Dual lipolytic control of body fat storage and mobilization in Drosophila

Abstract

Energy homeostasis is a fundamental property of animal life, providing a genetically fixed balance between fat storage and mobilization. The importance of body fat regulation is emphasized by dysfunctions resulting in obesity and lipodystrophy in humans. Packaging of storage fat in intracellular lipid droplets, and the various molecules and mechanisms guiding storage-fat mobilization, are conserved between mammals and insects. We generated a Drosophila mutant lacking the receptor (AKHR) of the adipokinetic hormone signaling pathway, an insect lipolytic pathway related to ss-adrenergic signaling in mammals. Combined genetic, physiological, and biochemical analyses provide in vivo evidence that AKHR is as important for chronic accumulation and acute mobilization of storage fat as is the Brummer lipase, the homolog of mammalian adipose triglyceride lipase (ATGL). Simultaneous loss of Brummer and AKHR causes extreme obesity and blocks acute storage-fat mobilization in flies. Our data demonstrate that storage-fat mobilization in the fly is coordinated by two lipocatabolic systems, which are essential to adjust normal body fat content and ensure lifelong fat-storage homeostasis.

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). AKHR^G6244 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 (AKHR^rev) 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 AKHR^rev 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 [AKHR^rev or bmm^rev] 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 (AKHR^rev or bmm^rev). 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 (AKHR^rev bmm^rev) 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 (AKHR^rev) 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.