Effects of AMP and Fructose 2,6 Bisphosphate on Fructose 1,6 Bisphosphatase Activity in Trichoplusia ni Parasitized by the Insect Parasite, Hyposoter exiguae

Abstract

The host association of the insect parasite, Hyposoter exiguae (Viereck) (Ichneumonidae), is characterized by elevated host blood sugar and fat body glycogen levels during the early period of the parasite's development and prior to direct destruction of host tissues (Thompson, 1982, Parasitology 84: 491-510). Recently, Thompson and Binder (1984, Journal of Parasitology 70: 644-651) reported that parasitized fourth instar Trichoplusia ni (Hiibner) (Noctuidae) larvae displayed a marked elevation in the activity of fat body fructose 1,6 bisphosphatase (FBPase), 1 of the rate-controlling enzymes of the gluconeogenic pathway. The results were not demonstrative but strongly suggested that the increased carbohydrate reserves may arise from an increase in gluconeogenic flux. The increased enzyme activity could not be explained on the basis of changes in the adenylate pool. Indeed, the level of adenosine 5' monophosphate (AMP), a potent inhibitor of FBPase and the major modulator of the enzyme's activity (McClard and Atkinson, 1979, Archives of Biochemistry and Biophysics 194: 236-243), was increased in parasitized individuals. The present note summarizes the results of additional experiments on the effects of AMP as well as fructose 2,6 bisphosphate (F2, 6BP) on FBPase in T. ni fat body following parasitization by H. exiguae. Fat body tissue from fourth instar T. ni larvae, 72 hr after parasitization by H. exiguae, was excised as described previously (Thompson and Binder, 1984, loc. cit.). The tissue from 10 individuals was pooled for each experiment and homogenized in 0.2 M triethanolamine buffer, pH 7.5, containing 0.5 mM EDTA and 1.0 mM MgCl2, and the homogenate centrifuged at 37,000 g for 40 min. Unparasitized larvae at the same developmental stage were used for controls. The activity of FBPase in the supernatant was determined with the enzyme-coupled assay described by Rosen, Rosen and Horecker (1965, Archives of Biochemistry and Biophysics 112: 411-420) in which the product, fructose 6 phosphate, is isomerized to the glucose derivative and oxidized in the presence of glucose 6 phosphate dehydrogenase to phosphogluconolactone accompanied by the reduction of nicotinamide adenine dinucleotide phosphate (NADP). The rate of change in optical density at 340 nm was monitored continuously with a Beckman 25 spectrophotometer. A 1 ml standard assay mixture contained: 0.02 mmoles triethanolamine, 0.5 ,moles EDTA, 1.0 umoles MgCl2, 0.5 ,umoles NADP, 1.7 units glucose 6 phosphate dehydrogenase and 3.5 units phosphoglucose isomerase. The reaction was initiated by addition of 0.2 ,umoles fructose 1,6 bisphosphate. The effects of F2,6BP concentration without and with 10 ,M AMP and AMP concentration without and with 2.5 ,uM F2,6BP were determined in fat body preparations of parasitized and control insects. The experiments were duplicated and the results expressed as a percentage of the maximal activity observed without F2,6BP and AMP, respectively (Van Schaftingen and Hers, 1981, Proceedings of the National Academy of Sciences 78: 28612863). AMP and F2,6BP were inhibitors of FBPase from T. ni fat body (Figs. 1, 2). Moreover, the inhibition caused by each effector was enhanced in the presence of the other. Although it was not possible to quantify inhibition precisely because of the variability between experiments, the effective concentration ranges of the inhibitors and the approximate levels of inhibition observed were similar to those reported for other animal systems cited below. In all cases AMP and F2,6BP were less inhibitory in fat body preparations from parasitized insects. These results were consistent with and may explain previous findings demonstrating an elevated FBPase activity in parasitized T. ni when the AMP level was simultaneously elevated. A quantitative evaluation, however, was not possible because the actual intracellular concentration of AMP was not deter-