Proteome analysis and conditional deletion of the EAAT2 glutamate transporter provide evidence against a role of EAAT2 in pancreatic insulin secretion in mice [Neurobiology]

November 26th, 2013 by Zhou, Y., Waanders, L. F., Holmseth, S., Guo, C., Berger, U. V., Li, Y., Lehre, A.-C., Lehre, K. P., Danbolt, N. C.

Islet function is incompletely understood, in part because key steps in glutamate handling remain undetermined. Glutamate 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 knockout mice. Crossing with Nestin-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 7000 islet proteins of which more than 100 were transporters. While mitochondrial glutamate transporters and transporters for neutral amino acids were present at high levels, all transporters with known ability to transport glutamate were strikingly absent. Glutamate metabolising enzymes were abundant. The level of glutamine synthetase was two 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. Finally, some of the most abundant islet proteins have unknown functions.
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