to young adult wild-type (WT) and Chkb(-/-) mice. RESULTS: The deformations are specific to the radius and ulna during the late embryonic stage. The radius and ulna of Chkb(-/-) mice display expanded hypertrophic zones, unorganized proliferative columns in their growth plates, and delayed formation of primary ossification centers. The differentiation of chondrocytes of Chkb(-/-) mice was impaired, as was chondrocyte proliferation and expression of matrix metalloproteinases 9 and 13. In chondrocytes from Chkb(-/-) mice, phosphatidylcholine was slightly lower than in WT mice whereas the amount of phosphocholine was decreased by approximately 75%. In addition, the radius and ulna from Chkb(-/-) mice contained fewer osteoclasts along the cartilage/bone interface. CONCLUSIONS: Chkb has a critical role in the normal embryogenic formation of the radius and ulna in mice. GENERAL SIGNIFICANCE: Our data indicate that choline kinase beta plays an important role in endochondral bone formation by modulating growth plate physiology.
Asunto(s)
Diferenciación Celular/genética , Colina Quinasa/genética , Placa de Crecimiento/crecimiento & desarrollo , Osteogénesis/genética , Animales , Colina Quinasa/metabolismo , Condrocitos/enzimología , Embrión de Mamíferos/enzimología , Desarrollo Embrionario/genética , Miembro Anterior/embriología , Miembro Anterior/enzimología , Miembro Anterior/crecimiento & desarrollo , Placa de Crecimiento/enzimología , Humanos , Ratones , Ratones Noqueados , Fosfatidilcolinas/metabolismoRESUMEN
BACKGROUND & AIMS: Phosphatidylethanolamine N-methyltransferase (PEMT), a liver enriched enzyme, is responsible for approximately one third of hepatic phosphatidylcholine biosynthesis. When fed a high-fat diet (HFD), Pemt(-/-) mice are protected from HF-induced obesity; however, they develop steatohepatitis. The vagus nerve relays signals between liver and brain that regulate peripheral adiposity and pancreas function. Here we explore a possible role of the hepatic branch of the vagus nerve in the development of diet induced obesity and steatohepatitis in Pemt(-/-) mice. METHODS: 8-week old Pemt(-/-) and Pemt(+/+) mice were subjected to hepatic vagotomy (HV) or capsaicin treatment, which selectively disrupts afferent nerves, and were compared to sham-operated or vehicle-treatment, respectively. After surgery, mice were fed a HFD for 10 weeks. RESULTS: HV abolished the protection against the HFD-induced obesity and glucose intolerance in Pemt(-/-) mice. HV normalized phospholipid content and prevented steatohepatitis in Pemt(-/-) mice. Moreover, HV increased the hepatic anti-inflammatory cytokine interleukin-10, reduced chemokine monocyte chemotactic protein-1 and the ER stress marker C/EBP homologous protein. Furthermore, HV normalized the expression of mitochondrial electron transport chain proteins and of proteins involved in fatty acid synthesis, acetyl-CoA carboxylase and fatty acid synthase in Pemt(-/-) mice. However, disruption of the hepatic afferent vagus nerve by capsaicin failed to reverse either the protection against the HFD-induced obesity or the development of HF-induced steatohepatitis in Pemt(-/-) mice. CONCLUSIONS: Neuronal signals via the hepatic vagus nerve contribute to the development of steatohepatitis and protection against obesity in HFD fed Pemt(-/-) mice.
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Hígado Graso , Hígado , Fosfatidilcolinas/biosíntesis , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Vagotomía , Animales , Quimiocina CCL2/metabolismo , Dieta Alta en Grasa/efectos adversos , Dieta Alta en Grasa/métodos , Modelos Animales de Enfermedad , Hígado Graso/etiología , Hígado Graso/metabolismo , Hígado Graso/patología , Hígado Graso/fisiopatología , Interleucina-10/metabolismo , Hígado/inervación , Hígado/metabolismo , Hígado/patología , Ratones , Obesidad , Periodo Posoperatorio , Factor de Transcripción CHOP/metabolismo , Vagotomía/efectos adversos , Vagotomía/métodos , Nervio Vago/fisiopatologíaRESUMEN
Biosynthesis of hepatic choline via phosphatidylethanolamine N-methyltransferase (PEMT) plays an important role in the development of type 2 diabetes and obesity. We investigated the mechanism(s) by which choline modulates insulin sensitivity. PEMT wild-type (Pemt(+/+)) and knock-out (Pemt(-/-)) mice received either a high fat diet (HF; 60% kcal of fat) or a high fat, high choline diet (HFHC; 4 g of choline/kg of HF diet) for 1 week. Hepatic insulin signaling and glucose and lipid homeostasis were investigated. Glucose and insulin intolerance occurred in Pemt(-/-) mice fed the HFHC diet, but not in their Pemt(-/-) littermates fed the HF diet. Plasma glucagon was elevated in Pemt(-/-) mice fed the HFHC diet compared with Pemt(-/-) mice fed the HF diet, concomitant with increased hepatic expression of glucagon receptor, phosphorylated AMP-activated protein kinase (AMPK), and phosphorylated insulin receptor substrate 1 at serine 307 (IRS1-s307). Gluconeogenesis and mitochondrial oxidative stress were markedly enhanced, whereas glucose oxidation and triacylglycerol biosynthesis were diminished in Pemt(-/-) mice fed the HFHC diet. A glucagon receptor antagonist (2-aminobenzimidazole) attenuated choline-induced hyperglycemia and insulin intolerance and blunted up-regulation of phosphorylated AMPK and IRS1-s307. Choline induces glucose and insulin intolerance in Pemt(-/-) mice through modulating plasma glucagon and its action in liver.
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Colina/administración & dosificación , Glucagón/fisiología , Resistencia a la Insulina , Hígado/efectos de los fármacos , Fosfatidiletanolamina N-Metiltransferasa/metabolismo , Animales , Secuencia de Bases , Colina/farmacología , Cartilla de ADN , Gluconeogénesis/efectos de los fármacos , Prueba de Tolerancia a la Glucosa , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Fosfatidiletanolamina N-Metiltransferasa/genéticaRESUMEN
Phosphatidylethanolamine N-methyltransferase (PEMT) catalyzes the methylation of phosphatidylethanolamine to phosphatidylcholine (PC). This 22.3 kDa protein is localized to the endoplasmic reticulum and mitochondria associated membranes of liver. The supply of the substrates AdoMet and phosphatidylethanolamine, and the product AdoHcy, can regulate the activity of PEMT. Estrogen has been identified as a positive activator, and Sp1 as a negative regulator, of transcription of the PEMT gene. Targeted inactivation of the PEMT gene produced mice that had a mild phenotype when fed a chow diet. However, when Pemt(-/-) mice were fed a choline-deficient diet steatohepatitis and liver failure developed after 3 days. The steatohepatitis was due to a decreased ratio of PC to phosphatidylethanolamine that caused leakage from the plasma membrane of hepatocytes. Pemt(-/-) mice exhibited attenuated secretion of very low-density lipoproteins and homocysteine. Pemt(-/-) mice bred with mice that lacked the low-density lipoprotein receptor, or apolipoprotein E were protected from high fat/high cholesterol-induced atherosclerosis. Surprisingly, Pemt(-/-) mice were protected from high fat diet-induced obesity and insulin resistance compared to wildtype mice. If the diet were supplemented with additional choline, the protection against obesity/insulin resistance in Pemt(-/-) mice was eliminated. Humans with a Val-to-Met substitution in PEMT at residue 175 may have increased susceptibility to nonalcoholic liver disease. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.
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Retículo Endoplásmico/metabolismo , Hígado Graso/metabolismo , Hígado/metabolismo , Mitocondrias Hepáticas/metabolismo , Fosfatidiletanolamina N-Metiltransferasa/deficiencia , Animales , Colina/metabolismo , Dieta Alta en Grasa/efectos adversos , Retículo Endoplásmico/patología , Estrógenos/metabolismo , Hígado Graso/etiología , Hígado Graso/patología , Humanos , Hígado/patología , Ratones , Ratones Noqueados , Mitocondrias Hepáticas/patología , Fosfatidilcolinas/metabolismo , Fosfatidiletanolamina N-Metiltransferasa/genética , Fosfatidiletanolaminas/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Factor de Transcripción Sp1/genética , Factor de Transcripción Sp1/metabolismoRESUMEN
Primary rodent hepatocytes and hepatoma cell lines are commonly used as model systems to elucidate and study potential drug targets for metabolic diseases such as obesity and atherosclerosis. However, if therapies are to be developed, it is essential that our knowledge gained from these systems is translatable to that of human. Here, we have characterized lipid and lipoprotein metabolism in primary human hepatocytes for comparison to rodent primary hepatocytes and human hepatoma cell lines. Primary human hepatocytes were maintained in collagen coated dishes in confluent monolayers for up to 3 days. We found primary human hepatocytes were viable, underwent lipid synthesis, and were able to secret lipoproteins up to 3 days in culture. Furthermore, the lipoprotein profile secreted by primary human hepatocytes was comparable to that found in human plasma; this contrasts with primary rodent hepatocytes and human hepatoma cells. We also investigated the pharmacological effects of nicotinic acid (niacin, NA), a potent dyslipidemic drug, on hepatic lipid synthesis and lipoprotein secretion. We found NA increased the expression of ATP-binding cassette transporter A1 in primary human hepatocytes, which may potentially explain how NA increases plasma high-density lipoproteins in humans. In conclusion, primary human hepatocytes are a more relevant model to study lipid synthesis and lipoprotein secretion than hepatoma cells or rodent primary hepatocyte models. Further research needs to be done to maintain liver specific functions of primary human hepatocytes in prolonged cultures for these cells to be a viable model.