Figure 4. CG8093 and CG6277 are novel lipases in the gut activated by AKT/FOXO in dcerk¹.

(A). Relative TAG level in ad libitum fed dcerk¹ is 80% of w¹¹¹⁸ and increases to 120% when AKT is reduced. Relative TAG level in 20 hr starved dcerk¹ is 50% of w¹¹¹⁸ and significantly increases when AKT is reduced. n = 3, error bars represent standard deviation. (B). Free fatty acids were measured in lysates prepared from starved w¹¹¹⁸ and dcerk¹ flies. Mutants show increased free fatty acids compared to w¹¹¹⁸. (C). Glycerol was measured in lysates prepared from starved w¹¹¹⁸ and dcerk¹ flies. Mutant shows increased glycerol compared to w¹¹¹⁸. (D). w¹¹¹⁸, dcerk¹, akt/+ and dcerk¹.akt/dcerk¹ flies are subjected to starvation and the number of surviving flies is recorded at 6 hr intervals. 200 flies divided into 10 groups for each genotype are used in one experiment. Three independent experiments are performed and the average percentage survival relative to w¹¹¹⁸ is shown for each time interval. Reduction in AKT increases the starvation sensitivity of dcerk¹ flies. (E). QPCR analysis shows transcript levels of two lipases CG8093 and CG6277 (identified by microarray analysis) are increased in dcerk¹ relative to w¹¹¹⁸ both in the fed and 20 h starved state. Both transcript levels are decreased upon reduction of AKT in dcerk¹. n = 3, error bars represent standard deviation. (F). Overexpression of constitutively active FOXO in the midgut decreases whole animal transcript levels of CG8093 and CG6277 while fat body specific expression does not significantly affect them in dcerk¹. FOXO overexpression with each driver is quantified by QPCR using FOXO primers and normalized. n = 3, error bars represent standard deviation. (G). Midgut specific overexpression of constitutively active FOXO increases whole animal TAG level in dcerk¹. The TAG level in dcerk¹ expressing esg-GAL4 driver is taken as 100%. (H). Constitutive FOXO represses CG8093 in luciferase reporter assays. A 2.4 kb fragment (genomic region between 1773 bp to 4177 bp from transcription start site) containing FOXO core motifs upstream of CG8093 in a luciferase reporter vector or a 1.3 kb fragment (region between 2842 bp and 4177 bp) deficient of the FOXO motifs was cotransfected with constitutive dFOXO A3. FOXO represses transcription of the 2.4 kb fragment while the repression is less when the binding sites are deleted. (I). Dissected adult midguts stained with Oil Red O show significantly reduced lipid staining in starved dcerk¹ compared to w¹¹¹⁸. dcerk¹ mutants with reduced AKT show comparatively more Oil Red O staining in the midgut. 15–20 guts are examined for each genotype in one experiment, which is carried out three times. Scale bar represents 50 µm. Magnified view of dissected adult midguts from w¹¹¹⁸ and dcerk¹.akt/dcerk¹ show many Oil Red O stained puncta while they are significantly reduced in dcerk¹. Scale bar represents 50 µm.