Takashi Furuyashiki

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.

Comparative analysis of carbohydrate-binding specificities of two anti-glycogen monoclonal antibodies using ELISA and surface plasmon resonance

For immunological experiments on glycogens, anti-glycogen antibodies are indispensable to capture, detect, and visualize sugar molecules. An anti-glycogen monoclonal antibody (IV58B6) and newly constructed antibody (ESG1A9mAb) have a common immunoglobulin type (IgM) and binding ability to glycogens, but overall possess different binding features. Therefore, they may prove useful for the construction of an advanced system of quantitative ELISA based on their molecular structures. For this purpose, detailed information on the carbohydrate-specificities of ESG1A9mAb and IV58B6 is first required, but their fine specificities for various types of glycogens have not been elucidated. To overcome this problem, we performed interaction analysis by ELISA of ESG1A9mAb and IV58B6 toward 15 glucose polymers, that is, 5 enzymatically-synthesized glycogens (ESGs), 6 natural source glycogens (NSGs), 3 enzymatically digested glycogens (EDGs), and soluble starch. To provide a more detailed analysis, we determined the association constants (K(a)) of the two antibodies toward these glycogens by surface plasmon resonance. The results indicated that the carbohydrate-binding properties toward NSGs of ESG1A9mAb and IV58B6 were similar, but markedly differed for ESGs and EDGs. ESG1A9mAb showed significant affinity for all the ESGs and NSGs tested, whereas IV58B6 had only slight affinity for ESGs, although the affinities were increased when the ESGs were enzymatically digested. This information should be helpful for the design of both in vitro and in vivo immunological assays.

Application of branching enzyme in starch processing.

Abstract Branching enzyme (BE; EC 2.4.1.18; (1→4)-α-D-glucan:(1→4)-α-D-glucan 6-α-D-[(1→4)-α-D-glucano]-transferase) is a glucan transferase which is responsible for synthesis of α-1,6-glucosidic bonds in starch and glycogen in vivo. It has been demonstrated that at least three BEs catalyze cyclization reactions in addition to the branching reaction in vitro. A thermostable BE from Bacillus stearothermophilus has been used industrially for starch processing to synthesize a food ingredient, highly branched cyclic dextrin (HBCD). HBCD has a narrow molecular-weight distribution and relatively long side chains compared with conventional dextrins, partially hydrolyzed starch produced with α-amylase. In order to further evaluate the potential of BE in starch processing, the action of BEs from several sources on amylopectin was analyzed. Many BEs tested in this study catalyzed similar reactions to B. stearothermophilus BE to produce HBCD. BE from Bacillus cereus catalyzed cyclization of amylopectin, but the chain-length distribution of the product was significantly different from HBCD. Noticeably, BE from kidney bean produced much larger molecules with shorter side chains than HBCD.

In vitro synthesis of glycogen: the structure, properties, and physiological function of enzymatically-synthesized glycogen

This review describes a new enzymatic method for in vitro glycogen synthesis and its structure and properties. In this method, short-chain amylose is used as the substrate for branching enzymes (BE, EC 2.4.1.18). Although a kidney bean BE and Bacillus cereus BE could not synthesize high-molecular weight glucan, BEs from 6 other bacterial sources produced enzymatically synthesized glycogen (ESG). The BE from Aquifex aeolicus was the most suitable for the production of glycogen with a weight-average molecular weight (Mw) of 3,000–30,000 k. The molecular weight of the ESG is controllable by changing the concentration of the substrate amylose. Furthermore, the addition of amylomaltase (AM, EC 2.4.1.25) significantly enhanced the efficiency of this process, and the yield of ESG reached approximately 65%. Typical preparations of ESG obtained by this method were subjected to structural analyses. The average chain length, interior chain length, and exterior chain length of the ESGs were 8.2–11.6, 2.0–3.3, and 4.2–7.6, respectively. Transmission electron microscopy and intrinsic viscosity measurement showed that the ESG molecules formed spherical particles. Unlike starch, the ESGs were barely degraded by pullulanase. Solutions of ESG were opalescent (milky-white and slightly bluish), and gave a reddishbrown color on the addition of iodine. These analyses revealed that ESG shares similar molecular shapes and solution properties with natural-source glycogen. Moreover, ESG had macrophage-stimulating activity and its activity depends on the molecular weight of ESG. We successfully achieved large scale production of ESG. ESG could lead to new industrial applications, such as in the food, chemical, and pharmaceutical fields.

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.

Enzymatically synthesized glycogen reduces lipid accumulation in diet-induced obese rats

Based on a recent study indicating that enzymatically synthesized glycogen (ESG) possesses a dietary, fiber-like action, we hypothesized that ESG can reduce the risk of obesity. In this study, the antiobesity effects of ESG were investigated in a model of diet-induced obesity. Male Sprague-Dawley rats were divided into 4 groups and fed a normal or high-fat diet, with or without 20% ESG, for 4 weeks. Body weight, food intake, lipid deposition in the white adipose tissues and liver, fecal lipid excretion, and plasma lipid profiles were measured. At week 3, the body fat mass was measured using an x-ray computed tomography system, which showed that ESG significantly suppressed the high-fat diet-induced lipid accumulation. Similar results were observed in the weight of the adipose tissue after the experiment. Moreover, ESG significantly suppressed the lipid accumulation in the liver but increased fecal lipid excretion. The plasma concentrations of triacylglycerol and nonesterified fatty acid were lowered after a high-fat diet, whereas the total bile acid concentration was increased by ESG. However, the hepatic messenger RNA (mRNA) levels of enzymes related to lipid metabolism were not affected by ESG. Conversely, the mRNA levels of long-chain acyl-CoA dehydrogenase and medium-chain acyl-CoA dehydrogenase were up-regulated by ESG in the muscle. These results suggest that the combined effects of increased fecal lipid excretion, increased mRNA levels of enzymes that oxidize fatty acids in the muscle, and increased total bile acid concentration in the plasma mediate the inhibitory effect of ESG on lipid accumulation.

Enzymatically synthesized glycogen inhibits colitis through decreasing oxidative stress

Abstracts Inflammatory bowel diseases are a group of chronic inflammation conditions of the gastrointestinal tract. Disruption of the mucosal immune response causes accumulation of oxidative stress, resulting in the induction of inflammatory bowel disease. In this study, we investigated the effect of enzymatically synthesized glycogen (ESG), which is produced from starch, on dextran sulfate sodium (DSS)‐ and 2,4,6‐trinitrobenzenesulfonic acid (TNBS)‐induced colitis in C57BL/6 mice. Oral administration of ESG suppressed DSS‐ and TNBS‐induced shortening of large intestine in female mice and significant decreased DSS‐induced oxidative stress and TNBS‐induced pro‐inflammatory cytokine expression in the large intestine. ESG increase in the expression levels of heme oxygenase‐1 (HO‐1) and NF‐E2‐related factor‐2 (Nrf2), a transcription factor for HO‐1 expressed in the large intestine. Furthermore, ESG‐induced HO‐1 and Nrf2 were expressed mainly in intestinal macrophages. ESG is considered to be metabolized to resistant glycogen (RG) during digestion with &agr;‐amylase in vivo. In mouse macrophage RAW264.7 cells, RG, but not ESG decreased 2,2′‐azobis(2‐amidinopropane) dihydrochloride (AAPH)‐induced reactive oxygen species (ROS). Knockdown of Nrf2 inhibited RG‐induced HO‐1 expression and negated the decrease in AAPH‐induced ROS brought about by RG. RG up‐regulated the protein stability of Nrf2 to decrease the formation of Nrf2‐Keap1 complexes. RG‐induced phosphorylation of Nrf2 at Ser40 was suppressed by ERK1/2 and JNK inhibitors. Our data indicate that ESG, digested with &agr;‐amylase to RG, suppresses DSS‐ and TNBS‐induced colitis by increasing the expression of HO‐1 in the large intestine of mice. Furthermore, we demonstrate that RG induces HO‐1 expression by promoting phosphorylation of Nrf2 at Ser40 through activation of the ERK1/2 and JNK cascade in macrophages. Graphical abstract Figure. No Caption available. HighlightsESG prevented DSS‐induced colitis in mice by decreasing oxidative stress.ESG administration induced Nrf2 and HO‐1 expression in colon tissues of mice.ESG metabolite RG induced Nrf2 and HO‐1 expression in RAW264.7 macrophages.Knockdown of Nrf2 and HO‐1 in RAW264.7 cells negated RG‐induced antioxidant effect.

Effects of ingesting highly branched cyclic dextrin during endurance exercise on rating of perceived exertion and blood components associated with energy metabolism

We compared the effect of relatively low doses (15 g) of highly branched cyclic dextrin (HBCD) with that of maltodextrin during endurance exercise on the rating of perceived exertion (RPE) in a crossover, double-blind study of healthy volunteers. The RPE increased during exercise and its increase was significantly less at 30 and 60 min after ingesting HBCD than maltodextrin.

Immunomodulatory activity of enzymatically synthesized glycogen and its digested metabolite in a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages

Previously, we developed enzymatically synthesized glycogen (ESG) from starch, and showed its immunomodulatory and dietary fiber-like activities. In this study, we investigated the metabolism of ESG and its immunomodulatory activity using differentiated Caco-2 cells as a model of the intestinal barrier. In a co-culture system consisting of differentiated Caco-2 cells and RAW264.7 macrophages, mRNA expression of IL-6, IL-8, IL-1β and BAFF cytokines was up-regulated in Caco-2 cells and IL-8 production in basolateral medium was induced after 24 h apical treatment with 5 mg ml(-1) of ESG. The mRNA level of iNOS was also up-regulated in RAW264.7 macrophages. After characterization of the binding of anti-glycogen monoclonal antibodies (IV58B6 and ESG1A9) to ESG and its digested metabolite resistant glycogen (RG), an enzyme-linked immunosorbent assay (ELISA) system was developed to quantify ESG and RG. Using this system, we investigated the metabolism of ESG in differentiated Caco-2 cells. When ESG (7000 kDa, 5 mg ml(-1)) was added to the apical side of Caco-2 monolayers, ESG disappeared and RG (about 3000 kDa, 3.5 mg ml(-1)) appeared in the apical solution during a 24 h incubation. Neither ESG nor RG was detected in the basolateral solution. In addition, both ESG and RG were bound to TLR2 in Caco-2 cells. In conclusion, we suggest that ESG is metabolized to a RG-like structure in the intestine, and this metabolite activates the immune system via stimulation of the intestinal epithelium, although neither ESG nor its metabolite could permeate the intestinal cells under our experimental conditions. These results provide evidence for the beneficial function of ESG as a food ingredient.

Qualitative and Quantitative Analyses of Glycogen in Human Milk

Identification as well as a detailed analysis of glycogen in human milk has not been shown yet. The present study confirmed that glycogen is contained in human milk by qualitative and quantitative analyses. High-performance anion exchange chromatography (HPAEC) and high-performance size exclusion chromatography with a multiangle laser light scattering detector (HPSEC-MALLS) were used for qualitative analysis of glycogen in human milk. Quantitative analysis was carried out by using samples obtained from the individual milks. The result revealed that the concentration of human milk glycogen varied depending on the mothers condition-such as the period postpartum and inflammation. The amounts of glycogen in human milk collected at 0 and 1-2 months postpartum were higher than in milk collected at 3-14 months postpartum. In the milk from mothers with severe mastitis, the concentration of glycogen was about 40 times higher than that in normal milk.