RESUMO
Chromogranin B (CgB, also known as CHGB) is abundantly expressed in dense core secretory granules of multiple endocrine tissues and has been suggested to regulate granule biogenesis in some cell types, including the pancreatic islet ß-cell, though the mechanisms are poorly understood. Here, we demonstrate a critical role for CgB in regulating secretory granule trafficking in the ß-cell. Loss of CgB impairs glucose-stimulated insulin secretion, impedes proinsulin processing to yield increased proinsulin content, and alters the density of insulin-containing granules. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin, we show that loss of CgB impairs Golgi budding of proinsulin-containing secretory granules, resulting in a substantial delay in trafficking of nascent granules to the plasma membrane with an overall decrease in total plasma membrane-associated granules. These studies demonstrate that CgB is necessary for efficient trafficking of secretory proteins into the budding granule, which impacts the availability of insulin-containing secretory granules for exocytic release.This article has an associated First Person interview with the first author of the paper.
Assuntos
Cromogranina B/metabolismo , Grânulos Citoplasmáticos/metabolismo , Complexo de Golgi/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cromogranina B/deficiência , Grânulos Citoplasmáticos/efeitos dos fármacos , Glucose/farmacologia , Complexo de Golgi/efeitos dos fármacos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , RNA Interferente Pequeno/metabolismo , Ratos , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/metabolismoRESUMO
Genetic manipulation via transgene overexpression, RNAi, or Cas9-based methods is central to biomedical research. Unfortunately, use of these tools is often limited by vector options. We have created a modular platform (pMVP) that allows a gene of interest to be studied in the context of an array of promoters, epitope tags, conditional expression modalities, and fluorescent reporters, packaged in 35 custom destination vectors, including adenovirus, lentivirus, PiggyBac transposon, and Sleeping Beauty transposon, in aggregate >108,000 vector permutations. We also used pMVP to build an epigenetic engineering platform, pMAGIC, that packages multiple gRNAs and either Sa-dCas9 or x-dCas9(3.7) fused to one of five epigenetic modifiers. Importantly, via its compatibility with adenoviral vectors, pMAGIC uniquely enables use of dCas9/LSD1 fusions to interrogate enhancers within primary cells. To demonstrate this, we used pMAGIC to target Sa-dCas9/LSD1 and modify the epigenetic status of a conserved enhancer, resulting in altered expression of the homeobox transcription factor PDX1 and its target genes in pancreatic islets and insulinoma cells. In sum, the pMVP and pMAGIC systems empower researchers to rapidly generate purpose-built, customized vectors for manipulation of gene expression, including via targeted epigenetic modification of regulatory elements in a broad range of disease-relevant cell types.
Assuntos
Sistemas CRISPR-Cas/genética , Engenharia Genética/métodos , Vetores Genéticos/genética , Proteínas de Homeodomínio/genética , Transativadores/genética , Transgenes/genética , Adenoviridae/genética , Animais , Elementos de DNA Transponíveis/genética , Elementos Facilitadores Genéticos/genética , Epigenômica/métodos , Edição de Genes/métodos , Regulação da Expressão Gênica/genética , Células HEK293 , Histona Desmetilases/genética , Humanos , Insulinoma/metabolismo , Ilhotas Pancreáticas/metabolismo , Lentivirus/genética , Camundongos , Regiões Promotoras Genéticas/genética , RNA Guia de Cinetoplastídeos/genética , RatosRESUMO
Recent studies have shown that the pyruvate-isocitrate cycling pathway, involving the mitochondrial citrate/isocitrate carrier and the cytosolic NADP-dependent isocitrate dehydrogenase (ICDc), is involved in control of glucose-stimulated insulin secretion (GSIS). Here we demonstrate that pyruvate-isocitrate cycling regulates expression of the voltage-gated potassium channel family member Kv2.2 in islet ß-cells. siRNA-mediated suppression of ICDc, citrate/isocitrate carrier, or Kv2.2 expression impaired GSIS, and the effect of ICDc knockdown was rescued by re-expression of Kv2.2. Moreover, chronic exposure of ß-cells to elevated fatty acids, which impairs GSIS, resulted in decreased expression of Kv2.2. Surprisingly, knockdown of ICDc or Kv2.2 increased rather than decreased outward K(+) current in the 832/13 ß-cell line. Immunoprecipitation studies demonstrated interaction of Kv2.1 and Kv2.2, and co-overexpression of the two channels reduced outward K(+) current compared with overexpression of Kv2.1 alone. Also, siRNA-mediated knockdown of ICDc enhanced the suppressive effect of the Kv2.1-selective inhibitor stromatoxin1 on K(+) currents. Our data support a model in which a key function of the pyruvate-isocitrate cycle is to maintain levels of Kv2.2 expression sufficient to allow it to serve as a negative regulator of Kv channel activity.
Assuntos
Regulação da Expressão Gênica/fisiologia , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Isocitratos/metabolismo , Ácido Pirúvico/metabolismo , Canais de Potássio Shab/biossíntese , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/genética , Secreção de Insulina , Células Secretoras de Insulina/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Ativação do Canal Iônico/fisiologia , Transporte de Íons/efeitos dos fármacos , Transporte de Íons/fisiologia , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Masculino , Modelos Biológicos , Peptídeos/farmacologia , Potássio/metabolismo , Ratos , Ratos Sprague-Dawley , Canais de Potássio Shab/antagonistas & inibidores , Canais de Potássio Shab/genética , Venenos de Aranha/farmacologiaRESUMO
OBJECTIVE: All forms of diabetes result from insufficient functional ß-cell mass. Thus, achieving the therapeutic goal of expanding ß-cell mass requires a better mechanistic understanding of how ß-cells proliferate. Glucose is a natural ß-cell mitogen that mediates its effects in part through the glucose-responsive transcription factor, carbohydrate response element binding protein (ChREBP) and the anabolic transcription factor, MYC. However, mechanistic details by which glucose activates Myc at the transcriptional level are poorly understood. METHODS: Here, siRNA was used to test the role of ChREBP in the glucose response of MYC, ChIP and ChIPseq to identify potential regulatory binding sites, chromatin conformation capture to identify DNA/DNA interactions, and an adenovirus was constructed to expresses x-dCas9 and an sgRNA that specifically disrupts the recruitment of ChREBP to a specific targeted ChoRE. RESULTS: We found that ChREBP is essential for glucose-mediated transcriptional induction of Myc, and for increases in Myc mRNA and protein abundance. Further, ChIPseq revealed that the carbohydrate response element (ChoRE) nearest to the Myc transcriptional start site (TSS) is immediately upstream of the gene encoding the lncRNA, Pvt1, 60,000 bp downstream of the Myc gene. Chromatin Conformation Capture (3C) confirmed a glucose-dependent interaction between these two sites. Transduction with an adenovirus expressing x-dCas9 and an sgRNA specifically targeting the highly conserved Pvt1 ChoRE, attenuates ChREBP recruitment, decreases Myc-Pvt1 DNA/DNA interaction, and decreases expression of the Pvt1 and Myc genes in response to glucose. Importantly, isolated and dispersed rat islet cells transduced with the ChoRE-disrupting adenovirus also display specific decreases in ChREBP-dependent, glucose-mediated expression of Pvt1 and Myc, as well as decreased glucose-stimulated ß-cell proliferation. CONCLUSIONS: The mitogenic glucose response of Myc is mediated via glucose-dependent recruitment of ChREBP to the promoter of the Pvt1 gene and subsequent DNA looping with the Myc promoter.
Assuntos
Genes myc , Glucose , Animais , Ratos , Cromatina/genética , DNA , Glucose/metabolismo , RNA Guia de Sistemas CRISPR-Cas , Fatores de Transcrição/metabolismo , Ativação Transcricional/genética , Proteínas Proto-Oncogênicas c-mycRESUMO
Carbohydrate response element-binding protein (ChREBP) is a carbohydrate-sensing transcription factor that regulates both adaptive and maladaptive genomic responses in coordination of systemic fuel homeostasis. Genetic variants in the ChREBP locus associate with diverse metabolic traits in humans, including circulating lipids. To identify novel ChREBP-regulated hepatokines that contribute to its systemic metabolic effects, we integrated ChREBP ChIP-Seq analysis in mouse liver with human genetic and genomic data for lipid traits and identified hepatocyte growth factor activator (HGFAC) as a promising ChREBP-regulated candidate in mice and humans. HGFAC is a protease that activates the pleiotropic hormone hepatocyte growth factor. We demonstrate that HGFAC-KO mice had phenotypes concordant with putative loss-of-function variants in human HGFAC. Moreover, in gain- and loss-of-function genetic mouse models, we demonstrate that HGFAC enhanced lipid and glucose homeostasis, which may be mediated in part through actions to activate hepatic PPARγ activity. Together, our studies show that ChREBP mediated an adaptive response to overnutrition via activation of HGFAC in the liver to preserve glucose and lipid homeostasis.
Assuntos
Glucose , Fatores de Transcrição , Animais , Humanos , Camundongos , Glucose/metabolismo , Homeostase , Lipídeos , Fatores de Transcrição/metabolismoRESUMO
Paracrine interactions between pancreatic islet cells have been proposed as a mechanism to regulate hormone secretion and glucose homeostasis. Here, we demonstrate the importance of proglucagon-derived peptides (PGDPs) for α to ß cell communication and control of insulin secretion. Signaling through this system occurs through both the glucagon-like peptide receptor (Glp1r) and glucagon receptor (Gcgr). Loss of PGDPs, or blockade of their receptors, decreases insulin secretion in response to both metabolic and nonmetabolic stimulation of mouse and human islets. This effect is due to reduced ß cell cAMP and affects the quantity but not dynamics of insulin release, indicating that PGDPs dictate the magnitude of insulin output in an isolated islet. In healthy mice, additional factors that stimulate cAMP can compensate for loss of PGDP signaling; however, input from α cells is essential to maintain glucose tolerance during the metabolic stress induced by high-fat feeding. These findings demonstrate an essential role for α cell regulation of ß cells, raising the possibility that abnormal paracrine signaling contributes to impaired insulin secretion in diabetes. Moreover, these findings support reconsideration of the role for α cells in postprandial glucose control.
Assuntos
AMP Cíclico/metabolismo , Células Secretoras de Insulina/metabolismo , Proglucagon/metabolismo , Transdução de Sinais , Animais , Feminino , Polipeptídeo Inibidor Gástrico/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Homeostase , Humanos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Current obesity interventions suffer from lack of durable effects and undesirable complications. Fumagillin, an inhibitor of methionine aminopeptidase-2, causes weight loss by reducing food intake, but with effects on weight that are superior to pair-feeding. Here, we show that feeding of rats on a high-fat diet supplemented with fumagillin (HF/FG) suppresses the aggressive feeding observed in pair-fed controls (HF/PF) and alters expression of circadian genes relative to the HF/PF group. Multiple indices of reduced energy expenditure are observed in HF/FG but not HF/PF rats. HF/FG rats also exhibit changes in gut hormones linked to food intake, increased energy harvest by gut microbiota, and caloric spilling in the urine. Studies in gnotobiotic mice reveal that effects of fumagillin on energy expenditure but not feeding behavior may be mediated by the gut microbiota. In sum, fumagillin engages weight loss-inducing behavioral and physiologic circuits distinct from those activated by simple caloric restriction.
Assuntos
Bactérias/isolamento & purificação , Cicloexanos/administração & dosagem , Metabolismo Energético/efeitos dos fármacos , Ácidos Graxos Insaturados/administração & dosagem , Microbioma Gastrointestinal/efeitos dos fármacos , Obesidade/tratamento farmacológico , Aminopeptidases/antagonistas & inibidores , Animais , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Comportamento Animal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Fezes/microbiologia , Comportamento Alimentar/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Vida Livre de Germes/efeitos dos fármacos , Vida Livre de Germes/fisiologia , Glicoproteínas/antagonistas & inibidores , Humanos , Masculino , Metionil Aminopeptidases , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Obesidade/metabolismo , Ratos , Ratos Wistar , Sesquiterpenos/administração & dosagem , Resultado do Tratamento , Redução de Peso/efeitos dos fármacosRESUMO
Branched-chain amino acids (BCAA) are strongly associated with dysregulated glucose and lipid metabolism, but the underlying mechanisms are poorly understood. We report that inhibition of the kinase (BDK) or overexpression of the phosphatase (PPM1K) that regulates branched-chain ketoacid dehydrogenase (BCKDH), the committed step of BCAA catabolism, lowers circulating BCAA, reduces hepatic steatosis, and improves glucose tolerance in the absence of weight loss in Zucker fatty rats. Phosphoproteomics analysis identified ATP-citrate lyase (ACL) as an alternate substrate of BDK and PPM1K. Hepatic overexpression of BDK increased ACL phosphorylation and activated de novo lipogenesis. BDK and PPM1K transcript levels were increased and repressed, respectively, in response to fructose feeding or expression of the ChREBP-ß transcription factor. These studies identify BDK and PPM1K as a ChREBP-regulated node that integrates BCAA and lipid metabolism. Moreover, manipulation of the BDK:PPM1K ratio relieves key metabolic disease phenotypes in a genetic model of severe obesity.
Assuntos
3-Metil-2-Oxobutanoato Desidrogenase (Lipoamida)/metabolismo , ATP Citrato (pro-S)-Liase/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Lipogênese , Obesidade/metabolismo , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Modelos Animais de Doenças , Feminino , Células HEK293 , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Fosfatase 2C , Ratos , Ratos Wistar , Ratos ZuckerRESUMO
Increased ß-cell death coupled with the inability to replicate existing ß-cells drives the decline in ß-cell mass observed in the progression of both major forms of diabetes. Understanding endogenous mechanisms of islet cell survival could have considerable value for the development of novel strategies to limit ß-cell loss and thereby promote ß-cell recovery. Insulinoma cells have provided useful insight into ß-cell death pathways but observations made in cell lines sometimes fail to translate to primary islets. Here, we report dramatic differences in the temporal regulation and engagement of the apoptotic program in primary rodent islets relative to the INS-1 derived 832/13 cell line. As expected, 832/13 cells rapidly induced cell stress markers in response to ER stress or DNA damage and were fully committed to apoptosis, resulting in >80% cell death within 24 h. In contrast, primary rat islets were largely refractory to cell death in response to ER stress and DNA damage, despite rapid induction of stress markers, such as XBP-1(s), CHOP, and PUMA. Gene expression profiling revealed a general suppression of pro-apoptotic machinery, such as Apaf-1 and caspase 3, and sustained levels of pro-survival factors, such as cIAP-1, cIAP-2, and XIAP, in rat islets. Furthermore, we observed sustained induction of autophagy following chronic ER stress and found that inhibition of autophagy rendered islet ß-cells highly vulnerable to ER stress-induced cell death. We propose that islet ß-cells dampen the apoptotic response to delay the onset of cell death, providing a temporal window in which autophagy can be activated to limit cellular damage and promote survival.
Assuntos
Apoptose/fisiologia , Autofagia/fisiologia , Ilhotas Pancreáticas/citologia , Animais , Fator Apoptótico 1 Ativador de Proteases , Caspase 3/metabolismo , Linhagem Celular , Sobrevivência Celular/fisiologia , Células Cultivadas , Estresse do Retículo Endoplasmático , Glucose/metabolismo , Proteínas Inibidoras de Apoptose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Insulinoma/patologia , Ilhotas Pancreáticas/fisiologia , Neoplasias Pancreáticas/patologia , RatosRESUMO
The homeodomain transcription factor Pdx-1 has important roles in pancreas and islet development as well as in ß-cell function and survival. We previously reported that Pdx-1 overexpression stimulates islet cell proliferation, but the mechanism remains unclear. Here, we demonstrate that overexpression of Pdx-1 triggers proliferation largely by a non-cell-autonomous mechanism mediated by soluble factors. Consistent with this idea, overexpression of Pdx-1 under the control of a ß-cell-specific promoter (rat insulin promoter [RIP]) stimulates proliferation of both α and ß cells, and overexpression of Pdx-1 in islets separated by a Transwell membrane from islets lacking Pdx-1 overexpression activates proliferation in the untreated islets. Microarray and gene ontology (GO) analysis identified inhibin beta-B (Inhbb), an activin subunit and member of the transforming growth factor ß (TGF-ß) superfamily, as a Pdx-1-responsive gene. Overexpression of Inhbb or addition of activin B stimulates rat islet cell and ß-cell proliferation, and the activin receptors RIIA and RIIB are required for the full proliferative effects of Pdx-1 in rat islets. In human islets, Inhbb overexpression stimulates total islet cell proliferation and potentiates Pdx-1-stimulated proliferation of total islet cells and ß cells. In sum, this study identifies a mechanism by which Pdx-1 induces a soluble factor that is sufficient to stimulate both rat and human islet cell proliferation.
Assuntos
Proteínas de Homeodomínio/metabolismo , Subunidades beta de Inibinas/genética , Insulina/genética , Ilhotas Pancreáticas/citologia , Transativadores/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Perfilação da Expressão Gênica , Células Secretoras de Glucagon/citologia , Células Secretoras de Glucagon/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Regiões Promotoras Genéticas , RatosRESUMO
The homeodomain transcription factor Pdx-1 has important roles in pancreatic development and ß-cell function and survival. In the present study, we demonstrate that adenovirus-mediated overexpression of Pdx-1 in rat or human islets also stimulates cell replication. Moreover, cooverexpression of Pdx-1 with another homeodomain transcription factor, Nkx6.1, has an additive effect on proliferation compared to either factor alone, implying discrete activating mechanisms. Consistent with this, Nkx6.1 stimulates mainly ß-cell proliferation, whereas Pdx-1 stimulates both α- and ß-cell proliferation. Furthermore, cyclins D1/D2 are upregulated by Pdx-1 but not by Nkx6.1, and inhibition of cdk4 blocks Pdx-1-stimulated but not Nkx6.1-stimulated islet cell proliferation. Genes regulated by Pdx-1 but not Nkx6.1 were identified by microarray analysis. Two members of the transient receptor potential cation (TRPC) channel family, TRPC3 and TRPC6, are upregulated by Pdx-1 overexpression, and small interfering RNA (siRNA)-mediated knockdown of TRPC3/6 or TRPC6 alone inhibits Pdx-1-induced but not Nkx6.1-induced islet cell proliferation. Pdx-1 also stimulates extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation, an effect partially blocked by knockdown of TRPC3/6, and blockade of ERK1/2 activation with a MEK1/2 inhibitor partially impairs Pdx-1-stimulated proliferation. These studies define a pathway by which overexpression of Pdx-1 activates islet cell proliferation that is distinct from and additive to a pathway activated by Nkx6.1.