RESUMO
With the rapid development of gene therapy technology in recent years, its abuse as a method of sports doping in athletics has become a concern. However, there is still room for improvement in gene-doping testing methods, and a robust animal model needs to be developed. Therefore, the purposes of this study were to establish a model of gene doping using recombinant adeno-associated virus vector-9, including the human erythropoietin gene (rAAV9-hEPO), and to establish a relevant testing method. First, it was attempted to establish the model using rAAV9-hEPO on mice. The results showed a significant increase in erythrocyte volume accompanied by an increase in spleen weight, confirming the validity of the model. Next, we attempted to detect proof of gene doping by targeting DNA and RNA. Direct proof of gene doping was detected using a TaqMan-qPCR assay with certain primers/probes. In addition, some indirect proof was identified in RNAs through the combination of a TB Green qPCR assay with RNA sequencing. Taken together, these results could provide the foundation for an effective test for gene doping in human athletes in the future.
Assuntos
Dependovirus , Dopagem Esportivo , Eritropoetina , Vetores Genéticos , Eritropoetina/genética , Animais , Camundongos , Dopagem Esportivo/métodos , Dependovirus/genética , Humanos , Vetores Genéticos/genética , Masculino , Terapia Genética/métodos , Modelos AnimaisRESUMO
Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl ß-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3ß,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.
Assuntos
Ferroptose , Hepatopatias , Oxirredutases atuantes sobre Doadores de Grupo CH-CH , Camundongos , Animais , Humanos , Dicloridrato de trans-1,4-Bis(2-clorobenzaminometil)ciclo-hexano , Oxirredutases atuantes sobre Doadores de Grupo CH-CH/metabolismoRESUMO
ELOVL fatty acid elongase 6 (ELOVL6) controls cellular fatty acid (FA) composition by catalyzing the elongation of palmitate (C16:0) to stearate (C18:0) and palmitoleate (C16:1n-7) to vaccinate (C18:1n-7). Although the transcriptional regulation of ELOVL6 has been well studied, the post-transcriptional regulation of ELOVL6 is not fully understood. Therefore, this study aims to evaluate the role of microRNAs (miRNAs) in regulating human ELOVL6. Bioinformatic analysis identified five putative miRNAs: miR-135b-5p, miR-135a-5p, miR-125a-5p, miR-125b-5p, and miR-22-3p, which potentially bind ELOVL6 3'-untranslated region (UTR). Results from dual-luciferase assays revealed that these miRNAs downregulate ELOVL6 by directly interacting with the 3'-UTR of ELOVL6 mRNA. Moreover, miR-135b-5p and miR-135a-5p suppress cell proliferation and migration in glioblastoma multiforme cells by inhibiting ELOVL6 at the mRNA and protein levels. Taken together, our results provide novel regulatory mechanisms for ELOVL6 at the post-transcriptional level and identify potential candidates for the treatment of patients with glioblastoma multiforme.
RESUMO
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
Local cryotherapy is widely used as a treatment for sports-related skeletal muscle injuries. The molecular mechanisms are unknown. To clarify these mechanisms, we applied one to three 15-min cold stimulations at 4 °C to various cell lines (in vitro), the tibialis anterior (TA) muscle (ex vivo), and mouse limbs (in vivo). In the in vitro assay, cyclic AMP (cAMP) response element binding protein 1 (CREB1) was markedly phosphorylated (p-CREB1), and the CREB-binding protein (CBP) was recruited to p-CREB-1 in response to two or three cold stimulations. In a reporter assay with the cAMP-responsive element, the signals significantly increased after two to three cold stimulations at 4 °C. In the ex vivo study, CREB-targeting genes were significantly upregulated following two or three cold stimulations. The in vivo experiment disclosed that cold stimulation of a mouse limb for 9 days significantly increased mitochondrial DNA copy number and upregulated genes involved in mitochondrial biogenesis. The results suggest that local cryotherapy increases CREB transcription and upregulates CREB-targeting genes, in a manner dependent on cold stimulation frequency and duration. This information will inform further investigations into local cryotherapy as a treatment for sports-related skeletal muscle trauma.
Assuntos
Crioterapia/métodos , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Músculo Esquelético/fisiologia , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/genética , Masculino , Camundongos , Camundongos Endogâmicos CBA , Músculo Esquelético/citologia , Músculo Esquelético/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Fosforilação , Transdução de Sinais , Ativação TranscricionalRESUMO
BACKGROUND: Renal hypouricemia (RHUC) is a hereditary disorder where mutations in SLC22A12 gene and SLC2A9 gene cause RHUC type 1 (RHUC1) and RHUC type 2 (RHUC2), respectively. These genes regulate renal tubular reabsorption of urates while there exist other genes counterbalancing the net excretion of urates including ABCG2 and SLC17A1. Urate metabolism is tightly interconnected with glucose metabolism, and SLC2A9 gene may be involved in insulin secretion from pancreatic ß-cells. On the other hand, a myriad of genes are responsible for the impaired insulin secretion independently of urate metabolism. CASE PRESENTATION: We describe a 67 year-old Japanese man who manifested severe hypouricemia (0.7 mg/dl (3.8-7.0 mg/dl), 41.6 µmol/l (226-416 µmol/l)) and diabetes with impaired insulin secretion. His high urinary fractional excretion of urate (65.5%) and low urinary C-peptide excretion (25.7 µg/day) were compatible with the diagnosis of RHUC and impaired insulin secretion, respectively. Considering the fact that metabolic pathways regulating urates and glucose are closely interconnected, we attempted to delineate the genetic basis of the hypouricemia and the insulin secretion defect observed in this patient using whole exome sequencing. Intriguingly, we found homozygous Trp258* mutations in SLC22A12 gene causing RHUC1 while concurrent mutations reported to be associated with hyperuricemia were also discovered including ABCG2 (Gln141Lys) and SLC17A1 (Thr269Ile). SLC2A9, that also facilitates glucose transport, has been implicated to enhance insulin secretion, however, the non-synonymous mutations found in SLC2A9 gene of this patient were not dysfunctional variants. Therefore, we embarked on a search for causal mutations for his impaired insulin secretion, resulting in identification of multiple mutations in HNF1A gene (MODY3) as well as other genes that play roles in pancreatic ß-cells. Among them, the Leu80fs in the homeobox gene NKX6.1 was an unreported mutation. CONCLUSION: We found a case of RHUC1 carrying mutations in SLC22A12 gene accompanied with compensatory mutations associated with hyperuricemia, representing the first report showing coexistence of the mutations with opposed potential to regulate urate concentrations. On the other hand, independent gene mutations may be responsible for his impaired insulin secretion, which contains novel mutations in key genes in the pancreatic ß-cell functions that deserve further scrutiny.
Assuntos
Complicações do Diabetes/genética , Proteínas Facilitadoras de Transporte de Glucose/genética , Transportadores de Ânions Orgânicos/genética , Proteínas de Transporte de Cátions Orgânicos/genética , Erros Inatos do Transporte Tubular Renal/genética , Cálculos Urinários/genética , Idoso , Complicações do Diabetes/complicações , Complicações do Diabetes/patologia , Glucose/metabolismo , Fator 1-alfa Nuclear de Hepatócito/genética , Heterozigoto , Proteínas de Homeodomínio/genética , Homozigoto , Humanos , Insulina/biossíntese , Insulina/genética , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Masculino , Mutação/genética , Erros Inatos do Transporte Tubular Renal/complicações , Erros Inatos do Transporte Tubular Renal/patologia , Ácido Úrico/metabolismo , Cálculos Urinários/complicações , Cálculos Urinários/patologia , Sequenciamento do ExomaRESUMO
The aim of this study was to clarify degradation characteristics in each tissue of the knee complex of a medial meniscectomy (MMx)-induced knee osteoarthritis (KOA) animal model using classical methods and an alternative comprehensive evaluation method called contrast-enhanced X-ray micro-computed tomography (CEX-µCT), which was developed in the study. Surgical MMx was performed in the right knee joints of five male Wistar rats to induce KOA. At four weeks post-surgery, the synovitis was evaluated using quantitative polymerase chain reaction (qPCR). Degradations of the articular cartilage of the tibial plateau were evaluated using classical methods and CEX-µCT. Evaluation of the synovitis demonstrated significantly increased expression levels of inflammation-associated marker genes in MMx-treated knees compared with those in sham-treated knees. Evaluation of the articular cartilage using classical methods showed that MMx fully induced degradation of the cartilage. Evaluation using CEX-µCT showed that local areas of the medial cartilage of the tibial plateau were significantly reduced in MMx-treated knees compared with those in sham-treated knees. On the other hand, total cartilage volumes were significantly increased in MMx-treated knees. On the basis of the findings of this study, the method could be relevant to study new treatments in KOA research.
RESUMO
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
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
With the rapid progress of genetic engineering and gene therapy, the World Anti-Doping Agency has been alerted to gene doping and prohibited its use in sports. However, there is no standard method available yet for the detection of transgenes delivered by recombinant adenoviral (rAdV) vectors. Here, we aim to develop a detection method for transgenes delivered by rAdV vectors in a mouse model that mimics gene doping. These rAdV vectors containing the mCherry gene was delivered in mice through intravenous injection or local muscular injection. After five days, stool and whole blood samples were collected, and total DNA was extracted. As additional experiments, whole blood was also collected from the mouse tail tip until 15 days from injection of the rAdv vector. Transgene fragments from different DNA samples were analyzed using semi-quantitative PCR (sqPCR), quantitative PCR (qPCR), and droplet digital PCR (ddPCR). In the results, transgene fragments could be directly detected from blood cell fraction DNA, plasma cell-free DNA, and stool DNA by qPCR and ddPCR, depending on specimen type and injection methods. We observed that a combination of blood cell fraction DNA and ddPCR was more sensitive than other combinations used in this model. These results could accelerate the development of detection methods for gene doping.
Assuntos
Adenoviridae/isolamento & purificação , Dopagem Esportivo , Vetores Genéticos/isolamento & purificação , Transgenes/genética , Adenoviridae/genética , Animais , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Humanos , CamundongosRESUMO
Glucocorticoids have various medical uses but are accompanied by side effects. The glucocorticoid receptor (GR) has been reported to regulate the clock genes, but the underlying mechanisms are incompletely understood. In this study, we focused on the suppressive effect of the GR on the expression of Rev-erbα (Nr1d1), an important component of the clock regulatory circuits. Here we show that the GR suppresses Rev-erbα expression via the formation of a complex with CLOCK and BMAL1, which binds to the E-boxes in the Nr1d1 promoter. In this GR-CLOCK-BMAL1 complex, the GR does not directly bind to DNA, which is referred to as tethering. These findings provide new insights into the role of the GR in the control of circadian rhythm.
Assuntos
Fatores de Transcrição ARNTL/metabolismo , Proteínas CLOCK/metabolismo , Dexametasona/administração & dosagem , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/genética , Receptores de Glucocorticoides/metabolismo , Animais , Ritmo Circadiano/efeitos dos fármacos , Dexametasona/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células HEK293 , Células Hep G2 , Humanos , Masculino , Camundongos , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/química , Membro 1 do Grupo D da Subfamília 1 de Receptores Nucleares/metabolismo , Regiões Promotoras Genéticas , Receptores de Glucocorticoides/agonistasRESUMO
Sterol regulatory element-binding protein (SREBP)-1a is a key transcription factor that activates the expression of genes involved in the synthesis of fatty acids, triglycerides (TGs), and cholesterol. Transgenic mice that overexpress the nuclear form of SREBP-1a under the control of the phosphoenolpyruvate carboxykinase promoter (Tg-1a) were previously shown to display a lipodystrophic phenotype characterized by enlarged and fatty livers, diminished peripheral white adipose tissue (WAT), and insulin resistance. In the current study, we crossed these Tg-1a mice with genetically obese (ob/ob) mice (Tg-1a;ob/ob) and examined change in fat distribution between liver and adipose tissues in severe obesity and mechanism underlying the lipodystrophic phenotype in mice with Tg-1a. Tg-1a;ob/ob mice developed more severe steatohepatitis but had reduced WAT mass and body weight compared with ob/ob mice. The reduction of WAT mass in Tg-1a and Tg-1a;ob/ob mice was accompanied by enhanced lipogenesis and lipid uptake in the liver, reduced plasma lipid levels, impaired adipocyte differentiation, reduced food intake, enhanced energy expenditure, and extended macrophage infiltration and fibrosis in WAT. Despite the improved glucose tolerance, Tg-1a;ob/ob mice showed severe peripheral insulin resistance. Adenoviral hepatic expression of SREBP-1a mimicked these phenotypes. The "fat steal"-like lipodystrophy phenotype of the Tg-1a;ob/ob model demonstrates that hepatic SREBP-1a activation has a strong impact on the partition of TG accumulation, resulting in adipose-tissue remodeling by inflammation and fibrosis and insulin resistance.
Assuntos
Resistência à Insulina/genética , Lipodistrofia/genética , Obesidade/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Animais , Células Cultivadas , Progressão da Doença , Feminino , Lipodistrofia/complicações , Lipodistrofia/patologia , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Camundongos Transgênicos , Obesidade/complicações , Obesidade/patologiaRESUMO
OBJECTIVE: A new opportunistic community-based strategy was launched in Japan in April 2014 to detect lifestyle-related diseases, including diabetes, by creating Specimen Measurement Offices (SMOs). SMOs offer walk-in fingertip HbA1c testing. This article aimed to assess the value-for-money of HbA1c testing services at SMOs by conducting a cost-effectiveness analysis. RESEARCH DESIGN AND METHODS: We compared two scenarios: 1) status quo, defined as HbA1c testing that is available only through conventional screening, and 2) HbA1c testing available at SMOs as a complement to the status quo scenario. The model consisted of a screening module with a decision tree and a disease progression module with a Markov model. We calculated incremental cost-effectiveness ratios (i.e., cost per quality-adjusted life-years [QALYs]) over the lifetime analytic horizon as the primary end point of the cost-effectiveness analysis. In this model, we assumed the participant cohort to be people 40-74 years of age who sought walk-in fingertip HbA1c testing at SMOs on the premises of community pharmacies. Costs and outcomes were discounted at a rate of 3%. The cost-effectiveness was analyzed from a societal perspective. RESULTS: The incremental cost per individual for those 40-74 years of age was estimated to be -527 U.S. dollars (USD) (-52,722 Japanese yen [JPY]) for HbA1c testing at SMOs compared with the status quo. Incremental effectiveness was estimated to be 0.0203 QALYs for HbA1c testing at SMOs compared with the status quo. Therefore, this cost-effectiveness analysis showed that compared with the status quo, HbA1c testing at SMOs was more effective and had lower cost for the population studied. CONCLUSIONS: We consider our results to be robust because most simulations were under the threshold of USD 50,000 (JPY 5,000,000) per QALYs gained, by sensitivity analysis. These results will be useful to managers of pharmacies or other health institutions and/or policy makers in local government.
Assuntos
Diabetes Mellitus Tipo 2/diagnóstico , Hemoglobinas Glicadas/análise , Programas de Rastreamento/economia , Farmácias/economia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Instituições de Assistência Ambulatorial/economia , Instituições de Assistência Ambulatorial/estatística & dados numéricos , Análise Custo-Benefício , Diabetes Mellitus Tipo 2/economia , Diabetes Mellitus Tipo 2/epidemiologia , Progressão da Doença , Feminino , Hemoglobinas Glicadas/economia , Humanos , Japão/epidemiologia , Masculino , Programas de Rastreamento/métodos , Programas de Rastreamento/estatística & dados numéricos , Pessoa de Meia-Idade , Farmácias/estatística & dados numéricos , Anos de Vida Ajustados por Qualidade de Vida , Características de Residência , Adulto JovemRESUMO
The SNP rs7903146 at the transcription factor 7-like 2 (TCF7L2) locus is established as the strongest known genetic marker for type 2 diabetes via genome-wide association studies. However, the functional SNPs regulating TCF7L2 expression remain unclear. Here, we show that the SNP rs7074440 is a candidate functional SNP highly linked with rs7903146. A reporter plasmid with rs7074440 normal allele sequence exhibited 15-fold higher luciferase activity compared with risk allele sequence in hepatocytes, demonstrating a strong enhancer activity at rs7074440. Additionally, we identified C-FOS as an activator binding to the rs7074440 enhancer using a TFEL genome-wide screen method. Consistently, knockdown of C-FOS significantly reduced TCF7L2 expression in hepatocytes. Collectively, a novel enhancer regulating TCF7L2 expression was revealed through searching for functional SNPs.
Assuntos
Diabetes Mellitus Tipo 2/genética , Hepatócitos/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Animais , Linhagem Celular , Feminino , Expressão Gênica , Células HEK293 , Células Hep G2 , Hepatócitos/citologia , Humanos , Masculino , CamundongosRESUMO
AIM: Myriad biological effects of leptin may lead to broad therapeutic applications for various metabolic diseases, including diabetes and its complications; however, in contrast to its anorexic effect, the molecular mechanisms underlying adipopenic and glucose-lowering effects of leptin have not been fully understood. Here we aim to clarify the role of hormone-sensitive lipase (HSL) in leptin's action. METHODS: Wild-type (WT) and HSL-deficient (HSLKO) mice were made hyperleptinemic by two commonly-used methods: adenovirus-mediated overexpression of leptin and continuous subcutaneous infusion of leptin by osmotic pumps. The amount of food intake, body weights, organ weights, and parameters of glucose and lipid metabolism were measured. RESULTS: Hyperleptinemia equally suppressed the food intake in WT and HSLKO mice. On the other hand, leptin-mediated fat loss and glucose-lowering were significantly blunted in the absence of HSL when leptin was overexpressed by recombinant adenovirus carrying leptin. By osmotic pumps, the fat-losing and glucose-lowering effects of leptin were milder due to lower levels of hyperleptinemia; although the difference between WT and HSLKO mice did not reach statistical significance, HSLKO mice had a tendency to retain more fat than WT mice in the face of hyperleptinemia. CONCLUSIONS: We clarify for the first time the role of HSL in leptin's effect using a genetic model: leptin-promoted fat loss and glucose-lowering are at least in part mediated via HSL-mediated lipolysis. Further studies to define the pathophysiological role of adipocyte lipases in leptin action may lead to a new therapeutic approach to circumvent leptin resistance.
Assuntos
Tecido Adiposo/patologia , Glucose/metabolismo , Leptina/farmacologia , Lipase/fisiologia , Lipólise/efeitos dos fármacos , Esterol Esterase/fisiologia , Tecido Adiposo/efeitos dos fármacos , Animais , Feminino , Leptina/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Fatty acid synthase (Fasn) is a key component of energy metabolism that is dynamically induced by food intake. Although extensive studies have revealed a number of transcription factors involved in the fasting/refeeding transition of Fasn expression in hepatocytes, much less evidence is available for adipocytes. Using the in vivo Ad-luc analytical system, we identified the inverted CCAAT element (ICE) around -100 nucleotides in the Fasn promoter as a critical cis-element for the refeeding response in adipocytes. Electrophoretic mobility shift assays and chromatin immunoprecipitation show that nuclear factor Y (NF-Y) binds to ICE specifically in refeeding states. Notably, the NF-Y binding to ICE is differently regulated between adipocytes and hepatocytes. These findings provide insights into the specific mechanisms controlling energy metabolism in adipocytes.
Assuntos
Adipócitos/metabolismo , Fator de Ligação a CCAAT/metabolismo , Ácido Graxo Sintases/metabolismo , Comportamento Alimentar , Células 3T3-L1 , Adenoviridae/genética , Adipócitos/citologia , Tecido Adiposo Branco/metabolismo , Animais , Sequência de Bases , Fator de Ligação a CCAAT/genética , Imunoprecipitação da Cromatina , Ensaio de Desvio de Mobilidade Eletroforética , Ácido Graxo Sintases/genética , Regulação da Expressão Gênica , Immunoblotting , Fígado/metabolismo , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Mutação , Regiões Promotoras Genéticas/genética , Ligação Proteica , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
CREB3L3 is involved in fatty acid oxidation and ketogenesis in a mutual manner with PPARα. To evaluate relative contribution, a combination of knockout and transgenic mice was investigated. On a ketogenic-diet (KD) that highlights capability of hepatic ketogenesis, Creb3l3-/- mice exhibited reduction of expression of genes for fatty oxidation and ketogenesis comparable to Ppara-/- mice. Most of the genes were further suppressed in double knockout mice indicating independent contribution of hepatic CREB3L3. During fasting, dependency of ketogenesis on CREB3L3 is lesser extents than Ppara-/- mice suggesting importance of adipose PPARα for supply of FFA and hyperlipidemia in Creb3l3-/- mice. In conclusion CREB3L3 plays a crucial role in hepatic adaptation to energy starvation via two pathways: direct related gene regulation and an auto-loop activation of PPARα. Furthermore, as KD-fed Creb3l3-/- mice exhibited severe fatty liver, activating inflammation, CREB3L3 could be a therapeutic target for NAFLD.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Ácidos Graxos/química , PPAR alfa/genética , Adenoviridae/genética , Animais , Glicemia/análise , Carnitina O-Palmitoiltransferase/química , Carnitina O-Palmitoiltransferase/genética , Carnitina O-Palmitoiltransferase/metabolismo , Linhagem Celular Tumoral , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/deficiência , Dieta Cetogênica , Metabolismo Energético , Ácidos Graxos/metabolismo , Fígado Gorduroso/etiologia , Fígado Gorduroso/metabolismo , Fatores de Crescimento de Fibroblastos/sangue , Expressão Gênica , Peroxidação de Lipídeos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , PPAR alfa/deficiência , Plasmídeos/genética , Plasmídeos/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , Triglicerídeos/sangueRESUMO
OBJECTIVE: The transcription factor cyclic AMP-responsive element-binding protein H (CREBH, encoded by Creb3l3) is highly expressed in the liver and small intestine. Hepatic CREBH contributes to glucose and triglyceride metabolism by regulating fibroblast growth factor 21 (Fgf21) expression. However, the intestinal CREBH function remains unknown. METHODS: To investigate the influence of intestinal CREBH on cholesterol metabolism, we compared plasma, bile, fecal, and tissue cholesterol levels between wild-type (WT) mice and mice overexpressing active human CREBH mainly in the small intestine (CREBH Tg mice) under different dietary conditions. RESULTS: Plasma cholesterol, hepatic lipid, and cholesterol crystal formation in the gallbladder were lower in CREBH Tg mice fed a lithogenic diet (LD) than in LD-fed WTs, while fecal cholesterol output was higher in the former. These results suggest that intestinal CREBH overexpression suppresses cholesterol absorption, leading to reduced plasma cholesterol, limited hepatic supply, and greater excretion. The expression of Niemann-Pick C1-like 1 (Npc1l1), a rate-limiting transporter mediating intestinal cholesterol absorption, was reduced in the small intestine of CREBH Tg mice. Adenosine triphosphate-binding cassette transporter A1 (Abca1), Abcg5/8, and scavenger receptor class B, member 1 (Srb1) expression levels were also reduced in CREBH Tg mice. Promoter assays revealed that CREBH directly regulates Npc1l1 expression. Conversely, CREBH null mice exhibited higher intestinal Npc1l1 expression, elevated plasma and hepatic cholesterol, and lower fecal output. CONCLUSION: Intestinal CREBH regulates dietary cholesterol flow from the small intestine by controlling the expression of multiple intestinal transporters. We propose that intestinal CREBH could be a therapeutic target for hypercholesterolemia.
Assuntos
Colesterol na Dieta/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Hipercolesterolemia/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Membro 5 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 8 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP , Animais , Humanos , Hipercolesterolemia/tratamento farmacológico , Lipoproteínas , Proteínas de Membrana , CamundongosRESUMO
Hepatic lipogenesis is nutritionally regulated (i.e., downregulated during fasting and upregulated during the postprandial state) as an adaptation to the nutritional environment. While alterations in the expression level of the transcription factor SREBP-1c are known to be critical for nutritionally regulated lipogenesis, upstream mechanisms governing Srebf1 expression remain unclear. Here, we show that the fasting-induced transcription factor KLF15, a key regulator of gluconeogenesis, forms a complex with LXR/RXR, specifically on the Srebf1 promoter. This complex recruits the corepressor RIP140 instead of the coactivator SRC1, resulting in reduced Srebf1 and thus downstream lipogenic enzyme expression during the early and euglycemic period of fasting prior to hypoglycemia and PKA activation. Through this mechanism, KLF15 overexpression specifically ameliorates hypertriglyceridemia without affecting LXR-mediated cholesterol metabolism. These findings reveal a key molecular link between glucose and lipid metabolism and have therapeutic implications for the treatment of hyperlipidemia.
Assuntos
Proteínas de Ligação a DNA/genética , Genoma , Gluconeogênese/genética , Hepatócitos/metabolismo , Lipogênese/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Fatores de Transcrição/genética , Animais , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Jejum , Genes Reporter , Hepatócitos/citologia , Fatores de Transcrição Kruppel-Like , Fígado/citologia , Fígado/metabolismo , Receptores X do Fígado/genética , Receptores X do Fígado/metabolismo , Luciferases/genética , Luciferases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Correpressor 1 de Receptor Nuclear/genética , Correpressor 1 de Receptor Nuclear/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Ligação Proteica , Receptores X de Retinoides/genética , Receptores X de Retinoides/metabolismo , Transdução de Sinais , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Fatores de Transcrição/metabolismo , Ativação TranscricionalRESUMO
Non-alcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, can progress to steatohepatitis (NASH) and advanced liver damage, such as that from liver cirrhosis and cancer. Recent studies have shown the benefits of consuming n-3 polyunsaturated fatty acids (PUFAs) for the treatment of NAFLD. In the present study, we investigated and compared the effects of the major n-3 PUFAs-eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6)-in preventing atherogenic high-fat (AHF) diet-induced NAFLD. Mice were fed the AHF diet supplemented with or without EPA or DHA for four weeks. Both EPA and DHA reduced the pathological features of AHF diet-induced NASH pathologies such as hepatic lobular inflammation and elevated serum transaminase activity. Intriguingly, EPA had a greater hepatic triacylglycerol (TG)-reducing effect than DHA. In contrast, DHA had a greater suppressive effect than EPA on AHF diet-induced hepatic inflammation and ROS generation, but no difference in fibrosis. Both EPA and DHA could be effective for treatment of NAFLD and NASH. Meanwhile, the two major n-3 polyunsaturated fatty acids might differ in a relative contribution to pathological intermediate steps towards liver fibrosis.