Amino Acid And Lipid Metabolism In The Fat Body Of The Tsetse Fly Glossina Morsitans

Abstract

Flight metabolism in tsetse flies of the genus Glossina is largely supported by the partial oxidation of proline to alanine. Reconstitution of proline from alanine supposedly occurs in the fat body of Glossina, using alanine, lipid and bicarbonate carbons. Comparison of enzyme activities in the fat body of the proline metabolising Glossina morsitans and carbohydrate metabolising fleshfly Sarcophaga tibialis showed that the activixYes of alanine aminotransferase and 3-hydroxyacyl-CoA de~ydrogenase were much higher in G. morsitans. Also, enzymes of the oxaloacetatecitrate- rr-oxoglutarate section of the tricarboxylic acid cycle, namely citrate synthase, aconitase and in particular NADP-dependent isocitrate dehydrogenase were more active in G. morsitans. Conversely, certain enzymes of the rr-oxoglutarate-succinate-fumarate-malateoxaloaGetate section of the cycle, rr-oxoglutarate dehydrogenase, fumarase and malate dehydrogenase were more active in S. tibialis. NAD-dependent isocitrate dehydrogenase was also more active in S. tibialis. Some enzymes of carbohydrate metabolism investigated, namely hexokinase, phosphofructokinase, aldolase and pyruvate kinase were all much more active in S. tibialis. The above observations conform with the idea that proline synthesis in the fat body of G. morsitans would involve B oxidation of fatty acids and the section of the tricarboxylic acid cycle from oxaloacetate to -oxoglutarate, and that carbohydrate metabolism 1S not as important in the fat body of G. morsitans as in S. tibialis. Whereas NADP-dependent isocitrate dehydrogenase and alanine aminotransferase were exclusively cytosolic in S. tibialis, these two enzymes were largely mitochondrial in G. morsitans. This observation, and the particularly low activity of NAD-dependent isocitrate dehydrogenase in G. morsitans strongly suggested that the mitochondrial oxidation of isocitrate to - oxoglutarate is catalysed mainly by NADP-dependent isocitrate dehydrogenase. In G. morsitans fat body, NADP-malic enzyme was exclusively cytosolic and appeared to be better geared to working in the direction of pyruvate formation than pyruvate carboxylase. On the other hand, pyruvate carboxylase was largely mitochondrial, and its Km values for pyruvate and bicarbonate were lower and nearer the likely physiological concentrations of these substrates than the Kms of NADP-malic enzyme. Pyruvate carboxylase activity was also totally dependent on acetyl-CoA. These observations indicated that carboxylation of pyruvate in G. morsitans is probably catalysed physiologically by pyruvate carboxylase. NADP-malic enzyme would probably be implicated in cytosolic lipogenesis. Dependenceof pyruvate carboxylase activity on acetyl-CoA concentration could also offer a mechanism whereby proline synthesis is increased by S oxidation of fatty acids . Kinetic studies of .alanine aminotransferase from the flight muscle and the fat body of G. morsisitans showed that the fat b6dy enzyme had lower Km values for alanine and -oxoglutarate than the flight muscle enzyme, while Km values for pyruvate and glutamate were slightly lower for the flight muscle enzyme. This indicated that the enzymes from the two tissues are different proteins. In vitro studies showed that the fat body cells of G. morsitans are capable of synthesizing proline from glutamate, and to a lesser extent from alanine. The rates of synthesis were increased by addition of NADH, NADPH or ATP. Addition of all three coenzymes together more than doubled the rate obtained in their absence. Isocitrate in the presence of coenzymes produces a further stimulation of synthesis. Citrate had a similar but lesser stimulatory effect, but cr-OXOglutarate had none. Isocitrate could substitute and augment the effects of added NADPH. Stimulation of proline synthesis by isocitrate was probably due to the generation of NADPH via the NADP-dependent isocitrate dehydrogenase. The in vitro formation of cr-oxoglutarate from isocitrate by fat boqy·cells in G. morsitans was much less in the absence of ~lutamate (the substrate for proline synthetase) thgn in its presence. This suggested that isocitrate oxidation via NADP-dependent isocitrate dehydrogenase is coupled to proline synthesis, so that reoxidation of mitochondrial NADPH by the proline synthetase reaction provides the coenzyme that allows the oxidation to continue efficiently.