Figure 3. Starved dHNF4 mutants have excess stored lipid and increased free fatty acids.

Late second instar larvae that were transheterozygous for precise excisions of the EP2449 and KG08976 P-elements (control) or dHNF4^Δ33/dHNF4^Δ17 mutant larvae (dHNF4⁻) were either fed or starved for 24 hrs, after which homogenates were subjected to (A) glycogen or (B) TAG assays. Amounts of glycogen (in μg) and TAG (in mg) were normalized to the amount of protein (in mg), shown on the y axis. Glycogen amounts drop to similar levels in starved control and dHNF4 mutant larvae, while TAG levels remain relatively high in starved dHNF4 mutants. N=25 animals for each sample, with each experiment using six samples. A representative of four (glycogen) or six (TAG) experiments is depicted. Error bars are +/− S.E. *p<0.01, **p<1×10⁻⁴, ***p<1×10⁻⁸ (C–F) Oil Red O staining of midguts and (G–J) oenocytes dissected from fed or 24 hour starved, control or dHNF4 mutant larvae. Whereas lipid levels drop significantly in the midguts of control larvae upon starvation (C,E), lipid is inefficiently depleted from the midguts of starved dHNF4 mutants (D,F). An increase in the lipid content of oenocytes occurs normally in starved dHNF4 mutants relative to starved controls (I,J). Fed dHNF4 mutants, however, display a slight increase in lipids in both the midgut and oenocytes relative to fed controls (C,D,G,H). (K–Q) Nile Red staining of larval fat bodies. Whereas the large lipid droplets in the fat bodies of fed controls become smaller upon starvation (K,M), lipid droplets appear to aggregate in starved dHNF4 mutant fat bodies (L,N). (O–Q) Tissue-specific rescue of lipid droplet morphology as assayed by Nile Red stains of fat bodies from starved animals. Starved UAS-dHNF4 dHNF4⁻/+ control animals (O) display wild-type lipid droplet morphology (compare to M). Starved dHNF4^Δ33/fb-GAL4 dHNF4^Δ17 mutants display lipid droplet aggregation (P), and this phenotype is largely rescued in starved UAS-dHNF4 dHNF4^Δ33/fb-GAL4 dHNF4^Δ17 larvae (Q). (R) GC/MS analyses of lipid extracts reveal that dHNF4 mutant early third instar larvae contain relatively higher levels of most free long chain fatty acids upon starvation than control larvae. N=20 larvae for each sample, with eight samples tested. Similar results were obtained from an independent experiment. The Y axis represents the concentration of each free fatty acid (FFA) relative to a ¹³C-labeled tridecanoate internal standard. Error bars are +/− S.E. *p<1×10⁻³, **p<1×10⁻⁴, ***p<1×10⁻⁵.