RESUMO
The Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report provides a summary of the proceedings from the workshop. The goals of the workshop were to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into six major theme areas, including 1) pancreas anatomy and physiology, 2) diabetes in the setting of exocrine disease, 3) metabolic influences on the exocrine pancreas, 4) genetic drivers of pancreatic diseases, 5) tools for integrated pancreatic analysis, and 6) implications of exocrine-endocrine cross talk. For each theme, multiple presentations were followed by panel discussions on specific topics relevant to each area of research; these are summarized here. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.
Assuntos
Diabetes Mellitus , Ilhotas Pancreáticas , Pâncreas Exócrino , Pancreatopatias , Humanos , Diabetes Mellitus/metabolismo , Pâncreas , Pancreatopatias/metabolismoRESUMO
Associations between human genetic variation and clinical phenotypes have become a foundation of biomedical research. Most repositories of these data seek to be disease-agnostic and therefore lack disease-focused views. The Type 2 Diabetes Knowledge Portal (T2DKP) is a public resource of genetic datasets and genomic annotations dedicated to type 2 diabetes (T2D) and related traits. Here, we seek to make the T2DKP more accessible to prospective users and more useful to existing users. First, we evaluate the T2DKP's comprehensiveness by comparing its datasets with those of other repositories. Second, we describe how researchers unfamiliar with human genetic data can begin using and correctly interpreting them via the T2DKP. Third, we describe how existing users can extend their current workflows to use the full suite of tools offered by the T2DKP. We finally discuss the lessons offered by the T2DKP toward the goal of democratizing access to complex disease genetic results.
Assuntos
Diabetes Mellitus Tipo 2 , Humanos , Diabetes Mellitus Tipo 2/genética , Acesso à Informação , Estudos Prospectivos , Genômica/métodos , FenótipoRESUMO
ABSTRACT: The "Integrated Physiology of the Exocrine and Endocrine Compartments in Pancreatic Diseases" Workshop was a 1.5-day scientific conference at the National Institutes of Health (Bethesda, MD) that engaged clinical and basic science investigators interested in diseases of the pancreas. This report summarizes the workshop proceedings. The goal of the workshop was to forge connections and identify gaps in knowledge that could guide future research directions. Presentations were segregated into 6 major themes, including (a) Pancreas Anatomy and Physiology; (b) Diabetes in the Setting of Exocrine Disease; (c) Metabolic Influences on the Exocrine Pancreas; (d) Genetic Drivers of Pancreatic Diseases; (e) Tools for Integrated Pancreatic Analysis; and (f) Implications of Exocrine-Endocrine Crosstalk. For each theme, there were multiple presentations followed by panel discussions on specific topics relevant to each area of research; these are summarized herein. Significantly, the discussions resulted in the identification of research gaps and opportunities for the field to address. In general, it was concluded that as a pancreas research community, we must more thoughtfully integrate our current knowledge of the normal physiology as well as the disease mechanisms that underlie endocrine and exocrine disorders so that there is a better understanding of the interplay between these compartments.
Assuntos
Diabetes Mellitus , Ilhotas Pancreáticas , Pâncreas Exócrino , Pancreatopatias , Humanos , Diabetes Mellitus/terapia , Diabetes Mellitus/metabolismo , Ilhotas Pancreáticas/metabolismo , Pâncreas/metabolismo , Pâncreas Exócrino/metabolismo , Pancreatopatias/diagnóstico , Pancreatopatias/terapia , Pancreatopatias/metabolismoRESUMO
Pancreatic ß-cells are a critical cell type in the pathology of diabetes. Models of genetic syndromes featuring diabetes can provide novel mechanistic insights into regulation of ß-cells in the context of disease. We previously examined ß-cell mass in models of two ciliopathies, Alström Syndrome (AS) and Bardet-Biedl Syndrome (BBS), which are similar in the presence of metabolic phenotypes, including obesity, but exhibit strikingly different rates of diabetes. Zebrafish models of these disorders show deficient ß-cells with diabetes in AS models and an increased ß-cells absent diabetes in BBS models, indicating ß-cell generation or maintenance that correlates with disease prevalence. Using transcriptome analyses, differential expression of several exocrine pancreas proteases with directionality that was consistent with ß-cell numbers were identified. Based on these lines of evidence, we hypothesized that pancreatic proteases directly impact ß-cells. In the present study, we examined this possibility and found that pancreatic protease genes contribute to proper maintenance of normal ß-cell numbers, proliferation in larval zebrafish, and regulation of AS and BBS ß-cell phenotypes. Our data suggest that these proteins can be taken up directly by cultured ß-cells and ex vivo murine islets, inducing proliferation in both. Endogenous uptake of pancreatic proteases by ß-cells was confirmed in vivo using transgenic zebrafish and in intact murine pancreata. Taken together, these findings support a novel proliferative signaling role for exocrine pancreas proteases through interaction with endocrine ß-cells.
Assuntos
Ciliopatias/etiologia , Ciliopatias/metabolismo , Células Secretoras de Insulina/metabolismo , Pâncreas Exócrino/enzimologia , Peptídeo Hidrolases/metabolismo , Animais , Animais Geneticamente Modificados , Proliferação de Células , Quimotripsina/genética , Quimotripsina/metabolismo , Ciliopatias/patologia , Suscetibilidade a Doenças , Expressão Gênica , Camundongos , Mutação , Peptídeo Hidrolases/genética , Peixe-ZebraRESUMO
Genome analysis of diverse human populations has contributed to the identification of novel genomic loci for diseases of major clinical and public health impact. Here, we report a genome-wide analysis of type 2 diabetes (T2D) in sub-Saharan Africans, an understudied ancestral group. We analyze ~18 million autosomal SNPs in 5,231 individuals from Nigeria, Ghana and Kenya. We identify a previously-unreported genome-wide significant locus: ZRANB3 (Zinc Finger RANBP2-Type Containing 3, lead SNP p = 2.831 × 10-9). Knockdown or genomic knockout of the zebrafish ortholog results in reduction in pancreatic ß-cell number which we demonstrate to be due to increased apoptosis in islets. siRNA transfection of murine Zranb3 in MIN6 ß-cells results in impaired insulin secretion in response to high glucose, implicating Zranb3 in ß-cell functional response to high glucose conditions. We also show transferability in our study of 32 established T2D loci. Our findings advance understanding of the genetics of T2D in non-European ancestry populations.
Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Diabetes Mellitus Tipo 2/genética , Estudos de Associação Genética , Predisposição Genética para Doença/genética , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , África do Norte , Animais , Apoptose , Sequência de Bases , Glicemia , Sistemas CRISPR-Cas , Modelos Animais de Doenças , Feminino , Edição de Genes , Técnicas de Inativação de Genes , Genótipo , Gana , Glucose/metabolismo , Homozigoto , Humanos , Quênia , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação , Nigéria , Polimorfismo de Nucleotídeo Único , RNA Interferente Pequeno , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Transcriptoma , Peixe-ZebraRESUMO
Alström syndrome (OMIM #203800) is an autosomal recessive obesity ciliopathy caused by loss-of-function mutations in the ALMS1 gene. In addition to multi-organ dysfunction, such as cardiomyopathy, retinal degeneration and renal dysfunction, the disorder is characterized by high rates of obesity, insulin resistance and early-onset type 2 diabetes mellitus (T2DM). To investigate the underlying mechanisms of T2DM phenotypes, we generated a loss-of-function deletion of alms1 in the zebrafish. We demonstrate conservation of hallmark clinical characteristics alongside metabolic syndrome phenotypes, including a propensity for obesity and fatty livers, hyperinsulinemia and glucose response defects. Gene expression changes in ß-cells isolated from alms1-/- mutants revealed changes consistent with insulin hypersecretion and glucose sensing failure, which were corroborated in cultured murine ß-cells lacking Alms1. We also found evidence of defects in peripheral glucose uptake and concomitant hyperinsulinemia in the alms1-/- animals. We propose a model in which hyperinsulinemia is the primary and causative defect underlying generation of T2DM associated with alms1 deficiency. These observations support the alms1 loss-of-function zebrafish mutant as a monogenic model for mechanistic interrogation of T2DM phenotypes.
Assuntos
Síndrome de Alstrom/genética , Diabetes Mellitus Tipo 2/genética , Resistência à Insulina/genética , Insuficiência Renal/genética , Degeneração Retiniana/genética , Peixe-Zebra/genética , Síndrome de Alstrom/fisiopatologia , Animais , Animais Geneticamente Modificados , Linhagem Celular , Modelos Animais de Doenças , Intolerância à Glucose , Hiperinsulinismo/genética , Células Secretoras de Insulina/metabolismo , Camundongos , Modelos Biológicos , Obesidade/genética , Fenótipo , Peixe-Zebra/embriologiaRESUMO
Trisomy for human chromosome 21 (Hsa21) results in Down syndrome (DS), one of the most genetically complex conditions compatible with human survival. Assessment of the physiological consequences of dosage-driven overexpression of individual Hsa21 genes during early embryogenesis and the resulting contributions to DS pathology in mammals are not tractable in a systematic way. A recent study looked at loss-of-function of a subset of Caenorhabditis elegans orthologs of Hsa21 genes and identified ten candidates with behavioral phenotypes, but the equivalent over-expression experiment has not been done. We turned to zebrafish as a developmental model and, using a number of surrogate phenotypes, we screened Hsa21 genes for effects on early embyrogenesis. We prepared a library of 164 cDNAs of conserved protein coding genes, injected mRNA into early embryos and evaluated up to 5 days post-fertilization (dpf). Twenty-four genes produced a gross morphological phenotype, 11 of which could be reproduced reliably. Seven of these gave a phenotype consistent with down regulation of the sonic hedgehog (Shh) pathway; two showed defects indicative of defective neural crest migration; one resulted consistently in pericardial edema; and one was embryonic lethal. Combinatorial injections of multiple Hsa21 genes revealed both additive and compensatory effects, supporting the notion that complex genetic relationships underlie end phenotypes of trisomy that produce DS. Together, our data suggest that this system is useful in the genetic dissection of dosage-sensitive gene effects on early development and can inform the contribution of both individual loci and their combinatorial effects to phenotypes relevant to the etiopathology of DS.
Assuntos
Cromossomos Humanos Par 21 , Regulação da Expressão Gênica no Desenvolvimento , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Animais , Modelos Animais de Doenças , Síndrome de Down/genética , Dosagem de Genes , Biblioteca Gênica , Estudos de Associação Genética , Teste de Complementação Genética , Humanos , FenótipoRESUMO
BACKGROUND: Elevated levels of low-density lipoprotein cholesterol (LDL-C) are a major risk factor for cardiovascular disease via its contribution to the development and progression of atherosclerotic lesions. Although the genetic basis of LDL-C has been studied extensively, currently known genetic variants account for only ≈20% of the variation in LDL-C levels. METHODS: Through an array-based association analysis in 1102 Amish subjects, we identified a variant strongly associated with LDL-C levels. Using a combination of genetic analyses, zebrafish models, and in vitro experiments, we sought to identify the causal gene driving this association. RESULTS: We identified a founder haplotype associated with a 15 mg/dL increase in LDL-C on chromosome 5. After recombination mapping, the associated region contained 8 candidate genes. Using a zebrafish model to evaluate the relevance of these genes to cholesterol metabolism, we found that expression of the transcribed pseudogene, APOOP1, increased LDL-C and vascular plaque formation. CONCLUSIONS: Based on these data, we propose that APOOP1 regulates levels of LDL-C in humans, thus identifying a novel mechanism of lipid homeostasis.
Assuntos
Amish/genética , Aterosclerose/genética , LDL-Colesterol/sangue , Cromossomos Humanos Par 5 , Dislipidemias/genética , Pseudogenes , Animais , Animais Geneticamente Modificados , Aterosclerose/sangue , Aterosclerose/diagnóstico , Aterosclerose/etnologia , Dislipidemias/sangue , Dislipidemias/diagnóstico , Dislipidemias/etnologia , Efeito Fundador , Estudos de Associação Genética , Predisposição Genética para Doença , Haplótipos , Humanos , Fenótipo , Recombinação Genética , Fatores de Risco , Peixe-Zebra/genéticaRESUMO
The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.
Assuntos
Perfilação da Expressão Gênica/métodos , RNA/genética , Análise de Sequência de RNA/métodos , Animais , RNA/química , Peixe-ZebraRESUMO
Bardet-Biedl syndrome is a model ciliopathy. Although the characterization of BBS proteins has evidenced their involvement in cilia, extraciliary functions for some of these proteins are also being recognized. Importantly, understanding both cilia and cilia-independent functions of the BBS proteins is key to fully dissect the cellular basis of the syndrome. Here we characterize a functional interaction between BBS4 and the secreted protein FSTL1, a protein linked to adipogenesis and inflammation among other functions. We show that BBS4 and cilia regulate FSTL1 mRNA levels, but BBS4 also modulates FSTL1 secretion. Moreover, we show that FSTL1 is a novel regulator of ciliogenesis thus underscoring a regulatory loop between FSTL1 and cilia. Finally, our data indicate that BBS4, cilia and FSTL1 are coordinated during the differentiation of 3T3-L1 cells and that FSTL1 plays a role in this process, at least in part, by modulating ciliogenesis. Therefore, our findings are relevant to fully understand the development of BBS-associated phenotypes such as obesity.
Assuntos
Diferenciação Celular/genética , Cílios/genética , Cílios/metabolismo , Proteínas Relacionadas à Folistatina/biossíntese , Proteínas Relacionadas à Folistatina/genética , Regulação da Expressão Gênica , Proteínas/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Adipogenia/genética , Animais , Técnicas de Silenciamento de Genes , Espaço Intracelular/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos , Proteínas/genéticaRESUMO
INTRODUCTION: Transplant surgeons conventionally select against livers displaying high degrees (>30%) of macrosteatosis (MaS), out of concern for primary non-function or severe graft dysfunction. As such, there is relatively limited experience with such livers, and the natural history remains incompletely characterized. We present our experience of transplanted livers with high degrees of MaS and microsteatosis (MiS), with a focus on the histopathologic and clinical outcomes. METHODS: Twenty-nine cases were identified with liver biopsies available from both the donor and the corresponding liver transplant recipient. Donor liver biopsies displayed either MaS or MiS ≥15%, while all recipients received postoperative liver biopsies for cause. RESULTS: The mean donor MaS and MiS were 15.6% (range 0%-60%) and 41.3% (7.5%-97.5%), respectively. MaS decreased significantly from donor (M=15.6%) to recipient postoperative biopsies (M=0.86%), P<.001. Similarly, MiS decreased significantly from donor biopsies (M=41.3%) to recipient postoperative biopsies (M=1.8%), P<.001. At a median of 68 days postoperatively (range 4-384), full resolution of MaS and MiS was observed in 27 of 29 recipients. CONCLUSIONS: High degrees of MaS and MiS in donor livers resolve in recipients following liver transplantation. Further insight into the mechanisms responsible for treating fatty liver diseases could translate into therapeutic targets.
Assuntos
Seleção do Doador , Hepatectomia , Transplante de Fígado , Doadores Vivos , Hepatopatia Gordurosa não Alcoólica/cirurgia , Adulto , Idoso , Biópsia , Feminino , Humanos , Fígado/patologia , Fígado/cirurgia , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica/diagnóstico , Hepatopatia Gordurosa não Alcoólica/patologia , Avaliação de Resultados em Cuidados de Saúde , Estudos Retrospectivos , Transplante HomólogoRESUMO
The transmembrane 6 superfamily member 2 (TM6SF2) loss-of-function variant rs58542926 is a genetic risk factor for nonalcoholic fatty liver disease and progression to fibrosis but is paradoxically associated with lower levels of hepatically derived triglyceride-rich lipoproteins. TM6SF2 is expressed predominantly in liver and small intestine, sites for triglyceride-rich lipoprotein biogenesis and export. In light of this, we hypothesized that TM6SF2 may exhibit analogous effects on both liver and intestine lipid homeostasis. To test this, we genotyped rs58542926 in 983 bariatric surgery patients from the Geisinger Medical Center for Nutrition and Weight Management, Geisinger Health System, in Pennsylvania and from 3,556 study participants enrolled in the Amish Complex Disease Research Program. Although these two cohorts have different metabolic profiles, carriers in both cohorts had improved fasting lipid profiles. Importantly, following a high-fat challenge, carriers in the Amish Complex Disease Research Program cohort exhibited significantly lower postprandial serum triglycerides, suggestive of a role for TM6SF2 in the small intestine. To gain further insight into this putative role, effects of TM6SF2 deficiency were studied in a zebrafish model and in cultured human Caco-2 enterocytes. In both systems TM6SF2 deficiency resulted in defects in small intestine metabolism in response to dietary lipids, including significantly increased lipid accumulation, decreased lipid clearance, and increased endoplasmic reticulum stress. CONCLUSIONS: These data strongly support a role of TM6SF2 in the regulation of postprandial lipemia, potentially through a similar function for TM6SF2 in the lipidation and/or export of both hepatically and intestinally derived triglyceride-rich lipoproteins. (Hepatology 2017;65:1526-1542).
Assuntos
Estresse do Retículo Endoplasmático , Intestino Delgado/metabolismo , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Proteínas de Membrana/genética , Animais , Sequência de Bases , Células CACO-2 , Enterócitos/metabolismo , Fígado Gorduroso/genética , Feminino , Hepatócitos/metabolismo , Homeostase , Humanos , Intestino Delgado/ultraestrutura , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Polimorfismo de Nucleotídeo Único , Período Pós-Prandial , Triglicerídeos/biossíntese , Triglicerídeos/sangue , Tunicamicina , Peixe-ZebraRESUMO
FOXE3 is a lens-specific transcription factor that has been associated with anterior segment ocular dysgenesis. To determine the transcriptional target(s) of FOXE3 that are indispensable for the anterior segment development, we examined the transcriptome and the proteome of cells expressing truncated FOXE3 responsible for Peters anomaly identified through linkage-coupled next-generation whole-exome sequencing. We found that DNAJB1, an autophagy-associated protein, was the only candidate exhibiting differential expression in both screens. We confirmed the candidacy of DNAJB1 through chromatin immunoprecipitation and luciferase assays while knockdown of DNAJB1 in human lens epithelial cells resulted in a mitotic arrest. Subsequently, we targeted dnajb1a in zebrafish through injection of a splice-blocking morpholino. The dnajb1a morphants exhibited underdeveloped cataractous lenses with persistent apoptotic nuclei. In conclusion, here we report DNAJB1 is a transcriptional target of FOXE3 in a novel pathway that is crucial for the development of the anterior segment of the eye.
Assuntos
Autofagia/genética , Opacidade da Córnea/genética , Anormalidades do Olho/genética , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica , Proteínas de Choque Térmico HSP40/genética , Animais , Opacidade da Córnea/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Anormalidades do Olho/metabolismo , Saúde da Família , Feminino , Fatores de Transcrição Forkhead/metabolismo , Perfilação da Expressão Gênica/métodos , Técnicas de Silenciamento de Genes , Células HEK293 , Proteínas de Choque Térmico HSP40/metabolismo , Humanos , Cristalino/patologia , Masculino , Linhagem , Sequenciamento do Exoma/métodos , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
BACKGROUND: Bardet-Biedl Syndrome (BBS) and Alström Syndrome are two pleiotropic ciliopathies with significant phenotypic overlap between them across many tissues. Although BBS and Alström genes are necessary for the proper function of primary cilia, their role in defects across multiple organ systems is unclear. METHODS: To provide insight into the pathways underlying BBS and Alström phenotypes, we carried out whole organism transcriptome analysis by RNA sequencing in established zebrafish models of the syndromes. RESULTS: We analyzed all genes that were significantly differentially expressed and found enrichment of phenotypically significant pathways in both models. These included multiple pathways shared between the two disease models as well as those unique to each model. Notably, we identified significant downregulation of genes in pathways relevant to visual system deficits and obesity in both disorders, consistent with those shared phenotypes. In contrast, neuronal pathways were significantly downregulated only in the BBS model but not in the Alström model. Our observations also suggested an important role for G-protein couple receptor and calcium signaling defects in both models. DISCUSSION: Pathway network analyses of both models indicate that visual system defects may be driven by genetic mechanisms independent of other phenotypes whereas the majority of other phenotypes are a result of genetic players that contribute to multiple pathways simultaneously. Additionally, examination of genes differentially expressed in opposing directions between the two models suggest a deficit in pancreatic function in the Alström model, that is not present in the BBS model. CONCLUSIONS: These findings provide important novel insight into shared and divergent phenotypes between two similar but distinct genetic syndromes.
Assuntos
Síndrome de Alstrom/genética , Síndrome de Bardet-Biedl/genética , Perfilação da Expressão Gênica , Fenótipo , Transcriptoma , Peixe-Zebra/genética , Síndrome de Alstrom/diagnóstico , Animais , Síndrome de Bardet-Biedl/diagnóstico , Biologia Computacional/métodos , Modelos Animais de Doenças , Regulação da Expressão Gênica , Ontologia Genética , Redes Reguladoras de Genes , Transdução de Sinal Luminoso , Vias Neurais , Vias VisuaisRESUMO
Type 2 diabetes (T2D) has been associated with a large number of genomic loci, many of which encompass multiple genes without a definitive causal gene. This complexity has hindered efforts to clearly identify functional candidate genes and interpret their role in mediating susceptibility to disease. Here we examined the relevance of individual genes found at T2D-associated loci by assessing their potential contribution to a phenotype relevant to the disease state: production and maintenance of ß-cell mass. Using transgenic zebrafish in which ß-cell mass could be rapidly visualized in vivo, we systematically suppressed the expression of orthologs of genes found at T2D-associated genomic loci. Overall, we tested 67 orthologs, many of which had no known relevance to ß-cell mass, at 62 human T2D-associated loci, including eight loci with multiple candidate genes. In total we identified 25 genes that were necessary for proper ß-cell mass, providing functional evidence for their role in a physiological phenotype directly related to T2D. Of these, 16 had not previously been implicated in the regulation of ß-cell mass. Strikingly, we identified single functional candidate genes at the majority of the loci for which multiple genes were analyzed. Further investigation into the contribution of the 25 genes to the adaptive capacity of ß-cells suggested that the majority of genes were not required for glucose-induced expansion of ß-cell mass but were significantly necessary for the regeneration of ß-cells. These findings suggest that genetically programmed deficiencies in ß-cell mass may be related to impaired maintenance. Finally, we investigated the relevance of our findings to human T2D onset in diabetic individuals from the Old Order Amish and found that risk alleles in ß-cell mass genes were associated with significantly younger age of onset and lower body mass index. Taken together, our study offers a functional approach to assign relevance to genes at T2D-associated loci and offers experimental evidence for the defining role of ß-cell mass maintenance in genetic susceptibility to T2D onset.
Assuntos
Diabetes Mellitus Tipo 2/genética , Células Secretoras de Insulina/fisiologia , Adulto , Amish/genética , Animais , Tamanho Celular , Mapeamento Cromossômico , Estudos de Associação Genética , Loci Gênicos , Predisposição Genética para Doença , Glucose/metabolismo , Homeostase , Humanos , Redes e Vias Metabólicas , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único , Peixe-ZebraRESUMO
Rare genetic syndromes characterized by early-onset type 2 diabetes have revealed the importance of pancreatic ß-cells in genetic susceptibility to diabetes. However, the role of genetic regulation of ß-cells in disorders that are also characterized by highly penetrant obesity, a major additional risk factor, is unclear. In this study, we investigated the contribution of genes associated with two obesity ciliopathies, Bardet-Biedl Syndrome and Alstrom Syndrome, to the production and maintenance of pancreatic ß-cells. Using zebrafish models of these syndromes, we identified opposing effects on production of ß-cells. Loss of the Alstrom gene, alms1, resulted in a significant decrease in ß-cell production whereas loss of BBS genes, bbs1 or bbs4, resulted in a significant increase. Examination of the regulatory program underlying ß-cell production suggested that these effects were specific to ß-cells. In addition to the initial production of ß-cells, we observed significant differences in their continued maintenance. Under prolonged exposure to high glucose conditions, alms1-deficient ß-cells were unable to continually expand as a result of decreased proliferation and increased cell death. Although bbs1-deficient ß-cells were similarly susceptible to apoptosis, the overall maintenance of ß-cell number in those animals was sustained likely due to increased proliferation. Taken together, these findings implicate discrepant production and maintenance of ß-cells in the differential susceptibility to diabetes found between these two genetic syndromes.
Assuntos
Síndrome de Alstrom/genética , Síndrome de Bardet-Biedl/genética , Células Secretoras de Insulina/patologia , Animais , Morte Celular , Proliferação de Células , Modelos Animais de Doenças , Glucose , Hiperglicemia/patologia , Proteínas Associadas aos Microtúbulos/genética , Morfolinos/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/genéticaRESUMO
Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Células Ciliadas Auditivas/metabolismo , Audição/fisiologia , Fatores de Transcrição/metabolismo , Animais , Animais Recém-Nascidos , Evolução Biológica , Imunoprecipitação da Cromatina , Feminino , Regulação da Expressão Gênica , Células Ciliadas Auditivas/ultraestrutura , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Família Multigênica , Fatores de Transcrição de Fator Regulador X , Fator Regulador X1 , Análise de Sequência de DNA , Transcriptoma , Peixe-ZebraRESUMO
Hyperlipidemia and arterial cholesterol accumulation are primary causes of cardiovascular events. Monogenic forms of hyperlipidemia and recent genome-wide association studies indicate that genetics plays an important role. Zebrafish are a useful model for studying the genetic susceptibility to hyperlipidemia owing to conservation of many components of lipoprotein metabolism, including those related to LDL, ease of genetic manipulation, and in vivo observation of lipid transport and vascular calcification. We sought to develop a genetic model for lipid metabolism in zebrafish, capitalizing on one well-understood player in LDL cholesterol (LDL-c) transport, the LDL receptor (ldlr), and an established in vivo model of hypercholesterolemia. We report that morpholinos targeted against the gene encoding ldlr effectively suppressed its expression in embryos during the first 8 days of development. The ldlr morphants exhibited increased LDL-c levels that were exacerbated by feeding a high cholesterol diet. Increased LDL-c was ameliorated in morphants upon treatment with atorvastatin. Furthermore, we observed significant vascular and liver lipid accumulation, vascular leakage, and plaque oxidation in ldlr-deficient embryos. Finally, upon transcript analysis of several cholesterol-regulating genes, we observed changes similar to those seen in mammalian systems, suggesting that cholesterol regulation may be conserved in zebrafish. Taken together, these observations indicate conservation of ldlr function in zebrafish and demonstrate the utility of transient gene knockdown in embryos as a genetic model for hyperlipidemia.
Assuntos
Vasos Sanguíneos/metabolismo , LDL-Colesterol/metabolismo , Hipercolesterolemia/genética , Hipercolesterolemia/metabolismo , Receptores de LDL/deficiência , Receptores de LDL/genética , Peixe-Zebra , Animais , Atorvastatina , Sequência de Bases , Vasos Sanguíneos/efeitos dos fármacos , Modelos Animais de Doenças , Embrião não Mamífero , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hepatomegalia/complicações , Ácidos Heptanoicos/farmacologia , Hipercolesterolemia/complicações , Fígado/efeitos dos fármacos , Fígado/metabolismo , Dados de Sequência Molecular , Morfolinos/genética , Oxirredução/efeitos dos fármacos , Pirróis/farmacologia , Veias/efeitos dos fármacos , Veias/metabolismoRESUMO
Primary cilia regulate an expanding list of signaling pathways in many different cell types. It is likely that identification of the full catalog of pathways associated with cilia will be necessary to fully understand their role in regulation of signaling and the implications for diseases associated with their dysfunction, ciliopathies. Bardet-Biedl Syndrome (BBS) is one such ciliopathy which is characterized by a spectrum of phenotypes. These include neural defects such as impaired cognitive development, centrally mediated hyperphagia and peripheral sensory defects. Here we investigate potential defects in a signaling pathway associated with neuronal function, brain derived neurotrophic factor (BDNF) signaling. Upon loss of BBS4 expression in cultured cells, we observed decreased phosphorylation and activation by BDNF of its target receptor, TrkB. Assessment of ciliary localization revealed that, TrkB localized to the axonemes or basal bodies of cilia only in the presence of BDNF. Axonemal localization, specifically, was abrogated with loss of BBS4. Finally, we present evidence that loss of the ciliary axoneme through depletion of KIF3A impedes activation of TrkB. Taken together, these data suggest the possibility of a previously uninvestigated pathway associated with perturbation of ciliary proteins.