RESUMO
Obesity and diabetes affect more than half a billion individuals worldwide. Interestingly, the two conditions do not always coincide and the molecular determinants of "healthy" versus "unhealthy" obesity remain ill-defined. Chronic metabolic inflammation (metaflammation) is believed to be pivotal. Here, we tested a hypothesized anti-inflammatory role for heme oxygenase-1 (HO-1) in the development of metabolic disease. Surprisingly, in matched biopsies from "healthy" versus insulin-resistant obese subjects we find HO-1 to be among the strongest positive predictors of metabolic disease in humans. We find that hepatocyte and macrophage conditional HO-1 deletion in mice evokes resistance to diet-induced insulin resistance and inflammation, dramatically reducing secondary disease such as steatosis and liver toxicity. Intriguingly, cellular assays show that HO-1 defines prestimulation thresholds for inflammatory skewing and NF-κB amplification in macrophages and for insulin signaling in hepatocytes. These findings identify HO-1 inhibition as a potential therapeutic strategy for metabolic disease.
Assuntos
Heme Oxigenase-1/metabolismo , Resistência à Insulina , Proteínas de Membrana/metabolismo , Obesidade/complicações , Tecido Adiposo/metabolismo , Animais , Dieta Hiperlipídica , Hepatócitos/metabolismo , Humanos , Inflamação/metabolismo , Fígado/metabolismo , Macrófagos/metabolismo , Doenças Metabólicas/metabolismo , Doenças Metabólicas/fisiopatologia , Camundongos , Camundongos Knockout , Obesidade/fisiopatologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
The transcriptional regulator peroxisome proliferator activated receptor gamma coactivator 1A (PGC-1α), encoded by PPARGC1A, has been linked to neurodegenerative diseases. Recently discovered CNS-specific PPARGC1A transcripts are initiated far upstream of the reference promoter, spliced to exon 2 of the reference gene, and are more abundant than reference gene transcripts in post-mortem human brain samples. The proteins translated from the CNS and reference transcripts differ only at their N-terminal regions. To dissect functional differences between CNS-specific isoforms and reference proteins, we used clustered regularly interspaced short palindromic repeats transcriptional activation (CRISPRa) for selective endogenous activation of the CNS or the reference promoters in SH-SY5Y cells. Expression and/or exon usage of the targets was ascertained by RNA sequencing. Compared to controls, more differentially expressed genes were observed after activation of the CNS than the reference gene promoter, while the magnitude of alternative exon usage was comparable between activation of the two promoters. Promoter-selective associations were observed with canonical signaling pathways, mitochondrial and nervous system functions and neurological diseases. The distinct N-terminal as well as the shared downstream regions of PGC-1α isoforms affect the exon usage of numerous genes. Furthermore, associations of risk genes of amyotrophic lateral sclerosis and Parkinson's disease were noted with differentially expressed genes resulting from the activation of the CNS and reference gene promoter, respectively. Thus, CNS-specific isoforms markedly amplify the biological functions of PPARGC1A and CNS-specific isoforms and reference proteins have common, complementary and selective functions relevant for neurodegenerative diseases.
Assuntos
Redes Reguladoras de Genes , Doenças Neurodegenerativas/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Regiões Promotoras Genéticas , Ativação Transcricional , Linhagem Celular Tumoral , Éxons , Células HEK293 , Humanos , Neurônios/metabolismo , Motivos de Nucleotídeos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , TranscriptomaRESUMO
Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. PGC-1α, encoded by PPARGC1A, is a transcriptional co-activator that has been implicated in the pathogenesis of neurodegenerative disorders. We recently discovered multiple new PPARGC1A transcripts that initiate from a novel promoter located far upstream of the reference gene promoter, are CNS-specific and are more abundant than reference gene transcripts in whole brain. These CNS-specific transcripts encode two main full-length and several truncated isoforms via alternative splicing. Truncated CNS-isoforms include 17â¯kDa proteins that lack the second LXXLL motif serving as an interaction site for several nuclear receptors. We now determined expression levels of CNS- and reference gene transcripts in 5 brain regions of 21, 8, and 13 deceased subjects with idiopathic PD, Lewy body dementia and controls without neurodegenerative disorders, respectively. We observed reductions of CNS-specific transcripts (encoding full-length isoforms) only in the substantia nigra pars compacta of PD and Lewy body dementia. However, in the substantia nigra and globus pallidus of PD cases we found an up-regulation of transcripts encoding the 17â¯kDa proteins that inhibited the co-activation of several transcription factors by full-length PGC-1α proteins in transfection assays. In two established animal models of PD, the PPARGC1A expression profiles differed from the profile in human PD in that the levels of CNS- and reference gene transcripts were decreased in several brain regions. Furthermore, we identified haplotypes in the CNS-specific region of PPARGC1A that appeared protective for PD in a clinical cohort and a post-mortem sample (Pâ¯=â¯.0002). Thus, functional and genetic studies support a role of the CNS-specific PPARGC1A locus in PD.
Assuntos
Encéfalo/metabolismo , Doença de Parkinson/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Idoso , Idoso de 80 Anos ou mais , Animais , Feminino , Loci Gênicos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Isoformas de Proteínas/genéticaRESUMO
BACKGROUND/AIMS: In the human genome, more than 400 genes encode ion channels, which are ubiquitously expressed and often coexist and participate in almost all physiological processes. Therefore, ion channel blockers represent fundamental tools in discriminating the contribution of individual channel types to a physiological phenomenon. However, unspecific effects of these compounds may represent a confounding factor. Three commonly used chloride channel inhibitors, i.e. 4,4'-diisothiocyano-2,2'-stilbene-disulfonic acid (DIDS), 5-nitro-2-[(3-phenylpropyl) amino]benzoic acid (NPPB) and the anti-inflammatory drug niflumic acid were tested to identify the lowest concentration effective on Cl- channels and ineffective on K+ channels. METHODS: The activity of the above mentioned compounds was tested by whole cell patch-clamp on the swelling-activated Cl- current ICl,swell and on the endogenous voltage-dependent, outwardly rectifying K+ selective current in human kidney cell lines (HEK 293/HEK 293 Phoenix). RESULTS: Micromolar (1-10 µM) concentrations of DIDS and NPPB could not discriminate between the Cl- and K+ selective currents. Specifically, 1 µM DIDS only affected the K+ current and 10 µM NPPB equally affected the Cl- and K+ currents. Only relatively high (0.1-1 mM) concentrations of DIDS and prolonged (5 minutes) exposure to 0.1-1 mM NPPB preferentially suppressed the Cl- current. Niflumic acid preferentially inhibited the Cl- current, but also significantly affected the K+ current. The endogenous voltage-dependent, outwardly rectifying K+ selective current in HEK 293/HEK 293 Phoenix cells was shown to arise from the Kv 3.1 channel, which is extensively expressed in brain and is involved in neurological diseases. CONCLUSION: The results of the present study underscore that sensitivity of a given physiological phenomenon to the Cl- channel inhibitors NPPB, DIDS and niflumic acid may actually arise from an inhibition of Cl- channels but can also result from an inhibition of voltage-dependent K+ channels, including the Kv 3.1 channel. The use of niflumic acid as anti-inflammatory drug in patients with concomitant Kv 3.1 dysfunction may result contraindicated.
Assuntos
Potenciais de Ação/efeitos dos fármacos , Canal de Potássio Kv1.3/metabolismo , Bloqueadores dos Canais de Potássio/farmacologia , Potássio/metabolismo , Animais , Cloretos/metabolismo , Células Epiteliais/citologia , Células HEK293 , Humanos , Túbulos Renais Proximais/citologia , Canal de Potássio Kv1.3/antagonistas & inibidores , Canal de Potássio Kv1.3/genética , Camundongos , Células NIH 3T3 , Ácido Niflúmico/química , Ácido Niflúmico/farmacologia , Nitrobenzoatos/química , Nitrobenzoatos/farmacologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/química , Interferência de RNA , RNA Interferente Pequeno/metabolismoRESUMO
The prevalence and spectrum of sequence alterations in the SLC26A4 gene, which codes for the anion exchanger pendrin, are population-specific and account for at least 50% of cases of non-syndromic hearing loss associated with an enlarged vestibular aqueduct. A cohort of nineteen patients from Austria with hearing loss and a radiological alteration of the vestibular aqueduct underwent Sanger sequencing of SLC26A4 and GJB2, coding for connexin 26. The pathogenicity of sequence alterations detected was assessed by determining ion transport and molecular features of the corresponding SLC26A4 protein variants. In this group, four uncharacterized sequence alterations within the SLC26A4 coding region were found. Three of these lead to protein variants with abnormal functional and molecular features, while one should be considered with no pathogenic potential. Pathogenic SLC26A4 sequence alterations were only found in 12% of patients. SLC26A4 sequence alterations commonly found in other Caucasian populations were not detected. This survey represents the first study on the prevalence and spectrum of SLC26A4 sequence alterations in an Austrian cohort and further suggests that genetic testing should always be integrated with functional characterization and determination of the molecular features of protein variants in order to unequivocally identify or exclude a causal link between genotype and phenotype.
Assuntos
Perda Auditiva Neurossensorial/patologia , Proteínas de Membrana Transportadoras/genética , Aqueduto Vestibular/anormalidades , População Branca/genética , Adolescente , Adulto , Idoso , Alelos , Áustria , Criança , Conexina 26/genética , Retículo Endoplasmático/metabolismo , Feminino , Genótipo , Células HEK293 , Perda Auditiva Neurossensorial/genética , Humanos , Masculino , Proteínas de Membrana Transportadoras/metabolismo , Pessoa de Meia-Idade , Fenótipo , Polimorfismo de Nucleotídeo Único , Transportadores de Sulfato , Aqueduto Vestibular/patologia , Adulto JovemRESUMO
BACKGROUND/AIM: Accurate genotyping of CYP2D6 is challenging due to its inherent genetic variation, copy number variation (duplications and deletions) and hybrid formation with highly homologous pseudogenes. Because a relatively high percentage (â¼25%) of clinically prescribed drugs are substrates for this enzyme, accurate determination of its genotype for phenotype prediction is essential. METHODS: A cohort of 365 patient samples was genotyped for CYP2D6 using Sanger sequencing (as the gold standard), hydrolysis probe assays or pyrosequencing. RESULTS: A discrepant result between the three genotyping methods for the loss of function CYP2D6*3 (g.2549delA, rs35742686) genetic variant was found in one of the samples. This sample also contained the CYP2D6 g.2470T>C (rs17002852) variation, which had an allele frequency of 2.47% in our cohort. Redesign of the CYP2D6*3 pyrosequencing and hydrolysis probe assays to avoid CYP2D6 g.2470 corrected the anomaly. CONCLUSION: To evidence allele drop out and increase the accuracy of genotyping, intra-patient validation of the same genetic variation with at least two separate methods should be considered.
Assuntos
Citocromo P-450 CYP2D6/genética , Variações do Número de Cópias de DNA , Técnicas de Genotipagem/métodos , Alelos , Estudos de Coortes , DNA/química , DNA/isolamento & purificação , DNA/metabolismo , Análise Discriminante , Frequência do Gene , Genótipo , Haplótipos , Humanos , Fenótipo , Análise de Sequência de DNARESUMO
Pendrin is upregulated in bronchial epithelial cells following IL-4 stimulation via binding of STAT6 to an N4 GAS motif. Basal CpG methylation of the pendrin promoter is cell-specific. We studied if a correlation exists between IL-4 sensitivity and the CpG methylation status of the pendrin promoter in human bronchial epithelial cell models. METHODS: Real-time PCR and pyrosequencing were used to respectively quantify pendrin mRNA levels and methylation of pendrin promoter, with and without IL-4 stimulation, in healthy and diseased primary HBE cells, as well as NCI-H292 cells. RESULTS: Increases in pendrin mRNA after IL-4 stimulation was more robust in NCI-H292 cells than in primary cells. The amount of gDNA methylated varied greatly between the cell types. In particular, CpG site 90 located near the N4 GAS motif was highly methylated in the primary cells. An additional CpG site (90bis), created by a SNP, was found only in the primary cells. IL-4 stimulation resulted in dramatic demethylation of CpG sites 90 and 90bis in the primary cells. CONCLUSIONS: IL-4 induces demethylation of specific CpG sites within the pendrin promoter. These epigenetic alterations are cell type specific, and may in part dictate pendrin mRNA transcription.
Assuntos
Brônquios/metabolismo , Ilhas de CpG , Metilação de DNA , Células Epiteliais/metabolismo , Interleucina-4/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Elementos de Resposta , Brônquios/citologia , Linhagem Celular , Epigênese Genética , Células Epiteliais/citologia , Feminino , Humanos , Pessoa de Meia-Idade , RNA Mensageiro/biossíntese , Transportadores de SulfatoRESUMO
OBJECTIVES: Non-alcoholic fatty liver disease (NAFLD) is closely linked to obesity; however, 5-8% of lean subjects also have evidence of NAFLD. We aimed to investigate clinical, genetic, metabolic and lifestyle characteristics in lean Caucasian subjects with NAFLD. METHODS: Data from 187 subjects allocated to one of the three groups according to body mass index (BMI) and hepatic steatosis on ultrasound were obtained: lean healthy (BMI≤25 kg/m2, no steatosis, N=71), lean NAFLD (BMI≤25 kg/m2, steatosis, N=55), obese NAFLD (BMI≥30 kg/m2, steatosis; N=61). All subjects received a detailed clinical and laboratory examination including oral glucose tolerance test. The serum metabolome was assessed using the Metabolomics AbsoluteIDQ p180 kit (BIOCRATES Life Sciences). Genotyping for single-nucleotide polymorphisms (SNPs) associated with NAFLD was performed. RESULTS: Lean NAFLD subjects had fasting insulin concentrations similar to lean healthy subjects but had markedly impaired glucose tolerance. Lean NAFLD subjects had a higher rate of the mutant PNPLA3 CG/GG variant compared to lean controls (P=0.007). Serum adiponectin concentrations were decreased in both NAFLD groups compared to controls (P<0.001 for both groups) The metabolomics study revealed a potential role for various lysophosphatidylcholines (lyso-PC C18:0, lyso-PC C17:0) and phosphatidylcholines (PCaa C36:3; false discovery rate (FDR)-corrected P-value<0.001) as well as lysine, tyrosine, and valine (FDR<0.001). CONCLUSIONS: Lean subjects with evidence of NAFLD have clinically relevant impaired glucose tolerance, low adiponectin concentrations and a distinct metabolite profile with an increased rate of PNPLA3 risk allele carriage.
Assuntos
Adiponectina/metabolismo , Intolerância à Glucose/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/metabolismo , Adulto , Idoso , Alelos , Índice de Massa Corporal , Estudos de Casos e Controles , Feminino , Genótipo , Intolerância à Glucose/epidemiologia , Humanos , Insulina/metabolismo , Resistência à Insulina , Lipase/genética , Fígado/diagnóstico por imagem , Fígado/metabolismo , Lisina/metabolismo , Lisofosfatidilcolinas/metabolismo , Masculino , Proteínas de Membrana/genética , Metabolômica , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/complicações , Hepatopatia Gordurosa não Alcoólica/diagnóstico por imagem , Hepatopatia Gordurosa não Alcoólica/epidemiologia , Obesidade/complicações , Fosfatidilcolinas/metabolismo , Polimorfismo de Nucleotídeo Único , Tirosina/metabolismo , Ultrassonografia , Valina/metabolismo , População BrancaRESUMO
BACKGROUND & AIMS: Liver biopsy (LB) is performed if non-invasive work-up of liver disease is inconclusive. The examination of liver tissue occasionally reveals normal histology. Long-term follow-up of such patients has not been performed. METHODS: We identified a total 70 subjects from our LB database with elevated liver tests and normal liver histology after a mean of 90.5 ± 52.3 (range 15-216) months and conducted reassessment of medical history, physical examination, laboratory testing, ultrasound, transient elastography and LB if indicated. RESULTS: At follow-up examination, 15 (7 females (f)/8 males (m); 21.4%) subjects had normal liver tests and no further evidence of liver disease. A subset of 37 (29 f/8 m; 52.9%) subjects had persistently elevated liver tests without evidence indicating progressive liver disease but the cause thereof remained unexplained also at the follow-up visit. Three (0 f/3 m; 4.3%) subjects had consumed excessive alcohol with indicators of alcoholic liver disease. Eleven subjects (4 f/7 m; 15.7%) had developed steatosis on ultrasound examination along with weight gain and/or biochemical features of the metabolic syndrome. In addition, three (2 f/1 m) patients developed autoimmune hepatitis, one female presented with primary biliary cirrhosis. One male was diagnosed with cholangiocellular carcinoma 3 months after the initial evaluation. CONCLUSION: The clinical course of most patients was benign, but in approximately 20% of the subjects a liver disease developed. Particular attention should be given to autoimmune liver diseases in subjects with positive autoantibodies. In addition, lifestyle factors such as weight gain and alcohol consumption were associated with the manifestation of liver diseases.
Assuntos
Consumo de Bebidas Alcoólicas , Hepatite Autoimune , Hepatopatias Alcoólicas , Hepatopatias , Fígado/patologia , Aumento de Peso , Adulto , Áustria/epidemiologia , Feminino , Seguimentos , Hepatite Autoimune/diagnóstico , Hepatite Autoimune/epidemiologia , Humanos , Hepatopatias/diagnóstico , Hepatopatias/epidemiologia , Hepatopatias/patologia , Hepatopatias Alcoólicas/diagnóstico , Hepatopatias Alcoólicas/epidemiologia , Testes de Função Hepática/métodos , Masculino , Pessoa de Meia-Idade , Fatores de RiscoRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating, adult-onset neurodegenerative disorder of the upper and lower motor systems. It leads to paresis, muscle wasting and inevitably to death, typically within 3-5 years. However, disease onset and survival vary considerably ranging in extreme cases from a few months to several decades. The genetic and environmental factors underlying this variability are of great interest as potential therapeutic targets. In ALS, men are affected more often and have an earlier age of onset than women. This gender difference is recapitulated in transgenic rodent models, but no underlying mechanism has been elucidated. Here we report that SNPs in the brain-specific promoter region of the transcriptional co-activator PGC-1α, a master regulator of metabolism, modulate age of onset and survival in two large and independent ALS populations and this occurs in a strictly male-specific manner. In complementary animal studies, we show that deficiency of full-length (FL) Pgc-1α leads to a significantly earlier age of onset and a borderline shortened survival in male, but not in female ALS-transgenic mice. In the animal model, FL Pgc-1α-loss is associated with reduced mRNA levels of the trophic factor Vegf-A in males, but not in females. In summary, we indentify PGC-1α as a novel and clinically relevant disease modifier of human and experimental ALS and report a sex-dependent effect of PGC-1α in this neurodegenerative disorder.
Assuntos
Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Adulto , Idade de Início , Idoso , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Polimorfismo de Nucleotídeo Único , Caracteres Sexuais , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
PGC-1α has been implicated in the pathogenesis of neurodegenerative disorders. Several single-nucleotide polymorphisms (SNPs) located in two separate haplotype blocks of PPARGC1A have shown associations with Huntington's disease (HD) and Parkinson's disease, but causative SNPs have not been identified. One SNP (rs7665116) was located in a highly conserved 233 bp region of intron 2. To determine whether rs7665116 is located in an alternative exon, we performed 5'-RLM-RACE from exon 3 and discovered multiple new transcripts that initiated from a common novel promoter located 587 kb upstream of exon 2, but did not contain the conserved region harboring rs7665116. Using real-time polymerase chain reaction, RNase protection assays and northern blotting, we show that the majority of these transcripts are brain specific and are at least equally or perhaps more abundant than the reference sequence PPARGC1A transcripts in whole brain. Two main transcripts containing independent methionine start codons encode full-length brain-specific PGC-1α proteins that differ only at their N-termini (NTs) from PGC-1α, encoded by the reference sequence. Additional truncated isoforms containing these NTs that are similar to NT-PGC-1α exist. Other transcripts may encode potential dominant negative forms, as they are predicted to lack the second LXXLL motif that serves as an interaction site for several nuclear receptors. Furthermore, we show that the new promoter is active in neuronal cell lines and describe haplotypes encompassing this region that are associated with HD age of onset. The discovery of such a large PPARGC1A genomic locus and multiple isoforms in brain warrants further functional studies and may provide new tissue-specific targets for treating neurodegenerative diseases.
Assuntos
Encéfalo/metabolismo , Genoma Humano , Proteínas de Choque Térmico/genética , Doença de Huntington/epidemiologia , Doença de Huntington/genética , Fatores de Transcrição/genética , Idade de Início , Éxons , Genômica , Proteínas de Choque Térmico/metabolismo , Humanos , Íntrons , Dados de Sequência Molecular , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/metabolismoRESUMO
BACKGROUND: Genetic modifiers are important clues for the identification of therapeutic targets in neurodegenerative diseases. Huntington disease (HD) is one of the most common autosomal dominant inherited neurodegenerative diseases. The clinical symptoms include motor abnormalities, cognitive decline and behavioral disturbances. Symptom onset is typically between 40 and 50 years of age, but can vary by several decades in extreme cases and this is in part determined by modifying genetic factors. The metabolic master regulator PGC-1α, coded by the PPARGC1A gene, coordinates cellular respiration and was shown to play a role in neurodegenerative diseases, including HD. METHODS: Using a candidate gene approach we analyzed a large European cohort (n = 1706) from the REGISTRY study for associations between PPARGC1A genotype and age at onset (AO) in HD. RESULTS: We report that a coding variant (rs3736265) in PPARGC1A is associated with an earlier motor AO in men but not women carrying the HD mutation. CONCLUSIONS: These results further strengthen the evidence for a role of PGC-1α in HD and unexpectedly suggest a gender effect.
Assuntos
Doença de Huntington/epidemiologia , Doença de Huntington/genética , Fases de Leitura Aberta/genética , Polimorfismo de Nucleotídeo Único/genética , Fatores de Transcrição/genética , Idade de Início , Estudos de Coortes , Europa (Continente)/epidemiologia , Feminino , Humanos , Doença de Huntington/diagnóstico , Masculino , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Sistema de RegistrosRESUMO
We identified a locus on chromosome 6q16.3-q24.2 (ref. 1) associated with childhood obesity that includes 2.4 Mb common to eight genome scans for type 2 diabetes (T2D) or obesity. Analysis of the gene ENPP1 (also called PC-1), a candidate for insulin resistance, in 6,147 subjects showed association between a three-allele risk haplotype (K121Q, IVS20delT-11 and A-->G+1044TGA; QdelTG) and childhood obesity (odds ratio (OR) = 1.69, P = 0.0006), morbid or moderate obesity in adults (OR = 1.50, P = 0.006 or OR = 1.37, P = 0.02, respectively) and T2D (OR = 1.56, P = 0.00002). The Genotype IBD Sharing Test suggested that this obesity-associated ENPP1 risk haplotype contributes to the observed chromosome 6q linkage with childhood obesity. The haplotype confers a higher risk of glucose intolerance and T2D to obese children and their parents and associates with increased serum levels of soluble ENPP1 protein in children. Expression of a long ENPP1 mRNA isoform, which includes the obesity-associated A-->G+1044TGA SNP, was specific for pancreatic islet beta cells, adipocytes and liver. These findings suggest that several variants of ENPP1 have a primary role in mediating insulin resistance and in the development of both obesity and T2D, suggesting that an underlying molecular mechanism is common to both conditions.
Assuntos
Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Teste de Tolerância a Glucose , Obesidade/genética , Diester Fosfórico Hidrolases/genética , Pirofosfatases/genética , Adulto , Estudos de Casos e Controles , Criança , Haplótipos , Humanos , RNA Mensageiro/genéticaRESUMO
One of the most pressing challenges in the post genomic era is the identification and characterization of protein-protein interactions (PPIs), as these are essential in understanding the cellular physiology of health and disease. Experimental techniques suitable for characterizing PPIs (X-ray crystallography or nuclear magnetic resonance spectroscopy, among others) are usually laborious, time-consuming and often difficult to apply to membrane proteins, and therefore require accurate prediction of the candidate interacting partners. High-throughput experimental methods (yeast two-hybrid and affinity purification) succumb to the same shortcomings, and can also lead to high rates of false positive and negative results. Therefore, reliable tools for predicting PPIs are needed. The use of the operon structure in the eukaryote Caenorhabditis elegans genome is a valuable, though underserved, tool for identifying physically or functionally interacting proteins. Based on the concept that genes organized in the same operon may encode physically or functionally related proteins, this algorithm is easy to be applied and, importantly, gives a limited number of candidate partners of a given protein, allowing for focused experimental verification. Moreover, this approach can be successfully used to predict PPIs in the human system, including those of membrane proteins.
Assuntos
Proteínas de Membrana/metabolismo , Óperon/genética , Mapas de Interação de Proteínas/genética , Animais , Caenorhabditis elegans/genética , Biologia Computacional , Genoma , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
BACKGROUND: Pendrin, an anion exchanger associated with the inner ear, thyroid and kidney, plays a significant role in respiratory tissues and diseases, where its expression is increased following IL-4 and IL-13 exposure. The mechanism leading to increased pendrin expression is in part due to binding of STAT6 to a consensus sequence (N4 GAS motif) located in the pendrin promoter. As retrospective analyses of the 5' upstream sequence of the human pendrin promoter revealed an additional N4 GAS motif (1660 base pairs upstream of the one previously identified), we set out to define its contribution to IL-4 stimulated changes in pendrin promoter activity. METHODS AND RESULTS: Electrophoretic mobility shift assays showed that STAT6 bound to oligonucleotides corresponding to both N4 GAS motifs in vitro, while dual luciferase promoter assays revealed that only one of the N4 GAS motifs was necessary for IL-4 -stimulated increases in pendrin promoter activity in living cells. We then examined the ability of STAT6 to bind each of the N4 GAS motifs in vivo with a site-specific ChIP assay, the results of which showed that STAT6 interacted with only the N4 GAS motif that was functionally implicated in increasing the activity of the pendrin promoter following IL-4 treatment. CONCLUSIONS: Of the two N4 GAS motifs located in the human pendrin promoter region analyzed in this study (nucleotides -3906 to +7), only the one located nearest to the first coding ATG participates in IL-4 stimulated increases in promoter activity.
Assuntos
Proteínas de Membrana Transportadoras/genética , Motivos de Nucleotídeos/genética , Regiões Promotoras Genéticas , Fator de Transcrição STAT6/genética , Sítios de Ligação , Humanos , Interleucina-13/metabolismo , Interleucina-4/genética , Interleucina-4/metabolismo , Proteínas de Membrana Transportadoras/biossíntese , Proteínas de Membrana Transportadoras/química , Ligação Proteica , Fator de Transcrição STAT6/química , Transportadores de SulfatoRESUMO
OBJECTIVE: Genetic studies implicated upstream stimulatory factor 1 (USF1) in familial combined hyperlipidemia because the rs2073658 minor allele was associated with reduced risk of familial combined hyperlipidemia and related disorders. The molecular mechanisms whereby rs2073658 influences trait expression have remained elusive. METHODS AND RESULTS: Plasma lipids, rs2073658 genotypes (N=372), and hepatic transcript levels (N=96) of USF1 and genes involved in hepatic lipoprotein production were determined in obese subjects. The rs2073658 minor allele was associated with reduced plasma triglycerides (TGs) (P<0.001), hepatic USF1 (P<0.01), and microsomal TG transfer protein transcript levels (P<0.05). Functional studies in human hepatocellular carcinoma cells showed that rs2073658 is located in a forkhead box A2 (FOXA2) binding site and that major allele constructs displayed higher transcriptional activity than minor allele constructs. Knockdown of FOXA2 reduced the activity of major, but not minor allele constructs. Furthermore, an interaction between hepatic FOXA2 transcript levels and rs2073658 minor allele carrier status on hepatic USF1 transcript levels was observed in vivo (P<0.05). USF1 activated the transcription of FOXA2 and FOXA2 strongly activated the transcription of microsomal TG transfer protein. CONCLUSIONS: A feed-forward loop comprising activation of USF1 transcription by FOXA2 and activation of FOXA2 transcription by USF1, driving microsomal TG transfer protein expression, is modulated by rs2073658. Hence, rs2073658 likely influences hepatic TG secretion.
Assuntos
Hiperlipidemia Familiar Combinada/genética , Fígado/metabolismo , Obesidade/genética , Polimorfismo de Nucleotídeo Único , Fatores Estimuladores Upstream/genética , Adulto , Idoso , Análise de Variância , Áustria , Sítios de Ligação , Proteínas de Transporte/metabolismo , Distribuição de Qui-Quadrado , Retroalimentação Fisiológica , Feminino , Frequência do Gene , Predisposição Genética para Doença , Células Hep G2 , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Humanos , Hiperlipidemia Familiar Combinada/sangue , Modelos Lineares , Desequilíbrio de Ligação , Masculino , Pessoa de Meia-Idade , Obesidade/sangue , Fenótipo , Regiões Promotoras Genéticas , Interferência de RNA , RNA Mensageiro/metabolismo , Ativação Transcricional , Transfecção , Triglicerídeos/sangueRESUMO
Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) is a transcriptional coactivator that contributes to the regulation of numerous transcriptional programs including the hepatic response to fasting. Mechanisms at transcriptional and post-transcriptional levels allow PGC-1α to support distinct biological pathways. Here we describe a novel human liver-specific PGC-1α transcript that results from alternative promoter usage and is induced by FOXO1 as well as glucocorticoids and cAMP-response element-binding protein signaling but is not present in other mammals. Hepatic tissue levels of novel and wild-type transcripts were similar but were only moderately associated (p < 0.003). Novel mRNA levels were associated with a polymorphism located in its promoter region, whereas wild-type transcript levels were not. Furthermore, hepatic PCK1 mRNA levels exhibited stronger associations with the novel than with the wild-type transcript levels. Except for a deletion of 127 amino acids at the N terminus, the protein, termed L-PGC-1α, is identical to PGC-1α. L-PGC-1α was localized in the nucleus and showed coactivation properties that overlap with those of PGC-1α. Collectively, our data support a role of L-PGC-1α in gluconeogenesis, but functional differences predicted from the altered structure suggest that L-PGC-1α may have arisen to adapt PGC-1α to more complex metabolic pathways in humans.
Assuntos
Proteínas de Choque Térmico/metabolismo , Fígado/metabolismo , Isoformas de Proteínas/metabolismo , Fatores de Transcrição/metabolismo , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Northern Blotting , Imunoprecipitação da Cromatina , Dexametasona/farmacologia , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/genética , Genótipo , Proteínas de Choque Térmico/genética , Células Hep G2 , Humanos , Immunoblotting , Técnicas In Vitro , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo , Isoformas de Proteínas/genética , RNA Mensageiro/genética , Ratos , Fatores de Transcrição/genéticaRESUMO
A recent study reported an association between the brain natriuretic peptide (BNP) promoter T-381C polymorphism (rs198389) and protection against type 2 diabetes (T2D). As replication in several studies is mandatory to confirm genetic results, we analyzed the T-381C polymorphism in seven independent case-control cohorts and in 291 T2D-enriched pedigrees totalling 39 557 subjects of European origin. A meta-analysis of the seven case-control studies (n = 39 040) showed a nominal protective effect [odds ratio (OR) = 0.86 (0.79-0.94), P = 0.0006] of the CC genotype on T2D risk, consistent with the previous study. By combining all available data (n = 49 279), we further confirmed a modest contribution of the BNP T-381C polymorphism for protection against T2D [OR = 0.86 (0.80-0.92), P = 1.4 x 10(-5)]. Potential confounders such as gender, age, obesity status or family history were tested in 4335 T2D and 4179 normoglycemic subjects and they had no influence on T2D risk. This study provides further evidence of a modest contribution of the BNP T-381C polymorphism in protection against T2D and illustrates the difficulty of unambiguously proving modest-sized associations even with large sample sizes.
Assuntos
Diabetes Mellitus Tipo 2/genética , Peptídeo Natriurético Encefálico/genética , Polimorfismo de Nucleotídeo Único , Idoso , Estudos de Casos e Controles , Estudos de Coortes , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/metabolismo , Europa (Continente)/epidemiologia , Estudo de Associação Genômica Ampla , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Peptídeo Natriurético Encefálico/metabolismo , Linhagem , Fatores de Risco , População Branca/genéticaRESUMO
Pendrin (SLC26A4, PDS) is an electroneutral anion exchanger transporting I(-), Cl(-), HCO(3)(-), OH(-), SCN(-) and formate. In the thyroid, pendrin is expressed at the apical membrane of the follicular epithelium and may be involved in mediating apical iodide efflux into the follicle; in the inner ear, it plays a crucial role in the conditioning of the pH and ion composition of the endolymph; in the kidney, it may exert a role in pH homeostasis and regulation of blood pressure. Mutations of the pendrin gene can lead to syndromic and non-syndromic hearing loss with EVA (enlarged vestibular aqueduct). Functional tests of mutated pendrin allelic variants found in patients with Pendred syndrome or non-syndromic EVA (ns-EVA) revealed that the pathological phenotype is due to the reduction or loss of function of the ion transport activity. The diagnosis of Pendred syndrome and ns-EVA can be difficult because of the presence of phenocopies of Pendred syndrome and benign polymorphisms occurring in the general population. As a consequence, defining whether or not an allelic variant is pathogenic is crucial. Recently, we found that the two parameters used so far to assess the pathogenic potential of a mutation, i.e. low incidence in the control population, and substitution of evolutionary conserved amino acids, are not always reliable for predicting the functionality of pendrin allelic variants; actually, we identified mutations occurring with the same frequency in the cohort of hearing impaired patients and in the control group of normal hearing individuals. Moreover, we identified functional polymorphisms affecting highly conserved amino acids. As a general rule however, we observed a complete loss of function for all truncations and amino acid substitutions involving a proline. In this view, clinical and radiological studies should be combined with genetic and molecular studies for a definitive diagnosis. In performing genetic studies, the possibility that the mutation could affect regions other than the pendrin coding region, such as its promoter region and/or the coding regions of functionally related genes (FOXI1, KCNJ10), should be taken into account. The presence of benign polymorphisms in the population suggests that genetic studies should be corroborated by functional studies; in this context, the existence of hypo-functional variants and possible differences between the I(-)/Cl(-) and Cl(-)/HCO(3)(-) exchange activities should be carefully evaluated.
Assuntos
Alelos , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Ânions/metabolismo , Bócio Nodular/genética , Bócio Nodular/patologia , Perda Auditiva Neurossensorial/genética , Perda Auditiva Neurossensorial/patologia , Humanos , Transporte de Íons , Mutação , Polimorfismo de Nucleotídeo Único , Transportadores de Sulfato , Aqueduto Vestibular/fisiologiaRESUMO
Organic anion transporters (OATs) are anion exchangers that transport small hydrophilic anions and diuretics, antibiotics, nonsteroidal anti-inflammatory drugs, antiviral nucleoside analogs, and antitumor drugs across membrane barriers of epithelia of diverse organs. Three OATs are present in human liver: OAT2, OAT5, and OAT7. Given that hepatocyte nuclear factor-1α (HNF-1α) has previously been shown to regulate the expression of several hepatocellular transporter genes, we investigated whether the liver-specific human OAT genes are also regulated by HNF-1α. Short interfering RNAs targeting HNF-1α reduced endogenous expression of OAT5 and OAT7, but not OAT2, in human liver-derived Huh7 cells. Luciferase reporter gene constructs containing the OAT5 (SLC22A10) and OAT7 (SLC22A9) promoter regions were transactivated by HNF-1α in HepG2 cells. Two putative HNF-1α binding elements in the proximal OAT5 promoter, located at nucleotides -68/-56 and -173/-160, and one element in the OAT7 promoter, located at nucleotides -14/-2 relative to the transcription start site, were shown to bind HNF-1α in electromobility shift assays, and these promoter regions also interacted with HNF-1α in chromatin immunoprecipitation assays. A correlation between HNF-1α and OAT5 (r = 0.134, P < 0.05) or OAT7 (r = 0.461, P < 0.001) mRNA expression levels in surgical liver biopsies from 75 patients further supported an important role of HNF-1α in the regulation of OAT gene expression.