Whole Brain ACC Activity Following Intraperitoneal Injection of PBS, Glucagon, Insulin and Insulin-Glucagon Challenge. Figure 4a (ACC, assayed without citrate). Figure 4b (ACC assayed, with 0.05 mM citrate). ACC activity in glucagon injected mice decreased significantly in the absence of citrate and increased in the presence of citrate. ACC activity in insulin injected mice shows activity regardless of citrate presence. Retired male, BALB/C breeder mice received intra-peritoneal (i.p) injection of glucagon (2.5 μU in 0.2 mL) or insulin (25 μU in 0.2 mL). Controls received PBS. For insulin-glucagon challenge group, 20 min after insulin (25 μU/0.2 mL) injection, they received glucagon (2.5 μU/0.2 mL) injection. Whole brain was dissected, homogenized and the extracts were assayed for ACC activity in the absence of citrate. Each extract was assayed in duplicate. The ACC activity assay mixture contained, 5.0 mM HEPES, 0.25 mM MnCl2, 0.2 mM DTT, 0.01235 mM acetyl CoA, 0.4 mM ATP, 0.075 mg/mL BSA, 2.51 mM NaHCO3- (containing 1.11 × 105 cpm/nmol). Results are expressed as the mean ± SD of assays of the brain extracts from four independent animals. A one-way analysis of variance revealed no significant differences in the presence of citrate between among the injection groups [F(3,12) = 1.01, p = 0.422]. When citrate was absent, significant differences between groups were found to be present [F(3,12) = 5.60, p < 0.012].