RESUMEN
The phosphorylation of the transmembrane protein nephrin has been shown to play an important role in signaling in kidney podocytes, and it has now been shown to also play a key role in regulating pancreatic ß-cell function. Williamson et al have recently shown that the loss of nephrin tyrosine phosphorylation on its 3 cytoplasmic YDxV motifs can enhance insulin release in aged female mice. These studies suggest that blocking nephrin phosphorylation may be an effective treatment option for improving ß-cell function.
Asunto(s)
Células Secretoras de Insulina , Proteínas de la Membrana , Animales , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Fosforilación/efectos de los fármacos , Ratones , Femenino , Humanos , Insulina/metabolismo , Podocitos/metabolismo , Podocitos/efectos de los fármacosRESUMEN
OBJECTIVE: Lipotoxic injury from renal lipid accumulation in obesity and type 2 diabetes (T2D) is implicated in associated kidney damage. However, models examining effects of renal ectopic lipid accumulation independent of obesity or T2D are lacking. We generated renal tubule-specific adipose triglyceride lipase knockout (RT-SAKO) mice to determine if this targeted triacylglycerol (TAG) over-storage affects glycemic control and kidney health. METHODS: Male and female RT-SAKO mice and their control littermates were tested for changes in glycemic control at 10-12 and 16-18 weeks of age. Markers of kidney health and blood lipid and hormone concentrations were analyzed. Kidney and blood lysophosphatidic acid (LPA) levels were measured, and a role for LPA in mediating impaired glycemic control was evaluated using the LPA receptor 1/3 inhibitor Ki-16425. RESULTS: All groups remained insulin sensitive, but 16- to 18-week-old male RT-SAKO mice became glucose intolerant, without developing kidney inflammation or fibrosis. Rather, these mice displayed lower circulating insulin and glucagon-like peptide 1 (GLP-1) levels. Impaired first-phase glucose-stimulated insulin secretion was detected and restored by Exendin-4. Kidney and blood LPA levels were elevated in older male but not female RT-SAKO mice, associated with increased kidney diacylglycerol kinase epsilon. Inhibition of LPA-mediated signaling restored serum GLP-1 levels, first-phase insulin secretion, and glucose tolerance. CONCLUSIONS: TAG over-storage alone is insufficient to cause renal tubule lipotoxicity. This work is the first to show that endogenously derived LPA modulates GLP-1 levels in vivo, demonstrating a new mechanism of kidney-gut-pancreas crosstalk to regulate insulin secretion and glucose homeostasis.
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Diabetes Mellitus Tipo 2 , Péptido 1 Similar al Glucagón , Animales , Femenino , Masculino , Ratones , Diabetes Mellitus Tipo 2/metabolismo , Péptido 1 Similar al Glucagón/metabolismo , Glucosa/metabolismo , Inflamación/metabolismo , Insulina/metabolismo , Secreción de Insulina , Riñón/metabolismo , Metabolismo de los Lípidos , Lípidos , Obesidad/metabolismoRESUMEN
Insulin, a key hormone in the regulation of glucose homoeostasis, is secreted by pancreatic ß-cells in response to elevated glucose levels. Insulin is released in a biphasic manner in response to glucose metabolism in ß-cells. The first phase of insulin secretion is triggered by an increase in the ATP:ADP ratio; the second phase occurs in response to both a rise in ATP:ADP and other key metabolic signals, including a rise in the NADPH:NADP+ ratio. Experimental evidence indicates that pyruvate-cycling pathways play an important role in the elevation of the NADPH:NADP+ ratio in response to glucose. The authors developed a kinetic model for the tricarboxylic acid cycle and pyruvate cycling pathways. The authors successfully validated the model against experimental observations and performed a sensitivity analysis to identify key regulatory interactions in the system. The model predicts that the dicarboxylate carrier and the pyruvate transporter are the most important regulators of pyruvate cycling and NADPH production. In contrast, the analysis showed that variation in the pyruvate carboxylase flux was compensated by a response in the activity of mitochondrial isocitrate dehydrogenase (ICDm ) resulting in minimal effect on overall pyruvate cycling flux. The model predictions suggest starting points for further experimental investigation, as well as potential drug targets for the treatment of type 2 diabetes.
Asunto(s)
Diabetes Mellitus Tipo 2 , Insulina , Humanos , Insulina/metabolismo , Ácido Pirúvico/metabolismo , NADP/metabolismo , Glucosa/metabolismo , Adenosina TrifosfatoRESUMEN
Oxidative stress caused by the exposure of pancreatic ß-cells to high levels of fatty acids impairs insulin secretion. This lipotoxicity is thought to play an important role in ß-cell failure in type 2 diabetes and can be prevented by antioxidants. Gamma-hydroxybutyrate (GHB), an endogenous antioxidant and energy source, has previously been shown to protect mice from streptozotocin and alloxan-induced diabetes; both compounds are generators of oxidative stress and yield models of type-1 diabetes. We sought to determine whether GHB could protect mouse islets from lipotoxicity caused by palmitate, a model relevant to type 2 diabetes. We found that GHB prevented the generation of palmitate-induced reactive oxygen species and the associated lipotoxic inhibition of glucose-stimulated insulin secretion while increasing the NADPH/NADP+ ratio. GHB may owe its antioxidant and insulin secretory effects to the formation of NADPH.
Asunto(s)
Diabetes Mellitus Tipo 2 , Islotes Pancreáticos , Oxibato de Sodio , Animales , Antioxidantes/farmacología , Ratones , NADP , Palmitatos/farmacología , Oxibato de Sodio/farmacologíaRESUMEN
Pancreatic ß-cells can secrete insulin via 2 pathways characterized as KATP channel -dependent and -independent. The KATP channel-independent pathway is characterized by a rise in several potential metabolic signaling molecules, including the NADPH/NADP+ ratio and α-ketoglutarate (αKG). Prolyl hydroxylases (PHDs), which belong to the αKG-dependent dioxygenase superfamily, are known to regulate the stability of hypoxia-inducible factor α. In the current study, we assess the role of PHDs in vivo using the pharmacological inhibitor dimethyloxalylglycine (DMOG) and generated ß-cell-specific knockout (KO) mice for all 3 isoforms of PHD (ß-PHD1 KO, ß-PHD2 KO, and ß-PHD3 KO mice). DMOG inhibited in vivo insulin secretion in response to glucose challenge and inhibited the first phase of insulin secretion but enhanced the second phase of insulin secretion in isolated islets. None of the ß-PHD KO mice showed any significant in vivo defects associated with glucose tolerance and insulin resistance except for ß-PHD2 KO mice which had significantly increased plasma insulin during a glucose challenge. Islets from both ß-PHD1 KO and ß-PHD3 KO had elevated ß-cell apoptosis and reduced ß-cell mass. Isolated islets from ß-PHD1 KO and ß-PHD3 KO had impaired glucose-stimulated insulin secretion and glucose-stimulated increases in the ATP/ADP and NADPH/NADP+ ratio. All 3 PHD isoforms are expressed in ß-cells, with PHD3 showing the most distinct expression pattern. The lack of each PHD protein did not significantly impair in vivo glucose homeostasis. However, ß-PHD1 KO and ß-PHD3 KO mice had defective ß-cell mass and islet insulin secretion, suggesting that these mice may be predisposed to developing diabetes.
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Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Prolil Hidroxilasas/metabolismo , Isoformas de Proteínas/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Regulación de la Expresión Génica , Glucosa/metabolismo , Prueba de Tolerancia a la Glucosa , Homeostasis , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Ácidos Cetoglutáricos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADP/metabolismo , Fosforilación Oxidativa , Consumo de Oxígeno , Fenotipo , Dominios ProteicosRESUMEN
The NAD-dependent deacetylase SIRT1 improves ß cell function. Accordingly, nicotinamide mononucleotide (NMN), the product of the rate-limiting step in NAD synthesis, prevents ß cell dysfunction and glucose intolerance in mice fed a high-fat diet. The current study was performed to assess the effects of NMN on ß cell dysfunction and glucose intolerance that are caused specifically by increased circulating free fatty acids (FFAs). NMN was intravenously infused, with or without oleate, in C57BL/6J mice over a 48-h-period to elevate intracellular NAD levels and consequently increase SIRT1 activity. Administration of NMN in the context of elevated plasma FFA levels considerably improved glucose tolerance. This was due not only to partial protection from FFA-induced ß cell dysfunction but also, unexpectedly, to a significant decrease in insulin clearance. However, in conditions of normal FFA levels, NMN impaired glucose tolerance due to decreased ß cell function. The presence of this dual action of NMN suggests caution in its proposed therapeutic use in humans.
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Ácidos Grasos no Esterificados/sangre , Intolerancia a la Glucosa/tratamiento farmacológico , Glucosa/efectos adversos , Insulina/metabolismo , Mononucleótido de Nicotinamida/administración & dosificación , Ácido Oléico/efectos adversos , Animales , Intolerancia a la Glucosa/sangre , Intolerancia a la Glucosa/inducido químicamente , Células Hep G2 , Humanos , Infusiones Intravenosas , Masculino , Ratones , Ratones Endogámicos C57BL , NAD/metabolismo , Mononucleótido de Nicotinamida/farmacología , Sirtuina 1/metabolismo , Regulación hacia ArribaRESUMEN
The α-ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is an HIF target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. Although PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about the effects of this highly conserved enzyme in insulin-secreting ß cells in vivo. Here, we show that the deletion of PHD3 specifically in ß cells (ßPHD3KO) was associated with impaired glucose homeostasis in mice fed a high-fat diet. In the early stages of dietary fat excess, ßPHD3KO islets energetically rewired, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in ßPHD3KO islets was associated with impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes, and insulin secretion. Thus, PHD3 might be a pivotal component of the ß cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress.
Asunto(s)
Ácidos Grasos/efectos adversos , Glucosa/metabolismo , Resistencia a la Insulina , Células Secretoras de Insulina/enzimología , Procolágeno-Prolina Dioxigenasa/metabolismo , Animales , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Glucólisis , Humanos , Secreción de Insulina , Metabolismo de los Lípidos , Masculino , Ratones , Ratones Noqueados , Oxidación-Reducción , Procolágeno-Prolina Dioxigenasa/genéticaRESUMEN
The transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor (HIF)-1ß (ARNT/HIF1ß) plays a key role in maintaining ß-cell function and has been shown to be one of the most downregulated transcription factors in islets from patients with type 2 diabetes. We have shown a role for ARNT/HIF1ß in glucose sensing and insulin secretion in vitro and no defects in in vivo glucose homeostasis. To gain a better understanding of the role of ARNT/HIF1ß in the development of diabetes, we placed control (+/+/Cre) and ß-cell-specific ARNT/HIF1ß knockout (fl/fl/Cre) mice on a high-fat diet (HFD). Unlike the control (+/+/Cre) mice, HFD-fed fl/fl/Cre mice had no impairment in in vivo glucose tolerance. The lack of impairment in HFD-fed fl/fl/Cre mice was partly due to an improved islet glucose-stimulated NADPH/NADP+ ratio and glucose-stimulated insulin secretion. The effects of the HFD-rescued insulin secretion in fl/fl/Cre islets could be reproduced by treating low-fat diet (LFD)-fed fl/fl/Cre islets with the lipid signaling molecule 1-monoacylglcyerol. This suggests that the defects seen in LFD-fed fl/fl/Cre islet insulin secretion involve lipid signaling molecules. Overall, mice lacking ARNT/HIF1ß in ß-cells have altered lipid signaling in vivo and are resistant to an HFD's ability to induce diabetes.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Diabetes Mellitus Experimental/metabolismo , Células Secretoras de Insulina/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Diabetes Mellitus Experimental/etiología , Dieta Alta en Grasa , Diglicéridos , Glucosa/metabolismo , Homeostasis , Secreción de Insulina , Masculino , Ratones Noqueados , NADP/metabolismoRESUMEN
Tre-2/USP6, BUB2, cdc16 domain family, member 1 (TBC1D1), a Rab-GTPase activating protein, is a paralogue of AS160, and has been implicated in the canonical insulin-signaling cascade in peripheral tissues. More recently, TBC1D1 was identified in rat and human pancreatic islets; however, the islet function of TBC1D1 remains not fully understood. We examined the role of TBC1D1 in glucose homeostasis and insulin secretion utilizing a rat knockout (KO) model. Chow-fed TBC1D1 KO rats had improved insulin action but impaired glucose-tolerance tests (GTT) and a lower insulin response during an intraperitoneal GTT compared with wild-type (WT) rats. The in vivo data suggest there may be an islet defect. Glucose-stimulated insulin secretion was higher in isolated KO rat islets compared with WT animals, suggesting TBC1D1 is a negative regulator of insulin secretion. Moreover, KO rats displayed reduced ß-cell mass, which likely accounts for the impaired whole-body glucose homeostasis. This ß-cell mass reduction was associated with increased active caspase 3, and unaltered Ki67 or urocortin 3, suggesting the induction of apoptosis rather than decreased proliferation or dedifferentiation may account for the decline in islet mass. A similar phenotype was observed in TBC1D1 heterozygous animals, highlighting the sensitivity of the pancreas to subtle reductions in TBC1D1 protein. An 8-week pair-fed high-fat diet did not further alter ß-cell mass or apoptosis in KO rats, suggesting that dietary lipids per se, do not lead to a further impairment in glucose homeostasis. The present study establishes a fundamental role for TBC1D1 in maintaining in vivo ß-cell mass.
Asunto(s)
Glucemia/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Homeostasis , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/metabolismo , Proteínas/metabolismo , Animales , Apoptosis , Caspasa 3/genética , Caspasa 3/metabolismo , Hormona Liberadora de Corticotropina/genética , Hormona Liberadora de Corticotropina/metabolismo , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Femenino , Intolerancia a la Glucosa/genética , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/sangre , Insulina/metabolismo , Resistencia a la Insulina , Secreción de Insulina , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Proteínas/genética , Ratas , Transducción de Señal , Urocortinas/genética , Urocortinas/metabolismoRESUMEN
Type 2 diabetes is associated with impaired nutrient-regulated anaplerosis and insulin secretion in pancreatic ß-cells. One key anaplerotic substrate that may be involved in regulating insulin release is α-ketoglutarate (αKG). Since prolyl hydroxylase domain proteins (PHDs) can metabolize cytosolic αKG, we sought to explore the role of this enzyme in the regulation of ß-cell function. The oxygen-sensing PHDs regulate the stability of hypoxia-inducible factor 1α (HIF1α) as well as other proline-containing proteins by catalyzing the hydroxylation of proline residues. This reaction is dependent on sufficient levels of oxygen, iron, and αKG. In the present study, we utilized both pharmacological and genetic approaches to assess the impact of inhibiting PHD activity on ß-cell function. We demonstrate that ethyl-3,4-dihydroxybenzoate (EDHB), a PHD inhibitor, significantly blunted glucose-stimulated insulin secretion (GSIS) from 832/13 clonal cells, rat, and human islets. EDHB reduced glucose utilization, ATP/ADP ratio, and key TCA cycle intermediates such as pyruvate, citrate, fumarate, and malate. siRNA-mediated knockdown of PHD1 and PHD3 inhibited GSIS, whereas siRNA-mediated knockdown of PHD2 had no effect on GSIS. Taken together, the current results demonstrate an important role for PHDs as mediators of islet insulin secretion.
Asunto(s)
Prolina Dioxigenasas del Factor Inducible por Hipoxia/fisiología , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animales , Línea Celular , Técnicas de Silenciamiento del Gen/métodos , Humanos , Secreción de Insulina , Estructura Terciaria de Proteína/fisiología , RatasRESUMEN
AIMS/HYPOTHESIS: It has been suggested that the transcription factor ARNT/HIF1ß is critical for maintaining in vivo glucose homeostasis and pancreatic beta cell glucose-stimulated insulin secretion (GSIS). Our goal was to gain more insights into the metabolic defects seen after the loss of ARNT/HIF1ß in beta cells. METHODS: The in vivo and in vitro consequences of the loss of ARNT/HIF1ß were investigated in beta cell specific Arnt/Hif1ß knockout mice (ß-Arnt (fl/fl/Cre) mice). RESULTS: The only in vivo defects found in ß-Arnt (fl/fl/Cre) mice were significant increases in the respiratory exchange ratio and in vivo carbohydrate oxidation, and a decrease in lipid oxidation. The mitochondrial oxygen consumption rate was unaltered in mouse ß-Arnt (fl/fl/Cre) islets upon glucose stimulation. ß-Arnt (fl/fl/Cre) islets had an impairment in the glucose-stimulated increase in Ca(2+) signalling and a reduced insulin secretory response to glucose in the presence of KCl and diazoxide. The glucose-stimulated increase in the NADPH/NADP(+) ratio was reduced in ß-Arnt (fl/fl/Cre) islets. The reduced GSIS and NADPH/NADP(+) levels in ß-Arnt (fl/fl/Cre) islets could be rescued by treatment with membrane-permeable tricarboxylic acid intermediates. Small interfering (si)RNA mediated knockdown of ARNT/HIF1ß in human islets also inhibited GSIS. These results suggest that the regulation of GSIS by the KATP channel-dependent and -independent pathways is affected by the loss of ARNT/HIF1ß in islets. CONCLUSIONS/INTERPRETATION: This study provides three new insights into the role of ARNT/HIF1ß in beta cells: (1) ARNT/HIF1ß deletion in mice impairs GSIS ex vivo; (2) ß-Arnt (fl/fl/Cre) mice have an increased respiratory exchange ratio; and (3) ARNT/HIF1ß is required for GSIS in human islets.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Glucosa/metabolismo , Homeostasis/genética , Células Secretoras de Insulina/enzimología , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/deficiencia , Prueba de Tolerancia a la Glucosa , Hormona de Crecimiento Humana/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Potencial de la Membrana Mitocondrial , Ratones , Ratones Noqueados , NADP/metabolismo , Consumo de Oxígeno , Intercambio Gaseoso PulmonarRESUMEN
Treatment regimens for acute myeloid leukemia (AML) continue to offer weak clinical outcomes. Through a high-throughput cell-based screen, we identified avocatin B, a lipid derived from avocado fruit, as a novel compound with cytotoxic activity in AML. Avocatin B reduced human primary AML cell viability without effect on normal peripheral blood stem cells. Functional stem cell assays demonstrated selectivity toward AML progenitor and stem cells without effects on normal hematopoietic stem cells. Mechanistic investigations indicated that cytotoxicity relied on mitochondrial localization, as cells lacking functional mitochondria or CPT1, the enzyme that facilitates mitochondria lipid transport, were insensitive to avocatin B. Furthermore, avocatin B inhibited fatty acid oxidation and decreased NADPH levels, resulting in ROS-dependent leukemia cell death characterized by the release of mitochondrial proteins, apoptosis-inducing factor, and cytochrome c. This study reveals a novel strategy for selective leukemia cell eradication based on a specific difference in mitochondrial function.
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Leucemia Mieloide Aguda/tratamiento farmacológico , Mitocondrias/efectos de los fármacos , Extractos Vegetales/farmacología , Aceites de Plantas/farmacología , Animales , Apoptosis/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Cromatografía Liquida/métodos , Frutas/química , Ensayos Analíticos de Alto Rendimiento/métodos , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Espectrometría de Masas/métodos , Ratones , Mitocondrias/metabolismo , Oxidación-Reducción , Persea/química , Especies Reactivas de Oxígeno/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Circulating free fatty acids (FFAs) are elevated in obesity and cause insulin resistance. The objective of the current study was to determine whether the antioxidant N-acetyl-l-cysteine (NAC) prevented hepatic and peripheral insulin resistance caused by prolonged elevation of plasma FFAs. Chronically cannulated Wistar rats received saline (SAL), Intralipid plus heparin (IH), IH plus NAC, or NAC i.v. infusion for 48âh. Insulin sensitivity was determined using the hyperinsulinemic-euglycemic clamp with tritiated glucose tracer. IH induced hepatic and peripheral insulin resistance (P<0.05). NAC co-infusion did not prevent insulin resistance in the liver, although it was able to prevent peripheral insulin resistance. Prolonged IH infusion did not appear to induce oxidative stress in the liver because hepatic content of protein carbonyl, malondialdehyde, and reduced to oxidized glutathione ratio did not differ across treatment groups. In alignment with our insulin sensitivity results, IH augmented skeletal muscle protein carbonyl content and this was prevented by NAC co-infusion. Taken together, our results indicate that oxidative stress mediates peripheral, but not hepatic, insulin resistance resulting from prolonged plasma FFA elevation. Thus, in states of chronic plasma FFA elevation, such as obesity, antioxidants may protect against peripheral but not hepatic insulin resistance.
Asunto(s)
Acetilcisteína/farmacología , Ácidos Grasos no Esterificados/sangre , Resistencia a la Insulina/fisiología , Fosfolípidos/administración & dosificación , Aceite de Soja/administración & dosificación , Animales , Biomarcadores , Glucemia , Emulsiones/administración & dosificación , Femenino , Depuradores de Radicales Libres/farmacología , Glucosa/metabolismo , Heparina/administración & dosificación , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/fisiología , Ratas , Ratas WistarRESUMEN
Uncontrolled glycemia is a hallmark of diabetes mellitus and promotes morbidities like neuropathy, nephropathy, and retinopathy. With the increasing prevalence of diabetes, both immune-mediated type 1 and obesity-linked type 2, studies aimed at delineating diabetes pathophysiology and therapeutic mechanisms are of critical importance. The ß-cells of the pancreatic islets of Langerhans are responsible for appropriately secreting insulin in response to elevated blood glucose concentrations. In addition to glucose and other nutrients, the ß-cells are also stimulated by specific hormones, termed incretins, which are secreted from the gut in response to a meal and act on ß-cell receptors that increase the production of intracellular cyclic adenosine monophosphate (cAMP). Decreased ß-cell function, mass, and incretin responsiveness are well-understood to contribute to the pathophysiology of type 2 diabetes, and are also being increasingly linked with type 1 diabetes. The present mouse islet isolation and cAMP determination protocol can be a tool to help delineate mechanisms promoting disease progression and therapeutic interventions, particularly those that are mediated by the incretin receptors or related receptors that act through modulation of intracellular cAMP production. While only cAMP measurements will be described, the described islet isolation protocol creates a clean preparation that also allows for many other downstream applications, including glucose stimulated insulin secretion, [3(H)]-thymidine incorporation, protein abundance, and mRNA expression.
Asunto(s)
Separación Celular/métodos , AMP Cíclico/análisis , Técnicas para Inmunoenzimas/métodos , Islotes Pancreáticos/química , Islotes Pancreáticos/citología , Animales , AMP Cíclico/metabolismo , Islotes Pancreáticos/metabolismo , RatonesRESUMEN
It is well known that mitochondrial metabolism of pyruvate is critical for insulin secretion; however, we know little about how pyruvate is transported into mitochondria in ß-cells. Part of the reason for this lack of knowledge is that the carrier gene was only discovered in 2012. In the current study, we assess the role of the recently identified carrier in the regulation of insulin secretion. Our studies show that ß-cells express both mitochondrial pyruvate carriers (Mpc1 and Mpc2). Using both pharmacological inhibitors and siRNA-mediated knockdown of the MPCs we show that this carrier plays a key role in regulating insulin secretion in clonal 832/13 ß-cells as well as rat and human islets. We also show that the MPC is an essential regulator of both the ATP-regulated potassium (KATP) channel-dependent and -independent pathways of insulin secretion. Inhibition of the MPC blocks the glucose-stimulated increase in two key signaling molecules involved in regulating insulin secretion, the ATP/ADP ratio and NADPH/NADP(+) ratio. The MPC also plays a role in in vivo glucose homeostasis as inhibition of MPC by the pharmacological inhibitor α-cyano-ß-(1-phenylindol-3-yl)-acrylate (UK5099) resulted in impaired glucose tolerance. These studies clearly show that the newly identified mitochondrial pyruvate carrier sits at an important branching point in nutrient metabolism and that it is an essential regulator of insulin secretion.
Asunto(s)
Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocondrias/metabolismo , Ácido Pirúvico/metabolismo , Acrilatos/farmacología , Adenosina Difosfato/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/genética , Adenosina Trifosfato/metabolismo , Animales , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Línea Celular Tumoral , Femenino , Técnicas de Silenciamiento del Gen , Glucosa/genética , Humanos , Insulina/genética , Secreción de Insulina , Células Secretoras de Insulina/citología , Masculino , Mitocondrias/genética , Proteínas de Transporte de Membrana Mitocondrial , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Transportadores de Ácidos Monocarboxílicos , NADP/genética , NADP/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Biphasic glucose-stimulated insulin secretion involves a rapid first phase followed by a prolonged second phase of insulin secretion. The biochemical pathways that control these 2 phases of insulin secretion are poorly defined. In this study, we used a gas chromatography mass spectroscopy-based metabolomics approach to perform a global analysis of cellular metabolism during biphasic insulin secretion. A time course metabolomic analysis of the clonal ß-cell line 832/13 cells showed that glycolytic, tricarboxylic acid, pentose phosphate pathway, and several amino acids were strongly correlated to biphasic insulin secretion. Interestingly, first-phase insulin secretion was negatively associated with L-valine, trans-4-hydroxy-L-proline, trans-3-hydroxy-L-proline, DL-3-aminoisobutyric acid, L-glutamine, sarcosine, L-lysine, and thymine and positively with L-glutamic acid, flavin adenine dinucleotide, caprylic acid, uridine 5'-monophosphate, phosphoglycerate, myristic acid, capric acid, oleic acid, linoleic acid, and palmitoleic acid. Tricarboxylic acid cycle intermediates pyruvate, α-ketoglutarate, and succinate were positively associated with second-phase insulin secretion. Other metabolites such as myo-inositol, cholesterol, DL-3-aminobutyric acid, and L-norleucine were negatively associated metabolites with the second-phase of insulin secretion. These studies provide a detailed analysis of key metabolites that are either negatively or positively associated with biphasic insulin secretion. The insights provided by these data set create a framework for planning future studies in the assessment of the metabolic regulation of biphasic insulin secretion.
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Ciclo del Ácido Cítrico , Glucólisis , Hiperglucemia/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Vía de Pentosa Fosfato , Animales , Línea Celular , Células Clonales , Cromatografía de Gases y Espectrometría de Masas , Glucosa/metabolismo , Hipoglucemia/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Cinética , Masculino , Metaboloma , Concentración Osmolar , Ratas , Ratas Sprague-Dawley , Técnicas de Cultivo de TejidosRESUMEN
Our understanding of adult human ß-cells is advancing, but we know little about the function and plasticity of ß-cells from infants. We therefore characterized islets and single islet cells from human infants after isolation and culture. Although islet morphology in pancreas biopsies was similar to that in adults, infant islets after isolation and 24-48 hours of culture had less insulin staining, content, and secretion. The cultured infant islets expressed pancreatic and duodenal homeobox 1 and several (Glut1, Cav1.3, Kir6.2) but not all (syntaxin 1A and synaptosomal-associated protein 25) markers of functional islets, suggesting a loss of secretory phenotype in culture. The activity of key ion channels was maintained in isolated infant ß-cells, whereas exocytosis was much lower than in adults. We examined whether a functional exocytotic phenotype could be reestablished under conditions thought to promote ß-cell differentiation. After a 24- to 28-day expansion and maturation protocol, we found preservation of endocrine markers and hormone expression, an increased proportion of insulin-positive cells, elevated expression of syntaxin 1A and synaptosomal-associated protein 25, and restoration of exocytosis to levels comparable with that in adult ß-cells. Thus, human infant islets are prone to loss of their exocytotic phenotype in culture but amenable to experimental approaches aimed at promoting expansion and functional maturation. Control of exocytotic protein expression may be an important mechanism underlying the plasticity of the secretory machinery, an increased understanding of which may lead to improved regenerative approaches to treat diabetes.
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Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Canales de Calcio Tipo L/metabolismo , Diferenciación Celular/fisiología , Células Cultivadas , Exocitosis/fisiología , Femenino , Glucagón/metabolismo , Transportador de Glucosa de Tipo 1/metabolismo , Humanos , Lactante , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/fisiología , Islotes Pancreáticos/crecimiento & desarrollo , Masculino , Persona de Mediana Edad , Técnicas de Placa-Clamp , Fenotipo , Canales de Potasio de Rectificación Interna/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Proteína 25 Asociada a Sinaptosomas/metabolismo , Sintaxina 1/metabolismoRESUMEN
Mitochondrial oxidative stress significantly contributes to the underlying pathology of several devastating neurodegenerative disorders. Mitochondria are highly sensitive to the damaging effects of reactive oxygen and nitrogen species; therefore, these organelles are equipped with a number of free radical scavenging systems. In particular, the mitochondrial glutathione (GSH) pool is a critical antioxidant reserve that is derived entirely from the larger cytosolic pool via facilitated transport. The mechanism of mitochondrial GSH transport has not been extensively studied in the brain. However, the dicarboxylate (DIC) and 2-oxoglutarate (OGC) carriers localized to the inner mitochondrial membrane have been established as GSH transporters in liver and kidney. Here, we investigated the role of these carriers in protecting neurons from oxidative and nitrosative stress. Immunoblot analysis of DIC and OGC in primary cultures of rat cerebellar granule neurons (CGNs) and cerebellar astrocytes showed differential expression of these carriers, with CGNs expressing only DIC and astrocytes expressing both DIC and OGC. Consistent with these findings, butylmalonate specifically reduced mitochondrial GSH in CGNs, whereas both butylmalonate and phenylsuccinate diminished mitochondrial GSH in astrocytes. Moreover, preincubation with butylmalonate but not phenylsuccinate significantly enhanced susceptibility of CGNs to oxidative and nitrosative stressors. This increased vulnerability was largely prevented by incubation with cell-permeable GSH monoethylester but not malate. Finally, knockdown of DIC with adenoviral siRNA also rendered CGNs more susceptible to oxidative stress. These findings demonstrate that maintenance of the mitochondrial GSH pool via sustained mitochondrial GSH transport is essential to protect neurons from oxidative and nitrosative stress.
Asunto(s)
Glutatión/metabolismo , Mitocondrias/metabolismo , Neuronas/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Transporte Biológico , Sistema Nervioso Central/metabolismo , Cerebelo/citología , Citosol/metabolismo , Radicales Libres , Malonatos/farmacología , Modelos Biológicos , Nitrógeno/metabolismo , Estrés Oxidativo , Ratas , Ratas Sprague-Dawley , Succinatos/farmacologíaRESUMEN
Defining the key metabolic pathways that are important for fuel-regulated insulin secretion is critical to providing a complete picture of how nutrients regulate insulin secretion. We have performed a detailed metabolomics study of the clonal ß-cell line 832/13 using a gas chromatography-mass spectrometer (GC-MS) to investigate potential coupling factors that link metabolic pathways to insulin secretion. Mid-polar and polar metabolites, extracted from the 832/13 ß-cells, were derivatized and then run on a GC/MS to identify and quantify metabolite concentrations. Three hundred fifty-five out of 527 chromatographic peaks could be identified as metabolites by our metabolomic platform. These identified metabolites allowed us to perform a systematic analysis of key pathways involved in glucose-stimulated insulin secretion (GSIS). Of these metabolites, 41 were consistently identified as biomarker for GSIS by orthogonal partial least-squares (OPLS). Most of the identified metabolites are from common metabolic pathways including glycolytic, sorbitol-aldose reductase pathway, pentose phosphate pathway, and the TCA cycle suggesting these pathways play an important role in GSIS. Lipids and related products were also shown to contribute to the clustering of high glucose sample groups. Amino acids lysine, tyrosine, alanine and serine were upregulated by glucose whereas aspartic acid was downregulated by glucose suggesting these amino acids might play a key role in GSIS. In summary, a coordinated signaling cascade elicited by glucose metabolism in pancreatic ß-cells is revealed by our metabolomics platform providing a new conceptual framework for future research and/or drug discovery.
Asunto(s)
Diabetes Mellitus Tipo 1/metabolismo , Glucosa/administración & dosificación , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animales , Línea Celular , Cromatografía de Gases y Espectrometría de Masas/métodos , Glucosa/metabolismo , Secreción de Insulina , Metabolómica/métodos , Análisis de Componente Principal , RatasRESUMEN
The ability of the pancreatic ß-cells to adapt the rate of insulin release in accordance to changes in circulating glucose levels is essential for glucose homeostasis. Two spatial barriers imposed by the plasma membrane and inner mitochondrial membrane need to be overcome in order to achieve stringent coupling between the different steps in the stimulus-secretion cascade. The first spatial barrier is overcome by the presence of a glucose transporter (GLUT) in the plasma membrane, whereas a low affinity hexokinase IV (glucokinase, GK) in the cytosol conveys glucose availability into a metabolic flux that triggers and accelerates insulin release. The mitochondrial inner membrane comprises a second spatial barrier that compartmentalizes glucose metabolism into glycolysis (cytosol) and tricarboxylate (TCA) cycle (mitochondrial matrix). The exchange of metabolites between cytosol and mitochondrial matrix is mediated via a set of mitochondrial carriers, including the aspartate-glutamate carrier (aralar1), α- ketoglutarate carrier (OGC), ATP/ADP carrier (AAC), glutamate carrier (GC1), dicarboxylate carrier (DIC) and citrate/isocitrate carrier (CIC). The scope of this review is to provide an overview of the role these carriers play in stimulus-secretion coupling and discuss the importance of these findings in the context of the exquisite glucose responsive state of the pancreatic ß-cell.