Drosophila HNF4 regulates lipid mobilization and beta-oxidation

Abstract

Drosophila HNF4 (dHNF4) is the single ancestral ortholog of a highly conserved subfamily of nuclear receptors that includes two mammalian receptors, HNFalpha and HNFgamma, and 269 members in C. elegans. We show here that dHNF4 null mutant larvae are sensitive to starvation. Starved mutant larvae consume glycogen normally but retain lipids in their midgut and fat body and have increased levels of long-chain fatty acids, suggesting that they are unable to efficiently mobilize stored fat for energy. Microarray studies support this model, indicating reduced expression of genes that control lipid catabolism and beta-oxidation. A GAL4-dHNF4;UAS-lacZ ligand sensor can be activated by starvation or exogenous long-chain fatty acids, suggesting that dHNF4 is responsive to dietary signals. Taken together, our results support a feed-forward model for dHNF4, in which fatty acids released from triglycerides activate the receptor, inducing enzymes that drive fatty acid oxidation for energy production.

Figure 1. dHNF4 is expressed in tissues that control metabolic homeostasis

Affinity-purified antibodies directed against dHNF4 were used to determine its spatial pattern of expression in organs dissected from third instar larvae. (A) dHNF4 protein is localized to the nucleus and is present at relatively high levels in the gastric caeca (GC) and anterior midgut (MG) of the digestive system, with lower levels in the proventriculus (PV). (B,C) Relatively high levels of expression are also seen in the fat body (FB), Malpighian tubules (MT), and hindgut imaginal ring (IR), with lower levels of expression in the salivary gland (SG). (D) dHNF4 is highly expressed in the larval oenocytes (arrows), with low level expression in the surrounding epidermis. (E) No expression is detectable in tissues dissected from dHNF4 mutant tissues, indicating that the antibody is specific for dHNF4 protein.

Figure 2. dHNF4 null mutants are sensitive to starvation

(A) A schematic representation of the dHNF4 locus is depicted, with the gene structure shown below along with three 3′-flanking genes, CG9298, CG9296, and CG9289. The locations of the two P-element insertions in dHNF4, EP2449 and KG08976 are shown, along with the dHNF4^Δ17 and dHNF4^Δ33 deletions. (B) A time course of the viability of starved control and dHNF4 mutant larvae, with and without a wild type hs-dHNF4 transgene. Whereas most larvae that are transheterozygous for precise excisions of the EP2449 and KG08976 P-elements (control) can survive two days of starvation, most dHNF4^Δ33/dHNF4^Δ17 mutants (dHNF4⁻) die. Heat treatment (+hs) has no effect on these animals. Heat treatment of dHNF4^Δ33/dHNF4^Δ17 mutants that carry a hs-dHNF4 rescue construct (hs-dHNF4, dHNF4⁻ +hs), however, regain their starvation resistance. (C) Control (cont) or dHNF4 mutant (dHNF4⁻) late second instar larvae were either maintained on moist filter paper or on a 20% sucrose diet (sucrose) for two days at 25°C, after which the percent of surviving animals was scored and plotted. Whereas dHNF4 mutant larvae are sensitive to starvation, they survive normally on a sucrose diet. A representative of four independent experiments is presented, with N=10–40 animals in each experiment. Error bars are +/− S.E. * p = 1.3×10⁻³.

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.

Figure 4. dHNF4 regulates all levels of the β-oxidation pathway

(A) Second instar larvae that were transheterozygous for precise excisions of the EP2449 and KG08976 P-elements (control), or dHNF4^Δ33/dHNF4^Δ17 mutants (dHNF4⁻), were fed or starved for 24 hours, after which RNA was isolated and analyzed by northern blot hybridization. dHNF4 mRNA is induced upon starvation, with no transcript seen in mutant animals. CG5321, CG2107, fatp, yip2, CG9577, CG6178, and Acox57D-d, which function in β-oxidation, are significantly down-regulated in the dHNF4 mutant, consistent with the microarray results. CG3523, which encodes a predicted fatty acid synthetase, desat1, which encodes a predicted stearoyl-CoA desaturase, and CG11198, which encodes a predicted acetyl-CoA carboxylase, are all down-regulated upon starvation in both control larvae and dHNF4 mutants, and their overall level of expression is reduced in the mutant. (B) A schematic representation of the mitochondrial β-oxidation pathway is depicted. At the top, stored lipid in the form of triglycerides are hydrolyzed into free fatty acids by lipases. Acyl-CoA synthetases, which reside in the outer mitochondrial membrane, convert fatty acids into acyl-CoA for entry into the β-oxidation pathway. The acyl group is transported through the outer and inner mitochondrial membranes via a carnitine intermediate, and then processed through four enzymatic steps, as depicted. Each cycle generates one FADH₂ and one NADH, which donate their high energy electrons to the electron transport chain for ATP production. Each cycle of β-oxidation results in an acyl-CoA that is shortened by two carbons. This acyl-CoA can be processed through successive cycles to produce more FADH₂ and NADH. The names of genes that are down-regulated in dHNF4 mutants are listed next to their predicted enzymatic or transport functions.

Figure 5. GAL4-dHNF4 can be activated by starvation and exogenous long chain fatty acids

(A) Fed larvae carrying both the GAL4-dHNF4 and UAS-nlacZ transgenes were heat-treated, allowed to recover for ~6 hours, and dissected organs were stained with X-gal for β-galactosidase activity. Low levels of ligand sensor activity are seen in the fat body, midgut, and epidermis of late second instar (late L2) or early third instar larvae (early L3), while high levels of activity are detected in mid-third instar larvae (mid L3). GAL4-dHNF4 activity in the mid-third instar midgut is restricted to the anterior region, adjacent to the proventriculus (arrow). No ligand sensor activity is detected in the oenocytes, although negative results with the ligand sensor system are difficult to interpret (Palanker et al., 2006). (B) Late second instar GAL4-dHNF4; UAS-nlacZ larvae were fasted or fed for 3 hrs, heat-treated, and fasted or fed for another 3– 4 hrs, after which dissected organs were stained with X-gal for β-galactosidase activity. Starvation leads to strong activation of GAL4-dHNF4 in the fat body and epidermis of early third instar larvae compared to fed controls (A, early L3). (C) Late second instar GAL4-dHNF4; UAS-nlacZ larvae were heat-treated to induce GAL4-dHNF4 expression, allowed to recover, and then bisected, inverted, and cultured overnight in either control medium or medium supplemented with fatty acids as shown. Palmitic acid (C16:0), oleic acid (C18:1), and the very long chain fatty acid, lignoceric acid (C24:0), activate the ligand sensor in epidermis. This activation, however, is not seen in animals that express either the L279Q or R285G GAL4-dHNF4 mutant ligand sensors. Activation is also seen in the trachea, as shown in the C16:0-treated control tissue.

Figure 6. A feed-forward model for dHNF4 in fatty acid β-oxidation

In the absence of dietary nutrients, triglycerides (TAGs) are cleaved by lipases to yield long chain fatty acids (black circles). We propose that these fatty acids can directly or indirectly activate dHNF4 in the nucleus, inducing target gene transcription. The dHNF4 target genes encode lipases as well as enzymes and transporters that use fatty acids as a substrate for β-oxidation in the mitochondria, removing the activating signal for dHNF4 and maintaining energy homeostasis. See text for more details.