Rare sugar L-sorbose exerts antitumor activity by impairing glucose metabolism.

Rare sugars are monosaccharides with low natural abundance. They are structural isomers of dietary sugars, but hardly be metabolized. Here, we report that rare sugar L-sorbose induces apoptosis in various cancer cells. As a C-3 epimer of D-fructose, L-sorbose is internalized via the transporter GLUT5 and phosphorylated by ketohexokinase (KHK) to produce L-sorbose-1-phosphate (S-1-P). Cellular S-1-P inactivates the glycolytic enzyme hexokinase resulting in attenuated glycolysis. Consequently, mitochondrial function is impaired and reactive oxygen species are produced. Moreover, L-sorbose downregulates the transcription of KHK-A, a splicing variant of KHK. Since KHK-A is a positive inducer of antioxidation genes, the antioxidant defense mechanism in cancer cells can be attenuated by L-sorbose-treatment. Thus, L-sorbose performs multiple anticancer activities to induce cell apoptosis. In mouse xenograft models, L-sorbose enhances the effect of tumor chemotherapy in combination with other anticancer drugs. These results demonstrate L-sorbose as an attractive therapeutic reagent for cancer treatment.

L-sorbose is not only a substrate for 2-keto-L-gulonic acid production in the artificial microbial ecosystem of two strains mixed fermentation.

The co-culture system of the fermentation process of vitamin C can be regarded as an artificial microbial ecosystem (AME). To extend our understanding of this AME, an investigation of the relationship between strains, substrate and product was carried out in this study. The results showed that both Ketogulonicigenium vulgare and 2-keto-L-gulonic acid (2-KLG, the precursor of vitamin C) can inhibit the growth of the helper strain, while the helper strain promoted the growth of K. vulgare and 2-KLG production. Moreover, L-sorbose is not only a substrate for 2-KLG production in the AME, but also a promoter of K. vulgare and an inhibitor of the helper strain. In the earlier stage of fermentation, the inhibition of L-sorbose on the helper strain's growth is a key factor for ensuring an efficient fermentation. In the condition of adding the extra helper strain (OD: 0.57, ratio of inoculation: 2%), the yields of 2-KLG is increased by 9% in the 14% L-sorbose medium. To the best of our knowledge, this is the first report about the inhibition of substrate in the AME of 2-KLG production.

D-sorbose inhibits disaccharidase activity and demonstrates suppressive action on postprandial blood levels of glucose and insulin in the rat.

In an attempt to develop D-sorbose as a new sweetener that could help in preventing lifestyle-related diseases, we investigated the inhibitory effect of D-sorbose on disaccharidase activity, using the brush border membrane vesicles of rat small intestines. The inhibitory effect was compared with that of L-sorbose and other rare sugars, and the small intestinal disaccharidases in rats was compared with that of humans as well. In humans and the small intestines of rats, d-sorbose strongly inhibited sucrase activity and weakly inhibited maltase activity. Inhibition by D-sorbose of sucrase activity was similar to that of L-arabinose, and the K(i) of D-sorbose was 7.5 mM. Inhibition by D-sorbose was very strong in comparison with that of L-sorbose (K(i), 60.8 mM), whereas inhibition of d-tagatose was between that of D-sorbose and L-sorbose. The inhibitory mode of D-sorbose for sucrose and maltase was uncompetitive, and that of L-sorbose was competitive. To determine a suppressive effect on postprandial blood levels of glucose and insulin via inhibition of sucrase activity, sucrose solution with or without D-sorbose was administered to rats. Increments in the blood levels of glucose and insulin were suppressed significantly after administration of sucrose solution with D-sorbose to rats, in comparison to administration of sucrose solution without D-sorbose. In contrast, the suppressive effect of L-sorbose on postprandial blood levels of glucose and insulin was very weak. These results suggest that D-sorbose may have an inhibitory effect on disaccharidase activity and could be used as a sweetener to suppress the postprandial elevation of blood levels of glucose and insulin. The use of D-sorbose as a sweetener may contribute to the prevention of lifestyle-related diseases, such as type 2 diabetes mellitus.

Dietary D-sorbose decreases serum insulin levels in growing Sprague-Dawley rats.

D-Sorbose is naturally occurring rare sugar. In this study, we examined the effects of dietary D-sorbose in rats. Four-week-old male Sprague-Dawley rats were fed either an AIN-93G-based control diet or a 3% D-sorbose diet for 28 d. Body weight and body fat accumulation were not different between the two diet groups. Dietary supplementation of D-sorbose lowered the serum insulin level (*p<0.05) significantly compared to the control, although the glucose was not changed. In addition, the relative weight of the cecum increased significantly in the D-sorbose group (**p<0.01). These findings suggest that intake of D-sorbose may improve the glucose metabolism by reducing insulin secretion, and D-sorbose can be used as a food ingredient.

The oxygenase CAO-1 of Neurospora crassa is a resveratrol cleavage enzyme.

The genome of the ascomycete Neurospora crassa encodes CAO-1 and CAO-2, two members of the carotenoid cleavage oxygenase family that target double bonds in different substrates. Previous studies demonstrated the role of CAO-2 in cleaving the C40 carotene torulene, a key step in the synthesis of the C35 apocarotenoid pigment neurosporaxanthin. In this work, we investigated the activity of CAO-1, assuming that it may provide retinal, the chromophore of the NOP-1 rhodopsin, by cleaving ß-carotene. For this purpose, we tested CAO-1 activity with carotenoid substrates that were, however, not converted. In contrast and consistent with its sequence similarity to family members that act on stilbenes, CAO-1 cleaved the interphenyl Cα-Cß double bond of resveratrol and its derivative piceatannol. CAO-1 did not convert five other similar stilbenes, indicating a requirement for a minimal number of unmodified hydroxyl groups in the stilbene background. Confirming its biological function in converting stilbenes, adding resveratrol led to a pronounced increase in cao-1 mRNA levels, while light, a key regulator of carotenoid metabolism, did not alter them. Targeted Δcao-1 mutants were not impaired by the presence of resveratrol, a phytoalexin active against different fungi, which did not significantly affect the growth and development of wild-type Neurospora. However, under partial sorbose toxicity, the Δcao-1 colonies exhibited faster radial growth than control strains in the presence of resveratrol, suggesting a moderate toxic effect of resveratrol cleavage products.

A rare sugar, d-allose, confers resistance to rice bacterial blight with upregulation of defense-related genes in Oryza sativa.

We investigated responses of rice plant to three rare sugars, d-altrose, d-sorbose, and d-allose, due to establishment of mass production methods for these rare sugars. Root growth and shoot growth were significantly inhibited by d-allose but not by the other rare sugars. A large-scale gene expression analysis using a rice microarray revealed that d-allose treatment causes a high upregulation of many defense-related, pathogenesis-related (PR) protein genes in rice. The PR protein genes were not upregulated by other rare sugars. Furthermore, d-allose treatment of rice plants conferred limited resistance of the rice against the pathogen Xanthomonas oryzae pv. oryzae but the other tested sugars did not. These results indicate that d-allose has a growth inhibitory effect but might prove to be a candidate elicitor for reducing disease development in rice.

Potential anthelmintic: D-psicose inhibits motility, growth and reproductive maturity of L1 larvae of Caenorhabditis elegans.

No anthelmintic sugars have yet been identified. Eight ketohexose stereoisomers (D- and L-forms of psicose, fructose, tagatose and sorbose), along with D-galactose and D-glucose, were examined for potency against L1 stage Caenorhabditis elegans fed Escherichia coli. Of the sugars, D-psicose specifically inhibited the motility, growth and reproductive maturity of the L1 stage. D-Psicose probably interferes with the nematode nutrition. The present results suggest that D-psicose, one of the rare sugars, is a potential anthelmintic.

Glucose uptake in germinating Aspergillus nidulans conidia: involvement of the creA and sorA genes.

D-Glucose uptake in germinating wild-type Aspergillus nidulans conidia is an energy-requiring process mediated by at least two transport systems of differing affinities for glucose: a low-affinity system (K(m) approximately 1.4 mM) and a high-affinity system (K(m) approximately 16 micro M). The low-affinity system is inducible by glucose; the high-affinity system is subject to glucose repression effected by the carbon catabolite repressor CreA and is absent in sorA3 mutant conidia, which exhibit resistance to L-sorbose toxicity. An intermediate-affinity system (K(m) approximately 400 micro M) is present in sorA3 conidia germinating in derepressing conditions. creA derepressed mutants show enhanced sensitivity to L-sorbose. The high-affinity uptake system appears to be responsible for the uptake of this toxic sugar.

Inhibition of the D-fructose transporter protein GLUT5 by fused-ring glyco-1,3-oxazolidin-2-thiones and -oxazolidin-2-ones.

The glucose transporter 5 (GLUT5)-a specific D-fructose transporter-belongs to a family of facilitating sugar transporters recently enlarged by the human genome sequencing. Prompted by the need to develop specific photolabels of these isoforms, we have studied the interaction of conformationally locked D-fructose and L-sorbose derived 1,3-oxazolidin-2-thiones and 1,3-oxazolidin-2-ones to provide a rational basis for an interaction model. The inhibition properties of the D-fructose transporter GLUT5 by glyco-1,3-oxazolidin-2-thiones and glyco-1,3-oxazolidin-2-ones is now reported. In vitro, the fused-rings systems tested showed an efficient inhibition of GLUT5, thus bringing new insights on the interaction of D-fructose with GLUT5.

L-Sorbose induces cellulase gene transcription in the cellulolytic fungus Trichoderma reesei.

L-Sorbose has previously been assumed to stimulate cellulase formation in an indirect manner, different from that of sophorose in Trichoderma reesei. Through Northern blot analysis however, L-sorbose was found to regulate coordinately six cellulase genes (including eg13, whose behavior has not been studied so far) at transcriptional level, as is the case with sophorose in T. reesei strains PC-3-7 and QM9414. Dot blot analysis showed that the proportions of each cellulase mRNA to cbh1 mRNA, the largest amount of mRNA transcribed in T. reesei, did not change when L-sorbose or sophorose was used as an inducer in the PC-3-7 and QM9414 strains. cbh2 and egl1 mRNAs were about 45-60% and 20-30% of the cbh1 transcript, whereas small amounts of mRNA, 1-2% of cbh1, were observed on other endoglucanase genes. Furthermore, the PC-3-7 strain showed an enhanced level of cellulase gene transcription, about two- and four- to six-fold higher than that of the QM9414 strain with sophorose and L-sorbose, respectively.

Inactivation of Cu,Zn-superoxide dismutase by intermediates of Maillard reaction and glycolytic pathway and some sugars.

Human Cu,Zn-superoxide dismutase (SOD) was incubated with various intermediates of the Maillard reaction and glycolytic pathway (arabinose, glyoxal, glycolaldehyde, glyceraldehyde, glyceraldehyde 3-phosphate, and dihydroxyacetone) and some reducing sugars (sorbose, xylose, and ribose). The change of the activity and the molecular weight were measured and compared with that of SOD incubated with glucose or fructose. Sorbose, xylose, and ribose decreased the activity with a rate comparable to fructose. Site-specific and random fragmentation were observed upon the incubation with them. Arabinose showed a similar inactivation rate as glucose. The intermediates other than arabinose had a high inactivation rate. Especially, glyceraldehyde, glycolaldehyde, and glyoxal most strongly lowered the activity in a concentration-dependent manner and a significant inactivation was recognized even at 1 mM level. SDS-PAGE band patterns indicated that the inactivation by those carbonyl compounds occurred by both crosslinking and site-specific fragmentation of SOD.

Molecular analysis of the phosphoenolpyruvate-dependent L-sorbose: phosphotransferase system from Klebsiella pneumoniae and of its multidomain structure.

We have cloned a 3.4 kb DNA fragment from the chromosome of Klebsiella pneumoniae that codes for a phosphoenolpyruvate-dependent L-sorbose: phosphotransferase system (PTS). The cloned fragment was sequenced and four open reading frames coding for 135 (sorF), 164 (sorB), 266 (sorA) and 274 (sorM) amino acids, respectively, were found. The corresponding proteins could be detected in a T7 overexpression system, which yielded molecular masses of about 14,000 for SorF, 19,000 for SorB, 25,000 for SorA and 27,000 for SorM. SorF and SorB have all the characteristics of soluble and intracellular proteins in accordance with their functions as EIIASor and EIIBSor domains of the L-sorbose PTS. SorA and SorM, by contrast, are strongly hydrophobic, membrane-bound proteins with two to five putative transmembrane helices that alternate with a series of hydrophilic loops. They correspond to domains EIICSor and EIIDSor. The four proteins of the L-sorbose PTS resemble closely (27%-60%) the four subunits of a D-fructose PTS (EIIALev, EIIBLev, EIICLev, and EIIDLev) from Bacillus subtilis and the three subunits of the D-mannose PTS (EIIA,BMan, EIICMan, and EIIDMan) from Escherichia coli K-12. The three systems constitute a new PTS family, and sequence comparisons revealed highly conserved structures for the membrane-bound proteins. A consensus sequence for the membrane proteins was used to postulate a model for their integration into the membrane.

Influence of dietary sorbose on diabetes in nonobese diabetic mice.

The effect of dietary sorbose on diabetes after the incidence of the syndrome in the nonobese diabetic mouse was investigated in the animals from 8 to 14 weeks of age. When sucrose (200 g/kg diet) in a control diet was replaced by sorbose, the body weight and the blood glucose concentration were significantly reduced, but the serum insulin concentration was unchanged. The urinary glucose concentration was the same for both sucrose and sorbose diets. It is suggested that after the incidence of diabetes, dietary sorbose could not improve urinary excretion of glucose, even though sorbose could reduce the blood glucose concentration.

Mechanism of hemolysis of canine erythrocytes induced by L-sorbose.

The cause of species difference in the susceptibility of erythrocytes to L-sorbose, and the difference in the hemolytic effect of sorbose on high potassium-containing (HK) and low potassium-containing (LK) canine erythrocytes were examined. L-Sorbose was phosphorylated in canine erythrocytes, but not in human erythrocytes. Furthermore, sorbose-1-phosphate, a metabolite of L-sorbose, strongly inhibited the hexokinase of LK canine erythrocytes, but not that of HK canine erythrocytes. These results strongly indicated that inhibition of hexokinase by sorbose-1-phosphate in LK erythrocytes induced severe glycolytic limitation in these cells, resulting in hemolysis, and that HK erythrocytes are resistant to sorbose-induced hemolysis because these cells have a high hexokinase activity.

Reduced plasma cholesterol and lipoprotein in laying hens without concomitant reduction of egg cholesterol in response to dietary sorbose.

Experiments were conducted to determine the effect of sorbose on feed consumption, egg production and size, and cholesterol metabolism of laying hens. In Experiment 1, 87-wk-old laying hens (10 per treatment) were fed diets containing 0, 10, or 20% sorbose for 4 wk. In a second experiment, 108-wk-old laying hens (eight per treatment) were fed a control diet, a diet with 10% added sorbose, or the control diet with intake restricted to the level of sorbose-treated hens for 4 wk. Feed consumption and egg production were recorded daily. Plasma and egg cholesterol levels were determined at 0, 2, and 4 wk. Plasma and egg very low density lipoprotein (VLDL) concentrations were determined after 4 wk. Egg production, feed intake, and body weight gain were significantly reduced by dietary sorbose. Egg and yolk weight and percentage yolk decreased in response to sorbose. Sorbose significantly reduced plasma cholesterol and VLDL by approximately 50%, compared with the hens fed a control diet. Egg cholesterol concentration (milligrams per gram of yolk) was significantly increased, although the reduction in yolk size resulted in similar total egg cholesterol (milligrams per egg). Restricting feed intake of laying hens significantly lowered plasma cholesterol, but not to levels comparable to that of sorbose-treated hens. The data indicate that substantial reduction of plasma cholesterol and VLDL by dietary sorbose was not accompanied by reduced egg cholesterol.

Dietary sorbose prevents and improves hyperglycemia in genetically diabetic mice.

The effect of dietary sorbose on the prevention (Experiment 1) and amelioration (Experiment 2) of diabetes was investigated in the genetically diabetic mouse [C57BL/KsJ (db/db)] for 6 wk. When sucrose (200 g/kg diet) in a control diet was replaced by sorbose, the blood glucose concentration was dramatically lower, but the serum insulin concentrations did not differ. When mice were fed the diets before the onset of diabetic symptoms, glucose excretion in urine was prevented in the mice fed the sorbose diet, but mice fed the control diet excreted glucose in the urine, and the concentration increased with age. When dietary treatment began after the development of diabetic symptoms, dietary sorbose greatly reduced the incidence of hyperglycemia and lowered urinary glucose excretion, compared with mice fed the sucrose-containing diet. These results suggest that dietary sorbose might be useful in patients with, or at risk of developing, noninsulin-dependent diabetes, both before and after exhibiting the syndrome.

L-sorbose does not cause hemolysis in dog erythrocytes with inherited high Na, K-ATPase activity.

1. The hemolytic effect of L-sorbose on canine erythrocytes characterized by inherited high Na, K-ATPase activity and a high potassium concentration (HK RBCs) was compared with that on normal canine erythrocytes (LK RBCs). 2. Dogs having HK RBCs (HK dogs) revealed no clinical and hematological changes after administration of L-sorbose, whereas normal dogs (LK dogs) developed severe hemolytic anemia associated with hemoglobinuria and marked decreases of erythrocyte ATP concentrations. 3. In vitro, L-sorbose induced hemolysis in LK RBCs along with the depression of both ATP and lactate formation in these cells, but not in HK RBCs. The inhibition of glycolysis by L-sorbose in LK RBCs, however, was not observed when glucose-6-phosphate was used as a substrate instead of glucose. 4. These results suggest that the disparity of susceptibility to sorbose-induced hemolysis may be due to the difference in erythrocyte metabolism between HK and LK RBCs, especially the high activity of hexokinase in HK cells, which was 2-fold greater than that in LK RBCs.

Influence of dietary sorbose on lipogenesis in gold thioglucose-injected obese mice.

1. The influence of dietary sorbose on food intake and fatty acid synthesis of the liver and epididymal white adipose tissue (EWAT) was investigated in gold thioglucose (GTG)-injected obese mice from 12 to 14 weeks of age. 2. Sorbose was supplemented to a semi-purified diet at a level of 200 g/kg diet at the expense of sucrose. 3. On the last day of the experiment, fatty acids synthesis in the liver and EWAT was measured using an i.p. injection [1-14C]sodium acetate. 4. The decreases in body weight and food intake by dietary sorbose in GTG-injected obese mice were greater than those in control mice. 5. Lipid content and fatty acid synthesis in the liver and EWAT of control mice were not influenced by dietary sorbose. 6. In GTG-injected obese mice, the reduction of food intake by dietary sorbose suppressed fatty acid synthesis and lipid deposition in both liver and EWAT.

Effect of dietary sorbose on lipid metabolism in male and female broilers.

Male and female broilers were given diets (6 males and 6 females per diet) containing varying percentages of sorbose (0, 3, 6, and 9%) and fed for ad libitum access from 28 to 56 days of age. Body weight gain and feed intake were decreased with increasing dietary sorbose, particularly in male birds fed diets containing 9% sorbose, although feed efficiency and N retention rate were not influenced by dietary treatments. Absolute and relative abdominal fat weights were higher in females than in males and decreased with the increasing levels of dietary sorbose in both sexes. Fat content in the pectoral muscle also decreased as dietary sorbose increased. Dietary sorbose did not have significant effects on serum glucose, triglyceride, total cholesterol, low density lipoprotein, very low density lipoprotein, and chylomicron levels in either male or female birds. The ME values of diets decreased as dietary sorbose increased. Palmitic acid content of abdominal fat was significantly lower in birds fed the 9% sorbose diet than in birds fed the control diet. The reverse was true for linoleic acid content. It was concluded that dietary sorbose can be used as a potential regulator of lipid deposition in broilers.

In vitro inhibition of stable 1,3-beta-D-glucan synthase activity from Neurospora crassa.

Glucan synthase activity of Neurospora crassa was isolated by treatment of protoplast lysates with 0.1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate and 0.5% octylglucoside in 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer, pH 7.4, containing 5 mM EDTA, 1 mM phenylmethylsulfonylfluoride, 200 mM inorganic phosphate, 10 microM GTP, 1 mM DTT, 10 mM sodium fluoride, and 600 mM glycerol. Resulting activity was partially purified by sucrose gradient density sedimentation. Approximately 70% of enzyme activity in the sucrose gradient peak fraction was soluble and enzyme activity was purified 7.3-fold. Partially purified enzyme activity had a half-life of several weeks at 4 degrees C, and a Km(app) of 1.66 +/- 0.28 mM. Inhibitors (Cilofungin, papulacandin B, aculeacin A, echinocandin B, sorbose and UDP) of 1,3-beta-D-glucan synthase activity were tested against crude particulate and detergent treated enzyme fractions and the Ki(app) of each inhibitor determined. It seems likely that this stable preparation of glucan synthase activity may be useful for in vitro enzyme screens for new glucan synthase inhibitors.