FIGURE 2:

Requirement of GDH for secretory responses of glutamine combined with l-leucine and derivatives in control and βGlud1^−/− islets. After an overnight culture in RPMI-1640 medium, islets isolated from control and βGlud1^−/− mice were handpicked and perifused with KRBH at 2.8 mM glucose (Basal). (A) Islets were sequentially stimulated for 15 min with 5 mM of glutamine (Gln) and Gln plus 10 mM BCH. (B) l-Leucine (Leu) is deaminated to KIC by BCATm, transferring the amino group to α-KG and thereby producing glutamate (Glut). Glutamate can also be produced by deamidation of Gln. KIC can generate acetyl-CoA, the latter being also produced by pyruvate (Pyr), a cytosolic product of glucose (Glc) catabolism. GDH can be allosterically activated by l-leucine or its nonmetabolized analogue BCH. (C) Secretory responses of βGlud1^−/− islets to l-leucine and KIC. Insulin secretion was tested in islets over a 1-h incubation period at basal and 22.8 mM glucose, and at basal plus 2 mM glutamine with either 10 mM l-leucine or KIC. (D) Islets isolated from control and βGlud1^−/− mice were transduced with Ad-lacZ and Ad-GDH adenoviruses, respectively. After overnight culture, islets were perifused with KRBH at 2.8 mM glucose (Basal) and then stimulated for 15 min with 5 mM Gln and 15 min with Gln plus 10 mM BCH. (B and C) Values are means ± SE of three independent experiments for each group. *, p < 0.05, **, p < 0.01 vs. basal of corresponding genotype; §, p < 0.05, §§, p < 0.01 control under corresponding stimulation condition; ##, p < 0.01 vs. 22.8 mM glucose of corresponding genotype.