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First published online September 19, 2008
Journal of Experimental Biology 211, 3103-3110 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.016451
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A glucagon-like endocrine pathway in Drosophila modulates both lipid and carbohydrate homeostasis

K. N. Bharucha1,*, P. Tarr2 and S. L. Zipursky2

1 Department of Pediatrics and Pharmacology, University of Texas Southwestern Medical School, Dallas, Texas 75390, USA
2 Department of Biological Chemistry, David Geffen School of Medicine, University of California and the Howard Hughes Medical Institute, Los Angeles, CA 90095, USA


Figure 1
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  		Fig. 1. Generation of Akhr mutants. (A) Schematic representation of Akhr alleles. Akhrp (EY11371) contains a P element inserted in exon 1, upstream of the start of translation. Akhrrev is a P-element excision allele; an ~20 bp remnant of the P element remains. Akhrnull removes the entire coding sequence of the Akhr gene, but does not disrupt the adjacent CG11188 and Tsp genes. The Akhrnull line also retains an insert of the P element (~400 bp) and removes ~150 bp 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 Akhrrev (left lane for each primer pair) but not in Akhrnull (right lane for each primer pair) as assessed using RT-PCR. Akhr mRNA was also detected in y1w67c23 and Akhrp (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).

 

Figure 2
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  		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 Akhrnull flies are shown in blue and those for Akhrrev flies in yellow. (A) Akhrnull flies have higher total body triglyceride content than Akhrrev (ad libitum fed). Triglyceride values are averages of triplicate measurements, with corresponding standard deviations. (B) Akhrnull 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 Akhrnull (right panel) and Akhrrev (left panel) flies. Scale bars, 10 µm. (C) Akhrnull flies have higher glycogen content (measurements obtained from whole body homogenates). Differences between Akhrnull and Akhrrev 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 Akhrnull 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 Akhrnull and Akhrrev were indistinguishable. Measurements for wing size, wing cell size, mesothorax size and foreleg femur length are shown. (F) Akhrnull 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., 2006Go). With prior starvation, Akhrnull flies had lower food ingestion during the first 30 min (Student's t-test, *P<0.05).

 

Figure 3
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  		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 Akhrnull flies and Akhrrev 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. Akhrrev flies have starvation resistance that is comparable to y1w67c23 flies, the genetic background from which the Akhrp line was generated. Akhrnull flies were markedly starvation resistant when compared to Akhrrev control flies, and Akhrp 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) Akhrnull flies have enhanced starvation resistance when compared to age-matched Akhrrev control flies (Student's t-test, *P<0.05). (C) Akhrnull 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 Akhrrev and Akhrnull using the DAMS. No gross defects in locomotor activity or circadian rhythm were observed in Akhrnull 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.

 

Figure 4
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  		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 Akhrnull flies restores wild-type starvation resistance (Student's t-test, *P<0.05). All genetic rescue experiments were done in an Akhrnull 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 Akhrnull flies. (H) Expression of AKHR in the fat body (in an otherwise Akhrnull background) dramatically reduces total body triglyceride content; the triglyceride content of Akhrnull flies is shown for comparison.

 

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© The Company of Biologists Ltd 2008