ABSTRACT
Sulprostone is a potent prostaglandin E2 (PGE2) analogue and one of the first identified selective G-protein-coupled receptor 3 (EP3) agonists. It has been investigated as a potential antiulcer agent and frequently used in the research of EP3 antagonist. To assist pharmacokinetic and pharmacodynamic studies, a rapid and sensitive LC-MS/MS method was developed and qualified for the quantitation of sulprostone in monkey plasma. Using electrospray ionization mass spectrometry, an ammonium adduct in positive mode was chosen for analysis which had seven times of the sensitivity of the depronated ion in negative mode. Latanoprost, a prostaglandin F2α analogue, was used as the internal standard while good sensitivity and chromatography were obtained on a 2.6⯵m core-shell column with pentafluorophenyl stationary phase. An assay dynamic range of 2 to 4000â¯ng/mL was achieved with a sample volume of 25⯵L plasma on a Sciex API4000 instrument with simple protein precipitation. Several esterase inhibitors including sodium fluoride (NaF), phenylmethanesulfonyl fluoride (PMSF), diisopropylfluorophosphate (DFP), paraoxon and dichlorvos as well as wet ice conditions were explored for the stabilization of sulprostone in monkey plasma. The developed method was successfully applied for the evaluation of pharmacokinetics of sulprostone after intravenous administration of 0.5â¯mg/kg to cynomolgus monkey.
Subject(s)
Chromatography, Liquid/methods , Dinoprostone/analogs & derivatives , Tandem Mass Spectrometry/methods , Animals , Dinoprostone/blood , Dinoprostone/chemistry , Dinoprostone/pharmacokinetics , Drug Stability , Linear Models , Macaca fascicularis , Male , Receptors, Prostaglandin E, EP3 Subtype/agonists , Reproducibility of Results , Sensitivity and SpecificityABSTRACT
Insulin secretion from ß cells of the pancreatic islets of Langerhans controls metabolic homeostasis and is impaired in individuals with type 2 diabetes (T2D). Increases in blood glucose trigger insulin release by closing ATP-sensitive K+ channels, depolarizing ß cells, and opening voltage-dependent Ca2+ channels to elicit insulin exocytosis. However, one or more additional pathway(s) amplify the secretory response, likely at the distal exocytotic site. The mitochondrial export of isocitrate and engagement with cytosolic isocitrate dehydrogenase (ICDc) may be one key pathway, but the mechanism linking this to insulin secretion and its role in T2D have not been defined. Here, we show that the ICDc-dependent generation of NADPH and subsequent glutathione (GSH) reduction contribute to the amplification of insulin exocytosis via sentrin/SUMO-specific protease-1 (SENP1). In human T2D and an in vitro model of human islet dysfunction, the glucose-dependent amplification of exocytosis was impaired and could be rescued by introduction of signaling intermediates from this pathway. Moreover, islet-specific Senp1 deletion in mice caused impaired glucose tolerance by reducing the amplification of insulin exocytosis. Together, our results identify a pathway that links glucose metabolism to the amplification of insulin secretion and demonstrate that restoration of this axis rescues ß cell function in T2D.
Subject(s)
Diabetes Mellitus, Type 2/physiopathology , Endopeptidases/physiology , Insulin/metabolism , Islets of Langerhans/metabolism , Isocitrates/metabolism , Animals , Catalytic Domain , Cell Membrane/metabolism , Cysteine Endopeptidases , Diabetes Mellitus, Type 2/pathology , Endopeptidases/biosynthesis , Endopeptidases/deficiency , Endopeptidases/genetics , Exocytosis/drug effects , Exocytosis/physiology , Gene Knockout Techniques , Glucose/metabolism , Glucose/pharmacology , Glutathione/pharmacology , HEK293 Cells , Homeostasis , Humans , Insulin/pharmacology , Insulin Secretion , Islets of Langerhans/physiopathology , Isocitrate Dehydrogenase/physiology , Isocitrates/pharmacology , Male , Membrane Potentials , Mice , Mice, Inbred C57BL , NADP/metabolism , Organ Specificity , RNA Interference , Recombinant Fusion Proteins/metabolism , Secretory Vesicles/metabolism , Signal Transduction , SumoylationABSTRACT
We have used a previously unavailable model of pancreatic development, derived in vitro from human embryonic stem cells, to capture a time-course of gene, miRNA and histone modification levels in pancreatic endocrine cells. We investigated whether it is possible to better understand, and hence control, the biological pathways leading to pancreatic endocrine formation by analysing this information and combining it with the available scientific literature to generate models using a casual reasoning approach. We show that the embryonic stem cell differentiation protocol is highly reproducible in producing endocrine precursor cells and generates cells that recapitulate many aspects of human embryonic pancreas development, including maturation into functional endocrine cells when transplanted into recipient animals. The availability of whole genome gene and miRNA expression data from the early stages of human pancreatic development will be of great benefit to those in the fields of developmental biology and diabetes research. Our causal reasoning algorithm suggested the involvement of novel gene networks, such as NEUROG3/E2F1/KDM5B and SOCS3/STAT3/IL-6, in endocrine cell development We experimentally investigated the role of the top-ranked prediction by showing that addition of exogenous IL-6 could affect the expression of the endocrine progenitor genes NEUROG3 and NKX2.2.
Subject(s)
Cell Differentiation/genetics , Cell Lineage/genetics , Gene Regulatory Networks , Islets of Langerhans/metabolism , Algorithms , Animals , Gene Expression Profiling , Gene Expression Regulation, Developmental , Glucose Tolerance Test , Homeobox Protein Nkx-2.2 , Homeodomain Proteins , Humans , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/metabolism , Interleukin-6/genetics , Interleukin-6/metabolism , Islets of Langerhans/embryology , Mice , Nuclear Proteins , Transcription FactorsABSTRACT
Orphan G protein-coupled receptors (oGPCRs) are a class of integral membrane proteins for which endogenous ligands or transmitters have not yet been discovered. Transgenic animal technologies have uncovered potential roles for many of these oGPCRs, providing new targets for the treatment of various diseases. Understanding signaling pathways of oGPCRs and validating these receptors as potential drug targets requires the identification of chemical probe compounds to be used in place of endogenous ligands to interrogate these receptors. A novel chemical probe identification platform was created in which GPCR-focused libraries were screened against sets of oGPCR targets, with a goal of discovering fit-for-purpose chemical probes for the more druggable members of the set. Application of the platform to a set of oGPCRs resulted in the discovery of the first reported small molecule agonists for GPR39, a receptor implicated in the regulation of insulin secretion and preservation of beta cells in the pancreas. Compound 1 stimulated intracellular calcium mobilization in recombinant and native cells in a GPR39-specific manner but did not potentiate glucose-stimulated insulin secretion in human islet preparations.
ABSTRACT
Insulin receptor substrate-2 (Irs2) integrates insulin-like signals with glucose and cAMP agonists to regulate beta-cell growth, function, and survival. This study investigated whether increased Irs2 concentration in beta-cells could reduce beta-cell destruction and the incidence of type 1 diabetes in nonobese diabetic (NOD) mice. NOD mice were intercrossed with C57BL/6 mice overexpressing Irs2 specifically in beta-cells to create NOD(Irs2) mice. After backcrossing NOD(Irs2) mice for 12 generations, glucose homeostasis and diabetes incidence were compared against NOD littermates. Compared with 12-wk-old NOD mice, the progression of severe insulitis was reduced and islet mass was increased in NOD(Irs2) mice. Moreover, the risk of diabetes decreased 50% in NOD(Irs2) mice until the experiment was terminated at 40 wk of age. Nondiabetic NOD(Irs2) mice displayed better glucose tolerance than nondiabetic NOD mice throughout the duration of the study and up to the age of 18 months. The effect of Irs2 to increase islet mass and improve glucose tolerance raised the possibility that NOD(Irs2) mice might have an increased capacity to respond to anti-CD3 antibody, which can induce remission of overt diabetes in some NOD mice. Anti-CD3 antibody injections restored glucose tolerance in newly diabetic NOD and NOD(Irs2) mice; however, anti-CD3-treated NOD(Irs2) mice were less likely than NOD mice to relapse during the experimental period because they displayed 10-fold greater beta-cell mass and mitogenesis. In conclusion, increased Irs2 attenuated the progression of beta-cell destruction, promoted beta-cell mitogenesis, and reduced diabetes incidence in NOD(Irs2) mice.
Subject(s)
Diabetes Mellitus, Type 1/metabolism , Insulin Receptor Substrate Proteins/metabolism , Insulin-Secreting Cells/physiology , Animals , CD3 Complex/immunology , Diabetes Mellitus, Type 1/immunology , Diabetes Mellitus, Type 1/therapy , Glucose/metabolism , Immunotherapy , Mice , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Transgenic , MitosisABSTRACT
The human GH family consists of five genes, including the placental chorionic somatomammotropins (CS), within a single locus on chromosome 17. Based on nuclease sensitivity, the entire GH/CS locus is accessible in pituitary chromatin, yet only GH-N is expressed. Previously, we reported a P sequence element (263P) capable of repressing placental CS-A promoter activity in transfected pituitary (GC) cells, and our data indicated a possible role for nuclear factor-1 (NF-1) and regulatory factor X1 in this repression. In this study we show the formation of two independent pituitary complexes in vitro: a repressor complex containing NF-1 and a nonfunctional complex containing regulatory factor X1. In vitro repressor function is stabilized by the presence of P sequence element C (PSE-C), downstream of the previously characterized PSE-A and PSE-B. Repressor function is also dependent on an intact Pit-1 binding site in the CS-A promoter. EMSAs with PSE-C reveal binding of the hepatocyte nuclear factor-3/forkhead (HNF-3/fkh) family of transcription factors in rat pituitary GC cells. This observation is extended to human pituitary tissue, where HNF-3alpha's association with P sequences is confirmed by chromatin immunoprecipitation. Furthermore, protein-protein interactions between HNF-3alpha and NF-1 family members are demonstrated. These results identify HNF-3alpha as an additional member of the pituitary P sequence regulatory complex, implicating it in tissue-specific expression of the human GH/CS family.
Subject(s)
Hepatocyte Nuclear Factor 3-alpha/metabolism , Human Growth Hormone/metabolism , Pituitary Gland/metabolism , Animals , Base Sequence , Binding Sites , CCAAT-Enhancer-Binding Proteins/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , Cells, Cultured , Chromatin/genetics , Chromatin/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Hepatocyte Nuclear Factor 3-alpha/genetics , Human Growth Hormone/genetics , Humans , Molecular Sequence Data , Multiprotein Complexes , NFI Transcription Factors/genetics , NFI Transcription Factors/metabolism , Pituitary Gland/cytology , Placental Lactogen/genetics , Placental Lactogen/metabolism , Promoter Regions, Genetic , Rats , Regulatory Factor X Transcription Factors , Regulatory Sequences, Nucleic Acid , Transcription Factor Pit-1/genetics , Transcription Factor Pit-1/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The human GH gene family is specifically expressed in somatotrophs of the anterior pituitary and placental syncytiotrophoblast. Two nuclease-hypersensitive sites, HS III and HS V, are associated with a region of chromatin located 28 and 30 kb upstream of the pituitary GH gene transcription initiation site (+1) in both pituitary and placenta nuclei. A role for this region in pituitary GH gene expression has been reported, but the potential relevance to placental gene expression has not been determined. Deletion analysis of a 5.2-kb region (nucleotides - 27,568/-32,746) containing HS III to V-related sequences localized significant enhancer activity to a 574-bp HS III fragment (nucleotides -27,676/-28,249) in multiple transfected cell lines. Four nuclease-protected regions [footprints (FP) 1-4] were identified in the 574-bp fragment. FP2 and FP3 were detected with placenta cell nuclear protein, whereas FP1 and FP4 were observed with placental and nonplacental cell nuclear extract. Disruption of FP1 had no effect on heterologous promoter activity in transfected pituitary and placental cells, whereas loss of FP2 and FP3 resulted in modest increases in placental cells, reflecting the presence of repressor activity associated with these regions in vitro. In contrast, disruption of the FP4 region by mutation or deletion significantly reduced enhancer activity. As a result, 30-fold enhancer activity was localized to a 41-bp region in transfected placental tumor cells. Binding of candidate proteins, activator protein (AP)-2 (FP3) and Elk-1 (FP4), was confirmed using competition assays with specific oligonucleotides and antibodies. Moreover, these factors were associated with the hyperacetylated HS III region in human pituitary [activator protein 2 (AP-2) and Elk-1] and term placenta (AP-2) chromatin. These data implicate AP-2 and ETS-domain family members in the regulation of the GH/CS locus and raise the possibility that different complexes form in the HS III region in placenta and pituitary cells.
Subject(s)
DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Human Growth Hormone/genetics , Placental Lactogen/genetics , Proto-Oncogene Proteins/metabolism , Transcription Factors/metabolism , Acetylation , Base Sequence , Binding Sites , Cell Line , Chromatin/genetics , Chromatin/metabolism , DNA/metabolism , DNA-Binding Proteins/genetics , Female , Human Growth Hormone/metabolism , Humans , Molecular Sequence Data , Mutation , Pituitary Gland/physiology , Placenta/cytology , Placenta/metabolism , Placental Lactogen/metabolism , Pregnancy , Protein Structure, Tertiary , Proto-Oncogene Proteins/genetics , Regulatory Sequences, Nucleic Acid , Transcription Factor AP-2 , Transcription Factors/genetics , ets-Domain Protein Elk-1ABSTRACT
The human GH family consists of five genes, including the placental chorionic somatomammotropins (CS), within a single locus on chromosome 17. Based on nuclease sensitivity, the entire GH/CS locus is accessible in pituitary chromatin, yet only GH-N is expressed. Previously, we reported a P sequence element (263P) capable of repressing placental CS promoter activity in transfected pituitary (GC) cells. Regions of protein binding within 263P include P sequence elements A and B (PSE-A and PSE-B), and we reported nuclear factor-1 (NF-1) recognition of PSE-B. We now provide evidence for multiple interactions on PSE-A, including binding of the regulatory factor X (RFX) family. Disruption of the RFX site within 263P blunts repressor activity in transfected GC cells; however, repression is only abolished when both PSE-A/RFX and PSE-B/NF-1 sites are mutated. The capacity of RFX and NF-1 to participate in a novel common complex is further suggested by coimmunoprecipitation of RFX1 and epitope-tagged NF-1 family members. Finally, we confirm the association of NF-1 and RFX1 with P sequences in human pituitary tissue by chromatin immunoprecipitation. Taken together, our data suggest that an inverse relationship exists between 263P and CS promoter histone hyperacetylation and the association of these factors in vivo.
Subject(s)
CCAAT-Enhancer-Binding Proteins/metabolism , Chromatin/metabolism , DNA-Binding Proteins/metabolism , Human Growth Hormone/genetics , Pituitary Gland/metabolism , Transcription Factors/metabolism , Animals , Base Sequence , Binding Sites/physiology , Gene Expression Regulation/physiology , Humans , In Vitro Techniques , Molecular Sequence Data , NFI Transcription Factors , Nuclear Proteins , Oligonucleotide Probes , Placental Hormones/genetics , Placental Hormones/metabolism , Promoter Regions, Genetic/physiology , Rats , Regulatory Factor X Transcription Factors , Regulatory Factor X1 , Y-Box-Binding Protein 1ABSTRACT
The prevalence of type 2 diabetes mellitus in the Oji-Cree of northwestern Ontario is the third highest in the world. A private mutation, G319S, in HNF1A, which encodes hepatic nuclear factor-1alpha (HNF-1alpha), was associated with Oji-Cree type 2 diabetes and was found in approximately 40% of affected subjects. The G319S mutation reduced the in vitro ability of HNF-1alpha to activate transcription by approximately 50%, with no effect on DNA binding or protein stability. There was no evidence of a dominant negative effect of the mutant protein. The impact of the G319S mutation at the population level was assessed by classifying subjects with type 2 diabetes according to HNF1A genotype and plotting the cumulative age of onset of diabetes. Disease onset was modeled satisfactorily by two-parameter sigmoidal functions for all diabetic subjects and all three HNF1A genotypes. Pairwise statistical comparisons showed significant between-genotype differences in t50 (all P < 0.00001), corresponding to the age at which half the subjects had become diabetic. Each dose of G319S accelerated median disease onset by approximately 7 years. Thus, the transactivation-deficient HNF1A G319S mutation affects the dynamics of disease onset. The demonstration of a functional consequence for HNF1A G319S provides a mechanistic basis for its strong association with Oji-Cree type 2 diabetes and its unparalleled specificity for diabetes prediction in these people, in whom diabetes presents a significant public health dilemma. The findings also show that HNF1A mutations can be associated with typical adult-onset insulin-resistant obesity-related diabetes in addition to maturity-onset diabetes of the young.