RESUMO
Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.
Assuntos
Doenças Mitocondriais , Ubiquinona , Animais , Testes Genéticos , Camundongos , Doenças Mitocondriais/genética , Oxirredução , Fosfatidiletanolamina N-Metiltransferase , Fosfolipídeos , Ubiquinona/metabolismoRESUMO
Diurnal regulation of whole-body lipid metabolism plays a vital role in metabolic health. Although changes in lipid levels across the diurnal cycle have been investigated, the system-wide molecular responses to both short-acting fasting-feeding transitions and longer-timescale circadian rhythms have not been explored in parallel. Here, we perform time-series multi-omics analyses of liver and plasma revealing that the majority of molecular oscillations are entrained by adaptations to fasting, food intake, and the postprandial state. By developing algorithms for lipid structure enrichment analysis and lipid molecular crosstalk between tissues, we find that the hepatic phosphatidylethanolamine (PE) methylation pathway is diurnally regulated, giving rise to two pools of oscillating phosphatidylcholine (PC) molecules in the circulation, which are coupled to secretion of either very low-density lipoprotein (VLDL) or high-density lipoprotein (HDL) particles. Our work demonstrates that lipid molecular timeline profiling across tissues is key to disentangling complex metabolic processes and provides a critical resource for the study of whole-body lipid metabolism.
Assuntos
Metabolismo dos Lipídeos/genética , Fígado/fisiologia , Animais , Ritmo Circadiano , CamundongosRESUMO
Phosphatidylethanolamine N-methyltransferase (PEMT) is a small integral membrane protein that converts phosphatidylethanolamine (PE) into phosphatidylcholine (PC). It has been previously reported that, unexpectedly, PEMT deficiency protected from high-fat diet (HFD)-induced obesity and insulin resistance, pointing to a possible role of this enzyme in the regulation of adipose cell metabolism. Using mouse 3T3-L1 preadipocytes as a biological system, we demonstrate that PEMT expression is strongly increased during the differentiation of preadipocytes into mature adipose cells. Knockdown of PEMT reduced the expression of early and late adipogenic markers, inhibited lipid droplet formation, reduced triacylglycerol content and decreased the levels of leptin release from the adipocytes, suggesting that PEMT is a novel and relevant regulator of adipogenesis. Investigation into the mechanisms whereby PEMT regulates adipocyte differentiation revealed that extracellularly regulated kinases (ERK1/2) and AKT are essential factors in this process. Specifically, the activities of ERK1/2 and AKT, which are decreased during adipocyte differentiation, were elevated upon Pemt knockdown. Moreover, treatment of cells with exogenous ceramide 1-phosphate (C1P), which we reported to be a negative regulator of adipogenesis, decreased PEMT expression, suggesting that PEMT is also a relevant factor in the anti-adipogenic action of C1P. Altogether, the data presented here identify PEMT as a novel regulator of adipogenesis and a mediator of the anti-adipogenic action of C1P.
Assuntos
Adipócitos/fisiologia , Adipogenia/fisiologia , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Células 3T3-L1 , Animais , Diferenciação Celular/fisiologia , Ceramidas/metabolismo , Meios de Cultura/metabolismo , Técnicas de Silenciamento de Genes , Gotículas Lipídicas/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidiletanolamina N-Metiltransferase/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Regulação para CimaRESUMO
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.
Assuntos
Resistência à Insulina/fisiologia , Fígado/metabolismo , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Animais , Dieta Hiperlipídica , Camundongos Transgênicos , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolaminas/metabolismoRESUMO
Phosphatidylethanolamine N-methyltransferase (PEMT) is a hepatic integral membrane protein localized to the endoplasmic reticulum (ER). PEMT catalyzes approximately 30% of hepatic phosphatidylcholine (PC) biosynthesis. Pemt-/- mice fed a high-fat diet (HFD) develop steatohepatitis. Interestingly, portions of the ER located close to the canaliculus are enriched in PEMT. Phospholipid balance and asymmetrical distribution by adenosine triphosphatase phospholipid transporting 8B1 (ATP8B1) on the canalicular membrane is required for membrane integrity and biliary processes. We hypothesized that PEMT is an important supplier of PC to the canaliculus and that PEMT activity is critical for the maintenance of canalicular membrane integrity and bile formation following HFD feeding when there is an increase in overall hepatic PC demand. Pemt+/+ and Pemt-/- mice were fed a chow diet, an HFD, or a choline-supplemented HFD. Plasma and hepatic indices of liver function and parameters of bile formation were determined. Pemt-/- mice developed cholestasis, i.e, elevated plasma bile acid (BA) concentrations and decreased biliary secretion rates of BAs and PC, during HFD feeding. The maximal BA secretory rate was reduced more than 70% in HFD-fed Pemt-/- mice. Hepatic ABCB11/bile salt export protein, responsible for BA secretion, was decreased in Pemt-/- mice and appeared to be retained intracellularly. Canalicular membranes of HFD-fed Pemt-/- mice contained fewer invaginations and displayed a smaller surface area than Pemt+/+ mice. Choline supplementation (CS) prevented and reversed the development of HFD-induced cholestasis. Conclusion: We propose that hepatic PC availability is critical for bile formation. Dietary CS might be a potential noninvasive therapy for a specific subset of patients with cholestasis.
RESUMO
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.
Assuntos
Insulina/metabolismo , Fígado/metabolismo , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Animais , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Phosphatidylethanolamine N-methyltransferase (PEMT) converts phosphatidylethanolamine (PE) to phosphatidylcholine (PC), mainly in the liver. Pemt-/- mice are protected from high-fat diet (HFD)-induced obesity and insulin resistance, but develop severe non-alcoholic fatty liver disease (NAFLD) when fed a HFD, mostly due to impaired VLDL secretion. Oxidative stress is thought to be an essential factor in the progression from simple steatosis to steatohepatitis. Vitamin E is an antioxidant that has been clinically used to improve NAFLD pathology. Our aim was to determine whether supplementation of the diet with vitamin E could attenuate HFD-induced hepatic steatosis and its progression to NASH in Pemt-/- mice. Treatment with vitamin E (0.5â¯g/kg) for 3â¯weeks improved VLDL-TG secretion and normalized cholesterol metabolism, but failed to reduce hepatic TG content. Moreover, vitamin E treatment was able to reduce hepatic oxidative stress, inflammation and fibrosis. We also observed abnormal ceramide metabolism in Pemt-/- mice fed a HFD, with elevation of ceramides and other sphingolipids and higher expression of mRNAs for acid ceramidase (Asah1) and ceramide kinase (Cerk). Interestingly, vitamin E supplementation restored Asah1 and Cerk mRNA and sphingolipid levels. Together this study shows that vitamin E treatment efficiently prevented the progression from simple steatosis to steatohepatitis in mice lacking PEMT.
Assuntos
Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Vitamina E/metabolismo , Vitamina E/farmacologia , Ceramidase Ácida , Animais , Antioxidantes/farmacologia , Colesterol/metabolismo , Dieta Hiperlipídica , Suplementos Nutricionais , Modelos Animais de Doenças , Progressão da Doença , Fígado Gorduroso/metabolismo , Fibrose/tratamento farmacológico , Inflamação/tratamento farmacológico , Resistência à Insulina , Metabolismo dos Lipídeos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfotransferases (Aceptor do Grupo Álcool) , RNA Mensageiro , Vitamina E/administração & dosagemRESUMO
The biophysical environment of membrane phospholipids affects structure, function, and stability of membrane-bound proteins.1,2 Obesity can disrupt membrane lipids, and in particular, alter the activity of sarco/endoplasmic reticulum (ER/SR) Ca2+-ATPase (SERCA) to affect cellular metabolism.3-5 Recent evidence suggests that transport efficiency (Ca2+ uptake / ATP hydrolysis) of skeletal muscle SERCA can be uncoupled to increase energy expenditure and protect mice from diet-induced obesity.6,7 In isolated SR vesicles, membrane phospholipid composition is known to modulate SERCA efficiency.8-11 Here we show that skeletal muscle SR phospholipids can be altered to decrease SERCA efficiency and increase whole-body metabolic rate. The absence of skeletal muscle phosphatidylethanolamine (PE) methyltransferase (PEMT) promotes an increase in skeletal muscle and whole-body metabolic rate to protect mice from diet-induced obesity. The elevation in metabolic rate is caused by a decrease in SERCA Ca2+-transport efficiency, whereas mitochondrial uncoupling is unaffected. Our findings support the hypothesis that skeletal muscle energy efficiency can be reduced to promote protection from obesity.
Assuntos
Cálcio/metabolismo , Metabolismo Energético , Músculo Esquelético/metabolismo , Fosfolipídeos/metabolismo , Animais , Dieta Hiperlipídica , Transporte de Íons , Metilação , Camundongos , Camundongos Knockout , Músculo Esquelético/enzimologia , Obesidade/enzimologia , Obesidade/genética , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
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.
Assuntos
Aterosclerose , Colina-Fosfato Citidililtransferase , Resistência à Insulina , Obesidade , Fosfatidilcolinas , Fosfatidiletanolamina N-Metiltransferase , Animais , Aterosclerose/enzimologia , Aterosclerose/genética , Aterosclerose/patologia , Colina-Fosfato Citidililtransferase/genética , Colina-Fosfato Citidililtransferase/metabolismo , Dieta/efeitos adversos , Embrião de Mamíferos/enzimologia , Embrião de Mamíferos/patologia , Marcação de Genes , História do Século XX , História do Século XXI , Humanos , Camundongos , Camundongos Knockout , Obesidade/induzido quimicamente , Obesidade/enzimologia , Obesidade/genética , Obesidade/patologia , Fosfatidilcolinas/biossíntese , Fosfatidilcolinas/genética , Fosfatidiletanolamina N-Metiltransferase/genética , Fosfatidiletanolamina N-Metiltransferase/metabolismoRESUMO
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.
Assuntos
Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/metabolismo , Animais , Fígado Gorduroso Alcoólico/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Lipoproteínas VLDL/metabolismo , Fígado/metabolismo , Regeneração Hepática , Doenças Metabólicas/metabolismo , Mitocôndrias/metabolismo , Músculo Esquelético/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
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.
Assuntos
Fenofibrato/administração & dosagem , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Obesidade/tratamento farmacológico , Fosfatidiletanolamina N-Metiltransferase/genética , Animais , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Ezetimiba/administração & dosagem , Humanos , Resistência à Insulina/genética , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipoproteínas VLDL/metabolismo , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , Oxirredução/efeitos dos fármacos , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Triglicerídeos/metabolismoRESUMO
BACKGROUND: The development of atherosclerosis is strongly linked to disorders of cholesterol metabolism. Matrix metalloproteinases (MMPs) are dysregulated in patients and animal models with atherosclerosis. Whether systemic MMP activity influences cholesterol metabolism is unknown. METHODS AND RESULTS: We examined MMP-9-deficient (Mmp9-/-) mice and found them to have abnormal lipid gene transcriptional responses to dietary cholesterol supplementation. As opposed to Mmp9+/+ (wild-type) mice, Mmp9-/- mice failed to decrease the hepatic expression of sterol regulatory element binding protein 2 pathway genes, which control hepatic cholesterol biosynthesis and uptake. Furthermore, Mmp9-/- mice failed to increase the expression of genes encoding the rate-limiting enzymes in biliary cholesterol excretion (eg, Cyp7a and Cyp27a). In contrast, MMP-9 deficiency did not impair intestinal cholesterol absorption, as shown by the 14C-cholesterol and 3H-sitostanol absorption assay. Similar to our earlier study on Mmp2-/- mice, we observed that Mmp9-/- mice had elevated plasma secreted phospholipase A2 activity. Pharmacological inhibition of systemic circulating secreted phospholipase A2 activity (with varespladib) partially normalized the hepatic transcriptional responses to dietary cholesterol in Mmp9-/- mice. Functional studies with mice deficient in other MMPs suggested an important role for the MMP system, as a whole, in modulation of cholesterol metabolism. CONCLUSIONS: Our results show that MMP-9 modulates cholesterol metabolism, at least in part, through a novel MMP-9-plasma secreted phospholipase A2 axis that affects the hepatic transcriptional responses to dietary cholesterol. Furthermore, the data suggest that dysregulation of the MMP system can result in metabolic disorder, which could lead to atherosclerosis and coronary heart disease.
Assuntos
Colesterol/metabolismo , Regulação da Expressão Gênica/genética , Absorção Intestinal/genética , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Metaloproteinase 9 da Matriz/genética , Fosfolipases A2/metabolismo , Acetatos/farmacologia , Animais , Colestanotriol 26-Mono-Oxigenase/efeitos dos fármacos , Colestanotriol 26-Mono-Oxigenase/genética , Colestanotriol 26-Mono-Oxigenase/metabolismo , Colesterol 7-alfa-Hidroxilase/efeitos dos fármacos , Colesterol 7-alfa-Hidroxilase/genética , Colesterol 7-alfa-Hidroxilase/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Indóis/farmacologia , Cetoácidos , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/efeitos dos fármacos , Masculino , Camundongos , Camundongos Knockout , Inibidores de Fosfolipase A2/farmacologia , Proteína de Ligação a Elemento Regulador de Esterol 2/efeitos dos fármacos , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismoRESUMO
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.
Assuntos
Anti-Infecciosos/farmacologia , Hepatite/prevenção & controle , Cirrose Hepática Experimental/prevenção & controle , Fígado/efeitos dos fármacos , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , PPAR gama/agonistas , Fosfatidiletanolamina N-Metiltransferase/deficiência , Tiazolidinedionas/farmacologia , Actinas/genética , Actinas/metabolismo , Adipócitos Brancos/efeitos dos fármacos , Adipócitos Brancos/enzimologia , Adipócitos Brancos/patologia , Tecido Adiposo Branco/efeitos dos fármacos , Tecido Adiposo Branco/enzimologia , Tecido Adiposo Branco/patologia , Adiposidade/efeitos dos fármacos , Animais , Proliferação de Células/efeitos dos fármacos , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadeia alfa 1 do Colágeno Tipo I , Dieta Hiperlipídica , Predisposição Genética para Doença , Hepatite/enzimologia , Hepatite/genética , Hepatite/patologia , Resistência à Insulina , Fígado/enzimologia , Fígado/patologia , Cirrose Hepática Experimental/enzimologia , Cirrose Hepática Experimental/genética , Cirrose Hepática Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Obesidade/enzimologia , Obesidade/genética , Obesidade/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , PPAR gama/metabolismo , Fenótipo , Fosfatidiletanolamina N-Metiltransferase/genética , Pioglitazona , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Inibidor Tecidual de Metaloproteinase-1/genética , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
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.
Assuntos
Ácidos Graxos/metabolismo , Fígado/enzimologia , Músculo Esquelético/enzimologia , Obesidade/enzimologia , Fosfatidilcolinas/metabolismo , Fosfatidiletanolamina N-Metiltransferase/deficiência , Fosfatidiletanolaminas/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Gorduras na Dieta/efeitos adversos , Metabolismo Energético , Expressão Gênica , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/enzimologia , Células Musculares/citologia , Células Musculares/enzimologia , Obesidade/etiologia , Obesidade/genética , Oxirredução , Consumo de Oxigênio , Fosfatidiletanolamina N-Metiltransferase/genética , Cultura Primária de CélulasRESUMO
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.
RESUMO
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.
Assuntos
Metabolismo Energético/genética , Hipotermia/genética , Obesidade/metabolismo , Fosfatidiletanolamina N-Metiltransferase/genética , Animais , Temperatura Baixa , Dieta Hiperlipídica , Glucose/metabolismo , Humanos , Hipotermia/metabolismo , Hipotermia/patologia , Lipólise/genética , Fígado/metabolismo , Fígado/patologia , Camundongos , Obesidade/genética , Obesidade/patologia , Consumo de Oxigênio/genéticaRESUMO
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.
Assuntos
Biocatálise , Esfingomielinas/biossíntese , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo , Animais , Encéfalo/citologia , Encéfalo/enzimologia , Encéfalo/metabolismo , Domínio Catalítico , Sobrevivência Celular , Ativação Enzimática , Éxons/genética , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Fígado/citologia , Fígado/enzimologia , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Mutação Puntual , Transporte Proteico , Esfingomielinas/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/deficiência , Transferases (Outros Grupos de Fosfato Substituídos)/genéticaRESUMO
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.
Assuntos
Tecido Adiposo Branco/enzimologia , Dieta Hiperlipídica , Lipogênese , Obesidade/prevenção & controle , Fosfatidiletanolamina N-Metiltransferase/deficiência , Adipócitos Brancos/enzimologia , Adipogenia , Tecido Adiposo Branco/fisiopatologia , Adiposidade , Animais , Biomarcadores/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Genótipo , Lipídeos/sangue , Lipólise , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/sangue , Obesidade/enzimologia , Obesidade/genética , Obesidade/fisiopatologia , Fenótipo , Fosfatidiletanolamina N-Metiltransferase/genética , Fatores de Proteção , Fatores de Tempo , Aumento de PesoRESUMO
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.
Assuntos
Fígado Gorduroso , Fígado , Fosfatidilcolinas/biossíntese , Fosfatidiletanolamina N-Metiltransferase/metabolismo , Vagotomia , Animais , Quimiocina CCL2/metabolismo , Dieta Hiperlipídica/efeitos adversos , Dieta Hiperlipídica/métodos , Modelos Animais de Doenças , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Fígado Gorduroso/fisiopatologia , Interleucina-10/metabolismo , Fígado/inervação , Fígado/metabolismo , Fígado/patologia , Camundongos , Obesidade , Período Pós-Operatório , Fator de Transcrição CHOP/metabolismo , Vagotomia/efeitos adversos , Vagotomia/métodos , Nervo Vago/fisiopatologiaRESUMO
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.