RESUMO
Regulation of the pyruvate dehydrogenase (PDH) complex by the pyruvate dehydrogenase kinase PDK4 enables the heart to respond to fluctuations in energy demands and substrate availability. Retinoic acid, the transcriptionally active form of vitamin A, is known to be involved in the regulation of cardiac function and growth during embryogenesis as well as under pathological conditions. Whether retinoic acid also maintains cardiac health under physiological conditions is unknown. However, vitamin A status and intake of its carotenoid precursor ß-carotene have been linked to the prevention of heart diseases. Here, we provide in vitro and in vivo evidence that retinoic acid regulates cardiac Pdk4 expression and thus PDH activity. Furthermore, we show that mice lacking ß-carotene 9',10'-oxygenase (BCO2), the only enzyme of the adult heart that cleaves ß-carotene to generate retinoids (vitamin A and its derivatives), displayed cardiac retinoic acid insufficiency and impaired metabolic flexibility linked to a compromised PDK4/PDH pathway. These findings provide novel insights into the functions of retinoic acid in regulating energy metabolism in adult tissues, especially the heart.
Assuntos
Dioxigenases , beta Caroteno , Animais , Dioxigenases/metabolismo , Camundongos , Camundongos Knockout , Oxigenases , Proteínas Quinases , Complexo Piruvato Desidrogenase/metabolismo , Tretinoína , Vitamina ARESUMO
Background As breastfeeding awareness and social acceptance are increased, maternal nutritional deficiency requires more investigation. Methods A prospective cohort study was conducted to determine if vitamin A deficiency is more common in pregnant, lactating post-bariatric surgery women in an inner city population. Antepartum, women after bariatric surgery and controls with no history of malabsorption were recruited. Third trimester, postpartum maternal blood and cord blood were collected as well as three breast milk samples: colostrum, transitional and mature milk. A nutritional survey of diet was completed. Each serum sample was analyzed for total retinol and ß-carotene; breast milk samples were analyzed for retinol and retinyl esters, total retinol and ß-carotene. Results Fifty-three women after bariatric surgery and 66 controls were recruited. Postpartum serum retinol was significantly higher in women after bariatric surgery in the univariate analysis (P<0.0001) and confirmed in the multiple linear mixed model (P=0.0001). Breast milk colostrum retinol and transitional milk total retinol were significantly greater in the bariatric surgery group in the univariate analysis (P=0.03 and P=0.02, respectively), but not after adjusting for confounders. Serum ß-carotene in the third trimester and postpartum were lower (P<0.0001 and P=0.003, respectively) in the bariatric surgery group but not after adjusting for confounders. Vitamin A deficiency was high in both groups in serum and breast milk samples. Conclusion Nutritional deficiencies in breastfeeding women after bariatric surgeries may in fact be less common than in control women in an inner city.
Assuntos
Cirurgia Bariátrica/efeitos adversos , Aleitamento Materno/estatística & dados numéricos , Leite Humano/química , Deficiência de Vitamina A , Vitamina A , beta Caroteno , Adulto , Cirurgia Bariátrica/métodos , Feminino , Humanos , Lactação/fisiologia , Avaliação Nutricional , Distúrbios Nutricionais/diagnóstico , Distúrbios Nutricionais/epidemiologia , Distúrbios Nutricionais/etiologia , Obesidade/cirurgia , Assistência Perinatal/métodos , Assistência Perinatal/estatística & dados numéricos , Gravidez , Terceiro Trimestre da Gravidez/sangue , Estados Unidos/epidemiologia , População Urbana/estatística & dados numéricos , Vitamina A/análise , Vitamina A/sangue , Deficiência de Vitamina A/diagnóstico , Deficiência de Vitamina A/epidemiologia , Deficiência de Vitamina A/etiologia , beta Caroteno/análise , beta Caroteno/sangueRESUMO
ß-Carotene is an important source of vitamin A for the mammalian embryo, which depends on its adequate supply to achieve proper organogenesis. In mammalian tissues, ß-carotene 15,15'-oxygenase (BCO1) converts ß-carotene to retinaldehyde, which is then oxidized to retinoic acid, the biologically active form of vitamin A that acts as a transcription factor ligand to regulate gene expression. ß-Carotene can also be cleaved by ß-carotene 9',10'-oxygenase (BCO2) to form ß-apo-10'-carotenal, a precursor of retinoic acid and a transcriptional regulator per se The mammalian embryo obtains ß-carotene from the maternal circulation. However, the molecular mechanisms that enable its transfer across the maternal-fetal barrier are not understood. Given that ß-carotene is transported in the adult bloodstream by lipoproteins and that the placenta acquires, assembles, and secretes lipoproteins, we hypothesized that the aforementioned process requires placental lipoprotein biosynthesis. Here we show that ß-carotene availability regulates transcription and activity of placental microsomal triglyceride transfer protein as well as expression of placental apolipoprotein B, two key players in lipoprotein biosynthesis. We also show that ß-apo-10'-carotenal mediates the transcriptional regulation of microsomal triglyceride transfer protein via hepatic nuclear factor 4α and chicken ovalbumin upstream promoter transcription factor I/II. Our data provide the first in vivo evidence of the transcriptional regulatory activity of ß-apocarotenoids and identify microsomal triglyceride transfer protein and its transcription factors as the targets of their action. This study demonstrates that ß-carotene induces a feed-forward mechanism in the placenta to enhance the assimilation of ß-carotene for proper embryogenesis.
Assuntos
Proteínas de Transporte/biossíntese , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas da Gravidez/biossíntese , Gravidez/metabolismo , beta Caroteno/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Proteínas de Transporte/genética , Dioxigenases/genética , Dioxigenases/metabolismo , Feminino , Camundongos , Camundongos Knockout , Gravidez/genética , Proteínas da Gravidez/genética , beta-Caroteno 15,15'-Mono-Oxigenase/genética , beta-Caroteno 15,15'-Mono-Oxigenase/metabolismoRESUMO
We previously defined that the mitochondria-localized PKCδ signaling complex stimulates the conversion of pyruvate to acetyl-coenzyme A by the pyruvate dehydrogenase complex. We demonstrated in vitro and ex vivo that retinol supplementation enhances ATP synthesis in the presence of the PKCδ signalosome. Here, we tested in vivo if a persistent oversupply of retinol would further impair glucose metabolism in a mouse model of diet-induced insulin resistance. We crossed mice overexpressing human retinol-binding protein (hRBP) under the muscle creatine kinase (MCK) promoter (MCKhRBP) with the PKCδ(-/-) strain to generate mice with a different status of the PKCδ signalosome and retinoid levels. Mice with a functional PKCδ signalosome and elevated retinoid levels (PKCδ(+/+)hRBP) developed the most advanced stage of insulin resistance. In contrast, elevation of retinoid levels in mice with inactive PKCδ did not affect remarkably their metabolism, resulting in phenotypic similarity between PKCδ(-/-)hRBP and PKCδ(-/-) mice. Therefore, in addition to the well-defined role of PKCδ in the etiology of metabolic syndrome, we present a novel PKCδ signaling pathway that requires retinol as a metabolic cofactor and is involved in the regulation of fuel utilization in mitochondria. The distinct role in whole-body energy homeostasis establishes the PKCδ signalosome as a promising target for therapeutic intervention in metabolic disorders.
Assuntos
Resistência à Insulina/fisiologia , Obesidade/metabolismo , Proteína Quinase C-delta/metabolismo , Vitamina A/metabolismo , Animais , Dieta/efeitos adversos , Modelos Animais de Doenças , Glucose/metabolismo , Homeostase/fisiologia , Humanos , Masculino , Síndrome Metabólica/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Regiões Promotoras Genéticas/fisiologia , Complexo Piruvato Desidrogenase/metabolismo , Retinoides/metabolismo , Proteínas de Ligação ao Retinol/metabolismo , Transdução de Sinais/fisiologiaRESUMO
We provide novel insights into the function(s) of ß-carotene-15,15'-oxygenase (CMOI) during embryogenesis. By performing in vivo and in vitro experiments, we showed that CMOI influences not only lecithin:retinol acyltransferase but also acyl CoA:retinol acyltransferase reaction in the developing tissues at mid-gestation. In addition, LC/MS lipidomics analysis of the CMOI-/- embryos showed reduced levels of four phosphatidylcholine and three phosphatidylethanolamine acyl chain species, and of eight triacylglycerol species with four or more unsaturations and fifty-two or more carbons in the acyl chains. Cholesteryl esters of arachidonate, palmitate, linoleate, and DHA were also reduced to less than 30% of control. Analysis of the fatty acyl CoA species ruled out a loss in fatty acyl CoA synthetase capability. Comparison of acyl species suggested significantly decreased 18:2, 18:3, 20:1, 20:4, or 22:6 acyl chains within the above lipids in CMOI-null embryos. Furthermore, LCAT, ACAT1 and DGAT2 mRNA levels were also downregulated in CMOI-/- embryos. These data strongly support the notion that, in addition to cleaving ß-carotene to generate retinoids, CMOI serves an additional function(s) in retinoid and lipid metabolism and point to its role in the formation of specific lipids, possibly for use in nervous system tissue.
Assuntos
Colesterol/metabolismo , Diglicerídeos/metabolismo , Embrião de Mamíferos/enzimologia , Metabolismo dos Lipídeos/fisiologia , Vitamina A/metabolismo , beta-Caroteno 15,15'-Mono-Oxigenase/metabolismo , Acetil-CoA C-Acetiltransferase/biossíntese , Acetil-CoA C-Acetiltransferase/genética , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Animais , Colesterol/genética , Diacilglicerol O-Aciltransferase/biossíntese , Diacilglicerol O-Aciltransferase/genética , Diglicerídeos/genética , Regulação para Baixo/fisiologia , Esterificação/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Camundongos , Camundongos Knockout , Tecido Nervoso/embriologia , Tecido Nervoso/enzimologia , Vitamina A/genética , beta-Caroteno 15,15'-Mono-Oxigenase/genéticaRESUMO
In mammals, ß-carotene-15,15'-oxygenase (BCO1) is the main enzyme that cleaves ß-carotene, the most abundant vitamin A precursor, to generate retinoids (vitamin A derivatives), both in adult and developing tissues. We previously reported that, in addition to this function, BCO1 can also influence the synthesis of retinyl esters, the storage form of retinoids, in the mouse embryo at mid-gestation. Indeed, lack of embryonic BCO1 impaired both lecithin-dependent and acyl CoA-dependent retinol esterification, mediated by lecithin:retinol acyltransferase (LRAT) and acyl CoA:retinol acyltransferase (ARAT), respectively. Furthermore, embryonic BCO1 also influenced the ester pools of cholesterol and diacylglycerol. In this report, we gained novel insights into this alternative function of BCO1 by investigating whether BCO1 influenced embryonic retinoid and lipid metabolism in a tissue-dependent manner. To this end, livers and brains from wild-type and BCO1-/- embryos at mid-gestation were analyzed for retinoid and lipid content, as well as gene expression levels. We also asked whether or not the role of BCO1 as a regulator of lecithin- and acyl CoA-dependent retinol esterification was exclusively restricted to the developing tissues. Thus, a survey of retinol and retinyl ester levels in adult tissues of wild-type, BCO1-/-, LRAT-/- and LRAT-/-BCO1-/- mice was performed. We showed that the absence of BCO1 affects embryonic retinoid and lipid homeostasis in a tissue-specific manner and that retinyl ester formation is also influenced by BCO1 in a few adult tissues (pancreas, lung, heart and adipose) in a sex-dependent manner.
Assuntos
Metabolismo dos Lipídeos , Retinoides/metabolismo , Caracteres Sexuais , beta-Caroteno 15,15'-Mono-Oxigenase/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Dioxigenases/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Inativação de Genes , Genótipo , Homeostase , Fígado/embriologia , Fígado/metabolismo , Masculino , Camundongos , Especificidade de Órgãos , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , beta-Caroteno 15,15'-Mono-Oxigenase/deficiência , beta-Caroteno 15,15'-Mono-Oxigenase/genéticaRESUMO
The requirement of the developing mammalian embryo for retinoic acid is well established. Retinoic acid, the active form of vitamin A, can be generated from retinol and retinyl ester obtained from food of animal origin, and from carotenoids, mainly ß-carotene, from vegetables and fruits. The mammalian embryo relies on retinol, retinyl ester and ß-carotene circulating in the maternal bloodstream for its supply of vitamin A. The maternal-fetal transfer of retinoids and carotenoids, as well as the metabolism of these compounds in the developing tissues are still poorly understood. The existing knowledge in this field has been summarized in this review in reference to our basic understanding of the transport and metabolism of retinoids and carotenoids in adult tissues. The need for future research on the metabolism of these essential lipophilic nutrients during development is highlighted. This article is part of a Special Issue entitled: Retinoid and Lipid Metabolism.
Assuntos
Desenvolvimento Embrionário , Troca Materno-Fetal , Vitamina A/metabolismo , beta Caroteno/metabolismo , Animais , Transporte Biológico , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Absorção Intestinal , Camundongos , Camundongos Knockout , Oxigenases/metabolismo , Gravidez , Proteínas de Ligação ao Retinol/metabolismo , Tretinoína/metabolismoRESUMO
Brassinosteroids (BRs) are a group of steroidal hormones involved in plant development. Although the BR biosynthesis pathways are well characterized, the BR inactivation process, which contributes to BR homeostasis, is less understood. Here, we show that a member of the BAHD (for benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, and deacetylvindoline 4-O-acetyltransferase) acyltransferase family may play a role in BR homeostasis in Arabidopsis (Arabidopsis thaliana). We isolated two gain-of-function mutants, brassinosteroid inactivator1-1Dominant (bia1-1D) and bia1-2D, in which a novel BAHD acyltransferase-like protein was transcriptionally activated. Both mutants exhibited dwarfism, reduced male fertility, and deetiolation in darkness, which are typical phenotypes of plants defective in BR biosynthesis. Exogenous BR treatment rescued the phenotypes of the bia1-1D mutant. Endogenous levels of BRs were reduced in the bia1-1D mutant, demonstrating that BIA1 regulates endogenous BR levels. When grown in darkness, the bia1 loss-of-function mutant showed a longer hypocotyl phenotype and was more responsive to exogenous BR treatment than the wild-type plant. BIA1 expression was predominantly observed in the root, where low levels of BRs were detected. These results indicate that the BAHD acyltransferase family member encoded by BIA1 plays a role in controlling BR levels, particularly in the root and hypocotyl in darkness. Taken together, our study provides new insights into a mechanism that maintains BR homeostasis in Arabidopsis, likely via acyl conjugation of BRs.
Assuntos
Aciltransferases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Brassinosteroides/biossíntese , Aciltransferases/genética , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Brassinosteroides/farmacologia , Escuridão , Fertilidade , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Homeostase , Hipocótilo/efeitos dos fármacos , Hipocótilo/enzimologia , Hipocótilo/genética , Dados de Sequência Molecular , Fenótipo , Filogenia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plasmídeos/genética , Plasmídeos/metabolismo , Protoplastos/metabolismo , Transdução de Sinais , Ativação TranscricionalRESUMO
This Perspective discusses how retinol catalyzes resonance energy transfer (RET) reactions pivotally important for mitochondrial energy homeostasis by protein kinase C δ (PKCδ). PKCδ signals to the pyruvate dehydrogenase complex, controlling oxidative phosphorylation. The PKCδ-retinol complex reversibly responds to the redox potential of cytochrome c, that changes with the electron transfer chain workload. In contrast, the natural retinoid anhydroretinol irreversibly activates PKCδ. Its elongated conjugated-double-bond system limits the energy quantum absorbed by RET. Consequently, while capable of triggering the exergonic activating pathway, anhydroretinol fails to activate the endergonic silencing path, trapping PKCδ in the ON position and causing harmful levels of reactive oxygen species. However, physiological retinol levels displace anhydroretinol, buffer cyotoxicity and potentially render anhydroretinol useful for rapid energy generation. Intriguingly, apocarotenoids, the primary products of the mitochondrial ß-carotene,9'-10'-oxygenase, have all the anhydroretinol-like features, including modulation of energy homeostasis. We predict significant conceptual advances to stem from further understanding of the retinoid-catalyzed RET.
Assuntos
Retinoides , Vitamina A , beta Caroteno , BiologiaRESUMO
Background/Aims: Non-time-sensitive gastrointestinal endoscopy was deferred because of the risk of exposure to coronavirus disease 2019 (COVID-19), but no population-based studies have quantified the adverse impact on gastrointestinal procedures. This study examined the impact of the COVID-19 pandemic on the performance of esophagogastroduodenoscopy (EGD), colonoscopy, ERCP, and abdominal ultrasonography (US) in South Korea. Methods: This nationwide, population-based study compared the claim data of EGD, colonoscopy, ERCP, and abdominal US in 2020 and 2021 (COVID-19 era) with those in 2019 (before the COVID-19 era). Results: During the first year (2020) of the COVID-19 pandemic, the annual claim data of EGD and colonoscopy were reduced by 6.3% and 6.9%, respectively, but those of ERCP and abdominal US were increased by 1.0% and 2.9%, compared to those in 2019. During the first surge (March and April 2020) of COVID-19, the monthly claim data of EGD, colonoscopy, ERCP, and abdominal US were reduced by 28.8%, 43.8%, 5.1%, and 21.6%, respectively, in March 2020, and also reduced by 17.2%, 32.8%, 4.4%, and 9.5%, respectively, in April 2020, compared to those in March and April 2019. During March and April 2020, the monthly claims of ERCP, compared with those in 2019, declined less significantly than those of EGD and colonoscopy (both p<0.001). Conclusions: The claims of EGD and colonoscopy were reduced more significantly than those of ERCP and abdominal US during the COVID-19 pandemic because ERCPs are time-sensitive procedures and abdominal USs are non-aerosolized procedures.
Assuntos
COVID-19 , Pandemias , Humanos , COVID-19/epidemiologia , Endoscopia Gastrointestinal , Colonoscopia/métodos , Endoscopia do Sistema Digestório/métodos , Colangiopancreatografia Retrógrada EndoscópicaRESUMO
The mammalian embryo relies on maternal circulating retinoids (vitamin A derivatives) for development. ß-Carotene is the major human dietary provitamin A. ß-Carotene-15,15'-oxygenase (CMOI) has been proposed as the main enzyme generating retinoid from ß-carotene in vivo. CMOI is expressed in embryonic tissues, suggesting that ß-carotene provides retinoids locally during development. We performed loss of CMOI function studies in mice lacking retinol-binding protein (RBP), an established model of embryonic vitamin A deficiency (VAD). We show that, unexpectedly, lack of CMOI in the developing tissues further exacerbates the severity of VAD and thus the embryonic malformations of RBP(-/-) mice. Since ß-carotene was not present in any of the mouse diets, we unveiled a novel action of CMOI independent from its ß-carotene cleavage activity. We also show for the first time that CMOI exerts an additional function on retinoid metabolism by influencing retinyl ester formation via modulation of lecithin:retinol acyltransferase (LRAT) activity, at least in developing tissues. Finally, we demonstrate unequivocally that ß-carotene can serve as an alternative vitamin A source for the in situ synthesis of retinoids in developing tissues by the action of CMOI.
Assuntos
Embrião de Mamíferos/metabolismo , Retinoides/metabolismo , beta Caroteno/metabolismo , beta-Caroteno 15,15'-Mono-Oxigenase/metabolismo , Aciltransferases/metabolismo , Animais , Western Blotting , Cromatografia Líquida de Alta Pressão , Embrião de Mamíferos/enzimologia , Feminino , Camundongos , Camundongos Knockout , Gravidez , Proteínas de Ligação ao Retinol/genética , Proteínas de Ligação ao Retinol/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tretinoína/metabolismo , Vitamina A/metabolismo , beta-Caroteno 15,15'-Mono-Oxigenase/genéticaRESUMO
Dietary ß-carotene is the most abundant vitamin A precursor. Once absorbed by the enterocytes, the provitamin A carotenoid can either be cleaved into retinoids (vitamin A and its derivatives) or incorporated in its intact form within chylomicrons to be distributed throughout the body for utilization and/or storage by other tissues. From the liver, together with endogenous lipids, intact ß-carotene can also be incorporated within very low-density lipoprotein/low-density lipoprotein (VLDL/LDL) for transport to other tissues and organs. Microsomal triglyceride transfer protein (MTP) is a key regulator of lipoprotein biosynthesis in intestine and liver as it facilitates the incorporation of dietary and endogenous lipids into nascent lipoproteins. MTP is also critical for transferring ß-carotene into lipoprotein particles for secretion. Here, we present an in vitro method to assess the transfer of ß-carotene by MTP from donor to acceptor vesicles. This transfer can be assessed by precipitating donor vesicles and measuring amounts of ß-carotene transferred to acceptor vesicles. The levels of transferred ß-carotene are quantified by HPLC analysis and intrinsic fluorescence of ß-carotene. This chapter demonstrates the feasibility of this method which is also useful to study the role of MTP for incorporation of other carotenoids that are known to be carried within VLDL/LDL and chylomicrons for organ distribution.
Assuntos
Vitamina A , beta Caroteno , Carotenoides , Proteínas de Transporte , Quilomícrons , Lipoproteínas , Lipoproteínas LDL , Lipoproteínas VLDL/metabolismo , beta Caroteno/metabolismoRESUMO
Vitamin A deficiency (VAD) results in intestinal inflammation, increased redox stress and reactive oxygen species (ROS) levels, imbalanced inflammatory and immunomodulatory cytokines, compromised barrier function, and perturbations of the gut microbiome. To combat VAD dietary interventions with ß-carotene, the most abundant precursor of vitamin A, are recommended. However, the impact of ß-carotene on intestinal health during VAD has not been fully clarified, especially regarding the VAD-associated intestinal dysbiosis. Here we addressed this question by using Lrat-/-Rbp-/- (vitamin A deficient) mice deprived of dietary preformed vitamin A and supplemented with ß-carotene as the sole source of the vitamin, alongside with WT (vitamin A sufficient) mice. We found that dietary ß-carotene impacted intestinal vitamin A status, barrier integrity and inflammation in both WT and Lrat-/-Rbp-/- (vitamin A deficient) mice on the vitamin A-free diet. However, it did so to a greater extent under overt VAD. Dietary ß-carotene also modified the taxonomic profile of the fecal microbiome, but only under VAD. Given the similarity of the VAD-associated intestinal phenotypes with those of several other disorders of the gut, collectively known as Inflammatory Bowel Disease (IBD) Syndrome, these findings are broadly relevant to the effort of developing diet-based intervention strategies to ameliorate intestinal pathological conditions.
Assuntos
Enteropatias , Deficiência de Vitamina A , Animais , Modelos Animais de Doenças , Disbiose/complicações , Disbiose/tratamento farmacológico , Inflamação/complicações , Inflamação/tratamento farmacológico , Camundongos , Vitamina A/uso terapêutico , Deficiência de Vitamina A/complicações , Deficiência de Vitamina A/tratamento farmacológico , Deficiência de Vitamina A/patologia , beta Caroteno/farmacologia , beta Caroteno/uso terapêuticoRESUMO
The review focuses on the role of vitamin A (retinol) in the control of energy homeostasis, and on the manner in which certain retinoids subvert this process, leading potentially to disease. In eukaryotic cells, the pyruvate dehydrogenase complex (PDHC) is negatively regulated by four pyruvate dehydrogenase kinases (PDKs) and two antagonistically acting pyruvate dehydrogenase phosphatases (PDPs). The second isoform, PDK2, is regulated by an autonomous mitochondrial signal cascade that is anchored on protein kinase Cδ (PKCδ), where retinoids play an indispensible co-factor role. Along with its companion proteins p66Shc, cytochrome c, and vitamin A, the PKCδ/retinol complex is located in the intermembrane space of mitochondria. At this site, and in contrast to cytosolic locations, PKCδ is activated by the site-specific oxidation of its cysteine-rich activation domain (CRD) that is configured into a complex RING-finger. Oxidation involves the transfer of electrons from cysteine moieties to oxidized cytochrome c, a step catalyzed by vitamin A. The PKCδ/retinol signalosome monitors the internal cytochrome c redox state that reflects the workload of the respiratory chain. Upon sensing demands for energy PKCδ signals the PDHC to increase glucose-derived fuel flux entering the KREBS cycle. Conversely, if excessive fuel flux surpasses the capacity of the respiratory chain, threatening the release of damaging reactive oxygen species (ROS), the polarity of the cytochrome c redox system is reversed, resulting in the chemical reduction of the PKCδ CRD, restoration of the RING-finger, refolding of PKCδ into the inactive, globular form, and curtailment of PDHC output, thereby constraining the respiratory capacity within safe margins. Several retinoids, notably anhydroretinol and fenretinide, capable of displacing retinol from binding sites on PKCδ, can co-activate PKCδ signaling but, owing to their extended system of conjugated double bonds, are unable to silence PKCδ in a timely manner. Left in the ON position, PKCδ causes chronic overload of the respiratory chain leading to mitochondrial dysfunction. This review explores how defects in the PKCδ signal machinery potentially contribute to metabolic and degenerative diseases.
Assuntos
Metabolismo Energético/genética , Mitocôndrias/genética , Proteína Quinase C-delta/genética , Piruvato Desidrogenase (Lipoamida)-Fosfatase/genética , Glucose/metabolismo , Homeostase/genética , Humanos , Mitocôndrias/metabolismo , Fosforilação Oxidativa , Proteína Quinase C-delta/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src/genética , Vitamina A/genética , Vitamina A/metabolismoRESUMO
STRA6 (stimulated by retinoic acid 6) is a 75kDa polytopic transmembrane protein that facilitates cellular retinol uptake from retinol-binding protein (RBP). Structural characterization of STRA6 from Danio rerio purified in detergent and reconstituted in amphipol A8-35 was achieved by single-particle cryo-electron microscopy (cryo-EM). This provided the first high-resolution snapshot of this protein, showing a novel topology of a tightly assembled homodimer, and an unexpected physiological association with calmodulin in addition to insights into its potential mechanism of function. Specifically, a large hydrophobic cavity in the center of STRA6 linked to the known site of interaction with RBP suggested a route of retinol entry into the cell by diffusion into the membrane through a lateral opening of the cavity directly into the bilayer. Moreover, the capability to produce pure and homogeneous protein has allowed previously unattainable functional characterization of STRA6 in a reconstituted system. Here, we describe detailed methods for Danio rerio STRA6 expression in insect cells, purification in detergent and reconstitution in amphipol for structural characterization by cryo-EM. Furthermore, we show reconstitution of the protein in liposomes for an in vitro proteoliposome-based assay of STRA6-mediated retinol uptake. Finally, we present methods and preliminary cryo-EM data for STRA6 incorporated in lipid-filled nanodiscs, a close to native milieu to study membrane protein structure and function.
Assuntos
Proteínas de Membrana , Proteínas de Ligação ao Retinol , Calmodulina , Microscopia Crioeletrônica , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Ligação Proteica , Proteínas de Ligação ao Retinol/metabolismoRESUMO
BACKGROUND: Cytochrome P450 1b1 (Cyp1b1) deletion and dietary retinol deficiency during pregnancy (GVAD) affect perinatal liver functions regulated by Srebp. Cyp1b1 is not expressed in perinatal liver but appears in the E9.5 embryo, close to sites of retinoic acid (RA) signaling. HYPOTHESIS: Parallel effects of Cyp1b1 and retinol on postnatal Srebp derive from effects in the developing liver or systemic signaling. APPROACH: Cluster postnatal increases in hepatic genes in relation to effects of GVAD or Cyp1b1 deletion. Sort expression changes in relation to genes regulated by Srebp1 and Srebp2.Test these treatments on embryos at E9.5, examining changes at the site of liver initiation. Use in situ hybridization to resolve effects on mRNA distributions of Aldh1a2 and Cyp26a1 (RA homeostasis); Hoxb1 and Pax6 (RA targets). Assess mice lacking Lrat and Rbp4 (DKO mice) that severely limits retinol supply to embryos. RESULTS: At birth, GVAD and Cyp1b1 deletion stimulate gene markers of hepatic stellate cell (HSC) activation but also suppress Hamp. These treatments then selectively prevent the postnatal onset of genes that synthesize cholesterol (Hmgcr, Sqle) and fatty acids (Fasn, Scd1), but also direct cholesterol transport (Ldlr, Pcsk9, Stard4) and retinoid synthesis (Aldh1a1, Rdh11). Extensive support by Cyp1b1 is implicated, but with distinct GVAD interventions for Srebp1 and Srebp2. At E9.5, Cyp1b1 is expressed in the septum transversum mesenchyme (STM) with ß-carotene oxygenase (Bco1) that generates retinaldehyde. STM provides progenitors for the HSC and supports liver expansion. GVAD and Cyp1b1-/- do not affect RA-dependent Hoxb1 and Pax6. In DKO embryos, RA-dependent Cyp26a1 is lost but Hoxb1 is sustained with Cyp1b1 at multiple sites. CONCLUSION: Cyp1b1-/- suppresses genes supported by Srebp. GVAD effects distinguish Srebp1 and Srebp2 mediation. Srebp regulation overlaps appreciably in cholesterol and retinoid homeostasis. Bco1/Cyp1b1 partnership in the STM may contribute to this later liver regulation.
Assuntos
Colesterol/biossíntese , Citocromo P-450 CYP1B1/fisiologia , Desenvolvimento Fetal , Fígado/metabolismo , Proteínas de Ligação a Elemento Regulador de Esterol/fisiologia , Tretinoína/metabolismo , Animais , Animais Recém-Nascidos , Citocromo P-450 CYP1B1/genética , Embrião de Mamíferos , Feminino , Desenvolvimento Fetal/efeitos dos fármacos , Desenvolvimento Fetal/genética , Fígado/efeitos dos fármacos , Fígado/embriologia , Fígado/crescimento & desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Proteínas Plasmáticas de Ligação ao Retinol/genética , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , Tretinoína/farmacologiaRESUMO
We evaluated the ability of saucerneol D (SD), a tetrahydrofuran-type sesquilignan isolated from Saururus chinensis, to regulate the expression of inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-stimulated murine macrophage-like RAW264.7 cells. SD consistently inhibited nitric oxide (NO) production in a dose-dependent manner, with an IC(50) of 2.62 microM, and also blocked LPS-induced iNOS expression. SD potently suppressed both the reporter gene expression and DNA-binding activity of nuclear factor-kappaB (NF-kappaB). In addition, SD inhibited IkappaB-alpha degradation in a concentration- and time-dependent manner. SD also inhibited LPS-induced activation of various mitogen-activated protein kinases, including extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). These findings suggest that SD may inhibit LPS-induced iNOS expression by blocking NF-kappaB and MAPK activation.
Assuntos
Expressão Gênica/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , NF-kappa B/antagonistas & inibidores , Óxido Nítrico Sintase Tipo II/biossíntese , Animais , Células Cultivadas , Lignanas/farmacologia , Lipopolissacarídeos/toxicidade , Macrófagos/enzimologia , CamundongosRESUMO
Vitamin A deficiency is still a public health concern affecting millions of pregnant women and children. Retinoic acid, the active form of vitamin A, is critical for proper mammalian embryonic development. Embryos can generate retinoic acid from maternal circulating ß-carotene upon oxidation of retinaldehyde produced via the symmetric cleavage enzyme ß-carotene 15,15'-oxygenase (BCO1). Another cleavage enzyme, ß-carotene 9',10'-oxygenase (BCO2), asymmetrically cleaves ß-carotene in adult tissues to prevent its mitochondrial toxicity, generating ß-apo-10'-carotenal, which can be converted to retinoids (vitamin A and its metabolites) by BCO1. However, the role of BCO2 during mammalian embryogenesis is unknown. We found that mice lacking BCO2 on a vitamin A deficiency-susceptible genetic background (Rbp4-/-) generated severely malformed vitamin A-deficient embryos. Maternal ß-carotene supplementation impaired fertility and did not restore normal embryonic development in the Bco2-/-Rbp4-/- mice, despite the expression of BCO1. These data demonstrate that BCO2 prevents ß-carotene toxicity during embryogenesis under severe vitamin A deficiency. In contrast, ß-apo-10'-carotenal dose-dependently restored normal embryonic development in Bco2-/-Rbp4-/- but not Bco1-/-Bco2-/-Rbp4-/- mice, suggesting that ß-apo-10'-carotenal facilitates embryogenesis as a substrate for BCO1-catalyzed retinoid formation. These findings provide a proof of principle for the important role of BCO2 in embryonic development and invite consideration of ß-apo-10'-carotenal as a nutritional supplement to sustain normal embryonic development in vitamin A-deprived pregnant women.
Assuntos
Carotenoides/metabolismo , Desenvolvimento Embrionário , Retinoides/metabolismo , Deficiência de Vitamina A/complicações , Deficiência de Vitamina A/fisiopatologia , Animais , Dioxigenases/deficiência , Dioxigenases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Plasmáticas de Ligação ao Retinol/deficiência , Proteínas Plasmáticas de Ligação ao Retinol/metabolismo , beta-Caroteno 15,15'-Mono-Oxigenase/deficiência , beta-Caroteno 15,15'-Mono-Oxigenase/metabolismoRESUMO
Vitamin A regulates many essential mammalian biological processes, including embryonic development. ß-carotene is the main source of vitamin A in the human diet. Once ingested, it is packaged into lipoproteins, predominantly low-density lipoproteins (LDL), and transported to different sites within the body, including the liver and developing tissues, where it can either be stored or metabolized to retinoids (vitamin A and its derivatives). The molecular mechanisms of ß-carotene uptake by the liver or developing tissues remain elusive. Here, we investigated the role of the LDL receptor (LDLr) in ß-carotene uptake by maternal liver, placenta and embryo. We administered a single dose of ß-carotene to Ldlr+/- and Ldlr-/- pregnant mice via intraperitoneal injection at mid-gestation and monitored the changes in ß-carotene content among maternal lipoproteins and the liver, as well as the accumulation of ß-carotene in the placental-fetal unit. We showed an abnormal ß-carotene distribution among serum lipoproteins and reduced hepatic ß-carotene uptake in Ldlr-/- dams. These data strongly imply that LDLr significantly contributes to ß-carotene uptake in the adult mouse liver. In contrast, LDLr does not seem to mediate acquisition of ß-carotene by the placental-fetal unit.
Assuntos
Fígado/metabolismo , Receptores de LDL/metabolismo , beta Caroteno/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Genótipo , Lipoproteínas/química , Troca Materno-Fetal , Camundongos , Camundongos Knockout , Circulação Placentária , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de LDL/genética , Retinoides/química , Retinoides/metabolismo , beta Caroteno/sangueRESUMO
Vitamin A homeostasis is critical to normal cellular function. Retinol-binding protein (RBP) is the sole specific carrier in the bloodstream for hydrophobic retinol, the main form in which vitamin A is transported. The integral membrane receptor STRA6 mediates cellular uptake of vitamin A by recognizing RBP-retinol to trigger release and internalization of retinol. We present the structure of zebrafish STRA6 determined to 3.9-angstrom resolution by single-particle cryo-electron microscopy. STRA6 has one intramembrane and nine transmembrane helices in an intricate dimeric assembly. Unexpectedly, calmodulin is bound tightly to STRA6 in a noncanonical arrangement. Residues involved with RBP binding map to an archlike structure that covers a deep lipophilic cleft. This cleft is open to the membrane, suggesting a possible mode for internalization of retinol through direct diffusion into the lipid bilayer.