A glucagon-like endocrine pathway in Drosophila modulates both lipid and carbohydrate homeostasis

Abstract

The regulation of energy homeostasis is fundamental to all organisms. The Drosophila fat body serves as a repository for both triglycerides and glycogen, combining the energy storage functions of mammalian adipose and hepatic tissues, respectively. Here we show that mutation of the Drosophila adipokinetic hormone receptor (AKHR), a functional analog of the mammalian glucagon receptor, leads to abnormal accumulation of both lipid and carbohydrate. As a consequence of their obese phenotypes, AKHR mutants are markedly starvation resistant. We show that AKHR is expressed in the fat body, and, intriguingly, in a subset of gustatory neurons that mediate sweet taste. Genetic rescue experiments establish that the metabolic phenotypes arise exclusively from the fat body AKHR expression. Behavioral experiments demonstrate that AKHR mutants are neither sedentary nor hyperphagic, suggesting the metabolic abnormalities derive from a genetic propensity to retain energy stores. Taken together, our results indicate that a single endocrine pathway contributes to both lipid and carbohydrate catabolism in the Drosophila fat body.

Fig. 1

Generation of Akhr mutants. (A) Schematic representation of Akhr alleles. Akhr^p (EY11371) contains a P element inserted in exon 1, upstream of the start of translation. Akhr^rev is a P-element excision allele; an ~20bp remnant of the P element remains. Akhr^null removes the entire coding sequence of the Akhr gene, but does not disrupt the adjacent CG 11188 and Tsp genes. The Akhr^null line also retains an insert of the P element (~400bp) and removes ~150bp of genomic sequence downstream of Akhr. See the Materials and methods section for further details. (B) Akhr mRNA is expressed in the fat body of Akhr^rev (left lane for each primer pair) but not in Akhr^null (right lane for each primer pair) as assessed using RT-PCR. Akhr mRNA was also detected in y¹w^67c23 and Akhr^p (EY11371) lines (data not shown). All bands are of the expected molecular mass; see the Materials and methods section for a description of the PCR protocol. (C) Transgenic flies that expressed the yeast transcriptional activator Gal4 under the control of the Akhr promoter (Akhr-Gal4) were crossed to reporter flies (UAS-GFP). Expression in the fat body (white arrows) was observed throughout the adult body, including head (D) and dissected fat body tissue (E); expression in the larval fat body was also observed (data not shown).

Fig. 2

Akhr mutants have abnormal lipid and carbohydrate homeostasis. All experiments were performed with 1-week-old male flies unless otherwise noted. In the bar graphs data for Akhr^null flies are shown in blue and those for Akhr^rev flies in yellow. (A) Akhr^null flies have higher total body triglyceride content than Akhr^rev (ad libitum fed). Triglyceride values are averages of triplicate measurements, with corresponding standard deviations. (B) Akhr^null flies had larger lipid cells than control flies, consistent with the total body triglyceride measurements. Representative images are shown for Nile Red staining of fat body tissue from ~1-week-old male Akhr^null (right panel) and Akhr^rev (left panel) flies. Scale bars, 10 µm. (C) Akhr^null flies have higher glycogen content (measurements obtained from whole body homogenates). Differences between Akhr^null and Akhr^rev flies are accentuated after 24 h of starvation (Student’s t-test, *P<0.05). (D) Genetic rescue experiments demonstrate that glycogen levels decrease when Akhr is re-introduced in an otherwise Akhr^null background. Data from Gal4 flies are shown as white bars and those for UAS control flies as black bars; data from flies containing both Gal4 and UAS transgenes are shown in grey. Gal4 and UAS control flies have a higher glycogen content than flies that contain both transgenes (which can now express Akhr; Student’s t-test, *P<0.05). (E) Tissue and cell size in Akhr^null and Akhr^rev were indistinguishable. Measurements for wing size, wing cell size, mesothorax size and foreleg femur length are shown. (F) Akhr^null flies are not hyperphagic. The ingestion of FD&C No. 1 blue was quantified for both fed and starved (18–24 h) 1-week-old male flies. Food intake is shown in arbitrary units which are proportional to the measured absorbance of ingested dye as per a published protocol (Libert et al., 2006). With prior starvation, Akhr^null flies had lower food ingestion during the first 30 min (Student’s t-test, *P<0.05).

Fig. 3

Akhr mutants are starvation resistant. All experiments were performed on 1-week-old male flies, except when specifically noted otherwise. Throughout the figure, data for Akhr^null flies and Akhr^rev flies are represented in blue and yellow, respectively. (A) Starvation resistance of Akhr mutant flies. Starvation resistance profiles were quantified on the Trikinetics Drosophila Activity Monitoring System (DAMS), with individual monitoring tubes containing 2% agarose, but no other food source. The time of death of an individual male fly was defined to be the time of last recorded locomotor activity, which correlated well with starvation profiles obtained from direct observation. Average starvation resistances are given for each genotype (N=16) with their corresponding standard deviations. Akhr^rev flies have starvation resistance that is comparable to y¹w^67c23 flies, the genetic background from which the Akhr^p line was generated. Akhr^null flies were markedly starvation resistant when compared to Akhr^rev control flies, and Akhr^p flies showed an intermediate phenotype (Student’s t-test, *P<0.05 for both comparisons). Starvation resistance comparisons between genotypes were repeated at least three times. (B) Both young and older (1 week) Akhr^null flies have enhanced starvation resistance when compared to age-matched Akhr^rev control flies (Student’s t-test, *P<0.05). (C) Akhr^null flies are able to mobilize triglyceride stores, as reflected by the decreased lipid levels of flies starved for 72 h. (D) Akhr mutants do not have defective locomotor activity or circadian rhythm. Average number of midline crossings (N=16 for each genotype) were recorded every 30 min for fed Akhr^rev and Akhr^null using the DAMS. No gross defects in locomotor activity or circadian rhythm were observed in Akhr^null flies. (E) Total locomotor activity (counted as number of midline crossings) for the first 24 h of starvation for each genotype, with corresponding standard deviations (N=16 for each genotype). The differences in locomotor activity between all starved lines were not statistically significant.

Fig. 4

Fat body (but not neuronal) AKHR expression substantiates observed metabolic phenotypes. (A–C) The axon projection pattern of akhr-Gal4-expressing gustatory neurons in the adult subesophageal ganglion. The dark region at the top of the picture (white arrow) is the esophagus. Shown from left to right, Gr5a-Gal4/UAS-GFP, Akhr-RFP, and the merged images, demonstrating co-expression of Gr5a and AKHR. (D–F) Double-labeling experiments with Gr66a-expressing neurons demonstrated exclusion of AKHR. Additional Akhr fibers likely represent axon projections of other attractive taste neurons. Single sections are displayed (1 µm); similar results were obtained through all projection layers in the subesophageal ganglion. (G) Genetic rescue experiments demonstrate that AKHR expression in the fat body of Akhr^null flies restores wild-type starvation resistance (Student’s t-test, *P<0.05). All genetic rescue experiments were done in an Akhr^null background in 1-week-old male flies. R⁴-Gal4 was used as a fat body driver. Gr5a-Gal4 (which drives Gal4 expression in the majority of attractive-gustatory neurons) was used as the gustatory neuron driver. The results are average starvation resistances from separate experiments (N=4 for fat body rescue, with 16 flies for each experiment; N=3 for Gr5a rescue, with 16 flies for each experiment), and standard deviations are shown. The starvation resistance of neuronal rescue flies is indistinguishable from Akhr^null flies. (H) Expression of AKHR in the fat body (in an otherwise Akhr^null background) dramatically reduces total body triglyceride content; the triglyceride content of Akhr^null flies is shown for comparison.