Regulated inositol synthesis is critical for balanced metabolism and development in Drosophila melanogaster

Abstract

Myo-inositol is a precursor of the membrane phospholipid, phosphatidylinositol (PI). It is involved in many essential cellular processes including signal transduction, energy metabolism, endoplasmic reticulum stress, and osmoregulation. Inositol is synthesized from glucose-6-phosphate by myo-inositol-3-phosphate synthase (MIPSp). The Drosophila melanogaster Inos gene encodes MIPSp. Abnormalities in myo-inositol metabolism have been implicated in type 2 diabetes, cancer, and neurodegenerative disorders. Obesity and high blood (hemolymph) glucose are two hallmarks of diabetes, which can be induced in Drosophila melanogaster third-instar larvae by high-sucrose diets. This study shows that dietary inositol reduces the obese-like and high-hemolymph glucose phenotypes of third-instar larvae fed high-sucrose diets. Furthermore, this study demonstrates Inos mRNA regulation by dietary inositol; when more inositol is provided there is less Inos mRNA. Third-instar larvae with dysregulated high levels of Inos mRNA and MIPSp show dramatic reductions of the obese-like and high-hemolymph glucose phenotypes. These strains, however, also display developmental defects and pupal lethality. The few individuals that eclose die within two days with striking defects: structural alterations of the wings and legs, and heads lacking proboscises. This study is an exciting extension of the use of Drosophila melanogaster as a model organism for exploring the junction of development and metabolism.

Keywords: Developmental defect; Head; Inositol; Metabolism; Obesity; Proboscis.

Fig. 1.

Inos mRNA levels are regulated in response to dietary inositol. qRT-PCR experiments with wild-type Canton-S larvae (N≥10 per experimental condition per trial) grown on low- and high- sucrose semi-defined food (50 µM inositol supplementation as indicated). Normalized to RPL32. Mean±s.e. of five independent trials are represented. *P<0.05; ***P<0.0001 as indicated determined by two-tailed t-test.

Fig. 2.

Dietary inositol decreases larval obesity and reduces hemolymph glucose. Wild-type Canton-S larvae grown on low- and high- sucrose semi-defined food (50 µM inositol supplementation as indicated). (A) The percentage of larvae that float (gold segment of bars) and sink in a buoyancy assay are indicated. N=total number of larvae. (B) Larvae assayed for TAG levels, values indicated are normalized to total protein. N=6 per condition per trial. (C) Glucose (mg/dl) in the hemolymph of the larvae. N=5 per condition per trial. Mean±s.e. of three independent trials of each experiment are represented. *P<0.05; **P<0.005; ***P<0.0001 as indicated determined by two-tailed t-test.

Fig. 3.

Inos mRNA levels vary with promoter-GAL4 constructs and MIPS protein levels vary in concordance with Inos mRNA levels. (A) The D. melanogaster Inos gene and the surrounding genomic region of chromosome 2. Location of the P-element D{XP+} is indicated by a green triangle. The primers for exon 1 to exon 2 (qRT-PCR experiments) are blue arrows. The details of the XP+ element are not to scale. Exons are blue blocks, introns are black lines, UTRs are grey blocks. (B) qRT-PCR experiments with larvae (N≥10 per experimental condition per trial) grown on standard rich food (inositol supplementation as indicated). Normalized to RPL32. Three independent trials of (control) strain ActGal4-3/+ were indistinguishable from the wild-type control Canton-S results shown. Mean±s.e. of three-six independent trials are represented. *P<0.05; **P<0.005 as indicated determined by two-tailed t-test. (C) Western blot of crude lysates extracted from third instar larvae (N≥30 per trial) of the indicated genotypes grown on standard rich food (upper panel). Ponceau staining of the blot (lower panel). Lane 1 molecular weight markers, lane 2 Canton-S, lane 3 D{XP+}/D{XP+}, lane 4 D{XP+}/+; ActGAL4-3/+, lane 5 D{XP+}/+; TubGAL4-3/+, lane 6 D{XP+}/UbiGAL4-2, lane 7 D{XP+}/ActGAL4-2, lane 8 blank, lane 9 D{XP+}/+. One representative blot of five independent trials is shown.

Fig. 4.

Increased Inos mRNA levels decrease larval obesity and reduce hemolymph glucose. Larvae grown on standard rich food with inositol supplementation as indicated. (A) The percentage of larvae of the indicated genotypes that float (gold segment of bars) and sink in a buoyancy assay are displayed. N=total number of larvae. Mean ±s.e. of four independent trials are represented. (B) Larvae assayed for TAG levels, values indicated are normalized to total protein. N=6 per condition per trial. Mean ±s.e. of three independent trials are represented. (C) Glucose (mg/dl) in the hemolymph of the larvae. N=5 per condition per trial. Mean±s.e. of three independent trials are represented. *P<0.05; **P<0.005; ***P<0.0001 as indicated determined by two-tailed t-test. Three independent trials of control strains ActGal4-3/+, TubGal4-3/+, UbiGal4-2/+, ActGal4-2/+ were indistinguishable from the wild-type control Canton-S results shown for all three experiments.

Fig. 5.

Dysregulated increased Inos RNA levels result in developmental arrest as pharate adults. The percent of adults (lighter bars) eclosing from pupae (darker bars) on standard rich food. Strains as indicated. Three independent trials of control strains ActGal4-3/+, TubGal4-3/+, UbiGal4-2/+ were identical to the wild-type control Canton-S results shown. N=total number of individuals examined. Mean±s.e. of three trials are represented. *P<0.05; **P<0.005; ***P<0.0001 as indicated determined by two-tailed t-test.

Fig. 6.

Dysregulated increased Inos RNA levels result in developmental morphological defects. Representative scanning electron (A,B,E,F) and brightfield (C,D,G,H) microscope images of adult females after eclosion. Panels A-D are wild-type control Canton-S (N=4 for A and B, N=10 for C and D) and panels E-H are D{XP+}/+; ActGAL4-3/+ (N=5 for E and F, N=15 for G and H). The arrows in A and E indicate the proboscis in the wild-type or the region lacking the proboscis in the genetically modified strain.