Inositol in Disease and Development: Roles of Catabolism via myo-Inositol Oxygenase in Drosophila melanogaster

Abstract

Inositol depletion has been associated with diabetes and related complications. Increased inositol catabolism, via myo-inositol oxygenase (MIOX), has been implicated in decreased renal function. This study demonstrates that the fruit fly Drosophila melanogaster catabolizes myo-inositol via MIOX. The levels of mRNA encoding MIOX and MIOX specific activity are increased when fruit flies are grown on a diet with inositol as the sole sugar. Inositol as the sole dietary sugar can support D. melanogaster survival, indicating that there is sufficient catabolism for basic energy requirements, allowing for adaptation to various environments. The elimination of MIOX activity, via a piggyBac WH-element inserted into the MIOX gene, results in developmental defects including pupal lethality and pharate flies without proboscises. In contrast, RNAi strains with reduced levels of mRNA encoding MIOX and reduced MIOX specific activity develop to become phenotypically wild-type-appearing adult flies. myo-Inositol levels in larval tissues are highest in the strain with this most extreme loss of myo-inositol catabolism. Larval tissues from the RNAi strains have inositol levels higher than wild-type larval tissues but lower levels than the piggyBac WH-element insertion strain. myo-Inositol supplementation of the diet further increases the myo-inositol levels in the larval tissues of all the strains, without any noticeable effects on development. Obesity and blood (hemolymph) glucose, two hallmarks of diabetes, were reduced in the RNAi strains and further reduced in the piggyBac WH-element insertion strain. Collectively, these data suggest that moderately increased myo-inositol levels do not cause developmental defects and directly correspond to reduced larval obesity and blood (hemolymph) glucose.

Keywords: Drosophila; developmental defect; diabetes; head; inositol; metabolism; obesity; oxygenase; proboscis.

Figure 1

D.melanogaster MIOX (CG6910) is regulated in response to dietary myo-inositol. (A) The D. melanogaster myo-inositol catabolic gene (myo-inositol oxygenase) CG6910, the surrounding genomic region of chromosome 3, and the transcript (isoform B) is displayed. The 5′ and 3′ UTRs are peach, and the exons are orange. The locations of the MIOXi2 (KK102548, green) and MIOXi3 (GD12073, purple) sequences and the ~7.2 kb piggyBac WH-element insertion (red) are also displayed. The primer locations for the qRT-PCR experiments are blue arrows. (B) qRT-PCR experiments examining MIOX mRNA levels of larvae (left) and adults (right) grown on rich or semi-defined sucrose (CAA-S) or semi-defined myo-inositol (CAA-I) food. Normalized to RpL32, mean ± SE of three independent trials are represented. Control strains ActGal4-3/TbGFP and CyOGFP/+; ActGal4-3/+ were indistinguishable from the wild-type control Canton-S results. n.s. = not significant, * p < 0.05, ** p< 0.005, *** p < 0.0001 as indicated, determined by two-tailed t-test. (C) MIOX enzyme assays to determine myo-inositol oxygenase specific activity in crude lysates of larvae and adults (as indicated) grown on rich, CAA-S, or CAA-I food. Mean ± SE of three independent trials are represented. There was no detectable MIOX activity in crude lysates of the homozygous piggyBac WH-element insertion strain (P-miox^f01770/P-miox^f01770). Control strains ActGal4-3/TbGFP and CyOGFP/+: ActGal4-3/+ were indistinguishable from the wild-type control Canton-S results shown. * p < 0.05, *** p < 0.0001 as indicated, determined by two-tailed t-test.

Figure 2

Wild-type (CS) D. melanogaster adults, but not MIOXi2/+; +/Act5CGal4-3 and MIOXi3/Act5CGal4-3 adults, are viable on food with myo-inositol as the sole sugar (CAA-I). On CAA-S, all the strains survive, and without sugar (CAA-0), all the strains die. Survivals on three foods are displayed: CAA-S (●), CAA-I (x), and CAA-0 (▲). (A) Wild-type control strain Canton S. (B) MIOXi2/+; +/Act5CGal4-3. (C) MIOXi3/Act5CGal4-3. Mean ± SE of six independent trials with twenty flies (half male/half female) per trial. n.s. = not significant, *** p < 10⁻⁶ as indicated determined by Mantel–Cox (log rank) test.

Figure 3

Disruption of myo-inositol catabolism via piggyBac WH-element insertion in MIOX results in developmental defects. (A) The percent of adults (light grey bars) eclosing from pupae (medium grey bars), normalized to the number of embryos (dark grey bars) on standard rich food. Strains as indicated. Wild-type control (CS) results shown. N = total number of individuals examined. Mean ± SE of three independent trials are represented. n.s. = not significant, ** p < 0.005, as determined by two-tailed t-test. (B) Brightfield microscope images of adult flies after eclosion. On the left is the control heterozygous P-miox^f01770/TbGFP (N = 15) (indistinguishable from the wild-type control Canton-S), and on the right is P-miox^f01770/P-miox^f01770 (N = 16). The arrows indicate the proboscis in the wild-type or the region lacking the proboscis in the piggyBac WH-element insertion strain.

Figure 4

Reduced myo-inositol catabolism increases myo-inositol levels but decreases larval obesity and hemolymph glucose. Larvae grown on standard rich food with myo-inositol supplementation as indicated. (A) Larval carcasses assayed for myo-inositol; values indicated are normalized to total protein. N = 5 per condition per trial. (B) Buoyancy assay; the percentage of larvae that sink are displayed. N = 20 per condition per trial. (C) TAG assay; values indicated are normalized to total protein. N = 6 per condition per trial. (D) Glucose (mg/dL) assay of hemolymph. N = 5 per condition per trial. Mean ± SE 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. Control strains ActGal4-3/TbGFP and CyOGFP/+: ActGal4-3/+ were indistinguishable from the wild-type control Canton-S results shown for all four experiments.