RESUMO
AIMS/HYPOTHESIS: Pancreatic islets depend on cytosolic calcium (Ca2+) to trigger the secretion of glucoregulatory hormones and trigger transcriptional regulation of genes important for islet response to stimuli. To date, there has not been an attempt to profile Ca2+-regulated gene expression in all islet cell types. Our aim was to construct a large single-cell transcriptomic dataset from human islets exposed to conditions that would acutely induce or inhibit intracellular Ca2+ signalling, while preserving biological heterogeneity. METHODS: We exposed intact human islets from three donors to the following conditions: (1) 2.8 mmol/l glucose; (2) 16 mmol/l glucose and 40 mmol/l KCl to maximally stimulate Ca2+ signalling; and (3) 16 mmol/l glucose, 40 mmol/l KCl and 5 mmol/l EGTA (Ca2+ chelator) to inhibit Ca2+ signalling, for 1 h. We sequenced 68,650 cells from all islet cell types, and further subsetted the cells to form an endocrine cell-specific dataset of 59,373 cells expressing INS, GCG, SST or PPY. We compared transcriptomes across conditions to determine the differentially expressed Ca2+-regulated genes in each endocrine cell type, and in each endocrine cell subcluster of alpha and beta cells. RESULTS: Based on the number of Ca2+-regulated genes, we found that each alpha and beta cell cluster had a different magnitude of Ca2+ response. We also showed that polyhormonal clusters expressing both INS and GCG, or both INS and SST, are defined by Ca2+-regulated genes specific to each cluster. Finally, we identified the gene PCDH7 from the beta cell clusters that had the highest number of Ca2+-regulated genes, and showed that cells expressing cell surface PCDH7 protein have enhanced glucose-stimulated insulin secretory function. CONCLUSIONS/INTERPRETATION: Here we use our large-scale, multi-condition, single-cell dataset to show that human islets have cell-type-specific Ca2+-regulated gene expression profiles, some of them specific to subpopulations. In our dataset, we identify PCDH7 as a novel marker of beta cells having an increased number of Ca2+-regulated genes and enhanced insulin secretory function. DATA AVAILABILITY: A searchable and user-friendly format of the data in this study, specifically designed for rapid mining of single-cell RNA sequencing data, is available at https://lynnlab.shinyapps.io/Human_Islet_Atlas/ . The raw data files are available at NCBI Gene Expression Omnibus (GSE196715).
Assuntos
Células Secretoras de Insulina , Ilhotas Pancreáticas , Cálcio/metabolismo , Glucose/metabolismo , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismoRESUMO
Cytosolic calcium influx activates signaling pathways known to support pancreatic beta cell function and survival by modulating gene expression. Impaired calcium signaling leads to decreased beta cell mass and diabetes. To appreciate the causes of these cytotoxic perturbations, a more detailed understanding of the relevant signaling pathways and their respective gene targets is required. In this study, we examined the calcium-induced expression of the cytoprotective beta cell transcription factor Npas4. Pharmacological inhibition implicated the calcineurin, Akt/protein kinase B, and Ca(2+)/calmodulin-dependent protein kinase signaling pathways in the regulation of Npas4 transcription and translation. Both Npas4 mRNA and protein had high turnover rates, and, at the protein level, degradation was mediated via the ubiquitin-proteasome pathway. Finally, beta cell cytotoxicity of the calcineurin inhibitor and immunosuppressant tacrolimus (FK-506) was prevented by Npas4 overexpression. These results delineate the pathways regulating Npas4 expression and stability and demonstrate its importance in clinical settings such as islet transplantation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/biossíntese , Sinalização do Cálcio/efeitos dos fármacos , Citotoxinas/efeitos adversos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Tacrolimo/efeitos adversos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Calcineurina/genética , Calcineurina/metabolismo , Citotoxinas/farmacologia , Células Secretoras de Insulina/patologia , Masculino , Camundongos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Tacrolimo/farmacologia , Ubiquitina/genética , Ubiquitina/metabolismoRESUMO
AIMS/HYPOTHESIS: The sex-determining region Y (SRY)-related high mobility group (HMG) box (SOX) family of transcription factors is essential for normal organismal development. Despite the longstanding knowledge that many SOX family members are expressed during pancreas development, a role for many of these factors in the establishment of insulin-producing beta cell fate remains to be determined. The aim of this study is to elucidate the role of SOX4 during beta cell development. METHODS: We used pancreas and endocrine progenitor mouse knockouts of Sox4 to uncover the roles of SOX4 during pancreas development. Lineage tracing and in vitro models were used to determine how SOX4 regulates beta cell formation and understand the fate of Sox4-null endocrine lineage cells. RESULTS: This study demonstrates a progenitor cell-autonomous role for SOX4 in regulating the genesis of beta cells and shows that it is required at multiple stages of the process. SOX4 deletion in the multipotent pancreatic progenitors resulted in impaired endocrine progenitor cell differentiation. Deletion of SOX4 later in the Neurog3-expressing cells also caused reductions in beta cells. Lineage studies showed loss of Sox4 in endocrine progenitors resulted in a block in terminal islet cell differentiation that was attributed to reduction in the production of key beta cell specification factors. CONCLUSIONS/INTERPRETATION: These results demonstrate that SOX4 is essential for normal endocrine pancreas development both concomitant with, and downstream of, the endocrine fate decision. In conclusion, these studies position Sox4 temporally in the endocrine differentiation programme and provide a new target for improving in vitro differentiation of glucose-responsive pancreatic beta cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Ilhotas Pancreáticas/embriologia , Proteínas do Tecido Nervoso/metabolismo , Organogênese/genética , Fatores de Transcrição SOXC/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Diferenciação Celular/genética , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Fatores de Transcrição SOXC/genéticaRESUMO
Pancreatic ß cells secrete insulin in response to elevated levels of glucose. Stem cell derived ß (SCß) cells aim to replicate this glucose-stimulated insulin secretion (GSIS) function, but current preparations cannot provide the same level of insulin as natural ß cells. Here, we develop an assay to measure GSIS at the single cell level to investigate the functional heterogeneity of SCß cells and donor-derived islet cells. Our assay involves randomly depositing single cells and insulin capture microbeads in open-top nanowells (40 × 40 × 55 µm3) fabricated on glass-bottom imaging microwell plates. Insulin secreted from single cells is captured on microbeads and then stained using a detection antibody. The nanowell microstructure limits diffusion of secreted insulin. The glass substrate provides an optically flat surface for quantitative microscopy to measure the concentration of secreted insulin. We used this approach to measure GSIS from SCß cells and donor-derived islet cells after 15 minutes exposure to 3.3 mM and 16.7 mM glucose. Both cell types exhibited significant GSIS heterogeneity, where elite cells (<20%) produced the majority of the secreted insulin (55-78%). This assay provides an immediate readout of single cell glucose-stimulated insulin secretion in a flexible well plate-based format.
RESUMO
OBJECTIVE: Human embryonic stem cell (hESC; SC)-derived pancreatic ß cells can be used to study diabetes pathologies and develop cell replacement therapies. Although current differentiation protocols yield SCß cells with varying degrees of maturation, these cells still differ from deceased donor human ß cells in several respects. We sought to develop a reporter cell line that could be used to dynamically track SCß cell functional maturation. METHODS: To monitor SCß cell maturation in vitro, we created an IAPP-2A-mScar and INSULIN-2A-EGFP dual fluorescent reporter (INS2A-EGFP/+;IAPP2A-mScarlet/+) hESC line using CRISPR/Cas9. Pluripotent SC were then differentiated using a 7-stage protocol to islet-like cells. Immunohistochemistry, flow cytometry, qPCR, GSIS and electrophysiology were used to characterise resulting cell populations. RESULTS: We observed robust expression of EGFP and mScarlet fluorescent proteins in insulin- and IAPP-expressing cells without any compromise to their differentiation. We show that the proportion of insulin-producing cells expressing IAPP increases over a 4-week maturation period, and that a subset of insulin-expressing cells remain IAPP-free. Compared to this IAPP-free population, we show these insulin- and IAPP-expressing cells are less polyhormonal, more glucose-sensitive, and exhibit decreased action potential firing in low (2.8 mM) glucose. CONCLUSIONS: The INS2A-EGFP/+;IAPP2A-mScarlet/+ hESC line provides a useful tool for tracking populations of maturing hESC-derived ß cells in vitro. This tool has already been shared with 3 groups and is freely available to all.
RESUMO
Islet transplantation is an attractive approach for treating type-1 diabetes, but there is a massive loss of transplanted islets. It is currently only possible to estimate islet mass indirectly, through measurement of circulating C-peptide and insulin levels. This type of estimation, however, is not sufficiently sensitive or reproducible for follow-up of individuals who have undergone islet transplantation. Here we show that islet graft survival could be assessed for 1 month in diabetic NOD mice using 9-(4-[(18)F]-fluoro-3-hydroxymethylbutyl)guanine ([(18)F]FHBG)-positron emission tomography (PET) technology, the PET signal reflecting insulin secretory capacity of transplanted islets. Expression of the gene encoding viral interleukin-10 (vIL-10), was measurable in real time with PET scanning. Additionally, we addressed the clinical potential of this approach by visualizing transplanted islets in the liver, the preferred clinical transplantation site. We conclude that quantitative in vivo PET imaging is a valid method for facilitating the development of protocols for prolonging islet survival, with the potential for tracking human transplants.
Assuntos
Sobrevivência de Enxerto , Transplante das Ilhotas Pancreáticas/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Animais , Células Cultivadas , Diabetes Mellitus Experimental/diagnóstico por imagem , Estudos de Viabilidade , Feminino , Genes Reporter , Guanina/análogos & derivados , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Transplante IsogênicoRESUMO
Restoring functional ß cell mass is a potential therapy for those with diabetes. However, the pathways regulating ß cell mass are not fully understood. Previously, we demonstrated that Sox4 is required for ß cell proliferation during prediabetes. Here, we report that Sox4 regulates ß cell mass through modulating expression of the type 2 diabetes (T2D) susceptibility gene GRK5. ß cell-specific Grk5 knockout mice showed impaired glucose tolerance with reduced ß cell mass, which was accompanied by upregulation of cell cycle inhibitor gene Cdkn1a. Furthermore, we found that Grk5 may drive ß cell proliferation through a pathway that includes phosphorylation of HDAC5 and subsequent transcription of immediate-early genes (IEGs) such as Nr4a1, Fosb, Junb, Arc, Egr1, and Srf. Together, these studies suggest GRK5 is linked to T2D through regulation of ß cell growth and that it may be a target to preserve ß cells during the development of T2D.
RESUMO
Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that exerts insulinotropic and growth and survival effects on pancreatic ß-cells. Additionally, there is increasing evidence supporting an important role for GIP in the regulation of adipocyte metabolism. In the current study we examined the molecular mechanisms involved in the regulation of GIP receptor (GIPR) expression in 3T3-L1 cells. GIP acted synergistically with insulin to increase neutral lipid accumulation during progression of 3T3-L1 preadipocytes to the adipocyte phenotype. Both GIPR protein and mRNA expression increased during 3T3-L1 cell differentiation, and this increase was associated with upregulation of nuclear levels of sterol response element binding protein 1c (SREBP-1c) and peroxisome proliferator-activated receptor γ (PPARγ), as well as acetylation of histones H3/H4. The PPARγ receptor agonists LY171883 and rosiglitazone increased GIPR expression in differentiated 3T3-L1 adipocytes, whereas the antagonist GW9662 ablated expression. Additionally, both PPARγ and acetylated histones H3/H4 were shown to bind to a region of the GIPR promoter containing the peroxisome proliferator response element (PPRE). Knockdown of PPARγ in differentiated 3T3-L1 adipocytes, using RNA interference, reduced GIPR expression, supporting a functional regulatory role. Taken together, these studies show that GIP and insulin act in a synergistic manner on 3T3-L1 cell development and that adipocyte GIPR expression is upregulated through a mechanism involving interactions between PPARγ and a GIPR promoter region containing an acetylated histone region.
Assuntos
Adipócitos/metabolismo , Histonas/metabolismo , PPAR gama/metabolismo , Receptores dos Hormônios Gastrointestinais/metabolismo , Células 3T3-L1 , Acetilação , Adipócitos/citologia , Animais , Western Blotting , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Imunoprecipitação da Cromatina , Imunoprecipitação , Camundongos , PPAR gama/genética , Regiões Promotoras Genéticas , Ligação Proteica , Interferência de RNA , Receptores dos Hormônios Gastrointestinais/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína de Ligação a Elemento Regulador de Esterol 1/genética , Proteína de Ligação a Elemento Regulador de Esterol 1/metabolismoRESUMO
GIP (glucose-dependent insulinotropic polypeptide) is a gastrointestinal hormone that regulates pancreatic islet function. Additionally, emerging evidence suggests an important physiological role for GIP in the regulation of adipocyte metabolism. In previous studies on the lipogenic effects of GIP, it was shown to increase adipocyte lipoprotein lipase (LPL) activity in both differentiated 3T3-L1 cells and human adipocytes through a pathway involving activation of protein kinase B (PKB)/Akt. In the current study, we examined the effects of GIP on LPL gene expression. GIP in the presence of insulin increased LPL gene expression in human adipocytes and LPL promoter activity in GIP receptor-expressing HEK-293 cells, and both effects were greatly reduced by the transcription inhibitor actinomycin D. Subsequent studies established that GIP increased phosphorylation of Serine 133 in cAMP-response element binding protein (CREB) and the nuclear localization of cAMP-responsive CREB coactivator 2 (TORC2) through a pathway involving phosphatidylinositol 3-kinase (PI3-K), PKB, and AMP-activated protein kinase (AMPK). However, in the presence of insulin, GIP failed to activate the cAMP/PKA pathway. Knockdown of CREB and TORC2 using RNA interference reduced LPL expression, supporting a functional regulatory role. GIP-induced phospho-CREB and TORC2 were shown to bind to a cAMP-response element (-II) site in the human LPL promoter and GIP increased protein-protein interactions of these two factors. The lipogenic effects of GIP in the presence of insulin are therefore at least partially mediated by upregulation of adipocyte LPL gene transcription through a pathway involving PI3-K/PKB/AMPK-dependent CREB/TORC2 activation.
Assuntos
Adipócitos/efeitos dos fármacos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Lipase Lipoproteica/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Adipócitos/citologia , Adipócitos/enzimologia , Adipócitos/metabolismo , Adulto , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Feminino , Células HEK293 , Humanos , Pessoa de Meia-Idade , Fosfatidilinositol 3-Quinases/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Depolarization of neuroendocrine cells results in calcium influx, which induces vesicle exocytosis and alters gene expression. These processes, along with the restoration of resting membrane potential, are energy intensive. We hypothesized that cellular mechanisms exist to maximize energy production during excitation. Here, we demonstrate that NPAS4, an immediate early basic helix-loop-helix (bHLH)-PAS transcription factor, acts to maximize energy production by suppressing hypoxia-inducible factor 1α (HIF1α). As such, knockout of Npas4 from insulin-producing ß cells results in reduced OXPHOS, loss of insulin secretion, ß cell dedifferentiation, and type 2 diabetes. NPAS4 plays a similar role in the nutrient-sensing cells of the hypothalamus. Its knockout here results in increased food intake, reduced locomotor activity, and elevated peripheral glucose production. In conclusion, NPAS4 is critical for the coordination of metabolism during the stimulation of electrically excitable cells; its loss leads to the defects in cellular metabolism that underlie the cellular dysfunction that occurs in metabolic disease.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Hipotálamo/metabolismo , Células Neuroendócrinas/metabolismo , Fosforilação Oxidativa , Oxigênio/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipotálamo/citologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Camundongos , Camundongos Transgênicos , Células Neuroendócrinas/citologiaRESUMO
The high-mobility group box transcription factor SOX4 is the most highly expressed SOX family protein in pancreatic islets, and mutations in Sox4 are associated with an increased risk of developing type 2 diabetes. We used an inducible ß-cell knockout mouse model to test the hypothesis that Sox4 is essential for the maintenance of ß-cell number during the development of type 2 diabetes. Knockout of Sox4 at 6 weeks of age resulted in time-dependent worsening of glucose tolerance, impairment of insulin secretion, and diabetes by 30 weeks of age. Immunostaining revealed a decrease in ß-cell mass in knockout mice that was caused by a 39% reduction in ß-cell proliferation. Gene expression studies revealed that induction of the cell cycle inhibitor Cdkn1a was responsible for the decreased proliferation in the knockout animals. Altogether, this study demonstrates that SOX4 is necessary for adult ß-cell replication through direct regulation of the ß-cell cycle.
Assuntos
Proliferação de Células/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Diabetes Mellitus Tipo 2/genética , Células Secretoras de Insulina/fisiologia , Fatores de Transcrição SOXC/deficiência , Animais , Insulina/genética , Insulina/metabolismo , Secreção de Insulina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
During pancreatic development, proliferating pancreatic progenitors activate the proendocrine transcription factor neurogenin 3 (NEUROG3), exit the cell cycle, and differentiate into islet cells. The mechanisms that direct robust NEUROG3 expression within a subset of progenitor cells control the size of the endocrine population. Here we demonstrate that NEUROG3 is phosphorylated within the nucleus on serine 183, which catalyzes its hyperphosphorylation and proteosomal degradation. During progression through the progenitor cell cycle, NEUROG3 phosphorylation is driven by the actions of cyclin-dependent kinases 2 and 4/6 at G1/S cell-cycle checkpoint. Using models of mouse and human pancreas development, we show that lengthening of the G1 phase of the pancreatic progenitor cell cycle is essential for proper induction of NEUROG3 and initiation of endocrine cell differentiation. In sum, these studies demonstrate that progenitor cell-cycle G1 lengthening, through its actions on stabilization of NEUROG3, is an essential variable in normal endocrine cell genesis.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Pâncreas/citologia , Pâncreas/metabolismo , Células-Tronco/citologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Células Endócrinas/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Ilhotas Pancreáticas/citologia , Camundongos , Proteínas do Tecido Nervoso/genética , Fosforilação/fisiologia , Células-Tronco/metabolismoRESUMO
The therapeutic potential of glucose-dependent insulinotropic polypeptide (GIP) for improving glycemic control has largely gone unstudied. A series of synthetic GIP peptides modified at the NH(2)-terminus were screened in vitro for resistance to dipeptidyl peptidase IV (DP IV) degradation and potency to stimulate cyclic AMP and affinity for the transfected rat GIP receptor. In vitro experiments indicated that [D-Ala(2)]GIP possessed the greatest resistance to enzymatic degradation, combined with minimal effects on efficacy at the receptor. Thus, [D-Ala(2)]GIP(1--42) was selected for further testing in the perfused rat pancreas and bioassay in conscious Wistar and Zucker rats. When injected subcutaneously in normal Wistar, Fa/?, or fa/fa Vancouver Diabetic Fatty (VDF) Zucker rats, both GIP and [D-Ala(2)]GIP significantly reduced glycemic excursions during a concurrent oral glucose tolerance test via stimulation of insulin release. The latter peptide displayed greater in vivo effectiveness, likely because of resistance to enzymatic degradation. Hence, despite reduced bioactivity in diabetic models at physiological concentrations, GIP and analogs with improved plasma stability still improve glucose tolerance when given in supraphysiological doses, and thus may prove useful in the treatment of diabetic states.
Assuntos
Glicemia/metabolismo , Diabetes Mellitus/tratamento farmacológico , Dipeptidil Peptidase 4/metabolismo , Polipeptídeo Inibidor Gástrico/uso terapêutico , Teste de Tolerância a Glucose , Obesidade , Adenilil Ciclases/metabolismo , Animais , Ligação Competitiva , Glicemia/análise , AMP Cíclico/biossíntese , Diabetes Mellitus/sangue , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/tratamento farmacológico , Ativação Enzimática , Polipeptídeo Inibidor Gástrico/sangue , Polipeptídeo Inibidor Gástrico/química , Humanos , Insulina/sangue , Cinética , Masculino , Ratos , Ratos Wistar , Ratos Zucker , Receptores dos Hormônios Gastrointestinais/genética , Receptores dos Hormônios Gastrointestinais/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
Human embryonic stem cells (hESCs) have great promise as a source of unlimited transplantable cells for regenerative medicine. However, current progress on producing the desired cell type for disease treatment has been limited due to an insufficient understanding of the developmental processes that govern their differentiation, as well as a paucity of tools to systematically study differentiation in the lab. In order to overcome these limitations, cell-type reporter hESC lines will be required. Here we outline two strategies using Transcription Activator Like Effector Nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-Associated protein (Cas) to create OCT4-eGFP knock-in add-on hESC lines. Thirty-one and forty-seven percent of clones were correctly modified using the TALEN and CRISPR-Cas9 systems, respectively. Further analysis of three correctly targeted clones demonstrated that the insertion of eGFP in-frame with OCT4 neither significantly impacted expression from the wild type allele nor did the fusion protein have a dramatically different biological stability. Importantly, the OCT4-eGFP fusion was easily detected using microscopy, flow cytometry and western blotting. The OCT4 reporter lines remained equally competent at producing CXCR4+ definitive endoderm that expressed a panel of endodermal genes. Moreover, the genomic modification did not impact the formation of NKX6.1+/SOX9+ pancreatic progenitor cells following directed differentiation. In conclusion, these findings demonstrate for the first time that CRISPR-Cas9 can be used to modify OCT4 and highlight the feasibility of creating cell-type specific reporter hESC lines utilizing genome-editing tools that facilitate homologous recombination.
Assuntos
Diferenciação Celular/genética , Endoderma/citologia , Células-Tronco Embrionárias Humanas/citologia , Fator 3 de Transcrição de Octâmero/genética , Sistemas CRISPR-Cas , Linhagem Celular , Endoderma/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Introdução de Genes , Genoma Humano , Recombinação Homóloga/genética , Humanos , Fator 3 de Transcrição de Octâmero/biossínteseRESUMO
Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that also plays a regulatory role in fat metabolism. In 3T3-L1 cells, resistin was demonstrated to be a key mediator of GIP stimulation of lipoprotein lipase (LPL) activity, involving activation of protein kinase B (PKB) and reduced phosphorylation of liver kinase B1 (LKB1) and AMP-activated protein kinase (AMPK). The current study was initiated to determine whether resistin has additional roles in GIP-regulated adipocyte functions. Analysis of primary adipocytes isolated from Retn(-/-), Retn(+/-), and Retn(+/+) mice found that GIP stimulated the PKB/LKB1/AMPK/LPL pathway and fatty acid uptake only in Retn(+/+) adipocytes, suggesting that GIP signaling and/or GIP responsiveness were compromised in Retn(+/-) and Retn(-/-) adipocytes. GIP receptor (GIPR) protein and mRNA were decreased in Retn(+/-) and Retn(-/-) adipocytes, but resistin treatment rescued LPL responsiveness to GIP. In addition, genes encoding tumor necrosis factor (TNF), TNF receptor 2 (TNFR2), and the signaling proteins stress-activated protein kinase (SAPK)/Jun NH(2)-terminal kinase (JNK), were downregulated, and phosphorylated levels of SAPK/JNK/c-Jun were decreased in Retn(-/-) mice. Chromatin immunoprecipitation assays were used to identify a 12-O-tetradecanoylphorbol-13-acetate (TPA)-response element (TRE-III) responsible for c-Jun-mediated transcriptional activation of Gipr. Blunted GIP responsiveness in Retn(+/-) and Retn(-/-) adipocytes was therefore largely due to the greatly reduced GIPR expression associated with decreased c-Jun-mediated transcriptional activation of Gipr.
Assuntos
Adipócitos/metabolismo , Polipeptídeo Inibidor Gástrico/metabolismo , Receptores dos Hormônios Gastrointestinais/biossíntese , Resistina/metabolismo , Células 3T3-L1 , Proteínas Quinases Ativadas por AMP/metabolismo , Adipócitos/efeitos dos fármacos , Animais , Carcinógenos/farmacologia , Células Cultivadas , Ácidos Graxos/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Regulação da Expressão Gênica , Lipase Lipoproteica/metabolismo , Masculino , Camundongos , Camundongos Knockout , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridinas/farmacologia , Receptores Tipo II do Fator de Necrose Tumoral/biossíntese , Receptores Tipo II do Fator de Necrose Tumoral/genética , Resistina/genética , Resistina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologiaRESUMO
Cellular homeostasis requires intrinsic sensing mechanisms to temper function in the face of prolonged activity. In the pancreatic ß-cell, glucose is likely a physiological trigger that activates an adaptive response to stimulation, thereby maintaining cellular homeostasis. Immediate early genes (IEGs) are activated as a first line of defense in cellular homeostasis and are largely responsible for transmitting an environmental cue to a cellular response. Here we examine the regulation and function of the novel ß-cell IEG, neuronal PAS domain protein 4 (Npas4). Using MIN6 cells, mouse and human islets, as well as in vivo infusions, we demonstrate that Npas4 is expressed within pancreatic islets and is upregulated by ß-cell depolarizing agents. Npas4 tempers ß-cell function through a direct inhibitory interaction with the insulin promoter and by blocking the potentiating effects of GLP-1 without significantly reducing glucose-stimulated secretion. Finally, Npas4 expression is induced by classical endoplasmic reticulum (ER) stressors and can prevent thapsigargin- and palmitate-induced dysfunction and cell death. These results suggest that Npas4 is a key activity-dependent regulator that improves ß-cell efficiency in the face of stress. We posit that Npas4 could be a novel therapeutic target in type 2 diabetes that could both reduce ER stress and cell death and maintain basal cell function.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citoproteção/genética , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Linhagem Celular , Citoproteção/efeitos dos fármacos , Emulsões/farmacologia , Estresse do Retículo Endoplasmático/fisiologia , Peptídeo 1 Semelhante ao Glucagon/farmacologia , Glucose/farmacologia , Humanos , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Ilhotas Pancreáticas/efeitos dos fármacos , Camundongos , Fosfolipídeos/farmacologia , Regiões Promotoras Genéticas , Óleo de Soja/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that potentiates glucose-stimulated insulin secretion during a meal. Since GIP has also been shown to exert ß-cell prosurvival and adipocyte lipogenic effects in rodents, both GIP receptor agonists and antagonists have been considered as potential therapeutics in type 2 diabetes (T2DM). In the present study, we tested the hypothesis that chronically elevating GIP levels in a transgenic (Tg) mouse model would increase adipose tissue expansion and exert beneficial effects on glucose homeostasis. In contrast, although GIP Tg mice demonstrated enhanced ß-cell function, resulting in improved glucose tolerance and insulin sensitivity, they exhibited reduced diet-induced obesity. Adipose tissue macrophage infiltration and hepatic steatosis were both greatly reduced, and a number of genes involved in lipid metabolism/inflammatory signaling pathways were found to be down-regulated. Reduced adiposity in GIP Tg mice was associated with decreased energy intake, involving overexpression of hypothalamic GIP. Together, these studies suggest that, in the context of over-nutrition, transgenic GIP overexpression has the potential to improve hepatic and adipocyte function as well as glucose homeostasis.
Assuntos
Fígado Gorduroso/prevenção & controle , Polipeptídeo Inibidor Gástrico/biossíntese , Glucose/metabolismo , Homeostase , Obesidade/etiologia , Tecido Adiposo/metabolismo , Animais , Dieta Hiperlipídica , Metabolismo Energético , Polipeptídeo Inibidor Gástrico/farmacologia , Intolerância à Glucose/metabolismo , Teste de Tolerância a Glucose , Resistência à Insulina/fisiologia , Masculino , Camundongos , Camundongos Transgênicos , Obesidade/metabolismoRESUMO
OBJECTIVE: Treatment of NOD mice with the dipeptidyl peptidase-IV (DPP-IV) inhibitor sitagliptin preserved islet transplants through a pathway involving modulation of splenic CD4(+) T-cell migration. In the current study, effects of sitagliptin on migration of additional subsets of CD4(+) T-cells were examined and underlying molecular mechanisms were further defined. RESEARCH DESIGN AND METHODS: Effects of sitagliptin on migration of NOD mouse splenic, thymic, and lymph node CD4(+) T-cells were determined. Signaling modules involved in DPP-IV-, Sitagliptin- and incretin-mediated modulation of CD4(+) T-cell migration were studied using Western blot and Rac1 and nuclear factor-kappaB (NF-kappaB) activity assays. RESULTS: Migration of splenic and lymph node CD4(+) T-cells of diabetic NOD mice was reduced by sitagliptin treatment. In vitro treatment of splenic, but not thymic or lymph node CD4(+) T-cells, from nondiabetic NOD mice with soluble (s) DPP-IV increased migration. Sitagliptin abolished sDPP-IV effects on splenic CD4(+) T-cell migration, whereas incretins decreased migration of lymph node, but not splenic, CD4(+) T-cells. Splenic CD4(+) T-cells demonstrating increased in vitro migration in response to sDPP-IV and lymph node CD4(+) T-cells that were nonresponsive to incretins selectively infiltrated islets of NOD mice, after injection. Sitagliptin decreases migration of splenic CD4(+) T-cells through a pathway involving Rac1/vasodilator-stimulated phosphoprotein, whereas its inhibitory effects on the migration of lymph node CD4(+) T-cells involve incretin-activation of the NF-kappaB pathway. CONCLUSIONS: Benefits of sitagliptin treatment in diabetic NOD mice may be mediated through selective effects on subpopulations of T-cells that are related to autoimmunity.
Assuntos
Linfócitos T CD4-Positivos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Dipeptidil Peptidase 4/metabolismo , Incretinas/metabolismo , Pirazinas/farmacologia , Subpopulações de Linfócitos T/efeitos dos fármacos , Triazóis/farmacologia , Análise de Variância , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Movimento Celular/imunologia , Inibidores da Dipeptidil Peptidase IV/farmacologia , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Fosfato de Sitagliptina , Baço/efeitos dos fármacos , Baço/imunologia , Baço/metabolismo , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , Timo/efeitos dos fármacos , Timo/imunologia , Timo/metabolismoRESUMO
AIMS: The gastrointestinal hormone GIP promotes pancreatic islet function and exerts pro-survival actions on cultured beta-cells. However, GIP also promotes lipogenesis, thus potentially restricting its therapeutic use. The current studies evaluated the effects of a truncated GIP analog, D-Ala(2)-GIP(1-30) (D-GIP(1-30)), on glucose homeostasis and beta-cell mass in rat models of diabetes. MATERIALS AND METHODS: The insulinotropic and pro-survival potency of D-GIP(1-30) was evaluated in perfused pancreas preparations and cultured INS-1 beta-cells, respectively, and receptor selectivity evaluated using wild type and GIP receptor knockout mice. Effects of D-GIP(1-30) on beta-cell function and glucose homeostasis, in vivo, were determined using Lean Zucker rats, obese Vancouver diabetic fatty rats, streptozotocin treated rats, and obese Zucker diabetic fatty rats, with effects on beta-cell mass determined in histological studies of pancreatic tissue. Lipogenic effects of D-GIP(1-30) were evaluated on cultured 3T3-L1 adipocytes. RESULTS: Acutely, D-GIP(1-30) improved glucose tolerance and insulin secretion. Chronic treatment with D-GIP(1-30) reduced levels of islet pro-apoptotic proteins in Vancouver diabetic fatty rats and preserved beta-cell mass in streptozotocin treated rats and Zucker diabetic fatty rats, resulting in improved insulin responses and glycemic control in each animal model, with no change in body weight. In in vitro studies, D-GIP(1-30) exhibited equivalent potency to GIP(1-42) on beta-cell function and survival, but greatly reduced action on lipoprotein lipase activity in 3T3-L1 adipocytes. CONCLUSIONS: These findings demonstrate that truncated forms of GIP exhibit potent anti-diabetic actions, without pro-obesity effects, and that the C-terminus contributes to the lipogenic actions of GIP.