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1.
PLoS One ; 13(9): e0198383, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30235209

RESUMEN

The ABC transporter ABCG1 contributes to the regulation of cholesterol efflux from cells and to the distribution of cholesterol within cells. We showed previously that ABCG1 deficiency inhibits insulin secretion by pancreatic beta cells and, based on its immunolocalization to insulin granules, proposed its essential role in forming granule membranes that are enriched in cholesterol. While we confirm elsewhere that ABCG1, alongside ABCA1 and oxysterol binding protein OSBP, supports insulin granule formation, the aim here is to clarify the localization of ABCG1 within insulin-secreting cells and to provide added insight regarding ABCG1's trafficking and sites of function. We show that stably expressed GFP-tagged ABCG1 closely mimics the distribution of endogenous ABCG1 in pancreatic INS1 cells and accumulates in the trans-Golgi network (TGN), endosomal recycling compartment (ERC) and on the cell surface but not on insulin granules, early or late endosomes. Notably, ABCG1 is short-lived, and proteasomal and lysosomal inhibitors both decrease its degradation. Following blockade of protein synthesis, GFP-tagged ABCG1 first disappears from the ER and TGN and later from the ERC and plasma membrane. In addition to aiding granule formation, our findings raise the prospect that ABCG1 may act beyond the TGN to regulate activities involving the endocytic pathway, especially as the amount of transferrin receptor is increased in ABCG1-deficient cells. Thus, ABCG1 may function at multiple intracellular sites and the plasma membrane as a roving sensor and modulator of cholesterol distribution, membrane trafficking and cholesterol efflux.


Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/análisis , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Células Secretoras de Insulina/metabolismo , Animales , Línea Celular , Citoplasma/metabolismo , Citoplasma/ultraestructura , Degradación Asociada con el Retículo Endoplásmico , Endosomas/metabolismo , Endosomas/ultraestructura , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/ultraestructura , Lisosomas/metabolismo , Lisosomas/ultraestructura , Mesotelina , Ratones , Microscopía Confocal , Transporte de Proteínas , Proteolisis , Ratas , Red trans-Golgi/metabolismo , Red trans-Golgi/ultraestructura
2.
Mol Biol Cell ; 29(10): 1238-1257, 2018 05 15.
Artículo en Inglés | MEDLINE | ID: mdl-29540530

RESUMEN

In pancreatic ß-cells, insulin granule membranes are enriched in cholesterol and are both recycled and newly generated. Cholesterol's role in supporting granule membrane formation and function is poorly understood. ATP binding cassette transporters ABCG1 and ABCA1 regulate intracellular cholesterol and are important for insulin secretion. RNAi inter-ference-induced depletion in cultured pancreatic ß-cells shows that ABCG1 is needed to stabilize newly made insulin granules against lysosomal degradation; ABCA1 is also involved but to a lesser extent. Both transporters are also required for optimum glucose-stimulated insulin secretion, likely via complementary roles. Exogenous cholesterol addition rescues knockdown-induced granule loss (ABCG1) and reduced secretion (both transporters). Another cholesterol transport protein, oxysterol binding protein (OSBP), appears to act proximally as a source of endogenous cholesterol for granule formation. Its knockdown caused similar defective stability of young granules and glucose-stimulated insulin secretion, neither of which were rescued with exogenous cholesterol. Dual knockdowns of OSBP and ABC transporters support their serial function in supplying and concentrating cholesterol for granule formation. OSBP knockdown also decreased proinsulin synthesis consistent with a proximal endoplasmic reticulum defect. Thus, membrane cholesterol distribution contributes to insulin homeostasis at production, packaging, and export levels through the actions of OSBP and ABCs G1 and A1.


Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/metabolismo , Gránulos Citoplasmáticos/metabolismo , Insulina/metabolismo , Receptores de Esteroides/metabolismo , Transportador 1 de Casete de Unión a ATP/deficiencia , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 1/deficiencia , Animales , Colesterol/farmacología , Gránulos Citoplasmáticos/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Exocitosis/efectos de los fármacos , Fluorescencia , Técnicas de Silenciamiento del Gen , Glucosa/farmacología , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lisosomas/efectos de los fármacos , Lisosomas/metabolismo , Ratones , Modelos Biológicos , Interferencia de ARN , Ratas , Vías Secretoras/efectos de los fármacos , beta-Ciclodextrinas/farmacología
3.
J Clin Invest ; 120(7): 2575-89, 2010 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-20530872

RESUMEN

Cholesterol is a critical component of cell membranes, and cellular cholesterol levels and distribution are tightly regulated in mammals. Recent evidence has revealed a critical role for pancreatic beta cell-specific cholesterol homeostasis in insulin secretion as well as in beta cell dysfunction in diabetes and the metabolic response to thiazolidinediones (TZDs), which are antidiabetic drugs. The ATP-binding cassette transporter G1 (ABCG1) has been shown to play a role in cholesterol efflux, but its role in beta cells is currently unknown. In other cell types, ABCG1 expression is downregulated in diabetes and upregulated by TZDs. Here we have demonstrated an intracellular role for ABCG1 in beta cells. Loss of ABCG1 expression impaired insulin secretion both in vivo and in vitro, but it had no effect on cellular cholesterol content or efflux. Subcellular localization studies showed the bulk of ABCG1 protein to be present in insulin granules. Loss of ABCG1 led to altered granule morphology and reduced granule cholesterol levels. Administration of exogenous cholesterol restored granule morphology and cholesterol content and rescued insulin secretion in ABCG1-deficient islets. These findings suggest that ABCG1 acts primarily to regulate subcellular cholesterol distribution in mouse beta cells. Furthermore, islet ABCG1 expression was reduced in diabetic mice and restored by TZDs, implicating a role for regulation of islet ABCG1 expression in diabetes pathogenesis and treatment.


Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Colesterol/metabolismo , Transportadoras de Casetes de Unión a ATP/biosíntesis , Animales , Transporte Biológico/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Colesterol/genética , Citoplasma/genética , Citoplasma/metabolismo , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Regulación hacia Abajo , Células Secretoras de Insulina/metabolismo , Metabolismo de los Lípidos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Vías Secretoras
4.
Mol Biol Cell ; 20(6): 1816-32, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19158374

RESUMEN

The epidermal growth factor receptor (EGFR) is targeted for lysosomal degradation by ubiquitin-mediated interactions with the ESCRTs (endosomal-sorting complexes required for transport) in multivesicular bodies (MVBs). We show that secretory carrier membrane protein, SCAMP3, localizes in part to early endosomes and negatively regulates EGFR degradation through processes that involve its ubiquitylation and interactions with ESCRTs. SCAMP3 is multimonoubiquitylated and is able to associate with Nedd4 HECT ubiquitin ligases and the ESCRT-I subunit Tsg101 via its PY and PSAP motifs, respectively. SCAMP3 also associates with the ESCRT-0 subunit Hrs. Depletion of SCAMP3 in HeLa cells by inhibitory RNA accelerated degradation of EGFR and EGF while inhibiting recycling. Conversely, overexpression enhanced EGFR recycling unless ubiquitylatable lysines, PY or PSAP motifs in SCAMP3 were mutated. Notably, dual depletions of SCAMP3 and ESCRT subunits suggest that SCAMP3 has a distinct function in parallel with the ESCRTs that regulates receptor degradation. This function may affect trafficking of receptors from prelysosomal compartments as SCAMP3 depletion appeared to sustain the incidence of EGFR-containing MVBs detected by immunoelectron microscopy. Together, our results suggest that SCAMP3, its modification with ubiquitin, and its interactions with ESCRTs coordinately regulate endosomal pathways and affect the efficiency of receptor down-regulation.


Asunto(s)
Proteínas Portadoras/metabolismo , Regulación hacia Abajo , Receptores ErbB/metabolismo , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/química , Proteínas Portadoras/genética , Línea Celular , Proteínas de Unión al ADN/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/metabolismo , Humanos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Ratones , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Mutación/genética , Fosfoproteínas/metabolismo , Unión Proteica , Transporte de Proteínas , Interferencia de ARN , Alineación de Secuencia , Factores de Transcripción/metabolismo , Vesículas Transportadoras/metabolismo , Vesículas Transportadoras/ultraestructura , Ubiquitinación
5.
J Cell Sci ; 115(Pt 14): 2963-73, 2002 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-12082156

RESUMEN

Recently, we reported that the minor regulated and constitutive-like pathways are the main source of resting secretion by parotid acinar cells. Using tissue lobules biosynthetically labeled with [(35)S]amino acids, we now show that discharge of the minor regulated pathway precedes granule exocytosis stimulated by isoproterenol (> or =1 microM) or carbachol (2 microM). Stimulation of the minor regulated pathway by 40 nM carbachol as well as altering its trafficking, either by adding brefeldin A or by incubating in K(+)-free medium, cause potentiation of amylase secretion stimulated by isoproterenol, suggesting that the minor regulated pathway contributes to the mechanism of potentiation. Both exocytosis of the minor regulated pathway and the potentiation-inducing treatments induce relocation of immunostained subapical puncta of the SNARE protein syntaxin 3 into the apical plasma membrane. Rab11 and possibly VAMP2 may be concentrated in the same relocating foci. These results suggest that the minor regulated pathway and granule exocytosis are functionally linked and that the minor regulated pathway has a second role beyond contributing to resting secretion - providing surface docking/fusion sites for granule exocytosis. In the current model of salivary protein export, discharge of the minor regulated pathway by either beta-adrenergic or cholinergic stimulation is an obligatory first step. Ensuing granule exocytosis is controlled mainly by beta-adrenergic stimulation whereas cholinergic stimulation mainly regulates the number of surface sites where release occurs.


Asunto(s)
Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Exocitosis/fisiología , Glándula Parótida/metabolismo , Proteínas y Péptidos Salivales/metabolismo , Vesículas Secretoras/metabolismo , Agonistas Adrenérgicos beta/farmacología , Aminoácidos/metabolismo , Aminoácidos/farmacología , Animales , Brefeldino A/farmacología , Células Cultivadas , Agonistas Colinérgicos/farmacología , Relación Dosis-Respuesta a Droga , Células Epiteliales/citología , Isoproterenol/farmacología , Masculino , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Glándula Parótida/citología , Deficiencia de Potasio/metabolismo , Inhibidores de la Síntesis de la Proteína/farmacología , Transporte de Proteínas/efectos de los fármacos , Transporte de Proteínas/fisiología , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratas , Ratas Sprague-Dawley , Proteínas y Péptidos Salivales/biosíntesis , Vesículas Secretoras/ultraestructura , Transducción de Señal/fisiología , Proteínas de Unión al GTP rab/metabolismo
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