title: Proteome analysis and conditional deletion of the EAAT2 glutamate transporter provide evidence against a role of EAAT2 in pancreatic insulin secretion in mice.
abstract: Islet function is incompletely understood in part because key steps in glutamate handling remain undetermined. The glutamate (excitatory amino acid) transporter 2 (EAAT2; Slc1a2) has been hypothesized to (a) provide islet cells with glutamate, (b) protect islet cells against high extracellular glutamate concentrations, (c) mediate glutamate release, or (d) control the pH inside insulin secretory granules. Here we floxed the EAAT2 gene to produce the first conditional EAAT2 knock-out mice. Crossing with Nestin-cyclization recombinase (Cre) eliminated EAAT2 from the brain, resulting in epilepsy and premature death, confirming the importance of EAAT2 for brain function and validating the genetic construction. Crossing with insulin-Cre lines (RIP-Cre and IPF1-Cre) to obtain pancreas-selective deletion did not appear to affect survival, growth, glucose tolerance, or β-cell number. We found (using TaqMan RT-PCR, immunoblotting, immunocytochemistry, and proteome analysis) that the EAAT2 levels were too low to support any of the four hypothesized functions. The proteome analysis detected more than 7,000 islet proteins of which more than 100 were transporters. Although mitochondrial glutamate transporters and transporters for neutral amino acids were present at high levels, all other transporters with known ability to transport glutamate were strikingly absent. Glutamate-metabolizing enzymes were abundant. The level of glutamine synthetase was 2 orders of magnitude higher than that of glutaminase. Taken together this suggests that the uptake of glutamate by islets from the extracellular fluid is insignificant and that glutamate is intracellularly produced. Glutamine synthetase may be more important for islets than assumed previously.
authors.affiliation: From The Neurotransporter Group, Department of Anatomy, Institute of Basic Medical Sciences, University of Oslo, 0317 Oslo, Norway.
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhou
authors.forename: Leonie F
authors.initials: LF
authors.lastname: Waanders
authors.forename: Silvia
authors.initials: S
authors.lastname: Holmseth
authors.forename: Caiying
authors.initials: C
authors.lastname: Guo
authors.forename: Urs V
authors.initials: UV
authors.lastname: Berger
authors.forename: Yuchuan
authors.initials: Y
authors.lastname: Li
authors.forename: Anne-Catherine
authors.initials: AC
authors.lastname: Lehre
authors.forename: Knut P
authors.initials: KP
authors.lastname: Lehre
authors.forename: Niels C
authors.initials: NC
authors.lastname: Danbolt
mesh_terms: D000818:Animals; D003433:Crosses, Genetic; D027342:Excitatory Amino Acid Transporter 2 / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism*; D007328:Insulin / Q000235:genetics / Q000378:metabolism*; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D020543:Proteome / Q000235:genetics / Q000378:metabolism*
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D007328:Insulin; D020543:Proteome; C493396:Slc1a2 protein, mouse; D018698:Glutamic Acid
keywords: Excitatory Amino Acid Transporter; Glutamate Uptake; Glutamine Synthase; Immunohistochemistry; Insulin Secretion; Pancreatic Islets; Proteomics; Slc1a2; Transgenic Mice; VGLUT3