RESUMO
A hallmark of multiple sclerosis (MS) is the formation of multiple focal demyelinating lesions within the central nervous system (CNS). These lesions mainly consist of phagocytes that play a key role in lesion progression and remyelination, and therefore represent a promising therapeutic target in MS. We recently showed that unsaturated fatty acids produced by stearoyl-CoA desaturase-1 induce inflammatory foam cell formation during demyelination. These fatty acids are elongated by the "elongation of very long chain fatty acids" proteins (ELOVLs), generating a series of functionally distinct lipids. Here, we show that the expression and activity of ELOVLs are altered in myelin-induced foam cells. Especially ELOVL6, an enzyme responsible for converting saturated and monounsaturated C16 fatty acids into C18 species, was found to be up-regulated in myelin phagocytosing phagocytes in vitro and in MS lesions. Depletion of Elovl6 induced a repair-promoting phagocyte phenotype through activation of the S1P/PPARγ pathway. Elovl6-deficient foamy macrophages showed enhanced ABCA1-mediated lipid efflux, increased production of neurotrophic factors, and reduced expression of inflammatory mediators. Moreover, our data show that ELOVL6 hampers CNS repair, as Elovl6 deficiency prevented demyelination and boosted remyelination in organotypic brain slice cultures and the mouse cuprizone model. These findings indicate that targeting ELOVL6 activity may be an effective strategy to stimulate CNS repair in MS and other neurodegenerative diseases.
Assuntos
Esclerose Múltipla , Remielinização , Animais , Camundongos , Adipogenia , Modelos Animais de Doenças , Ácidos Graxos , Ácidos Graxos Monoinsaturados , Células EspumosasRESUMO
Maintenance of metabolic homeostasis is secured by metabolite-sensing systems, which can be overwhelmed by constant macronutrient surplus in obesity. Not only the uptake processes but also the consumption of energy substrates determine the cellular metabolic burden. We herein describe a novel transcriptional system in this context comprised of peroxisome proliferator-activated receptor alpha (PPARα), a master regulator for fatty acid oxidation, and C-terminal binding protein 2 (CtBP2), a metabolite-sensing transcriptional corepressor. CtBP2 interacts with PPARα to repress its activity, and the interaction is enhanced upon binding to malonyl-CoA, a metabolic intermediate increased in tissues in obesity and reported to suppress fatty acid oxidation through inhibition of carnitine palmitoyltransferase 1. In line with our preceding observations that CtBP2 adopts a monomeric configuration upon binding to acyl-CoAs, we determined that mutations in CtBP2 that shift the conformational equilibrium toward monomers increase the interaction between CtBP2 and PPARα. In contrast, metabolic manipulations that reduce malonyl-CoA decreased the formation of the CtBP2-PPARα complex. Consistent with these in vitro findings, we found that the CtBP2-PPARα interaction is accelerated in obese livers while genetic deletion of CtBP2 in the liver causes derepression of PPARα target genes. These findings support our model where CtBP2 exists primarily as a monomer in the metabolic milieu of obesity to repress PPARα, representing a liability in metabolic diseases that can be exploited to develop therapeutic approaches.
Assuntos
Oxirredutases do Álcool , Proteínas Correpressoras , Obesidade , PPAR alfa , Humanos , Ácidos Graxos/metabolismo , Fígado/metabolismo , Obesidade/genética , Obesidade/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismo , Oxirredutases do Álcool/metabolismo , Proteínas Correpressoras/metabolismo , Regulação AlostéricaRESUMO
BACKGROUND: Elongation of very-long-chain fatty acids protein 6 (ELOVL6), an enzyme regulating elongation of saturated and monounsaturated fatty acids with C12 to C16 to those with C18, has been recently indicated to affect various immune and inflammatory responses; however, the precise process by which ELOVL6-related lipid dysregulation affects allergic airway inflammation is unclear. OBJECTIVES: This study sought to evaluate the biological roles of ELOVL6 in allergic airway responses and investigate whether regulating lipid composition in the airways could be an alternative treatment for asthma. METHODS: Expressions of ELOVL6 and other isoforms were examined in the airways of patients who are severely asthmatic and in mouse models of asthma. Wild-type and ELOVL6-deficient (Elovl6-/-) mice were analyzed for ovalbumin-induced, and also for house dust mite-induced, allergic airway inflammation by cell biological and biochemical approaches. RESULTS: ELOVL6 expression was downregulated in the bronchial epithelium of patients who are severely asthmatic compared with controls. In asthmatic mice, ELOVL6 deficiency led to enhanced airway inflammation in which lymphocyte egress from lymph nodes was increased, and both type 2 and non-type 2 immune responses were upregulated. Lipidomic profiling revealed that the levels of palmitic acid, ceramides, and sphingosine-1-phosphate were higher in the lungs of ovalbumin-immunized Elovl6-/- mice compared with those of wild-type mice, while the aggravated airway inflammation was ameliorated by treatment with fumonisin B1 or DL-threo-dihydrosphingosine, inhibitors of ceramide synthase and sphingosine kinase, respectively. CONCLUSIONS: This study illustrates a crucial role for ELOVL6 in controlling allergic airway inflammation via regulation of fatty acid composition and ceramide-sphingosine-1-phosphate biosynthesis and indicates that ELOVL6 may be a novel therapeutic target for asthma.
Assuntos
Asma , Ceramidas , Animais , Camundongos , Modelos Animais de Doenças , Inflamação/tratamento farmacológico , Ovalbumina/efeitos adversosRESUMO
Renal cell carcinoma (RCC) features altered lipid metabolism and accumulated polyunsaturated fatty acids (PUFAs). Elongation of very long-chain fatty acid (ELOVL) family enzymes catalyze fatty acid elongation, and ELOVL5 is indispensable for PUFAs elongation, but its role in RCC progression remains unclear. Here, we show that higher levels of ELOVL5 correlate with poor RCC clinical prognosis. Liquid chromatography/electrospray ionization-tandem mass spectrometry analysis showed decreases in ELOVL5 end products (arachidonic acid and eicosapentaenoic acid) under CRISPR/Cas9-mediated knockout of ELOVL5 while supplementation with these fatty acids partially reversed the cellular proliferation and invasion effects of ELOVL5 knockout. Regarding cellular proliferation and invasion, CRISPR/Cas9-mediated knockout of ELOVL5 suppressed the formation of lipid droplets and induced apoptosis via endoplasmic reticulum stress while suppressing renal cancer cell proliferation and in vivo tumor growth. Furthermore, CRISPR/Cas9-mediated knockout of ELOVL5 inhibited AKT Ser473 phosphorylation and suppressed renal cancer cell invasion through chemokine (C-C motif) ligand-2 downregulation by AKT-mTOR-STAT3 signaling. Collectively, these results suggest that ELOVL5-mediated fatty acid elongation promotes not only cellular proliferation but also invasion in RCC.
Assuntos
Carcinoma de Células Renais , Elongases de Ácidos Graxos , Neoplasias Renais , Acetiltransferases/genética , Acetiltransferases/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/patologia , Proliferação de Células/genética , Elongases de Ácidos Graxos/genética , Ácidos Graxos , Humanos , Neoplasias Renais/genética , Neoplasias Renais/patologia , Proteínas Proto-Oncogênicas c-aktRESUMO
Sterol regulatory element-binding proteins (SREBPs) are master transcription factors for lipid synthesis, and SREBP-1 is important for fatty acid and triglyceride synthesis. SREBP-1 has two isoforms, SREBP-1a and SREBP-1c, which are splicing variants transcribed from the Srebf1 gene. Although SREBP-1a exhibits stronger transcriptional activity than SREBP-1c, hepatic SREBP-1c is considered more physiologically important. We generated SREBP-1a flox mice using the CRISPR/Cas9 system and hepatocyte- and macrophage-specific SREBP-1a knockout (KO) mice (LKO, liver-knockout; and mΦKO, macrophage-knockout). There were no significant differences among all the mouse genotypes upon feeding with a normal diet. However, feeding with a methionine- and choline-deficient (MCD) diet resulted in exacerbated liver injury in both KO mice. In LKO mice, fatty liver was unexpectedly exacerbated, leading to macrophage infiltration and inflammation. In contrast, in mΦKO mice, the fatty liver state was similar to that in flox mice, but the polarity of the macrophages in the liver was transformed into a proinflammatory M1 subtype, resulting in the exacerbation of inflammation. Taken together, we found that SREBP-1a does not contribute to hepatic lipogenesis, but in either hepatocytes or macrophages distinctly controls the onset of pathological conditions in MCD diet-induced hepatitis.NEW & NOTEWORTHY Hepatocyte- and macrophage-specific SREBP-1a knockout mice were generated for the first time. This study reveals that SREBP-1a does not contribute to hepatic lipogenesis, but in either hepatocytes or macrophages distinctly controls the onset of pathological conditions in methionine- and choline-deficient diet-induced hepatitis.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Camundongos , Animais , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Metionina , Colina/metabolismo , Camundongos Endogâmicos C57BL , Hepatócitos/metabolismo , Fígado/metabolismo , Camundongos Knockout , Dieta/efeitos adversos , Inflamação/metabolismo , Macrófagos/metabolismoRESUMO
cAMP responsive element-binding protein H (CREBH) is a hepatic transcription factor to be activated during fasting. We generated CREBH knock-in flox mice, and then generated liver-specific CREBH transgenic (CREBH L-Tg) mice in an active form. CREBH L-Tg mice showed a delay in growth in the postnatal stage. Plasma growth hormone (GH) levels were significantly increased in CREBH L-Tg mice, but plasma insulin-like growth factor 1 (IGF1) levels were significantly decreased, indicating GH resistance. In addition, CREBH overexpression significantly increased hepatic mRNA and plasma levels of FGF21, which is thought to be as one of the causes of growth delay. However, the additional ablation of FGF21 in CREBH L-Tg mice could not correct GH resistance at all. CREBH L-Tg mice sustained GH receptor (GHR) reduction and the increase of IGF binding protein 1 (IGFBP1) in the liver regardless of FGF21. As GHR is a first step in GH signaling, the reduction of GHR leads to impairment of GH signaling. These data suggest that CREBH negatively regulates growth in the postnatal growth stage via various pathways as an abundant energy response by antagonizing GH signaling.
Assuntos
Composição Corporal , Índice de Massa Corporal , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Fatores de Crescimento de Fibroblastos/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Hormônio do Crescimento/metabolismo , Fígado/metabolismo , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Transdução de SinaisRESUMO
While molecular oxygen is essential for aerobic organisms, its utilization is inseparably connected with generation of oxidative insults. To cope with the detrimental aspects, cells evolved antioxidative defense systems, and insufficient management of the oxidative insults underlies the pathogenesis of a wide range of diseases. A battery of genes for this antioxidative defense are regulated by the transcription factors nuclear factor-erythroid 2-like 1 and 2 (NRF1 and NRF2). While the regulatory steps for the activation of NRFs have been investigated with particular emphasis on nuclear translocation and proteosomal degradation, unknown redundancy may exist considering the indispensable nature of these defense systems. Here we unraveled that C-terminal binding protein 2 (CtBP2), a transcriptional cofactor with redox-sensing capability, is an obligate partner of NRFs. CtBP2 forms transcriptional complexes with NRF1 and NRF2 that is required to promote the expression of antioxidant genes in response to oxidative insults. Our findings illustrate a basis for understanding the transcriptional regulation of antioxidative defense systems that may be exploited therapeutically.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas Correpressoras/metabolismo , Fator 1 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Sequência de Aminoácidos , Antioxidantes/metabolismo , Regulação da Expressão Gênica , Humanos , Fator 1 Relacionado a NF-E2/química , Fator 1 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/química , Fator 2 Relacionado a NF-E2/genética , Estresse Oxidativo , Ligação Proteica , Transcrição GênicaRESUMO
High protein diet (HPD) is an affordable and positive approach in prevention and treatment of many diseases. It is believed that transcriptional regulation is responsible for adaptation after HPD feeding and Kruppel-like factor 15 (KLF15), a zinc finger transcription factor that has been proved to perform transcriptional regulation over amino acid, lipid and glucose metabolism, is known to be involved at least in part in this HPD response. To gain more insight into molecular mechanisms by which HPD controls expressions of genes involved in amino acid metabolism in the liver, we performed RNA-seq analysis of mice fed HPD for a short period (3 days). Compared to a low protein diet, HPD feeding significantly increased hepatic expressions of enzymes involved in the breakdown of all the 20 amino acids. Moreover, using KLF15 knockout mice and in vivo Ad-luc analytical system, we were able to identify Cth (cystathionine gamma-lyase) as a new target gene of KLF15 transcription as well as Ast (aspartate aminotransferase) as an example of KLF15-independent gene despite its remarkable responsiveness to HPD. These findings provide us with a clue to elucidate the entire transcriptional regulatory mechanisms of amino acid metabolic pathways.
Assuntos
Aspartato Aminotransferases/genética , Cistationina gama-Liase/genética , Dieta Rica em Proteínas/métodos , Fatores de Transcrição Kruppel-Like/genética , Transcrição Gênica , Adaptação Fisiológica/genética , Aminoácidos/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Cistationina gama-Liase/metabolismo , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genes Reporter , Glucose/metabolismo , Fatores de Transcrição Kruppel-Like/deficiência , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Luciferases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Análise de Sequência de RNA , Transdução de SinaisRESUMO
BACKGROUND AND AIMS: Dysfunctional hepatic lipid metabolism is a cause of nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disorder worldwide, and is closely associated with insulin resistance and type 2 diabetes. ELOVL fatty acid elongase 6 (Elovl6) is responsible for converting C16 saturated and monounsaturated fatty acids (FAs) into C18 species. We have previously shown that Elovl6 contributes to obesity-induced insulin resistance by modifying hepatic C16/C18-related FA composition. APPROACH AND RESULTS: To define the precise molecular mechanism by which hepatic Elovl6 affects energy homeostasis and metabolic disease, we generated liver-specific Elovl6 knockout (LKO) mice. Unexpectedly, LKO mice were not protected from high-fat diet-induced insulin resistance. Instead, LKO mice exhibited higher insulin sensitivity than controls when consuming a high-sucrose diet (HSD), which induces lipogenesis. Hepatic patatin-like phospholipase domain-containing protein 3 (Pnpla3) expression was down-regulated in LKO mice, and adenoviral Pnpla3 restoration reversed the enhancement in insulin sensitivity in HSD-fed LKO mice. Lipidomic analyses showed that the hepatic ceramide(d18:1/18:0) content was lower in LKO mice, which may explain the effect on insulin sensitivity. Ceramide(d18:1/18:0) enhances protein phosphatase 2A (PP2A) activity by interfering with the binding of PP2A to inhibitor 2 of PP2A, leading to Akt dephosphorylation. Its production involves the formation of an Elovl6-ceramide synthase 4 (CerS4) complex in the endoplasmic reticulum and a Pnpla3-CerS4 complex on lipid droplets. Consistent with this, liver-specific Elovl6 deletion in ob/ob mice reduced both hepatic ceramide(d18:1/18:0) and PP2A activity and ameliorated insulin resistance. CONCLUSIONS: Our study demonstrates the key role of hepatic Elovl6 in the regulation of the acyl-chain composition of ceramide and that C18:0-ceramide is a potent regulator of hepatic insulin signaling linked to Pnpla3-mediated NAFLD.
Assuntos
Ceramidas/metabolismo , Elongases de Ácidos Graxos/fisiologia , Resistência à Insulina/genética , Fígado/enzimologia , Animais , Ceramidas/química , Sacarose Alimentar/administração & dosagem , Regulação para Baixo , Elongases de Ácidos Graxos/genética , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Fosfolipases A2 Independentes de Cálcio/metabolismo , Proteína Fosfatase 2/metabolismo , Esfingosina N-Aciltransferase/metabolismoRESUMO
BACKGROUND AND AIM: The incidence of non-alcoholic steatohepatitis (NASH)-related hepatocellular carcinoma (HCC) is progressively increasing. However, the pathophysiology and etiology of NASH progression to HCC are unknown. We hypothesized that steatosis was the key factor in NASH-related hepatocarcinogenesis and aimed to evaluate the effects of long-term liver X receptor (LXR) agonist stimulation on hepatic steatosis induced by a high-fat diet and oxidative stress. METHODS: We used an LXR agonist (T0901317) and CCl4 to induce hepatic steatosis and oxidative stress, respectively. C57BL/6 mice fed with a high-fat diet were treated with either T0901317 + CCl4 (T09 + CCl4 group) or CCl4 alone (CCl4 group). T0901317 (2.5 mg/kg) and CCl4 (0.1 mL/kg) were intraperitoneally administered twice weekly for 24 weeks. RESULTS: The liver-to-body weight ratio was significantly higher in the T09 + CCl4 group than in the CCl4 group. Mice in the T09 + CCl4 group exhibited abnormal lipid metabolism and NASH-like histopathological features. Additionally, all mice in the T09 + CCl4 group developed liver tumors diagnosed as well-differentiated HCC. The genes identified via microarray analysis were related to NASH and HCC development. CONCLUSIONS: By combining long-term LXR agonist stimulation with oxidative stress and a high-fat diet, we successfully reproduced liver conditions in mice similar to those in humans with NASH and progression to HCC. Our results provide new insight into NASH-related HCC progression and therapy.
Assuntos
Carcinoma Hepatocelular/etiologia , Hidrocarbonetos Fluorados/efeitos adversos , Neoplasias Hepáticas/etiologia , Receptores X do Fígado/agonistas , Hepatopatia Gordurosa não Alcoólica/complicações , Estresse Oxidativo , Sulfonamidas/efeitos adversos , Animais , Tetracloreto de Carbono/administração & dosagem , Tetracloreto de Carbono/efeitos adversos , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Progressão da Doença , Hidrocarbonetos Fluorados/administração & dosagem , Injeções Intraperitoneais , Metabolismo dos Lipídeos/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Sulfonamidas/administração & dosagemRESUMO
The epithelial to mesenchymal transition (EMT) is a cell intrinsic program controlling cellular morphological and phenotypic remodeling in a wide range of biological processes. Despite the accumulating evidence, the transcriptional networks regulating EMT still remain to be elucidated. In this study, we demonstrate that C-terminal binding protein 2 (CtBP2), a critical transcriptional co-repressor harboring pyridine nucleotide sensing capability, orchestrates the EMT program at least in part through a novel transcriptional interaction with an octamer transcription factor, OCT1 (POU2F1, POU class 2 homeobox 1). We identified novel interactions of CtBP2 with several octamer transcription factors, and CtBP2 exhibits a direct interaction with OCT1 in particular. OCT1 accelerates the EMT program as reported, which is diminished by the mutation of the CtBP-binding motif in OCT1, suggesting OCT1 represses epithelial gene expression through recruiting the co-repressor CtBP2. In accordance with these findings, a canonical EMT activator transforming growth factor-ß (TGF-ß) promotes the formation of the CtBP2/OCT1 complex. Our observations illustrate the role of CtBP2 to orchestrate the EMT program through the interaction with OCT1 and highlight the potential of therapeutic exploitation of this new transcriptional system for a wide range of diseases.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas Correpressoras/metabolismo , Transição Epitelial-Mesenquimal/fisiologia , Fator 1 de Transcrição de Octâmero/metabolismo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/genética , Sequência de Aminoácidos , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proteínas Correpressoras/química , Proteínas Correpressoras/genética , Sequência Conservada , Transição Epitelial-Mesenquimal/genética , Feminino , Redes Reguladoras de Genes , Humanos , Células MCF-7 , Camundongos , Mutação , Fator 1 de Transcrição de Octâmero/química , Fator 1 de Transcrição de Octâmero/genética , Domínios e Motivos de Interação entre Proteínas , Ratos , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVE: Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1ß (interleukin 1ß) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation. APPROACH AND RESULTS: The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1ß release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1ß release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1ß release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1ß release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1ß release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1ß-deficient mice. CONCLUSIONS: These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.
Assuntos
Ácidos Graxos/toxicidade , Inflamassomos/agonistas , Inflamação/induzido quimicamente , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos Peritoneais/efeitos dos fármacos , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Células Cultivadas , Cristalização , Elongases de Ácidos Graxos , Ácidos Graxos/metabolismo , Inflamassomos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Inflamação/prevenção & controle , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
Peroxisome proliferator-activated receptor-α (PPARα) is a ligand-activated transcription factor involved in the regulation of lipid homeostasis and improves hypertriglyceridemia. Pemafibrate is a novel selective PPARα modulator (SPPARMα) that activates PPARα transcriptional activity. Here, we computationally constructed the structure of the human PPARα in a complex with pemafibrate, along with that of hPPARα complexed with the classical fenofibrate, and studied their interactions quantitatively by using the first-principles calculations-based fragment molecular orbital (FMO) method. Comprehensive structural and protein-ligand binding elucidation along with the in vitro luciferase analysis let us to identify pemafibrate as a novel SPPARMα. Unlike known fibrate ligands, which bind only with the arm I of the Y-shaped ligand binding pocket, the Y-shaped pemafibrate binds to the entire cavity region. This lock and key nature causes enhanced induced fit in pemafibrate-ligated PPARα. Importantly, this selective modulator allosterically changes PPARα conformation to form a brand-new interface, which in turn binds to PPARα co-activator, PGC-1α, resulting in the full activation of PPARα. The structural basis for the potent effects of pemafibrate on PPARα transcriptional activity predicted by the in silico FMO methods was confirmed by in vitro luciferase assay for mutants. The unique binding mode of pemafibrate reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering cues for improving the binding affinity and selectivity of ligand for better clinical consequences. The findings explain the high affinity and efficacy of pemafibrate, which is expected to be in the clinical use soon.
Assuntos
Benzoxazóis/química , Benzoxazóis/metabolismo , Butiratos/química , Butiratos/metabolismo , Modelos Moleculares , PPAR alfa/química , PPAR alfa/metabolismo , Fenofibrato/química , Fenofibrato/metabolismo , Células Hep G2 , Humanos , Ligantes , Luciferases/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismoRESUMO
Although several non-alcoholic steatohepatitis (NASH) models have been reported to date, few of these models fully reflect the histopathology and pathophysiology of human NASH. The aim of this study was to establish a novel NASH model by feeding a high-fat (HF) diet and administering both carbon tetrachloride (CCl4 ) and the Liver X receptor agonist T0901317. Male C57BL/6J mice were divided into four groups (each n = 5): HF, HF + CCl4 , HF + T0901317, and the novel NASH model (HF + CCl4 + T0901317). CCl4 (0.1 mL/kg) and T0901317 (2.5 mg/kg) were intraperitoneally administered four times and five times, respectively. The livers of the novel NASH model group presented a whitish colour. The serum levels of TNF-α and IL-6 were significantly increased in the novel NASH model group, and mice in this group exhibited histopathological features and insulin resistance reflective of NASH, i.e., macrovesicular hepatic steatosis, ballooning hepatocytes, Mallory-Denk bodies, lobular inflammation and fibrosis. The novel NASH model group presented significantly upregulated expression levels of mRNAs related to lipogenesis, oxidative stress, fibrosis and steatosis and significantly downregulated expression levels of mRNAs related to triglyceride export. We successfully established a novel experimental NASH model that exhibits similar histopathology and pathophysiology to human NASH.
Assuntos
Modelos Animais de Doenças , Hepatopatia Gordurosa não Alcoólica/patologia , Animais , Tetracloreto de Carbono/toxicidade , Dieta Hiperlipídica/efeitos adversos , Hidrocarbonetos Fluorados/toxicidade , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Sulfonamidas/toxicidadeRESUMO
Peroxisome proliferator-activated receptor α (PPARα) is a therapeutic target for hyperlipidemia. Pemafibrate (K-877) is a new selective PPARα modulator activating PPARα transcriptional activity. To determine the effects of pemafibrate on diet-induced obesity, wild-type mice were fed a high-fat diet (HFD) containing pemafibrate for 12 weeks. Like fenofibrate, pemafibrate significantly suppressed HFD-induced body weight gain; decreased plasma glucose, insulin and triglyceride (TG) levels; and increased plasma fibroblast growth factor 21 (FGF21). However, compared to the dose of fenofibrate, a relatively low dose of pemafibrate showed these effects. Pemafibrate activated PPARα transcriptional activity in the liver, increasing both hepatic expression and plasma levels of FGF21. Additionally, pemafibrate increased the expression of genes involved in thermogenesis and fatty acid oxidation, including Ucp1, Cidea and Cpt1b in inguinal adipose tissue (iWAT) and the mitochondrial marker Elovl3 in brown adipose tissue (BAT). Therefore, pemafibrate activates thermogenesis in iWAT and BAT by increasing plasma levels of FGF21. Additionally, pemafibrate induced the expression of Atgl and Hsl in epididymal white adipose tissue, leading to the activation of lipolysis. Taken together, pemafibrate suppresses diet-induced obesity in mice and improves their obesity-related metabolic abnormalities. We propose that pemafibrate may be useful for the suppression and improvement of obesity-induced metabolic abnormalities.
Assuntos
Fármacos Antiobesidade/uso terapêutico , Benzoxazóis/uso terapêutico , Butiratos/uso terapêutico , Obesidade/tratamento farmacológico , PPAR alfa/antagonistas & inibidores , Acetiltransferases/genética , Acetiltransferases/metabolismo , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Fármacos Antiobesidade/administração & dosagem , Fármacos Antiobesidade/farmacologia , Proteínas Reguladoras de Apoptose/genética , Proteínas Reguladoras de Apoptose/metabolismo , Benzoxazóis/administração & dosagem , Benzoxazóis/farmacologia , Glicemia/metabolismo , Butiratos/administração & dosagem , Butiratos/farmacologia , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Dieta Hiperlipídica/efeitos adversos , Elongases de Ácidos Graxos , Insulina/sangue , Lipólise , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/prevenção & controle , Triglicerídeos/sangue , Proteína Desacopladora 1/genética , Proteína Desacopladora 1/metabolismoRESUMO
Sodium-glucose cotransporter 2 (SGLT2) inhibitors have both anti-diabetic and anti-obesity effects. However, the precise mechanism of the anti-obesity effect remains unclear. We previously demonstrated that the glycogen depletion signal triggers lipolysis in adipose tissue via liver-brain-adipose neurocircuitry. In this study, therefore, we investigated whether the anti-obesity mechanism of SGLT2 inhibitor is mediated by this mechanism. Diet-induced obese mice were subjected to hepatic vagotomy (HVx) or sham operation and loaded with high fat diet containing 0.015% tofogliflozin (TOFO), a highly selective SGLT2 inhibitor, for 3 weeks. TOFO-treated mice showed a decrease in fat mass and the effect of TOFO was attenuated in HVx group. Although both HVx and sham mice showed a similar level of reduction in hepatic glycogen by TOFO treatment, HVx mice exhibited an attenuated response in protein phosphorylation by protein kinase A (PKA) in white adipose tissue compared with the sham group. As PKA pathway is known to act as an effector of the liver-brain-adipose axis and activate triglyceride lipases in adipocytes, these results indicated that SGLT2 inhibition triggered glycogen depletion signal and actuated liver-brain-adipose axis, resulting in PKA activation in adipocytes. Taken together, it was concluded that the effect of SGLT2 inhibition on weight loss is in part mediated via the liver-brain-adipose neurocircuitry.
Assuntos
Tecido Adiposo/fisiologia , Compostos Benzidrílicos/administração & dosagem , Encéfalo/fisiologia , Glucosídeos/administração & dosagem , Fígado/fisiologia , Inibidores do Transportador 2 de Sódio-Glicose , Transportador 2 de Glucose-Sódio/metabolismo , Redução de Peso/fisiologia , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/inervação , Animais , Fármacos Antiobesidade/administração & dosagem , Encéfalo/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/inervação , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Vagotomia , Nervo Vago/efeitos dos fármacos , Nervo Vago/fisiologia , Nervo Vago/cirurgiaRESUMO
Peroxisome proliferator-activated receptor α (PPARα) is a well-known therapeutic target for treating hyperlipidemia. K-877 is a novel selective PPARα modulator (SPPARMα) that enhances PPARα transcriptional activity with high selectivity and potency, resulting in reduced plasma lipid levels. This study aimed to evaluate the effects of K-877 on hyperlipidemia in low-density lipoprotein receptor knockout (Ldlr-/-) mice, a mouse model of atherosclerosis. We revealed that K-877 administration significantly decreased plasma triglyceride (TG) and total cholesterol (TC) levels and increased plasma high-density lipoprotein cholesterol (HDL-C) levels in Ldlr-/- mice. K-877 administration to Ldlr-/- mice efficiently increased the gene expression of PPARα and its target genes related to fatty acid oxidation in the liver and small intestine. The same treatment significantly increased ATP-binding cassette a1 gene expression in the liver and small intestine and reduced Niemann Pick C1-like 1 gene expression in the small intestine, suggesting that K-877 administration induced HDL-C production in the liver and small intestine and reduced cholesterol absorption in the small intestine. In conclusion, K-877 administration had pronounced effects on the liver and small intestine in Ldlr-/- mice. K-877 is an attractive PPARα-modulating drug for treating hyperlipidemia that works equally well in both the liver and small intestine.
Assuntos
Aterosclerose/tratamento farmacológico , Aterosclerose/genética , Benzoxazóis/farmacologia , Benzoxazóis/uso terapêutico , Butiratos/farmacologia , Butiratos/uso terapêutico , Expressão Gênica/efeitos dos fármacos , Hiperlipidemias/tratamento farmacológico , Hiperlipidemias/genética , Intestino Delgado/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , PPAR alfa/agonistas , PPAR alfa/genética , Receptores de LDL/genética , Animais , Aterosclerose/metabolismo , Colesterol/metabolismo , Modelos Animais de Doenças , Ácidos Graxos/metabolismo , Técnicas de Inativação de Genes , Hiperlipidemias/metabolismo , Absorção Intestinal/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Terapia de Alvo Molecular , Oxirredução/efeitos dos fármacosRESUMO
HMG-CoA reductase (HMGCR) catalyzes the conversion of HMG-CoA to mevalonic acid (MVA); this is the rate-limiting enzyme of the mevalonate pathway that synthesizes cholesterol. Statins, HMGCR inhibitors, are widely used as cholesterol-reducing drugs. However, statin-induced myopathy is the most adverse side effect of statins. To eludicate the mechanisms underlying statin the myotoxicity and HMGCR function in the skeletal muscle, we developed the skeletal muscle-specific HMGCR knockout mice. Knockout mice exhibited postnatal myopathy with elevated serum creatine kinase levels and necrosis. Myopathy in knockout mice was completely rescued by the oral administration of MVA. These results suggest that skeletal muscle toxicity caused by statins is dependent on the deficiencies of HMGCR enzyme activity and downstream metabolites of the mevalonate pathway in skeletal muscles rather than the liver or other organs.
Assuntos
Hidroximetilglutaril-CoA Redutases/deficiência , Inibidores de Hidroximetilglutaril-CoA Redutases/efeitos adversos , Músculo Esquelético/enzimologia , Rabdomiólise/enzimologia , Rabdomiólise/etiologia , Animais , Colesterol/metabolismo , Creatina Quinase/sangue , Modelos Animais de Doenças , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Masculino , Ácido Mevalônico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doenças Musculares/induzido quimicamente , Doenças Musculares/enzimologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Fatty acid elongase 5 (ELOVL5) is an enzyme involved in the synthesis of polyunsaturated fatty acids. Sterol Regulatory Element-binding Protein (SREBP)-1 activates ELOVL5 and increases polyunsaturated fatty acid synthesis, which in turn negatively affects SREBP-1 expression. Thus, ELOVL5 has been established as an SREBP-1 target gene and an important component of the negative feedback loop of de novo lipogenesis. However, the human ELOVL5 promoter/enhancer has not been fully analyzed and the location of SREBP biding sites around the ELOVL5 gene has yet to be defined. Here we performed a detailed promoter/enhancer analysis of human ELOVL5 gene, and identified two new SREBP binding sites, one in the 10 kb upstream region and one in the exon 1. These two SRE motifs are conserved among mammals and the mechanism found in the present study by which SREBP activates ELOVL5 is considered to be common in mammals. Through these findings, we clarified the molecular mechanism how SREBP activates ELOVL5, an important regulator of de novo lipogenesis.
Assuntos
Acetiltransferases/genética , Elementos Facilitadores Genéticos , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Animais , Sequência de Bases , Sítios de Ligação/genética , Éxons , Elongases de Ácidos Graxos , Ácidos Graxos Insaturados/metabolismo , Ácidos Graxos Insaturados/farmacologia , Células HEK293 , Humanos , Lipogênese/genética , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mutação , Regiões Promotoras Genéticas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Regulação para CimaRESUMO
Hepatocyte-specific Phosphatase and tensin homolog (Pten)-knockout (KO) mice exhibit hepatic lesions analogous to non-alcoholic steatohepatitis (NASH). 1,8-cineole is a monoterpene oxide and it has several biological effects including hepatoprotective effects. In this study we revealed that 1,8-cineole ameliorates NASH of Pten KO mice. Pten KO mice were assigned to a control group without any medication or to a 1,8-cineole group injected with 50 mg/kg i.p. twice per week for eight weeks. At eight weeks, livers from each group were processed to measure triglyceride (TG) content, gene expression analysis, western blot analysis, and histological examination including Oil red O staining. 1,8-cineole ameliorated hepatic steatosis in Pten KO mice, revealed by TG content and Oil red O staining. Moreover, 1,8-cineole downregulated collagen 1a1 expression and improved liver fibrosis. Thus, 1,8-cineole has potential as a candidate to treat NASH by inactivating the Akt/PI3-kinase pathway.