RESUMEN
Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been shown to increase ketone bodies in patients with type 2 diabetes; however, the underlying mechanisms have not been fully elucidated. Here we examined the effect of the SGLT2 inhibitor dapagliflozin (1 mg/kg/day, formulated in a water, PEG400, ethanol, propylene glycol solution, 4 weeks) on lipid metabolism in obese Zucker rats. Fasting FFA metabolism was assessed in the anesthetized state using a [9,10-3H(N)]-palmitic acid tracer by estimating rates of plasma FFA appearance (Ra), whole-body FFA oxidation (Rox), and nonoxidative disposal (Rst). In the liver, clearance (Kß-ox) and flux (Rß-ox) of FFA into ß-oxidation were estimated using [9,10-3H]-(R)-bromopalmitate/[U-14C]palmitate tracers. As expected, dapagliflozin induced glycosuria and a robust antidiabetic effect; treatment reduced fasting plasma glucose and insulin, lowered glycated hemoglobin, and increased pancreatic insulin content compared with vehicle controls. Dapagliflozin also increased plasma FFA, Ra, Rox, and Rst with enhanced channeling toward oxidation versus storage. In the liver, there was also enhanced channeling of FFA to ß-oxidation, with increased Kß-ox, Rß-ox and tissue acetyl-CoA, compared with controls. Finally, dapagliflozin increased hepatic HMG-CoA and plasma ß-hydroxybutyrate, consistent with a specific enhancement of ketogenesis. Since ketogenesis has not been directly measured, we cannot exclude an additional contribution of impaired ketone body clearance to the ketosis. In conclusion, this study provides evidence that the dapagliflozin-induced increase in plasma ketone bodies is driven by the combined action of FFA mobilization from adipose tissue and diversion of hepatic FFA toward ß-oxidation.
Asunto(s)
Diabetes Mellitus Tipo 2 , Cetosis , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Animales , Compuestos de Bencidrilo , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Grasos no Esterificados , Glucósidos , Humanos , Insulina/metabolismo , Cuerpos Cetónicos/metabolismo , Cetosis/inducido químicamente , Cetosis/metabolismo , Hígado/metabolismo , Ratas , Ratas Zucker , Inhibidores del Cotransportador de Sodio-Glucosa 2/efectos adversos , Inhibidores del Cotransportador de Sodio-Glucosa 2/metabolismoRESUMEN
AIMS/HYPOTHESIS: Insufficient insulin release and hyperglucagonaemia are culprits in type 2 diabetes. Cocaine- and amphetamine-regulated transcript (CART, encoded by Cartpt) affects islet hormone secretion and beta cell survival in vitro in rats, and Cart (-/-) mice have diminished insulin secretion. We aimed to test if CART is differentially regulated in human type 2 diabetic islets and if CART affects insulin and glucagon secretion in vitro in humans and in vivo in mice. METHODS: CART expression was assessed in human type 2 diabetic and non-diabetic control pancreases and rodent models of diabetes. Insulin and glucagon secretion was examined in isolated islets and in vivo in mice. Ca(2+) oscillation patterns and exocytosis were studied in mouse islets. RESULTS: We report an important role of CART in human islet function and glucose homeostasis in mice. CART was found to be expressed in human alpha and beta cells and in a subpopulation of mouse beta cells. Notably, CART expression was several fold higher in islets of type 2 diabetic humans and rodents. CART increased insulin secretion in vivo in mice and in human and mouse islets. Furthermore, CART increased beta cell exocytosis, altered the glucose-induced Ca(2+) signalling pattern in mouse islets from fast to slow oscillations and improved synchronisation of the oscillations between different islet regions. Finally, CART reduced glucagon secretion in human and mouse islets, as well as in vivo in mice via diminished alpha cell exocytosis. CONCLUSIONS/INTERPRETATION: We conclude that CART is a regulator of glucose homeostasis and could play an important role in the pathophysiology of type 2 diabetes. Based on the ability of CART to increase insulin secretion and reduce glucagon secretion, CART-based agents could be a therapeutic modality in type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatología , Glucagón/metabolismo , Insulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Animales , Western Blotting , Señalización del Calcio/fisiología , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/sangre , Electrofisiología , Exocitosis/genética , Exocitosis/fisiología , Femenino , Células Secretoras de Glucagón/metabolismo , Glucosa/metabolismo , Homeostasis , Humanos , Inmunohistoquímica , Hibridación in Situ , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
AIMS/HYPOTHESIS: The NEFA-responsive G-protein coupled receptor 120 (GPR120) has been implicated in the regulation of inflammation, in the control of incretin secretion and as a predisposing factor influencing the development of type 2 diabetes by regulation of islet cell apoptosis. However, there is still considerable controversy about the tissue distribution of GPR120 and, in particular, it remains unclear which islet cell types express this molecule. In the present study, we have addressed this issue by constructing a Gpr120-knockout/ß-galactosidase (LacZ) knock-in (KO/KI) mouse to examine the distribution and functional role of GPR120 in the endocrine pancreas. METHODS: A KO/KI mouse was generated in which exon 1 of the Gpr120 gene (also known as Ffar4) was replaced in frame by LacZ, thereby allowing for regulated expression of ß-galactosidase under the control of the endogenous GPR120 promoter. The distribution of GPR120 was inferred from expression studies detecting ß-galactosidase activity and protein production. Islet hormone secretion was measured from isolated mouse islets treated with selective GPR120 agonists. RESULTS: ß-galactosidase activity was detected as a surrogate for GPR120 expression exclusively in a small population of islet endocrine cells located peripherally within the islet mantle. Immunofluorescence analysis revealed co-localisation with somatostatin suggesting that GPR120 is preferentially produced in islet delta cells. In confirmation of this, glucose-induced somatostatin secretion was inhibited by a range of selective GPR120 agonists. This response was lost in GPR120-knockout mice. CONCLUSIONS/INTERPRETATION: The results imply that GPR120 is selectively present within the delta cells of murine islets and that it regulates somatostatin secretion.
Asunto(s)
Islotes Pancreáticos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Células Secretoras de Somatostatina/metabolismo , Somatostatina/metabolismo , Animales , Ratones , Ratones Mutantes , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Dapagliflozin is a sodium-glucose co-transporter 2 (SGLT2) inhibitor used for the treatment of diabetes. This study examines the effects of dapagliflozin on human islets, focusing on alpha and beta cell composition in relation to function in vivo, following treatment of xeno-transplanted diabetic mice. Mouse beta cells were ablated by alloxan, and dapagliflozin was provided in the drinking water while controls received tap water. Body weight, food and water intake, plasma glucose, and human C-peptide levels were monitored, and intravenous arginine/glucose tolerance tests (IVarg GTT) were performed to evaluate islet function. The grafted human islets were isolated at termination and stained for insulin, glucagon, Ki67, caspase 3, and PDX-1 immunoreactivity in dual and triple combinations. In addition, human islets were treated in vitro with dapagliflozin at different glucose concentrations, followed by insulin and glucagon secretion measurements. SGLT2 inhibition increased the animal survival rate and reduced plasma glucose, accompanied by sustained human C-peptide levels and improved islet response to glucose/arginine. SGLT2 inhibition increased both alpha and beta cell proliferation (Ki67+glucagon+ and Ki67+insulin+) while apoptosis was reduced (caspase3+glucagon+ and caspase3+insulin+). Alpha cells were fewer following inhibition of SGLT2 with increased glucagon/PDX-1 double-positive cells, a marker of alpha to beta cell transdifferentiation. In vitro treatment of human islets with dapagliflozin had no apparent impact on islet function. In summary, SGLT2 inhibition supported human islet function in vivo in the hyperglycemic milieu and potentially promoted alpha to beta cell transdifferentiation, most likely through an indirect mechanism.
RESUMEN
The generation of pancreatic cell types from renewable cell sources holds promise for cell replacement therapies for diabetes. Although most effort has focused on generating pancreatic beta cells, considerable evidence indicates that glucagon secreting alpha cells are critically involved in disease progression and proper glucose control. Here we report on the generation of stem cell-derived human pancreatic alpha (SC-alpha) cells from pluripotent stem cells via a transient pre-alpha cell intermediate. These pre-alpha cells exhibit a transcriptional profile similar to mature alpha cells and although they produce proinsulin protein, they do not secrete significant amounts of processed insulin. Compound screening identified a protein kinase c activator that promotes maturation of pre-alpha cells into SC-alpha cells. The resulting SC-alpha cells do not express insulin, share an ultrastructure similar to cadaveric alpha cells, express and secrete glucagon in response to glucose and some glucagon secretagogues, and elevate blood glucose upon transplantation in mice.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Secretoras de Glucagón/citología , Células Secretoras de Insulina/efectos de los fármacos , Células Madre Pluripotentes/citología , Western Blotting , Diferenciación Celular/fisiología , Línea Celular , Electrofisiología , Técnica del Anticuerpo Fluorescente , Humanos , Páncreas/citologíaRESUMEN
GPR44 expression has recently been described as highly ß-cell selective in the human pancreas and constitutes a tentative surrogate imaging biomarker in diabetes. A radiolabeled small-molecule GPR44 antagonist, [11C]AZ12204657, was evaluated for visualization of ß-cells in pigs and nonhuman primates by positron emission tomography as well as in immunodeficient mice transplanted with human islets under the kidney capsule. In vitro autoradiography of human and animal pancreatic sections from subjects without and with diabetes, in combination with insulin staining, was performed to assess ß-cell selectivity of the radiotracer. Proof of principle of in vivo targeting of human islets by [11C]AZ12204657 was shown in the immunodeficient mouse transplantation model. Furthermore, [11C]AZ12204657 bound by a GPR44-mediated mechanism in pancreatic sections from humans and pigs without diabetes, but not those with diabetes. In vivo [11C]AZ12204657 bound specifically to GPR44 in pancreas and spleen and could be competed away dose-dependently in nondiabetic pigs and nonhuman primates. [11C]AZ12204657 is a first-in-class surrogate imaging biomarker for pancreatic ß-cells by targeting the protein GPR44.
Asunto(s)
Diabetes Mellitus Tipo 1/diagnóstico por imagen , Diabetes Mellitus Tipo 2/diagnóstico por imagen , Células Secretoras de Insulina/patología , Islotes Pancreáticos/diagnóstico por imagen , Éteres Fenílicos/farmacocinética , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Animales , Autorradiografía , Biomarcadores/metabolismo , Biopsia , Radioisótopos de Carbono , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 1/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Humanos , Células Secretoras de Insulina/metabolismo , Eliminación Intestinal , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Trasplante de Islotes Pancreáticos/diagnóstico por imagen , Trasplante de Islotes Pancreáticos/patología , Ligandos , Macaca fascicularis , Imagen por Resonancia Magnética , Ratones Desnudos , Éteres Fenílicos/administración & dosificación , Tomografía Computarizada por Tomografía de Emisión de Positrones , Prueba de Estudio Conceptual , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Prostaglandina/antagonistas & inhibidores , Sus scrofa , Distribución Tisular , Trasplante Heterólogo , Trasplante HeterotópicoRESUMEN
AIMS/HYPOTHESIS: GPR44 (DP2, PTGDR2, CRTh2) is the receptor for the pro-inflammatory mediator prostaglandin D2 (PGD2) and it is enriched in human islets. In rodent islets, PGD2 is produced in response to glucose, suggesting that the PGD2-GPR44/DP2 axis may play a role in human islet function during hyperglycemia. Consequently, the aim of this work was to elucidate the insulinotropic role of GPR44 antagonism in vitro in human beta-cells and in type 2 diabetes (T2DM) patients. METHODS: We determined the drive on PGD2 secretion by glucose and IL-1beta, as well as, the impact on insulin secretion by pharmacological GPR44/DP2 antagonism (AZD1981) in human islets and beta-cells in vitro. To test if metabolic control would be improved by antagonizing a hyperglycemia-driven increased PGD2 tone, we performed a proof-of-mechanism study in 20 T2DM patients (average 54 years, HbA1c 9.4%, BMI 31.6 kg/m2). The randomized, double-blind, placebo-controlled cross-over study consisted of two three-day treatment periods (AZD1981 or placebo) separated by a three-day wash-out period. Mixed meal tolerance test (MMTT) and intravenous graded glucose infusion (GGI) was performed at start and end of each treatment period. Assessment of AZD1981 pharmacokinetics, glucose, insulin, C-peptide, glucagon, GLP-1, and PGD2 pathway biomarkers were performed. RESULTS: We found (1) that PGD2 is produced in human islet in response to high glucose or IL-1beta, but likely by stellate cells rather than endocrine cells; (2) that PGD2 suppresses both glucose and GLP-1 induced insulin secretion in vitro; and (3) that the GPR44/DP2 antagonist (AZD1981) in human beta-cells normalizes insulin secretion. However, AZD1981 had no impact on neither glucose nor incretin dependent insulin secretion in humans (GGI AUC C-peptide 1-2h and MMTT AUC Glucose 0-4h LS mean ratios vs placebo of 0.94 (80% CI of 0.90-0.98, p = 0.12) and 0.99 (90% CI of 0.94-1.05, p = 0.45), despite reaching the expected antagonist exposure. CONCLUSION/INTERPRETATION: Pharmacological inhibition of the PGD2-GPR44/DP2 axis has no major impact on the modulation of acute insulin secretion in T2DM patients. TRIAL REGISTRATION: ClinicalTrials.gov NCT02367066.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Insulina/metabolismo , Prostaglandina D2/metabolismo , Receptores Inmunológicos/metabolismo , Receptores de Prostaglandina/metabolismo , Factores de Transcripción/metabolismo , Acetatos/farmacología , Acetatos/uso terapéutico , Glucemia/metabolismo , Péptido C/sangre , Línea Celular , Proteínas de Unión al ADN/antagonistas & inhibidores , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Indoles/farmacología , Indoles/uso terapéutico , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Masculino , Persona de Mediana Edad , Prostaglandina D2/antagonistas & inhibidores , Receptores Inmunológicos/antagonistas & inhibidores , Receptores de Prostaglandina/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidoresRESUMEN
Beta cell dysfunction accompanies and drives the progression of type 2 diabetes mellitus (T2D), but there are few clinical biomarkers available to assess islet cell stress in humans. Secretagogin, a protein enriched in pancreatic islets, demonstrates protective effects on beta cell function in animals. However, its potential as a circulating biomarker released from human beta cells and islets has not been studied. In this study primary human islets, beta cells and plasma samples were used to explore secretion and expression of secretagogin in relation to the T2D pathology. Secretagogin was abundantly and specifically expressed and secreted from human islets. Furthermore, T2D patients had an elevated plasma level of secretagogin compared with matched healthy controls, which was confirmed in plasma of diabetic mice transplanted with human islets. Additionally, the plasma secretagogin level of the human cohort had an inverse correlation to clinical assessments of beta cell function. To explore the mechanism of secretagogin release in vitro, human beta cells (EndoC-ßH1) were exposed to elevated glucose or cellular stress-inducing agents. Secretagogin was not released in parallel with glucose stimulated insulin release, but was markedly elevated in response to endoplasmic reticulum stressors and cytokines. These findings indicate that secretagogin is a potential novel biomarker, reflecting stress and islet cell dysfunction in T2D patients.
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Islotes Pancreáticos/metabolismo , Secretagoginas/sangre , Adulto , Anciano , Animales , Biomarcadores/sangre , Núcleo Celular/metabolismo , Estudios de Cohortes , Citocinas/metabolismo , Citoplasma/metabolismo , Diabetes Mellitus Experimental/sangre , Retículo Endoplásmico/metabolismo , Ensayo de Inmunoadsorción Enzimática , Femenino , Glucagón/metabolismo , Glucosa/farmacología , Prueba de Tolerancia a la Glucosa , Humanos , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/fisiopatología , Trasplante de Islotes Pancreáticos , Masculino , Ratones , Persona de Mediana EdadRESUMEN
Apelin is the endogenous ligand of the G-protein coupled apj receptor. Apelin is expressed in the brain, the hypothalamus and the stomach and was recently shown also to be an adipokine secreted from the adipocytes. Although apelin has been suggested to be involved in the regulation of food intake, it is not known whether the peptide affects islet function and glucose homeostasis. We show here that the apj receptor is expressed in pancreatic islets and that intravenous administration of full-length apelin-36 (2 nmol/kg) inhibits the rapid insulin response to intravenous glucose (1 g/kg) by 35% in C57BL/6J mice. Thus, the acute (1-5 min) insulin response to intravenous glucose was 682+/-23 pmol/l after glucose alone (n=17) and 445+/-58 pmol/l after glucose plus apelin-36 (n=18; P=0.017). This was associated with impaired glucose elimination (the 5-20 min glucose elimination was 2.9+/-0.1%/min after glucose alone versus 2.3+/-0.2%/min after glucose plus apelin-36, P=0.008). Apelin (2 nmol/kg) also inhibited the insulin response to intravenous glucose in obese insulin resistant high-fat fed C57BL/6J mice (P=0.041). After 60 min incubation of isolated islets from normal mice, insulin secretion in the presence of 16.7 mmol/l glucose was inhibited by apelin-36 at 1 mumol/l, whereas apelin-36 did not significantly affect insulin secretion at 2.8 or 8.3 mmol/l glucose or after stimulation of insulin secretion by KCl. Islet glucose oxidation at 16.7 mmol/l was not affected by apelin-36. We conclude that the apj receptor is expressed in pancreatic islets and that apelin-36 inhibits glucose-stimulated insulin secretion both in vivo and in vitro. This may suggest that the islet beta-cells are targets for apelin-36.
Asunto(s)
Proteínas Portadoras/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Adipoquinas , Animales , Apelina , Receptores de Apelina , Femenino , Glucosa/metabolismo , Homeostasis , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Receptores Acoplados a Proteínas G/genéticaRESUMEN
Glucagon-like peptide-1 (GLP-1) has long-term effects on pancreatic islets by increasing the insulin secretory capacity and beta cell mass. The islet effects of GLP-1 are glucose dependent and therefore tied to glucose sensing and metabolism. We examined whether prevention of inactivation of GLP-1 by inhibiting dipeptidyl peptidase-4 (DPP-4) is sufficient to promote long-term augmentation of glucose-stimulated insulin secretion. We also explored whether a defective glucose sensing and metabolism could be overcome by DPP-4 inhibition. We administered the orally active and highly selective DPP-4 inhibitor (1-[[(3-hydroxy-1-adamantyl) amino] acetyl]-2-cyano-(S)-pyrrolidineP-4; vildagliptin; 3 mumol/mouse daily) to normal, wildtype, mice and to mice with a beta-cell targeted dominant-negative mutant hepatocyte nuclear factor-1alpha (HNF-1alpha); these mice have a defective islet response to glucose. After eight weeks, vildagliptin augmented the insulin response after gastric glucose (75 mg) by 5-fold in male mice (7.3+/-0.8 vs. 1.3+/-0.5 nmol/l, P<0.001) and 30-fold in female mice (26.5+/-5.8 vs. 0.9+/-0.3 nmol/l, P<0.001). Furthermore, glucose-stimulated insulin secretion from isolated islets was markedly enhanced by 9 weeks treatment with vildagliptin. In contrast, in transgenic mice, the severely suppressed insulin response was only marginally improved by vildagliptin in males, and not affected at all in females. We conclude that DPP-4 inhibition improves islet function and increases beta cell secretory responses on a long-term basis and that this is dependent on intact expression of HNF-1alpha.
Asunto(s)
Dipeptidil Peptidasa 4/metabolismo , Factor Nuclear 1-alfa del Hepatocito/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Adamantano/análogos & derivados , Adamantano/farmacología , Animales , Peso Corporal/efectos de los fármacos , Peso Corporal/genética , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Femenino , Genotipo , Glucosa/farmacología , Prueba de Tolerancia a la Glucosa , Factor Nuclear 1-alfa del Hepatocito/genética , Técnicas In Vitro , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Intubación Gastrointestinal , Masculino , Ratones , Ratones Endogámicos CBA , Ratones Endogámicos , Ratones Transgénicos , Nitrilos , Pirrolidinas , Factores de Tiempo , VildagliptinaRESUMEN
Lipids are thought to serve as coupling factors in insulin secretion. Hormone-sensitive lipase (HSL) is expressed in pancreatic beta-cells and could potentially regulate insulin secretion via mobilization of stored triglycerides. Here, we examined the impact of HSL deficiency on fuel metabolism and insulin secretion in mouse islets. Lack of HSL resulted in abrogation of neutral cholesterol ester hydrolase activity, whereas diglyceride lipase activity remained intact. Although glucose stimulates lipolysis in rat islets, elevation of glucose with or without addition of cAMP failed to increase lipolysis in mouse islets regardless of genotype, as indicated by release of glycerol from islets. Storage of lipids, assayed as total acylglycerides, was unaltered in HSL null islets, and oxidation of fatty acids or glucose was not different. The intracellular rise in Ca(2+) triggered by glucose and its subsequent oscillations was unaffected in HSL null islets. Accordingly, insulin secretion in static incubations of islets, in response to fuel- and nonfuel secretagogues, was in no instance significantly different between wild-type and HSL null mice. The lacking impact of HSL deficiency on insulin secretion may be attributed to the failure of insulin secretagogues to stimulate lipolysis. Consequently, a regulatory function of lipid mobilization in insulin secretion in the mouse appears unlikely.
Asunto(s)
Grasas/metabolismo , Glicéridos/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/enzimología , Lipólisis , Esterol Esterasa/metabolismo , Esterol Esterasa/fisiología , Animales , Calcio/metabolismo , Femenino , Glucosa/metabolismo , Secreción de Insulina , Ratones , Oxidación-Reducción , Palmitatos/metabolismoRESUMEN
BACKGROUND: Although it is known that the islet expression of glucagon receptors is increased in type 2 diabetes, its implication for beta-cell function is not known. OBJECTIVE: To determine whether increased beta cell glucagon receptor expression and action influences multiple aspects of beta cell function. MATERIALS/METHODS: Mice with beta cell specific overexpression of the glucagon receptor (RIP-Gcgr) were subjected to intravenous glucose tolerance tests with acute injections of glucagon or GLP-1. Mice were also subjected to intravenous arginine and carbachol tests and insulin secretory responses were evaluated. RESULTS: The specific beta-cell overexpression of glucagon receptors has a complex and diverse consequence with dissociated consequences on beta-cell secretion depending on the stimulatory secretagogue in that whereas the potentiating effects of GLP-1 and arginine on glucose-stimulated insulin secretion were completely lost, the response to the muscarinic receptor agonist carbachol was largely unaffected and the insulin secretory response to glucose was exaggerated. CONCLUSION: This suggests that glucagon receptor overexpression, which is seen in hyperglycemia, may have dissociated consequence on beta cell function in its regulation under fasting, after meal and in response to autonomic nervous activation.
Asunto(s)
Células Secretoras de Insulina/metabolismo , Receptores de Glucagón/biosíntesis , Animales , Arginina/farmacología , Peso Corporal/efectos de los fármacos , Carbacol/farmacología , Glucagón/farmacología , Péptido 1 Similar al Glucagón/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Prueba de Tolerancia a la Glucosa , Humanos , Hipoglucemiantes/farmacología , Insulina/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Ratones , Ratones Transgénicos , Receptores de Glucagón/efectos de los fármacos , Receptores de Glucagón/genética , Receptores Muscarínicos/efectos de los fármacosRESUMEN
Exogenous administration of islet amyloid polypeptide (IAPP) has been shown to inhibit both insulin and glucagon secretion. This study examined alpha-cell function in mice with beta-cell specific overexpression of human IAPP (hIAPP) after an oral protein gavage (75 mg whey protein/mouse). Baseline glucagon levels were higher in transgenic mice (41 +/- 4.0 pg/mL, n = 6) than in wildtype animals (19 +/- 5.1 pg/mL, n = 5, P = .015). In contrast, the glucagon response to protein was impaired in transgenic animals (21 +/- 2.7 pg/mL in transgenic mice versus 38 +/- 5.7 pg/mL in wildtype mice at 15 minutes; P = .027). Baseline insulin levels did not differ between the groups, while the insulin response, as the glucagon response, was impaired after protein challenge (P = .018). Glucose levels were not different between the groups and did not change significantly after protein gavage. Acetaminophen was given through gavage to the animals (2 mg/mouse) to estimate gastric emptying. The plasma acetaminophen profile was similar in the two groups of mice. We conclude that disturbances in glucagon secretion exist in mice with beta-cell specific overexpression of human IAPP, which are not secondary to changes in gastric emptying. The reduced glucagon response to protein challenge may reflect a direct inhibitory influence of hIAPP on glucagon secretion.
Asunto(s)
Amiloide/genética , Células Secretoras de Glucagón/fisiología , Células Secretoras de Insulina/metabolismo , Enfermedades Pancreáticas/genética , Acetaminofén/sangre , Acetaminofén/farmacología , Administración Oral , Amiloide/metabolismo , Animales , Glucemia/análisis , Glucemia/efectos de los fármacos , Femenino , Vaciamiento Gástrico/efectos de los fármacos , Glucagón/sangre , Células Secretoras de Glucagón/efectos de los fármacos , Humanos , Insulina/sangre , Células Secretoras de Insulina/efectos de los fármacos , Polipéptido Amiloide de los Islotes Pancreáticos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Especificidad de Órganos/genética , Enfermedades Pancreáticas/sangre , Enfermedades Pancreáticas/etiología , Pruebas de Función Pancreática/métodos , Proteínas/administración & dosificación , Regulación hacia Arriba/genéticaRESUMEN
Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 microM) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action.
Asunto(s)
Acilcoenzima A/farmacología , Adipocitos/enzimología , Islotes Pancreáticos/enzimología , Lipasa/metabolismo , Lipólisis/efectos de los fármacos , Animales , Citosol/enzimología , Inhibidor de la Unión a Diazepam/farmacología , Inhibidores Enzimáticos/farmacología , Ácidos Grasos no Esterificados/metabolismo , Insulina/metabolismo , Secreción de Insulina , Lipasa/antagonistas & inhibidores , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Palmitoil Coenzima A/farmacología , Fosforilación , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Esterol Esterasa/antagonistas & inhibidores , Esterol Esterasa/deficiencia , Esterol Esterasa/metabolismo , Triglicéridos/metabolismoRESUMEN
OBJECTIVE: To discover a possible absorption and/or secretion of enterostatin into the circulating blood, as well as to compare the levels of circulating enterostatin after high-fat feeding and low-fat feeding. RESEARCH METHODS AND PROCEDURES: Using a specific enzyme-linked immunosorbent assay, plasma enterostatin levels were determined after feeding a high-fat, a high-fat/-sucrose, or a low-fat meal to Sprague-Dawley rats deprived of food overnight. RESULTS: The enterostatin levels were increased by all diets; the response to the high-fat and the high-fat/-sucrose meals was greater in magnitude and duration than that to the low-fat meal. In addition, enterostatin levels correlated with the intake of dietary fat. Plasma enterostatin levels after high-fat feeding were found to be similar to those after intravenous administration of exogenous enterostatin known to inhibit high-fat food intake. Gel chromatography of pooled postprandial plasma extracts followed by high-performance liquid chromatography analysis showed that plasma enterostatin was identical to synthetic enterostatin. Affinity cross-linking of plasma proteins with 125I-enterostatin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by autoradiography, revealed a single band with a molecular weight of about 66 kDa, indicating the presence of a potential enterostatin-binding protein in plasma. DISCUSSION: The measurements of plasma enterostatin may be a sensitive indicator for the measurement of fat intake.
Asunto(s)
Colipasas/sangre , Alimentos , Precursores de Proteínas/sangre , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Colipasas/administración & dosificación , Dieta , Grasas de la Dieta/administración & dosificación , Sacarosa en la Dieta/administración & dosificación , Electroforesis en Gel de Poliacrilamida , Precursores Enzimáticos , Femenino , Privación de Alimentos , Inyecciones Intravenosas , Precursores de Proteínas/administración & dosificación , Ratas , Ratas Sprague-DawleyRESUMEN
The identity of the enzymes responsible for lipase and cholesterol esterase activities in the small intestinal mucosa is not known. Because hormone-sensitive lipase (HSL) catalyzes the hydrolysis of acylglycerols and cholesteryl esters, we sought to determine whether HSL could be involved. HSL mRNA and protein were detected in all segments of the small intestine by Northern and Western blot analyses, respectively. Immunocytochemistry experiments revealed that HSL was expressed in the differentiated enterocytes of the villi and was absent in the undifferentiated cells of the crypt. Diacylglycerol lipase and cholesterol esterase activities were found in the different segments. Analysis of gut from HSL-null mice showed that diacylglycerol lipase activity was unchanged in the duodenum and reduced in jejunum. Neutral cholesterol esterase activity was totally abolished in duodenum, jejunum, and ileum of HSL-null mice. Analysis of HSL mRNA structure showed two types of transcripts expressed in equal amounts with alternative 5'-ends transcribed from two exons. This work demonstrates that HSL is expressed in the mucosa of the small intestine. The results also reveal that the enzyme participates in acylglycerol hydrolysis in jejunal enterocytes and cholesteryl ester hydrolysis throughout the small intestine.
Asunto(s)
Mucosa Intestinal/enzimología , Esterol Esterasa/genética , Esterol Esterasa/metabolismo , Animales , Secuencia de Bases , Cartilla de ADN , Cinética , Lipoproteína Lipasa/metabolismo , Masculino , Ratones , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
Lipid metabolism plays an important role in glucose homeostasis under normal and pathological conditions. In adipocytes, skeletal muscle, and pancreatic beta-cells, lipids are mobilized from acylglycerides by the hormone-sensitive lipase (HSL). Here, the consequences of a targeted disruption of the HSL gene for glucose homeostasis were examined. HSL null mice were slightly hyperglycemic in the fasted, but not fed state, which was accompanied by moderate hyperinsulinemia. During glucose challenges, however, disposal of the sugar was not affected in HSL null mice, presumably because of release of increased amounts of insulin. Impaired insulin sensitivity was further indicated by retarded glucose disposal during an insulin tolerance test. A euglycemic hyperinsulinemic clamp revealed that hepatic glucose production was insufficiently blocked by insulin in HSL null mice. In vitro, insulin-stimulated glucose uptake into soleus muscle, and lipogenesis in adipocytes were moderately reduced, suggesting additional sites of insulin resistance. Morphometric analysis of pancreatic islets revealed a doubling of beta-cell mass in HSL null mice, which is consistent with an adaptation to insulin resistance. Insulin secretion in vitro, examined by perifusion of isolated islets, was not impacted by HSL deficiency. Thus, HSL deficiency results in a moderate impairment of insulin sensitivity in multiple target tissues of the hormone but is compensated by hyperinsulinemia.