RESUMEN
Artificial pancreas system (APS) is an emerging new treatment for type 1 diabetes mellitus. The aim of this study was to develop a rat APS as a research tool and demonstrate its application. We established a rat APS using Medtronic Minimed Pump 722, Medtronic Enlite sensor, and the open artificial pancreas system as a controller. We tested different dilutions of Humalog (100 units/ml) in saline ranged from 1:3 to 1:20 and determined that 1:7 dilution works well for rats with ~500g bodyweight. Blood glucose levels (BGL) of diabetic rats fed with chow diet (58% carbohydrate) whose BGL was managed by the closed-loop APS for the total duration of 207h were in euglycemic range (70-180 mg/dl) for 94.5% of the time with 2.1% and 3.4% for hyperglycemia (>180mg/dl) and hypoglycemia (<70 mg/dl), respectively. Diabetic rats fed with Sucrose pellets (94.8% carbohydrate) for the experimental duration of 175h were in euglycemic range for 61% of the time with 35% and 4% for hyperglycemia and hypoglycemia, respectively. Heathy rats fed with chow diet showed almost a straight line of BGL ~ 95 mg/dl (average 94.8 mg/dl) during the entire experimental period (281h), which was minimally altered by food intake. In the healthy rats, feeding sucrose pellets caused greater range of BGL in high and low levels but still within euglycemic range (99.9%). Next, to study how healthy and diabetic rats handle supra-physiological concentrations of glucose, we intraperitoneally injected various amounts of 50% dextrose (2, 3, 4g/kg) and monitored BGL. Duration of hyperglycemia after injection of 50% dextrose at all three different concentrations was significantly greater for healthy rats than diabetic rats, suggesting that insulin infusion by APS was superior in reducing BGL as compared to natural insulin released from pancreatic ß-cells. Ex vivo studies showed that islets isolated from diabetic rats were almost completely devoid of pancreatic ß-cells but with intact α-cells as expected. Lipid droplet deposition in the liver of diabetic rats was significantly lower with higher levels of triacylglyceride in the blood as compared to those of healthy rats, suggesting lipid metabolism was altered in diabetic rats. However, glycogen storage in the liver determined by Periodic acid-Schiff staining was not altered in diabetic rats as compared to healthy rats. A rat APS may be used as a powerful tool not only to study alterations of glucose and insulin homeostasis in real-time caused by diet, exercise, hormones, or antidiabetic agents, but also to test mathematical and engineering models of blood glucose prediction or new algorithms for closed-loop APS.
Asunto(s)
Glucemia/análisis , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/terapia , Insulina/administración & dosificación , Páncreas Artificial , Animales , Glucemia/efectos de los fármacos , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/diagnóstico , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/inducido químicamente , Diabetes Mellitus Tipo 1/diagnóstico , Hemoglobina Glucada/análisis , Humanos , Infusiones Intravenosas/instrumentación , Infusiones Intravenosas/métodos , Masculino , Ratas , Estreptozocina/administración & dosificación , Estreptozocina/toxicidadRESUMEN
Obesity is one of the primary causes of type 2 diabetes mellitus (T2DM). To better understand how obesity impairs glucose-insulin homeostasis, we tracked fasting blood glucose and insulin levels and the key components of glucose-insulin homeostasis for 7 months in high fat diet (HFD; 45% fat) fed mice (n = 8). Every 2 weeks we measured body weight, fasting blood glucose and insulin levels, and estimated 5 key rate constants of glucose-insulin homeostasis using the methods established previously (Heliyon 3: e00310, 2017). Mice gained weight steadily, more than doubling their weights after 7 months (23.6 ± 0.5 to 52.3 ± 1.4 g). Fasting (basal) insulin levels were elevated (221.3 ± 16.7 to 1043.1 ± 90.5 pmol l-1) but fasting blood glucose levels unexpectedly returned to the baseline levels (152.8 ± 7.0 to 152.0 ± 7.2 mg/dl) despite significantly elevated levels (216.8 ± 44.9 mg/dl, average of 3 highest values for 8 mice) during the experimental period. After 7 months of HFD feeding, the rate constants for insulin secretion (k1), insulin-independent glucose uptake (k3), and insulin concentration where liver switches from glucose uptake to release (Ipi) were significantly elevated. Insulin-dependent glucose uptake (k2) and rate constant of liver glucose transfer (k4) were lowered but no statistical significance was reached. The novel and key finding of this study is the wide range of fluctuations of the rate constants during the course of obesity, reflecting the body's compensatory responses against metabolic alterations caused by obesity.
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Artificial pancreas system (APS) is a viable option to treat diabetic patients. Researchers, however, have not conclusively determined the best control method for APS. Due to intra-/inter-variability of insulin absorption and action, an individualized algorithm is required to control blood glucose level (BGL) for each patient. To this end, we developed model predictive control (MPC) based on artificial neural networks (ANNs), which combines ANN for BGL prediction based on inputs and MPC for BGL control based on the ANN (NN-MPC). First, we developed a mathematical model for diabetic rats, which was used to identify individual virtual subjects by fitting to empirical data collected through an APS, including BGL data, insulin injection, and food intake. Then, the virtual subjects were used to generate datasets for training ANNs. The NN-MPC determines control actions (insulin injection) based on BGL predicted by the ANN. To evaluate the NN-MPC, we conducted experiments using four virtual subjects under three different scenarios. Overall, the NN-MPC maintained BGL within the normal range about 90% of the time with a mean absolute deviation of 4.7 mg/dl from a desired BGL. Our findings suggest that the NN-MPC can provide subject-specific BGL control in conjunction with a closed-loop APS. Graphical abstract á .
Asunto(s)
Diabetes Mellitus Experimental/prevención & control , Diabetes Mellitus Experimental/terapia , Diabetes Mellitus Tipo 1/prevención & control , Diabetes Mellitus Tipo 1/terapia , Redes Neurales de la Computación , Páncreas Artificial , Animales , Glucemia/análisis , Diabetes Mellitus Experimental/sangre , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Tipo 1/sangre , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Insulina/administración & dosificación , Insulina/uso terapéutico , Ratas Sprague-DawleyRESUMEN
Destruction of the insulin-producing ß-cells is the key determinant of diabetes mellitus regardless of their types. Due to their anatomical location within the islets of Langerhans scattered throughout the pancreas, it is difficult to monitor ß-cell function and mass clinically. To this end, we propose to use a mathematical model of glucose-insulin homeostasis to estimate insulin secretion, glucose uptake by tissues, and hepatic handling of glucose. We applied the mathematical model by Lombarte et al. (2013) to compare various rate constants representing glucose-insulin homeostasis between lean (11% fat)- and high fat diet (HFD; 45% fat)-fed mice. Mice fed HFD (n = 12) for 3 months showed significantly higher body weights (49.97 ± 0.52 g vs. 29.86 ± 0.46 g), fasting blood glucose levels (213.08 ± 10.35 mg/dl vs. 121.91 ± 2.26 mg/dl), and glucose intolerance compared to mice fed lean diet (n = 12). Mice were injected with 1 g/kg glucose intraperitoneally and blood glucose levels were measured at various intervals for 120 min. We performed simulation using Arena™ software based on the mathematical model and estimated the rate constants (9 parameters) for various terms in the differential equations using OptQuest™. The simulated data fit accurately to the observed data for both lean and obese mice, validating the use of the mathematical model in mice at different stages of diabetes progression. Among 9 parameters, 5 parameters including basal insulin, k2 (rate constant for insulin-dependent glucose uptake to tissues), k3 (rate constant for insulin-independent glucose uptake to tissues), k4 (rate constant for liver glucose transfer), and Ipi (rate constant for insulin concentration where liver switches from glucose release to uptake) were significantly different between lean- and HFD-fed mice. Basal blood insulin levels, k3, and Ipi were significantly elevated but k2 and k4 were reduced in mice fed a HFD compared to those fed a lean diet. Non-invasive assessment of the key components of glucose-insulin homeostasis including insulin secretion, glucose uptake by tissues, and hepatic handling of glucose may be helpful for individualized drug therapy and designing a customized control algorithm for the artificial pancreas.
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An injection of hydrogel-encapsulated islets that controls blood glucose levels over long term would provide a much needed alternative treatment for type 1 diabetes mellitus (T1DM). To this end, we tested the feasibility of using an injectable polyethylene glycol (PEG) hydrogel as a scaffold for islet encapsulation. Encapsulated islets cultured in vitro for 6 days showed excellent cell viability and released insulin with higher basal and stimulated insulin secretion than control islets. Host responses to PEG hydrogels were studied by injecting PEG hydrogels (no treatment and vehicle controls used) into the peritoneal cavities of B6D2F1 mice and monitoring alterations in body weight, food and water intake, and blood glucose levels. After 2 weeks, peritoneal cavity cells were harvested, followed by hydrogel retrieval, and extraction of spleens. Body weights, food and water intake, and blood glucose levels were unaltered in mice injected with hydrogels compared to no treatment and vehicle-injected control mice. Frozen sections of a hydrogel showed the presence of tissues and small number of immune cells surrounding the hydrogel but no cell infiltration into the hydrogel bulk. Spleen sizes were not significantly different under the experimental conditions. Peritoneal cavity cells were slightly higher in mice injected with hydrogels compared to control mice but no statistical difference between vehicle- and hydrogel-injected mice was noted. As an in vivo feasibility study, streptozotocin-induced diabetic mice were injected with vehicle or hydrogels containing 50 islets each into two sites, the peritoneal cavity and a subcutaneous site on the back. Transient control of blood glucose levels were observed in mice injected with hydrogels containing islets. In summary, we developed an injectable PEG hydrogel that supported islet function and survival in vitro and in vivo and elicited only a mild host response. Our work illustrates the feasibility of using injectable PEG hydrogels for islet encapsulation.
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Peroxynitrite has been implicated in type 2 diabetes and diabetic complications. As a follow-up study to our previous work on SR-135 (Arch Biochem Biophys 577-578: 49-59, 2015), we provide evidence that this series of compounds are effective when administered orally, and their mechanisms of actions extend to the peripheral tissues. A more soluble analogue of SR-135, SR-110 (from a new class of Mn(III) bis(hydroxyphenyl)-dipyrromethene complexes) was orally administered for 2 weeks to B6D2F1 mice fed a high fat-diet (HFD). Mice fed a HFD for 4 months gained significantly higher body weights compared to lean diet-fed mice (52 ± 1.5 g vs 34 ± 1.3 g). SR-110 (10 mg/kg daily) treatment significantly reduced fasting blood glucose and insulin levels, and enhanced glucose tolerance as compared to HFD control or vehicle (peanut butter) group. SR-110 treatment enhanced insulin signaling in the peripheral organs, liver, heart, and skeletal muscle, and reduced lipid accumulation in the liver. Furthermore, SR-110 increased insulin content, restored islet architecture, decreased islet size, and reduced tyrosine nitration. These results suggest that a peroxynitrite decomposing catalyst is effective in improving glucose homeostasis and restoring islet morphology and ß-cell insulin content under nutrient overload.
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Grasas de la Dieta/efectos adversos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Ácido Peroxinitroso/metabolismo , Porfobilinógeno/análogos & derivados , Transducción de Señal/efectos de los fármacos , Administración Oral , Animales , Glucemia/metabolismo , Grasas de la Dieta/farmacología , Homeostasis/efectos de los fármacos , Ratones , Porfobilinógeno/química , Porfobilinógeno/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
Peroxynitrite has been implicated in ß-cell dysfunction and insulin resistance in obesity. Chemical catalysts that destroy peroxynitrite, therefore, may have therapeutic value for treating type 2 diabetes. To this end, we have recently demonstrated that Mn(III) bis(hydroxyphenyl)-dipyrromethene complexes, SR-135 and its analogs, can effectively catalyze the decomposition of peroxynitrite in vitro and in vivo through a 2-electron mechanism (Rausaria et al., 2011). To study the effects of SR-135 on glucose homeostasis in obesity, B6D2F1 mice were fed with a high fat-diet (HFD) for 12 weeks and treated with vehicle, SR-135 (5mg/kg), or a control drug SRB for 2 weeks. SR-135 significantly reduced fasting blood glucose and insulin levels, and enhanced glucose tolerance as compared to HFD control, vehicle or SRB. SR-135 also enhanced glucose-stimulated insulin secretion based on ex vivo studies. Moreover, SR-135 increased insulin content, restored islet architecture, decreased islet size, and reduced tyrosine nitration and apoptosis. These results suggest that a peroxynitrite decomposing catalyst enhances ß-cell function and survival under nutrient overload.
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Diabetes Mellitus Tipo 2/etiología , Hipoglucemiantes/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Manganeso/farmacología , Obesidad/complicaciones , Ácido Peroxinitroso/metabolismo , Porfobilinógeno/análogos & derivados , Animales , Apoptosis/efectos de los fármacos , Glucemia/análisis , Glucemia/metabolismo , Supervivencia Celular/efectos de los fármacos , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/metabolismo , Dieta Alta en Grasa/efectos adversos , Prueba de Tolerancia a la Glucosa , Hipoglucemiantes/química , Insulina/sangre , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Masculino , Manganeso/química , Ratones , Ratones Endogámicos C57BL , Obesidad/sangre , Obesidad/metabolismo , Porfobilinógeno/química , Porfobilinógeno/farmacologíaRESUMEN
IQGAP1 has emerged as a key component in the regulation of cytoskeleton dynamics during cell migration, maintenance of adherens junctions, microbial pathogenesis and intracellular trafficking. IQGAP1 is known to localize to the protruding edge of lamellipodia in a variety of cell types and interact with regulators of actin dynamics. Here, we provide evidence suggesting a novel role of IQGAP1 in cell motility through cell edge retraction. In some of the cell lines examined, IQGAP1 was markedly separated from WAVE localization suggesting IQGAP1 may localize to retracting edges. B16F10 mouse melanoma cells exhibited the most restricted separation in which the appearance of GFP-IQGAP1 correlated with cell edge retraction velocity and the disappearance of mCherry-Arp3. These results demonstrate that in some cell types IQGAP1 may function to promote cell retraction not lamellipodium edge protrusion. In addition, we examined co-localization of IQGAP1 with adhesion site markers, myosin IIA, calmodulin and IQGAP2. In areas rich in IQGAP1 there was decreased immunofluorescence staining of vinculin, paxillin and phosphorylated-tyrosine indicating adhesion site disassembly. Interestingly, calmodulin, but not myosin IIA or IQGAP2, co-localized with IQGAP1 in areas of cell retraction. Overall these results suggest a new role of IQGAP1, distinct form IQGAP2, in cell migration through up regulation of contractility and downregulation of adhesion sites potentially through calmodulin interaction.
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Movimiento Celular/fisiología , Animales , Calmodulina/metabolismo , Adhesión Celular/fisiología , Línea Celular , Ratones , Miosina Tipo IIA no Muscular/metabolismo , Seudópodos/fisiología , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
The aim of this study was to assess multifactorial ß-cell responses to metabolic perturbations in primary rat and human islets. Treatment of dispersed rat islet cells with elevated glucose and free fatty acids (FFAs, oleate:palmitate = 1:1 v/v) resulted in increases in the size and the number of lipid droplets in ß-cells in a time- and concentration-dependent manner. Glucose and FFAs synergistically stimulated the nutrient sensor mammalian target of rapamycin complex 1 (mTORC1). A potent mTORC1 inhibitor, rapamycin (25 nM), significantly reduced triglyceride accumulation in rat islets. Importantly, lipid droplets accumulated only in ß-cells but not in α-cells in an mTORC1-dependent manner. Nutrient activation of mTORC1 upregulated the expression of adipose differentiation related protein (ADRP), known to stabilize lipid droplets. Rat islet size and new DNA synthesis also increased under nutrient overload. Insulin secretion into the culture medium increased steadily over a 4-day period without any significant difference between glucose (10 mM) alone and the combination of glucose (10 mM) and FFAs (240 µM). Insulin content and insulin biosynthesis, however, were significantly reduced under the combination of nutrients compared with glucose alone. Elevated nutrients also stimulated lipid droplet formation in human islets in an mTORC1-dependent manner. Unlike rat islets, however, human islets did not increase in size under nutrient overload despite a normal response to nutrients in releasing insulin. The different responses of islet cell growth under nutrient overload appear to impact insulin biosynthesis and storage differently in rat and human islets.
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Ácidos Grasos no Esterificados/administración & dosificación , Glucosa/administración & dosificación , Células Secretoras de Insulina/metabolismo , Animales , Western Blotting , Diabetes Mellitus Tipo 2/metabolismo , Ácidos Grasos no Esterificados/metabolismo , Glucosa/metabolismo , Humanos , Inmunohistoquímica , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/ultraestructura , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteínas de la Membrana/metabolismo , Microscopía de Contraste de Fase , Complejos Multiproteicos , Perilipina-2 , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Triglicéridos/metabolismoRESUMEN
The plus-ends of microtubules target the cell cortex to modulate actin protrusion dynamics and polarity, but little is known of the molecular mechanism that couples the interaction. EB1 protein associates with the plus-ends of microtubules, placing EB1 in an ideal spatial position to mediate microtubule-actin cross talk. The objective of the current study was to further understand intracellular signaling involved in EB1-dependent cell polarity and motility. B16F10 mouse melanoma cells were depleted of EB1 protein using short hair-pin RNA interference. Correlative live cell-immunofluorescence microscopy was performed to determine localization of WAVE2 and IQGAP1 to protruding versus retracting edges. EB1 knock down caused poor subcellular separation of WAVE2 and IQGAP1, and overall decreased localization. Activation of PKC corrected defects in WAVE2 and IQGAP1 localization, cell spreading and cell shape to levels observed in control cells, but did not correct defects in cell migration. Consistent with these findings, decreased PKC phosphorylation was observed in EB1 knock down cells. These findings support a model where EB1 protein links microtubules to actin protrusion and cell polarity through signaling pathways involving PKC.
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Polaridad Celular , Melanoma Experimental/patología , Proteínas Asociadas a Microtúbulos/metabolismo , Proteína Quinasa C/biosíntesis , Animales , Línea Celular Tumoral , Movimiento Celular , Activación Enzimática , Técnicas de Silenciamiento del Gen , Melanoma Experimental/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/genética , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Proteínas Activadoras de ras GTPasa/metabolismoRESUMEN
This perspective delineates approaches to develop therapeutic strategies to stimulate the proliferative potential of adult human ß-cells in vitro. Previous findings demonstrated that nutrients, through regulation of mTOR signaling, promote regenerative processes including DNA synthesis, cell cycle progression and ß-cell proliferation in rodent islets but rarely in human islets. Recently, we discovered that regulation of the Wnt/GSK-3/ß-catenin pathway by directly inhibiting GSK-3 with pharmacologic agents, in combination with nutrient activation of mTOR, was required to increase growth and proliferation in human islets. Studies also revealed that nuclear translocation of ß-catenin in response to GSK-3 inhibition regulated these processes and was rapamycin sensitive, indicating a role for mTOR. Human islets displayed a high level of insulin resistance consistent with the inability of exogenous insulin to activate Akt and engage the Wnt pathway by GSK-3 inhibition. This insulin resistance in human islets is not present in rodent islets and may explain the differential requirement in human islets to inhibit GSK-3 to enhance these regenerative processes. Human islets exhibited normal insulin secretion but a loss of insulin content, which was independent of all treatment conditions. The loss of insulin content may be related to insulin resistance, the isolation process or culture conditions. In this perspective, we provide strategies to enhance the proliferative capacity of adult human ß-cells and highlight important differences between human and rodent islets: the lack of a nutrient response, requirement for direct GSK-3 inhibition, insulin resistance and loss of insulin content that emphasize the physiological significance of conducting studies in human islets.
RESUMEN
OBJECTIVE: Our previous studies demonstrated that nutrient regulation of mammalian target of rapamycin (mTOR) signaling promotes regenerative processes in rodent islets but rarely in human islets. Our objective was to extend these findings by using therapeutic agents to determine whether the regulation of glycogen synthase kinase-3 (GSK-3)/beta-catenin and mTOR signaling represent key components necessary for effecting a positive impact on human beta-cell mass relevant to type 1 and 2 diabetes. RESEARCH DESIGN AND METHODS: Primary adult human and rat islets were treated with the GSK-3 inhibitors, LiCl and the highly potent 1-azakenpaullone (1-Akp), and with nutrients. DNA synthesis, cell cycle progression, and proliferation of beta-cells were assessed. Measurement of insulin secretion and content and Western blot analysis of GSK-3 and mTOR signaling components were performed. RESULTS: Human islets treated for 4 days with LiCl or 1-Akp exhibited significant increases in DNA synthesis, cell cycle progression, and proliferation of beta-cells that displayed varying degrees of sensitivity to rapamycin. Intermediate glucose (8 mmol/l) produced a striking degree of synergism in combination with GSK-3 inhibition to enhance bromodeoxyuridine (BrdU) incorporation and Ki-67 expression in human beta-cells. Nuclear translocation of beta-catenin responsible for cell proliferation was found to be particularly sensitive to rapamycin. CONCLUSIONS: A combination of GSK-3 inhibition and nutrient activation of mTOR contributes to enhanced DNA synthesis, cell cycle progression, and proliferation of human beta-cells. Identification of therapeutic agents that appropriately regulate GSK-3 and mTOR signaling may provide a feasible and available approach to enhance human islet growth and proliferation.
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Ciclo Celular/fisiología , División Celular/fisiología , Replicación del ADN , Diabetes Mellitus Tipo 1/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Glucógeno Sintasa Quinasa 3/metabolismo , Islotes Pancreáticos/citología , Islotes Pancreáticos/fisiología , Proteínas Quinasas/metabolismo , Adolescente , Adulto , Anciano , Animales , Replicación del ADN/efectos de los fármacos , Diabetes Mellitus Tipo 1/enzimología , Diabetes Mellitus Tipo 2/enzimología , Femenino , Glucógeno Sintasa Quinasa 3/antagonistas & inhibidores , Humanos , Insulina/farmacología , Islotes Pancreáticos/enzimología , Antígeno Ki-67/genética , Masculino , Persona de Mediana Edad , Proteínas Quinasas/genética , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Serina-Treonina Quinasas TOR , Adulto JovenRESUMEN
The aim of this study was to define metabolic signaling pathways that mediate DNA synthesis and cell cycle progression in adult rodent islets to devise strategies to enhance survival, growth, and proliferation. Since previous studies indicated that glucose-stimulated activation of mammalian target of rapamycin (mTOR) leads to [3H]thymidine incorporation and that mTOR activation is mediated, in part, through the K(ATP) channel and changes in cytosolic Ca2+, we determined whether glyburide, an inhibitor of K(ATP) channels that stimulates Ca2+ influx, modulates [3H]thymidine incorporation. Glyburide (10-100 nm) at basal glucose stimulated [3H]thymidine incorporation to the same magnitude as elevated glucose and further enhanced the ability of elevated glucose to increase [3H]thymidine incorporation. Diazoxide (250 microm), an activator of KATP channels, paradoxically potentiated glucose-stimulated [3H]thymidine incorporation 2-4-fold above elevated glucose alone. Cell cycle analysis demonstrated that chronic exposure of islets to basal glucose resulted in a typical cell cycle progression pattern that is consistent with a low level of proliferation. In contrast, chronic exposure to elevated glucose or glyburide resulted in progression from G0/G1 to an accumulation in S phase and a reduction in G2/M phase. Rapamycin (100 nm) resulted in an approximately 62% reduction of S phase accumulation. The enhanced [3H]thymidine incorporation with chronic elevated glucose or glyburide therefore appears to be associated with S phase accumulation. Since diazoxide significantly enhanced [3H]thymidine incorporation without altering S phase accumulation under chronic elevated glucose, this increase in DNA synthesis also appears to be primarily related to an arrest in S phase and not cell proliferation.
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Glucosa/metabolismo , Islotes Pancreáticos/citología , Canales de Potasio/química , Proteínas Quinasas/metabolismo , Animales , Western Blotting , Calcio/metabolismo , Ciclo Celular , División Celular , Proliferación Celular , Células Cultivadas , Citosol/metabolismo , Diazóxido/farmacología , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Fase G2 , Gliburida/química , Gliburida/metabolismo , Masculino , Nifedipino/farmacología , Potasio/química , Ratas , Ratas Sprague-Dawley , Fase S , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Timidina/metabolismoRESUMEN
Lipid and glucose metabolism are adversely affected by diabetes, a disease characterized by pancreatic beta-cell dysfunction. To clarify the role of lipids in insulin secretion, we generated mice with beta-cell-specific overexpression (betaLPL-TG) or inactivation (betaLPL-KO) of lipoprotein lipase (LPL), a physiologic provider of fatty acids. LPL enzyme activity and triglyceride content were increased in betaLPL-TG islets; decreased LPL enzyme activity in betaLPL-KO islets did not affect islet triglyceride content. Surprisingly, both betaLPL-TG and betaLPL-KO mice were strikingly hyperglycemic during glucose tolerance testing. Impaired glucose tolerance in betaLPL-KO mice was present at one month of age, whereas betaLPL-TG mice did not develop defective glucose homeostasis until approximately five months of age. Glucose-simulated insulin secretion was impaired in islets isolated from both mouse models. Glucose oxidation, critical for ATP production and triggering of insulin secretion mediated by the ATP-sensitive potassium (KATP) channel, was decreased in betaLPL-TG islets but increased in betaLPL-KO islets. Islet ATP content was not decreased in either model. Insulin secretion was defective in both betaLPL-TG and betaLPL-KO islets under conditions causing calcium-dependent insulin secretion independent of the KATP channel. These results show that beta-cell-derived LPL has two physiologically relevant effects in islets, the inverse regulation of glucose metabolism and the independent mediation of insulin secretion through effects distal to membrane depolarization.
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Glucosa/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/fisiología , Lipoproteína Lipasa/metabolismo , Animales , Membrana Celular/fisiología , ADN Complementario , Glucosa/farmacología , Humanos , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Cinética , Lipoproteína Lipasa/genética , Potenciales de la Membrana/fisiología , Ratones , Ratones Transgénicos , Valores de Referencia , Triglicéridos/metabolismoRESUMEN
Mammalian target of rapamycin (mTOR) is a protein kinase that integrates signals from mitogens and the nutrients, glucose and amino acids, to regulate cellular growth and proliferation. Previous findings demonstrated that glucose robustly activates mTOR in an amino acid-dependent manner in rodent and human islets. Furthermore, activation of mTOR by glucose significantly increases rodent islet DNA synthesis that is abolished by rapamycin. Glucagon-like peptide-1 (GLP-1) agonists, through the production of cAMP, have been shown to enhance glucose-dependent proinsulin biosynthesis and secretion and to stimulate cellular growth and proliferation. The objective of this study was to determine if the glucose-dependent and cAMP-mediated mechanism by which GLP-1 agonists enhance beta-cell growth and proliferation is mediated, in part, through mTOR. Our studies demonstrated that forskolin-generated cAMP resulted in activation of mTOR at basal glucose concentrations as assessed by phosphorylation of S6K1, a downstream effector of mTOR. Conversely, an adenylyl cyclase inhibitor partially blocked glucose-induced S6K1 phosphorylation. Furthermore, the GLP-1 receptor agonist, Exenatide, dose-dependently enhanced phosphorylation of S6K1 at an intermediate glucose concentration (8 mmol/l) in a rapamycin-sensitive manner. To determine the mechanism responsible for this potentiation of mTOR, the effects of intra- and extracellular Ca2+ were examined. Glyburide, an inhibitor of ATP-sensitive K+ channels (K(ATP) channels), provided partial activation of mTOR at basal glucose concentrations due to the influx of extracellular Ca2+, and diazoxide, an activator of KATP channels, resulted in partial inhibition of S6K1 phosphorylation by 20 mmol/l glucose. Furthermore, Exenatide or forskolin reversed the inhibition by diazoxide, probably through mobilization of intracellular Ca2+ stores by cAMP. BAPTA, a chelator of intracellular Ca2+, resulted in inhibition of glucose-stimulated S6K1 phosphorylation due to a reduction in cytosolic Ca2+ concentrations. Selective blockade of glucose-stimulated Ca2+ influx unmasked a protein kinase A (PKA)-sensitive component involved in the mobilization of intracellular Ca2+ stores, as revealed with the PKA inhibitor H-89. Overall, these studies support our hypothesis that incretin-derived cAMP participates in the metabolic activation of mTOR by mobilizing intracellular Ca2+ stores that upregulate mitochondrial dehydrogenases and result in enhanced ATP production. ATP can then modulate KATP channels, serve as a substrate for adenylyl cyclase, and possibly directly regulate mTOR activation.
Asunto(s)
Sustancias de Crecimiento/fisiología , Islotes Pancreáticos/fisiología , Proteínas Quinasas/fisiología , Transducción de Señal/fisiología , Animales , Colforsina/farmacología , AMP Cíclico/farmacología , Diazóxido/farmacología , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/efectos de los fármacos , Fenómenos Fisiológicos de la Nutrición , Fosforilación , Serina-Treonina Quinasas TORRESUMEN
Lipid accumulation in pancreatic beta-cells is thought to cause its dysfunction and/or destruction via apoptosis. Our studies show that incubation of the beta-cell line RINm5F with the saturated free fatty acids (FFA) palmitate caused apoptosis based on increases in caspase 3 activity, Annexin V staining, and cell death. Furthermore, exposure of RINm5F cells to cAMP-increasing agents, 3-isobutyl-1-methylxanthine (IBMX), and forskolin completely abolished palmitate-mediated caspase 3 activity and significantly inhibited Annexin V staining and cell death. The cyclic AMP analogs cpt-cAMP and dibutyryl-cAMP mimicked the protective effects of IBMX and forskolin, suggesting that cAMP is the mediator of the anti-apoptotic effects. The protective action of IBMX and forskolin was rapid and did not appear to require gene transcription or new protein synthesis. However, these protective effects were clearly independent of protein kinase A (PKA) because of the lack of inhibition by the PKA inhibitors H-89 and KT5720. In attempts to identify this PKA-independent mechanism, we found that the newly developed cAMP analog 8CPT-2Me-cAMP, which selectively activates the cAMP-dependent guanine nucleotide exchange factor (cAMP-GEF) pathway, mimicked the protective effects of IBMX and forskolin, suggesting that the cAMP-GEF pathway is involved. In addition, both glucagon-like peptide (GLP-1) and its receptor agonist, Exenatide, inhibited palmitate-mediated caspase 3 activation in a dose-dependent manner. Unexpectedly, H-89 partially reversed the protective effects of GLP-1 and Exenatide, suggesting that PKA may play a role in the protective effects of these incretins. To explain these seemingly conflicting results, we demonstrated that low concentrations of cAMP produced by GLP-1 and Exenatide preferentially activate the PKA pathway, whereas higher cAMP concentrations produced by IBMX and forskolin activate the more dominant cAMP-GEF pathway. Taken together, these results indicate that intracellular concentrations of cAMP may play a key role in determining divergent signaling pathways that lead to antiapoptotic responses.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/farmacología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Islotes Pancreáticos/metabolismo , Sulfonamidas , 1-Metil-3-Isobutilxantina/farmacología , Animales , Apoptosis/fisiología , Carbazoles/farmacología , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Indoles/farmacología , Islotes Pancreáticos/patología , Isoquinolinas/farmacología , Ratones , Palmitatos/farmacología , Pirroles/farmacología , Transducción de Señal/efectos de los fármacosRESUMEN
Mammalian target of rapamycin (mTOR) is a serine and threonine protein kinase that regulates numerous cellular functions, in particular, the initiation of protein translation. mTOR-mediated phosphorylation of both the translational repressor eukaryotic initiation factor 4E binding protein-1 and p70 S6 kinase are early events that control the translation initiation process. Rapamycin, an inhibitor of mTOR, is a potent immunosuppressant due, in part, to its ability to interfere with T-cell activation at the level of translation, and it has gained a prominent role in preventing the development and progression of rejection in pancreatic islet transplant recipients. The characterization of the insulin signaling cascade that modulates mTOR in insulin-sensitive tissues has been a major focus of investigation. Recently, the ability of nutrients, in particular the branched-chain amino acid leucine, to activate mTOR independent of insulin by a process designated as nutrient signaling has been identified. The beta-cell expresses components of the insulin signaling cascade and utilizes the metabolism of nutrients to affect insulin secretion. These combined transduction processes make the beta-cell an unique cell to study metabolic and autocrine regulation of mTOR signaling. Our studies have described the ability of insulin and IGFs in concert with the nutrients leucine, glutamine, and glucose to modulate protein translation through mTOR in beta-cells. These findings suggest that mitochondria-derived factors, ATP in particular, may be responsible for nutrient signaling. The significance of these findings is that the optimization of mitochondrial function is not only important for insulin secretion but may significantly impact the growth and proliferation of beta-cells through these mTOR signaling pathways.