The effect of orally administered glycogen on anti-tumor activity and natural killer cell activity in mice.

Natural killer (NK) cells, innate immune effectors that mediate rapid responses to various antigens, play an important role in potentiating host defenses through the clearance of tumor cells and virally infected cells. By using enzymatically synthesized glycogen (ESG) with the same characteristics as natural glycogen, we examined whether orally administered glycogen enhances the innate defense of tumor-implanted mice and the cytotoxicity of NK cells. Oral administration of ESG led to the suppression of tumor proliferation and the prolongation of survival times of tumor-bearing mice. Splenic NK activities of BALB/c mice treated orally with ESG were significantly higher than those of water-treated mice, which were used as a negative control. In addition, intraduodenal injections of ESG gradually and markedly lowered splenic sympathetic nerve activity, which has an inverse correlation with NK activity. Furthermore, ESG activated Peyer's patch cells to induce the production of macrophage inflammatory protein-2 (MIP-2), interleukin-6 (IL-6), and immunoglobulin A (IgA) from these cells. These results demonstrated that orally administrated glycogen significantly enhanced the cytotoxicity of NK cells by acting on Peyer's patch cells and autonomic nerves, and eventually induced the potentiation of host defenses. We propose that glycogen functions not only as an energy source for life support but also as an oral adjuvant for immunopotentiation.

Safety evaluation of an enzymatically-synthesized glycogen (ESG).

An enzymatically-synthesized glycogen (ESG), intended for use as a food ingredient, was investigated for potential toxicity. ESG is synthesized in vitro from short-chain amylose by the co-operative action of branching enzyme and amylomaltase. In an acute toxicity study, oral administration of ESG to Sprague-Dawley rats at a dose of 2000 mg/kg body weight did not result in any signs of toxicity. ESG did not exhibit mutagenic activity in an in vitro bacterial reverse mutation assay. In a subchronic toxicity study, increased cecal weights noted in the mid- (10%) and high-dose (30%) animals are common findings in rodents fed excess amounts of carbohydrates that increase osmotic value of the cecal contents, and thus were considered a physiological rather than toxicological response. The hematological and histopathological effects observed in the high-dose groups were of no toxicological concern as they were secondary to the physiological responses resulting from the high carbohydrate levels in the test diets. The no-observed-adverse-effect level for ESG in rats was therefore established to be 30% in the diet (equivalent to approximately 18 and 21 g/kg body weight/day for male and female rats, respectively). These results support the safety of ESG as a food ingredient for human consumption.

Structure and solution properties of enzymatically synthesized glycogen.

Recently, a new enzymatic process for glycogen production was developed. In this process, short-chain amylose is used as a substrate for branching enzymes (BE, EC 2.4.1.18). The molecular weight of the enzymatically synthesized glycogen (ESG) depends on the size and concentration of the substrate. Structural and physicochemical properties of ESG were compared to those of natural source glycogen (NSG). The average chain length, interior chain length, and exterior chain length of ESG were 8.2-11.6, 2.0-3.3, and 4.2-7.6, respectively. These values were within the range of variation of NSG. The appearances of both ESG and NSG in solution were opalescent (milky white and slightly bluish). Furthermore, transmission electron microscopy and atomic force microscopy showed that ESG molecules formed spherical particles, and that there were no differences between ESG and NSG. Viscometric analyses also showed the spherical nature of both glycogens. When ESG and NSG were treated with pullulanase, a glucan-hydrolyzing enzyme known to degrade glycogen only on its surface portion, both glycogens were similarly degraded. These analyses revealed that ESG shares similar molecular shapes and surface properties with NSG.

Safety evaluation of amylomaltase from Thermus aquaticus.

A recombinant amylomaltase, MQ-01, obtained by cultivation of Bacillus subtilis expressing the amylomaltase gene from Thermus aquaticus is to be used in the production of enzymatically-synthesized glycogen; which is intended for use as a food ingredient. In order to establish the safety of MQ-01, the enzyme was subjected to standard toxicological testing. In a battery of standard Salmonella typhimurium strains (TA98, TA100, TA1535, and TA1537) and in Escherichia coli WP2 uvrA, both with and without metabolic activation, MQ-01 failed to exhibit mutagenic activity. Similarly, MQ-01 did not display clastogenic properties in Chinese hamster lung fibroblast cells (CHL/IU), in an in vitro chromosomal aberration assay. In a 13-week subchronic toxicity study in rats, oral administration of MQ-01 at doses of up to 15 mL/kg body weight/day (corresponding to approximately 1230 mg/kg body weight/day) did not produce compound-related clinical signs or toxicity, changes in body weight gain, food consumption, hematology, clinical chemistry, urinalysis, organ weights, or in any gross and microscopic findings. The results of this study support the safety of MQ-01 in food production.

Fine structural properties of natural and synthetic glycogens.

Glycogen, highly branched (1-->4)(1-->6)-linked alpha-d-glucan, can be extracted from natural sources such as animal tissues or shellfish (natural source glycogen, NSG). Glycogen can also be synthesized in vitro from glucose-1-phosphate using the cooperative action of alpha-glucan phosphorylase (GP, EC 2.4.1.1) and branching enzyme (BE, EC 2.4.1.18), or from short-chain amylose by the cooperative action of BE and amylomaltase (AM, EC 2.4.1.25). It has been shown that enzymatically synthesized glycogen (ESG) has structural and physicochemical properties similar to those of NSG. In this study, the fine structures of ESG and NSG were analyzed using isoamylase and alpha-amylase. Isoamylase completely hydrolyzed the alpha-1,6 linkages of ESG and NSG. The unit-chain distribution (distribution of degrees of polymerization (DP) of alpha-1,4 linked chains) of ESG was slightly narrower than that of NSG. alpha-Amylase treatment revealed that initial profiles of hydrolyses of ESG and NSG were almost the same: both glycogens were digested slowly, compared with starch. The final products from NSG by alpha-amylase hydrolysis were glucose, maltose, maltotriose, branched oligosaccharides with DP4, and highly branched macrodextrin molecules with molecular weights of up to 10,000. When ESG was digested with excess amounts of alpha-amylase, much larger macrodextrins (molecular weight>10(6)) were detected. In contrast, oligosaccharides with DP 4-7 could not be detected from ESG. These results suggest that the alpha-1,6 linkages in ESG molecules are more regularly distributed than those in NSG molecules.

Molecular basis for specificities of reactivating factors for adenosylcobalamin-dependent diol and glycerol dehydratases.

Adenosylcobalamin-dependent diol and glycerol dehydratases are isofunctional enzymes and undergo mechanism-based inactivation by a physiological substrate glycerol during catalysis. Inactivated holoenzymes are reactivated by their own reactivating factors that mediate the ATP-dependent exchange of an enzyme-bound, damaged cofactor for free adenosylcobalamin through intermediary formation of apoenzyme. The reactivation takes place in two steps: (a) ADP-dependent cobalamin release and (b) ATP-dependent dissociation of the resulting apoenzyme-reactivating factor complexes. The in vitro experiments with purified proteins indicated that diol dehydratase-reactivating factor (DDR) cross-reactivates the inactivated glycerol dehydratase, whereas glycerol dehydratase-reactivating factor (GDR) did not cross-reactivate the inactivated diol dehydratase. We investigated the molecular basis of their specificities in vitro by using purified preparations of cognate and noncognate enzymes and reactivating factors. DDR mediated the exchange of glycerol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin, whereas GDR cannot mediate the exchange of diol dehydratase-bound cyanocobalamin for free adeninylpentylcobalamin. As judged by denaturing PAGE, the glycerol dehydratase-DDR complex was cross-formed, although the diol dehydratase-GDR complex was not formed. There were no specificities of reactivating factors in the ATP-dependent dissociation of enzyme-reactivating factor complexes. Thus, it is very likely that the specificities of reactivating factors are determined by the capability of reactivating factors to form complexes with apoenzymes. A modeling study based on the crystal structures of enzymes and reactivating factors also suggested why DDR cross-forms a complex with glycerol dehydratase, and why GDR does not cross-form a complex with diol dehydratase.

Relationship between structure and immunostimulating activity of enzymatically synthesized glycogen.

Glycogen acts as energy and carbon reserves in animal cells and in microorganisms. Although anti-tumor activity has recently been reported for shellfish glycogen and enzymatically synthesized glycogen, the activity of glycogen has not yet been fully clarified. We enzymatically prepared various sizes of glycogens with controlled structures to investigate the relationship between the structure and immunostimulating activity of glycogen. The results revealed that glycogens with a weight-average molecular weight (M(w)) of more than 10,000K hardly activated RAW264.7, a murine macrophage cell line, whereas glycogens of M(w) 5000K and 6500K strongly stimulated RAW264.7 in the presence of interferon-gamma (IFN-gamma), leading to augmented production of nitric oxide (NO), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6). Comparing the fine structure of the glycogens, the average-number of chain length, as well as the exterior and the interior chain lengths of the glycogens, had minor correlation between active and less-active glycogen derivatives. The available evidence suggests that the macrophage-stimulating activity of glycogen is strictly related to its molecular weight rather than to any fine structural property.

Anti-tumor activity of an enzymatically synthesized alpha-1,6 branched alpha-1,4-glucan, glycogen.

Oral administration of an enzymatically synthesized alpha-1,4:1,6-glycogen (ESG) at a dose of 50 mug/ml significantly prolonged the survival time of Meth A tumor-bearing mice. ESG also significantly stimulated macrophage-like cells (J774.1), leading to augmented production of nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha). The weight-average degree of polymerization (DPw) and the ratio of branch linkage (BL) of ESG were 149,000 and 8.1% respectively. beta-Amylase-treated ESG, however, lost J774.1-activating activity although inhibited subcutaneous growth of Meth A tumor cells admixed with it. Its DPw and BL changed to 126,000 and 20% respectively. Partially degraded amylopectin [(AP), DPw: 110,000, BL; 5.1] was also effective at stimulating J774.1, but its activity was lower than that of ESG. Other alpha-glucans [cycloamylose (CA), enzymatically synthesized amylose (ESA), highly branched cyclic dextrin (HBCD), and beta-amylase-treated HBCD], of which DPw was lower than that of ESG, showed no J774.1-activating activity and weaker anti-tumor activity.