AMPK supports growth in Drosophila by regulating muscle activity and nutrient uptake in the gut

Abstract

The larval phase of the Drosophila life cycle is characterized by constant food intake, resulting in a two hundred-fold increase in mass over four days. Here we show that the conserved energy sensor AMPK is essential for nutrient intake in Drosophila. Mutants lacking dAMPKalpha are small, with low triglyceride levels, small fat body cells and early pupal lethality. Using mosaic analysis, we find that dAMPKalpha functions as a nonautonomous regulator of cell growth. Nutrient absorption is impaired in dAMPKalpha mutants, and this defect stems not from altered gut epithelial cell polarity but from impaired peristaltic activity. Expression of a wild-type dAMPKalpha transgene or an activated version of the AMPK target myosin regulatory light chain (MRLC) in the dAMPKalpha mutant visceral musculature restores gut function and growth. These data suggest strongly that AMPK regulates visceral smooth muscle function through phosphorylation of MRLC. Furthermore, our data show that in Drosophila, AMPK performs an essential cell-nonautonomous function, serving the needs of the organism by promoting activity of the visceral musculature and, consequently, nutrient intake.

Figure 1. Generation of a dAMPKα mutant allele

A) Schematic of the dAMPKα locus (top) showing the dAMPKα gene (black), the P element (P) and the genes CG3719 and CG32813 (gray). Imprecise P element excision yielded the dAMPKα^Δ39 allele (bottom). B) Q-PCR for exons 1 and 2 (black bars) and the 3’UTR (gray bars) of dAMPKα was performed on RNA isolated from wild type (FM7, GFP/Y) and dAMPKα^Δ39/Y third instar larvae. dAMPKα transcript levels were normalized to Rp49 transcript levels. Data are presented as mean ± s.d., **p < 0.01 vs. FM7, GFP/Y. C) Western blot analyses of phosphorylated (p-Thr184, top) and total dAMPKα (middle) and tubulin (bottom) in lysates from FM7, GFP/Y and dAMPKα^Δ39/Y third instar larvae. D) FM7, GFP/Y (top) and dAMPKα^Δ39/Y larvae (bottom) at 72, 96 and 120 h AEL, scale bar = 1 mm. E) FM7, GFP/Y and dAMPKα^Δ39/Y pupae, scale bar = 1 mm. The axial ratio (length/width, mean ± s.d.) is greater in dAMPKα^Δ39 mutants compared with WT (p < 0.01).

Figure 2. Nutrient storage is decreased in dAMPKα mutant larvae

A) Western blot analyses of phosphorylated (p-Ser93, top) and total dACC (bottom) in lysates from FM7, GFP/Y and dAMPKα^Δ39/Y third instar larvae, n.s., non-specific. B) Whole-body triglyceride levels were measured in FM7, GFP/Y (black bars) and dAMPKα^Δ39/Y (gray bars) 108 h AEL larvae (n = 18/group) and white prepupae (n = 13/group) and normalized to protein. Data are presented as mean ± s.e.m., **p < 0.01 vs. FM7, GFP/Y. C) Fat bodies from 108 h AEL and wandering (FM7, GFP/Y: ~120 h AEL, dAMPKα^Δ39/Y: ~168 h AEL) larvae were stained with phalloidin and DAPI to label plasma membranes and nuclei. Scale bar = 50 µm. D-F) Heat shock was used to induce clones of fat body cells homozygous for the dAMPKα^Δ39 (D) or the dAMPKα¹ (E) mutations (GFP negative cells, left panels). Cell membranes were labeled with phalloidin (center panels). Merged images are shown in the right panels, scale bar = 50 µm. F) 24 h starvation of larvae bearing clones had no additional effect on dAMPKα^Δ39 homozygous cell size (arrows). Genotypes: D and F) UbiGFP, hsFLP, FRT19A/dAMPKα^Δ39, FRT19A. E) UbiGFP, hsFLP, FRT19A/dAMPKα¹, FRT19A.

Figure 3. Impaired nutrient absorption despite normal epithelial polarity in dAMPKα mutants

A) FM7, GFP/Y (black bars) and dAMPKα^Δ39/Y larvae (gray bars) were fed radiolabeled glucose for 4 or 14 h, and incorporation of radioactivity into glycogen and neutral lipids was measured. Data are presented as mean ± s.d., *p < 0.05 vs. FM7, GFP/Y at the same time point, †p < 0.05 vs. dAMPKα^Δ39/Y at 4 h. For FM7, GFP/Y, all 14 h values differ significantly from 4 h values, p < 0.05. B-E) Representative images of FM7, GFP/Y (top) and dAMPKα^Δ39/Y (bottom) midguts. B) Toluidine blue-stained transverse sections of third instar midguts showing the brush border (arrows) in both genotypes and vacuoles (v) in the dAMPKα^Δ39 midgut epithelium. C) Phalloidin labeling of the apical layer of filamentous actin (red, arrows) in sections of second instar guts. D, E) Immunocytochemistry for Na⁺/K⁺-ATPase (D, red, arrows), and Fasciclin III (E, white, arrows) in sections (D) or whole guts (E) from second instar larvae. Nuclei in C and D are stained with DAPI (blue), scale bars = 50 µm.

Figure 4. Visceral muscle function is impaired in dAMPKα mutants

A) Whole-mount immunocytochemistry for myosin heavy chain showing circular and longitudinal visceral muscle fibers in FM7, GFP/Y (left) and dAMPKα^Δ39/Y (right) third instar midguts, scale bar = 50 µm. B) Toluidine blue staining of transverse sections of FM7, GFP/Y (left) and dAMPKα^Δ39/Y (right) hindguts. Brackets indicate individual sarcomeres. C, D) Larvae were raised on food containing FD&C No. 1 Blue dye, transferred to Whatman paper at 96 h AEL and scored for the amount of food remaining in the gut over 24 h as described in the Materials and Methods. C) FM7, GFP/Y: n = 16, dAMPKα^Δ39/Y: n = 13. D) FM7, GFP/Y: n = 12, dAMPKα¹/Y: n = 11. Data are presented as mean ± s.d., **p < 0.01 vs. FM7, GFP/Y at the same time point. E) Still images of FM7, GFP/Y (left) and dAMPKα^Δ39/Y (right) third instar guts imaged live in HL3 buffer. Each frame is separated from the next by 12 seconds. The arrow points to constriction of wild type gut muscles.

Figure 5. Muscle-specific expression of dAMPKα rescues dAMPKα^Δ39 phenotypes

A–C) Visceral muscle function was tested using the blue food transit assay. Expression of wild type dAMPKα in muscle using 24B- (A) or dMef2-GAL4 (B) rescued the dAMPKα^Δ39 phenotype (compare the center set of bars: dAMPKα^Δ39 with UAS-dAMPKα alone, to the right set of bars: dAMPKα^Δ39 with GAL4 > dAMPKα). C) MHC-GAL4 was unable to rescue the dAMPKα^Δ39 gut transit phenotype. For A–C: FM7, GFP/Y, n = 23–24; dAMPKα^Δ39/Y; UAS-dAMPKα, n = 5–7; and dAMPKα^Δ39/Y; GAL4 > dAMPKα, n = 18–37. Data are presented as mean ± s.d., *p < 0.01 vs. FM7, GFP/Y, †p < 0.01 vs. dAMPKα^Δ39/Y; UAS-dAMPKα. 24B-, dMef2-, and MHC-GAL4-driven GFP expression in visceral muscle (top panels) and phalloidin counter-staining (bottom panels) are shown to the right of each graph. D) Triglyceride levels in 120 h AEL FM7, GFP/Y (black bars) and dAMPKα^Δ39/Y larvae (gray bars) with UAS-dAMPKα alone (FM7, GFP, n = 6, dAMPKα^Δ39, n = 4) or 24B > dAMPKα (FM7, GFP, n = 8, dAMPKα^Δ39, n = 11). Data are presented as mean ± s.d., *p < 0.01 and NS, not significant, vs. FM7, GFP/Y. E) dAMPKα^Δ39 mutants (left) died after pupariation, while 24B-GAL4-rescued dAMPKα^Δ39 mutants (right) underwent metamorphosis, as indicated by their red eyes (arrow), scale bar = 1 mm.

Figure 6. Expression of activated MRLC in muscle rescues the dAMPKα^Δ39 gut phenotype

A, B) Visceral muscle function in 96 h AEL wild type larvae was tested using the blue food transit assay. Data are presented as mean ± s.d. A) Larvae were fed 1% agar for 22 h, followed by 4 h of blue-dyed 1% agar (n = 9) or cornmeal/molasses food (n = 14). B) Larvae were fed 10 mM glucose and 1% yeast extract in blue-dyed 1% agar with vehicle (n = 13) or 50 mM 2-DG (n = 11). C) Visceral muscle function in lkb1^4B1–11/TM3, Ser, GFP (n = 34) and lkb1^4B1–11 (n = 52) larvae. Data are presented as mean ± s.e.m., *p < 0.05 vs. lkb1^4B1–11/TM3, Ser, GFP. D) Western blot analysis of phosphorylated (p-Thr184, top) and total dAMPKα (bottom) in lysates from lkb1^4B1–11/TM3, Ser, GFP and lkb1^4B1–11 120 h AEL larvae. E) 24B-GAL4 driven expression of an activated MRLC transgene (sqh^EE) provided a partial rescue of the dAMPKα^Δ39 gut transit phenotype. Compare the middle set of bars: dAMPKα^Δ39 with 24B > dAMPKα (n = 13), to the right set of bars: dAMPKα^Δ39 with 24B > sqh^EE (n = 15). Data are presented as mean ± s.e.m., NS, not significant, vs. FM7, GFP/Y. F) Whole-mount phalloidin labeling showing circular and longitudinal visceral muscle fibers in FM7, GFP/Y, dAMPKα^Δ39/Y; 24B-GAL4 / +, dAMPKα^Δ39/Y; 24B > dAMPKα and dAMPKα^Δ39/Y; 24B > sqh^EE third instar midguts.