Molecular basis for thermoprotection in Bemisia: structural differences between whitefly ketose reductase and other medium-chain dehydrogenases/reductases

The silverleaf whitefly (Bemisia argentifolii, Bellows and Perring) accumulates sorbitol as a thermoprotectant in response to elevated temperature. Sorbitol synthesis in this insect is catalyzed by an unconventional ketose reductase (KR) that uses NADPH to reduce fructose. A cDNA encoding the NADPH-KR from adult B. argentifolii was cloned and sequenced to determine the primary structure of this enzyme. The cDNA encoded a protein of 352 amino acids with a calculated molecular mass of 38.2 kDa. The deduced amino acid sequence of the cDNA shared 60% identity with sheep NAD(+)-dependent sorbitol dehydrogenase (SDH). Residues in SDH involved in substrate binding were conserved in the whitefly NADPH-KR. An important structural difference between the whitefly NADPH-KR and NAD(+)-SDHs occurred in the nucleotide-binding site. The Asp residue that coordinates the adenosyl ribose hydroxyls in NAD(+)-dependent dehydrogenases (including NAD(+)-SDH), was replaced by an Ala in the whitefly NADPH-KR. The whitefly NADPH-KR also contained two neutral to Arg substitutions within four residues of the Asp to Ala substitution. Molecular modeling indicated that addition of the Arg residues and loss of the Asp decreased the electric potential of the adenosine ribose-binding pocket, creating an environment favorable for NADPH-binding. Because of the ability to use NADPH, the whitefly NADPH-KR synthesizes sorbitol under physiological conditions, unlike NAD(+)-SDHs, which function in sorbitol catabolism.     

A thermoprotective role for sorbitol in the silverleaf whitefly,Bemisia argentifolii

Accumulation of polyols in insects is well known as a cold-hardening response related to overwintering or to protection against cold shock. The silverleaf whitefly (Bemisia argentifolii, Bellows and Perring) is a major insect pest in tropical and subtropical regions where heat stress and desiccation pose formidable threats to survival. We found that sorbitol levels increased ten-fold when whiteflies were exposed to elevated temperatures. Sorbitol levels rose from 0.16nmolwhitefly(-1) at 25 degrees C to 1.59nmolwhitefly(-1) at 42 degrees C. Sorbitol levels fluctuated diurnally under glasshouse and field conditions increasing ten-fold from morning to early afternoon. Feeding experiments on artificial diets showed that both temperature and dietary sucrose concentration were key factors influencing sorbitol accumulation. Cell free extracts prepared from adult whiteflies catalyzed NADPH-dependent fructose reduction, but were unable to reduce glucose with either NADPH or NADH. Radiotracer experiments with labeled glucose and fructose showed that fructose was the immediate precursor of sorbitol. Thus, sorbitol synthesis in the whitefly is apparently unconventional, involving conversion of fructose by a novel NADPH-dependent ketose reductase. We propose that sorbitol accumulation is a mechanism for thermoprotection and osmoregulation in the silverleaf whitefly, allowing the insect to thrive in environments conducive to thermal and osmotic stress.     

Distinct sucrose isomerases catalyze trehalulose synthesis in whiteflies,Bemisia argentifolii,and Erwinia rhapontici

Isomaltulose [alpha-D-glucopyranosyl-(1,6)-D-fructofuranose] and trehalulose [alpha-D-glucopyranosyl-(1,1)-D-fructofuranose] are commercially valuable sucrose-substitutes that are produced in several microorganisms by the palI gene product, a sucrose isomerase. Trehalulose also occurs in the silverleaf whitefly, Bemisia argentifoli, as the major carbohydrate in the insect's honeydew. To determine if the enzyme that synthesizes trehalulose in whiteflies was similar to the well-characterized sucrose isomerase from microbial sources, the properties of the enzymes from whiteflies and the bacterium, Erwinia rhapontici, were compared. Partial purification of both enzymes showed that the enzyme from whiteflies was a 116 kD membrane-associated polypeptide, in contrast to the enzyme from E. rhapontici, which was soluble and 66 kD. The enzyme from E. rhapontici converted sucrose to isomaltulose and trehalulose in a 5:1 ratio, whereas the enzyme from whiteflies produced only trehalulose. Unlike the E. rhapontici enzyme, the whitefly enzyme did not convert isomaltulose to trehalulose, but both enzymes catalyzed the transfer of fructose to trehalulose using sucrose as the glucosyl donor. The results indicate that trehalulose synthase from whiteflies is structurally and functionally distinct from the sucrose isomerases described in bacteria. The whitefly enzyme is the first reported case of an enzyme that converts sucrose to exclusively trehalulose.     

Detection and characterization of sorbitol dehydrogenase from apple callus tissue

Sorbitol dehydrogenase (l-iditol:NAD(+) oxidoreductase, EC 1.1.1.14) has been detected and characterized from apple (Malus domestica cv. Granny Smith) mesocarp tissue cultures. The enzyme oxidized sorbitol, xylitol, l-arabitol, ribitol, and l-threitol in the presence of NAD. NADP could not replace NAD. Mannitol was slightly oxidized (8% of sorbitol). Other polyols that did not serve as substrate were galactitol, myo-inositol, d-arabitol, erythritol, and glycerol. The dehydrogenase oxidized NADH in the presence of d-fructose or l-sorbose. No detectable activity was observed with d-tagatose. NADPH could partially substitute for NADH.Maximum rate of NAD reduction in the presence of sorbitol occurred in tris(hydroxymethyl)aminomethane-HCl buffer (pH 9), or in 2-amino-2-methyl-1,3-propanediol buffer (pH 9.5). Maximum rates of NADH oxidation in the presence of fructose were observed between pH 5.7 and 7.0 with phosphate buffer. Reaction rates increased with increasing temperature up to 60 C. The K(m) for sorbitol and xylitol oxidation were 86 millimolar and 37 millimolar, respectively. The K(m) for fructose reduction was 1.5 molar.Sorbitol oxidation was completely inhibited by heavy metal ions, iodoacetate, p-chloromercuribenzoate, and cysteine. ZnSO(4) (0.25 millimolar) reversed the cysteine inhibition. It is suggested that apple sorbitol dehydrogenase contains sulfhydryl groups and requires a metal ion for full activity.     

Presence of trehalulose and other oligosaccharides in hemipteran honeydew, particularly Aleyrodidae

Abstract. Production by a bacterium of the disaccharide trehalulose was reported 30 years ago. The association between the fabrication of trehalulose and Insecta was found more recently. It was initially discovered in the honeydew, excreta, of the whitefly Bemisia tabaci feeding on Euphorbia pulcherrima where it was the predominant sugar. In the present study, B. tabaci -produced trehalulose was again found in honeydew at significant levels, this time when the whitefly fed on 10 different plants. In seven of 10 Bemisia /host combinations, trehalulose accounted for more than 30% of the total carbohydrates found in their honeydew and was the principal oligosaccharide. Trehalulose constituents, glucose and fructose, were also present at lower levels, as were other oligosaccharides such as melezitose. Feeding by B. tabaci on the three other plant hosts also resulted in the production of trehalulose at relatively high levels (6.1–16.5%). Other whitefly species examined had little (e.g. B. afer and Aleurothrixus spp.), or no trehalulose (e.g. Trialeurodes spp. and Siphoninus phillyreae ), in their honeydew. Trehalulose was also found in the honeydew of two aphid and one scale insect species. In insects whose honeydew had low levels or no trehalulose, sucrose, its constituents, or larger sugars predominated. The trisaccharide bemisiose was also found in the honeydew of half the whitefly species examined. Bemisiose was discovered here for the first time in the honeydew of three aphid species and three species of scale insects. Reasons for the production of trehalulose are often linked to high levels of dietary sucrose in whiteflies. This is probably true in our case because cotton and cucurbits, at least, are known to contain a great deal of sucrose. Although other functional possibilities were explored, the data suggest that trehalulose, being less susceptible to hydrolysis than sucrose, is involved in osmoregulation.     

Steady-state kinetic properties of sorbitol dehydrogenase from chicken liver

The steady-state kinetic properties of partially purified chicken liver sorbitol dehydrogenase (SDH) were determined spectrophotometrically at 25 degrees C, in 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS) buffer, pH 8.0. In the sorbitol-to-fructose direction, analysis was based on initial rate data obtained at [NAD(+)](o)=0.1-0.4 mM and [sorbitol](o)=1.25-10 mM. The reverse process was analyzed by recording progress curves for NADH consumption, starting with [NADH](o)=0.2 mM and [fructose](o)=66.7-267 mM. The kinetics conformed to an ordered sequential model, with the cofactors adding first. The steady-state parameters in the forward direction, K(NAD(+)), K(iNAD(+)) and K(sorbitol), were found to be 210+/-62 muM, 220+/-69 microM and 3.2+/-0.54 mM, respectively. The corresponding parameters in the reverse direction were K(NADH)=240+/-58 microM, K(iNADH)=10+/-2.8 microM and K(fructose)=1000+/-140 mM. The results indicated a close parallelism with human SDH, yet up to 40-fold differences were observed when compared to related reports on other mammalian species. The structural and adaptive bases of the variation in substrate and cofactor affinities need to be accounted for.     

Effect of the alpha-glucosidase inhibitor, bromoconduritol, on carbohydrate metabolism in the silverleaf whitefly, Bemisia argentifolii

The involvement of alpha-glucosidase in the partitioning of ingested sucrose between excretion and incorporation was investigated in the silverleaf whitefly (Bemisia argentifolii). Approximately half of the alpha-glucosidase activity in adult whiteflies was soluble and the remainder was associated with membranes. In contrast, almost all of the trehalulose synthase was membrane-associated. Isoelectric focusing revealed that soluble and membrane-associated alpha-glucosidases were each composed of several isozymes in the pH 5 to 6.5 range, but the distribution of activity among the various isozymes was different. Bromoconduritol, an inhibitor of glucosidases, inhibited trehalulose synthase and alpha-glucosidase activities in whitefly extracts. Inhibition was greatest when bromoconduritol was incubated with extracts prior to the addition of sucrose, consistent with the irreversible nature of this inhibitor. Addition of bromoconduritol to artificial diets decreased the extractable trehalulose synthase and alpha-glucosidase activities by about 30 and 50%, respectively. Ingestion of bromoconduritol reduced the amount of carbohydrate excreted by about 80% without changing the distribution of the major honeydew sugars or causing an increase in the proportion of sucrose that was excreted. Ingestion of bromoconduritol did not affect respiration, the content and distribution of soluble carbohydrates in whitefly bodies, or the conversion of labeled sucrose into glucose, trehalose and isobemisiose. The results indicate that partitioning of ingested carbon between excretion and metabolism in whiteflies is highly regulated, probably involving multiple forms of alpha-glucosidase that facilitate a separation of the processes involved in the metabolic utilization of sucrose from those involved in excretion of excess carbohydrate. Arch. Insect Biochem. Physiol. 45:117-128, 2000. Published 2001 Wiley-Liss, Inc.     

Molecular evidence of sorbitol dehydrogenase in tomato, a non-Rosaceae plant

The enzyme NAD-dependent sorbitol dehydrogenase (SDH) is well characterized in the Rosaceae family of fruit trees, which synthesizes sorbitol as a translocatable photosynthate. Expressed sequence tags of SDH-like sequences have also been generated from various non-Rosaceae species that do not synthesize sorbitol as a primary photosynthetic product, but the physiological roles of the encoded proteins in non-Rosaceae plants are unknown. Therefore, we isolated an SDH-like cDNA (SDL) from tomato (Lycopersicon esculentum Mill.). Genomic Southern blot analysis suggested that SDL exists in the tomato genome as a single-copy gene. Northern blot analysis showed that SDL is ubiquitously expressed in tomato plants. Recombinant SDL protein was produced and purified for enzymatic characterization. SDL catalyzed the interconversion of sorbitol and fructose with NAD (H). SDL showed highest activity for sorbitol among the several substrates tested. SDL showed no activity with NADP+. Thus, SDL was identified as a SDH, although the Km values and substrate specificity of SDL were significantly different from those of SDH purified from the Japanese pear (Pyrus pyrifolia), a Rosaceae fruit tree. In addition, tomato was transformed with antisense SDL to evaluate the contribution of SDL to SDH activity in tomato. The transformation decreased SDH activity to approximately 50% on average. Taken together, these results provide molecular evidence of SDH in tomato, and SDL was renamed LeSDH.     

Relation between the oxidation state of nicotinamide–adenine dinucleotide and the metabolism of spermatozoa

1. The existing procedures for extraction of oxidized and reduced nicotinamide coenzymes were adapted to spermatozoa to overcome the coenzyme-degrading activity of seminal plasma. 2. The content of total NAD(+) and NADH was determined in the spermatozoa of ram, bull, boar, stallion and cock. NADP(+) and NADPH were not detected in ram spermatozoa. 3. The oxidation state of sperm NAD depended on the seminal plasma, the removal of which produced a change in the percentage oxidation state of the coenzyme, 100x[NAD(+)/(NAD(+)+NADH)], without altering the total content of NAD(+)+NADH. 4. In suspensions of washed ram spermatozoa, incubated anaerobically at 25 degrees C, the percentage oxidation state of NAD declined with increasing spermatozoa concentration. 5. When ram or boar spermatozoa that had been previously washed and resuspended in Ringer phosphate medium, were incubated anaerobically at 25 degrees C with various substances, pronounced effects on the percentage oxidation state of NAD could be observed with l-lactate, pyruvate, oxaloacetate, dihydroxyacetone, formaldehyde and glyceraldehyde; sorbitol and acetoacetate acted only on ram spermatozoa; fructose, glucose, mannose and acetaldehyde acted predominantly on boar spermatozoa. Formaldehyde lowered the (NAD(+)+NADH) content of ram spermatozoa, but none of the other substances had a comparable effect. 6. The percentage oxidation state of sperm NAD was not influenced by exogenous cysteine, cystine, ergothioneine or ascorbate. 7. A highly active sorbitol dehydrogenase could be prepared from ram, but not from boar, spermatozoa. 8. Sorbitol, acetoacetate and 3-hydroxybutyrate effectively supported the respiration of ram, but not boar, spermatozoa. 9. ;Cold shock', resulting from sudden cooling of spermatozoa, abolished motility completely and irreversibly but produced only a slow and partial decrease in the total NAD content. Slight over-heating, sufficient to produce loss of motility, had no adverse effect on the total NAD content. 10. Storage of ram sperm at 14 degrees C produced only a small decrease of NAD after 2 days, but subsequently the loss became greater.     

Carbohydrate availability modifies sorbitol dehydrogenase activity of apple fruit

Complete defoliation of the stem or spur and girdling the phloem (D/G) subtending a single apple ( Malus domestica Borkh.) fruit at 70 or more days after bloom resulted in reduction of fruit growth. Extractable sorbitol dehydrogenase (SDH) (enzyme code, 1.1.1.14) activity and sorbitol and starch content of the cortex tissue of fruit receiving D/G treatment subsequently declined. Co-extraction of control and D/G cortex tissues yielded expected levels of SDH activity, indicating that the loss of extractable SDH activity in D/G fruit was not due to presence of an inhibitor or proteolytic activity. Incubating cortex sections from D/G fruit in a buffered 200 m M sorbitol or glucose solution increased extractable SDH activity, and incubating cortex sections from control fruit in the sorbitol solution maintained the activity. However, neither 200 m M fructose or 27 m M PEG, the latter with the same osmotic potential as the sorbitol solution, affected extractable SDH activity of D/G fruit. The results indicate that carbohydrate availability may affect extractable SDH activity of apple fruit, and that specific carbohydrates such as sorbitol and glucose may be signals for modulating this activity.     

X-ray crystallographic and kinetic studies of human sorbitol dehydrogenase

Sorbitol dehydrogenase (hSDH) and aldose reductase form the polyol pathway that interconverts glucose and fructose. Redox changes from overproduction of the coenzyme NADH by SDH may play a role in diabetes-induced dysfunction in sensitive tissues, making SDH a therapeutic target for diabetic complications. We have purified and determined the crystal structures of human SDH alone, SDH with NAD(+), and SDH with NADH and an inhibitor that is competitive with fructose. hSDH is a tetramer of identical, catalytically active subunits. In the apo and NAD(+) complex, the catalytic zinc is coordinated by His69, Cys44, Glu70, and a water molecule. The inhibitor coordinates the zinc through an oxygen and a nitrogen atom with the concomitant dissociation of Glu70. The inhibitor forms hydrophobic interactions to NADH and likely sterically occludes substrate binding. The structure of the inhibitor complex provides a framework for developing more potent inhibitors of hSDH.     

Zymomonas mobilis mutants blocked in fructose utilization

Summary A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l^-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.     

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. Mol. Reprod. Dev. 79: 603–612, 2012. © 2012 Wiley Periodicals, Inc.     

Study of the polyol pathway in the porcine epididymis

Concentrations of D-glucose, D-fructose and D-sorbitol were quantified in porcine epididymal fluid by spectrofluorimetric assays and aldose reductase (AR) and sorbitol dehydrogenase (SDH) were located immunohistochemically in the epididymal epithelium. Glucose and fructose concentrations were low (<1 mM) and decreased in the cauda whereas sorbitol concentration (4-7 mM) was rather uniform along the duct. AR was luminally located on microvilli in the caput and corpus with less presence distally and was present in the lumen. SDH was present apically and basally in epithelial cells throughout the epididymis and in the lumen. The observations are consistent with diffusion of circulating glucose into the lumen, its conversion via AR to sorbitol which accumulates in the lumen and the action of SDH on sorbitol to produce fructose. Sperm metabolism of glucose and fructose may explain their lower concentrations in the cauda and sorbitol could be a metabolic substrate or osmolyte required for volume regulation.     

A study of the coenzyme-binding characteristics of rabbit muscle l-glycerol 3-phosphate dehydrogenase

1. The binding of NAD(+) and NADH to glycerol 3-phosphate dehydrogenase was studied in the pH range 6.0-9.0 at 25 degrees C and in the temperature range 16-43 degrees C at pH7.0. 2. The second-order velocity constants for the combination of NADH with the enzyme in the pH range 6.0-9.0 and for the combination of NAD(+) with the enzyme at pH6.0 were determined. 3. The velocity constant for the dissociation of the enzyme-NAD(+) complex at pH6.0 was measured.     

Temperature and the regulation of enzyme activity in poikilotherms. Regulatory properties of fructose diphosphatase from muscle of the Alaskan king-crab

1. The properties of fructose diphosphatase from skeletal muscle of the Alaskan king-crab (Paralithodes camtschatica) were examined over the physiological temperature range of the animal. 2. King-crab muscle fructose diphosphatase is first activated by Na(+) and NH(4) (+) and is then partially inhibited by these cations at concentrations higher than 10mm at 0 degrees , 8 degrees and 15 degrees C. Enzyme activity is stimulated by K(+) at 0 degrees C, but is curtailed at 8 degrees C and 15 degrees C, an effect that could render rate independent of temperature. 3. Affinity for substrate increases with decreasing temperature; below the temperature of acclimatization, K(m) for fructose 1,6-diphosphate increases, resulting in a complex U-shaped temperature-K(m) curve. 4. King-crab muscle fructose diphosphatase is inhibited by low concentrations of AMP. As with enzymes of other poikilotherms, inhibition by AMP is sensitive to temperature; the enzyme is least sensitive to inhibition by AMP near the temperature of acclimatization. 5. The affinity of fructose diphosphatase for fructose 1,6-diphosphate is enhanced by phosphoenolpyruvate, and this activation is temperature-sensitive; 0.5mm-phosphoenolpyruvate causes a sevenfold decrease in K(m) for fructose 1,6-diphosphate at 15 degrees C but a 25-fold decrease at 0 degrees C. 6. Phosphoenolpyruvate appears to decrease the affinity of king-crab muscle fructose diphosphatase for AMP at low temperature, whereas at the higher temperature it appears to enhance inhibition by AMP. Phosphoenolpyruvate was not observed to cause a reversal of inhibition of fructose diphosphatase activity by AMP. The identification of phosphoenolpyruvate as an activator of a rate-limiting step in gluconeogenesis permits the suggestion of a coupling of the controlling mechanisms of several steps in the glycolytic and gluconeogenic chains. 7. These findings suggest mechanisms for the maintenance and regulation of control of fructose diphosphatase activity in king-crab skeletal muscle at low temperature and under conditions that favour concomitant activity of phosphofructokinase.     

Complete protection by alpha-crystallin of lens sorbitol dehydrogenase undergoing thermal stress

Sorbitol dehydrogenase (l-iditol:NAD(+) 2-oxidoreductase, E.C. 1.1.1. 14) (SDH) was significantly protected from thermally induced inactivation and aggregation by bovine lens alpha-crystallin. An alpha-crystallin/SDH ratio as low as 1:2 in weight was sufficient to preserve the transparency of the enzyme solution kept for at least 2 h at 55 degrees C. Moreover, an alpha-crystallin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55 degrees C for at least 40 min. The protection by alpha-crystallin of SDH activity was essentially unaffected by high ionic strength (i.e. 0.5 m NaCl). On the other hand, the transparency of the protein solution was lost at a high salt concentration because of the precipitation of the alpha-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by alpha-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of alpha-crystallin against the inactivation of SDH induced at 55 degrees C by thiol blocking agents or EDTA together with the additive effect of NADH in stabilizing the enzyme in the presence of alpha-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with alpha-crystallin. Two different adducts between alpha-crystallin and SDH were isolated by gel filtration chromatography. One adduct was characterized by a high M(r) of approximately 800,000 and carried exclusively inactive SDH. A second adduct, carrying active SDH, had a size consistent with an interaction of the enzyme with monomers or low M(r) aggregates of alpha-crystallin. Even though it had a reduced efficiency with respect to alpha-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of alpha-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of alpha-crystallin may not be a necessary requirement for the stabilization of the enzyme activity.     

Diabetic neuropathies. Metabolism of sorbitol in sciatic nerve tissue in streptozotocin diabetes

The increase of sorbitol and fructose levels caused by aldose reductase activation and sorbitol dehydrogenase inhibition were observed in sciatic nerve of streptozotocin-diabetic rats. Elevated polyol pathway activity has been implicated in the development of diabetic complications such as neuropathy. The regulation of polyol pathway enzymes is based on the changes of redox state of free nicotinamide nucleotides. The decrease of the NADP+/NADPH ratio in cytosolic compartment of sciatic nerve cells activated aldose reductase and the decrease of the NAD+/NADH ratio inhibited sorbitol dehydrogenase. Nicotinamide as a precursor of NAD+ biosynthesis increased the free NADP+/NADPH and NAD+/NADH ratios and inhibited the activity of polyol pathway. The sorbitol level decreased in sciatic nerve of nicotinamide-treated streptozotocin-diabetic rats as compared to non-treated ones. Thus, the data provide evidence for important role of nicotinamide, as an antidiabetic drug, in prevention or correction of diabetic neuropathy.