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

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.

Porting the synthetic D-glucaric acid pathway from Escherichia coli to Saccharomyces cerevisiae.

D-Glucaric acid can be produced as a value-added chemical from biomass through a de novo pathway in Escherichia coli. However, previous studies have identified pH-mediated toxicity at product concentrations of 5 g/L and have also found the eukaryotic myo-inositol oxygenase (MIOX) enzyme to be rate-limiting. We ported this pathway to Saccaromyces cerevisiae, which is naturally acid-tolerant and evaluate a codon-optimized MIOX homologue. We constructed two engineered yeast strains that were distinguished solely by their MIOX gene - either the previous version from Mus musculus or a homologue from Arabidopsis thaliana codon-optimized for expression in S. cerevisiae - in order to identify the rate-limiting steps for D-glucaric acid production both from a fermentative and non-fermentative carbon source. myo-Inositol availability was found to be rate-limiting from glucose in both strains and demonstrated to be dependent on growth rate, whereas the previously used M. musculus MIOX activity was found to be rate-limiting from glycerol. Maximum titers were 0.56 g/L from glucose in batch mode, 0.98 g/L from glucose in fed-batch mode, and 1.6 g/L from glucose supplemented with myo-inositol. Future work focusing on the MIOX enzyme, the interplay between growth and production modes, and promoting aerobic respiration should further improve this pathway.

Disruption of renal tubular mitochondrial quality control by Myo-inositol oxygenase in diabetic kidney disease.

Diabetic kidney disease (DKD) is associated with oxidative stress and mitochondrial injury. Myo-inositol oxygenase (MIOX), a tubular-specific enzyme, modulates redox imbalance and apoptosis in tubular cells in diabetes, but these mechanisms remain unclear. We investigated the role of MIOX in perturbation of mitochondrial quality control, including mitochondrial dynamics and autophagy/mitophagy, under high-glucose (HG) ambience or a diabetic state. HK-2 or LLC-PK1 cells subjected to HG exhibited an upregulation of MIOX accompanied by mitochondrial fragmentation and depolarization, inhibition of autophagy/mitophagy, and altered expression of mitochondrial dynamic and mitophagic proteins. Furthermore, dysfunctional mitochondria accumulated in the cytoplasm, which coincided with increased reactive oxygen species generation, Bax activation, cytochrome C release, and apoptosis. Overexpression of MIOX in LLC-PK1 cells enhanced the effects of HG, whereas MIOX siRNA or d-glucarate, an inhibitor of MIOX, partially reversed these perturbations. Moreover, decreasing the expression of MIOX under HG ambience increased PTEN-induced putative kinase 1 expression and the dependent mitofusin-2-Parkin interaction. In tubules of diabetic mice, increased MIOX expression and mitochondrial fragmentation and defective autophagy were observed. Dietary supplementation of d-glucarate in diabetic mice decreased MIOX expression, attenuated tubular damage, and improved renal functions. Notably, d-glucarate administration also partially attenuated mitochondrial fragmentation, oxidative stress, and apoptosis and restored autophagy/mitophagy in the tubular cells of these mice. These results suggest a novel mechanism linking MIOX to impaired mitochondrial quality control during tubular injury in the pathogenesis of DKD and suggest d-glucarate as a potential therapeutic agent for the amelioration of DKD.

The ethylene response factor AtERF98 enhances tolerance to salt through the transcriptional activation of ascorbic acid synthesis in Arabidopsis.

Ascorbic acid (AsA) is an important antioxidant in plants, and its biosynthesis is finely regulated through developmental and environmental cues; however, the regulatory mechanism remains unclear. In this report, the knockout and knockdown mutants of Arabidopsis AtERF98 decreased the AsA level, whereas the overexpression of AtERF98 increased it, which suggests that AtERF98 plays an important role in regulating AsA biosynthesis. AtERF98-overexpressing plants showed enhanced expression of AsA synthesis genes in the d-mannose/l-galactose (d-Man/l-Gal) pathway and the myo-inositol pathway gene MIOX4, as well as of AsA turnover genes. In contrast, AtERF98 mutants showed decreased expression of AsA synthesis genes in the d-Man/l-Gal pathway but not of the myo-inositol pathway gene or AsA turnover genes. In addition, the role of AtERF98 in regulating AsA production was significantly impaired in the d-Man/l-Gal pathway mutant vtc1-1, but the expression of the myo-inositol pathway gene or AsA turnover genes was not affected, which indicates that the regulation of AtERF98 in AsA synthesis is primarily mediated by the d-Man/l-Gal pathway. Transient expression and chromatin immunoprecipitation assays further showed that AtERF98 binds to the promoter of VTC1, which indicates that AtERF98 modulates AsA biosynthesis by directly regulating the expression of the AsA synthesis genes. Moreover, the knockout mutant aterf98-1 displayed decreased salt-induced AsA synthesis and reduced tolerance to salt. The supplementation of exogenous AsA increased the salt tolerance of aterf98-1; coincidently, the enhanced salt tolerance of AtERF98-overexpressing plants was impaired in vtc1-1. Thus, our data provide evidence that the regulation of AtERF98 in AsA biosynthesis contributes to enhanced salt tolerance in Arabidopsis.

Myo-inositol oxygenase genes are involved in the development of syncytia induced by Heterodera schachtii in Arabidopsis roots.

* In plants, UDP-glucuronic acid is synthesized by the oxidation of UDP-glucose by UDP-glucose dehydrogenase or the oxygenation of free myo-inositol by myo-inositol oxygenase (MIOX). In Arabidopsis, myo-inositol oxygenase is encoded by four genes. Transcriptome analysis of syncytia induced by the cyst nematode Heterodera schachtii in Arabidopsis roots revealed that MIOX genes are among the most strongly upregulated genes. * We have used beta-glucuronidase (GUS) analysis, in situ reverse transcription polymerase chain reaction (RT-PCR), and real-time RT-PCR to study the expression of all four MIOX genes in syncytia induced by H. schachtii in Arabidopsis roots. All these methods showed that MIOX genes are strongly induced in syncytia. GeneChip data were analysed for the expression of genes related to the MIOX pathway (mapman). * Two complementary double mutants were used to study the importance of MIOX genes. Results of the infection assay with double mutants in two combinations (Deltamiox1+2, Deltamiox4+5) showed a significant reduction (P < 0.05) in the number of females per plant when compared with the wild-type. Furthermore, syncytia in double mutants were significantly smaller than in wild-type plants. * Our data demonstrate an important role of the MIOX genes for syncytium development and for the development of female nematodes.