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 β-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 β-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 β-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.Functions of mitochondria require regulated flux of molecules across the two membranes surrounding the matrix. Mitochondrial solute carriers (SLC25) are a large family of nuclearly encoded membrane-embedded proteins that promote solute transport across the inner mitochondrial membrane (1–4). The human genome contains 48 members of the SLC25 family, among them about 30 have been identified and characterized biochemically (1, 5–8). In particular, very little is known on solute carrier proteins transporting metabolites, such as glutamate. The two isoforms of the glutamate carrier GC1 and GC2 (encoded by SLC25A22 and SLC25A18, respectively) catalyze the transport of glutamate across the inner mitochondrial membrane, either by proton co-transport or in exchange for hydroxyl ions. To date, one human pathology has been associated with GC1, exhibiting a correlation between GC1 mutation and neonatal myoclonic epilepsy (9). Of interest, the high K_m isoform GC1 was shown to be expressed in different tissues, especially in the brain, liver, and pancreas (10). Despite the importance of these studies, we still lack subcellular localization and demonstration of the physiological function of glutamate carriers. The elevated expression levels in the pancreas triggered our interest, given that the glutamate pathway has been highlighted over the last years in the endocrine pancreas in general and the β-cell in particular (11). Still, the putative mechanisms responsible for mitochondrial glutamate transport have not yet been characterized in specialized tissues such as insulin-secreting cells. Only two carriers involved in mitochondrial shuttles have been shown to play an important role in the control of insulin secretion, i.e. the aspartate/glutamate carrier (AGC1 or Aralar1) (12) and the citrate/isocitrate carrier (13).It is well founded that mitochondrial metabolism is crucial in pancreatic β-cells by generating signals involved in metabolism-secretion coupling (14). Upon glucose stimulation, generation of ATP through mitochondrial activation leads to the closure of ATP-sensitive K⁺ channels and depolarization of the plasma membrane (15). This, in turn, induces the opening of voltage-dependent calcium channels resulting in elevation of cytosolic Ca²⁺ (16). Ca²⁺ is necessary but not sufficient for the full development of the insulin secretory response (17). Other messengers have been proposed to contribute to stimulation of insulin exocytosis, such as protein kinases A and C, long chain acyl-CoAs, nucleotides, and glutamate (18). The involvement of the latter amino acid was deduced from experiments performed under conditions of intracellular Ca²⁺] clamped at permissive concentrations, during which intracellular provision of glutamate directly stimulated insulin exocytosis (19–21). Based on these results, it was proposed that glutamate could act downstream of mitochondrial function, participating in the coupling of glucose metabolism to insulin secretion (21). The importance of the glutamate pathway for β-cell function is illustrated in transgenic mice (named βGlud1^−/−) with conditional β-cell-specific deletion of the mitochondrial enzyme glutamate dehydrogenase, resulting in about 40% reduction of glucose-stimulated insulin secretion (22). The exact role of glutamate in β-cell function is still debated as the glutamate pathway might raise insulin release by participating in the amplifying pathway (19–21) and/or by relaying signals of protein abundance to mitochondria (23–25). In both models, glutamate should be transported in and out of the mitochondria by some putative mitochondrial carrier that remains to be identified in β-cells. Overall, better characterization of mitochondrial glutamate handling will contribute to our comprehension of mechanisms implicated in the control of insulin secretion.In this study, we identified glutamate carrier GC1 as being expressed in the inner mitochondrial membrane of insulinoma INS-1E cells as well as in primary rat islets. Adenovirus-mediated knockdown of GC1 by shRNA² demonstrated physiological functionality of GC1 in insulin secretion.