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1.
Genome Biol ; 22(1): 252, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34465366

RESUMO

Detecting multiplets in single nucleus (sn)ATAC-seq data is challenging due to data sparsity and limited dynamic range. AMULET (ATAC-seq MULtiplet Estimation Tool) enumerates regions with greater than two uniquely aligned reads across the genome to effectively detect multiplets. We evaluate the method by generating snATAC-seq data in the human blood and pancreatic islet samples. AMULET has high precision, estimated via donor-based multiplexing, and high recall, estimated via simulated multiplets, compared to alternatives and identifies multiplets most effectively when a certain read depth of 25K median valid reads per nucleus is achieved.

2.
Nat Commun ; 12(1): 5242, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34475398

RESUMO

Genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs) at >250 loci in the human genome to type 2 diabetes (T2D) risk. For each locus, identifying the functional variant(s) among multiple SNPs in high linkage disequilibrium is critical to understand molecular mechanisms underlying T2D genetic risk. Using massively parallel reporter assays (MPRA), we test the cis-regulatory effects of SNPs associated with T2D and altered in vivo islet chromatin accessibility in MIN6 ß cells under steady state and pathophysiologic endoplasmic reticulum (ER) stress conditions. We identify 1,982/6,621 (29.9%) SNP-containing elements that activate transcription in MIN6 and 879 SNP alleles that modulate MPRA activity. Multiple T2D-associated SNPs alter the activity of short interspersed nuclear element (SINE)-containing elements that are strongly induced by ER stress. We identify 220 functional variants at 104 T2D association signals, narrowing 54 signals to a single candidate SNP. Together, this study identifies elements driving ß cell steady state and ER stress-responsive transcriptional activation, nominates causal T2D SNPs, and uncovers potential roles for repetitive elements in ß cell transcriptional stress response and T2D genetics.


Assuntos
Diabetes Mellitus Tipo 2/genética , Estresse do Retículo Endoplasmático/genética , Células Secretoras de Insulina/patologia , Polimorfismo de Nucleotídeo Único , Ativação Transcricional/genética , Alelos , Animais , Linhagem Celular , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/patologia , Estudo de Associação Genômica Ampla , Humanos , Camundongos , Locos de Características Quantitativas , Elementos Nucleotídeos Curtos e Dispersos/genética
3.
Nat Commun ; 12(1): 5074, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417463

RESUMO

ß cells may participate and contribute to their own demise during Type 1 diabetes (T1D). Here we report a role of their expression of Tet2 in regulating immune killing. Tet2 is induced in murine and human ß cells with inflammation but its expression is reduced in surviving ß cells. Tet2-KO mice that receive WT bone marrow transplants develop insulitis but not diabetes and islet infiltrates do not eliminate ß cells even though immune cells from the mice can transfer diabetes to NOD/scid recipients. Tet2-KO recipients are protected from transfer of disease by diabetogenic immune cells.Tet2-KO ß cells show reduced expression of IFNγ-induced inflammatory genes that are needed to activate diabetogenic T cells. Here we show that Tet2 regulates pathologic interactions between ß cells and immune cells and controls damaging inflammatory pathways. Our data suggests that eliminating TET2 in ß cells may reduce activating pathologic immune cells and killing of ß cells.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Diabetes Mellitus Tipo 1/patologia , Inflamação/patologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Proteínas Proto-Oncogênicas/metabolismo , Animais , Sequência de Bases , Citotoxicidade Imunológica , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/imunologia , Progressão da Doença , Feminino , Humanos , Imunidade , Inflamação/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Linfócitos T/imunologia , Transcrição Genética
4.
Diabetes ; 68(6): 1168-1177, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30936147

RESUMO

Patterns of abdominal fat distribution (for example, a high vs. low visceral adipose tissue [VAT]/[VAT + subcutaneous adipose tissue (SAT)] ratio), independent of obesity, during adolescence carry a high risk for insulin resistance and type 2 diabetes. Longitudinal follow-up of a cohort of obese adolescents has recently revealed that a high ratio (high VAT/[VAT + SAT]) is a major determinant of fatty liver and metabolic impairment over time, with these effects being more pronounced in girls than in boys. To unravel the underlying metabolic alterations associated with the unfavorable VAT/(VAT + SAT) phenotype, we used the 2H2O labeling method to measure the turnover of adipose lipids and cells in the subcutaneous abdominal and gluteal/femoral adipose tissue (SAT) of weight-stable obese adolescent girls with a similar level of obesity but discordant VAT/(VAT + SAT) ratios. Girls with the unfavorable (high VAT/[VAT + SAT]) phenotype exhibited higher in vivo rates of triglyceride (TG) turnover (representing both lipolysis and synthesis at steady state), without significant differences in de novo lipogenesis in both abdominal and gluteal depots, compared with obese girls with the favorable phenotype. Moreover, mature adipocytes had higher turnover, with no difference in stromal vascular cell proliferation in both depots in the metabolically unfavorable phenotype. The higher TG turnover rates were significantly correlated with higher intrahepatic fat stores. These findings are contrary to the hypothesis that impaired capacity to deposit TGs or proliferation of new mature adipocytes are potential mechanisms for ectopic fat distribution in this setting. In summary, these results suggest that increased turnover of TGs (lipolysis) and of mature adipocytes in both abdominal and gluteal SAT may contribute to metabolic impairment and the development of fatty liver, even at this very early stage of disease.


Assuntos
Adipócitos/metabolismo , Distribuição da Gordura Corporal , Obesidade/metabolismo , Gordura Subcutânea/metabolismo , Triglicerídeos/metabolismo , Absorciometria de Fóton , Adolescente , Óxido de Deutério , Feminino , Humanos , Gordura Intra-Abdominal/diagnóstico por imagem , Metabolismo dos Lipídeos , Lipogênese , Imageamento por Ressonância Magnética , Obesidade Abdominal/diagnóstico por imagem , Obesidade Abdominal/metabolismo , Gordura Subcutânea/diagnóstico por imagem , Adulto Jovem
5.
Cell Rep ; 26(3): 788-801.e6, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30650367

RESUMO

EndoC-ßH1 is emerging as a critical human ß cell model to study the genetic and environmental etiologies of ß cell (dys)function and diabetes. Comprehensive knowledge of its molecular landscape is lacking, yet required, for effective use of this model. Here, we report chromosomal (spectral karyotyping), genetic (genotyping), epigenomic (ChIP-seq and ATAC-seq), chromatin interaction (Hi-C and Pol2 ChIA-PET), and transcriptomic (RNA-seq and miRNA-seq) maps of EndoC-ßH1. Analyses of these maps define known (e.g., PDX1 and ISL1) and putative (e.g., PCSK1 and mir-375) ß cell-specific transcriptional cis-regulatory networks and identify allelic effects on cis-regulatory element use. Importantly, comparison with maps generated in primary human islets and/or ß cells indicates preservation of chromatin looping but also highlights chromosomal aberrations and fetal genomic signatures in EndoC-ßH1. Together, these maps, and a web application we created for their exploration, provide important tools for the design of experiments to probe and manipulate the genetic programs governing ß cell identity and (dys)function in diabetes.


Assuntos
Redes Reguladoras de Genes/genética , Células Secretoras de Insulina/metabolismo , Linhagem Celular , Humanos
6.
Diabetes ; 67(11): 2466-2477, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30181159

RESUMO

Type 2 diabetes (T2D) is a complex disorder in which both genetic and environmental risk factors contribute to islet dysfunction and failure. Genome-wide association studies (GWAS) have linked single nucleotide polymorphisms (SNPs), most of which are noncoding, in >200 loci to islet dysfunction and T2D. Identification of the putative causal variants and their target genes and whether they lead to gain or loss of function remains challenging. Here, we profiled chromatin accessibility in pancreatic islet samples from 19 genotyped individuals and identified 2,949 SNPs associated with in vivo cis-regulatory element use (i.e., chromatin accessibility quantitative trait loci [caQTL]). Among the caQTLs tested (n = 13) using luciferase reporter assays in MIN6 ß-cells, more than half exhibited effects on enhancer activity that were consistent with in vivo chromatin accessibility changes. Importantly, islet caQTL analysis nominated putative causal SNPs in 13 T2D-associated GWAS loci, linking 7 and 6 T2D risk alleles, respectively, to gain or loss of in vivo chromatin accessibility. By investigating the effect of genetic variants on chromatin accessibility in islets, this study is an important step forward in translating T2D-associated GWAS SNP into functional molecular consequences.


Assuntos
Cromatina/metabolismo , Diabetes Mellitus Tipo 2/genética , Ilhotas Pancreáticas/metabolismo , Alelos , Cromatina/genética , Diabetes Mellitus Tipo 2/metabolismo , Predisposição Genética para Doença , Genótipo , Humanos
7.
Am J Hum Genet ; 102(4): 620-635, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29625024

RESUMO

Genome-wide association studies (GWASs) and functional genomics approaches implicate enhancer disruption in islet dysfunction and type 2 diabetes (T2D) risk. We applied genetic fine-mapping and functional (epi)genomic approaches to a T2D- and proinsulin-associated 15q22.2 locus to identify a most likely causal variant, determine its direction of effect, and elucidate plausible target genes. Fine-mapping and conditional analyses of proinsulin levels of 8,635 non-diabetic individuals from the METSIM study support a single association signal represented by a cluster of 16 strongly associated (p < 10-17) variants in high linkage disequilibrium (r2 > 0.8) with the GWAS index SNP rs7172432. These variants reside in an evolutionarily and functionally conserved islet and ß cell stretch or super enhancer; the most strongly associated variant (rs7163757, p = 3 × 10-19) overlaps a conserved islet open chromatin site. DNA sequence containing the rs7163757 risk allele displayed 2-fold higher enhancer activity than the non-risk allele in reporter assays (p < 0.01) and was differentially bound by ß cell nuclear extract proteins. Transcription factor NFAT specifically potentiated risk-allele enhancer activity and altered patterns of nuclear protein binding to the risk allele in vitro, suggesting that it could be a factor mediating risk-allele effects. Finally, the rs7163757 proinsulin-raising and T2D risk allele (C) was associated with increased expression of C2CD4B, and possibly C2CD4A, both of which were induced by inflammatory cytokines, in human islets. Together, these data suggest that rs7163757 contributes to genetic risk of islet dysfunction and T2D by increasing NFAT-mediated islet enhancer activity and modulating C2CD4B, and possibly C2CD4A, expression in (patho)physiologic states.


Assuntos
Proteínas de Ligação ao Cálcio/genética , Sequência Conservada , Elementos Facilitadores Genéticos/genética , Evolução Molecular , Ilhotas Pancreáticas/patologia , Mutação/genética , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Idoso , Alelos , Animais , Sequência de Bases , Proteínas de Ligação ao Cálcio/metabolismo , Linhagem Celular , Cromatina/metabolismo , Cromossomos Humanos Par 15/genética , Citocinas/metabolismo , DNA Intergênico/genética , Humanos , Mediadores da Inflamação/metabolismo , Camundongos , Pessoa de Meia-Idade , Fatores de Transcrição NFATC/metabolismo , Mapeamento Físico do Cromossomo , Polimorfismo de Nucleotídeo Único/genética , Proinsulina/metabolismo , Ratos , Fatores de Risco
8.
Horm Mol Biol Clin Investig ; 33(2)2018 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-29596053

RESUMO

Obesity has become a major global health challenge of the 21st century, as it is associated with the onset of type 2 diabetes (T2D) and cardiovascular complications, even at a very early age in life. The root causes of pediatric obesity remain incompletely understood. The obesity epidemic together with the relationship of obesity to the growing population burden of chronic disease presents unprecedented research opportunities and challenges. Decades of obesity-related research funded by governments around the world have yielded many important discoveries about both etiological pathways and preventive or therapeutic interventions. Yet, there is a sense that the problem is outpacing these research efforts. Obesity poses a significant risk for the development of cardiovascular disease (CVD) , diabetes and certain cancers thereby shortening life expectancy. Nevertheless, many obese individuals do not develop any of these comorbidities. One hypothesis explaining this dilemma is that total body fat is not the culprit of adverse health in obesity rather the relative proportion of lipids in various fat depots is what determines the metabolic risk. In this review, we describe the role of altered fat partitioning in youth onset obesity and its relation to fatty liver and T2D during adolescence.


Assuntos
Tecido Adiposo/metabolismo , Resistência à Insulina , Obesidade/metabolismo , Tecido Adiposo/patologia , Adolescente , Distribuição da Gordura Corporal , Diabetes Mellitus Tipo 2/epidemiologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Fígado Gorduroso/epidemiologia , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Humanos , Inflamação/epidemiologia , Inflamação/metabolismo , Inflamação/patologia , Lipogênese , Obesidade/complicações , Obesidade/epidemiologia , Obesidade/patologia
9.
Sci Rep ; 7(1): 11959, 2017 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-28931935

RESUMO

Alpha TC1 (αTC1) and Beta-TC-6 (ßTC6) mouse islet cell lines are cellular models of islet (dys)function and type 2 diabetes (T2D). However, genomic characteristics of these cells, and their similarities to primary islet alpha and beta cells, are undefined. Here, we report the epigenomic (ATAC-seq) and transcriptomic (RNA-seq) landscapes of αTC1 and ßTC6 cells. Each cell type exhibits hallmarks of its primary islet cell counterpart including cell-specific expression of beta (e.g., Pdx1) and alpha (e.g., Arx) cell transcription factors (TFs), and enrichment of binding motifs for these TFs in αTC1/ßTC6 cis-regulatory elements. αTC1/ßTC6 transcriptomes overlap significantly with the transcriptomes of primary mouse/human alpha and beta cells. Our data further indicate that ATAC-seq detects cell-specific regulatory elements for cell types comprising ≥ 20% of a mixed cell population. We identified αTC1/ßTC6 cis-regulatory elements orthologous to those containing type 2 diabetes (T2D)-associated SNPs in human islets for 33 loci, suggesting these cells' utility to dissect T2D molecular genetics in these regions. Together, these maps provide important insights into the conserved regulatory architecture between αTC1/ßTC6 and primary islet cells that can be leveraged in functional (epi)genomic approaches to dissect the genetic and molecular factors controlling islet cell identity and function.


Assuntos
Diabetes Mellitus Tipo 2/patologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ilhotas Pancreáticas/patologia , Animais , Células Cultivadas , Camundongos , Transcrição Genética
10.
Diabetes ; 66(9): 2521-2530, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28684635

RESUMO

Molecular mechanisms remain unknown for most type 2 diabetes genome-wide association study identified loci. Variants associated with type 2 diabetes and fasting glucose levels reside in introns of ADCY5, a gene that encodes adenylate cyclase 5. Adenylate cyclase 5 catalyzes the production of cyclic AMP, which is a second messenger molecule involved in cell signaling and pancreatic ß-cell insulin secretion. We demonstrated that type 2 diabetes risk alleles are associated with decreased ADCY5 expression in human islets and examined candidate variants for regulatory function. rs11708067 overlaps a predicted enhancer region in pancreatic islets. The type 2 diabetes risk rs11708067-A allele showed fewer H3K27ac ChIP-seq reads in human islets, lower transcriptional activity in reporter assays in rodent ß-cells (rat 832/13 and mouse MIN6), and increased nuclear protein binding compared with the rs11708067-G allele. Homozygous deletion of the orthologous enhancer region in 832/13 cells resulted in a 64% reduction in expression level of Adcy5, but not adjacent gene Sec22a, and a 39% reduction in insulin secretion. Together, these data suggest that rs11708067-A risk allele contributes to type 2 diabetes by disrupting an islet enhancer, which results in reduced ADCY5 expression and impaired insulin secretion.


Assuntos
Adenilil Ciclases/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Regulação da Expressão Gênica/fisiologia , Variação Genética , Estudo de Associação Genômica Ampla , Ilhotas Pancreáticas/metabolismo , Adenilil Ciclases/genética , Diabetes Mellitus Tipo 2/genética , Humanos , Insulina/metabolismo , Secreção de Insulina
11.
Diabetes Care ; 40(8): 1082-1089, 2017 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-28611053

RESUMO

OBJECTIVE: In this study, we aimed to explore the mechanism by which TCF7L2 rs7903146 risk allele confers susceptibility to impaired glucose tolerance (IGT) or type 2 diabetes (T2D) in obese adolescents. RESEARCH DESIGN AND METHODS: The rs7903146 variant in the TCF7L2 gene was genotyped in a multiethnic cohort of 955 youths. All subjects underwent an oral glucose tolerance test with the use of the Oral Minimal Model to assess insulin secretion, and 33 subjects underwent a hyperinsulinemic-euglycemic clamp. In 307 subjects, a follow-up oral glucose tolerance test was repeated after 3.11 ± 2.36 years. RESULTS: The TCF7L2 rs7903146 risk allele was associated with higher 2-h glucose levels in Caucasians (P = 0.006) and African Americans (P = 0.009), and a trend was seen also in Hispanics (P = 0.072). Also, the T allele was associated with decreased ß-cell responsivity and IGT (P < 0.05). Suppression of endogenous hepatic glucose production was lower in subjects with the risk variant (P = 0.006). Finally, the odds of showing IGT/T2D at follow-up were higher in subjects carrying the minor allele (odds ratio 2.224; 95% CI 1.370-3.612; P = 0.0012). CONCLUSIONS: The rs7903146 variant in the TCF7L2 gene increases the risk of IGT/T2D in obese adolescents by impairing ß-cell function, and hepatic insulin sensitivity predicts the development of IGT/T2D over time.


Assuntos
Diabetes Mellitus Tipo 2/genética , Obesidade Pediátrica/genética , Estado Pré-Diabético/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Adolescente , Alelos , Índice de Massa Corporal , Criança , Estudos de Coortes , Grupos de Populações Continentais/genética , Diabetes Mellitus Tipo 2/sangue , Feminino , Seguimentos , Predisposição Genética para Doença , Intolerância à Glucose/sangue , Intolerância à Glucose/genética , Teste de Tolerância a Glucose , Humanos , Insulina/sangue , Insulina/metabolismo , Resistência à Insulina , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Fígado/metabolismo , Estudos Longitudinais , Masculino , Obesidade Pediátrica/sangue , Estado Pré-Diabético/sangue , Fatores de Risco
12.
Proc Natl Acad Sci U S A ; 114(9): 2301-2306, 2017 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-28193859

RESUMO

Genome-wide association studies (GWAS) have identified >100 independent SNPs that modulate the risk of type 2 diabetes (T2D) and related traits. However, the pathogenic mechanisms of most of these SNPs remain elusive. Here, we examined genomic, epigenomic, and transcriptomic profiles in human pancreatic islets to understand the links between genetic variation, chromatin landscape, and gene expression in the context of T2D. We first integrated genome and transcriptome variation across 112 islet samples to produce dense cis-expression quantitative trait loci (cis-eQTL) maps. Additional integration with chromatin-state maps for islets and other diverse tissue types revealed that cis-eQTLs for islet-specific genes are specifically and significantly enriched in islet stretch enhancers. High-resolution chromatin accessibility profiling using assay for transposase-accessible chromatin sequencing (ATAC-seq) in two islet samples enabled us to identify specific transcription factor (TF) footprints embedded in active regulatory elements, which are highly enriched for islet cis-eQTL. Aggregate allelic bias signatures in TF footprints enabled us de novo to reconstruct TF binding affinities genetically, which support the high-quality nature of the TF footprint predictions. Interestingly, we found that T2D GWAS loci were strikingly and specifically enriched in islet Regulatory Factor X (RFX) footprints. Remarkably, within and across independent loci, T2D risk alleles that overlap with RFX footprints uniformly disrupt the RFX motifs at high-information content positions. Together, these results suggest that common regulatory variations have shaped islet TF footprints and the transcriptome and that a confluent RFX regulatory grammar plays a significant role in the genetic component of T2D predisposition.


Assuntos
Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença , Genoma Humano , Ilhotas Pancreáticas/metabolismo , Locos de Características Quantitativas , Transcriptoma , Alelos , Sequência de Bases , Sítios de Ligação , Cromatina/química , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Epigênese Genética , Perfilação da Expressão Gênica , Variação Genética , Estudo de Associação Genômica Ampla , Impressão Genômica , Humanos , Ilhotas Pancreáticas/patologia , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Transcrição de Fator Regulador X/genética , Fatores de Transcrição de Fator Regulador X/metabolismo
13.
Gastroenterology ; 152(7): 1638-1646, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28192105

RESUMO

As a consequence of the global rise in the prevalence of adolescent obesity, an unprecedented phenomenon of type 2 diabetes has emerged in pediatrics. At the heart of the development of type 2 diabetes lies a key metabolic derangement: insulin resistance (IR). Despite the widespread occurrence of IR affecting an unmeasurable number of youths worldwide, its pathogenesis remains elusive. IR in obese youth is a complex phenomenon that defies explanation by a single pathway. In this review we first describe recent data on the prevalence, severity, and racial/ethnic differences in pediatric obesity. We follow by elucidating the initiating events associated with the onset of IR, and describe a distinct "endophenotype" in obese adolescents characterized by a thin superficial layer of abdominal subcutaneous adipose tissue, increased visceral adipose tissue, marked IR, dyslipidemia, and fatty liver. Further, we provide evidence for the cellular and molecular mechanisms associated with this peculiar endophenotype and its relations to IR in the obese adolescent.


Assuntos
Resistência à Insulina , Obesidade Pediátrica/epidemiologia , Obesidade Pediátrica/fisiopatologia , Gordura Subcutânea Abdominal/fisiopatologia , Adolescente , Índice de Massa Corporal , Endofenótipos , Humanos , Inflamassomos/fisiologia , Inflamação/fisiopatologia , Gordura Intra-Abdominal/fisiopatologia , Fígado/fisiopatologia , Prevalência
14.
Genome Res ; 27(2): 208-222, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-27864352

RESUMO

Blood glucose levels are tightly controlled by the coordinated action of at least four cell types constituting pancreatic islets. Changes in the proportion and/or function of these cells are associated with genetic and molecular pathophysiology of monogenic, type 1, and type 2 (T2D) diabetes. Cellular heterogeneity impedes precise understanding of the molecular components of each islet cell type that govern islet (dys)function, particularly the less abundant delta and gamma/pancreatic polypeptide (PP) cells. Here, we report single-cell transcriptomes for 638 cells from nondiabetic (ND) and T2D human islet samples. Analyses of ND single-cell transcriptomes identified distinct alpha, beta, delta, and PP/gamma cell-type signatures. Genes linked to rare and common forms of islet dysfunction and diabetes were expressed in the delta and PP/gamma cell types. Moreover, this study revealed that delta cells specifically express receptors that receive and coordinate systemic cues from the leptin, ghrelin, and dopamine signaling pathways implicating them as integrators of central and peripheral metabolic signals into the pancreatic islet. Finally, single-cell transcriptome profiling revealed genes differentially regulated between T2D and ND alpha, beta, and delta cells that were undetectable in paired whole islet analyses. This study thus identifies fundamental cell-type-specific features of pancreatic islet (dys)function and provides a critical resource for comprehensive understanding of islet biology and diabetes pathogenesis.


Assuntos
Proteínas de Transporte/genética , Diabetes Mellitus Tipo 2/genética , Análise de Célula Única , Transcriptoma/genética , Diabetes Mellitus Tipo 2/patologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/genética , Humanos , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Transdução de Sinais/genética
15.
Nat Commun ; 7: 12639, 2016 08 31.
Artigo em Inglês | MEDLINE | ID: mdl-27577745

RESUMO

Insulin resistance is a key driver of type 2 diabetes (T2D) and is characterized by defective insulin receptor (INSR) signalling. Although surface INSR downregulation is a well-established contributor to insulin resistance, the underlying molecular mechanisms remain obscure. Here we show that the E3 ubiquitin ligase MARCH1 impairs cellular insulin action by degrading cell surface INSR. Using a large-scale RNA interference screen, we identify MARCH1 as a negative regulator of INSR signalling. March1 loss-of-function enhances, and March1 overexpression impairs, hepatic insulin sensitivity in mice. MARCH1 ubiquitinates INSR to decrease cell surface INSR levels, but unlike other INSR ubiquitin ligases, MARCH1 acts in the basal state rather than after insulin stimulation. Thus, MARCH1 may help set the basal gain of insulin signalling. MARCH1 expression is increased in white adipose tissue of obese humans, suggesting that MARCH1 contributes to the pathophysiology of T2D and could be a new therapeutic target.


Assuntos
Antígenos CD/metabolismo , Diabetes Mellitus Tipo 2/patologia , Resistência à Insulina/fisiologia , Insulina/metabolismo , Obesidade/patologia , Receptor de Insulina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Tecido Adiposo Branco/patologia , Adolescente , Animais , Antígenos CD/genética , Biópsia , Linhagem Celular , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/terapia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutagênese Sítio-Dirigida , Obesidade/sangue , Obesidade/etiologia , Obesidade/terapia , Oligonucleotídeos Antissenso/administração & dosagem , Oligonucleotídeos Antissenso/genética , Fosforilação , RNA Interferente Pequeno/metabolismo , Receptor de Insulina/genética , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Regulação para Cima
16.
Diabetes ; 65(3): 610-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26718495

RESUMO

The innate immune cell sensor leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome controls the activation of caspase-1, and the release of proinflammatory cytokines interleukin (IL)-1ß and IL-18. The NLRP3 inflammasome is implicated in adipose tissue inflammation and the pathogenesis of insulin resistance. Herein, we tested the hypothesis that adipose tissue inflammation and NLRP3 inflammasome are linked to the downregulation of subcutaneous adipose tissue (SAT) adipogenesis/lipogenesis in obese adolescents with altered abdominal fat partitioning. We performed abdominal SAT biopsies on 58 obese adolescents and grouped them by MRI-derived visceral fat to visceral adipose tissue (VAT) plus SAT (VAT/VAT+SAT) ratio (cutoff 0.11). Adolescents with a high VAT/VAT+SAT ratio showed higher SAT macrophage infiltration and higher expression of the NLRP3 inflammasome-related genes (i.e., TLR4, NLRP3, IL1B, and CASP1). The increase in inflammation markers was paralleled by a decrease in genes related to insulin sensitivity (ADIPOQ, GLUT4, PPARG2, and SIRT1) and lipogenesis (SREBP1c, ACC, LPL, and FASN). Furthermore, SAT ceramide concentrations correlated with the expression of CASP1 and IL1B. Infiltration of macrophages and upregulation of the NLRP3 inflammasome together with the associated high ceramide content in the plasma and SAT of obese adolescents with a high VAT/VAT+SAT may contribute to the limited expansion of the subcutaneous abdominal adipose depot and the development of insulin resistance.


Assuntos
Adipogenia/genética , Proteínas de Transporte/genética , Gordura Intra-Abdominal/metabolismo , Lipogênese/genética , Obesidade/metabolismo , Gordura Subcutânea/metabolismo , Abdome , Acetil-CoA Carboxilase/genética , Acetil-CoA Carboxilase/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Adolescente , Proteínas de Transporte/imunologia , Caspase 1/genética , Caspase 1/metabolismo , Criança , Regulação para Baixo , Ácido Graxo Sintase Tipo I/genética , Ácido Graxo Sintase Tipo I/metabolismo , Feminino , Perfilação da Expressão Gênica , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Humanos , Inflamassomos , Resistência à Insulina , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Gordura Intra-Abdominal/patologia , Leptina/metabolismo , Lipase Lipoproteica/genética , Lipase Lipoproteica/metabolismo , Macrófagos/imunologia , Imageamento por Ressonância Magnética , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR , Obesidade/imunologia , PPAR gama/genética , PPAR gama/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismo , Gordura Subcutânea/imunologia , Gordura Subcutânea/patologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo
17.
J Immunol ; 196(1): 437-47, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26582949

RESUMO

Inflammation is well established to significantly impact metabolic diseases. The inflammatory protease caspase-1 has been implicated in metabolic dysfunction; however, a potential role for the related inflammatory caspases is currently unknown. In this study, we investigated a role for caspase-11 and caspase-12 in obesity and insulin resistance. Loss of caspase-12 in two independently generated mouse strains predisposed mice to develop obesity, metabolic inflammation, and insulin resistance, whereas loss of caspase-11 had no effect. The use of bone marrow chimeras determined that deletion of caspase-12 in the radio-resistant compartment was responsible for this metabolic phenotype. The Nlrp3 inflammasome pathway mediated the metabolic syndrome of caspase-12-deficient mice as ablation of Nlrp3 reversed Casp12(-/-) mice obesity phenotype. Although the majority of people lack a functional caspase-12 because of a T(125) single nucleotide polymorphism that introduces a premature stop codon, a fraction of African descendents express full-length caspase-12. Expression of caspase-12 was linked to decreased systemic and adipose tissue inflammation in a cohort of African American obese children. However, analysis of the Dallas Heart Study African American cohort indicated that the coding T(125)C single nucleotide polymorphism was not associated with metabolic parameters in humans, suggesting that host-specific differences mediate the expressivity of metabolic disease.


Assuntos
Caspase 12/fisiologia , Caspases/fisiologia , Resistência à Insulina/genética , Obesidade/genética , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/imunologia , Caspase 12/genética , Caspases/genética , Caspases Iniciadoras , Intolerância à Glucose/genética , Humanos , Inflamação/genética , Inflamação/imunologia , Masculino , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR , Polimorfismo de Nucleotídeo Único/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor , Proteína Serina-Treonina Quinases de Interação com Receptores/genética
18.
Curr Diab Rep ; 15(9): 66, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26272056

RESUMO

Islets of Langerhans contain multiple hormone-producing endocrine cells controlling glucose homeostasis. Transcription establishes and maintains islet cellular fates and identities. Genetic and environmental disruption of islet transcription triggers cellular dysfunction and disease. Early transcriptional regulation studies of specific islet genes, including insulin (INS) and the transcription factor PDX1, identified the first cis-regulatory DNA sequences and trans-acting factors governing islet function. Here, we review how human islet "omics" studies are reshaping our understanding of transcriptional regulation in islet (dys)function and diabetes. First, we highlight the expansion of islet transcript number, form, and function and of DNA transcriptional regulatory elements controlling their production. Next, we cover islet transcriptional effects of genetic and environmental perturbation. Finally, we discuss how these studies' emerging insights should empower our diabetes research community to build mechanistic understanding of diabetes pathophysiology and to equip clinicians with tailored, precision medicine options to prevent and treat islet dysfunction and diabetes.


Assuntos
Regulação da Expressão Gênica , Ilhotas Pancreáticas/metabolismo , Transcrição Genética , Animais , Variação Genética , Humanos , Insulina/genética , Regiões Promotoras Genéticas
19.
Obesity (Silver Spring) ; 23(3): 653-61, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25645909

RESUMO

OBJECTIVE: Fructose consumption has risen alongside obesity and diabetes. Gut hormones involved in hunger and satiety (ghrelin and PYY) may respond differently to fructose compared with glucose ingestion. This study evaluated the effects of glucose and fructose ingestion on ghrelin and PYY in lean and obese adolescents with differing insulin sensitivity. METHODS: Adolescents were divided into lean (n = 14), obese insulin sensitive (n = 12) (OIS), and obese insulin resistant (n = 15) (OIR). In a double-blind, cross-over design, subjects drank 75 g of glucose or fructose in random order, serum was obtained every 10 minutes for 60 minutes. RESULTS: Baseline acyl-ghrelin was highest in lean and lowest in OIR (P = 0.02). After glucose ingestion, acyl-ghrelin decreased similarly in lean and OIS but was lower in OIR (vs. lean, P = 0.03). Suppression differences were more pronounced after fructose (lean vs. OIS, P = 0.008, lean vs. OIR, P < 0.001). OIS became significantly hungrier after fructose (P = 0.015). PYY was not significantly different at baseline, varied minimally after glucose, and rose after fructose. CONCLUSIONS: Compared with lean, OIS adolescents have impaired acyl-ghrelin responses to fructose but not glucose, whereas OIR adolescents have blunted responses to both. Diminished suppression of acyl-ghrelin in childhood obesity, particularly if accompanied by insulin resistance, may promote hunger and overeating.


Assuntos
Frutose/farmacologia , Grelina/metabolismo , Resistência à Insulina/fisiologia , Obesidade Pediátrica/metabolismo , Acilação , Adolescente , Método Duplo-Cego , Ingestão de Alimentos/fisiologia , Feminino , Hormônios Gastrointestinais/sangue , Glucose/farmacologia , Humanos , Fome/efeitos dos fármacos , Hiperfagia/sangue , Hiperfagia/metabolismo , Insulina/sangue , Masculino , Obesidade Pediátrica/sangue , Peptídeo YY/sangue , Período Pós-Prandial/fisiologia
20.
Cell ; 160(4): 745-758, 2015 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-25662011

RESUMO

Impaired insulin-mediated suppression of hepatic glucose production (HGP) plays a major role in the pathogenesis of type 2 diabetes (T2D), yet the molecular mechanism by which this occurs remains unknown. Using a novel in vivo metabolomics approach, we show that the major mechanism by which insulin suppresses HGP is through reductions in hepatic acetyl CoA by suppression of lipolysis in white adipose tissue (WAT) leading to reductions in pyruvate carboxylase flux. This mechanism was confirmed in mice and rats with genetic ablation of insulin signaling and mice lacking adipose triglyceride lipase. Insulin's ability to suppress hepatic acetyl CoA, PC activity, and lipolysis was lost in high-fat-fed rats, a phenomenon reversible by IL-6 neutralization and inducible by IL-6 infusion. Taken together, these data identify WAT-derived hepatic acetyl CoA as the main regulator of HGP by insulin and link it to inflammation-induced hepatic insulin resistance associated with obesity and T2D.


Assuntos
Acetilcoenzima A/metabolismo , Resistência à Insulina , Fígado/metabolismo , Paniculite/metabolismo , Tecido Adiposo Branco/química , Adolescente , Animais , Diabetes Mellitus Tipo 2 , Dieta Hiperlipídica , Glucose/metabolismo , Humanos , Hiperglicemia , Interleucina-6/análise , Lipólise , Masculino , Camundongos , Obesidade/metabolismo , Ratos Sprague-Dawley
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