RESUMEN
Glucagon-like peptide-1 receptor (GLP-1R) agonists are efficacious antidiabetic medications that work by enhancing glucose-dependent insulin secretion and improving energy balance. Currently approved GLP-1R agonists are peptide based, and it has proven difficult to obtain small-molecule activators possessing optimal pharmaceutical properties. We report the discovery and mechanism of action of LY3502970 (OWL833), a nonpeptide GLP-1R agonist. LY3502970 is a partial agonist, biased toward G protein activation over ß-arrestin recruitment at the GLP-1R. The molecule is highly potent and selective against other class B G protein-coupled receptors (GPCRs) with a pharmacokinetic profile favorable for oral administration. A high-resolution structure of LY3502970 in complex with active-state GLP-1R revealed a unique binding pocket in the upper helical bundle where the compound is bound by the extracellular domain (ECD), extracellular loop 2, and transmembrane helices 1, 2, 3, and 7. This mechanism creates a distinct receptor conformation that may explain the partial agonism and biased signaling of the compound. Further, interaction between LY3502970 and the primate-specific Trp33 of the ECD informs species selective activity for the molecule. In efficacy studies, oral administration of LY3502970 resulted in glucose lowering in humanized GLP-1R transgenic mice and insulinotropic and hypophagic effects in nonhuman primates, demonstrating an effect size in both models comparable to injectable exenatide. Together, this work determined the molecular basis for the activity of an oral agent being developed for the treatment of type 2 diabetes mellitus, offering insights into the activation of class B GPCRs by nonpeptide ligands.
Asunto(s)
Receptor del Péptido 1 Similar al Glucagón/agonistas , Hipoglucemiantes/farmacología , Dominios Proteicos/genética , Administración Oral , Aminopiridinas/farmacología , Animales , Fármacos Antiobesidad/farmacología , Benzamidas/farmacología , Microscopía por Crioelectrón , Receptor del Péptido 1 Similar al Glucagón/genética , Receptor del Péptido 1 Similar al Glucagón/metabolismo , Receptor del Péptido 1 Similar al Glucagón/ultraestructura , Células HEK293 , Humanos , Incretinas/farmacología , Macaca fascicularis , Masculino , Ratones , Ratones Transgénicos , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Ratas , Especificidad de la Especie , Porcinos , Triptófano/genéticaRESUMEN
Although mitogen-activated protein kinase kinase (MEK) is a key signaling molecule and a negative regulator of insulin action, it is still uncertain whether MEK can be a therapeutic target for amelioration of insulin resistance (IR) in type 2 diabetes (T2D) in vivo. To clarify whether MEK inhibition improves T2D, we examined the effect of continuous MEK inhibition with two structurally different MEK inhibitors, RO5126766 and RO4987655, in mouse models of T2D. RO5126766 and RO4987655 were administered via dietary admixture. Both compounds decreased blood glucose and improved glucose tolerance in doses sufficient to sustain inhibition of extracellular signal-regulated kinase (ERK)1/2 phosphorylation downstream of MEK in insulin-responsive tissues in db/db mice. A hyperinsulinemic-euglycemic clamp test showed increased glucose infusion rate (GIR) in db/db mice treated with these compounds, and about 60% of the increase was attributed to the inhibition of endogenous glucose production, suggesting that the liver is responsible for the improvement of IR. By means of adenovirus-mediated Mek1 shRNA expression, we confirmed that blood glucose levels are reduced by suppression of MEK1 expression in the liver of db/db mice. Taken together, these results suggested that the MEK signaling pathway could be a novel therapeutic target for novel antidiabetic agents.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Resistencia a la Insulina , Hígado/metabolismo , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 1/metabolismo , Adenoviridae , Animales , Benzamidas/uso terapéutico , Glucemia/análisis , Peso Corporal , Cumarinas/uso terapéutico , Dieta , Modelos Animales de Enfermedad , Glucosa/uso terapéutico , Técnica de Clampeo de la Glucosa , Prueba de Tolerancia a la Glucosa , Hipoglucemiantes/uso terapéutico , Insulina/sangre , Masculino , Ratones , Oxazinas/uso terapéutico , Fosforilación , ARN Interferente Pequeño/metabolismo , Transducción de SeñalRESUMEN
Sodium glucose cotransporter 2 inhibitors have attracted attention as they exert antidiabetic and antiobesity effects. In this study, we investigated the effects of tofogliflozin on glucose homeostasis and its metabolic consequences and clarified the underlying molecular mechanisms. C57BL/6 mice were fed normal chow containing tofogliflozin (0.005%) for 20 weeks or a high-fat diet containing tofogliflozin (0.005%) for 8 weeks ad libitum. In addition, the animals were pair-fed in relation to controls to exclude the influence of increased food intake. Tofogliflozin reduced the body weight gain, mainly because of fat mass reduction associated with a diminished adipocyte size. Glucose tolerance and insulin sensitivity were ameliorated. The serum levels of nonesterified fatty acid and ketone bodies were increased and the respiratory quotient was decreased in the tofogliflozin-treated mice, suggesting the acceleration of lipolysis in the white adipose tissue and hepatic ß-oxidation. In fact, the phosphorylation of hormone-sensitive lipase and the adipose triglyceride lipase protein levels in the white adipose tissue as well as the gene expressions related to ß-oxidation, such as Cpt1α in the liver, were significantly increased. The hepatic triglyceride contents and the expression levels of lipogenic genes were decreased. Pair-fed mice exhibited almost the same results as mice fed an high-fat diet ad libitum. Moreover, a hyperinsulinemic-euglycemic clamp revealed that tofogliflozin improved insulin resistance by increasing glucose uptake, especially in the skeletal muscle, in pair-fed mice. Taken together, these results suggest tofogliflozin ameliorates insulin resistance and obesity by increasing glucose uptake in skeletal muscle and lipolysis in adipose tissue.
Asunto(s)
Tejido Adiposo Blanco/efectos de los fármacos , Compuestos de Bencidrilo/farmacología , Glucósidos/farmacología , Hipoglucemiantes/farmacología , Resistencia a la Insulina , Lipólisis/efectos de los fármacos , Músculo Esquelético/efectos de los fármacos , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Dieta Alta en Grasa , Ácidos Grasos no Esterificados/sangre , Expresión Génica/efectos de los fármacos , Glucosa/metabolismo , Técnica de Clampeo de la Glucosa , Cuerpos Cetónicos/sangre , Lipasa/efectos de los fármacos , Lipasa/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Lipogénesis/efectos de los fármacos , Lipogénesis/genética , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Ratones , Músculo Esquelético/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Esterol Esterasa/efectos de los fármacos , Esterol Esterasa/metabolismo , Aumento de Peso/efectos de los fármacosRESUMEN
Sodium-glucose cotransporter 2 (SGLT2) inhibitors showed a glucose lowering effect in type 2 diabetes patients through inducing renal glucose excretion. Detailed analysis of the mechanism of the glucosuric effect of SGLT2 inhibition, however, has been hampered by limitations of clinical study. Here, we investigated the mechanism of urinary glucose excretion using nonhuman primates with SGLT inhibitors tofogliflozin and phlorizin, both in vitro and in vivo. In cells overexpressing cynomolgus monkey SGLT2 (cSGLT2), both tofogliflozin and phlorizin competitively inhibited uptake of the substrate (α-methyl-d-glucopyranoside; AMG). Tofogliflozin was found to be a selective cSGLT2 inhibitor, inhibiting cSGLT2 more strongly than did phlorizin, with selectivity toward cSGLT2 1,000 times that toward cSGLT1; phlorizin was found to be a nonselective cSGLT1/2 inhibitor. In a glucose titration study in cynomolgus monkeys under conditions of controlled plasma drug concentration, both tofogliflozin and phlorizin increased fractional excretion of glucose (FEG) by up to 50% under hyperglycemic conditions. By fitting the titration curve using a newly introduced method that avoids variability in estimating the threshold of renal glucose excretion, we found that tofogliflozin and phlorizin lowered the threshold and extended the splay in a dose-dependent manner without significantly affecting the tubular transport maximum for glucose (TmG). Our results demonstrate the contribution of SGLT2 to renal glucose reabsorption (RGR) in cynomolgus monkeys and demonstrate that competitive inhibition of cSGLT2 exerts a glucosuric effect by mainly extending splay and lowering threshold without affecting TmG.
Asunto(s)
Compuestos de Bencidrilo/farmacología , Glucosa/metabolismo , Glucósidos/farmacología , Macaca fascicularis/orina , Florizina/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Transportador 2 de Sodio-Glucosa/efectos de los fármacos , Animales , Células COS/metabolismo , Células COS/patología , Chlorocebus aethiops , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Riñón/metabolismo , Riñón/patología , Masculino , Metilglucósidos/metabolismo , Modelos Animales , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Transportador 1 de Sodio-Glucosa/efectos de los fármacos , Transportador 1 de Sodio-Glucosa/genética , Transportador 2 de Sodio-Glucosa/genéticaRESUMEN
Although excessive fructose intake is epidemiologically linked with dyslipidemia, obesity, and diabetes, the mechanisms regulating plasma fructose are not well known. Cells transfected with sodium/glucose cotransporter 5 (SGLT5), which is expressed exclusively in the kidney, transport fructose in vitro; however, the physiological role of this transporter in fructose metabolism remains unclear. To determine whether SGLT5 functions as a fructose transporter in vivo, we established a line of mice lacking the gene encoding SGLT5. Sodium-dependent fructose uptake disappeared in renal brush border membrane vesicles from SGLT5-deficient mice, and the increased urinary fructose in SGLT5-deficient mice indicated that SGLT5 was the major fructose reabsorption transporter in the kidney. From this, we hypothesized that urinary fructose excretion induced by SGLT5 deficiency would ameliorate fructose-induced hepatic steatosis. To test this hypothesis we compared SGLT5-deficient mice with wild-type mice under conditions of long-term fructose consumption. Paradoxically, however, fructose-induced hepatic steatosis was exacerbated in the SGLT5-deficient mice, and the massive urinary fructose excretion was accompanied by reduced levels of plasma triglycerides and epididymal fat but fasting hyperinsulinemia compared with fructose-fed wild-type mice. There was no difference in food consumption, water intake, or plasma fructose between the two types of mice. No compensatory effect by other transporters reportedly involved in fructose uptake in the liver and kidney were indicated at the mRNA level. These surprising findings indicated a previously unrecognized link through SGLT5 between renal fructose reabsorption and hepatic lipid metabolism.
Asunto(s)
Hígado Graso/metabolismo , Fructosa/metabolismo , Riñón/metabolismo , Proteínas de Transporte de Sodio-Glucosa/metabolismo , Animales , Células COS , Chlorocebus aethiops , Hígado Graso/inducido químicamente , Fructosa/toxicidad , Genotipo , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Noqueados , Proteínas de Transporte de Sodio-Glucosa/genéticaRESUMEN
To understand the risk of hypoglycemia associated with urinary glucose excretion (UGE) induced by sodium-glucose cotransporter (SGLT) inhibitors, it is necessary to know the relationship between the ratio of contribution of SGLT2 vs. SGLT1 to renal glucose reabsorption (RGR) and the glycemic levels in vivo. To examine the contributions of SGLT2 and SGLT1 in normal rats, we compared the RGR inhibition by tofogliflozin, a highly specific SGLT2 inhibitor, and phlorizin, an SGLT1 and SGLT2 (SGLT1/2) inhibitor, at plasma concentrations sufficient to completely inhibit rat SGLT2 (rSGLT2) while inhibiting rSGLT1 to different degrees. Under hyperglycemic conditions by glucose titration, tofogliflozin and phlorizin achieved ≥50% inhibition of RGR. Under hypoglycemic conditions by hyperinsulinemic clamp, RGR was reduced by 20-50% with phlorizin and by 1-5% with tofogliflozin, suggesting the smaller contribution of rSGLT2 to RGR under hypoglycemic conditions than under hyperglycemic conditions. Next, to evaluate the hypoglycemic potentials of SGLT1/2 inhibition, we measured the plasma glucose (PG) and endogenous glucose production (EGP) simultaneously after UGE induction by SGLT inhibitors. Tofogliflozin (400 ng/ml) induced UGE of about 2 mg·kg⻹·min⻹ and increased EGP by 1-2 mg·kg⻹·min⻹, resulting in PG in the normal range. Phlorizin (1,333 ng/ml) induced UGE of about 6 mg·kg⻹·min⻹ and increased EGP by about 4 mg·kg⻹·min⻹; this was more than with tofogliflozin, but the minimum PG was lower. These results suggest that the contribution of SGLT1 to RGR is greater under lower glycemic conditions than under hyperglycemic conditions and that SGLT2-selective inhibitors pose a lower risk of hypoglycemia than SGLT1/2 inhibitors.
Asunto(s)
Compuestos de Bencidrilo/efectos adversos , Glucósidos/efectos adversos , Glucosuria/inducido químicamente , Hipoglucemiantes/efectos adversos , Riñón/efectos de los fármacos , Bloqueadores de los Canales de Sodio/efectos adversos , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Absorción/efectos de los fármacos , Animales , Compuestos de Bencidrilo/administración & dosificación , Compuestos de Bencidrilo/farmacocinética , Compuestos de Bencidrilo/uso terapéutico , Glucemia/análisis , Creatinina/metabolismo , Creatinina/orina , Relación Dosis-Respuesta a Droga , Gluconeogénesis/efectos de los fármacos , Glucósidos/administración & dosificación , Glucósidos/farmacocinética , Glucósidos/uso terapéutico , Glucosuria/etiología , Hiperglucemia/sangre , Hiperglucemia/tratamiento farmacológico , Hiperglucemia/metabolismo , Hiperglucemia/orina , Hipoglucemia/sangre , Hipoglucemia/inducido químicamente , Hipoglucemia/metabolismo , Hipoglucemia/orina , Hipoglucemiantes/administración & dosificación , Hipoglucemiantes/farmacocinética , Hipoglucemiantes/uso terapéutico , Riñón/metabolismo , Masculino , Florizina/administración & dosificación , Florizina/efectos adversos , Florizina/farmacocinética , Florizina/uso terapéutico , Ratas , Ratas Wistar , Bloqueadores de los Canales de Sodio/administración & dosificación , Bloqueadores de los Canales de Sodio/farmacocinética , Bloqueadores de los Canales de Sodio/uso terapéutico , Transportador 1 de Sodio-Glucosa/metabolismo , Transportador 2 de Sodio-Glucosa/metabolismoRESUMEN
Sodium/glucose cotransporter 2 (SGLT2) is the predominant mediator of renal glucose reabsorption and is an emerging molecular target for the treatment of diabetes. We identified a novel potent and selective SGLT2 inhibitor, tofogliflozin (CSG452), and examined its efficacy and pharmacological properties as an antidiabetic drug. Tofogliflozin competitively inhibited SGLT2 in cells overexpressing SGLT2, and K(i) values for human, rat, and mouse SGLT2 inhibition were 2.9, 14.9, and 6.4 nM, respectively. The selectivity of tofogliflozin toward human SGLT2 versus human SGLT1, SGLT6, and sodium/myo-inositol transporter 1 was the highest among the tested SGLT2 inhibitors under clinical development. Furthermore, no interaction with tofogliflozin was observed in any of a battery of tests examining glucose-related physiological processes, such as glucose uptake, glucose oxidation, glycogen synthesis, hepatic glucose production, glucose-stimulated insulin secretion, and glucosidase reactions. A single oral gavage of tofogliflozin increased renal glucose clearance and lowered the blood glucose level in Zucker diabetic fatty rats. Tofogliflozin also improved postprandial glucose excursion in a meal tolerance test with GK rats. In db/db mice, 4-week tofogliflozin treatment reduced glycated hemoglobin and improved glucose tolerance in the oral glucose tolerance test 4 days after the final administration. No blood glucose reduction was observed in normoglycemic SD rats treated with tofogliflozin. These findings demonstrate that tofogliflozin inhibits SGLT2 in a specific manner, lowers blood glucose levels by increasing renal glucose clearance, and improves pathological conditions of type 2 diabetes with a low hypoglycemic potential.
Asunto(s)
Compuestos de Bencidrilo/farmacología , Glucemia/metabolismo , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Glucósidos/farmacología , Hemoglobina Glucada/metabolismo , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Diabetes Mellitus Tipo 2/sangre , Relación Dosis-Respuesta a Droga , Intolerancia a la Glucosa/tratamiento farmacológico , Prueba de Tolerancia a la Glucosa , Humanos , Hiperglucemia/sangre , Hiperglucemia/tratamiento farmacológico , Riñón/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos , Ratas , Ratas Sprague-Dawley , Ratas ZuckerRESUMEN
Granulocyte-colony stimulating factor (G-CSF) has been reported to mobilize bone marrow multi-potent stem cells, which differentiate into cardiac myocytes after myocardial infarction (MI). However, there have not been any reports regarding the effect of G-CSF on stem cell infiltration in the MI site. Hearts of mice that had undergone coronary occlusion were isolated and digested with collagenase. Infiltrating cells in the heart were collected using Percoll density gradients. The infiltrating cells were sorted for side population (SP) cells using Hoechst 33342 dye. Hundreds of infiltrating SP cells were found in the heart from 1 to 14 d after MI. There were only a few SP cells in hearts without infarction. Infiltrating SP cells were increased in the 4-d G-CSF treated group compared with the vehicle group (1106 +/- 106 vs. 323 +/- 26/heart, P < 0.05). The infiltration of inflammatory cells was not influenced by the G-CSF treatment. In a separate series of experiments, we confirmed that the infiltrating SP cells were derived from bone marrow. That is, SP cells in the infarcted hearts of mice, which had been transplanted with bone marrow from ROSA 26 (beta-galactosidase transgenic) mice, were positive for beta-galactosidase. In the immunohistochemical examination, Sca-1(+)/CD45(-) cells were existed in the infarcted site after MI. Therefore, SP cells may infiltrate into infarcted heart. G-CSF augmented this kind of stem cell infiltration without increasing inflammatory cells. These results suggest that G-CSF may enhance myocardial regeneration without aggravated inflammation in the infarcted heart.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Factor Estimulante de Colonias de Granulocitos/farmacología , Células Madre Multipotentes/efectos de los fármacos , Infarto del Miocardio/patología , Animales , Recuento de Células , Citometría de Flujo , Humanos , Inflamación/tratamiento farmacológico , Inflamación/patología , Ratones , Células Madre Multipotentes/citología , Infarto del Miocardio/tratamiento farmacológicoRESUMEN
The anti-anginal effect of CP-060S, a new cardioprotective agent that prevents myocardial Na+-, Ca2+-overload and has Ca2+-channel blocking activity, was evaluated in a rat model of arginine8-vasopressin (AVP)-induced cardiac ischaemia. Infusion of AVP (0.2 IU kg(-1)) depressed the electrocardiogram (ECG) ST segment, an index of myocardial ischaemia. Vehicle, CP-060S and diltiazem were given orally 1, 2, 4, 8, 12 and 24 h before the administration of AVP. CP-060S, at 3 mg kg(-1) and 10 mg kg(-1), suppressed AVP-induced ST-segment depression for 2 h and 12 h, respectively. In contrast, diltiazem, at 10 and 30 mg kg(-1), suppressed AVP-induced ST-segment depression for only 1 h. The persistent suppression of the AVP-induced ST-segment depression by CP-060S correlated with the time course of changes in its plasma concentration. The minimum effective concentration of CP-060S was estimated to be 30 ng mL(-1) (approximately 50 nM), consistent with its vasorelaxant potency in rat isolated aortic strips (concentration producing 50% relaxation of KCl contraction, IC50 = 32.6+/-8.3 nM). Intravenously administered CP-060S, at 300 microg kg(-1) and diltiazem at 500 microg kg(-1) showed similar haemodynamic changes, whereas CP-060S, at 300 microg kg(-1), significantly suppressed AVP-induced ST-segment depression and diltiazem, at 500 microg kg(-1), had no effect on AVP-induced ST-segment depression. In summary, orally administered CP-060S exerted a long-lasting anti-anginal effect proportionate to the time course of changes in its plasma concentration in a rat model of AVP-induced ischaemia.