RESUMO
Clec16a has been identified as a disease susceptibility gene for type 1 diabetes, multiple sclerosis, and adrenal dysfunction, but its function is unknown. Here we report that Clec16a is a membrane-associated endosomal protein that interacts with E3 ubiquitin ligase Nrdp1. Loss of Clec16a leads to an increase in the Nrdp1 target Parkin, a master regulator of mitophagy. Islets from mice with pancreas-specific deletion of Clec16a have abnormal mitochondria with reduced oxygen consumption and ATP concentration, both of which are required for normal ß cell function. Indeed, pancreatic Clec16a is required for normal glucose-stimulated insulin release. Moreover, patients harboring a diabetogenic SNP in the Clec16a gene have reduced islet Clec16a expression and reduced insulin secretion. Thus, Clec16a controls ß cell function and prevents diabetes by controlling mitophagy. This pathway could be targeted for prevention and control of diabetes and may extend to the pathogenesis of other Clec16a- and Parkin-associated diseases.
Assuntos
Diabetes Mellitus Tipo 1/genética , Ilhotas Pancreáticas/patologia , Lectinas Tipo C/metabolismo , Mitofagia , Proteínas de Transporte de Monossacarídeos/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Transporte/química , Diabetes Mellitus Tipo 1/patologia , Predisposição Genética para Doença , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Lectinas Tipo C/química , Lectinas Tipo C/genética , Lisossomos/química , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Dados de Sequência Molecular , Proteínas de Transporte de Monossacarídeos/química , Proteínas de Transporte de Monossacarídeos/genética , Polimorfismo de Nucleotídeo Único , Ubiquitina-Proteína LigasesRESUMO
The stress response and cell survival are necessary for normal pancreatic ß-cell function, glucose homeostasis, and prevention of diabetes. The homeodomain transcription factor and human diabetes gene pancreas/duodenum homeobox protein 1 (Pdx1) regulates ß-cell survival and endoplasmic reticulum stress susceptibility, in part through direct regulation of activating transcription factor 4 (Atf4). Here we show that Atf5, a close but less-studied relative of Atf4, is also a target of Pdx1 and is critical for ß-cell survival under stress conditions. Pdx1 deficiency led to decreased Atf5 transcript, and primary islet ChIP-sequencing localized PDX1 to the Atf5 promoter, implicating Atf5 as a PDX1 target. Atf5 expression was stress inducible and enriched in ß cells. Importantly, Atf5 deficiency decreased survival under stress conditions. Loss-of-function and chromatin occupancy experiments positioned Atf5 downstream of and parallel to Atf4 in the regulation of eIF4E-binding protein 1 (4ebp1), a mammalian target of rapamycin (mTOR) pathway component that inhibits protein translation. Accordingly, Atf5 deficiency attenuated stress suppression of global translation, likely enhancing the susceptibility of ß cells to stress-induced apoptosis. Thus, we identify ATF5 as a member of the transcriptional network governing pancreatic ß-cell survival during stress.
Assuntos
Fatores Ativadores da Transcrição/genética , Apoptose/genética , Estresse do Retículo Endoplasmático/genética , Células Secretoras de Insulina/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Fatores Ativadores da Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Animais , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Células Cultivadas , Fatores de Iniciação em Eucariotos , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Regiões Promotoras Genéticas/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
Type 2 diabetes mellitus (T2DM) results from pancreatic beta cell failure in the setting of insulin resistance. Heterozygous mutations in the gene encoding the beta cell transcription factor pancreatic duodenal homeobox 1 (Pdx1) are associated with both T2DM and maturity onset diabetes of the young (MODY4), and low levels of Pdx1 accompany beta cell dysfunction in experimental models of glucotoxicity and diabetes. Here, we find that Pdx1 is required for compensatory beta cell mass expansion in response to diet-induced insulin resistance through its roles in promoting beta cell survival and compensatory hypertrophy. Pdx1-deficient beta cells show evidence of endoplasmic reticulum (ER) stress both in the complex metabolic milieu of high-fat feeding as well as in the setting of acutely reduced Pdx1 expression in the Min6 mouse insulinoma cell line. Further, Pdx1 deficiency enhances beta cell susceptibility to ER stress-associated apoptosis. The results of high throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates a broad array of genes involved in diverse functions of the ER, including proper disulfide bond formation, protein folding, and the unfolded protein response. These findings suggest that Pdx1 deficiency leads to a failure of beta cell compensation for insulin resistance at least in part by impairing critical functions of the ER.
Assuntos
Apoptose/genética , Diabetes Mellitus Tipo 2/metabolismo , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica/genética , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/metabolismo , Transativadores/metabolismo , Animais , Crescimento Celular , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Inativação Gênica , Proteínas de Homeodomínio/genética , Resistência à Insulina/fisiologia , Camundongos , Camundongos Transgênicos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Transativadores/genéticaRESUMO
The calcium-regulated phosphatase calcineurin intersects with both calcium and cAMP-mediated signaling pathways in the pancreatic ß-cell. Pharmacologic calcineurin inhibition, necessary to prevent rejection in the setting of organ transplantation, is associated with post-transplant ß-cell failure. We sought to determine the effect of calcineurin inhibition on ß-cell replication and survival in rodents and in isolated human islets. Further, we assessed whether the GLP-1 receptor agonist and cAMP stimulus, exendin-4 (Ex-4), could rescue ß-cell replication and survival following calcineurin inhibition. Following treatment with the calcineurin inhibitor tacrolimus, human ß-cell apoptosis was significantly increased. Although we detected no human ß-cell replication, tacrolimus significantly decreased rodent ß-cell replication. Ex-4 nearly normalized both human ß-cell survival and rodent ß-cell replication when co-administered with tacrolimus. We found that tacrolimus decreased Akt phosphorylation, suggesting that calcineurin could regulate replication and survival via the PI3K/Akt pathway. We identify insulin receptor substrate-2 (Irs2), a known cAMP-responsive element-binding protein target and upstream regulator of the PI3K/Akt pathway, as a novel calcineurin target in ß-cells. Irs2 mRNA and protein are decreased by calcineurin inhibition in both rodent and human islets. The effect of calcineurin on Irs2 expression is mediated at least in part through the nuclear factor of activated T-cells (NFAT), as NFAT occupied the Irs2 promoter in a calcineurin-sensitive manner. Ex-4 restored Irs2 expression in tacrolimus-treated rodent and human islets nearly to baseline. These findings reveal calcineurin as a regulator of human ß-cell survival in part through regulation of Irs2, with implications for the pathogenesis and treatment of diabetes following organ transplantation.
Assuntos
Calcineurina/farmacologia , Proliferação de Células/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Calcineurina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , AMP Cíclico/metabolismo , Diabetes Mellitus/etiologia , Diabetes Mellitus/metabolismo , Diabetes Mellitus/patologia , Exenatida , Regulação da Expressão Gênica/efeitos dos fármacos , Receptor do Peptídeo Semelhante ao Glucagon 1 , Humanos , Hipoglicemiantes/farmacologia , Imunossupressores/farmacologia , Proteínas Substratos do Receptor de Insulina/metabolismo , Células Secretoras de Insulina/patologia , Camundongos , Fatores de Transcrição NFATC/metabolismo , Transplante de Órgãos/efeitos adversos , Peptídeos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Glucagon/agonistas , Receptores de Glucagon/metabolismo , Tacrolimo/farmacologia , Peçonhas/farmacologiaRESUMO
OBJECTIVE: Adult obesity risk is influenced by alterations to fetal and neonatal environments. Modifying neonatal gut or neurohormone signaling pathways can have negative metabolic consequences in adulthood. Here we characterize the effect of neonatal activation of glucagon like peptide-1 (GLP-1) receptor (GLP1R) signaling on adult adiposity and metabolism. METHODS: Wild type C57BL/6 mice were injected with 1 nmol/kg Exendin-4 (Ex-4), a GLP1R agonist, for 6 consecutive days after birth. Growth, body composition, serum analysis, energy expenditure, food intake, and brain and fat pad histology and gene expression were assessed at multiple time points through 42 weeks. Similar analyses were conducted in a Glp1r conditional allele crossed with a Sim1Cre deleter strain to produce Sim1Cre;Glp1rloxP/loxP mice and control littermates. RESULTS: Neonatal administration of Ex-4 reduced adult body weight and fat mass, increased energy expenditure, and conferred protection from diet-induced obesity in female mice. This was associated with induction of brown adipose genes and increased noradrenergic fiber density in parametrial white adipose tissue (WAT). We further observed durable alterations in orexigenic and anorexigenic projections to the paraventricular hypothalamic nucleus (PVH). Genetic deletion of Glp1r in the PVH by Sim1-Cre abrogated the impact of neonatal Ex-4 on adult body weight, WAT browning, and hypothalamic architecture. CONCLUSION: These observations suggest that the acute activation of GLP1R in neonates durably alters hypothalamic architecture to limit adult weight gain and adiposity, identifying GLP1R as a therapeutic target for obesity prevention.
Assuntos
Adiposidade , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Hipotálamo/crescimento & desenvolvimento , Animais , Exenatida , Feminino , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Hipotálamo/citologia , Incretinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Orexinas/genética , Orexinas/metabolismo , Peptídeos/farmacologia , Peçonhas/farmacologiaRESUMO
The recognition of ß cell dedifferentiation in type 2 diabetes raises the translational relevance of mechanisms that direct and maintain ß cell identity. LIM domain-binding protein 1 (LDB1) nucleates multimeric transcriptional complexes and establishes promoter-enhancer looping, thereby directing fate assignment and maturation of progenitor populations. Many terminally differentiated endocrine cell types, however, remain enriched for LDB1, but its role is unknown. Here, we have demonstrated a requirement for LDB1 in maintaining the terminally differentiated status of pancreatic ß cells. Inducible ablation of LDB1 in mature ß cells impaired insulin secretion and glucose homeostasis. Transcriptomic analysis of LDB1-depleted ß cells revealed the collapse of the terminally differentiated gene program, indicated by a loss of ß cell identity genes and induction of the endocrine progenitor factor neurogenin 3 (NEUROG3). Lineage tracing confirmed that LDB1-depleted, insulin-negative ß cells express NEUROG3 but do not adopt alternate endocrine cell fates. In primary mouse islets, LDB1 and its LIM homeodomain-binding partner islet 1 (ISL1) were coenriched at chromatin sites occupied by pancreatic and duodenal homeobox 1 (PDX1), NK6 homeobox 1 (NKX6.1), forkhead box A2 (FOXA2), and NK2 homeobox 2 (NKX2.2) - factors that co-occupy active enhancers in 3D chromatin domains in human islets. Indeed, LDB1 was enriched at active enhancers in human islets. Thus, LDB1 maintains the terminally differentiated state of ß cells and is a component of active enhancers in both murine and human islets.
Assuntos
Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas com Domínio LIM/metabolismo , Fatores de Transcrição/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células Secretoras de Insulina/patologia , Proteínas com Domínio LIM/genética , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Proteínas de Peixe-ZebraRESUMO
The homeodomain transcription factor Pdx1 controls pancreas organogenesis, specification of endocrine pancreas progenitors, and the postnatal growth and function of pancreatic ß-cells. Pdx1 expression in human-derived stem cells is used as a marker for induced pancreatic precursor cells. Unfortunately, the differentiation efficiency of human pancreatic progenitors into functional ß-cells is poor. In order to gain insight into the genes that Pdx1 regulates during differentiation, we performed Pdx1 chromatin immunoprecipitation followed by high-throughput sequencing of embryonic day (e) 13.5 and 15.5 mouse pancreata. From this, we identified the transcription factor Teashirt zinc finger 1 (Tshz1) as a direct Pdx1 target. Tshz1 is expressed in developing and adult insulin- and glucagon-positive cells. Endocrine cells are properly specified in Tshz1-null embryos, but critical regulators of ß-cell (Pdx1 and Nkx6.1) and α-cell (MafB and Arx) formation and function are downregulated. Adult Tshz1(+/-) mice display glucose intolerance due to defects in glucose-stimulated insulin secretion associated with reduced Pdx1 and Clec16a expression in Tshz1(+/-) islets. Lastly, we demonstrate that TSHZ1 levels are reduced in human islets of donors with type 2 diabetes. Thus, we position Tshz1 in the transcriptional network of maturing ß-cells and suggest that its dysregulation could contribute to the islet phenotype of human type 2 diabetes.
Assuntos
Diferenciação Celular/genética , Diabetes Mellitus Tipo 2/metabolismo , Células Secretoras de Insulina/metabolismo , Organogênese/genética , Pâncreas/metabolismo , Proteínas Repressoras/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Redes Reguladoras de Genes , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/citologia , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Camundongos , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo , Pâncreas/citologia , Proteínas Repressoras/genética , Transativadores/genética , Transativadores/metabolismoRESUMO
Mitophagy is a critical regulator of mitochondrial quality control and is necessary for elimination of dysfunctional mitochondria to maintain cellular respiration. Here, we report that the homeodomain transcription factor Pdx1, a gene associated with both type 2 diabetes and monogenic diabetes of the young, regulates mitophagy in pancreatic ß-cells. Loss of Pdx1 leads to abnormal mitochondrial morphology and function as well as impaired mitochondrial turnover. High-throughput expression microarray and chromatin occupancy analyses reveal that Pdx1 regulates the expression of Clec16a, a type 1 diabetes gene and itself a key mediator of mitophagy through regulation of the E3 ubiquitin ligase Nrdp1. Indeed, expression of Clec16a and Nrdp1 are both reduced in Pdx1 haploinsufficient islets, and reduction of Pdx1 impairs fusion of autophagosomes containing mitochondria to lysosomes during mitophagy. Importantly, restoration of Clec16a expression after Pdx1 loss of function restores mitochondrial trafficking during mitophagy and improves mitochondrial respiration and glucose-stimulated insulin release. Thus, Pdx1 orchestrates nuclear control of mitochondrial function in part by controlling mitophagy through Clec16a. The novel Pdx1-Clec16a-Nrdp1 pathway we describe provides a genetic basis for the pathogenesis of mitochondrial dysfunction in multiple forms of diabetes that could be targeted for future therapies to improve ß-cell function.
Assuntos
Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/metabolismo , Células Secretoras de Insulina/fisiologia , Lectinas Tipo C/metabolismo , Mitofagia/fisiologia , Proteínas de Transporte de Monossacarídeos/metabolismo , Transativadores/metabolismo , Animais , Proteínas de Transporte/genética , DNA/genética , DNA/metabolismo , Proteínas de Homeodomínio/genética , Humanos , Lectinas Tipo C/genética , Camundongos , Mitocôndrias/fisiologia , Proteínas de Transporte de Monossacarídeos/genética , Análise Serial de Proteínas , RNA/genética , RNA/metabolismo , Transativadores/genética , Ubiquitina-Proteína LigasesRESUMO
OBJECTIVE: The Polycomb Repressive Complexes (PRC) 1 and 2 function to epigenetically repress target genes. The PRC1 component, Bmi1, plays a crucial role in maintenance of glucose homeostasis and beta cell mass through repression of the Ink4a/Arf locus. Here we have explored the role of Bmi1 in regulating glucose homeostasis in the adult animal, which had not been previously reported due to poor postnatal survival of Bmi1 (-/-) mice. METHODS: The metabolic phenotype of Bmi1 (+/-) mice was characterized, both in vivo and ex vivo. Glucose and insulin tolerance tests and hyperinsulinemic-euglycemic clamps were performed. The insulin signaling pathway was assessed at the protein and transcript level. RESULTS: Here we report a negative correlation between Bmi1 levels and insulin sensitivity in two models of insulin resistance, aging and liver-specific insulin receptor deficiency. Further, heterozygous loss of Bmi1 results in increased insulin sensitivity in adult mice, with no impact on body weight or composition. Hyperinsulinemic-euglycemic clamp reveals increased suppression of hepatic glucose production and increased glucose disposal rate, indicating elevated glucose uptake to peripheral tissues, in Bmi1 (+/-) mice. Enhancement of insulin signaling, specifically an increase in Akt phosphorylation, in liver and, to a lesser extent, in muscle appears to contribute to this phenotype. CONCLUSIONS: Together, these data define a new role for Bmi1 in regulating insulin sensitivity via enhancement of Akt phosphorylation.
RESUMO
The homeodomain transcription factor pancreatic duodenal homeobox 1 (Pdx1) is a major mediator of insulin transcription and a key regulator of the ß cell phenotype. Heterozygous mutations in PDX1 are associated with the development of diabetes in humans. Understanding how Pdx1 expression levels are controlled is therefore of intense interest in the study and treatment of diabetes. Pdx1 C terminus-interacting factor-1 (Pcif1, also known as SPOP) is a nuclear protein that inhibits Pdx1 transactivation. Here, we show that Pcif1 targets Pdx1 for ubiquitination and proteasomal degradation. Silencing of Pcif1 increased Pdx1 protein levels in cultured mouse ß cells, and Pcif1 heterozygosity normalized Pdx1 protein levels in Pdx1(+/-) mouse islets, thereby increasing expression of key Pdx1 transcriptional targets. Remarkably, Pcif1 heterozygosity improved glucose homeostasis and ß cell function and normalized ß cell mass in Pdx1(+/-) mice by modulating ß cell survival. These findings indicate that in adult mouse ß cells, Pcif1 limits Pdx1 protein accumulation and thus the expression of insulin and other gene targets important in the maintenance of ß cell mass and function. They also provide evidence that targeting the turnover of a pancreatic transcription factor in vivo can improve glucose homeostasis.