Osmotic expression of aldose reductase in retinal pigment epithelial cells: involvement of NFAT5.

BACKGROUND: Diabetic retinopathy is associated with osmotic stress resulting from hyperglycemia and intracellular sorbitol accumulation. Systemic hypertension is a risk factor of diabetic retinopathy. High intake of dietary salt increases extracellular osmolarity resulting in systemic hypertension. We determined the effects of extracellular hyperosmolarity, chemical hypoxia, and oxidative stress on the gene expression of enzymes involved in sorbitol production and conversion in cultured human retinal pigment epithelial (RPE) cells. METHODS: Alterations in the expression of aldose reductase (AR) and sorbitol dehydrogenase (SDH) genes were examined with real-time RT-PCR. Protein levels were determined with Western blot analysis. Nuclear factor of activated T cell 5 (NFAT5) was knocked down with siRNA. RESULTS: AR gene expression in RPE cells was increased by high (25 mM) extracellular glucose, CoCl2 (150 µM)-induced chemical hypoxia, H2O2 (20 µM)-induced oxidative stress, and extracellular hyperosmolarity induced by addition of NaCl or sucrose. Extracellular hyperosmolarity (but not hypoxia) also increased AR protein level. SDH gene expression was increased by hypoxia and oxidative stress, but not extracellular hyperosmolarity. Hyperosmolarity and hypoxia did not alter the SDH protein level. The hyperosmotic AR gene expression was dependent on activation of metalloproteinases, autocrine/paracrine TGF-ß signaling, activation of p38 MAPK, ERK1/2, and PI3K signal transduction pathways, and the transcriptional activity of NFAT5. Knockdown of NAFT5 or inhibition of AR decreased the cell viability under hyperosmotic (but not hypoxic) conditions and aggravated the hyperosmotic inhibition of cell proliferation. CONCLUSIONS: The data suggest that sorbitol accumulation in RPE cells occurs under hyperosmotic, but not hypoxic and oxidative stress conditions. NFAT5- and AR-mediated sorbitol accumulation may protect RPE cells under conditions of osmotic stress.

Enhanced production of L-sorbose from D-sorbitol by improving the mRNA abundance of sorbitol dehydrogenase in Gluconobacter oxydans WSH-003.

BACKGROUND: Production of L-sorbose from D-sorbitol by Gluconobacter oxydans is the first step to produce L-ascorbic acid on industrial scale. The sldhAB gene, which encodes the sorbitol dehydrogenase (SLDH), was overexpressed in an industrial strain G. oxydans WSH-003 with a strong promoter, P tufB . To enhance the mRNA abundance, a series of artificial poly(A/T) tails were added to the 3'-terminal of sldhAB gene. Besides, their role in sldhAB overexpression and their subsequent effects on L-sorbose production were investigated. RESULTS: The mRNA abundance of the sldhAB gene could be enhanced in G. oxydans by suitable poly(A/T) tails. By self-overexpressing the sldhAB gene in G. oxydans WSH-003 with an optimal poly(A/T) tail under the constitutive promoter P tufB , the titer and the productivity of L-sorbose were enhanced by 36.3% and 25.0%, respectively, in a 1-L fermenter. Immobilization of G. oxydans-sldhAB6 cells further improved the L-sorbose titer by 33.7% after 20 days of semi-continuous fed-batch fermentation. CONCLUSIONS: The artificial poly(A/T) tails could significantly enhance the mRNA abundance of the sldhAB. Immobilized G. oxydans-sldhAB6 cells could further enlarge the positive effect caused by enhanced mRNA abundance of the sldhAB.

The polyol pathway in the bovine oviduct.

The oviducts likely provide optimized micro-environments for the final maturation of gametes, fertilization, and early embryo development. Hexoses, including glucose, fructose, and sorbitol, are involved in these critical reproductive events. Monosaccharide production is controlled, in part, by the polyol pathway and requires two enzymes: an aldose reductase (AR) that reduces glucose into sorbitol, followed by its oxidation into fructose by sorbitol dehydrogenase (SDH). We analyzed the expression of AR and SDH in the isthmus and ampulla of the bovine oviduct at the proliferative, mid-luteal, and late-luteal phases of the estrous cycle by quantitative PCR and immunoblots. Immunochemistry and an assay of SDH activity were also performed. The quantity of hexoses in whole sections of isthmus and ampulla were determined by liquid chromatography coupled to mass spectrometry. In sum, AR expression was restricted to the isthmus, while SDH was mostly expressed in the isthmic-ampullary junction and the ampulla, specifically concentrated in the luminal epithelium of the oviduct. The estrous cycle had no impact on protein expression of AR and SDH. Instead, the levels of AR and SDH expression were associated with higher ratios of sorbitol to fructose in the isthmus (1.6) than in the ampulla (4.1; P = 0.005). These results are discussed in light of physiological events occurring in the oviduct.

Sorbitol can fuel mouse sperm motility and protein tyrosine phosphorylation via sorbitol dehydrogenase.

Energy sources that can be metabolized to yield ATP are essential for normal sperm functions such as motility. Two major monosaccharides, sorbitol and fructose, are present in semen. Furthermore, sorbitol dehydrogenase (SORD) can convert sorbitol to fructose, which can then be metabolized via the glycolytic pathway in sperm to make ATP. Here we characterize Sord mRNA and SORD expression during mouse spermatogenesis and examine the ability of sorbitol to support epididymal sperm motility and tyrosine phosphorylation. Sord mRNA levels increased during the course of spermatogenic differentiation. SORD protein, however, was first detected at the condensing spermatid stage. By indirect immunofluorescence, SORD was present along the length of the flagella of caudal epididymal sperm. Furthermore, immunoelectron microscopy showed that SORD was associated with mitochondria and the plasma membranes of sperm. Sperm incubated with sorbitol maintained motility, indicating that sorbitol was utilized as an energy source. Sorbitol, as well as glucose and fructose, were not essential to induce hyperactive motility. Protein tyrosine phosphorylation increased in a similar manner when sorbitol was substituted for glucose in the incubation medium used for sperm capacitation. These results indicate that sorbitol can serve as an alternative energy source for sperm motility and protein tyrosine phosphorylation.

Expression, purification and preliminary crystallographic analysis of human sorbitol dehydrogenase.

Human sorbitol dehydrogenase (SDH) was expressed in Escherichia coli BL21 cells and purified using ammonium sulfate precipitation and anion-exchange and dye-affinity chromatography. Purified SDH was crystallized from polyethylene glycol solutions using the hanging-drop vapour-diffusion method. X-ray data were collected to 2.75 A resolution. The crystals belong to the monoclinic C2 space group, with unit-cell parameters a = 145.9, b = 52.3, c = 169.0 A, beta = 101.8 degrees. This is the first crystallization report of human sorbitol dehydrogenase.

Analysis of gene expression of aldose reductase and sorbitol dehydrogenase in rat Schwann cells by competitive RT-PCR method using non-homologous DNA standards.

Aldose reductase (AR) and sorbitol dehydrogenase (SDH) are the enzymes constituting the polyol pathway, an alternate route of glucose metabolism. A wealth of experimental data has indicated the involvement of the polyol pathway in the pathogenesis of diabetic complications. However, there has been surprisingly little research on the relative abundance of SDH to AR in the tissues affected in diabetes. We therefore developed a competitive RT-PCR system to simultaneously determine the mRNA levels of these two enzymes in small amounts of samples, and studied their expression in Schwann cells isolated from adult rat sciatic nerves. Although both AR and SDH mRNA were expressed in the Schwann cells, the levels of SDH cDNA were much lower than those of AR cDNA. The induction of AR mRNA expression in the Schwann cells under hyperosmotic conditions was similarly detected by Northern blot analysis and our competitive RT-PCR method. The RT-PCR system developed in this study may be a useful tool in ascertaining the relative contributions of AR and SDH to the metabolic derangements resulting from the acceleration of polyol pathway activity in the target organ of diabetic complications.

Expression of aldose reductase, sorbitol dehydrogenase and Na+/myo-inositol and Na+/Cl-/betaine transporter mRNAs in individual cells of the kidney during changes in the diuretic state.

The effect of changes in medullary extracellular tonicity on mRNA expression for aldose reductase (AR), sorbitol dehydrogenase (SDH), Na+/Cl-/betaine (BGT) and Na+/myo-inositol (SMIT) cotransporter in different kidney zones was studied using Northern blot analysis and non-radioactive in situ hybridization in four groups of rats: controls, acute diuresis (the loop diuretic furosemide was administered), chronic diuresis (5 days of diuresis), and antidiuresis [5 days of diuresis followed by 24 h deamino-Cys1, d-Arg8 vasopressin (dDAVP)]. Acute administration of the loop diuretic furosemide significantly reduced AR, SMIT and BGT gene expression in the inner and outer medulla compared with controls. Administration of dDAVP to chronically diuretic rats raised the expression of these three mRNAs in the inner but not the outer medulla compared with the chronically diuretic rats. None of these alterations in medullary tonicity significantly changed SDH expression. The in situ hybridization studies showed AR, BGT and SMIT mRNAs to be expressed in both epithelial and non-epithelial cells of the outer and inner medulla. The various cell types (epithelial, endothelial and interstitial cells) differed in their expression pattern and intensity of AR, SDH, BGT and SMIT mRNA, but the inner medullary cells responded uniformly to a decrease in extracellular tonicity with a reduction, and to an increase with enhancement of their AR, BGT and SMIT expression.

Expression of Na+/Cl-/betaine and Na+/myo-inositol transporters, aldose reductase and sorbitol dehydrogenase in macula densa cells of the kidney.

It has been suggested that macula densa cells may be exposed to hyperosmotic stress. Since chronic exposure to hypertonic stress causes the amount of intracellular organic osmolytes to increase, the expression of transporters and enzymes that participate in the intracellular accumulation of organic osmolytes was examined using non-radioactive in situ hybridization in the macula densa region of control rats and furosemide-treated animals. Both the sodium- and chloride-dependent betaine transporter (BGT) and sodium-dependent myo-inositol transporter (SMIT) were expressed preferentially in macula densa cells and for both mRNAs the signal intensity was visibly reduced by furosemide. The enzymes aldose reductase (which mediates the conversion of glucose to sorbitol) and sorbitol dehydrogenase (which converts sorbitol into fructose) were expressed not only in macula densa cells but also in the surrounding tubular cells, and the expression was insensitive to furosemide. Thus it remains unclear whether the expression of BGT and SMIT is related to a putative hypertonic juxtaglomerular region.

Effects of sorbitol dehydrogenase deficiency on nerve conduction in experimental diabetic mice.

In this report, we made use of sorbitol dehydrogenase (SDH)-deficient mutant mice (C57BL/LiA) to test whether there is a close correlation between the level of polyol accumulation and the degree of reduction in motor nerve conduction velocity (MNCV) associated with diabetes. The C57BL/LiA mouse has SDH deficiency due to a G-to-A mutation at the +1 position of intron 8, thus producing only aberrant SDH transcripts. These C57BL/LiA mice should have higher levels of polyol accumulation in the peripheral nerve because of the inability to further metabolize sorbitol to fructose. Here, we confirm by Western blot analysis and high-performance liquid chromatography that these mice lack SDH in the sciatic nerve and other various tissues, whereas normal mice possess SDH. These C57BL/LiA mice do not display any obvious phenotype that includes peripheral neuropathy in the normal laboratory environment and breed normally as described previously, although the tissues that normally contain SDH accumulate more sorbitol. This finding suggested that C57BL/LiA mouse strain is a valid model for studying the role in diabetic neuropathy of the polyol pathway, which consists of two enzymes-aldose reductase for converting glucose to sorbitol and SDH for converting sorbitol to fructose. Sorbitol levels in the sciatic nerve of diabetic C57BL/10N, nondiabetic, and diabetic C57BL/LiA mice were increased 4.3-, 16.6-, and 38.1-fold, respectively, above that of nondiabetic C57BL/10N. The fructose level in the sciatic nerve was increased 2.4-fold in diabetic C57BL/10N mice compared with that of nondiabetic and diabetic C57BL/LiA mice. Diabetic SDH-deficient mice showed an MNCV reduction similar in magnitude to that of diabetic C57BL/10N mice, despite greater nerve sorbitol accumulation and the lack of fructose in the former. The present data suggest that the levels of sorbitol and fructose in the sciatic nerve of mice do not correlate with the severity of MNCV deficit associated with diabetes.

Cloning of NAD-dependent sorbitol dehydrogenase from apple fruit and gene expression.

Partial amino acid sequences of NAD-dependent sorbitol dehydrogenase (NAD-SDH) were used to identify a full-length cDNA from apple fruit. This clone consisted of 1,433 bp containing an open reading frame of 1,137 bp that could code for a polypeptide with 379 amino acids. To our knowledge, this is the first report about cloning of NAD-SDH cDNA from a plant source. The deduced amino acids from cDNA revealed 43.7% identity to human NAD-SDH. The activity of this enzyme to convert sorbitol to fructose with the reduction of NAD was certified by the fusion protein of this clone expressed in Escherichia coli. Northern blot analysis showed that the mRNA was expressed in matured apple fruit.

Increased white cell aldose reductase mRNA levels in diabetic patients.

This paper examines the question of whether diabetes in humans is associated with changes in aldose reductase and sorbitol dehydrogenase gene expression. The polyol pathway, which comprises the enzymes aldose reductase and sorbitol dehydrogenase, is recognised to play a central role in the pathogenesis of the diabetic complications. Whilst it is known that experimental diabetes in the rat is associated with increased aldose reductase gene expression, possibly as an osmoregulatory response to hyperglycaemia, little is known about aldose reductase and sorbitol dehydrogenase gene expression in diabetes in humans. White cell aldose reductase mRNA levels were increased in patients with insulin-dependent (by 135%, P < 0.05) and non-insulin-dependent (by 132%, P < 0.05) diabetes compared to levels in healthy volunteers. Levels of glycosylated haemoglobin were also increased (P < 0.001) in diabetes but there was no correlation between white cell aldose reductase mRNA and glycosylated haemoglobin levels. In contrast to aldose reductase, levels of white cell sorbitol dehydrogenase mRNA were not affected by diabetes. These results establish for the first time that diabetic patients show increases in white cell aldose reductase mRNA levels, possibly consistent with increased aldose reductase gene expression. This finding may have implications for the use of aldose reductase inhibitors in the treatment of the diabetic complications.

Regulation of aldose reductase, sorbitol dehydrogenase, and taurine cotransporter mRNA in rat medulla.

The regulation of mRNA for aldose reductase, sorbitol dehydrogenase, and the Na+/Cl-/taurine cotransporter was studied with three in vivo models in which urinary concentration is reduced: Sprague-Dawley rats undergoing a water diuresis or fed a low-protein diet or Brattleboro rats. In Sprague-Dawley rats, 3 days of water diuresis reduced inner medullary aldose reductase mRNA abundance 6.5-fold compared with untreated rats, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. When water diuretic rats were acutely deprived of water, urine osmolality increased significantly after 4 h but aldose reductase mRNA did not increase until 12 h. Heat shock protein-70 mRNA was not increased by water deprivation. Second, in rats fed a low-protein diet for 3 wk, aldose reductase mRNA increased two-fold, whereas sorbitol dehydrogenase and taurine cotransporter mRNA were unchanged. Finally, in Brattleboro rats, urine osmolality and levels of aldose reductase and taurine cotransporter mRNA increased in response to 1 day of water deprivation, whereas sorbitol dehydrogenase mRNA was unchanged. Administering vasopressin (1 U/day) to Brattleboro rats for 8 days also increased urine osmolality and aldose reductase mRNA but did not alter sorbitol dehydrogenase or taurine cotransporter mRNA. This result is consistent with the hypothesis that changes in urine osmolality induce changes in aldose reductase mRNA abundance that are independent of vasopressin. It was concluded that, in rat inner medulla: (1) aldose reductase mRNA abundance varies with changes in water balance or dietary protein, whereas sorbitol dehydrogenase and taurine cotransporter mRNA do not; and (2) heat shock protein-70 mRNA abundance is not increased during acute osmotic stress.

Structural organization of the human sorbitol dehydrogenase gene (SORD).

The primary structure of human sorbitol dehydrogenase (SORD) was determined by cDNA and genomic cloning. The nucleotide sequence of the mRNA covers 2471 bp including an open reading frame that yields a protein of 356 amino acid residues. The gene structure of SORD spans approximately 30 kb divided into 9 exons and 8 introns. The gene was localized to chromosome 15q21.1 by in situ hybridization. Two transcription initiation sites were detected. Three Sp1 sites and a repetitive sequence (CAAA)5 were observed in the 5' noncoding region; no classical TATAA or CCAAT elements were found. The related alcohol dehydrogenases and zeta-crystallin have the same gene organization split by 8 introns, but no splice points coincide between SORD and these gene types. The deduced amino acid sequence of the SORD structure differs at a few positions from the directly determined protein sequence, suggesting allelic forms of the enzyme. High levels of SORD transcripts were observed in lens and kidney, as judged from Northern blot analysis.

Glucitol induction in Bacillus subtilis is mediated by a regulatory factor, GutR.

Expression of the glucitol dehydrogenase gene (gutB) is suggested to be regulated both positively and negatively in Bacillus subtilis. A mutation in the gutR locus results in the constitutive expression of gutB. The exact nature of this mutation and the function of gutR are still unknown. Cloning and characterization of gutR indicated that this gene is located immediately upstream of gutB and is transcribed in the opposite direction relative to gutB. GutR is suggested to be a 95-kDa protein with a putative helix-turn-helix motif and a nucleotide binding domain at the N-terminal region. At the C-terminal region, a short sequence of GutR shows homology with two proteins, Cyc8 (glucose repression mediator protein) and GsiA (glucose starvation-inducible protein), known to be directly or indirectly involved in catabolite repression. Part of the C-terminal conserved sequence from these proteins shows all the features observed in the tetratricopeptide motif found in many eucaryotic proteins. To study the functional role of gutR, chromosomal gutR was insertionally inactivated. A total loss of glucitol inducibility was observed. Reintroduction of a functional gutR to the GutR-deficient strain through integration at the amyE locus restores the inducibility. Therefore, GutR serves as a regulatory factor to modulate glucitol induction. The nature of the gutR1 mutation was also determined. A single amino acid change (serine-289 to arginine-289) near the putative nucleotide binding motif B in GutR is responsible for the observed phenotype. Possible models for the action of GutR are discussed.

Cloning and sequence determination of the gene encoding sorbitol dehydrogenase from Saccharomyces cerevisiae.

The identification of a sorbitol-induced sorbitol dehydrogenase (SDH) activity from Saccharomyces cerevisiae is described. The SDH1 structural gene was isolated from a lambda gt11 yeast genomic library using an antibody to a 40-kDa protein induced in yeast cells growing in medium containing sorbitol. The gene encodes a 357-amino-acid (aa) protein deduced from the nucleotide sequence. Comparison of the aa sequence of the yeast SDH1 with that of sheep liver SDH reveals a 63% overall similarity. Yeast transformants containing the cloned gene carried on a multicopy plasmid express high levels of SDH1 only when grown on sorbitol, suggesting that the cloned gene contains both regulatory and coding sequences.

Biosynthesis of NAD-sorbitol dehydrogenase is induced by acclimation at 5 degrees C in diapause eggs of the silkworm, Bombyx mori.

1. In diapausing eggs of the silkworm, Bombyx mori, activity of NAD-sorbitol dehydrogenase (EC 1.1.1.14, SDH) is almost negligible, but is increased by acclimation at 5 degrees C (Yaginuma et al., 1990, J. comp. Physiol. B160, 277-285). To elucidate the mechanism regulating SDH activity, the following experiments were conducted. Anti-SDH serum was made in a mouse using purified sheep liver SDH. 2. This antiserum reacted with Bombyx egg SDH purified partially by Blue Sepharose CL-6B and Sephacryl S-300 column chromatographies. 3. SDS-PAGE and immunoblotting analyses using the antiserum showed that SDH activity was correlated with the amount of the enzyme protein. 4. These results indicate that biosynthesis of SDH is induced by acclimation at 5 degrees C in diapause eggs of B. mori.

Renal sorbitol accumulation and associated enzyme activities in diabetes.

Streptozotocin-induced diabetes increased sorbitol levels in the rat renal medulla. The activities of renal medullary aldose reductase and sorbitol dehydrogenase, responsible for the formation and metabolism of sorbitol, favor sorbitol formation and did not change in diabetes. The elevated sorbitol concentration appears to be due to an increase in medullary glucose concentration.

Concentration-related changes in blood and tissue parameters of hepatotoxicity and their interdependence in rats exposed to bromobenzene and 1,2-dichlorobenzene.

Liver damage resulting from 4 h exposure to bromobenzene (BB) (146-957 ppm) and 1,2-dichlorobenzene (DCB) (245-739 ppm) as model toxicants was evaluated in rats. The modifications considered were the increases in serum glutamate dehydrogenase (GLDH) and sorbitol dehydrogenase (SDH) activities and the decreases in centrolobular liver-cell glucose-6-phosphatase (G6-Pase) staining intensity. A linear inverse relationship was established between the logarithmic values of blood enzyme activities and liver G6-Pase staining intensity. In addition, the levels of exposure to each test chemical were found to be linearly related to liver G6-Pase staining intensity and to the logarithmic values of blood enzyme activities.

Catabolite repression of enzyme synthesis does not prevent sporulation.

In the presence of excess glucose, a decrease of guanine nucleotides in Bacillus subtilis initiated sporulation but did not prevent catabolite repression of three enzymes. Therefore, the ultimate mechanism(s) repressing enzyme synthesis differs from that suppressing sporulation.