RESUMO
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.
Assuntos
Estrogênios de Catecol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Canal de Cátion TRPA1/metabolismo , Animais , Células Cultivadas , Glucose/metabolismo , Humanos , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , RatosRESUMO
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.
Assuntos
Benzoatos/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Hipoglicemiantes/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Receptores de Somatostatina/antagonistas & inibidores , Compostos de Espiro/farmacologia , Animais , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Células HEK293 , Humanos , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Ratos Sprague-Dawley , Ratos Zucker , Receptores de Somatostatina/genética , Via Secretória/efeitos dos fármacosRESUMO
The G protein-coupled receptor 40 (GPR40) also known as free fatty acid receptor 1 (FFAR1) is highly expressed in pancreatic, islet ß-cells and responds to endogenous fatty acids, resulting in amplification of insulin secretion only in the presence of elevated glucose levels. Hypothesis driven structural modifications to endogenous FFAs, focused on breaking planarity and reducing lipophilicity, led to the identification of spiropiperidine and tetrahydroquinoline acid derivatives as GPR40 agonists with unique pharmacology, selectivity, and pharmacokinetic properties. Compounds 1 (LY2881835), 2 (LY2922083), and 3 (LY2922470) demonstrated potent, efficacious, and durable dose-dependent reductions in glucose levels along with significant increases in insulin and GLP-1 secretion during preclinical testing. A clinical study with 3 administered to subjects with T2DM provided proof of concept of 3 as a potential glucose-lowering therapy. This manuscript summarizes the scientific rationale, medicinal chemistry, preclinical, and early development data of this new class of GPR40 agonists.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Descoberta de Drogas , Hipoglicemiantes/farmacologia , Piperidinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Compostos de Espiro/farmacologia , Animais , Relação Dose-Resposta a Droga , Teste de Tolerância a Glucose , Células HEK293 , Humanos , Hipoglicemiantes/síntese química , Hipoglicemiantes/química , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Estrutura Molecular , Piperidinas/síntese química , Piperidinas/química , Ratos , Ratos Zucker , Compostos de Espiro/síntese química , Compostos de Espiro/química , Relação Estrutura-AtividadeRESUMO
Vertical sleeve gastrectomy (VSG) produces high rates of type 2 diabetes remission; however, the mechanisms responsible for this remain incompletely defined. Glucagon-like peptide-1 (GLP-1) is a gut hormone that contributes to the maintenance of glucose homeostasis and is elevated after VSG. VSG-induced increases in postprandial GLP-1 secretion have been proposed to contribute to the glucoregulatory benefits of VSG; however, previous work has been equivocal. In order to test the contribution of enhanced ß-cell GLP-1 receptor (GLP-1R) signaling we used a ß-cell-specific tamoxifen-inducible GLP-1R knockout mouse model. Male ß-cell-specific Glp-1r(ß-cell+/+) wild type (WT) and Glp-1r(ß-cell-/-) knockout (KO) littermates were placed on a high-fat diet for 6 weeks and then switched to high-fat diet supplemented with tamoxifen for the rest of the study. Mice underwent sham or VSG surgery after 2 weeks of tamoxifen diet and were fed ad libitum postoperatively. Mice underwent oral glucose tolerance testing at 3 weeks and were euthanized at 6 weeks after surgery. VSG reduced body weight and food intake independent of genotype. However, glucose tolerance was only improved in VSG WT compared with sham WT, whereas VSG KO had impaired glucose tolerance relative to VSG WT. Augmentation of glucose-stimulated insulin secretion during the oral glucose tolerance test was blunted in VSG KO compared with VSG WT. Therefore, our data suggest that enhanced ß-cell GLP-1R signaling contributes to improved glucose regulation after VSG by promoting increased glucose-stimulated insulin secretion.
Assuntos
Gastrectomia , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Transtornos do Metabolismo de Glucose/cirurgia , Células Secretoras de Insulina/metabolismo , Animais , Peso Corporal , Ingestão de Alimentos , Teste de Tolerância a Glucose , Insulina/metabolismo , Secreção de Insulina , Masculino , Camundongos Knockout , TamoxifenoRESUMO
Class B G protein-coupled receptors (GPCRs) are important regulators of endocrine physiology, and peptide-based therapeutics targeting some of these receptors have proven effective at treating disorders such as hypercalcemia, osteoporosis, and type 2 diabetes mellitus (T2DM). As next generation efforts attempt to develop novel non-peptide, orally available molecules for these GPCRs, new animal models expressing human receptor orthologs may be required because small molecule ligands make fewer receptor contacts, and thus, the impact of amino acid differences across species may be substantially greater. The objective of this report was to generate and characterize a new mouse model of the human glucagon-like peptide-1 receptor (hGLP-1R), a class B GPCR for which established peptide therapeutics exist for the treatment of T2DM. hGLP-1R knock-in mice express the receptor from the murine Glp-1r locus. Glucose tolerance tests and gastric emptying studies show hGLP-1R mice and their wild-type littermates display similar physiological responses for glucose metabolism, insulin secretion, and gastric transit, and treatment with the GLP-1R agonist, exendin-4, elicits similar responses in both groups. Further, ex vivo assays show insulin secretion from humanized islets is glucose-dependent and enhanced by GLP-1R agonists. To enable additional utility, the targeting construct of the knock-in line was engineered to contain both flanking LoxP sites and a C-terminal FLAG epitope. Anti-FLAG affinity purification shows strong expression of hGLP-1R in islets, lung, and stomach. We crossed the hGLP-1R line with Rosa26Cre mice and generated global Glp-1r-/- animals. Immunohistochemistry of pancreas from humanized and knock-out mice identified a human GLP-1R-specific antibody that detects the GLP-1R in human pancreas as well as in the pancreas of hGLP-1r knock-in mice. This new hGLP-1R model will allow tissue-specific deletion of the GLP-1R, purification of potential GLP-1R partner proteins, and testing of novel therapeutic agents targeting the hGLP-1R.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Receptores de Glucagon/metabolismo , Animais , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animais de Doenças , Receptor do Peptídeo Semelhante ao Glucagon 1 , Glucose/metabolismo , Teste de Tolerância a Glucose , Humanos , Insulina/metabolismo , Camundongos , Camundongos Transgênicos , Pâncreas/metabolismo , Receptores de Glucagon/genéticaRESUMO
Hypercholesterolemia is a major risk factor for coronary artery disease. Oxysterols are known to inhibit cholesterol biosynthesis and have been explored as potential antihypercholesterolemic agents. The ability of 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one (15-ketosterol) to lower non-HDL cholesterol has been demonstrated in rodent and primate models, but the mechanisms of action remain poorly understood. Here we show in a coactivator recruitment assay and cotransfection assays that the 15-ketosterol is a partial agonist for liver X receptor-alpha and -beta (LXRalpha and LXRbeta). The binding affinity for the LXRs was comparable to those of native oxysterols. In a macrophage cell line of human origin, the 15-ketosterol elevated ATP binding cassette transporter ABCA1 mRNA in a concentration-dependent fashion with a potency similar to those of other oxysterols. We further found that in human embryonic kidney HEK 293 cells, the 15-ketosterol suppressed sterol-responsive element binding protein processing activity and thus inhibited mRNA expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor, and PCSK9. Our data thus provide a molecular basis for the hypocholesterolemic activity of the 15-ketosterol and further suggest its potential antiatherosclerotic benefit as an LXR agonist.
Assuntos
Colestenonas/farmacologia , Proteínas de Ligação a DNA/metabolismo , Hidroximetilglutaril-CoA Redutases/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/antagonistas & inibidores , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/biossíntese , Células Cultivadas , Proteínas de Ligação a DNA/agonistas , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Receptores X do Fígado , Receptores Nucleares Órfãos , Pró-Proteína Convertase 9 , Pró-Proteína Convertases , Receptores Citoplasmáticos e Nucleares/agonistas , Serina Endopeptidases/biossínteseRESUMO
Peroxisomes are the exclusive site for the beta-oxidation of very-long-chain fatty acids of more than 20 carbons in length (VLCFAs). Although the bulk of dietary long-chain fatty acids are oxidized in the mitochondria, VLCFAs cannot be catabolized in mitochondria and must be shortened first by peroxisomal beta-oxidation. The regulation of peroxisomal, mitochondrial, and microsomal fatty acid oxidation systems in liver is mediated principally by peroxisome proliferator-activated receptor alpha (PPARalpha). In this study we provide evidence that the liver X receptor (LXR) regulates the expression of the genetic program for peroxisomal beta-oxidation in liver. The genes encoding the three enzymes of the classic peroxisomal beta-oxidation cycle, acyl-coenzyme A (acyl-CoA) oxidase, enoyl-CoA hydratase/L-3-hydroxyacyl-CoA dehydrogenase, and 3-ketoacyl-CoA thiolase, are activated by the LXR ligand, T0901317. Accordingly, administration of T0901317 in mice promoted a dose-dependent and greater than 2-fold increase in the rate of peroxisomal beta-oxidation in the liver. The LXR effect is independent of PPARalpha, because T0901317-induced peroxisomal beta-oxidation in the liver of PPARalpha-null mice. Interestingly, T0901317-induced peroxisomal beta-oxidation is dependent on the LXRalpha isoform, but not the LXRbeta isoform. We propose that induction of peroxisomal beta-oxidation by LXR agonists may serve as a counterregulatory mechanism for responding to the hypertriglyceridemia and liver steatosis that is promoted by potent LXR agonists in vivo; however, additional studies are warranted.