RESUMEN
The homeobox transcription factor Nkx6.1 is sufficient to increase functional ß-cell mass, where functional ß-cell mass refers to the combination of ß-cell proliferation, glucose-stimulated insulin secretion (GSIS) and ß-cell survival. Here, we demonstrate that the histone deacetylase 1 (HDAC1), which is an early target of Nkx6.1, is sufficient to increase functional ß-cell mass. We show that HDAC activity is necessary for Nkx6.1-mediated proliferation, and that HDAC1 is sufficient to increase ß-cell proliferation in primary rat islets and the INS-1 832/13 ß-cell line. The increase in HDAC1-mediated proliferation occurs while maintaining GSIS and increasing ß-cell survival in response to apoptotic stimuli. We demonstrate that HDAC1 overexpression results in decreased expression of the cell cycle inhibitor Cdkn1b/p27 which is essential for inhibiting the G1 to S phase transition of the cell cycle. This corresponds with increased expression of key cell cycle activators, such as Cyclin A2, Cyclin B1 and E2F1, which are activated by activation of the Cdk4/Cdk6/Cyclin D holoenzymes due to down-regulation of Cdkn1b/p27. Finally, we demonstrate that overexpression of Cdkn1b/p27 inhibits HDAC1-mediated ß-cell proliferation. Our data suggest that HDAC1 is critical for the Nkx6.1-mediated pathway that enhances functional ß-cell mass.
Asunto(s)
Proliferación Celular/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Regulación hacia Abajo/fisiología , Regulación Enzimológica de la Expresión Génica , Histona Desacetilasa 1/biosíntesis , Células Secretoras de Insulina/metabolismo , Animales , Células Cultivadas , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/antagonistas & inhibidores , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Histona Desacetilasa 1/genética , Humanos , Masculino , Ratas , Ratas WistarRESUMEN
The clinical benefit of ketosis has historically and almost exclusively centered on neurological conditions, lending insight into how ketones alter mitochondrial function in neurons. However, there is a gap in our understanding of how ketones influence mitochondria within skeletal muscle cells. The purpose of this study was to elucidate the specific effects of ß-hydroxybutyrate (ß-HB) on muscle cell mitochondrial physiology. In addition to increased cell viability, murine myotubes displayed beneficial mitochondrial changes evident in reduced H2O2 emission and less mitochondrial fission, which may be a result of a ß-HB-induced reduction in ceramides. Furthermore, muscle from rats in sustained ketosis similarly produced less H2O2 despite an increase in mitochondrial respiration and no apparent change in mitochondrial quantity. In sum, these results indicate a general improvement in muscle cell mitochondrial function when ß-HB is provided as a fuel.
Asunto(s)
Ácido 3-Hidroxibutírico/farmacología , Ceramidas/metabolismo , Mitocondrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Animales , Ratones , Músculo Esquelético/citologíaRESUMEN
ß-Cell insulin secretion is dependent on proper mitochondrial function. Various studies have clearly shown that the Nr4a family of orphan nuclear receptors is essential for fuel utilization and mitochondrial function in liver, muscle, and adipose. Previously, we have demonstrated that overexpression of Nr4a1 or Nr4a3 is sufficient to induce proliferation of pancreatic ß-cells. In this study, we examined whether Nr4a expression impacts pancreatic ß-cell mitochondrial function. Here, we show that ß-cell mitochondrial respiration is dependent on the nuclear receptors Nr4a1 and Nr4a3. Mitochondrial respiration in permeabilized cells was significantly decreased in ß-cells lacking Nr4a1 or Nr4a3. Furthermore, respiration rates of intact cells deficient for Nr4a1 or Nr4a3 in the presence of 16 mM glucose resulted in decreased glucose mediated oxygen consumption. Consistent with this reduction in respiration, a significant decrease in glucose-stimulated insulin secretion rates is observed with deletion of Nr4a1 or Nr4a3. Interestingly, the changes in respiration and insulin secretion occur without a reduction in mitochondrial content, suggesting decreased mitochondrial function. We establish that knockdown of Nr4a1 and Nr4a3 results in decreased expression of the mitochondrial dehydrogenase subunits Idh3g and Sdhb. We demonstrate that loss of Nr4a1 and Nr4a3 impedes production of ATP and ultimately inhibits glucose-stimulated insulin secretion. These data demonstrate for the first time that the orphan nuclear receptors Nr4a1 and Nr4a3 are critical for ß-cell mitochondrial function and insulin secretion.
Asunto(s)
Respiración de la Célula/genética , Proteínas de Unión al ADN/genética , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , ARN Mensajero/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Línea Celular Tumoral , Supervivencia Celular , Técnicas de Silenciamiento del Gen , Glucosa/metabolismo , Immunoblotting , Secreción de Insulina , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Succinato Deshidrogenasa/genética , Succinato Deshidrogenasa/metabolismoRESUMEN
Decreased ß-cell mass is a hallmark of type 1 and type 2 diabetes. Islet transplantation as a method of diabetes therapy is hampered by the paucity of transplant ready islets. Understanding the pathways controlling islet proliferation may be used to increase functional ß-cell mass through transplantation or by enhanced growth of endogenous ß-cells. We have shown that the transcription factor Nkx6.1 induces ß-cell proliferation by upregulating the orphan nuclear hormone receptors Nr4a1 and Nr4a3. Using expression analysis to define Nkx6.1-independent mechanisms by which Nr4a1 and Nr4a3 induce ß-cell proliferation, we demonstrated that cyclin-dependent kinase 5 regulatory subunit 1 (Cdk5r1) is upregulated by Nr4a1 and Nr4a3 but not by Nkx6.1. Overexpression of Cdk5r1 is sufficient to induce primary rat ß-cell proliferation while maintaining glucose stimulated insulin secretion. Overexpression of Cdk5r1 in ß-cells confers protection against apoptosis induced by etoposide and thapsigargin, but not camptothecin. The Cdk5 kinase complex inhibitor roscovitine blocks islet proliferation, suggesting that Cdk5r1 mediated ß-cell proliferation is a kinase dependent event. Overexpression of Cdk5r1 results in pRb phosphorylation, which is inhibited by roscovitine treatment. These data demonstrate that activation of the Cdk5 kinase complex is sufficient to induce ß-cell proliferation while maintaining glucose stimulated insulin secretion.
Asunto(s)
Proliferación Celular , Células Secretoras de Insulina/metabolismo , Fosfotransferasas/metabolismo , Animales , Apoptosis , Células Cultivadas , Quinasa 5 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 5 Dependiente de la Ciclina/genética , Quinasa 5 Dependiente de la Ciclina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Glucosa/farmacología , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/patología , Masculino , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Fosforilación , Fosfotransferasas/genética , Cultivo Primario de Células , Inhibidores de Proteínas Quinasas/farmacología , Interferencia de ARN , Ratas Wistar , Proteína de Retinoblastoma/metabolismo , Transducción de Señal , Transfección , Regulación hacia ArribaRESUMEN
Type 1 and type 2 diabetes are ultimately characterized by depleted ß-cell mass. Characterization of the molecular pathways that control ß-cell proliferation could be harnessed to restore these cells. The homeobox ß-cell transcription factor Nkx6.1 induces ß-cell proliferation by activating the orphan nuclear receptors Nr4a1 and Nr4a3. Here, we demonstrate that Nkx6.1 localizes to the promoter of the mitotic kinase AURKA (Aurora Kinase A) and induces its expression. Adenovirus mediated overexpression of AURKA is sufficient to induce proliferation in primary rat islets while maintaining glucose stimulated insulin secretion. Furthermore, AURKA is necessary for Nkx6.1 mediated ß-cell proliferation as demonstrated by shRNA mediated knock down and pharmacological inhibition of AURKA kinase activity. AURKA preferentially induces DNA replication in ß-cells as measured by BrdU incorporation, and enhances the rate of histone H3 phosphorylation in primary ß-cells, demonstrating that AURKA induces the replicative and mitotic cell cycle phases in rat ß-cells. Finally, overexpression of AURKA results in phosphorylation of the cell cycle regulator p53, which targets p53 for degradation and permits cell cycle progression. These studies define a pathway by which AURKA upregulation by Nkx6.1 results in phosphorylation and degradation of p53, thus removing a key inhibitory factor and permitting engagement of the ß-cell proliferation pathway.