ABSTRACT
Estrogen hormones play an important role in controlling glucose homeostasis and pancreatic ß-cell function. Despite the significance of estrogen hormones for regulation of glucose metabolism, little is known about the roles of endogenous estrogen metabolites in modulating pancreatic ß-cell function. In this study, we evaluated the effects of major natural estrogen metabolites, catechol estrogens, on insulin secretion in pancreatic ß-cells. We show that catechol estrogens, hydroxylated at positions C2 and C4 of the steroid A ring, rapidly potentiated glucose-induced insulin secretion via a nongenomic mechanism. 2-Hydroxyestrone, the most abundant endogenous estrogen metabolite, was more efficacious in stimulating insulin secretion than any other tested catechol estrogens. In insulin-secreting cells, catechol estrogens produced rapid activation of calcium influx and elevation in cytosolic free calcium. Catechol estrogens also generated sustained elevations in cytosolic free calcium and evoked inward ion current in HEK293 cells expressing the transient receptor potential A1 (TRPA1) cation channel. Calcium influx and insulin secretion stimulated by estrogen metabolites were dependent on the TRPA1 activity and inhibited with the channel-specific pharmacological antagonists or the siRNA. Our results suggest the role of estrogen metabolism in a direct regulation of TRPA1 activity with potential implications for metabolic diseases.
Subject(s)
Estrogens, Catechol/pharmacology , Gene Expression Regulation/drug effects , Insulin Secretion/drug effects , Insulin-Secreting Cells/metabolism , TRPA1 Cation Channel/metabolism , Animals , Cells, Cultured , Glucose/metabolism , Humans , Insulin-Secreting Cells/cytology , Insulin-Secreting Cells/drug effects , Male , Mice , Mice, Inbred C57BL , RatsABSTRACT
Extracellular ATP released from pancreatic ß-cells acts as a potent insulinotropic agent through activation of P2 purinergic receptors. Ectonucleotidases, a family of membrane-bound nucleotide-metabolizing enzymes, regulate extracellular ATP levels by degrading ATP and related nucleotides. Ectonucleotidase activity affects the relative proportion of ATP and its metabolites, which in turn will impact the level of purinergic receptor stimulation exerted by extracellular ATP. Therefore, we investigated the expression and role of ectonucleotidases in pancreatic ß-cells. Of the ectonucleotidases studied, only ENTPD3 (gene encoding the NTPDase3 enzyme) mRNA was detected at fairly abundant levels in human and mouse pancreatic islets as well as in insulin-secreting MIN6 cells. ARL67156, a selective ectonucleotidase inhibitor, blocked degradation of extracellular ATP that was added to MIN6 cells. The compound also decreased degradation of endogenous ATP released from cells. Measurements of insulin secretion in MIN6 cells as well as in mouse and human pancreatic islets demonstrated that ARL67156 potentiated glucose-dependent insulin secretion. Downregulation of NTPDase3 expression in MIN6 cells with the specific siRNA replicated the effects of ARL67156 on extracellular ATP hydrolysis and insulin secretion. Our results demonstrate that NTPDase3 is the major ectonucleotidase in pancreatic ß-cells in multiple species and that it modulates insulin secretion by controlling activation of purinergic receptors.
Subject(s)
Glucose/metabolism , Insulin-Secreting Cells/enzymology , Insulin/metabolism , Pyrophosphatases/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenosine Triphosphate/pharmacology , Animals , Cells, Cultured , Enzyme Inhibitors/pharmacology , Glucose/pharmacology , Humans , Insulin Secretion , Insulin-Secreting Cells/chemistry , Insulin-Secreting Cells/drug effects , Male , Mice , Mice, Inbred C57BL , Pyrophosphatases/analysis , Pyrophosphatases/antagonists & inhibitors , RNA, Messenger/analysis , RNA, Messenger/metabolism , Tissue DistributionABSTRACT
We identify biomarkers for disease progression in three type 2 diabetes cohorts encompassing 2,973 individuals across three molecular classes, metabolites, lipids and proteins. Homocitrulline, isoleucine and 2-aminoadipic acid, eight triacylglycerol species, and lowered sphingomyelin 42:2;2 levels are predictive of faster progression towards insulin requirement. Of ~1,300 proteins examined in two cohorts, levels of GDF15/MIC-1, IL-18Ra, CRELD1, NogoR, FAS, and ENPP7 are associated with faster progression, whilst SMAC/DIABLO, SPOCK1 and HEMK2 predict lower progression rates. In an external replication, proteins and lipids are associated with diabetes incidence and prevalence. NogoR/RTN4R injection improved glucose tolerance in high fat-fed male mice but impaired it in male db/db mice. High NogoR levels led to islet cell apoptosis, and IL-18R antagonised inflammatory IL-18 signalling towards nuclear factor kappa-B in vitro. This comprehensive, multi-disciplinary approach thus identifies biomarkers with potential prognostic utility, provides evidence for possible disease mechanisms, and identifies potential therapeutic avenues to slow diabetes progression.
Subject(s)
Diabetes Mellitus, Type 2 , Islets of Langerhans , Mice , Animals , Male , Diabetes Mellitus, Type 2/metabolism , Blood Glucose/metabolism , Islets of Langerhans/metabolism , Insulin/metabolism , Lipids , Biomarkers/metabolism , Cell Adhesion Molecules/metabolism , Extracellular Matrix Proteins/metabolismABSTRACT
The GPR119 receptor plays an important role in the secretion of incretin hormones in response to nutrient consumption. We have studied the ability of an array of naturally occurring endocannabinoid-like lipids to activate GPR119 and have identified several lipid receptor agonists. The most potent receptor agonists identified were three N-acylethanolamines: oleoylethanolamine (OEA), palmitoleoylethanolamine, and linoleylethanolamine (LEA), all of which displayed similar potency in activating GPR119. Another lipid, 2-oleoylglycerol (2-OG), also activated GPR119 receptor but with significantly lower potency. Endogenous levels of endocannabinoid-like lipids were measured in intestine in fasted and refed mice. Of the lipid GPR119 agonists studied, the intestinal levels of only OEA, LEA, and 2-OG increased significantly upon refeeding. Intestinal levels of OEA and LEA in the fasted mice were low. In the fed state, OEA levels only moderately increased, whereas LEA levels rose drastically. 2-OG was the most abundant of the three GPR119 agonists in intestine, and its levels were radically elevated in fed mice. Our data suggest that, in lean mice, 2-OG and LEA may serve as physiologically relevant endogenous GPR119 agonists that mediate receptor activation upon nutrient uptake.
Subject(s)
Cannabinoid Receptor Agonists/metabolism , Endocannabinoids/metabolism , Receptors, G-Protein-Coupled/agonists , Amides , Animals , Cannabinoid Receptor Agonists/chemistry , Cannabinoid Receptor Agonists/pharmacology , Cannabinoid Receptor Antagonists/pharmacology , Cell Line , Endocannabinoids/antagonists & inhibitors , Endocrine Cells/drug effects , Endocrine Cells/metabolism , Ethanolamines/antagonists & inhibitors , Ethanolamines/metabolism , Fasting/metabolism , Glucagon-Like Peptide 1/metabolism , Glycerides/antagonists & inhibitors , Glycerides/metabolism , Humans , Intestinal Mucosa/metabolism , Male , Mice , Mice, Inbred C57BL , Oleic Acids/antagonists & inhibitors , Oleic Acids/metabolism , Organ Specificity , Palmitic Acids/antagonists & inhibitors , Palmitic Acids/metabolism , Random Allocation , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Recombinant Proteins/agonists , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/metabolism , Thinness/metabolism , Up-RegulationABSTRACT
Phenotypic screening is a neoclassical approach for drug discovery. We conducted phenotypic screening for insulin secretion enhancing agents using INS-1E insulinoma cells as a model system for pancreatic beta-cells. A principal regulator of insulin secretion in beta-cells is the metabolically regulated potassium channel Kir6.2/SUR1 complex. To characterize hit compounds, we developed an assay to quantify endogenous potassium channel activity in INS-1E cells. We quantified ligand-regulated potassium channel activity in INS-1E cells using fluorescence imaging and thallium flux. Potassium channel activity was metabolically regulated and coupled to insulin secretion. The pharmacology of channel opening agents (diazoxide) and closing agents (sulfonylureas) was used to validate the applicability of the assay. A precise high-throughput assay was enabled, and phenotypic screening hits were triaged to enable a higher likelihood of discovering chemical matter with novel and useful mechanisms of action.
Subject(s)
Diazoxide/pharmacology , Insulin-Secreting Cells/drug effects , Potassium Channels, Inwardly Rectifying/metabolism , Secretagogues/pharmacology , Sulfonylurea Compounds/pharmacology , Sulfonylurea Receptors/metabolism , Cells, Cultured , Drug Evaluation, Preclinical , High-Throughput Screening Assays , Humans , Insulin Secretion/drug effects , Insulin-Secreting Cells/metabolism , Optical Imaging , PhenotypeABSTRACT
Type 2 diabetes (T2D) is associated with defective insulin secretion and reduced ß cell mass. Available treatments provide a temporary reprieve, but secondary failure rates are high, making insulin supplementation necessary. Reversibility of ß cell failure is a key translational question. Here, we reverse engineered and interrogated pancreatic islet-specific regulatory networks to discover T2D-specific subpopulations characterized by metabolic inflexibility and endocrine progenitor/stem cell features. Single-cell gain- and loss-of-function and glucose-induced Ca2+ flux analyses of top candidate master regulatory (MR) proteins in islet cells validated transcription factor BACH2 and associated epigenetic effectors as key drivers of T2D cell states. BACH2 knockout in T2D islets reversed cellular features of the disease, restoring a nondiabetic phenotype. BACH2-immunoreactive islet cells increased approximately 4-fold in diabetic patients, confirming the algorithmic prediction of clinically relevant subpopulations. Treatment with a BACH inhibitor lowered glycemia and increased plasma insulin levels in diabetic mice, and restored insulin secretion in diabetic mice and human islets. The findings suggest that T2D-specific populations of failing ß cells can be reversed and indicate pathways for pharmacological intervention, including via BACH2 inhibition.
Subject(s)
Basic-Leucine Zipper Transcription Factors/antagonists & inhibitors , Basic-Leucine Zipper Transcription Factors/metabolism , Calcium Signaling , Diabetes Mellitus, Type 2/metabolism , Epigenesis, Genetic , Insulin-Secreting Cells/metabolism , Basic-Leucine Zipper Transcription Factors/genetics , Diabetes Mellitus, Type 2/drug therapy , Diabetes Mellitus, Type 2/genetics , HEK293 Cells , HumansABSTRACT
OBJECTIVE: GPR142 agonists are being pursued as novel diabetes therapies by virtue of their insulin secretagogue effects. But it is undetermined whether GPR142's functions in pancreatic islets are limited to regulating insulin secretion. The current study expands research on its action. METHODS AND RESULTS: We demonstrated by in situ hybridization and immunostaining that GPR142 is expressed not only in ß cells but also in a subset of α cells. Stimulation of GPR142 by a selective agonist increased glucagon secretion in both human and mouse islets. More importantly, the GPR142 agonist also potentiated glucagon-like peptide-1 (GLP-1) production and its release from islets through a mechanism that involves upregulation of prohormone convertase 1/3 expression. Strikingly, stimulation of insulin secretion and increase in insulin content via GPR142 engagement requires intact GLP-1 receptor signaling. Furthermore, GPR142 agonist increased ß cell proliferation and protected both mouse and human islets against stress-induced apoptosis. CONCLUSIONS: Collectively, we provide here evidence that local GLP-1 release from α cells defines GPR142's beneficial effects on improving ß cell function and mass, and we propose that GPR142 agonism may have translatable and durable efficacy for the treatment of type 2 diabetes.
Subject(s)
Glucagon-Like Peptide 1/metabolism , Glucagon-Secreting Cells/metabolism , Insulin-Secreting Cells/metabolism , Receptors, G-Protein-Coupled/metabolism , Animals , Apoptosis , Cell Proliferation , Cells, Cultured , Glucagon-Like Peptide-1 Receptor/metabolism , Humans , Insulin Secretion , Insulin-Secreting Cells/physiology , Male , Mice , Mice, Inbred C57BL , Proprotein Convertase 1/metabolismABSTRACT
Adiponectin/adiponectin receptors (AdipoR) are involved in energy homeostasis and inflammatory pathways. To investigate the role of AdipoR2 in metabolic control, we studied the lipid and glucose metabolic phenotypes in AdipoR2-deficient mice. AdipoR2 deletion diminished high-fat diet-induced dyslipidemia and insulin resistance yet deteriorated glucose homeostasis as high-fat feeding continued, which resulted from the failure of pancreatic beta-cells to adequately compensate for the moderate insulin resistance. A defect in the AdipoR2 gene may represent a mechanism underlying the etiology of certain subgroups of type 2 diabetic patients who eventually develop overt diabetes, whereas other obese patients do not.
Subject(s)
Diabetes Mellitus, Type 2/etiology , Diet , Insulin Resistance , Receptors, Cell Surface/deficiency , Animals , Blood Glucose/metabolism , Dietary Fats/administration & dosage , Dietary Fats/pharmacology , Dose-Response Relationship, Drug , Dyslipidemias/physiopathology , Energy Intake , Glucose/metabolism , Insulin-Secreting Cells/metabolism , Lipid Metabolism/genetics , Liver/metabolism , Male , Mice , Mice, Knockout , Obesity/blood , Obesity/physiopathology , Receptors, Adiponectin , Weight GainABSTRACT
Incretin and insulin responses to nutrient loads are suppressed in persons with diabetes, resulting in decreased glycemic control. Agents including sulfonylureas and dipeptidyl peptidase-4 inhibitors (DPP4i) partially reverse these effects and provide therapeutic benefit; however, their modes of action limit efficacy. Because somatostatin (SST) has been shown to suppress insulin and glucagonlike peptide-1 (GLP-1) secretion through the Gi-coupled SST receptor 5 (SSTR5) isoform in vitro, antagonism of SSTR5 may improve glycemic control via intervention in both pathways. Here, we show that a potent and selective SSTR5 antagonist reverses the blunting effects of SST on insulin secretion from isolated human islets, and demonstrate that SSTR5 antagonism affords increased levels of systemic GLP-1 in vivo. Knocking out Sstr5 in mice provided a similar increase in systemic GLP-1 levels, which were not increased further by treatment with the antagonist. Treatment of mice with the SSTR5 antagonist in combination with a DPP4i resulted in increases in systemic GLP-1 levels that were more than additive and resulted in greater glycemic control compared with either agent alone. In isolated human islets, the SSTR5 antagonist completely reversed the inhibitory effect of exogenous SST-14 on insulin secretion. Taken together, these data suggest that SSTR5 antagonism should increase circulating GLP-1 levels and stimulate insulin secretion (directly and via GLP-1) in humans, improving glycemic control in patients with diabetes.
Subject(s)
Benzoates/pharmacology , Glucagon-Like Peptide 1/metabolism , Hypoglycemic Agents/pharmacology , Insulin/metabolism , Islets of Langerhans/drug effects , Receptors, Somatostatin/antagonists & inhibitors , Spiro Compounds/pharmacology , Animals , CHO Cells , Cells, Cultured , Cricetinae , Cricetulus , HEK293 Cells , Humans , Insulin Secretion , Islets of Langerhans/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Rats , Rats, Sprague-Dawley , Rats, Zucker , Receptors, Somatostatin/genetics , Secretory Pathway/drug effectsABSTRACT
Prostaglandins E1 and E2 are synthesized in the intestine and mediate a range of gastrointestinal functions via activation of the prostanoid E type (EP) family of receptors. We examined the potential role of EP receptors in the regulation of gut hormone secretion from L cells. Analysis of mRNA expression in mouse enteroendocrine GLUTag cells demonstrated the abundant expression of EP4 receptor, whereas expression of other EP receptors was much lower. Prostaglandin E1 and E2, nonselective agonists for all EP receptor subtypes, triggered glucagon like peptide 1 (GLP-1) secretion from GLUTag cells, as did the EP4-selective agonists CAY10580 and TCS2510. The effect of EP4 agonists on GLP-1 secretion was blocked by incubation of cells with the EP4-selective antagonist L161,982 or by down-regulating EP4 expression with specific small interfering RNA. Regulation of gut hormone secretion with EP4 agonists was further studied in mice. Administration of EP4 agonists to mice produced a significant elevation of plasma levels of GLP-1, glucagon like peptide 2 (GLP-2) and peptide YY (PYY), whereas gastric inhibitory peptide (GIP) levels were not increased. Thus, our data demonstrate that activation of the EP4 receptor in enteroendocrine L cells triggers secretion of gut hormones.
Subject(s)
Glucagon-Like Peptide 1/blood , Glucagon-Like Peptide 2/blood , Peptide YY/blood , Receptors, Prostaglandin E, EP4 Subtype/metabolism , Animals , Cells, Cultured , Gastric Inhibitory Polypeptide/blood , Intestinal Mucosa/metabolism , Mice , Real-Time Polymerase Chain Reaction , Receptors, Prostaglandin E, EP4 Subtype/antagonists & inhibitors , Receptors, Prostaglandin E, EP4 Subtype/genetics , Thiophenes/pharmacology , Triazoles/pharmacologyABSTRACT
The Drosophila ovary is a model system for examining the genetic control of epithelial morphogenesis. The somatic follicle cells form a polarized epithelium surrounding the 16-cell germ line cyst. The integrity of this epithelium is essential for the successful completion of oogenesis. Reciprocal signaling between germ line and somatic cells establishes embryonic and eggshell polarity. The follicle cells are responsible for shaping the egg and secreting the eggshell. Follicle cells at the boundary between the nurse cells and the oocyte migrate centripetally to cover the anterior end of the oocyte and secrete the operculum. Dorsal anterior main body follicle cells undergo elaborate patterning to produce the dorsal appendages. We have examined the expression of the Toll-like receptor, 18-wheeler (18w), in the ovary and find it to be restricted to subpopulations of follicle cells. Females carrying loss-of-function 18w mutant clones in their ovaries show delayed follicle cell migrations. The eggs laid by such females also show morphological defects in egg shape and dorsal appendage morphology. We propose that the 18W protein plays an adhesive or signaling role in regions of the epithelium engaged in cell migration.