RESUMEN
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.
Asunto(s)
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
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
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
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
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 beta-cell function. Williamson et al. (2024) have recently shown that the loss of nephrin tyrosine phosphorylation on its three 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.
RESUMEN
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.
Asunto(s)
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
The metabolic pathways that are involved in regulating insulin secretion from pancreatic ß-cells are still incompletely understood. One potential regulator of the metabolic phenotype of ß-cells is the transcription factor aryl hydrocarbon receptor nuclear translocator (ARNT)/hypoxia-inducible factor (HIF)-1ß. ARNT/HIF-1ß levels are profoundly reduced in islets obtained from type 2 diabetic patients. However, no study to date has investigated key pathways involved in regulating insulin release in ß-cells that lack ARNT/HIF-1ß. In this study, we confirm that siRNA-mediated knockdown of ARNT/HIF-1ß inhibits glucose-stimulated insulin secretion. We next investigated the metabolic consequence of the loss of ARNT/HIF-1ß knockdown. We demonstrate that ß-cells with reduced ARNT/HIF-1ß expression levels exhibit a 31% reduction in glycolytic flux without significant changes in glucose oxidation or the ATP:ADP ratio. Metabolic profiling of ß-cells treated with siRNAs against the ARNT/HIF-1ß gene revealed that glycolysis, anaplerosis, and glucose-induced fatty acid production were down-regulated, and all are key events involved in glucose-stimulated insulin secretion. In addition, both first and second phase insulin secretion in islets were significantly reduced after ARNT/HIF-1ß knockdown. Together, our data suggest an important role for ARNT/HIF-1ß in anaplerosis, and it may play a critical role in maintaining normal secretion competence of ß-cells.
Asunto(s)
Translocador Nuclear del Receptor de Aril Hidrocarburo/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Translocador Nuclear del Receptor de Aril Hidrocarburo/genética , Línea Celular Tumoral , Ciclo del Ácido Cítrico/fisiología , Diabetes Mellitus Tipo 2/genética , Ácidos Grasos no Esterificados/metabolismo , Cromatografía de Gases y Espectrometría de Masas , Perfilación de la Expresión Génica , Glucosa/farmacología , Células Secretoras de Insulina/citología , Insulinoma , Metabolómica , Oxidación-Reducción , Neoplasias Pancreáticas , Vía de Pentosa Fosfato/fisiología , ARN Interferente Pequeño , RatasRESUMEN
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.
Asunto(s)
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
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
Glucose-stimulated insulin secretion from pancreatic islet beta-cells is dependent in part on pyruvate cycling through the pyruvate/isocitrate pathway, which generates cytosolic alpha-ketoglutarate, also known as 2-oxoglutarate (2OG). Here, we have investigated if mitochondrial transport of 2OG through the 2-oxoglutarate carrier (OGC) participates in control of nutrient-stimulated insulin secretion. Suppression of OGC in clonal pancreatic beta-cells (832/13 cells) and isolated rat islets by adenovirus-mediated delivery of small interfering RNA significantly decreased glucose-stimulated insulin secretion. OGC suppression also reduced insulin secretion in response to glutamine plus the glutamate dehydrogenase activator 2-amino-2-norbornane carboxylic acid. Nutrient-stimulated increases in glucose usage, glucose oxidation, glutamine oxidation, or ATP:ADP ratio were not affected by OGC knockdown, whereas suppression of OGC resulted in a significant decrease in the NADPH:NADP(+) ratio during stimulation with glucose but not glutamine + 2-amino-2-norbornane carboxylic acid. Finally, OGC suppression reduced insulin secretion in response to a membrane-permeant 2OG analog, dimethyl-2OG. These data reveal that the OGC is part of a mechanism of fuel-stimulated insulin secretion that is common to glucose, amino acid, and organic acid secretagogues, involving flux through the pyruvate/isocitrate cycling pathway. Although the components of this pathway must remain intact for appropriate stimulus-secretion coupling, production of NADPH does not appear to be the universal second messenger signal generated by these reactions.
Asunto(s)
Glucosa/metabolismo , Glutamina/metabolismo , Insulina/metabolismo , Ácidos Cetoglutáricos/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mitocondrias/metabolismo , Animales , Citosol/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Modelos Biológicos , NADP/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
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
Gemini surfactants have demonstrated significant potential for use in constructing non-viral transfection vectors for the delivery of genes into cells to induce protein expression. Previously, two asymmetric gemini surfactants containing pyrenyl groups in one of the alkyl tails of the surfactants were synthesized as fluorescence probes for use in mechanistic studies of the transfection process. Here we present the results of a thermodynamic investigation of the binding interaction(s) between the pyrenyl-modified surfactants and DNA. The thermodynamics of the interactions have been examined using isothermal titration calorimetry, light scattering, zeta potential, and circular dichroism measurements. Distinct differences are observed between the interaction of 12-s-12 vs. the pyrene modified py-s-12 surfactants with DNA; an intercalated binding is found for the py-s-12 surfactants that disrupts the typical interactions observed between DNA and gemini surfactants.
Asunto(s)
ADN/metabolismo , Tensoactivos/metabolismo , Animales , Calorimetría , Dicroismo Circular , ADN/química , Modelos Moleculares , Conformación Molecular , Salmón , Tensoactivos/química , TermodinámicaRESUMEN
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
Uncoupling protein 2 (UCP2) may act as an important regulator of insulin secretion. In this study, beta-cell function in UCP2-deficient mice was examined after a 45% high-fat diet (HFD) to assess its role during the development of diet-induced type 2 diabetes. HFD-fed UCP2 (-/-) mice have lower fasting blood glucose and elevated insulin levels when compared with wild-type (WT) mice. UCP2 (-/-) mice also have enhanced beta-cell glucose sensitivity compared with WT mice after HFD, a result that is due in part to the deterioration of glucose responsiveness in WT mice. HFD-fed UCP2 (-/-) mice have increased insulin secretory capacity as a result of increased pancreatic beta-cell mass and insulin content per islet. Islets from WT mice exposed to 0.5 mmol/l palmitate for 48 h have significantly reduced mitochondrial membrane potential, ATP concentrations, and glucose responsiveness compared with UCP2 (-/-) islets, suggesting that elevated UCP2 in WT mice increases proton leak and decreases mitochondrial ATP production. Highly increased carnitine palmitoyl transferase-1 gene expression in UCP2 (-/-) mice is suggestive of enhanced fatty acid oxidizing capacity, particularly after HFD stress. These results further establish UCP2 as a component in glucose sensing and suggest a possible new aspect of UCP2 function during the progression of type 2 diabetes.
Asunto(s)
Grasas de la Dieta/farmacología , Insulina/metabolismo , Proteínas de Transporte de Membrana , Proteínas Mitocondriales , Animales , Secuencia de Bases , Glucemia/metabolismo , Cartilla de ADN , Diabetes Mellitus Tipo 2/fisiopatología , Modelos Animales de Enfermedad , Ácidos Grasos no Esterificados/sangre , Glucagón/sangre , Insulina/sangre , Secreción de Insulina , Canales Iónicos , Ratones , Ratones Noqueados , Proteínas/genética , Proteínas/fisiología , Triglicéridos/sangre , Proteína Desacopladora 2 , Aumento de PesoRESUMEN
Abnormalities in insulin action are the characteristics of type 2 diabetes. Dominant-negative muscle-specific IGF-I receptor (MKR) mice exhibit elevated lipid levels at an early age and eventually develop type 2 diabetes. To evaluate the role of elevated lipids in the progression of the diabetic state, MKR mice were treated with WY14,643, a peroxisome proliferator-activated receptor (PPAR)-alpha agonist. WY14,643 treatment markedly reduced serum fatty acid and triglyceride levels within a few days, as well as muscle triglyceride levels, and subsequently normalized glucose and insulin levels in MKR mice. Hyperinsulinemic-euglycemic clamp analysis showed that WY14,643 treatment enhanced muscle and adipose tissue glucose uptake by improving whole-body insulin sensitivity. Insulin suppression of endogenous glucose production by the liver of MKR mice was also improved. The expression of genes involved in fatty acid oxidation was increased in liver and skeletal muscle, whereas gene expression levels of hepatic gluconeogenic enzymes were decreased in WY14,643-treated MKR mice. WY14,643 treatment also improved the pattern of glucose-stimulated insulin secretion from the perfused pancreata of MKR mice and reduced the beta-cell mass. Taken together, these findings suggest that the reduction in circulating or intracellular lipids by activation of PPAR-alpha improved insulin sensitivity and the diabetic condition of MKR mice.
Asunto(s)
Diabetes Mellitus Tipo 2/sangre , Glucosa/metabolismo , Receptor IGF Tipo 1/fisiología , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/agonistas , Factores de Transcripción/genética , Triglicéridos/metabolismo , Animales , Gluconeogénesis/efectos de los fármacos , Técnica de Clampeo de la Glucosa , Homeostasis , Cinética , Lípidos/sangre , Hígado/efectos de los fármacos , Hígado/metabolismo , Ratones , Ratones Endogámicos , Ratones Transgénicos , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Pirimidinas/farmacología , ARN Mensajero/genética , ARN Ribosómico 18S/efectos de los fármacos , ARN Ribosómico 18S/genética , Receptor IGF Tipo 1/genética , Receptores Citoplasmáticos y Nucleares/efectos de los fármacos , Factores de Tiempo , Factores de Transcripción/efectos de los fármacosRESUMEN
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
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.