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1.
J Biol Chem ; 296: 100646, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33839150

RESUMEN

Dysregulated glucagon secretion deteriorates glycemic control in type 1 and type 2 diabetes. Although insulin is known to regulate glucagon secretion via its cognate receptor (insulin receptor, INSR) in pancreatic alpha cells, the role of downstream proteins and signaling pathways underlying insulin's activities are not fully defined. Using in vivo (knockout) and in vitro (knockdown) studies targeting insulin receptor substrate (IRS) proteins, we compared the relative roles of IRS1 and IRS2 in regulating alpha cell function. Alpha cell-specific IRS1-knockout mice exhibited glucose intolerance and inappropriate glucagon suppression during glucose tolerance tests. In contrast, alpha cell-specific IRS2-knockout animals manifested normal glucose tolerance and suppression of glucagon secretion after glucose administration. Alpha cell lines with stable IRS1 knockdown could not repress glucagon mRNA expression and exhibited a reduction in phosphorylation of AKT Ser/Thr kinase (AKT, at Ser-473 and Thr-308). AlphaIRS1KD cells also displayed suppressed global protein translation, including reduced glucagon expression, impaired cytoplasmic Ca2+ response, and mitochondrial dysfunction. This was supported by the identification of novel IRS1-specific downstream target genes, Trpc3 and Cartpt, that are associated with glucagon regulation in alpha cells. These results provide evidence that IRS1, rather than IRS2, is a dominant regulator of pancreatic alpha cell function.


Asunto(s)
Células Secretoras de Glucagón/patología , Glucagón/metabolismo , Intolerancia a la Glucosa/patología , Proteínas Sustrato del Receptor de Insulina/fisiología , Resistencia a la Insulina , Animales , Femenino , Células Secretoras de Glucagón/metabolismo , Intolerancia a la Glucosa/etiología , Intolerancia a la Glucosa/metabolismo , Masculino , Ratones , Ratones Noqueados , Fosforilación , Transducción de Señal
2.
Anal Chem ; 90(8): 5171-5178, 2018 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-29578696

RESUMEN

Microfluidics have been used to create "body-on-chip" systems to mimic in vivo cellular interactions with a high level of control. Most such systems rely on optical observation of cells as a readout. In this work we integrated a cell-cell interaction chip with online microchip electrophoresis immunoassay to monitor the effects of the interaction on protein secretion dynamics. The system was used to investigate the effects of adipocytes on insulin secretion. Chips were loaded with 190 000 3T3-L1 adipocytes and a single islet of Langerhans in separate chambers. The chambers were perfused at 300-600 nL/min so that adipocyte secretions flowed over the islets for 3 h. Adipocytes produced 80 µM of nonesterified fatty acids (NEFAs), a factor known to impact insulin secretion, at the islets. After perfusion, islets were challenged with a step change in glucose from 3 to 11 mM while monitoring insulin secretion at 8 s intervals by online immunoassay. Adipocyte treatment augmented insulin secretion by 6-fold compared to controls. The effect was far greater than comparable concentrations of NEFA applied to the islets demonstrating that adipocytes release multiple factors that can strongly potentiate insulin secretion. The experiments reveal that integration of chemical analysis with cell-cell interaction can provide valuable insights into cellular functions.


Asunto(s)
Adipocitos/citología , Electroforesis por Microchip , Inmunoensayo , Islotes Pancreáticos/citología , Técnicas Analíticas Microfluídicas , Células 3T3-L1 , Animales , Comunicación Celular , Células Cultivadas , Ácidos Grasos no Esterificados/análisis , Ácidos Grasos no Esterificados/biosíntesis , Ratones , Tamaño de la Partícula , Propiedades de Superficie
3.
Methods Mol Biol ; 1547: 57-67, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-28044287

RESUMEN

It has been demonstrated that microfluidic systems allow integration of sampling, reagent mixing, and rapid electrophoretic analysis. They have also proven useful for culturing cells wherein control over the environment allows novel and automated experiments. Here, we describe a microchip-based electrophoresis assay that allows cell culture and hormone monitoring. An online gradient generator can control cell culture condition precisely. This system has been applied for Pancreas islets' glucose sensitivity studies.


Asunto(s)
Electroforesis Capilar/métodos , Hormonas , Inmunoensayo/métodos , Animales , Electroforesis Capilar/instrumentación , Electroforesis por Microchip/instrumentación , Electroforesis por Microchip/métodos , Diseño de Equipo , Glucosa , Hormonas/sangre , Hormonas/metabolismo , Inmunoensayo/instrumentación , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Ratones , Microfluídica/instrumentación , Microfluídica/métodos
4.
Diabetes ; 65(3): 699-709, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26647386

RESUMEN

Human proinsulin with C-peptide-bearing Superfolder Green Fluorescent Protein (CpepSfGFP) has been expressed in transgenic mice, driven by the Ins1 promoter. The protein, expressed exclusively in ß-cells, is processed and stored as CpepSfGFP and human insulin comprising only ∼0.04% of total islet proinsulin plus insulin, exerting no metabolic impact. The kinetics of the release of insulin and CpepSfGFP from isolated islets appear identical. Upon a single acute stimulatory challenge in vitro, fractional release of insulin does not detectably deplete islet fluorescence. In vivo, fluorescence imaging of the pancreatic surface allows, for the first time, visual assessment of pancreatic islet insulin content, and we demonstrate that CpepSfGFP visibly declines upon diabetes progression in live lepR(db/db) mice. In anesthetized mice, after intragastric or intravenous saline delivery, pancreatic CpepSfGFP (insulin) content remains undiminished. Remarkably, however, within 20 min after acute intragastric or intravenous glucose delivery (with blood glucose concentrations reaching >15 mmol/L), a small subset of islets shows rapid dispossession of a major fraction of their stored CpepSfGFP (insulin) content, whereas most islets exhibit no demonstrable loss of CpepSfGFP (insulin). These studies strongly suggest that there are "first responder" islets to an in vivo glycemic challenge, which cannot be replicated by islets in vitro.


Asunto(s)
Péptido C/metabolismo , Diabetes Mellitus/metabolismo , Células Secretoras de Insulina/metabolismo , Proinsulina/metabolismo , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Péptido C/genética , Progresión de la Enfermedad , Glucosa/farmacología , Proteínas Fluorescentes Verdes/genética , Humanos , Técnicas In Vitro , Células Secretoras de Insulina/efectos de los fármacos , Ratones , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Proinsulina/efectos de los fármacos
5.
Endocrinology ; 157(2): 611-23, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26697721

RESUMEN

Mouse islets exhibit glucose-dependent oscillations in electrical activity, intracellular Ca(2+) and insulin secretion. We developed a mathematical model in which a left shift in glucose threshold helps compensate for insulin resistance. To test this experimentally, we exposed isolated mouse islets to varying glucose concentrations overnight and monitored their glucose sensitivity the next day by measuring intracellular Ca(2+), electrical activity, and insulin secretion. Glucose sensitivity of all oscillation modes was increased when overnight glucose was greater than 2.8mM. To determine whether threshold shifts were a direct effect of glucose or involved secreted insulin, the KATP opener diazoxide (Dz) was coapplied with glucose to inhibit insulin secretion. The addition of Dz or the insulin receptor antagonist s961 increased islet glucose sensitivity, whereas the KATP blocker tolbutamide tended to reduce it. This suggests insulin and glucose have opposing actions on the islet glucose threshold. To test the hypothesis that the threshold shifts were due to changes in plasma membrane KATP channels, we measured cell KATP conductance, which was confirmed to be reduced by high glucose pretreatment and further reduced by Dz. Finally, treatment of INS-1 cells with glucose and Dz overnight reduced high affinity sulfonylurea receptor (SUR1) trafficking to the plasma membrane vs glucose alone, consistent with insulin increasing KATP conductance by altering channel number. The results support a role for metabolically regulated KATP channels in the maintenance of glucose homeostasis.


Asunto(s)
Señalización del Calcio/efectos de los fármacos , Glucosa/farmacología , Hiperglucemia/metabolismo , Islotes Pancreáticos/efectos de los fármacos , Islotes Pancreáticos/metabolismo , Animales , Células Cultivadas , Glucosa/administración & dosificación , Intolerancia a la Glucosa/metabolismo , Hiperinsulinismo/metabolismo , Insulina/metabolismo , Resistencia a la Insulina , Secreción de Insulina , Masculino , Ratones , Modelos Teóricos , Factores de Tiempo
6.
Diabetes ; 62(7): 2439-49, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23493568

RESUMEN

Patients with type 2 diabetes (T2D) often exhibit hyperglucagonemia despite hyperglycemia, implicating defective α-cell function. Although endoplasmic reticulum (ER) stress has been suggested to underlie ß-cell dysfunction in T2D, its role in α-cell biology remains unclear. X-box binding protein 1 (XBP1) is a transcription factor that plays a crucial role in the unfolded protein response (UPR), and its deficiency in ß-cells has been reported to impair insulin secretion, leading to glucose intolerance. To evaluate the role of XBP1 in α-cells, we created complementary in vivo (α-cell-specific XBP1 knockout [αXBPKO] mice) and in vitro (stable XBP1 knockdown α-cell line [αXBPKD]) models. The αXBPKO mice exhibited glucose intolerance, mild insulin resistance, and an inability to suppress glucagon secretion after glucose stimulation. αXBPKD cells exhibited activation of inositol-requiring enzyme 1, an upstream activator of XBP1, leading to phosphorylation of Jun NH2-terminal kinase. Interestingly, insulin treatment of αXBPKD cells reduced tyrosine phosphorylation of insulin receptor substrate 1 (IRS1) (pY(896)) and phosphorylation of Akt while enhancing serine phosphorylation (pS(307)) of IRS1. Consequently, the αXBPKD cells exhibited blunted suppression of glucagon secretion after insulin treatment in the presence of high glucose. Together, these data indicate that XBP1 deficiency in pancreatic α-cells induces altered insulin signaling and dysfunctional glucagon secretion.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , Células Secretoras de Glucagón/metabolismo , Glucagón/metabolismo , Intolerancia a la Glucosa/metabolismo , Resistencia a la Insulina/genética , Insulina/metabolismo , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/genética , Glucagón/genética , Células Secretoras de Glucagón/efectos de los fármacos , Intolerancia a la Glucosa/genética , Insulina/farmacología , Proteínas Sustrato del Receptor de Insulina/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Masculino , Ratones , Ratones Noqueados , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Factores de Transcripción del Factor Regulador X , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Factores de Transcripción/genética , Proteína 1 de Unión a la X-Box
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