Plasma mannose level, a putative indicator of glycogenolysis, and glucose tolerance in Japanese individuals.

AIMS/INTRODUCTION: Mannose is a monosaccharide constituent of glycoproteins and glycolipids. Experiments in rats have shown previously that the plasma mannose level decreases after glucose load, but does not decrease in diabetic rats, and that hepatic glycogenolysis is a source of this plasma mannose; however, these results are not fully elucidated in humans. Plasma mannose levels before/after glucose loading in humans with various degrees of glucose intolerance were examined to analyze their association with clinical factors. MATERIALS AND METHODS: The 75-g oral glucose tolerance test was carried out in Japanese individuals not taking diabetes medications. Participants were classified into normal glucose tolerance, impaired glucose metabolism and diabetes mellitus groups. Insulinogenic index as an index of insulin secretion, and Matsuda Index as an index of insulin sensitivity were calculated. Mannose was assayed by the established method using high-performance liquid chromatography after labeling. RESULTS: After glucose load, the plasma mannose level decreased gradually in the normal glucose tolerance group, but did not decrease in the diabetes mellitus group. Plasma mannose changes during 120 min from baseline (M120 -M0 ) were significantly different among the three groups (normal glucose tolerance: -16.7 ± 1.7; impaired glucose metabolism: -9.0 ± 1.9; diabetes mellitus: -1.4 ± 1.8 µmol/L [n = 25 in each group], P < 0.0001). Plasma glucose 120 min after glucose loading (R2  = 0.412) or loge -insulinogenic index, loge -Matsuda Index and age (R2  = 0.230) were determinants of M120 -M0 in multiple regression analyses. CONCLUSIONS: We clarified the relationship between plasma mannose level and glucose tolerance in humans. The present results are compatible with those using rats, in which mannose derived from glycogenolysis plays an important role in the alteration of mannose levels after glucose loading.

Sustained high plasma mannose less sensitive to fluctuating blood glucose in glycogen storage disease type Ia children.

Plasma mannose is suggested to be largely generated from liver glycogen-oriented glucose-6-phosphate. This study examined plasma mannose in glycogen storage disease type Ia (GSD Ia) lacking conversion of glucose-6-phosphate to glucose in the liver. We initially examined fasting--and postprandial 2 h--plasma mannose and other blood carbohydrates and lipids for seven GSD Ia children receiving dietary interventions using cornstarch and six healthy age-matched children. Next, one-day successive intra-individual parameter changes were examined for six affected and two control children. Although there were no significant differences in fasting--and postprandial 2 h--glucose and insulin levels, the mannose level of the affected group was invariably much higher than that of the control group (p < 0.001): the fasting level of the affected group was about two-fold that of the control group; the postprandial-2 h level remained almost unchanged in the affected group, although it was one-half of the fasting level in the control group. Inter-individual analyses revealed that the GSD Ia group mannose level was significantly and positively correlated with lactate and triglycerides levels at both time points (p < 0.01). In each control, mannose levels fluctuated greatly, maintaining strong and significant negative correlations with glucose and insulin levels (p < 0.001). Correlations were lower or nonexistent in GSD Ia children. In individuals with high lactate and triglycerides levels, strikingly high mannose levels never changed against glucose and insulin fluctuations. Plasma mannose is less sensitive to blood glucose and insulin in GSD Ia children. Its basal level and the fluctuation pattern differ by their metabolic activity.

Ingestion of a moderate high-sucrose diet results in glucose intolerance with reduced liver glucokinase activity and impaired glucagon-like peptide-1 secretion.

UNLABELLED: Aims/Introduction: Excessive intake of sucrose can cause severe health issues, such as diabetes mellitus. In animal studies, consumption of a high-sucrose diet (SUC) has been shown to cause obesity, insulin resistance and glucose intolerance. However, several in vivo experiments have been carried out using diets with much higher sucrose contents (50-70% of the total calories) than are typically ingested by humans. In the present study, we examined the effects of a moderate SUC on glucose metabolism and the underlying mechanism. MATERIALS AND METHODS: C57BL/6J mice received a SUC (38.5% sucrose), a high-starch diet (ST) or a control diet for 5 weeks. We assessed glucose tolerance, incretin secretion and liver glucose metabolism. RESULTS: An oral glucose tolerance test (OGTT) showed that plasma glucose levels in the early phase were significantly higher in SUC-fed mice than in ST-fed or control mice, with no change in plasma insulin levels at any stage. SUC-fed mice showed a significant improvement in insulin sensitivity. Glucagon-like peptide-1 (GLP-1) secretion 15 min after oral glucose administration was significantly lower in SUC-fed mice than in ST-fed or control mice. Hepatic glucokinase (GCK) activity was significantly reduced in SUC-fed mice. During the OGTT, the accumulation of glycogen in the liver was suppressed in SUC-fed mice in a time-dependent manner. CONCLUSIONS: These results indicate that mice that consume a moderate SUC show glucose intolerance with a reduction in hepatic GCK activity and impairment in GLP-1 secretion. (J Diabetes Invest, doi: 10.1111/j.2040-1124.2012.00208.x, 2012).

Glucokinase and IRS-2 are required for compensatory beta cell hyperplasia in response to high-fat diet-induced insulin resistance.

Glucokinase (Gck) functions as a glucose sensor for insulin secretion, and in mice fed standard chow, haploinsufficiency of beta cell-specific Gck (Gck(+/-)) causes impaired insulin secretion to glucose, although the animals have a normal beta cell mass. When fed a high-fat (HF) diet, wild-type mice showed marked beta cell hyperplasia, whereas Gck(+/-) mice demonstrated decreased beta cell replication and insufficient beta cell hyperplasia despite showing a similar degree of insulin resistance. DNA chip analysis revealed decreased insulin receptor substrate 2 (Irs2) expression in HF diet-fed Gck(+/-) mouse islets compared with wild-type islets. Western blot analyses confirmed upregulated Irs2 expression in the islets of HF diet-fed wild-type mice compared with those fed standard chow and reduced expression in HF diet-fed Gck(+/-) mice compared with those of HF diet-fed wild-type mice. HF diet-fed Irs2(+/-) mice failed to show a sufficient increase in beta cell mass, and overexpression of Irs2 in beta cells of HF diet-fed Gck(+/-) mice partially prevented diabetes by increasing beta cell mass. These results suggest that Gck and Irs2 are critical requirements for beta cell hyperplasia to occur in response to HF diet-induced insulin resistance.

Decline in glucokinase activity in the arcuate nucleus of streptozotocin-induced diabetic rats.

Glucokinase (GK) is known to be the critical glucose sensor of pancreatic B-cells. However, the localization and functional role of GK in the brain remains to be elucidated. In this study, we measured both the activity and mRNA level of GK in the hypothalamic nuclei and the cortex of rats injected intraperitoneally with streptozotocin or vehicle. GK activity was measured by a fluorometric assay; and the GK mRNA level, by use of the real-time reverse transcription polymerase chain reaction. GK activity in vehicle-treated rats was high in the arcuate nucleus, moderate or low in the ventromedial nucleus, lateral hypothalamic area, and paraventricular nucleus, and very low in the cortex. The order of GK mRNA level was almost the same as that of GK activity. GK activity and GK mRNA level only in the arcuate nucleus of streptozotocin-treated rats at 7 d, but not at 2 d, after treatment were lower than those of vehicle-treated rats. The results suggest that prolonged hyperglycemia induced by diabetes decreased the activity of GK in the arcuate nucleus.

Effect of chitobiose and chitotriose on carbon tetrachloride-induced acute hepatotoxicity in rats.

The purpose of the present study was to examine whether chitobiose and chitotriose can protect rats from CCl4-induced hepatic toxicity when given orally. We studied the effects of the 2 chitooligosaccharides given orally to rats on the acute hepatotoxicity induced by CCl4-dependent lipid peroxidation. The increase in the sum of malondialdehyde and 4-hydroxy-2-alkenals, a marker of lipid peroxidation, in both plasma and liver of CCl4-treated rats was suppressed by oral administration of chitobiose or chitotriose. The elevation in the levels of plasma aspartate transaminase and alanine transaminase activities, markers of hepatic injury, induced by CCl4 intoxication was also counteracted by oral administration of either chitooligosaccharide. The results indicate that chitobiose and chitotriose have the ability to exert a protective action against CCl4-induced acute hepatoxicity, probably by their antioxidant activity.

Pharmacokinetics of chitobiose and chitotriose administered intravenously or orally to rats.

Chitooligosaccharides have attracted much attention as new biomedical materials. The biologic availability of each of these chitooligosaccharides, however, has not yet been studied. In the present study, we found that chitobiose and chitotriose appeared in the blood of rats with maximum plasma concentrations at around 1 h after administration when given orally at a dose of 30 mg/kg. However, chitotetraose and chitopentaose did not appear in the blood when given at a dose of 300 mg/kg. Pharmacokinetic analysis of chitobiose and chitotriose after intravenous administration at 100 mg/kg revealed that both sugars were eliminated from the body following a one-compartment model and that the former relative to the latter was higher for both the total body clearance (224+/-43 vs. 155+/-26 ml/h/kg) and the distribution volume (107+/-15 vs. 65+/-9 ml/kg). The absolute oral bioavailability of chitobiose was higher than that of chitotriose at all doses (30, 100, and 300 mg/kg) examined. The first-order absorption rate constants for chitobiose and chitotriose at all doses were less than 1.0 h(-1) and smaller than the elimination rate constants (2.2+/-0.3, 2.7+/-0.1 h(-1), respectively). The absorption was slow, resulting in flip-flop kinetics. This study indicates that among various chitooligosaccharides, only chitobiose and chitotriose can be appreciably absorbed from the gastrointestinal tract.

A series of maturity onset diabetes of the young, type 2 (MODY2) mouse models generated by a large-scale ENU mutagenesis program.

Mutant mouse models are indispensable tools for clarifying the functions of genes and for elucidating the underlying pathogenic mechanisms of human diseases. Currently, several large-scale mutagenesis projects that employ the chemical mutagen N-ethyl-N-nitrosourea (ENU) are underway worldwide. One specific aim of our ENU mutagenesis project is to generate diabetic mouse models. We screened 9375 animals for dominant traits using a clinical biochemical test and thereby identified 11 mutations in the glucokinase (Gk) gene that were associated with hyperglycemia. GK is a key regulator of insulin secretion in the pancreatic beta-cell. Approximately 190 heterozygous mutations in the human GK gene have been reported to cause maturity onset diabetes of the young, type 2 (MODY2). In addition, five mutations have been reported to cause permanent neonatal diabetes mellitus (PNDM) when present on both alleles. The mutations in our 11 hyperglycemic mutants are located at different positions in Gk. Four have also been found in human MODY2 patients, and another mutant bears its mutation at the same location that is mutated in a PNDM patient. Thus, ENU mutagenesis is effective for developing mouse models for various human genetic diseases, including diabetes mellitus. Some of our Gk mutant lines displayed impaired glucose-responsive insulin secretion and the mutations had different effects on Gk mRNA levels and/or the stability of the GK protein. This collection of Gk mutants will be valuable for understanding GK gene function, for dissecting the function of the enzyme and as models of human MODY2 and PNDM.

Antioxidant activity of a Schiff base of pyridoxal and aminoguanidine.

We recently reported that PL-AG, a Schiff base of pyridoxal and aminoguanidine, was more effective than aminoguanidine (AG), a well-known anti-diabetic-complication compound, in preventing nephropathy in diabetic mice and presented brief data indicating the antioxidant activity of the adduct. In the present study, we additionally investigated the inhibitory activity of PL-AG in comparison with that of AG against in vitro and in vivo oxidation. PL-AG was more potent than AG and reference compounds such as pyridoxal and pyridoxamine in any of the five antioxidant activities examined in vitro, i.e., hydrogen peroxide-scavenging, hydroxyl radical-scavenging, superoxide radical-scavenging, ascorbic acid-autoxidation inhibitory, and low-density lipoprotein (LDL)-oxidation inhibitory activities, the last two of which were assessed in the presence of Cu(2+). Unlike AG, PL-AG did not show the pro-oxidant activity. The inhibitory activity of PL-AG against lipid peroxidation in diabetic rats was higher than that of AG, for example, the amounts of malondialdehyde in erythrocytes (nmol/g hemoglobin; mean +/- SD) in normal, untreated diabetic, AG-treated diabetic, and PL-AG-treated diabetic rats were 3.53 +/- 0.35, 4.99 +/- 0.23, 4.65 +/- 0.45, and 4.06 +/- 0.35, respectively. A fluorescent substance different from PL-AG was found in the plasma and urine of rats treated with PL-AG. The chemical structure of this substance, i.e., oxidized PL-AG, was determined by a combination of nuclear magnetic resonance, mass, and infrared spectrometry. AG dramatically decreased the pyridoxal phosphate level in the diabetic rat liver, whereas PL-AG only moderately affected it. Our results indicate that the antioxidant activity of PL-AG is due to its chelation with transition metal ions and to scavenging of reactive oxygen species. They also suggest that PL-AG is more promising for the treatment of diabetic complications than AG.

Antioxidant activities of chitobiose and chitotriose.

Chitooligosaccharides, the oligomers made up of beta-1,4-linked D-glucosamine, are obtained by partial hydrolysis of chitosan, a deacetylation product of chitin. The antioxidant activity of various chitooligosaccharides was tested in vitro with aminoguanidine, pyridoxamine, and Trolox as reference compounds. Hydroxylation of benzoate to salicylate by H2O2 in the presence of Cu(2+) was effectively inhibited by chitobiose, chitotriose, aminoguanidine, pyridoxamine, and Trolox (their IC(50) values=18, 80, 85, 10, and 95 microM, respectively), whereas glucosamine and N-acetylchito-oligosaccharides (di-N-acetylchitobiose and tri-N-acetylchitotriose) did not show any inhibitory activity. Chitobiose and chitotriose were more potent than the 3 reference compounds in scavenging hydroxyl radicals produced by photolysis of zinc oxide: IC(50) values of the 2 oligomers were 30 and 55 microM, respectively. Such a scavenging activity of these 2 chitooligomers was also shown by the use of another system, a mixture of Fe(3+)/EDTA/ascorbate/H2O2, for producing hydroxyl radicals. Only chitobiose and Trolox, of the 10 compounds tested, had the ability to scavenge superoxide radicals generated by a non-enzymatic system using phenazine methosulfate and NADH. Taken together with our unpublished observation that chitobiose and chitotriose are appreciably absorbed from the intestine of rats, the present results suggest that these 2 chitooligosaccharides would act as effective antioxidants in vivo when orally ingested.

Two new nitric oxide synthase inhibitors: pyridoxal aminoguanidine and 8-quinolinecarboxylic hydrazide selectively inhibit basal but not agonist-stimulated release of nitric oxide in rat aorta.

Structural modification at one of the guanidine nitrogens of L-arginine has led to the development of a number of compounds N(G)-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine (L-NOARG), N(G)-nitro-L-arginine methyl ester (L-NAME) that competitively inhibit nitric oxide synthase (NOS). It was reported that another chemically related compound known as a glycation inhibitor, aminoguanidine also inhibits NOS. Recently, two new glycation inhibitors, structurally related to aminoguanidine (AG), pyridoxal aminoguanidine (PLAG) and 8-quinoline carboxylic hydrazide (8Q) were synthesized. In this study, the effects of these two inhibitors on responses mediated by constitutive nitric oxide (NO) were investigated in vitro. For this purpose, in the present study vascular responses to phenylephrine and acetylcholine in isolated aortas were evaluated. Incubation (15 min) with PLAG and 8Q (10(-4)M for each) induced potentiation of phenylephrine-induced contraction in endothelium intact but not in endothelium denuded rings of rat aorta. The ability of PLAG or 8Q to augment phenylephrine-induced tone in endothelium containing rings was completely prevented by preincubation with L-arginine (1mM), but not with D-arginine. Both compounds (PLAG, 8Q) did not affect acetylcholine-induced relaxation. These results suggest that both of the new compounds produced a selective inhibition of basal but not agonist stimulated production of nitric oxide in rat aorta.

Glyceraldehyde metabolism in human erythrocytes in comparison with that of glucose and dihydroxyacetone.

Metabolism of D-glyceraldehyde in human erythrocytes in comparison with that of glucose and dihydroxyacetone was studied. Both trioses were metabolized to produce L-lactate at rates comparable to that of L-lactate formation from glucose. Almost complete inactivation of glyceraldehyde-3-phosphate dehydrogenase by treatment of cells with iodoacetate resulted in a 95% decrease in L-lactate formation from the ketotriose as well as from glucose, whereas L-lactate formation from the aldotriose was only partially reduced (60%). D-Lactate was produced faster from either the aldotriose or the ketotriose than from glucose, but the ability of the two trioses to produce D-lactate was far lower than that to produce L-lactate. Almost complete inhibition of aldehyde dehydrogenase by disulfiram and of both aldose reductase and aldehyde reductase II by sorbinil, had no effect on L-lactate formation from D-glyceraldehyde. The present study suggests that D-glyceraldehyde is metabolized via two or more pathways including the glycolytic pathway after its phosphorylation by triokinase, and that neither oxidation to D-glyceric acid nor reduction to glycerol is a prerequisite for D-glyceraldehyde metabolism.

An improved quantitative assay of glycogen in erythrocytes.

BACKGROUND: Current methods for the quantitative determination of glycogen in erythrocytes are unsatisfactory. METHODS: Erythrocytes were deproteinized with perchloric acid either after haemolysing by freezing and thawing twice or without freezing and thawing, and the glycogen content was determined by an enzymatic method. RESULTS: Freezing and thawing resulted in a significantly higher glycogen content, and the recovery of added glycogen using this method was nearly 100%. The mean erythrocyte glycogen content in healthy volunteers (n = 17) was 69.5 microg/g haemoglobin, a value much higher than the previously reported values in healthy subjects. CONCLUSION: Freezing and thawing improves the assay of erythrocyte glycogen.