Figure 1.
Molecular Organization, Mutants, and Fat Body Expression of the AKHR Gene
(A) Genomic organization of the AKHR gene represented by the AKHR cDNA comprised of seven exons (grey boxes: coding exons; open boxes: UTRs). AKHRG6244 flies carrying a P{w+mC = EP} insertion in the first AKHR exon were used to generate AKHR deletion mutants (AKHR1, AKHR2) and genetically matched control flies (AKHRrev) having an intact AKHR gene.
(B–F) In situ hybridization showing expression of the fat body marker gene Adh (B) and AKHR expression in fat body tissue during late embryonic (C) and third instar larval stages (E) lacking in AKHR1 mutants ([D] and[F]). All embryos are depicted in dorsal view, anterior is left. Scale bar represents 50 μm. br, brain; fb, fat body; g, gut.
Figure 2.
AKH-Dependent Storage Fat Mobilization Strictly Depends on AKHR, but Not on brummer Lipase Function
(A and B) Organismal fat content (A) and cellular phenotype of fat-storage tissue visualized by Nile red staining of lipid storage droplets (B) show excessive fat storage in AKHR1 mutants and in flies lacking AKH-positive neuroendocrine cells by reaper-induced apoptosis (AKH-ZD mutants; for details see Materials and Methods) compared to the AKHRrev control. AKH-dependent depletion of fat storage (compare AKH induced vs. AKH uninduced control in [A] and [B]) is blocked in flies lacking AKHR function (compare AKHR1 mutant AKH induced vs. AKHR1 mutant AKH uninduced control in [A] and [B]). Scale bar represents 25 μm.
(C) AKH induction reduces fat storage in bmm mutants (compare bmm1 AKH induced vs. bmm1 AKH uninduced control).
Figure 3.
Severe Obesity and Impaired Lipid Mobilization in AKHR brummer Double-Mutant Flies
(A and B) Organismal fat content (A) and Nile red staining of lipid storage droplets in fat body tissue (B) demonstrate extreme obesity of ad libitum–fed AKHR1 bmm1 double mutants (glyceride content doubled compared to AKHR1 or bmm1 single mutants, quadrupled compared to genetically matched controls [AKHRrev or bmmrev] having wild-type AKHR and bmm function; filled bars). Induced storage-fat mobilization in response to starvation is impaired in AKHR1 and bmm1 single mutants, but blocked in AKHR1 bmm1 double mutants (open bars in [A]).
(C) Survival curves demonstrate starvation resistance of obese AKHR1 and bmm1 single mutants, but starvation sensitivity of extremely obese AKHR1 bmm1 mutants compared to genetically matched controls (AKHRrev or bmmrev). Scale bar represents 25 μm. Note: Except where given, p is less than 0.001 for all comparisons between mutant and control, and fed versus starved to death conditions.
Figure 4.
Impaired Basal and Blocked Starvation-Induced TAG Lipolysis in Fat Body Cells Lacking Both AKHR and brummer Gene Function
Fat body cells of control flies (AKHRrev bmmrev) exhibit basal TAG lipolysis, which is doubled by starvation-induced lipolysis after 6 h or 12 h of food deprivation. bmm mutant cells have reduced basal lipolysis and lack induced lipolysis after 12 h starvation. AKHR mutant cells lack early (6 h) induced lipolysis, but show strong starvation-induced lipolysis after 12 h food deprivation. AKHR bmm double mutants have reduced basal lipolysis and lack starvation-induced lipolysis altogether.
Figure 5.
Antagonistic Transcriptional Regulation of brummer Lipase in Response to AKH/AKHR Lipolytic Signaling
(A) Moderate transcriptional up-regulation of bmm in control flies (AKHRrev) after 6 h food deprivation, but starvation-induced hyperstimulation of bmm transcription in obese AKHR mutants (AKHR1) and flies lacking the AKH-producing neuroendocrine cells (AKH-ZD). By contrast, bmm transcription in lean flies chronically expressing AKH in the fat body (B) is strongly reduced.
Table 1.
Names, Genotypes, and References of Fly Stocks