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.