Drosophila melanogaster Acetyl-CoA-carboxylase sustains a fatty acid-dependent remote signal to waterproof the respiratory system

Abstract

Fatty acid (FA) metabolism plays a central role in body homeostasis and related diseases. Thus, FA metabolic enzymes are attractive targets for drug therapy. Mouse studies on Acetyl-coenzymeA-carboxylase (ACC), the rate-limiting enzyme for FA synthesis, have highlighted its homeostatic role in liver and adipose tissue. We took advantage of the powerful genetics of Drosophila melanogaster to investigate the role of the unique Drosophila ACC homologue in the fat body and the oenocytes. The fat body accomplishes hepatic and storage functions, whereas the oenocytes are proposed to produce the cuticular lipids and to contribute to the hepatic function. RNA-interfering disruption of ACC in the fat body does not affect viability but does result in a dramatic reduction in triglyceride storage and a concurrent increase in glycogen accumulation. These metabolic perturbations further highlight the role of triglyceride and glycogen storage in controlling circulatory sugar levels, thereby validating Drosophila as a relevant model to explore the tissue-specific function of FA metabolic enzymes. In contrast, ACC disruption in the oenocytes through RNA-interference or tissue-targeted mutation induces lethality, as does oenocyte ablation. Surprisingly, this lethality is associated with a failure in the watertightness of the spiracles-the organs controlling the entry of air into the trachea. At the cellular level, we have observed that, in defective spiracles, lipids fail to transfer from the spiracular gland to the point of air entry. This phenotype is caused by disrupted synthesis of a putative very-long-chain-FA (VLCFA) within the oenocytes, which ultimately results in a lethal anoxic issue. Preventing liquid entry into respiratory systems is a universal issue for air-breathing animals. Here, we have shown that, in Drosophila, this process is controlled by a putative VLCFA produced within the oenocytes.

Figure 1. Immunodetection of ACC.

Direct GFP fluorescence (A,C,E,G), and staining to ACC (B,D,F,H,J) and nuclei (B,D). ACC expression was very high in the FB (B) and in the oenocytes (D) of L3 larvae, and thus detection was performed with very low laser intensity. The ACC signal was lower in the gut (F) and in the imaginal discs (H) of L3 larvae, and thus detection was performed with high laser intensity. Specificity of the ACC signal was monitored by generating homozygote FRT-ACC^B131 clones (identified by the lack of GFP staining; arrowheads in A,C,E,G), which do not express the ACC protein in the corresponding pictures (arrowheads in B,D,F,H respectively). In the embryo, the strongest ACC staining was observed in the oenocytes (J) co-labeled to GFP driven by BO-Gal4 (I). Scale bars: 20 µm.

Figure 2. Metabolic defects due to ACC disruption.

(A, B) LD contents labeled by Nile red staining (B) in the FB of well fed animals. Note the drop of LD accumulation (B) in homozygote FRT-ACC^B131 mutant clones marked by the absence of GFP (A); the dotted yellow line surrounds the GFP-negative clone (Scale bar: 20 µm). (C) Mean weight (mg) of 0–4 h prepupal female. (D–H) Concentration of TGs (D), proteins (E), trehalose (F), glucose (G) and glycogen (H) levels in prepupae. The values represent the concentration of each metabolite in µg per mg of 0–4 h prepupae. (I–N) Relative concentration (arbitrary units) of tetradecanoic (I), palmitic (J) palmitoleic (K), stearic (L), oleic (M) and linoleic (N) acid in 100 mg of 0–4 h prepupae. Color symbols (C–N): control (white bar) or expressing an ACC-RNAi in the FB (grey bar). T test: *: P<0.05; **: P<0.01; ***: P<0.001.

Figure 3. ACC disruption in the oenocytes.

(A) Growth phenotype at the L2/L3 transition (larvae at the top of the panel) and 2 days later (larvae at the bottom of the panel) corresponding to 71 h- and 119 h-old larvae, respectively. Symbols used: (BO) BO-Gal4/+ control; (grim) BO>grim; (ACC-Ri) BO>ACC-RNAi; (svp80) svp-Gal80;BO>ACC-RNAi; (Resc) ACC^B131;da-Gal4>UAS-ACC, ACC mutant rescued by ubiquitous ACC-cDNA expression; (oeTD) ACC^oeTD, ACC mutant targeted to the oenocytes. The differences in size observed at 71 h are not representative of all individuals; 71 h is a median value after a 5 h collection of newly-hatched larvae. (B) Survival of animals in hours after egg-laying. Symbols used as in A: (black square) BO-Gal4/+ control; (empty square) BO>grim; (black triangle) BO>ACC-RNAi; (empty diamond) svp-Gal80;BO>ACC-RNAi; (empty circle) ACC^B131;da-Gal4>UAS-ACC; (black circle) ACC^oeTD. (C) Feeding behavior of late L2 larvae. The percentage indicates the proportion of larvae that stray away from the food 1 h after being placed in a small piece of food in the middle of an agar plate. Symbols used as in A. (D–G) Oenocytes stained with Oil-Red-O. The oenocytes of BO>ACC-RNAi; (E) and ACC^oeTD (F) late L2 larvae accumulate high levels of LDs compared to control oenocytes (D). (G) An ACC^B131 mutant oenocyte labeled by the absence of GFP (arrow) accumulate high levels of LDs compared to the neighboring control oenocytes (GFP labeled). Scale bars: 20 µm.

Figure 4. Oenocytes ACC signals to the tracheal system.

(A) Dorsal view of a wild-type larva showing the two dorsal main trunks of the tracheal system, which extend from the anterior to the posterior spiracles (arrowheads). Air-filling phenotypes in the tracheal systems of BO>grim (B), BO>ACC-RNAi (C), ACC^oeTD mutant (D), ACC^B131 mutant rescued by ubiquitous UAS-ACC (E) and BO>KAR-RNAi (F) animals. The portions of the dorsal main trunks filled with an aqueous solution are difficult to distinguish (arrows). The larvae have been selected early after the L2/L3 molting transition (prior to death in B,C,D,F). Larvae oriented anterior to the top. (G) Higher magnification of tracheal branches in early L3 ACC^oeTD larvae; a branch that originates from the air-filled part of the main trunk is entirely filled with air (arrow) whereas a branch that originates from the liquid-filled part of the main trunk is filled with liquid in its proximal end but not in the distal sub-branches (arrowhead). Scale bars: 100 µm. (H) Transcriptional expression of the charybdis and scylla hypoxic-responsive genes in control, cell-ablated, ACC or KAR deficient animals. Symbols used: (WT) control; (grim) BO>grim; (ACC-Ri) BO>ACC-RNAi; (oeTD) ACC^oeTD; (Resc) ACC^B131;da-Gal4>UAS-ACC; (KAR-Ri) BO-Gal4>KAR-RNAi. Genotypes behaved differently in their hypoxic response (ANOVA: genotype: F5,32 = 36.19 P<10⁻³; gene F1,32 = 1.24 P = 0.27; genotype×gene: F5,32 = 0.47 P = 0.795). Dunnet T test: *: P<0.05; **: P<0.01; ***: P<0.001.

Figure 5. Water-tightness defect of the spiracles.

(A–L) Tests for liquid entry into the tracheal system after transfer of larvae onto a semi-liquid media stained with Brilliant Blue FCF. Control larvae were maintained for 36 h inside the tinted feeding medium and analyzed at various times (A,B,G,H,K) Mutant larvae without any visible tracheal defect were transferred onto the tinted feeding medium and analyzed as they strayed away (C,D,E,F,I,J,L). Larvae of the following genotypes and stages: (A) BO control late L2, (B) BO control early L3, (C) BO>ACC-RNAi early L3, (D) BO>grim late L2, (E) BO>CG6660-RNAi early L3, (F) BO>FAS^CG17374-RNAi early L3, (G) promE control at the L2/L3 transition, (H) promE control early L3, (C) promE>ACC-RNAi early L3, (D) promE>grim at the L2/L3 transition, (K) ACC^B131 mutant rescue and (L) ACC^oeTD. Note that the staining of the gut due to the tinted food is not visible in the hemolymph, which confirms that Brilliant Blue FCF does not easily cross the epithelia. Entry of tinted liquid into the anterior and posterior spiracles (arrowheads in C) and extension into the main trunks (arrow in D) are indicated. Larvae oriented anterior to the top.

Figure 6. Lipid transfer within the anterior spiracles.

(A–P) Oil-Red-O staining of anterior spiracles. (A–D) Control larvae at the late L2 (A), early L3 (B), mid L3 (C), late L3 (D) stages contain LD clusters in their spiracular glands that form lipid-containing ducts (arrowheads); note the accumulation of lipids at the spiracular opening of late L3 larvae (arrow in D). (F–L) Spiracles at the late L2 stage (E,G) and after the L2/L3 molt (F,H,I,J,K,L) of the following larvae: (E–F) BO>grim, (G–H) BO>ACC-RNAi, (I) promE>grim, (J) promE>ACC-RNAi, (K) ACC^oeTD and (L) ACC^B131 mutant rescue. (M–P) Presence (M,P) or absence (N,O) of lipid-containing ducts in L3 spiracular terminal branch of the following larvae: (M) control, (N) BO>ACC-RNAi, (O) ACC^oeTD; (P) ACC^B131 mutant rescue.

Figure 7. A VLCFA–dependent remote signal from the oenocytes controls lipid transfer within the spiracles.

The default of VLCFA synthesis within the oenocytes provokes the failure to transfer lipids through the spiracular ducts from the spiracular gland to the spiracular opening. The gene products identified to be involved in this metabolic pathway within the oenocytes are indicated (oval forms surrounded in black). The signal running from the oenocytes to the spiracles is yet unidentified.