RESUMO
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.
Assuntos
Diabetes Mellitus Tipo 2 , Ilhotas Pancreáticas , Camundongos , Animais , Masculino , Diabetes Mellitus Tipo 2/metabolismo , Glicemia/metabolismo , Ilhotas Pancreáticas/metabolismo , Insulina/metabolismo , Lipídeos , Biomarcadores/metabolismo , Moléculas de Adesão Celular/metabolismo , Proteínas da Matriz Extracelular/metabolismoRESUMO
We report that intra-islet glucagon secreted from α-cells signals through ß-cell glucagon and GLP-1 receptors (GcgR and GLP-1R), thereby conferring to rat islets their competence to exhibit first-phase glucose-stimulated insulin secretion (GSIS). Thus, in islets not treated with exogenous glucagon or GLP-1, first-phase GSIS is abolished by a GcgR antagonist (LY2786890) or a GLP-1R antagonist (Ex[9-39]). Mechanistically, glucose competence in response to intra-islet glucagon is conditional on ß-cell cAMP signaling because it is blocked by the cAMP antagonist prodrug Rp-8-Br-cAMPS-pAB. In its role as a paracrine hormone, intra-islet glucagon binds with high affinity to the GcgR, while also exerting a "spillover" effect to bind with low affinity to the GLP-1R. This produces a right shift of the concentration-response relationship for the potentiation of GSIS by exogenous glucagon. Thus, 0.3 nM glucagon fails to potentiate GSIS, as expected if similar concentrations of intra-islet glucagon already occupy the GcgR. However, 10 to 30 nM glucagon effectively engages the ß-cell GLP-1R to potentiate GSIS, an action blocked by Ex[9-39] but not LY2786890. Finally, we report that the action of intra-islet glucagon to support insulin secretion requires a step-wise increase of glucose concentration to trigger first-phase GSIS. It is not measurable when GSIS is stimulated by a gradient of increasing glucose concentrations, as occurs during an oral glucose tolerance test in vivo. Collectively, such findings are understandable if defective intra-islet glucagon action contributes to the characteristic loss of first-phase GSIS in an intravenous glucose tolerance test that is diagnostic of type 2 diabetes in the clinical setting.
Assuntos
Diabetes Mellitus Tipo 2 , Receptor do Peptídeo Semelhante ao Glucagon 1 , Glucagon , Glucose , Secreção de Insulina , Ilhotas Pancreáticas , Animais , Diabetes Mellitus Tipo 2/metabolismo , Glucagon/metabolismo , Glucagon/farmacologia , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucose/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RatosRESUMO
Tirzepatide (LY3298176), a dual GIP and GLP-1 receptor (GLP-1R) agonist, delivered superior glycemic control and weight loss compared with GLP-1R agonism in patients with type 2 diabetes. However, the mechanism by which tirzepatide improves efficacy and how GIP receptor (GIPR) agonism contributes is not fully understood. Here, we show that tirzepatide is an effective insulin sensitizer, improving insulin sensitivity in obese mice to a greater extent than GLP-1R agonism. To determine whether GIPR agonism contributes, we compared the effect of tirzepatide in obese WT and Glp-1r-null mice. In the absence of GLP-1R-induced weight loss, tirzepatide improved insulin sensitivity by enhancing glucose disposal in white adipose tissue (WAT). In support of this, a long-acting GIPR agonist (LAGIPRA) was found to enhance insulin sensitivity by augmenting glucose disposal in WAT. Interestingly, the effect of tirzepatide and LAGIPRA on insulin sensitivity was associated with reduced branched-chain amino acids (BCAAs) and ketoacids in the circulation. Insulin sensitization was associated with upregulation of genes associated with the catabolism of glucose, lipid, and BCAAs in brown adipose tissue. Together, our studies show that tirzepatide improved insulin sensitivity in a weight-dependent and -independent manner. These results highlight how GIPR agonism contributes to the therapeutic profile of dual-receptor agonism, offering mechanistic insights into the clinical efficacy of tirzepatide.
Assuntos
Tecido Adiposo Branco/metabolismo , Polipeptídeo Inibidor Gástrico/farmacologia , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Resistência à Insulina , Obesidade/metabolismo , Tecido Adiposo Branco/patologia , Aminoácidos de Cadeia Ramificada/genética , Aminoácidos de Cadeia Ramificada/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Peso Corporal/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/genética , Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Camundongos , Camundongos Knockout , Obesidade/tratamento farmacológico , Obesidade/genética , Obesidade/patologiaRESUMO
AIM: To examine the mechanism of action of γ-aminobutyric acid (GABA) on ß-cell proliferation and investigate if co-treatment with Ly49, a novel GABA type A receptor positive allosteric modulator (GABAA -R PAM), amplifies this effect. METHODS: Human or mouse islets were co-treated for 4-5 days with GABA and selected receptor or cell signalling pathway modulators. Immunofluorescence was used to determine protein co-localization, cell number or proliferation, and islet size. Osmotic minipumps were surgically implanted in mice to assess Ly49 effects on pancreatic ß-cells. RESULTS: Amplification of GABAA -R signalling enhanced GABA-stimulated ß-cell proliferation in cultured mouse islets. Co-treatment of GABA with an inhibitor specific for PI3K, mTORC1/2, or p70S6K, abolished GABA-stimulated ß-cell proliferation in mouse and human islets. Nuclear p-AktSer473 and p-p70S6KThr421/Ser424 expression in pancreatic ß-cells was increased in GABA-treated mice compared with vehicle-treated mice, an effect augmented with GABA and Ly49 co-treatment. Mice co-treated with GABA and Ly49 exhibited enhanced ß-cell area and proliferation compared with GABA-treated mice. Furthermore, S961 injection (an insulin receptor antagonist) resulted in enhanced plasma insulin in GABA and Ly49 co-treated mice compared with GABA-treated mice. Importantly, GABA co-treated with Ly49 increased ß-cell proliferation in human islets providing a potential application for human subjects. CONCLUSIONS: We show that GABA stimulates ß-cell proliferation via the PI3K/mTORC1/p70S6K pathway in both mouse and human islets. Furthermore, we show that Ly49 enhances the ß-cell regenerative effects of GABA, showing potential in the intervention of diabetes.
Assuntos
Receptores de GABA , Proteínas Quinases S6 Ribossômicas 70-kDa , Animais , Proliferação de Células , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Ácido gama-AminobutíricoRESUMO
The microtubule cytoskeleton of pancreatic islet ß-cells regulates glucose-stimulated insulin secretion (GSIS). We have reported that the microtubule-mediated movement of insulin vesicles away from the plasma membrane limits insulin secretion. High glucose-induced remodeling of microtubule network facilitates robust GSIS. This remodeling involves disassembly of old microtubules and nucleation of new microtubules. Here, we examine the mechanisms whereby glucose stimulation decreases microtubule lifetimes in ß-cells. Using real-time imaging of photoconverted microtubules, we demonstrate that high levels of glucose induce rapid microtubule disassembly preferentially in the periphery of individual ß-cells, and this process is mediated by the phosphorylation of microtubule-associated protein tau. Specifically, high glucose induces tau hyper-phosphorylation via glucose-responsive kinases GSK3, PKA, PKC, and CDK5. This causes dissociation of tau from and subsequent destabilization of microtubules. Consequently, tau knockdown in mouse islet ß-cells facilitates microtubule turnover, causing increased basal insulin secretion, depleting insulin vesicles from the cytoplasm, and impairing GSIS. More importantly, tau knockdown uncouples microtubule destabilization from glucose stimulation. These findings suggest that tau suppresses peripheral microtubules turning over to restrict insulin oversecretion in basal conditions and preserve the insulin pool that can be released following stimulation; high glucose promotes tau phosphorylation to enhance microtubule disassembly to acutely enhance GSIS.
Assuntos
Glucose/farmacologia , Secreção de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/efeitos dos fármacos , Microtúbulos/efeitos dos fármacos , Proteínas tau/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Quinase 5 Dependente de Ciclina/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Células Secretoras de Insulina/metabolismo , Camundongos , Microtúbulos/metabolismo , Fosforilação/efeitos dos fármacos , Proteína Quinase CRESUMO
γ-Aminobutyric acid (GABA) administration has been shown to increase ß-cell mass, leading to a reversal of type 1 diabetes in mice. Whether GABA has any effect on ß cells of healthy and prediabetic/glucose-intolerant obese mice remains unknown. In the present study, we show that oral GABA administration ( ad libitum) to mice indeed increased pancreatic ß-cell mass, which led to a modest enhancement in insulin secretion and glucose tolerance. However, GABA treatment did not further increase insulin-positive islet area in high fat diet-fed mice and was unable to prevent or reverse glucose intolerance and insulin resistance. Mechanistically, whether in vivo or in vitro, GABA treatment increased ß-cell proliferation. In vitro, the effect was shown to be mediated via the GABAA receptor. Single-cell RNA sequencing analysis revealed that GABA preferentially up-regulated pathways linked to ß-cell proliferation and simultaneously down-regulated those networks required for other processes, including insulin biosynthesis and metabolism. Interestingly, single-cell differential expression analysis revealed GABA treatment gave rise to a distinct subpopulation of ß cells with a unique transcriptional signature, including urocortin 3 ( ucn3), wnt4, and hepacam2. Taken together, this study provides new mechanistic insight into the proliferative nature of GABA but suggests that ß-cell compensation associated with prediabetes overlaps with, and negates, its proliferative effects.-Untereiner, A., Abdo, S., Bhattacharjee, A., Gohil, H., Pourasgari, F., Ibeh, N., Lai, M., Batchuluun, B., Wong, A., Khuu, N., Liu, Y., Al Rijjal, D., Winegarden, N., Virtanen, C., Orser, B. A., Cabrera, O., Varga, G., Rocheleau, J., Dai, F. F., Wheeler, M. B. GABA promotes ß-cell proliferation, but does not overcome impaired glucose homeostasis associated with diet-induced obesity.
Assuntos
Proliferação de Células , Glucose/metabolismo , Células Secretoras de Insulina/metabolismo , Obesidade/metabolismo , Transcriptoma , Ácido gama-Aminobutírico/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Homeostase , Humanos , Insulina/metabolismo , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/etiologia , Receptores de GABA-A/metabolismo , Urocortinas/metabolismoRESUMO
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
OBJECTIVE: A novel dual GIP and GLP-1 receptor agonist, LY3298176, was developed to determine whether the metabolic action of GIP adds to the established clinical benefits of selective GLP-1 receptor agonists in type 2 diabetes mellitus (T2DM). METHODS: LY3298176 is a fatty acid modified peptide with dual GIP and GLP-1 receptor agonist activity designed for once-weekly subcutaneous administration. LY3298176 was characterised in vitro, using signaling and functional assays in cell lines expressing recombinant or endogenous incretin receptors, and in vivo using body weight, food intake, insulin secretion and glycemic profiles in mice. A Phase 1, randomised, placebo-controlled, double-blind study was comprised of three parts: a single-ascending dose (SAD; doses 0.25-8 mg) and 4-week multiple-ascending dose (MAD; doses 0.5-10 mg) studies in healthy subjects (HS), followed by a 4-week multiple-dose Phase 1 b proof-of-concept (POC; doses 0.5-15 mg) in patients with T2DM (ClinicalTrials.gov no. NCT02759107). Doses higher than 5 mg were attained by titration, dulaglutide (DU) was used as a positive control. The primary objective was to investigate safety and tolerability of LY3298176. RESULTS: LY3298176 activated both GIP and GLP-1 receptor signaling in vitro and showed glucose-dependent insulin secretion and improved glucose tolerance by acting on both GIP and GLP-1 receptors in mice. With chronic administration to mice, LY3298176 potently decreased body weight and food intake; these effects were significantly greater than the effects of a GLP-1 receptor agonist. A total of 142 human subjects received at least 1 dose of LY3298176, dulaglutide, or placebo. The PK profile of LY3298176 was investigated over a wide dose range (0.25-15 mg) and supports once-weekly administration. In the Phase 1 b trial of diabetic subjects, LY3298176 doses of 10 mg and 15 mg significantly reduced fasting serum glucose compared to placebo (least square mean [LSM] difference [95% CI]: -49.12 mg/dL [-78.14, -20.12] and -43.15 mg/dL [-73.06, -13.21], respectively). Reductions in body weight were significantly greater with the LY3298176 1.5 mg, 4.5 mg and 10 mg doses versus placebo in MAD HS (LSM difference [95% CI]: -1.75 kg [-3.38, -0.12], -5.09 kg [-6.72, -3.46] and -4.61 kg [-6.21, -3.01], respectively) and doses of 10 mg and 15 mg had a relevant effect in T2DM patients (LSM difference [95% CI]: -2.62 kg [-3.79, -1.45] and -2.07 kg [-3.25, -0.88], respectively. The most frequent side effects reported with LY3298176 were gastrointestinal (vomiting, nausea, decreased appetite, diarrhoea, and abdominal distension) in both HS and patients with T2DM; all were dose-dependent and considered mild to moderate in severity. CONCLUSIONS: Based on these results, the pharmacology of LY3298176 translates from preclinical to clinical studies. LY3298176 has the potential to deliver clinically meaningful improvement in glycaemic control and body weight. The data warrant further clinical evaluation of LY3298176 for the treatment of T2DM and potentially obesity.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Polipeptídeo Inibidor Gástrico/uso terapêutico , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Hipoglicemiantes/uso terapêutico , Incretinas/uso terapêutico , Receptores dos Hormônios Gastrointestinais/agonistas , Adulto , Animais , Apetite/efeitos dos fármacos , Glicemia/metabolismo , Peso Corporal , Diarreia/etiologia , Feminino , Polipeptídeo Inibidor Gástrico/efeitos adversos , Polipeptídeo Inibidor Gástrico/farmacologia , Humanos , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/farmacologia , Incretinas/efeitos adversos , Incretinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Vômito/etiologiaRESUMO
As a part of our program to identify potent GPR40 agonists capable of being dosed orally once daily in humans, we incorporated fused heterocycles into our recently disclosed spiropiperidine and tetrahydroquinoline acid derivatives 1, 2, and 3 with the intention of lowering clearance and improving the maximum absorbable dose (Dabs). Hypothesis-driven structural modifications focused on moving away from the zwitterion-like structure. and mitigating the N-dealkylation and O-dealkylation issues led to triazolopyridine acid derivatives with unique pharmacology and superior pharmacokinetic properties. Compound 4 (LY3104607) demonstrated functional potency and glucose-dependent insulin secretion (GDIS) in primary islets from rats. Potent, efficacious, and durable dose-dependent reductions in glucose levels were seen during glucose tolerance test (GTT) studies. Low clearance, volume of distribution, and high oral bioavailability were observed in all species. The combination of enhanced pharmacology and pharmacokinetic properties supported further development of this compound as a potential glucose-lowering drug candidate.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Descoberta de Drogas , Hipoglicemiantes/farmacologia , Piridinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Triazóis/farmacologia , Administração Oral , Animais , Cães , Humanos , Hipoglicemiantes/administração & dosagem , Hipoglicemiantes/síntese química , Hipoglicemiantes/farmacocinética , Masculino , Piridinas/administração & dosagem , Piridinas/síntese química , Piridinas/farmacocinética , Ratos , Relação Estrutura-Atividade , Triazóis/administração & dosagem , Triazóis/síntese química , Triazóis/farmacocinéticaRESUMO
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
Type 1 diabetes (T1D) patients who receive pancreatic islet transplant experience significant improvement in their quality-of-life. This comes primarily through improved control of blood sugar levels, restored awareness of hypoglycemia, and prevention of serious and potentially life-threatening diabetes-associated complications, such as kidney failure, heart and vascular disease, stroke, nerve damage, and blindness. Therefore, beta cell replacement through transplantation of isolated islets is an important option in the treatment of T1D. However, lasting success of this promising therapy depends on durable survival and efficacy of the transplanted islets, which are directly influenced by the islet isolation procedures. Thus, isolating pancreatic islets with consistent and reliable quality is critical in the clinical application of islet transplantation.Quality of isolated islets is important in pre-clinical studies as well, as efforts to advance and improve clinical outcomes of islet transplant therapy have relied heavily on animal models ranging from rodents, to pigs, to nonhuman primates. As a result, pancreatic islets have been isolated from these and other species and used in a variety of in vitro or in vivo applications for this and other research purposes. Protocols for islet isolation have been somewhat similar across species, especially, in mammals. However, given the increasing evidence about the distinct structural and functional features of human and mouse islets, using similar methods of islet isolation may contribute to inconsistencies in the islet quality, immunogenicity, and experimental outcomes. This may also contribute to the discrepancies commonly observed between pre-clinical findings and clinical outcomes. Therefore, it is prudent to consider the particular features of pancreatic islets from different species when optimizing islet isolation protocols.In this chapter, we explore the structural and functional features of pancreatic islets from mice, pigs, nonhuman primates, and humans because of their prevalent use in nonclinical, preclinical, and clinical applications.
Assuntos
Ilhotas Pancreáticas/fisiologia , Animais , Humanos , Ilhotas Pancreáticas/irrigação sanguínea , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/inervação , Comunicação Parácrina , Transdução de SinaisRESUMO
Therapeutic intervention to activate the glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion and improves energy balance in patients with type 2 diabetes mellitus. Studies investigating mechanisms whereby peptide ligands activate GLP-1R have utilized mutagenesis, receptor chimeras, photo-affinity labeling, hydrogen-deuterium exchange, and crystallography of the ligand-binding ectodomain to establish receptor homology models. However, this has not enabled the design or discovery of drug-like non-peptide GLP-1R activators. Recently, studies investigating 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), a GLP-1R-positive allosteric modulator, determined that Cys-347 in the GLP-1R is required for positive allosteric modulator activity via covalent modification. To advance small molecule activation of the GLP-1R, we characterized the insulinotropic mechanism of BETP. In guanosine 5'-3-O-(thio)triphosphate binding and INS1 832-3 insulinoma cell cAMP assays, BETP enhanced GLP-1(9-36)-NH2-stimulated cAMP signaling. Using isolated pancreatic islets, BETP potentiated insulin secretion in a glucose-dependent manner that requires both the peptide ligand and GLP-1R. In studies of the covalent mechanism, PAGE fluorography showed labeling of GLP-1R in immunoprecipitation experiments from GLP-1R-expressing cells incubated with [(3)H]BETP. Furthermore, we investigated whether other reported GLP-1R activators and compounds identified from screening campaigns modulate GLP-1R by covalent modification. Similar to BETP, several molecules were found to enhance GLP-1R signaling in a Cys-347-dependent manner. These chemotypes are electrophiles that react with GSH, and LC/MS determined the cysteine adducts formed upon conjugation. Together, our results suggest covalent modification may be used to stabilize the GLP-1R in an active conformation. Moreover, the findings provide pharmacological guidance for the discovery and characterization of small molecule GLP-1R ligands as possible therapeutics.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Regulação Alostérica , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Receptor do Peptídeo Semelhante ao Glucagon 1/agonistas , Receptor do Peptídeo Semelhante ao Glucagon 1/química , Glucose/metabolismo , Células HEK293 , Humanos , Insulina/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Pirimidinas/química , Pirimidinas/farmacologia , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
cAMP-elevating agents such as the incretin hormone glucagon-like peptide-1 potentiate glucose-stimulated insulin secretion (GSIS) from pancreatic ß-cells. However, a debate has existed since the 1970s concerning whether or not cAMP signaling is essential for glucose alone to stimulate insulin secretion. Here, we report that the first-phase kinetic component of GSIS is cAMP-dependent, as revealed through the use of a novel highly membrane permeable para-acetoxybenzyl (pAB) ester prodrug that is a bioactivatable derivative of the cAMP antagonist adenosine-3',5'-cyclic monophosphorothioate, Rp-isomer (Rp-cAMPS). In dynamic perifusion assays of human or rat islets, a step-wise increase of glucose concentration leads to biphasic insulin secretion, and under these conditions, 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer, 4-acetoxybenzyl ester (Rp-8-Br-cAMPS-pAB) inhibits first-phase GSIS by up to 80%. Surprisingly, second-phase GSIS is inhibited to a much smaller extent (≤20%). Using luciferase, fluorescence resonance energy transfer, and bioluminescence resonance energy transfer assays performed in living cells, we validate that Rp-8-Br-cAMPS-pAB does in fact block cAMP-dependent protein kinase activation. Novel effects of Rp-8-Br-cAMPS-pAB to block the activation of cAMP-regulated guanine nucleotide exchange factors (Epac1, Epac2) are also validated using genetically encoded Epac biosensors, and are independently confirmed in an in vitro Rap1 activation assay using Rp-cAMPS and Rp-8-Br-cAMPS. Thus, in addition to revealing the cAMP dependence of first-phase GSIS from human and rat islets, these findings establish a pAB-based chemistry for the synthesis of highly membrane permeable prodrug derivatives of Rp-cAMPS that act with micromolar or even nanomolar potency to inhibit cAMP signaling in living cells.
Assuntos
8-Bromo Monofosfato de Adenosina Cíclica/análogos & derivados , AMP Cíclico/farmacologia , Glucose/farmacologia , Insulina/metabolismo , Pró-Fármacos/farmacologia , Tionucleotídeos/farmacologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Álcool Benzílico/farmacologia , Linhagem Celular , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citosol/metabolismo , Ativação Enzimática/efeitos dos fármacos , Esterases/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Holoenzimas/metabolismo , Humanos , Secreção de Insulina , Integrases/metabolismo , Luciferases/metabolismo , Masculino , Pessoa de Meia-Idade , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Mitochondrial GTP (mtGTP)-insensitive mutations in glutamate dehydrogenase (GDH(H454Y)) result in fasting and amino acid-induced hypoglycemia in hyperinsulinemia hyperammonemia (HI/HA). Surprisingly, hypoglycemia may occur in this disorder despite appropriately suppressed insulin. To better understand the islet-specific contribution, transgenic mice expressing the human activating mutation in ß-cells (H454Y mice) were characterized in vivo. As in the humans with HI/HA, H454Y mice had fasting hypoglycemia, but plasma insulin concentrations were similar to the controls. Paradoxically, both glucose- and glutamine-stimulated insulin secretion were severely impaired in H454Y mice. Instead, lack of a glucagon response during hypoglycemic clamps identified impaired counterregulation. Moreover, both insulin and glucagon secretion were impaired in perifused islets. Acute pharmacologic inhibition of GDH restored both insulin and glucagon secretion and normalized glucose tolerance in vivo. These studies support the presence of an mtGTP-dependent signal generated via ß-cell GDH that inhibits α-cells. As such, in children with activating GDH mutations of HI/HA, this insulin-independent glucagon suppression may contribute importantly to symptomatic hypoglycemia. The identification of a human mutation causing congenital hypoglucagonemic hypoglycemia highlights a central role of the mtGTP-GDH-glucagon axis in glucose homeostasis.
Assuntos
Aminoácidos/metabolismo , Células Secretoras de Glucagon/metabolismo , Glucagon/metabolismo , Glutamato Desidrogenase/genética , Guanosina Trifosfato/metabolismo , Hiperamonemia/genética , Hiperinsulinismo/genética , Hipoglicemia/genética , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocôndrias/metabolismo , Animais , Técnica Clamp de Glucose , Humanos , Secreção de Insulina , Camundongos , Camundongos Transgênicos , Mutação , SíndromeRESUMO
The autonomic nervous system is thought to modulate blood glucose homeostasis by regulating endocrine cell activity in the pancreatic islets of Langerhans. The role of islet innervation, however, has remained elusive because the direct effects of autonomic nervous input on islet cell physiology cannot be studied in the pancreas. Here, we used an in vivo model to study the role of islet nervous input in glucose homeostasis. We transplanted islets into the anterior chamber of the eye and found that islet grafts became densely innervated by the rich parasympathetic and sympathetic nervous supply of the iris. Parasympathetic innervation was imaged intravitally by using transgenic mice expressing GFP in cholinergic axons. To manipulate selectively the islet nervous input, we increased the ambient illumination to increase the parasympathetic input to the islet grafts via the pupillary light reflex. This reduced fasting glycemia and improved glucose tolerance. These effects could be blocked by topical application of the muscarinic antagonist atropine to the eye, indicating that local cholinergic innervation had a direct effect on islet function in vivo. By using this approach, we found that parasympathetic innervation influences islet function in C57BL/6 mice but not in 129X1 mice, which reflected differences in innervation densities and may explain major strain differences in glucose homeostasis. This study directly demonstrates that autonomic axons innervating the islet modulate glucose homeostasis.
Assuntos
Sistema Nervoso Autônomo/fisiologia , Olho/inervação , Ilhotas Pancreáticas/fisiologia , Modelos Biológicos , Animais , Proteínas de Fluorescência Verde/metabolismo , Iris/inervação , Iris/fisiologia , Transplante das Ilhotas Pancreáticas , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras NervosasRESUMO
Glucokinase is a key regulator of glucose homeostasis, and small molecule allosteric activators of this enzyme represent a promising opportunity for the treatment of type 2 diabetes. Systemically acting glucokinase activators (liver and pancreas) have been reported to be efficacious but in many cases present hypoglycaemia risk due to activation of the enzyme at low glucose levels in the pancreas, leading to inappropriately excessive insulin secretion. It was therefore postulated that a liver selective activator may offer effective glycemic control with reduced hypoglycemia risk. Herein, we report structure-activity studies on a carboxylic acid containing series of glucokinase activators with preferential activity in hepatocytes versus pancreatic ß-cells. These activators were designed to have low passive permeability thereby minimizing distribution into extrahepatic tissues; concurrently, they were also optimized as substrates for active liver uptake via members of the organic anion transporting polypeptide (OATP) family. These studies lead to the identification of 19 as a potent glucokinase activator with a greater than 50-fold liver-to-pancreas ratio of tissue distribution in rodent and non-rodent species. In preclinical diabetic animals, 19 was found to robustly lower fasting and postprandial glucose with no hypoglycemia, leading to its selection as a clinical development candidate for treating type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Ativadores de Enzimas/síntese química , Glucoquinase/metabolismo , Hepatócitos/metabolismo , Hipoglicemiantes/síntese química , Imidazóis/síntese química , Ácidos Nicotínicos/síntese química , Sítio Alostérico , Animais , Glicemia/metabolismo , Cães , Ativadores de Enzimas/farmacocinética , Ativadores de Enzimas/farmacologia , Haplorrinos , Humanos , Hipoglicemiantes/farmacocinética , Hipoglicemiantes/farmacologia , Imidazóis/farmacocinética , Imidazóis/farmacologia , Técnicas In Vitro , Células Secretoras de Insulina/metabolismo , Masculino , Modelos Moleculares , Ácidos Nicotínicos/farmacocinética , Ácidos Nicotínicos/farmacologia , Transportadores de Ânions Orgânicos/metabolismo , Ligação Proteica , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Estereoisomerismo , Relação Estrutura-Atividade , Distribuição TecidualRESUMO
BACKGROUND: Glucagon is an important hormone in the regulation of glucose homeostasis, particularly in the maintenance of euglycemia and prevention of hypoglycemia. In type 2 Diabetes Mellitus (T2DM), glucagon levels are elevated in both the fasted and postprandial states, which contributes to inappropriate hyperglycemia through excessive hepatic glucose production. Efforts to discover and evaluate glucagon receptor antagonists for the treatment of T2DM have been ongoing for approximately two decades, with the challenge being to identify an agent with appropriate pharmaceutical properties and efficacy relative to potential side effects. We sought to determine the hepatic & systemic consequence of full glucagon receptor antagonism through the study of the glucagon receptor knock-out mouse (Gcgr-/-) compared to wild-type littermates. RESULTS: Liver transcriptomics was performed using Affymetric expression array profiling, and liver proteomics was performed by iTRAQ global protein analysis. To complement the transcriptomic and proteomic analyses, we also conducted metabolite profiling (~200 analytes) using mass spectrometry in plasma. Overall, there was excellent concordance (R = 0.88) for changes associated with receptor knock-out between the transcript and protein analysis. Pathway analysis tools were used to map the metabolic processes in liver altered by glucagon receptor ablation, the most notable being significant down-regulation of gluconeogenesis, amino acid catabolism, and fatty acid oxidation processes, with significant up-regulation of glycolysis, fatty acid synthesis, and cholesterol biosynthetic processes. These changes at the level of the liver were manifested through an altered plasma metabolite profile in the receptor knock-out mice, e.g. decreased glucose and glucose-derived metabolites, and increased amino acids, cholesterol, and bile acid levels. CONCLUSIONS: In sum, the results of this study suggest that the complete ablation of hepatic glucagon receptor function results in major metabolic alterations in the liver, which, while promoting improved glycemic control, may be associated with adverse lipid changes.
Assuntos
Diabetes Mellitus/tratamento farmacológico , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Fígado/metabolismo , Proteômica , Receptores de Glucagon/antagonistas & inibidores , Receptores de Glucagon/genética , Aminoácidos/metabolismo , Animais , Metabolismo dos Carboidratos/genética , Diabetes Mellitus/metabolismo , Hipoglicemiantes/farmacologia , Hipoglicemiantes/uso terapêutico , Metabolismo dos Lipídeos/genética , Masculino , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glucagon/deficiênciaRESUMO
OBJECTIVE: To test the graft-promoting effects of mesenchymal stem cells (MSCs) in a cynomolgus monkey model of islet/bone marrow transplantation. RESEARCH DESIGN AND METHODS: Cynomolgus MSCs were obtained from iliac crest aspirate and characterized through passage 11 for phenotype, gene expression, differentiation potential, and karyotype. Allogeneic donor MSCs were cotransplanted intraportally with islets on postoperative day (POD) 0 and intravenously with donor marrow on PODs 5 and 11. Recipients were followed for stabilization of blood glucose levels, reduction of exogenous insulin requirement (EIR), C-peptide levels, changes in peripheral blood T regulatory cells, and chimerism. Destabilization of glycemia and increases in EIR were used as signs of rejection; additional intravenous MSCs were administered to test the effect on reversal of rejection. RESULTS: MSC phenotype and a normal karyotype were observed through passage 11. IL-6, IL-10, vascular endothelial growth factor, TGF-ß, hepatocyte growth factor, and galectin-1 gene expression levels varied among donors. MSC treatment significantly enhanced islet engraftment and function at 1 month posttransplant (n = 8), as compared with animals that received islets without MSCs (n = 3). Additional infusions of donor or third-party MSCs resulted in reversal of rejection episodes and prolongation of islet function in two animals. Stable islet allograft function was associated with increased numbers of regulatory T-cells in peripheral blood. CONCLUSIONS: MSCs may provide an important approach for enhancement of islet engraftment, thereby decreasing the numbers of islets needed to achieve insulin independence. Furthermore, MSCs may serve as a new, safe, and effective antirejection therapy.
Assuntos
Diabetes Mellitus Experimental/cirurgia , Transplante das Ilhotas Pancreáticas/fisiologia , Transplante de Células-Tronco Mesenquimais/métodos , Animais , Antígenos CD/análise , Glicemia/metabolismo , Diferenciação Celular , Meios de Cultura , Diabetes Mellitus Experimental/sangue , Fator de Crescimento Epidérmico/genética , Fatores de Transcrição Forkhead/análise , Galectina 1/genética , Fator de Crescimento de Hepatócito/genética , Antígenos de Histocompatibilidade Classe II/análise , Teste de Histocompatibilidade , Interleucinas/genética , Cariotipagem , Macaca fascicularis/imunologia , Macaca fascicularis/fisiologia , Complexo Principal de Histocompatibilidade , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Fenótipo , RNA/genética , RNA/isolamento & purificação , Fator de Crescimento Transformador beta/genética , Transplante HomólogoRESUMO
Extracellular ATP has been proposed as a paracrine signal in rodent islets, but it is unclear what role ATP plays in human islets. We now show the presence of an ATP signaling pathway that enhances the human beta cell's sensitivity and responsiveness to glucose fluctuations. By using in situ hybridization, RT-PCR, immunohistochemistry, and Western blotting as well as recordings of cytoplasmic-free Ca(2+) concentration, [Ca(2+)](i), and hormone release in vitro, we show that human beta cells express ionotropic ATP receptors of the P2X(3) type and that activation of these receptors by ATP coreleased with insulin amplifies glucose-induced insulin secretion. Released ATP activates P2X(3) receptors in the beta-cell plasma membrane, resulting in increased [Ca(2+)](i) and enhanced insulin secretion. Therefore, in human islets, released ATP forms a positive autocrine feedback loop that sensitizes the beta cell's secretory machinery. This may explain how the human pancreatic beta cell can respond so effectively to relatively modest changes in glucose concentration under physiological conditions in vivo.
Assuntos
Trifosfato de Adenosina/metabolismo , Comunicação Autócrina , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Receptores Purinérgicos P2/metabolismo , Cálcio/metabolismo , Humanos , Secreção de Insulina , Receptores Purinérgicos P2/genética , Receptores Purinérgicos P2X3 , Transdução de SinaisRESUMO
An important feature of glucose homeostasis is the effective release of glucagon from the pancreatic alpha cell. The molecular mechanisms regulating glucagon secretion are still poorly understood. We now demonstrate that human alpha cells express ionotropic glutamate receptors (iGluRs) that are essential for glucagon release. A lowering in glucose concentration results in the release of glutamate from the alpha cell. Glutamate then acts on iGluRs of the AMPA/kainate type, resulting in membrane depolarization, opening of voltage-gated Ca(2+) channels, increase in cytoplasmic free Ca(2+) concentration, and enhanced glucagon release. In vivo blockade of iGluRs reduces glucagon secretion and exacerbates insulin-induced hypoglycemia in mice. Hence, the glutamate autocrine feedback loop endows the alpha cell with the ability to effectively potentiate its own secretory activity. This is a prerequisite to guarantee adequate glucagon release despite relatively modest changes in blood glucose concentration under physiological conditions.