RESUMO
Glucagon-like peptide 1 (GLP1), which is mainly processed and cleaved from proglucagon in enteroendocrine cells (EECs) of the intestinal tract, acts on the GLP1 receptor in pancreatic cells to stimulate insulin secretion and to inhibit glucagon secretion. However, GLP1 processing is not fully understood. Here, we show that reticulon 4B (Nogo-B), an endoplasmic reticulum (ER)-resident protein, interacts with the major proglucagon fragment of proglucagon to retain proglucagon on the ER, thereby inhibiting PCSK1-mediated cleavage of proglucagon in the Golgi. Intestinal Nogo-B knockout in male type 2 diabetes mellitus (T2DM) mice increases GLP1 and insulin levels and decreases glucagon levels, thereby alleviating pancreatic injury and insulin resistance. Finally, we identify aberrantly elevated Nogo-B expression and inhibited proglucagon cleavage in EECs from diabetic patients. Our study reveals the subcellular regulatory processes involving Nogo-B during GLP1 production and suggests intestinal Nogo-B as a potential therapeutic target for T2DM.
Assuntos
Diabetes Mellitus Tipo 2 , Retículo Endoplasmático , Peptídeo 1 Semelhante ao Glucagon , Proteínas Nogo , Proglucagon , Pró-Proteína Convertase 1 , Animais , Humanos , Masculino , Camundongos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/genética , Retículo Endoplasmático/metabolismo , Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Complexo de Golgi/metabolismo , Células HEK293 , Insulina/metabolismo , Resistência à Insulina , Intestinos/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nogo/metabolismo , Proteínas Nogo/genética , Proglucagon/metabolismo , Proglucagon/genética , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 1/genética , Ligação Proteica , ProteóliseRESUMO
OBJECTIVES: To study the prevalence and influence on metabolic profile of the prohormone-convertase-1 (PCSK1) N221D variant in childhood obesity, proven its role in the leptin-melanocortin signaling pathway as in proinsulin and other prohormone cleavage. METHODS: Transversal study of 1066 children with obesity (mean age and BMI Z-score 10.38 ± 3.44 years and +4.38 ± 1.77, respectively), 51.4â¯% males, 54.4â¯% prepubertal, 71.5â¯% Caucasians and 20.8â¯% Latinos. Anthropometric and metabolic features were compared between patients carrying the N221D variant in PCSK1 and patients with no variants found after next generation sequencing analysis of 17 genes (CREBBP, CPE, HTR2C, KSR2, LEP, LEPR, MAGEL2, MC3R, MC4R, MRAP2, NCOA1, PCSK1, POMC, SH2B1, SIM1, TBX3 and TUB) involved in the leptin-melanocortin pathway. RESULTS: No variants were found in 531 patients (49.8â¯%), while 68 patients carried the PCSK1 N221D variant (42 isolately, and 26 with at least one additional gene variant). Its prevalence was higher in Caucasians vs. Latinos (χ2 7.81; p<0.01). Patients carrying exclusively the PCSK1 N221D variant (n=42) showed lower insulinemia (p<0.05), HOMA index (p<0.05) and area under the curve for insulin in the oral glucose tolerance test (p<0.001) and higher WBISI (p<0.05) than patients with no variants, despite similar obesity severity, age, sex and ethnic distribution. CONCLUSIONS: The N221D variant in PCSK1 is highly prevalent in childhood obesity, influenced by ethnicity. Indirect estimation of insulin resistance, based on insulinemia could be byassed in these patients and underestimate their type 2 diabetes mellitus risk.
Assuntos
Diabetes Mellitus Tipo 2 , Obesidade Infantil , Masculino , Humanos , Criança , Feminino , Obesidade Infantil/epidemiologia , Obesidade Infantil/genética , Leptina/genética , Leptina/metabolismo , Melanocortinas/metabolismo , Metaboloma , Proteínas , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 1/metabolismoRESUMO
OBJECTIVE: The hypothalamus regulates feeding and glucose homeostasis through the balanced action of different neuropeptides, which are cleaved and activated by the proprotein convertases PC1/3 and PC2. However, the recent association of polymorphisms in the proprotein convertase FURIN with type 2 diabetes, metabolic syndrome, and obesity, prompted us to investigate the role of FURIN in hypothalamic neurons controlling glucose and feeding. METHODS: POMC-Cre+/- mice were bred with Furinfl/fl mice to generate conditional knockout mice with Furin-deletion in neurons expressing proopiomelanocortin (POMCFurKO), and Furinfl/fl mice were used as controls. POMCFurKO and controls were periodically monitored on both normal chow diet and high fat diet (HFD) for body weight and glucose tolerance by established in-vivo procedures. Food intake was measured in HFD-fed FurKO and controls. Hypothalamic Pomc mRNA was measured by RT-qPCR. ELISAs quantified POMC protein and resulting peptides in the hypothalamic extracts of POMCFurKO mice and controls. The in-vitro processing of POMC was studied by biochemical techniques in HEK293T and CHO cell lines lacking FURIN. RESULTS: In control mice, Furin mRNA levels were significantly upregulated on HFD feeding, suggesting an increased demand for FURIN activity in obesogenic conditions. Under these conditions, the POMCFurKO mice were hyperphagic and had increased body weight compared to Furinfl/fl mice. Moreover, protein levels of POMC were elevated and ACTH concentrations markedly reduced. Also, the ratio of α-MSH/POMC was decreased in POMCFurKO mice compared to controls. This indicates that POMC processing was significantly reduced in the hypothalami of POMCFurKO mice, highlighting for the first time the involvement of FURIN in the cleavage of POMC. Importantly, we found that in vitro, the first stage in processing where POMC is cleaved into proACTH was achieved by FURIN but not by PC1/3 or the other proprotein convertases in cell lines lacking a regulated secretory pathway. CONCLUSIONS: These results suggest that FURIN processes POMC into proACTH before sorting into the regulated secretory pathway, challenging the dogma that PC1/3 and PC2 are the only convertases responsible for POMC cleavage. Furthermore, its deletion affects feeding behaviors under obesogenic conditions.
Assuntos
Diabetes Mellitus Tipo 2 , Comportamento Alimentar , Furina , Hipotálamo , Pró-Opiomelanocortina , Animais , Humanos , Camundongos , alfa-MSH/metabolismo , Peso Corporal , Dieta Hiperlipídica/efeitos adversos , Comportamento Alimentar/fisiologia , Furina/genética , Furina/metabolismo , Glucose , Células HEK293 , Hipotálamo/metabolismo , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/genética , Pró-Proteína Convertase 2/metabolismo , Pró-Proteína Convertases/genética , Pró-Proteína Convertases/metabolismo , RNA Mensageiro/metabolismo , Subtilisinas/genética , Subtilisinas/metabolismoRESUMO
Prader−Willi syndrome (PWS) is a complex neurodevelopmental disorder caused by the deletion or inactivation of paternally expressed imprinted genes at the chromosomal region 15q11−q13. The PWS-critical region (PWScr) harbors tandemly repeated non-protein coding IPW-A exons hosting the intronic SNORD116 snoRNA gene array that is predominantly expressed in brain. Paternal deletion of PWScr is associated with key PWS symptoms in humans and growth retardation in mice (PWScr model). Dysregulation of the hypothalamic−pituitary axis (HPA) is thought to be causally involved in the PWS phenotype. Here we performed a comprehensive reverse transcription quantitative PCR (RT-qPCR) analysis across nine different brain regions of wild-type (WT) and PWScr mice to identify stably expressed reference genes. Four methods (Delta Ct, BestKeeper, Normfinder and Genorm) were applied to rank 11 selected reference gene candidates according to their expression stability. The resulting panel consists of the top three most stably expressed genes suitable for gene-expression profiling and comparative transcriptome analysis of WT and/or PWScr mouse brain regions. Using these reference genes, we revealed significant differences in the expression patterns of Igfbp7, Nlgn3 and three HPA associated genes: Pcsk1, Pcsk2 and Nhlh2 across investigated brain regions of wild-type and PWScr mice. Our results raise a reasonable doubt on the involvement of the Snord116 in posttranscriptional regulation of Nlgn3 and Nhlh2 genes. We provide a valuable tool for expression analysis of specific genes across different areas of the mouse brain and for comparative investigation of PWScr mouse models to discover and verify different regulatory pathways affecting this complex disorder.
Assuntos
Síndrome de Prader-Willi , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Encéfalo/metabolismo , Modelos Animais de Doenças , Éxons , Impressão Genômica , Humanos , Camundongos , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/metabolismo , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 1/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismoRESUMO
Melanocortin-4 receptor (MC4R) plays an important role in energy balance regulation and insulin secretion. It has been demonstrated that in the pancreas, it is expressed in islet α and ß cells, wherein it is significantly correlated with insulin and glucagon-like peptide-1 (GLP-1) secretion. However, the molecular mechanism by which it regulates islet function is still unclear. Therefore, in this study, our aim was to clarify the signaling and target genes involved in the regulation of insulin and GLP-1 secretion by islet MC4R. The results obtained showed that in islet cells, the expression of prohormone convertase 1/3 (PC1/3), which is correlated with islet GLP-1 and insulin secretion, increased significantly under the action of the MC4R agonist, NDP-α-MSH, but decreased under the action of the MC4R antagonist, AgRP. Additionally, we observed that to exert their regulatory functions in the islets, cAMP and ß-arrestin-1 acted as important signaling mediators of MC4R, and compared with control islets, the cAMP, PKA, and ß-arrestin-1 levels corresponding to NDP-α-MSH-treated islets were significantly elevated; however, in AgRP-treated islets, their levels decreased significantly. Islets treated with the PKA inhibitor, H89, and the ERK1/2 inhibitor, PD98059, also showed significant decreases in PC1/3 expression level, indicating that the cAMP and ß-arrestin-1 pathways are significantly correlated with PC1/3 expression. These findings suggest that islet MC4R possibly affects PC1/3 expression via the cAMP and ß-arrestin-1 pathways to regulate GLP-1 and insulin secretion. These results provide a new theoretical basis for targeting the molecular mechanism of type 2 diabetes mellitus.
Assuntos
Diabetes Mellitus Tipo 2 , Ilhotas Pancreáticas , Animais , Camundongos , Proteína Relacionada com Agouti/metabolismo , beta-Arrestina 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Peptídeo 1 Semelhante ao Glucagon , Glucose/metabolismo , Insulina/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 1/metabolismo , Receptor Tipo 4 de Melanocortina/metabolismoRESUMO
OBJECTIVE: Prader-Willi syndrome (PWS) is a genomic imprinting disorder predominantly caused by the absence of paternally expressed imprinted genes at chromosome 15q11.2-q13. The PCSK1 gene is vital for the processing of hypothalamic POMC to ACTH and α-MSH, leading to food intake suppression and increased energy expenditure. The aim of this study was to investigate whether our PWS patient had a defect in genes involved in the hypothalamic melanocortin-4 receptor (MC4R) pathway. PATIENTS AND METHODS: A 27-year-old Greek man with PWS presented to the Adult Endocrine Clinic with morbid obesity and hyperphagia. He also had obstructive sleep apnea, growth hormone deficiency, gonadal failure and metabolic disturbances. At 6 years of age, chromosomal testing confirmed PWS with a deletion in the q11q13 region of the long arm of paternal chromosome 15. RESULTS: At the age of 27 years, further genetic testing was conducted, and next generation sequencing revealed a PCSK1_pN221D_HET mutation which was confirmed by Sanger sequencing. CONCLUSIONS: Our findings suggest that different genetic abnormalities may be present in an individual with PWS and that patients with PWS may need to be investigated for PCSK1 mutations, as the finding may potentially offer a novel treatment perspective for them.
Assuntos
Síndrome de Prader-Willi , Adulto , Impressão Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Mutação , Síndrome de Prader-Willi/diagnóstico , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/metabolismo , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 1/metabolismoRESUMO
BACKGROUND: Previously, we reported that Mof was highly expressed in α-cells, and its knockdown led to ameliorated fasting blood glucose (FBG) and glucose tolerance in non-diabetic mice, attributed by reduced total α-cell but enhanced prohormone convertase (PC)1/3-positive α-cell mass. However, how Mof and histone 4 lysine 16 acetylation (H4K16ac) control α-cell and whether Mof inhibition improves glucose handling in type 2 diabetes (T2DM) mice remain unknown. METHODS: Mof overexpression and chromatin immunoprecipitation sequence (ChIP-seq) based on H4K16ac were applied to determine the effect of Mof on α-cell transcriptional factors and underlying mechanism. Then we administrated mg149 to α-TC1-6 cell line, wild type, db/db and diet-induced obesity (DIO) mice to observe the impact of Mof inhibition in vitro and in vivo. In vitro, western blotting and TUNEL staining were used to examine α-cell apoptosis and function. In vivo, glucose tolerance, hormone levels, islet population, α-cell ratio and the co-staining of glucagon and PC1/3 or PC2 were examined. RESULTS: Mof activated α-cell-specific transcriptional network. ChIP-seq results indicated that H4K16ac targeted essential genes regulating α-cell differentiation and function. Mof activity inhibition in vitro caused impaired α-cell function and enhanced apoptosis. In vivo, it contributed to ameliorated glucose intolerance and islet dysfunction, characterized by decreased fasting glucagon and elevated post-challenge insulin levels in T2DM mice. CONCLUSION: Mof regulates α-cell differentiation and function via acetylating H4K16ac and H4K16ac binding to Pax6 and Foxa2 promoters. Mof inhibition may be a potential interventional target for T2DM, which led to decreased α-cell ratio but increased PC1/3-positive α-cells.
Assuntos
Diabetes Mellitus Tipo 2/enzimologia , Diabetes Mellitus Tipo 2/fisiopatologia , Células Secretoras de Glucagon/enzimologia , Células Secretoras de Glucagon/patologia , Intolerância à Glucose/enzimologia , Intolerância à Glucose/fisiopatologia , Histona Acetiltransferases/antagonistas & inibidores , Acetilação/efeitos dos fármacos , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Tipo 2/complicações , Dieta , Redes Reguladoras de Genes/efeitos dos fármacos , Células Secretoras de Glucagon/efeitos dos fármacos , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Lisina/metabolismo , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Pró-Proteína Convertase 1/metabolismo , Salicilatos/farmacologiaRESUMO
OBJECTIVE: The area postrema (AP) and nucleus tractus solitarius (NTS) located in the hindbrain are key nuclei that sense and integrate peripheral nutritional signals and consequently regulate feeding behaviour. While single-cell transcriptomics have been used in mice to reveal the gene expression profile and heterogeneity of key hypothalamic populations, similar in-depth studies have not yet been performed in the hindbrain. METHODS: Using single-nucleus RNA sequencing, we provide a detailed survey of 16,034 cells within the AP and NTS of mice in the fed and fasted states. RESULTS: Of these, 8,910 were neurons that group into 30 clusters, with 4,289 from mice fed ad libitum and 4,621 from overnight fasted mice. A total of 7,124 nuclei were from non-neuronal cells, including oligodendrocytes, astrocytes, and microglia. Interestingly, we identified that the oligodendrocyte population was particularly transcriptionally sensitive to an overnight fast. The receptors GLP1R, GIPR, GFRAL, and CALCR, which bind GLP1, GIP, GDF15, and amylin, respectively, are all expressed in the hindbrain and are major targets for anti-obesity therapeutics. We characterise the transcriptomes of these four populations and show that their gene expression profiles are not dramatically altered by an overnight fast. Notably, we find that roughly half of cells that express GIPR are oligodendrocytes. Additionally, we profile POMC-expressing neurons within the hindbrain and demonstrate that 84% of POMC neurons express either PCSK1, PSCK2, or both, implying that melanocortin peptides are likely produced by these neurons. CONCLUSION: We provide a detailed single-cell level characterisation of AP and NTS cells expressing receptors for key anti-obesity drugs that are either already approved for human use or in clinical trials. This resource will help delineate the mechanisms underlying the effectiveness of these compounds and also prove useful in the continued search for other novel therapeutic targets.
Assuntos
Ingestão de Alimentos , Jejum , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 2/genética , Rombencéfalo/metabolismo , Animais , Área Postrema/metabolismo , Comportamento Alimentar , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/metabolismo , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/metabolismo , Análise de Sequência de RNA , Núcleo Solitário/metabolismoRESUMO
Glucose-mediated signaling regulates the expression of a limited number of genes in human pancreatic ß-cells at the transcriptional level. However, it is unclear whether glucose plays a role in posttranscriptional RNA processing or translational control of gene expression. Here, we asked whether glucose affects posttranscriptional steps and regulates protein synthesis in human ß-cell lines. We first showed the involvement of the mTOR pathway in glucose-related signaling. We also used the surface sensing of translation technique, based on puromycin incorporation into newly translated proteins, to demonstrate that glucose treatment increased protein translation. Among the list of glucose-induced proteins, we identified the proconvertase PCSK1, an enzyme involved in the proteolytic conversion of proinsulin to insulin, whose translation was induced within minutes following glucose treatment. We finally performed global proteomic analysis by mass spectrometry to characterize newly translated proteins upon glucose treatment. We found enrichment in proteins involved in translation, glycolysis, TCA metabolism, and insulin secretion. Taken together, our study demonstrates that, although glucose minorly affects gene transcription in human ß-cells, it plays a major role at the translational level.
Assuntos
Metabolismo Energético/genética , Glucose/farmacologia , Secreção de Insulina/genética , Células Secretoras de Insulina/metabolismo , Biossíntese de Proteínas/genética , Linhagem Celular , Subunidade RIIalfa da Proteína Quinase Dependente de AMP Cíclico/metabolismo , Metabolismo Energético/efeitos dos fármacos , Humanos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Pró-Proteína Convertase 1/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismoRESUMO
Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates glucose-stimulated insulin secretion. GLP-1 is classically produced by gut L cells; however, under certain circumstances α cells can express the prohormone convertase required for proglucagon processing to GLP-1, prohormone convertase 1/3 (PC1/3), and can produce GLP-1. However, the mechanisms through which this occurs are poorly defined. Understanding the mechanisms by which α cell PC1/3 expression can be activated may reveal new targets for diabetes treatment. Here, we demonstrate that the GLP-1 receptor (GLP-1R) agonist, liraglutide, increased α cell GLP-1 expression in a ß cell GLP-1R-dependent manner. We demonstrate that this effect of liraglutide was translationally relevant in human islets through application of a new scRNA-seq technology, DART-Seq. We found that the effect of liraglutide to increase α cell PC1/3 mRNA expression occurred in a subcluster of α cells and was associated with increased expression of other ß cell-like genes, which we confirmed by IHC. Finally, we found that the effect of liraglutide to increase bihormonal insulin+ glucagon+ cells was mediated by the ß cell GLP-1R in mice. Together, our data validate a high-sensitivity method for scRNA-seq in human islets and identify a potentially novel GLP-1-mediated pathway regulating human α cell function.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células Secretoras de Glucagon/metabolismo , Células Secretoras de Insulina/metabolismo , Pró-Proteína Convertase 1/metabolismo , Animais , Feminino , Técnicas de Silenciamento de Genes , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/deficiência , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Células Secretoras de Glucagon/efeitos dos fármacos , Humanos , Hipoglicemiantes/farmacologia , Técnicas In Vitro , Células Secretoras de Insulina/efeitos dos fármacos , Liraglutida/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA-Seq , Transdução de SinaisRESUMO
The C2 domain containing protein extended synaptotagmin (E-Syt) plays important roles in both lipid homeostasis and the intracellular signaling; however, its role in physiology remains largely unknown. Here, we show that hypothalamic E-Syt3 plays a critical role in diet-induced obesity (DIO). E-Syt3 is characteristically expressed in the hypothalamic nuclei. Whole-body or proopiomelanocortin (POMC) neuron-specific ablation of E-Syt3 ameliorated DIO and related comorbidities, including glucose intolerance and dyslipidemia. Conversely, overexpression of E-Syt3 in the arcuate nucleus moderately promoted food intake and impaired energy expenditure, leading to increased weight gain. Mechanistically, E-Syt3 ablation led to increased processing of POMC to α-melanocyte-stimulating hormone (α-MSH), increased activities of protein kinase C and activator protein-1, and enhanced expression of prohormone convertases. These findings reveal a previously unappreciated role for hypothalamic E-Syt3 in DIO and related metabolic disorders.
Assuntos
Regulação da Expressão Gênica/fisiologia , Obesidade/induzido quimicamente , Obesidade/genética , Sinaptotagminas/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Predisposição Genética para Doença , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/genética , Pró-Proteína Convertase 2/metabolismo , Sinaptotagminas/genéticaRESUMO
BACKGROUND: Clinical trials and animal studies have shown that sodium-glucose co-transporter type 2 (SGLT2) inhibitors improve pancreatic beta cell function. Our study aimed to investigate the effect of dapagliflozin on islet morphology and cell phenotype, and explore the origin and possible reason of the regenerated beta cells. METHODS: Two diabetic mouse models, db/db mice and pancreatic alpha cell lineage-tracing (glucagon-ß-gal) mice whose diabetes was induced by high fat diet combined with streptozotocin, were used. Mice were treated by daily intragastric administration of dapagliflozin (1â¯mg/kg) or vehicle for 6â¯weeks. The plasma insulin, glucagon and glucagon-like peptide-1 (GLP-1) were determined by using ELISA. The evaluation of islet morphology and cell phenotype was performed with immunofluorescence. Primary rodent islets and αTC1.9, a mouse alpha cell line, were incubated with dapagliflozin (0.25-25⯵mol/L) or vehicle in the presence or absence of GLP-1 receptor antagonist for 24â¯h in regular or high glucose medium. The expression of specific markers and hormone levels were determined. RESULTS: Treatment with dapagliflozin significantly decreased blood glucose in the two diabetic models and upregulated plasma insulin and GLP-1 levels in db/db mice. The dapagliflozin treatment increased islet and beta cell numbers in the two diabetic mice. The beta cell proliferation as indicated by C-peptide and BrdU double-positive cells was boosted by dapagliflozin. The alpha to beta cell conversion, as evaluated by glucagon and insulin double-positive cells and confirmed by using alpha cell lineage-tracing, was facilitated by dapagliflozin. After the dapagliflozin treatment, some insulin-positive cells were located in the duct compartment or even co-localized with duct cell markers, suggestive of duct-derived beta cell neogenesis. In cultured primary rodent islets and αTC1.9 cells, dapagliflozin upregulated the expression of pancreatic endocrine progenitor and beta cell specific markers (including Pdx1) under high glucose condition. Moreover, dapagliflozin upregulated the expression of Pcsk1 (which encodes prohormone convertase 1/3, an important enzyme for processing proglucagon to GLP-1), and increased GLP-1 content and secretion in αTC1.9 cells. Importantly, the dapagliflozin-induced upregulation of Pdx1 expression was attenuated by GLP-1 receptor antagonist. CONCLUSIONS: Except for glucose-lowering effect, dapagliflozin has extra protective effects on beta cells in type 2 diabetes. Dapagliflozin enhances beta cell self-replication, induces alpha to beta cell conversion, and promotes duct-derived beta cell neogenesis. The promoting effects of dapagliflozin on beta cell regeneration may be partially mediated via GLP-1 secreted from alpha cells.
Assuntos
Compostos Benzidrílicos/farmacologia , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Células Endócrinas/efeitos dos fármacos , Células Secretoras de Glucagon/efeitos dos fármacos , Glucosídeos/farmacologia , Regeneração/efeitos dos fármacos , Animais , Glicemia/metabolismo , Peptídeo C/metabolismo , Modelos Animais de Doenças , Células Endócrinas/metabolismo , Glucagon/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Masculino , Camundongos , Pró-Proteína Convertase 1/metabolismo , Inibidores do Transportador 2 de Sódio-GlicoseRESUMO
BACKGROUND: Males absent on the first (Mof) is implicated in gene control of diverse biological processes, such as cell growth, differentiation, apoptosis and autophagy. However, the relationship between glucose regulation and Mof-mediated transcription events remains unexplored. We aimed to unravel the role of Mof in glucose regulation by using global and pancreatic α-cell-specific Mof-deficient mice in vivo and α-TC1-6 cell line in vitro. METHODS: We used tamoxifen-induced temporal Mof-deficient mice first to show Mof regulate glucose homeostasis, islet cell proportions and hormone secretion. Then we used α-cell-specific Mof-deficient mice to clarify how α-cell subsets and ß-cell mass were regulated and corresponding hormone level alterations. Ultimately, we used small interfering RNA (siRNA) to knockdown Mof in α-TC1-6 and unravel the mechanism regulating α-cell mass and glucagon secretion. RESULTS: Mof was mainly expressed in α-cells. Global Mof deficiency led to lower glucose levels, attributed by decreased α/ß-cell ratio and glucagon secretion. α-cell-specific Mof-deficient mice exhibited similar alterations, with more reduced prohormone convertase 2 (PC2)-positive α-cell mass, responsible for less glucagon, and enhanced prohormone convertase 1 (PC1/3)-positive α-cell mass, leading to more glucagon-like peptide-1 (GLP-1) secretion, thus increased ß-cell mass and insulin secretion. In vitro, increased DNA damage, dysregulated autophagy, enhanced apoptosis and altered cell fate factors expressions upon Mof knockdown were observed. Genes and pathways linked to impaired glucagon secretion were uncovered through transcriptome sequencing. CONCLUSION: Mof is a potential interventional target for glucose regulation, from the aspects of both α-cell subset mass and glucagon, intra-islet GLP-1 secretion. Upon Mof deficiency, Up-regulated PC1/3 but down-regulated PC2-positive α-cell mass, leads to more GLP-1 and insulin but less glucagon secretion, and contributed to lower glucose level.
Assuntos
Glicemia/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Células Secretoras de Glucagon/citologia , Glucagon/metabolismo , Histona Acetiltransferases/fisiologia , Homeostase , Animais , Linhagem Celular , Histona Acetiltransferases/deficiência , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/metabolismoRESUMO
Transcription factor cAMP responsive element-binding protein 3 like 1 (Creb3l1) is a non-classical endoplasmic reticulum stress molecule that is emerging as an important component for cellular homeostasis, particularly within cell types with high peptide secretory capabilities. We have previously shown that Creb3l1 serves an important role in body fluid homeostasis through its transcriptional control of the gene coding for antidiuretic hormone arginine vasopressin in the neuropeptide-rich magnocellular neurones of the supraoptic nucleus. In response to osmotic stimuli such as dehydration, vasopressin magnocellular neurones undergo remarkable transcriptome changes, including increased Creb3l1 expression, to ensure that the supply of vasopressin meets demand. To determine where else Creb3l1 fits into the secretory cell supply chain, we performed RNA-sequencing of Creb3l1 knockdown anterior pituitary mouse corticotroph cell line AtT20. The target chosen for further investigation was Pcsk1, which encodes proprotein convertase enzyme 1 (PC1/3). PC1/3 is crucial for processing of neuropeptides and peptide hormones such as pro-opiomelanocortin (POMC), proinsulin, proglucagon, vasopressin and oxytocin. Viral manipulations in supraoptic nuclei by over-expression of Creb3l1 increased Pcsk1, whereas Creb3l1 knockdown decreased Pcsk1 expression. In vitro promoter activity and binding studies showed that Creb3l1 was a transcription factor of the Pcsk1 gene binding directly to a G-box motif in the promoter. In the dehydrated rat anterior pituitary, Creb3l1 and Pcsk1 expression decreased in parallel compared to control, supporting our findings from manipulations in AtT20 cells and the supraoptic nucleus. No relationship was observed between Creb3l1 and Pcsk1 expression in the neurointermediate lobe of the pituitary, indicating a different mechanism of PC1/3 synthesis by these POMC-synthesising cells. Therefore, Creb3l1, by regulating the expression of Pcsk1, does not control the processing of POMC peptides in the intermediate lobe.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Pressão Osmótica/fisiologia , Hipófise/metabolismo , Pró-Proteína Convertase 1/metabolismo , Núcleo Supraóptico/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Masculino , Camundongos , Pró-Opiomelanocortina/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Glucose mediated insulin biosynthesis is tightly regulated and shared between insulin granule proteins such as its processing enzymes, prohormone convertases, PC1/3 and PC2. However, the molecular players involved in the co-ordinated translation remain elusive. The trans-acting factors like PABP (Poly A Binding Protein) and PDI (Protein Disulphide Isomerize) binds to a conserved sequence in the 5'UTR of insulin mRNA and regulates its translation. Here, we demonstrate that 5'UTR of PC1/3 and PC2 also associate with PDI and PABP. We show that a' and RRM 3-4 domains of PDI and PABP respectively, are necessary for RNA binding activity to the 5'UTRs of insulin and its processing enzymes.
Assuntos
Insulina/metabolismo , Proteínas de Ligação a Poli(A)/metabolismo , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/metabolismo , Biossíntese de Proteínas , Isomerases de Dissulfetos de Proteínas/metabolismo , Regiões 5' não Traduzidas , Animais , Linhagem Celular , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/metabolismo , Insulina/genética , Camundongos , Proteínas de Ligação a Poli(A)/genética , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 2/genética , Isomerases de Dissulfetos de Proteínas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Glucagon-like peptide-1 (GLP-1) is an important signal in the peripheral and neural systems, which contributes to the maintenance of glucose and energy homeostasis. In this study, 1H NMR validated polyphenols and polysaccharides extracted from sprouted quinoa yoghurt were used as isolates and conjugates to upregulate the stimulation of GLP-1 release in NCI-H716 cells. In addition, we explored their effect on proglucagon and prohormone convertase 3 mRNA expressions, HNF-3γ and CCK-2R gene protein expression, as well as cytosolic calcium release. Variations in concentration showed a dose-dependent GLP-1 stimulation, and were significantly optimized by germination. Proglucagon mRNA expression in NCI-H716 cells was upregulated, and was relatively highest with QYPSP1 treatments in a 2.68 fold. The results suggested that the conjugates had greater potential to stimulate GLP-1 release than their isolates. Sprouted quinoa yoghurt could therefore be a potential functional food useful to regulate glucose and energy homeostasis.
Assuntos
Chenopodium quinoa/química , Inibidores da Dipeptidil Peptidase IV/química , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Polifenóis/química , Polissacarídeos/química , Iogurte/análise , Cálcio/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Chenopodium quinoa/crescimento & desenvolvimento , Chenopodium quinoa/metabolismo , Cromatografia Líquida de Alta Pressão , Inibidores da Dipeptidil Peptidase IV/metabolismo , Inibidores da Dipeptidil Peptidase IV/farmacologia , Germinação , Humanos , Polifenóis/análise , Polifenóis/isolamento & purificação , Polissacarídeos/análise , Polissacarídeos/isolamento & purificação , Proglucagon/antagonistas & inibidores , Proglucagon/genética , Proglucagon/metabolismo , Pró-Proteína Convertase 1/antagonistas & inibidores , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 1/metabolismoRESUMO
The proprotein convertase subtilisin/Kexin type 1 (PCSK1/PC1) protein processes inactive pro-hormone precursors into biologically active hormones in a number of neuroendocrine and endocrine cell types. Patients with recessive mutations in PCSK1 exhibit a complex spectrum of traits including obesity, diarrhoea and endocrine disorders. We describe here a new mouse model with a point mutation in the Pcsk1 gene that exhibits obesity, hyperphagia, transient diarrhoea and hyperproinsulinaemia, phenotypes consistent with human patient traits. The mutation results in a pV96L amino acid substitution and changes the first nucleotide of mouse exon 3 leading to skipping of that exon and in homozygotes very little full-length transcript. Overexpression of the exon 3 deleted protein or the 96L protein results in ER retention in Neuro2a cells. This is the second Pcsk1 mouse model to display obesity phenotypes, contrasting knockout mouse alleles. This model will be useful in investigating the basis of endocrine disease resulting from prohormone processing defects.
Assuntos
Diabetes Mellitus/genética , Obesidade/genética , Pró-Proteína Convertase 1/genética , Alelos , Animais , Linhagem Celular , Diarreia/genética , Retículo Endoplasmático/metabolismo , Éxons , Feminino , Intolerância à Glucose/genética , Homozigoto , Hiperfagia/genética , Masculino , Camundongos , Mutação , Pró-Proteína Convertase 1/metabolismo , Splicing de RNARESUMO
Common mutations in the human prohormone convertase (PC)1/3 gene (PCKSI) are linked to increased risk of obesity. Previous work has shown that the rs6232 single-nucleotide polymorphism (N221D) results in slightly decreased activity, although whether this decrease underlies obesity risk is not clear. We observed significantly decreased activity of the N221D PC1/3 enzyme at the pH of the trans-Golgi network; at this pH, the mutant enzyme was less stable than wild-type enzyme. Recombinant N221D PC1/3 also showed enhanced susceptibility to heat stress. Enhanced susceptibility to tunicamycin-induced endoplasmic reticulum stress was observed in AtT-20/PC2 cell clones in which murine PC1/3 was replaced by human N221D PC1/3, as compared with wild-type human PC1/3. However, N221D PC1/3-expressing AtT-20/PC2 clones processed proopiomelanocortin to α-MSH similarly to wild-type PC1/3. We also generated a CRISPR-edited mouse line expressing the N221D mutation in the PCKSI gene. When homozygous N221D mice were fed either a standard or a high-fat diet, we found no increase in body weight compared with their wild-type sibling controls. Sexual dimorphism was observed in pituitary ACTH for both genotypes, with females exhibiting lower levels of pituitary ACTH. In contrast, hypothalamic α-MSH content for both genotypes was higher in females compared with males. Hypothalamic corticotropin-like intermediate peptide content was higher in wild-type females compared with wild-type, but not N221D, males. Taken together, these data suggest that the increased obesity risk linked to the N221D allele in humans may be due in part to PC1/3-induced loss of resilience to stressors rather than strictly to decreased enzymatic activity on peptide precursors.
Assuntos
Obesidade/genética , Pró-Proteína Convertase 1/metabolismo , Animais , Estresse do Retículo Endoplasmático , Estabilidade Enzimática , Feminino , Intolerância à Glucose , Humanos , Concentração de Íons de Hidrogênio , Hipotálamo/metabolismo , Masculino , Camundongos , Neuropeptídeo Y/metabolismo , Hipófise/metabolismo , Polimorfismo de Nucleotídeo Único , Pró-Opiomelanocortina/metabolismo , Pró-Proteína Convertase 1/genética , Caracteres Sexuais , alfa-MSH/metabolismoRESUMO
Metabolic reprogramming is a feature of cancer cells and crucial for tumor growth and metastasis. Interferon-γ (IFNγ) is a cytokine that plays a pivotal role in host antitumor immunity. However, little is known about the roles of metabolic reprogramming in immune responses. Here, we show that colon cancer cells reprogram metabolism to coordinate proper cellular responses to IFNγ by downregulating mitochondrial pyruvate carrier (MPC)1 and 2 via STAT3 signaling. Forced overexpression of MPC promote the production of reactive oxygen species and enhance the apoptosis induced by IFNγ in colon cancer cells. Moreover, inhibiting STAT3 sensitize the antitumor efficacy of IFN-γ against colon cancer cells. Our findings present a previously unrecognized mechanism that colon cancer manipulate to resist IFNγ mediated antitumor immunity that have implications for targeting a unique aspect of this disease.
Assuntos
Neoplasias do Colo/metabolismo , Interferon gama/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Piruvatos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Transporte Biológico , Linhagem Celular Tumoral , Neoplasias do Colo/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Camundongos , Mitocôndrias/genética , Modelos Biológicos , Oxirredução , Pró-Proteína Convertase 1/genética , Pró-Proteína Convertase 1/metabolismo , Pró-Proteína Convertase 2/genética , Pró-Proteína Convertase 2/metabolismoRESUMO
BACKGROUND Recent studies have illustrated that the transcription co-repressor, C-terminal binding protein 1 (CtBP1), links the metabolic alterations to transcription controls in proliferation, EMT, genome stability, metabolism, and lifespan, but whether CtBP1 affects the cellular redox homeostasis is unexplored. This study was designed to investigate the mechanism of CtBP1-mediated transcription repression that contributes to the metabolic reprogramming. MATERIAL AND METHODS Knockdown of CtBP1 in both mouse MEF cells and human melanoma cells changed cell redox homeostasis. Further, chromatin immunoprecipitation (ChIP) and luciferase reporter assay were performed for identification of CtBP1 downstream targets, pyruvate carrier 1 and 2 genes (MPC1 and MPC2), which contribute to redox homeostasis and are transcriptionally regulated by CtBP1. Moreover, blockage of the cellular NADH level with the glycolysis inhibitor 2-Deoxy-D-Glucose (2-DG) rescued MPC1 and MPC2 expression. MTT assay and scratch assay were performed to investigate the effect of MPC1 and MPC2 expression on malignant properties of melanoma cells. RESULTS The data demonstrated that CtBP1 directly bound to the promoters of MPC1 and MPC2 and transcriptionally repressed them, leading to increased levels of free NADH in the cytosol and nucleus, thus positively feeding back CtBP1's functions. Consequently, restoring MPC1 and MPC2 in human tumor cells decreases free NADH and inhibits melanoma cell proliferation and migration. CONCLUSIONS Our data indicate that MPC1 and MPC2 are principal mediators that link CtBP1-mediated transcription regulation to NADH production. The discovery of CtBP1 as an NADH regulator in addition to being an NADH sensor shows that CtBP1 is at the center of tumor metabolism and transcription control.