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1.
Yakugaku Zasshi ; 142(5): 457-463, 2022.
Artículo en Japonés | MEDLINE | ID: mdl-35491149

RESUMEN

Depression of lipid metabolism in ß-cells has been indicated to be one of the causes of impaired insulin secretion in type 2 diabetes. Diacylglycerol (DAG) is an important lipid mediator and is known to regulate insulin secretion in pancreatic ß-cells. Intracellular DAG accumulation is involved in ß-cell dysfunction in the pathogenesis of type 2 diabetes; thus, the regulation of intracellular DAG levels is likely important for maintaining the ß-cell function. We focused on diacylglycerol kinases (DGKs), which strictly regulate intracellular DAG levels, and analyzed the function of type I DGKs (DGKα, γ), which are activated by intracellular Ca2+ and expressed in the cytoplasm, in ß-cells. The suppression of the DGKα and γ expression decreased the insulin secretory response, and the decreased expression of DGKα and γ was observed in islets of diabetic model mice. In the pancreatic ß-cell line MIN6, 1 µM R59949 (a type I DGK inhibitor) and 10 µM DiC8 (a cell permeable DAG analog) enhanced glucose-induced [Ca2+]i oscillation in a PKC-dependent manner, while 10 µM R59949 and 100 µM DiC8 suppressed [Ca2+]i oscillation and voltage-dependent Ca2+ channel activity in a PKC-independent manner. These results suggest that the intracellular accumulation of DAG by the loss of the DGKα and γ functions regulates insulin secretion in a dual manner depending on the degree of DAG accumulation. The regulation of the insulin secretory response through DAG metabolism by type I DGKs may change depending on the degree of progression of type 2 diabetes.


Asunto(s)
Diabetes Mellitus Tipo 2 , Insulinas , Animales , Diabetes Mellitus Tipo 2/metabolismo , Diacilglicerol Quinasa/fisiología , Diglicéridos/metabolismo , Secreción de Insulina , Insulinas/metabolismo , Ratones
2.
FASEB J ; 35(5): e21420, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33774855

RESUMEN

Although an aberrant reduction in pancreatic ß-cell mass contributes to the pathogenesis of diabetes, the mechanism underlying the regulation of ß-cell mass is poorly understood. Here, we show that diacylglycerol kinase δ (DGKδ) is a key enzyme in the regulation of ß-cell mass. DGKδ expression was detected in the nucleus of ß-cells. We developed ß-cell-specific DGKδ knockout (ßDGKδ KO) mice, which showed lower blood glucose, higher plasma insulin levels, and better glucose tolerance compared to control mice. Moreover, an increased number of small islets and Ki-67-positive islet cells, as well as elevated cyclin B1 expression in the islets, were detected in the pancreas of ßDGKδ KO mice. DGKδ knockdown in the ß-cell line MIN6 induced significant increases in bromodeoxyuridine (BrdU) incorporation and cyclin B1 expression. Finally, we confirmed that streptozotocin-induced hyperglycemia and ß-cell loss were alleviated in ßDGKδ KO mice. Thus, suppressing the expression or enzymatic activity of DGKδ that functions as a suppressor of ß-cell proliferation could be a novel therapeutic approach to increase ß-cell mass for the treatment of diabetes.


Asunto(s)
Encéfalo/enzimología , Proliferación Celular , Diabetes Mellitus Experimental/complicaciones , Diacilglicerol Quinasa/fisiología , Hiperglucemia/prevención & control , Células Secretoras de Insulina/metabolismo , Animales , Hiperglucemia/etiología , Hiperglucemia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Transducción de Señal
3.
J Biol Chem ; 296: 100516, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33676892

RESUMEN

Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes, but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting that syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.


Asunto(s)
Movimiento Celular , Diacilglicerol Quinasa/fisiología , Proteínas Asociadas a la Distrofina/metabolismo , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/metabolismo , Neuropéptidos/metabolismo , Proteína Quinasa C-alfa/farmacología , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Animales , Diglicéridos/metabolismo , Proteínas Asociadas a la Distrofina/genética , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Ratones , Ratones Noqueados , Sustrato de la Proteína Quinasa C Rico en Alanina Miristoilada/genética , Neuropéptidos/genética , Dominios Proteicos , Proteína de Unión al GTP rac1/genética , Proteína de Unión al GTP rhoA/genética
4.
J Pharmacol Sci ; 140(2): 178-186, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-31279581

RESUMEN

The role of type I diacylglycerol kinases (DGKs) in the regulation of insulin secretion was investigated in MIN6 ß-cells. In intracellular Ca2+ concentration ([Ca2+]i) measurement experiments, 1 µM R59949, a type I DGK inhibitor, and 10 µM DiC8, a diacylglycerol (DAG) analog, amplified 22.2 mM glucose-induced [Ca2+]i oscillations in a protein kinase C (PKC)-dependent manner, whereas 10 µM R59949 and 100 µM DiC8 decreased [Ca2+]i independent of PKC. High concentrations of R59949 and DiC8 attenuated voltage-dependent Ca2+ channel currents. According to these results, 22.2 mM glucose-stimulated insulin secretion (GSIS) was potentiated by 1 µM R59949 but suppressed by 10 µM of the same. The DGKα inhibitor R59022 showed a similar dual effect. Conversely, DiC8 at 10 and 100 µM potentiated GSIS, although 100 µM DiC8 decreased [Ca2+]i. These results suggest that DAG accumulated through declined type I DGK activity shows a dual effect on insulin secretion depending on the degree of accumulation; a mild DAG accumulation induces a PKC-dependent stimulatory effect on insulin secretion, whereas an excessive DAG accumulation suppresses it in a PKC-independent manner, possibly via attenuation of VDCC activity.


Asunto(s)
Diacilglicerol Quinasa/fisiología , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/metabolismo , Células Cultivadas , Diacilglicerol Quinasa/antagonistas & inhibidores , Diglicéridos/metabolismo , Relación Dosis-Respuesta a Droga , Glucosa/farmacología , Secreción de Insulina/efectos de los fármacos , Ratones , Piperidinas/farmacología , Proteína Quinasa C/fisiología , Quinazolinonas/farmacología
5.
Sci Signal ; 11(530)2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29764991

RESUMEN

Skeletal muscle rapidly remodels in response to various stresses, and the resulting changes in muscle mass profoundly influence our health and quality of life. We identified a diacylglycerol kinase ζ (DGKζ)-mediated pathway that regulated muscle mass during remodeling. During mechanical overload, DGKζ abundance was increased and required for effective hypertrophy. DGKζ not only augmented anabolic responses but also suppressed ubiquitin-proteasome system (UPS)-dependent proteolysis. We found that DGKζ inhibited the transcription factor FoxO that promotes the induction of the UPS. This function was mediated through a mechanism that was independent of kinase activity but dependent on the nuclear localization of DGKζ. During denervation, DGKζ abundance was also increased and was required for mitigating the activation of FoxO-UPS and the induction of atrophy. Conversely, overexpression of DGKζ prevented fasting-induced atrophy. Therefore, DGKζ is an inhibitor of the FoxO-UPS pathway, and interventions that increase its abundance could prevent muscle wasting.


Asunto(s)
Diacilglicerol Quinasa/metabolismo , Diacilglicerol Quinasa/fisiología , Proteína Forkhead Box O3/metabolismo , Fibras Musculares Esqueléticas/patología , Atrofia Muscular/patología , Ubiquitina/metabolismo , Respuesta de Proteína Desplegada , Animales , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Femenino , Regulación de la Expresión Génica , Hipertrofia/etiología , Hipertrofia/metabolismo , Hipertrofia/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fibras Musculares Esqueléticas/metabolismo , Atrofia Muscular/etiología , Atrofia Muscular/metabolismo , FN-kappa B/metabolismo , Proteolisis , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo
6.
Neurosci Lett ; 630: 228-232, 2016 Sep 06.
Artículo en Inglés | MEDLINE | ID: mdl-27495014

RESUMEN

Diacylglycerol kinase (DGK) ß is a type 1 isozyme of the DGK family. We previously reported that DGKß was deeply involved in neurite spine formation, and DGKß knockout (KO) mice exhibited behavioral abnormalities concerning spine formation, such as cognitive, emotional, and attentional impairment. Moreover, some of these abnormalities were ameliorated by the administration of a mood stabilizer. However, there is no data about how memory-improving drugs used in the treatment of Alzheimer's disease affect DGKß KO mice. In the present study, we evaluated the effect of an anti-Alzheimer's drug, memantine on the working memory deficit observed in DGKß KO mice. In the Y-maze test, the administration of memantine significantly improved working memory of DGKß KO mice. We also found that the expression levels of the NR2A and NR2B N-methyl-d-aspartate (NMDA) receptor subunits were increased in the prefrontal cortex, but decreased in the hippocampus of DGKß KO mice. These altered expression levels of NR2 subunits might be related to the effect of an NMDA receptor antagonist, memantine. Taken together, these findings may support the hypothesis that DGKß has a pivotal role in cognitive function.


Asunto(s)
Diacilglicerol Quinasa/fisiología , Antagonistas de Aminoácidos Excitadores/administración & dosificación , Memantina/administración & dosificación , Memoria a Corto Plazo/efectos de los fármacos , Memoria a Corto Plazo/fisiología , Receptores de N-Metil-D-Aspartato/agonistas , Animales , Diacilglicerol Quinasa/genética , Hipocampo/metabolismo , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Corteza Prefrontal/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo
7.
Brain Res ; 1648(Pt A): 193-201, 2016 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-27423518

RESUMEN

Diacylglycerol kinase (DGK) is a lipid-metabolizing enzyme that phosphorylates diacylglycerol to produce phosphatidic acid. Previously, we reported that the δ isozyme of DGK was abundantly expressed in the mouse brain. However, the functions of DGKδ in the brain are still unclear. Because conventional DGKδ-knockout (KO) mice die within 24h after birth, we have generated brain-specific conditional DGKδ-KO mice to circumvent the lethality. In the novel object recognition test, the number of contacts in the DGKδ-KO mice to novel and familiar objects was greatly increased compared to the control mice, indicating that the DGKδ-KO mice showed irrational contacts with objects such as compulsive checking. In the marble burying test, which is used for analyzing obsessive-compulsive disorder (OCD)-like phenotypes, the DGKδ-KO mice buried more marbles than the control mice. Additionally, these phenotypes were significantly alleviated by the administration of an OCD remedy, fluoxetine. These results indicate that the DGKδ-KO mice showed OCD-like behaviors. Moreover, the number of long axon/neurites increased in both DGKδ-KO primary cortical neurons and DGKδ-knockdown neuroblastoma Neuro-2a cells compared to control cells. Conversely, overexpression of DGKδ decreased the number of long axon/neurites of Neuro-2a cells. Taken together, these results strongly suggest that a deficiency of DGKδ induces OCD-like behavior through enhancing axon/neurite outgrowth.


Asunto(s)
Conducta Animal/fisiología , Encéfalo/enzimología , Diacilglicerol Quinasa/fisiología , Trastorno Obsesivo Compulsivo/enzimología , Animales , Conducta Animal/efectos de los fármacos , Línea Celular Tumoral , Diacilglicerol Quinasa/genética , Femenino , Fluoxetina/administración & dosificación , Isoenzimas/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuritas/enzimología , Fenotipo , Reconocimiento en Psicología/fisiología , Inhibidores Selectivos de la Recaptación de Serotonina/administración & dosificación
8.
Yakugaku Zasshi ; 136(3): 461-5, 2016.
Artículo en Japonés | MEDLINE | ID: mdl-26935087

RESUMEN

The appropriate secretion of insulin from pancreatic ß-cells is essential for regulating blood glucose levels. Glucose-stimulated insulin secretion (GSIS) involves the following steps: Glucose uptake by pancreatic ß-cells is metabolized to produce ATP. Increased ATP levels result in the closure of ATP-sensitive K(+) (KATP) channels, resulting in membrane depolarization that activates voltage-dependent Ca(2+) channels to subsequently trigger insulin secretion. In addition to this primary mechanism through KATP channels, insulin secretion is regulated by cyclic AMP and diacylglycerol (DAG), which mediate the effects of receptor agonists such as GLP-1 and acetylcholine. Glucose by itself can also increase the levels of these second messengers. Recently, we have shown an obligatory role of diacylglycerol kinase (DGK), an enzyme catalyzing the conversion of DAG to phosphatidic acid, in GSIS. Of the 10 known DGK isoforms, we focused on type-I DGK isoforms (i.e., DGKα, DGKß, and DGKγ), which are activated by Ca(2+). The protein expression of DGKα and DGKγ was detected in mouse pancreatic islets and the pancreatic ß-cell line MIN6. Depletion of these DGKs by a specific inhibitor or siRNA decreased both [Ca(2+)]i and insulin secretion in MIN6 cells. Similar [Ca(2+)]i responses were induced by DiC8, a membrane-permeable DAG analog. These results suggest that DGKα and DGKγ play crucial roles in insulin secretion, and that their depletion impairs insulin secretion through DAG accumulation. In this article, we review the current understanding of the roles of DAG- and DGK-signaling in pancreatic ß-cells, and discuss their pathophysiological roles in the progression of type-2 diabetes.


Asunto(s)
Diabetes Mellitus Tipo 2/etiología , Diabetes Mellitus Tipo 2/metabolismo , Diacilglicerol Quinasa/fisiología , Descubrimiento de Drogas , Células Secretoras de Insulina/metabolismo , Metabolismo de los Lípidos , Terapia Molecular Dirigida , Adenosina Trifosfato/metabolismo , Animales , Canales de Calcio/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diglicéridos/metabolismo , Glucosa/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Canales KATP/metabolismo , Ratones
9.
Am J Physiol Endocrinol Metab ; 310(1): E51-60, 2016 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-26530149

RESUMEN

Decrease of AMPK-related signal transduction and insufficient lipid oxidation contributes to the pathogenesis of obesity and type 2 diabetes. Previously, we identified that diacylglycerol kinase-δ (DGKδ), an enzyme involved in triglyceride biosynthesis, is reduced in skeletal muscle from type 2 diabetic patients. Here, we tested the hypothesis that DGKδ plays a role in maintaining appropriate AMPK action in skeletal muscle and energetic aspects of contraction. Voluntary running activity was reduced in DGKδ(+/-) mice, but glycogen content and mitochondrial markers were unaltered, suggesting that DGKδ deficiency affects skeletal muscle energetics but not mitochondrial protein abundance. We next determined the role of DGKδ in AMPK-related signal transduction and lipid metabolism in isolated skeletal muscle. AMPK activation and signaling were reduced in DGKδ(+/-) mice, concomitant with impaired lipid oxidation and elevated incorporation of free fatty acids into triglycerides. Strikingly, DGKδ deficiency impaired work performance, as evident by altered force production and relaxation dynamics in response to repeated contractions. In conclusion, DGKδ deficiency impairs AMPK signaling and lipid metabolism, thereby highlighting the deleterious role of excessive lipid metabolites in the development of peripheral insulin resistance and type 2 diabetes pathogenesis. DGKδ deficiency also influences skeletal muscle energetics, which may lead to low physical activity levels in type 2 diabetes.


Asunto(s)
Adenilato Quinasa/metabolismo , Diacilglicerol Quinasa/fisiología , Metabolismo Energético/genética , Metabolismo de los Lípidos/genética , Músculo Esquelético/metabolismo , Animales , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Diacilglicerol Quinasa/genética , Masculino , Ratones , Ratones Transgénicos , Actividad Motora/genética , Condicionamiento Físico Animal/fisiología , Transducción de Señal/genética
10.
PLoS One ; 10(12): e0144942, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26701304

RESUMEN

Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis.


Asunto(s)
Diacilglicerol Quinasa/fisiología , Embrión de Mamíferos/metabolismo , Fibroblastos/metabolismo , Pinocitosis/fisiología , Proteína de Unión al GTP rac1/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/citología , Ratones , Ratones Noqueados , Microscopía Fluorescente , Fosforilación , Transducción de Señal , Imagen de Lapso de Tiempo
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