Mitochondrial glutamate carrier GC1 as a newly identified player in the control of glucose-stimulated insulin secretion.
The SLC25 carrier family mediates solute transport across the inner mitochondrial membrane, a process that is still poorly characterized regarding both the mechanisms and proteins implicated. This study investigated mitochondrial glutamate carrier GC1 in insulin-secreting beta-cells. GC1 was cloned from insulin-secreting cells, and sequence analysis revealed hydropathy profile of a six-transmembrane protein, characteristic of mitochondrial solute carriers. GC1 was found to be expressed at the mRNA and protein levels in INS-1E beta-cells and pancreatic rat islets. Immunohistochemistry showed that GC1 was present in mitochondria, and ultrastructural analysis by electron microscopy revealed inner mitochondrial membrane localization of the transporter. Silencing of GC1 in INS-1E beta-cells, mediated by adenoviral delivery of short hairpin RNA, reduced mitochondrial glutamate transport by 48% (p < 0.001). Insulin secretion at basal 2.5 mM glucose and stimulated either by intermediate 7.5 mM glucose or non-nutrient 30 mM KCl was not modified by GC1 silencing. Conversely, insulin secretion stimulated with optimal 15 mM glucose was reduced by 23% (p < 0.005) in GC1 knocked down cells compared with controls. Adjunct of cell-permeant glutamate (5 mM dimethyl glutamate) fully restored the secretory response at 15 mM glucose (p < 0.005). Kinetics of insulin secretion were investigated in perifused isolated rat islets. GC1 silencing in islets inhibited the secretory response induced by 16.7 mM glucose, both during first (-25%, p < 0.05) and second (-33%, p < 0.05) phases. This study demonstrates that insulin-secreting cells depend on GC1 for maximal glucose response, thereby assigning a physiological function to this newly identified mitochondrial glutamate carrier.
Casimir
Marina
M
Department of Cell Physiology and Metabolism, Faculty of Medicine, University of Geneva, 1211 Geneva 4, Switzerland.
Lasorsa
Francesco M
FM
Rubi
Blanca
B
Caille
Dorothée
D
Palmieri
Ferdinando
F
Meda
Paolo
P
Maechler
Pierre
P
D000818:Animals; D002460:Cell Line; D020868:Gene Silencing; D005947:Glucose / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000378:metabolism; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins / Q000378:metabolism / Q000502:physiology*; D008954:Models, Biological; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats
D007328:Insulin; D026901:Membrane Transport Proteins; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; D034741:RNA, Small Interfering; C499215:SLC25A22 protein, human; D018698:Glutamic Acid; D005947:Glucose