title: Requirement for aralar and its Ca2+-binding sites in Ca2+ signal transduction in mitochondria from INS-1 clonal beta-cells.
abstract: Aralar, the mitochondrial aspartate-glutamate carrier present in beta-cells, is a component of the malate-aspartate NADH shuttle (MAS). MAS is activated by Ca2+ in mitochondria from tissues with aralar as the only AGC isoform with an S0.5 of approximately 300 nm. We have studied the role of aralar and its Ca2+-binding EF-hand motifs in glucose-induced mitochondrial NAD(P)H generation by two-photon microscopy imaging in INS-1 beta-cells lacking aralar or expressing aralar mutants blocked for Ca2+ binding. Aralar knock-down in INS-1 beta-cell lines resulted in undetectable levels of aralar protein and complete loss of MAS activity in isolated mitochondria and in a 25% decrease in glucose-stimulated insulin secretion. MAS activity in mitochondria from INS-1 cells was activated 2-3-fold by extramitochondrial Ca2+, whereas aralar mutants were Ca2+ insensitive. In Ca2+-free medium, glucose-induced increases in mitochondrial NAD(P)H were small (1.3-fold) and unchanged regardless of the lack of aralar. In the presence of 1.5 mm Ca2+, glucose induced robust increases in mitochondrial NAD(P)H (approximately 2-fold) in cell lines with wild-type or mutant aralar. There was a approximately 20% reduction in NAD(P)H response in cells lacking aralar, illustrating the importance of MAS in glucose action. When small Ca2+ signals that resulted in extremely small mitochondrial Ca2+ transients were induced in the presence of glucose, the rise in mitochondrial NAD(P)H was maintained in cells with wild-type aralar but was reduced by approximately 50% in cells lacking or expressing mutant aralar. These results indicate that 1) glucose-induced activation of MAS requires Ca2+ potentiation; 2) Ca2+ activation of MAS represents a larger fraction of glucose-induced mitochondrial NAD(P)H production under conditions where suboptimal Ca2+ signals are associated with a glucose challenge (50 versus 20%, respectively); 3) inactivation of EF-hand motifs in aralar prevents activation of MAS by small Ca2+ signals. The results suggest a possible role for aralar and MAS in priming the beta-cell by Ca2+-mobilizing neurotransmitter or hormones.
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa CSIC-UAM, CIBER de Enfermedades Raras (CIBERER), Universidad Autónoma, 28049 Madrid, Spain, the Area de Bioquímica, Centro Regional de Investigaciones Biomádicas (CRIB), Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, 48071 Toledo, Spain, and the Department of Cell Physiology and Metabolism, University Medical Center, CH-1211 Geneva, Switzerland.
authors.forename: Patricia
authors.initials: P
authors.lastname: Mármol
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa CSIC-UAM, CIBER de Enfermedades Raras (CIBERER), Universidad Autónoma, 28049 Madrid, Spain, the Area de Bioquímica, Centro Regional de Investigaciones Biomádicas (CRIB), Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, 48071 Toledo, Spain, and the Department of Cell Physiology and Metabolism, University Medical Center, CH-1211 Geneva, Switzerland.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Pardo
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa CSIC-UAM, CIBER de Enfermedades Raras (CIBERER), Universidad Autónoma, 28049 Madrid, Spain, the Area de Bioquímica, Centro Regional de Investigaciones Biomádicas (CRIB), Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, 48071 Toledo, Spain, and the Department of Cell Physiology and Metabolism, University Medical Center, CH-1211 Geneva, Switzerland.
authors.forename: Andreas
authors.initials: A
authors.lastname: Wiederkehr
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa CSIC-UAM, CIBER de Enfermedades Raras (CIBERER), Universidad Autónoma, 28049 Madrid, Spain, the Area de Bioquímica, Centro Regional de Investigaciones Biomádicas (CRIB), Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, 48071 Toledo, Spain, and the Department of Cell Physiology and Metabolism, University Medical Center, CH-1211 Geneva, Switzerland; Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa CSIC-UAM, CIBER de Enfermedades Raras (CIBERER), Universidad Autónoma, 28049 Madrid, Spain, the Area de Bioquímica, Centro Regional de Investigaciones Biomádicas (CRIB), Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, 48071 Toledo, Spain, and the Department of Cell Physiology and Metabolism, University Medical Center, CH-1211 Geneva, Switzerland.
authors.forename: Araceli
authors.initials: A
authors.lastname: Del Arco
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa CSIC-UAM, CIBER de Enfermedades Raras (CIBERER), Universidad Autónoma, 28049 Madrid, Spain, the Area de Bioquímica, Centro Regional de Investigaciones Biomádicas (CRIB), Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, 48071 Toledo, Spain, and the Department of Cell Physiology and Metabolism, University Medical Center, CH-1211 Geneva, Switzerland.
authors.forename: Claes B
authors.initials: CB
authors.lastname: Wollheim
authors.affiliation: Departamento de Biología Molecular and Centro de Biología Molecular Severo Ochoa CSIC-UAM, CIBER de Enfermedades Raras (CIBERER), Universidad Autónoma, 28049 Madrid, Spain, the Area de Bioquímica, Centro Regional de Investigaciones Biomádicas (CRIB), Facultad de Ciencias del Medio Ambiente, Universidad de Castilla-La Mancha, 48071 Toledo, Spain, and the Department of Cell Physiology and Metabolism, University Medical Center, CH-1211 Geneva, Switzerland. Electronic address: jsatrustegui@cbm.uam.es.
authors.forename: Jorgina
authors.initials: J
authors.lastname: Satrústegui
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling / Q000502:physiology*; D002478:Cells, Cultured; D055785:Gene Knockdown Techniques; D005947:Glucose / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D009249:NADP / Q000096:biosynthesis; D018377:Neurotransmitter Agents / Q000378:metabolism; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism
chemical_list: D007328:Insulin; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D018377:Neurotransmitter Agents; D020033:Protein Isoforms; C491956:Slc25a12 protein, mouse; D009249:NADP; D005947:Glucose; D002118:Calcium