RESUMEN
The field of epigenetics broadly seeks to define heritable phenotypic modifications that occur within cells without changes to the underlying DNA sequence. These modifications allow for precise control and specificity of function between cell types-ultimately creating complex organ systems that all contain the same DNA but only have access to the genes and sequences necessary for their cell-type-specific functions. The pancreas is an organ that contains varied cellular compartments with functions ranging from highly regulated glucose-stimulated insulin secretion in the ß-cell to the pancreatic ductal cells that form a tight epithelial lining for the delivery of digestive enzymes. With diabetes cases on the rise worldwide, understanding the epigenetic mechanisms driving ß-cell identity, function, and even disease is particularly valuable. In this chapter, we will discuss the known epigenetic modifications in pancreatic islet cells, how they are deposited, and the environmental and metabolic contributions to epigenetic mechanisms. We will also explore how a deeper understanding of epigenetic effectors can be used as a tool for diabetes therapeutic strategies.
Asunto(s)
Epigénesis Genética , Páncreas , Humanos , Páncreas/embriología , Páncreas/metabolismo , Animales , Células Secretoras de Insulina/metabolismo , Metilación de ADN/genética , Diabetes Mellitus/genéticaRESUMEN
The role of aberrant glycosylation in pancreatic ductal adenocarcinoma (PDAC) remains an under-investigated area of research. In this study, we determined that ST6 ß-galactoside α2,6 sialyltransferase 1 (ST6GAL1), which adds α2,6-linked sialic acids to N-glycosylated proteins, was upregulated in patients with early-stage PDAC and was further increased in advanced disease. A tumor-promoting function for ST6GAL1 was elucidated using tumor xenograft experiments with human PDAC cells. Additionally, we developed a genetically engineered mouse (GEM) model with transgenic expression of ST6GAL1 in the pancreas and found that mice with dual expression of ST6GAL1 and oncogenic KRASG12D had greatly accelerated PDAC progression compared with mice expressing KRASG12D alone. As ST6GAL1 imparts progenitor-like characteristics, we interrogated ST6GAL1's role in acinar to ductal metaplasia (ADM), a process that fosters neoplasia by reprogramming acinar cells into ductal, progenitor-like cells. We verified ST6GAL1 promotes ADM using multiple models including the 266-6 cell line, GEM-derived organoids and tissues, and an in vivo model of inflammation-induced ADM. EGFR is a key driver of ADM and is known to be activated by ST6GAL1-mediated sialylation. Importantly, EGFR activation was dramatically increased in acinar cells and organoids from mice with transgenic ST6GAL1 expression. These collective results highlight a glycosylation-dependent mechanism involved in early stages of pancreatic neoplasia.
Asunto(s)
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Ratones , Animales , Neoplasias Pancreáticas/patología , Páncreas/patología , Carcinoma Ductal Pancreático/patología , Receptores ErbB/genética , Metaplasia/patología , Sialiltransferasas/genética , beta-D-Galactósido alfa 2-6-Sialiltransferasa , Antígenos CDRESUMEN
OBJECTIVE: Transcriptional complex activity drives the development and function of pancreatic islet cells to allow for proper glucose regulation. Prior studies from our lab and others highlighted that the LIM-homeodomain transcription factor (TF), Islet-1 (Isl1), and its interacting co-regulator, Ldb1, are vital effectors of developing and adult ß-cells. We further found that a member of the Single Stranded DNA-Binding Protein (SSBP) co-regulator family, SSBP3, interacts with Isl1 and Ldb1 in ß-cells and primary islets (mouse and human) to impact ß-cell target genes MafA and Glp1R in vitro. Members of the SSBP family stabilize TF complexes by binding directly to Ldb1 and protecting the complex from ubiquitin-mediated turnover. In this study, we hypothesized that SSBP3 has critical roles in pancreatic islet cell function in vivo, similar to the Isl1::Ldb1 complex. METHODS: We first developed a novel SSBP3 LoxP allele mouse line, where Cre-mediated recombination imparts a predicted early protein termination. We bred this mouse with constitutive Cre lines (Pdx1- and Pax6-driven) to recombine SSBP3 in the developing pancreas and islet (SSBP3ΔPanc and SSBP3ΔIslet), respectively. We assessed glucose tolerance and used immunofluorescence to detect changes in islet cell abundance and markers of ß-cell identity and function. Using an inducible Cre system, we also deleted SSBP3 in the adult ß-cell, a model termed SSBP3Δß-cell. We measured glucose tolerance as well as glucose-stimulated insulin secretion (GSIS), both in vivo and in isolated islets in vitro. Using islets from control and SSBP3Δß-cell we conducted RNA-Seq and compared our results to published datasets for similar ß-cell specific Ldb1 and Isl1 knockouts to identify commonly regulated target genes. RESULTS: SSBP3ΔPanc and SSBP3ΔIslet neonates present with hyperglycemia. SSBP3ΔIslet mice are glucose intolerant by P21 and exhibit a reduction of ß-cell maturity markers MafA, Pdx1, and UCN3. We observe disruptions in islet cell architecture with an increase in glucagon+ α-cells and ghrelin+ ε-cells at P10. Inducible loss of ß-cell SSBP3 in SSBP3Δß-cell causes hyperglycemia, glucose intolerance, and reduced GSIS. Transcriptomic analysis of 14-week-old SSBP3Δß-cell islets revealed a decrease in ß-cell function gene expression (Ins, MafA, Ucn3), increased stress and dedifferentiation markers (Neurogenin-3, Aldh1a3, Gastrin), and shared differentially expressed genes between SSBP3, Ldb1, and Isl1 in adult ß-cells. CONCLUSIONS: SSBP3 drives proper islet identity and function, where its loss causes altered islet-cell abundance and glucose homeostasis. ß-Cell SSBP3 is required for GSIS and glucose homeostasis, at least partially through shared regulation of Ldb1 and Isl1 target genes.
Asunto(s)
Hiperglucemia , Islotes Pancreáticos , Adulto , Ratones , Humanos , Animales , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Islotes Pancreáticos/metabolismo , Glucosa/metabolismo , Hiperglucemia/metabolismo , Homeostasis , Proteínas de Unión al ADN/metabolismo , Proteínas con Dominio LIM/metabolismoRESUMEN
Pancreatic islet cell development is regulated by transcription factors (TFs) that mediate embryonic progenitor differentiation toward mature endocrine cells. Prior studies from our lab and others showed that the islet-enriched TF, Islet-1 (Isl1), interacts with the broadly-expressed transcriptional co-regulator, Ldb1, to regulate islet cell maturation and postnhyperatal function (by embryonic day (E)18.5). However, Ldb1 is expressed in the developing pancreas prior to Isl1 expression, notably in multipotent progenitor cells (MPCs) marked by Pdx1 and endocrine progenitors (EPs) expressing Neurogenin-3 (Ngn3). MPCs give rise to the endocrine and exocrine pancreas, while Ngn3+ EPs specify pancreatic islet endocrine cells. We hypothesized that Ldb1 is required for progenitor identity in MPC and EP populations during development to impact islet appearance and function. To test this, we generated a whole-pancreas Ldb1 knockout, termed Ldb1ΔPanc , and observed severe developmental and postnatal pancreas defects including disorganized progenitor pools, a significant reduction of Ngn3-expressing EPs, Pdx1HI ß-cells, and early hormone+ cells. Ldb1ΔPanc neonates presented with severe hyperglycemia, hypoinsulinemia, and drastically reduced hormone expression in islets, yet no change in total pancreas mass. This supports the endocrine-specific actions of Ldb1. Considering this, we also developed an endocrine-enriched model of Ldb1 loss, termed Ldb1ΔEndo . We observed similar dysglycemia in this model, as well as a loss of islet identity markers. Through in vitro and in vivo chromatin immunoprecipitation experiments, we found that Ldb1 occupies key Pdx1 and Ngn3 promoter domains. Our findings provide insight into novel regulation of endocrine cell differentiation that may be vital toward improving cell-based diabetes therapies.
Asunto(s)
Proteínas de Homeodominio , Islotes Pancreáticos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Hormonas/metabolismo , Humanos , Recién Nacido , Islotes Pancreáticos/metabolismo , Proteínas con Dominio LIM/genética , Páncreas , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Type 1 diabetes is an autoimmune disease characterized by insulin-producing ß cell destruction. Although islet transplantation restores euglycemia and improves patient outcomes, an ideal transplant site remains elusive. Brown adipose tissue (BAT) has a highly vascularized and antiinflammatory microenvironment. Because these tissue features can promote islet graft survival, we hypothesized that islets transplanted into BAT will maintain islet graft and BAT function while delaying immune-mediated rejection. We transplanted syngeneic and allogeneic islets into BAT or under the kidney capsule of streptozotocin-induced diabetic NOD.Rag and NOD mice to investigate islet graft function, BAT function, metabolism, and immune-mediated rejection. Islet grafts within BAT restored euglycemia similarly to kidney capsule controls. Islets transplanted in BAT maintained expression of islet hormones and transcription factors and were vascularized. Compared with those in kidney capsule and euglycemic mock-surgery controls, no differences in glucose or insulin tolerance, thermogenic regulation, or energy expenditure were observed with islet grafts in BAT. Immune profiling of BAT revealed enriched antiinflammatory macrophages and T cells. Compared with the kidney capsule control, there were significant delays in autoimmune and allograft rejection of islets transplanted in BAT, possibly due to increased antiinflammatory immune populations. Our data support BAT as an alternative islet transplant site that may improve graft survival.
Asunto(s)
Tejido Adiposo Pardo/cirugía , Diabetes Mellitus Tipo 1/cirugía , Regulación de la Expresión Génica , Rechazo de Injerto/genética , Proteínas de Homeodominio/genética , Trasplante de Islotes Pancreáticos/métodos , Transactivadores/genética , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Pardo/patología , Animales , Diferenciación Celular , Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Rechazo de Injerto/inmunología , Rechazo de Injerto/metabolismo , Supervivencia de Injerto , Proteínas de Homeodominio/biosíntesis , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , ARN/genética , Transactivadores/biosíntesis , Trasplante HomólogoRESUMEN
Type 1 diabetes (T1D) is an autoimmune disease characterized by autoreactive T cell-mediated destruction of insulin-producing pancreatic beta-cells. Loss of beta-cells leads to insulin insufficiency and hyperglycemia, with patients eventually requiring lifelong insulin therapy to maintain normal glycemic control. Since T1D has been historically defined as a disease of immune system dysregulation, there has been little focus on the state and response of beta-cells and how they may also contribute to their own demise. Major hurdles to identifying a cure for T1D include a limited understanding of disease etiology and how functional and transcriptional beta-cell heterogeneity may be involved in disease progression. Recent studies indicate that the beta-cell response is not simply a passive aspect of T1D pathogenesis, but rather an interplay between the beta-cell and the immune system actively contributing to disease. Here, we comprehensively review the current literature describing beta-cell vulnerability, heterogeneity, and contributions to pathophysiology of T1D, how these responses are influenced by autoimmunity, and describe pathways that can potentially be exploited to delay T1D.
Asunto(s)
Autoinmunidad/inmunología , Diabetes Mellitus Tipo 1/inmunología , Células Secretoras de Insulina/inmunología , Animales , Diabetes Mellitus Tipo 1/patología , Humanos , Células Secretoras de Insulina/patologíaRESUMEN
BACKGROUND: Islet transplantation with neonatal porcine islets (NPIs) is a promising treatment for type 1 diabetes (T1D), but immune rejection poses a major hurdle for clinical use. Innate immune-derived reactive oxygen species (ROS) synthesis can facilitate islet xenograft destruction and enhance adaptive immune responses. METHODS: To suppress ROS-mediated xenograft destruction, we utilized nanothin encapsulation materials composed of multilayers of tannic acid (TA), an antioxidant, and a neutral polymer, poly(N-vinylpyrrolidone) (PVPON). We hypothesized that (PVPON/TA)-encapsulated NPIs will maintain euglycemia and dampen proinflammatory innate immune responses following xenotransplantation. RESULTS: (PVPON/TA)-encapsulated NPIs were viable and glucose-responsive similar to non-encapsulated NPIs. Transplantation of (PVPON/TA)-encapsulated NPIs into hyperglycemic C57BL/6.Rag or NOD.Rag mice restored euglycemia, exhibited glucose tolerance, and maintained islet-specific transcription factor levels similar to non-encapsulated NPIs. Gene expression analysis of (PVPON/TA)-encapsulated grafts post-transplantation displayed reduced proinflammatory Ccl5, Cxcl10, Tnf, and Stat1 while enhancing alternatively activated macrophage Retnla, Arg1, and Stat6 mRNA accumulation compared with controls. Flow cytometry analysis demonstrated significantly reduced innate immune infiltration, MHC-II, co-stimulatory molecule, and TNF expression with concomitant increases in arginase-1+ macrophages and dendritic cells. Similar alterations in immune responses were observed following xenotransplantation into immunocompetent NOD mice. CONCLUSION: Our data suggest that (PVPON/TA) encapsulation of NPIs is an effective strategy to decrease inflammatory innate immune signals involved in NPI xenograft responses through STAT1/6 modulation without compromising islet function.
Asunto(s)
Trasplante de Islotes Pancreáticos , Islotes Pancreáticos , Animales , Humanos , Inmunidad Innata , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Porcinos , Taninos , Trasplante HeterólogoRESUMEN
OBJECTIVE: Brown adipose tissue (BAT) is critical for thermogenesis and glucose/lipid homeostasis. Exploiting the energy uncoupling capacity of BAT may reveal targets for obesity therapies. This exploitation requires a greater understanding of the transcriptional mechanisms underlying BAT function. One potential regulator of BAT is the transcriptional co-regulator LIM domain-binding protein 1 (LDB1), which acts as a dimerized scaffold, allowing for the assembly of transcriptional complexes. Utilizing a global LDB1 heterozygous mouse model, we recently reported that LDB1 might have novel roles in regulating BAT function. However, direct evidence for the LDB1 regulation of BAT thermogenesis and substrate utilization has not been elucidated. We hypothesize that brown adipocyte-expressed LDB1 is required for BAT function. METHODS: LDB1-deficient primary cells and brown adipocyte cell lines were assessed via qRT-PCR and western blotting for altered mRNA and protein levels to define the brown adipose-specific roles. We conducted chromatin immunoprecipitation with primary BAT tissue and immortalized cell lines. Potential transcriptional partners of LDB1 were revealed by conducting LIM factor surveys via qRT-PCR in mouse and human brown adipocytes. We developed a Ucp1-Cre-driven LDB1-deficiency mouse model, termed Ldb1ΔBAT, to test LDB1 function in vivo. Glucose tolerance and uptake were assessed at thermoneutrality via intraperitoneal glucose challenge and glucose tracer studies. Insulin tolerance was measured at thermoneutrality and after stimulation with cold or the administration of the ß3-adrenergic receptor (ß3-AR) agonist CL316,243. Additionally, we analyzed plasma insulin via ELISA and insulin signaling via western blotting. Lipid metabolism was evaluated via BAT weight, histology, lipid droplet morphometry, and the examination of lipid-associated mRNA. Finally, energy expenditure and cold tolerance were evaluated via indirect calorimetry and cold challenges. RESULTS: Reducing Ldb1 in vitro and in vivo resulted in altered BAT-selective mRNA, including Ucp1, Elovl3, and Dio2. In addition, there was reduced Ucp1 induction in vitro. Impacts on gene expression may be due, in part, to LDB1 occupying Ucp1 upstream regulatory domains. We also identified BAT-expressed LIM-domain factors Lmo2, Lmo4, and Lhx8, which may partner with LDB1 to mediate activity in brown adipocytes. Additionally, we observed LDB1 enrichment in human brown adipose. In vivo analysis revealed LDB1 is required for whole-body glucose and insulin tolerance, in part through reduced glucose uptake into BAT. In Ldb1ΔBAT tissue, we found significant alterations in insulin-signaling effectors. An assessment of brown adipocyte morphology and lipid droplet size revealed larger and more unilocular brown adipocytes in Ldb1ΔBAT mice, particularly after a cold challenge. Alterations in lipid handling were further supported by reductions in mRNA associated with fatty acid oxidation and mitochondrial respiration. Finally, LDB1 is required for energy expenditure and cold tolerance in both male and female mice. CONCLUSIONS: Our findings support LDB1 as a regulator of BAT function. Furthermore, given LDB1 enrichment in human brown adipose, this co-regulator may have conserved roles in human BAT.
Asunto(s)
Tejido Adiposo Pardo/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas con Dominio LIM/metabolismo , Animales , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas con Dominio LIM/deficiencia , Proteínas con Dominio LIM/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , TranscriptomaRESUMEN
Glucagon regulates glucose and lipid metabolism and promotes weight loss. Thus, therapeutics stimulating glucagon receptor (GCGR) signaling are promising for obesity treatment; however, the underlying mechanism(s) have yet to be fully elucidated. We previously identified that hepatic GCGR signaling increases circulating fibroblast growth factor 21 (FGF21), a potent regulator of energy balance. We reported that mice deficient for liver Fgf21 are partially resistant to GCGR-mediated weight loss, implicating FGF21 as a regulator of glucagon's weight loss effects. FGF21 signaling requires an obligate coreceptor (ß-Klotho, KLB), with expression limited to adipose tissue, liver, pancreas, and brain. We hypothesized that the GCGR-FGF21 system mediates weight loss through a central mechanism. Mice deficient for neuronal Klb exhibited a partial reduction in body weight with chronic GCGR agonism (via IUB288) compared with controls, supporting a role for central FGF21 signaling in GCGR-mediated weight loss. Substantiating these results, mice with central KLB inhibition via a pharmacological KLB antagonist, 1153, also displayed partial weight loss. Central KLB, however, is dispensable for GCGR-mediated improvements in plasma cholesterol and liver triglycerides. Together, these data suggest GCGR agonism mediates part of its weight loss properties through central KLB and has implications for future treatments of obesity and metabolic syndrome.
Asunto(s)
Glucagón/metabolismo , Proteínas Klotho/metabolismo , Receptores de Glucagón/metabolismo , Transducción de Señal , Pérdida de Peso , Animales , Peso Corporal , Ingestión de Alimentos , Factores de Crecimiento de Fibroblastos/genética , Expresión Génica , Glucosa/metabolismo , Homeostasis , Proteínas Klotho/genética , Metabolismo de los Lípidos , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Obesidad/metabolismo , PéptidosRESUMEN
Glucagon (GCG) is an essential regulator of glucose and lipid metabolism that also promotes weight loss. We have shown that glucagon-receptor (GCGR) signaling increases fatty acid oxidation (FAOx) in primary hepatocytes and reduces liver triglycerides in diet-induced obese (DIO) mice; however, the mechanisms underlying this aspect of GCG biology remains unclear. Investigation of hepatic GCGR targets elucidated a potent and previously unknown induction of leptin receptor (Lepr) expression. Liver leptin signaling is known to increase FAOx and decrease liver triglycerides, similar to glucagon action. Therefore, we hypothesized that glucagon increases hepatic LEPR, which is necessary for glucagon-mediated reversal of hepatic steatosis. Eight-week-old control and liver-specific LEPR-deficient mice (LeprΔliver) were placed on a high-fat diet for 12 weeks and then treated with a selective GCGR agonist (IUB288) for 14 days. Liver triglycerides and gene expression were assessed in liver tissue homogenates. Administration of IUB288 in both lean and DIO mice increased hepatic Lepr isoforms a-e in acute (4 hours) and chronic (72 hours,16 days) (P < 0.05) settings. LeprΔliver mice displayed increased hepatic triglycerides on a chow diet alone (P < 0.05), which persisted in a DIO state (P < 0.001), with no differences in body weight or composition. Surprisingly, chronic administration of IUB288 in DIO control and LeprΔliver mice reduced liver triglycerides regardless of genotype (P < 0.05). Together, these data suggest that GCGR activation induces hepatic Lepr expression and, although hepatic glucagon and leptin signaling have similar liver lipid targets, these appear to be 2 distinct pathways.
Asunto(s)
Hígado Graso/tratamiento farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Metabolismo de los Lípidos/efectos de los fármacos , Péptidos/farmacología , Receptores de Glucagón/metabolismo , Receptores de Leptina/metabolismo , Animales , Área Bajo la Curva , Dieta Alta en Grasa , Homeostasis , Metabolismo de los Lípidos/fisiología , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Noqueados , Obesidad/inducido químicamente , Receptores de Glucagón/genética , Receptores de Leptina/genética , Transducción de SeñalRESUMEN
Diabetes is characterized by a loss of ß-cell mass, and a greater understanding of the transcriptional mechanisms governing ß-cell function is required for future therapies. Previously, we reported that a complex of the Islet-1 (Isl1) transcription factor and the co-regulator single-stranded DNA-binding protein 3 (SSBP3) regulates the genes necessary for ß-cell function, but few proteins are known to interact with this complex in ß-cells. To identify additional components, here we performed SSBP3 reverse-cross-linked immunoprecipitation (ReCLIP)- and MS-based experiments with mouse ß-cell extracts and compared the results with those from our previous Isl1 ReCLIP study. Our analysis identified the E3 ubiquitin ligases ring finger protein 20 (RNF20) and RNF40, factors that in nonpancreatic cells regulate transcription through imparting monoubiquitin marks on histone H2B (H2Bub1), a precursor to histone H3 lysine 4 trimethylation (H3K4me3). We hypothesized that RNF20 and RNF40 regulate similar genes as those regulated by Isl1 and SSBP3 and are important for ß-cell function. We observed that Rnf20 and Rnf40 depletion reduces ß-cell H2Bub1 marks and uncovered several target genes, including glucose transporter 2 (Glut2), MAF BZIP transcription factor A (MafA), and uncoupling protein 2 (Ucp2). Strikingly, we also observed that Isl1 and SSBP3 depletion reduces H2Bub1 and H3K4me3 marks, suggesting that they have epigenetic roles. We noted that the RNF complex is required for glucose-stimulated insulin secretion and normal mitochondrial reactive oxygen species levels. These findings indicate that RNF20 and RNF40 regulate ß-cell gene expression and insulin secretion and establish a link between Isl1 complexes and global cellular epigenetics.
Asunto(s)
Proteínas con Dominio LIM/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Transportador de Glucosa de Tipo 2/genética , Transportador de Glucosa de Tipo 2/metabolismo , Histonas/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Proteínas con Dominio LIM/química , Proteínas con Homeodominio LIM/antagonistas & inhibidores , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Factores de Transcripción Maf de Gran Tamaño/genética , Factores de Transcripción Maf de Gran Tamaño/metabolismo , Ratones , Unión Proteica , Dominios Proteicos , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
Pancreatic ß-cells undergo profound hyperplasia during pregnancy to maintain maternal euglycemia. Failure to reprogram ß-cells into a more replicative state has been found to underlie susceptibility to gestational diabetes mellitus (GDM). We recently identified a requirement for prolactin receptor (PRLR) signaling in the metabolic adaptations to pregnancy, where ß-cell-specific PRLR knockout (ßPRLRKO) mice exhibit a metabolic phenotype consistent with GDM. However, the underlying transcriptional program that is responsible for the PRLR-dependent metabolic adaptations during gestation remains incompletely understood. To identify PRLR signaling gene regulatory networks and target genes within ß-cells during pregnancy, we performed a transcriptomic analysis of pancreatic islets isolated from either ßPRLRKO mice or littermate controls in late gestation. Gene set enrichment analysis identified forkhead box protein M1 and polycomb repressor complex 2 subunits, Suz12 and enhancer of zeste homolog 2 (Ezh2), as novel candidate regulators of PRLR-dependent ß-cell adaptation. Gene ontology term pathway enrichment revealed both established and novel PRLR signaling target genes that together promote a state of increased cellular metabolism and/or proliferation. In contrast to the requirement for ß-cell PRLR signaling in maintaining euglycemia during pregnancy, PRLR target genes were not induced following high-fat diet feeding. Collectively, the current study expands our understanding of which transcriptional regulators and networks mediate gene expression required for islet adaptation during pregnancy. The current work also supports the presence of pregnancy-specific adaptive mechanisms distinct from those activated by nutritional stress.
Asunto(s)
Regulación de la Expresión Génica , Células Secretoras de Insulina/metabolismo , Receptores de Prolactina/genética , Transducción de Señal/genética , Animales , Línea Celular , Línea Celular Tumoral , Proliferación Celular/genética , Diabetes Gestacional/genética , Diabetes Gestacional/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Femenino , Perfilación de la Expresión Génica , Ontología de Genes , Redes Reguladoras de Genes , Células Secretoras de Insulina/citología , Ratones Endogámicos C57BL , Ratones Noqueados , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Embarazo , Receptores de Prolactina/metabolismoRESUMEN
The LIM-homeodomain (LIM-HD) transcription factor Islet-1 (Isl1) interacts with the LIM domain-binding protein 1 (Ldb1) coregulator to control expression of key pancreatic ß-cell genes. However, Ldb1 also has Isl1-independent effects, supporting that another LIM-HD factor interacts with Ldb1 to impact ß-cell development and/or function. LIM homeobox 1 (Lhx1) is an Isl1-related LIM-HD transcription factor that appears to be expressed in the developing mouse pancreas and in adult islets. However, roles for this factor in the pancreas are unknown. This study aimed to determine Lhx1 interactions and elucidate gene regulatory and physiological roles in the pancreas. Co-immunoprecipitation using ß-cell extracts demonstrated an interaction between Lhx1 and Isl1, and thus we hypothesized that Lhx1 and Isl1 regulate similar target genes. To test this, we employed siRNA-mediated Lhx1 knockdown in ß-cell lines and discovered reduced Glp1R mRNA. Chromatin immunoprecipitation revealed Lhx1 occupancy at a domain also known to be occupied by Isl1 and Ldb1. Through development of a pancreas-wide knockout mouse model ( Lhx1∆Panc), we demonstrate that aged Lhx1∆Panc mice have elevated fasting blood glucose levels, altered intraperitoneal and oral glucose tolerance, and significantly upregulated glucagon, somatostatin, pancreatic polypeptide, MafB, and Arx islet mRNAs. Additionally, Lhx1∆Panc mice exhibit significantly reduced Glp1R, an mRNA encoding the insulinotropic receptor for glucagon-like peptide 1 along with a concomitant dampened Glp1 response and mild glucose intolerance in mice challenged with oral glucose. These data are the first to reveal that the Lhx1 transcription factor contributes to normal glucose homeostasis and Glp1 responses.
Asunto(s)
Glucemia/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Factores de Transcripción/metabolismo , Animales , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/metabolismo , Técnicas de Silenciamiento del Gen , Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/genética , Proteínas de Homeodominio/genética , Homeostasis , Células Secretoras de Insulina/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteínas con Homeodominio LIM/genética , Factor de Transcripción MafB/genética , Ratones , Ratones Noqueados , Polipéptido Pancreático/genética , ARN Mensajero/metabolismo , Somatostatina/genética , Factores de Transcripción/genética , Regulación hacia ArribaRESUMEN
The two main types of diabetes mellitus have distinct etiologies, yet a similar outcome: loss of islet ß-cell function that is solely responsible for the secretion of the insulin hormone to reduce elevated plasma glucose toward euglycemic levels. Type 1 diabetes (T1D) has traditionally been characterized by autoimmune-mediated ß-cell death leading to insulin-dependence, whereas type 2 diabetes (T2D) has hallmarks of peripheral insulin resistance, ß-cell dysfunction, and cell death. However, a growing body of evidence suggests that, especially during T2D, key components of ß-cell failure involves: (1) loss of cell identity, specifically proteins associated with mature cell function (e.g., insulin and transcription factors like MAFA, PDX1, and NKX6.1), as well as (2) de-differentiation, defined by regression to a progenitor or stem cell-like state. New technologies have allowed the field to compare islet cell characteristics from normal human donors to those under pathophysiological conditions by single cell RNA-Sequencing and through epigenetic analysis. This has revealed a remarkable level of heterogeneity among histologically defined "insulin-positive" ß-cells. These results not only suggest that these ß-cell subsets have different responses to insulin secretagogues, but that defining their unique gene expression and epigenetic modification profiles will offer opportunities to develop cellular therapeutics to enrich/maintain certain subsets for correcting pathological glucose levels. In this review, we will summarize the recent literature describing how ß-cell heterogeneity and plasticity may be influenced in T2D, and various possible avenues of therapeutic intervention.
RESUMEN
Type 1 Diabetes (T1D) is a chronic pro-inflammatory autoimmune disease consisting of islet-infiltrating leukocytes involved in pancreatic ß-cell lysis. One promising treatment for T1D is islet transplantation; however, clinical application is constrained due to limited islet availability, adverse effects of immunosuppressants, and declining graft survival. Islet encapsulation may provide an immunoprotective barrier to preserve islet function and prevent immune-mediated rejection after transplantation. We previously demonstrated that a novel cytoprotective nanothin multilayer coating for islet encapsulation consisting of tannic acid (TA), an immunomodulatory antioxidant, and poly(N-vinylpyrrolidone) (PVPON), was efficacious in dampening in vitro immune responses involved in transplant rejection and preserving in vitro islet function. However, the ability of (PVPON/TA) to maintain islet function in vivo and reverse diabetes has not been tested. Recent evidence has demonstrated that modulation of redox status can affect pro-inflammatory immune responses. Therefore, we hypothesized that transplanted (PVPON/TA)-encapsulated islets can restore euglycemia to diabetic mice and provide an immunoprotective barrier. Our results demonstrate that (PVPON/TA) nanothin coatings can significantly decrease in vitro chemokine synthesis and diabetogenic T cell migration. Importantly, (PVPON/TA)-encapsulated islets restored euglycemia after transplantation into diabetic mice. Our results demonstrate that (PVPON/TA)-encapsulated islets may suppress immune responses and enhance islet allograft acceptance in patients with T1D.
Asunto(s)
Quimiocinas/biosíntesis , Materiales Biocompatibles Revestidos/farmacología , Mediadores de Inflamación/metabolismo , Islotes Pancreáticos/fisiología , Polifenoles/farmacología , Linfocitos T/citología , Animales , Antígeno B7-2/metabolismo , Biomarcadores/metabolismo , Quimiotaxis/efectos de los fármacos , Técnicas de Cocultivo , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/patología , Radicales Libres/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Trasplante de Islotes Pancreáticos , Activación de Macrófagos/efectos de los fármacos , Macrófagos/citología , Macrófagos/efectos de los fármacos , Ratones , Poli I-C/farmacología , Pirrolidinonas/síntesis química , Pirrolidinonas/química , Bazo/patología , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismoRESUMEN
The broadly expressed transcriptional coregulator LDB1 is essential for ß-cell development and glucose homeostasis. However, it is unclear whether LDB1 has metabolic roles beyond the ß-cell, especially under metabolic stress. Global Ldb1 deletion results in early embryonic lethality; thus, we used global heterozygous Ldb1+/- and inducible ß-cell-specific Ldb1-deficient (Ldb1Δß-cell) mice. We assessed glucose and insulin tolerance, body composition, feeding, and energy expenditure during high-fat diet exposure. Brown adipose tissue (BAT) biology was evaluated by thermogenic gene expression and LDB1 chromatin immunoprecipitation analysis. We found that partial loss of Ldb1 does not impair the maintenance of glucose homeostasis; rather, we observed improved insulin sensitivity in these mice. Partial loss of Ldb1 also uncovered defects in energy expenditure in lean and diet-induced obese (DIO) mice. This decreased energy expenditure during DIO was associated with significantly altered BAT gene expression, specifically Cidea, Elovl3, Cox7a1, and Dio2. Remarkably, the observed changes in energy balance during DIO were absent in Ldb1Δß-cell mice, despite a similar reduction in plasma insulin, suggesting a role for LDB1 in BAT. Indeed, LDB1 is expressed in brown adipocytes and occupies a regulatory domain of Elovl3, a gene crucial to normal BAT function. We conclude that LDB1 regulates energy homeostasis, in part through transcriptional modulation of critical regulators in BAT function.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Metabolismo Energético/genética , Homeostasis/genética , Proteínas con Dominio LIM/fisiología , Obesidad/genética , Tejido Adiposo Pardo/metabolismo , Animales , Proteínas de Unión al ADN/genética , Dieta Alta en Grasa , Regulación de la Expresión Génica , Heterocigoto , Proteínas con Dominio LIM/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Obesos , Ratones Transgénicos , Obesidad/etiología , Obesidad/metabolismo , Termogénesis/genéticaRESUMEN
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.
Asunto(s)
Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Células Secretoras de Insulina/metabolismo , Proteínas con Dominio LIM/metabolismo , Factores de Transcripción/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas de Unión al ADN/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Factor Nuclear 3-beta del Hepatocito/genética , Factor Nuclear 3-beta del Hepatocito/metabolismo , Proteína Homeobox Nkx-2.2 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Secretoras de Insulina/patología , Proteínas con Dominio LIM/genética , Proteínas con Homeodominio LIM/genética , Proteínas con Homeodominio LIM/metabolismo , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Proteínas de Pez CebraRESUMEN
Islet-1 (Isl1) is a Lin11, Isl1, Mec3 (LIM)-homeodomain transcription factor important for pancreatic islet cell development, maturation, and function, which largely requires interaction with the LIM domain-binding protein 1 (Ldb1) coregulator. In other tissues, Ldb1 and Isl1 interact with additional factors to mediate target gene transcription, yet few protein partners are known in ß-cells. Therefore, we hypothesize that Ldb1 and Isl1 participate in larger regulatory complexes to impact ß-cell gene expression. To test this, we used cross-linked immunoprecipitation and mass spectrometry to identify interacting proteins from mouse ß-cells. Proteomic datasets revealed numerous interacting candidates, including a member of the single-stranded DNA-binding protein (SSBP) coregulator family, SSBP3. SSBPs potentiate LIM transcription factor complex activity and stability in other tissues. However, nothing was known of SSBP3 interaction, expression, or activity in ß-cells. Our analyses confirmed that SSBP3 interacts with Ldb1 and Isl1 in ß-cell lines and in mouse and human islets and demonstrated SSBP3 coexpression with Ldb1 and Isl1 pancreas tissue. Furthermore, ß-cell line SSBP3 knockdown imparted mRNA deficiencies similar to those observed upon Ldb1 reduction in vitro or in vivo. This appears to be (at least) due to SSBP3 occupancy of known Ldb1-Isl1 target promoters, including MafA and Glp1r. This study collectively demonstrates that SSBP3 is a critical component of Ldb1-Isl1 regulatory complexes, required for expression of critical ß-cell target genes.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas con Dominio LIM/metabolismo , Proteínas con Homeodominio LIM/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Células Secretoras de Insulina , Proteínas con Dominio LIM/genética , Proteínas con Homeodominio LIM/genética , Ratones , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
Diabetes mellitus has rapidly become a 21st century epidemic with the promise to create vast economic and health burdens, if left unchecked. The 2 major forms of diabetes arise from unique causes, with outcomes being an absolute (type 1) or relative (type 2) loss of functional pancreatic islet ß-cell mass. Currently, patients rely on exogenous insulin and/or other pharmacologies that restore glucose homeostasis. Although these therapies have prolonged countless lives over the decades, the striking increases in both type 1 and type 2 diabetic diagnoses worldwide suggest a need for improved treatments. To this end, islet biologists are developing cell-based therapies by which a patient's lost insulin-producing ß-cell mass is replenished. Pancreatic or islet transplantation from cadaveric donors into diabetic patients has been successful, yet the functional islet demand far surpasses supply. Thus, the field has been striving toward transplantation of renewable in vitro-derived ß-cells that can restore euglycemia. Challenges have been numerous, but progress over the past decade has generated much excitement. In this review we will summarize recent findings that have placed us closer than ever to ß-cell replacement therapies. With the promise of cell-based diabetes therapies on the horizon, we will also provide an overview of cellular encapsulation technologies that will deliver critical protection of newly implanted cells.
Asunto(s)
Diferenciación Celular , Células Secretoras de Insulina/citología , Animales , Células Inmovilizadas/citología , Humanos , Células Madre Pluripotentes/citología , Trasplante de Células MadreRESUMEN
AIMS/HYPOTHESIS: Several forkhead box (FOX) transcription factor family members have important roles in controlling pancreatic cell fates and maintaining beta cell mass and function, including FOXA1, FOXA2 and FOXM1. In this study we have examined the importance of FOXP1, FOXP2 and FOXP4 of the FOXP subfamily in islet cell development and function. METHODS: Mice harbouring floxed alleles for Foxp1, Foxp2 and Foxp4 were crossed with pan-endocrine Pax6-Cre transgenic mice to generate single and compound Foxp mutant mice. Mice were monitored for changes in glucose tolerance by IPGTT, serum insulin and glucagon levels by radioimmunoassay, and endocrine cell development and proliferation by immunohistochemistry. Gene expression and glucose-stimulated hormone secretion experiments were performed with isolated islets. RESULTS: Only the triple-compound Foxp1/2/4 conditional knockout (cKO) mutant had an overt islet phenotype, manifested physiologically by hypoglycaemia and hypoglucagonaemia. This resulted from the reduction in glucagon-secreting alpha cell mass and function. The proliferation of alpha cells was profoundly reduced in Foxp1/2/4 cKO islets through the effects on mediators of replication (i.e. decreased Ccna2, Ccnb1 and Ccnd2 activators, and increased Cdkn1a inhibitor). Adult islet Foxp1/2/4 cKO beta cells secrete insulin normally while the remaining alpha cells have impaired glucagon secretion. CONCLUSIONS/INTERPRETATION: Collectively, these findings reveal an important role for the FOXP1, 2, and 4 proteins in governing postnatal alpha cell expansion and function.