Ichitomo Miwa

Glucokinase and IRS-2 are required for compensatory β 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.

PANCREATIC BETA -CELL-SPECIFIC TARGETED DISRUPTION OF GLUCOKINASE GENE : DIABETES MELLITUS DUE TO DEFECTIVE INSULIN SECRETION TO GLUCOSE

Mice carrying a null mutation in the glucokinase (GK) gene in pancreatic β-cells, but not in the liver, were generated by disrupting the β-cell-specific exon. Heterozygous mutant mice showed early-onset mild diabetes due to impaired insulin-secretory response to glucose. Homozygotes showed severe diabetes shortly after birth and died within a week. GK-deficient islets isolated from homozygotes showed defective insulin secretion in response to glucose, while they responded to other secretagogues: almost normally to arginine and to some extent to sulfonylureas. These data provide the first direct proof that GK serves as a glucose sensor molecule for insulin secretion and plays a pivotal role in glucose homeostasis. GK-deficient mice serve as an animal model of the insulin-secretory defect in human non-insulin-dependent diabetes mellitus.

Insulin Secretion by Anomers of D-Glucose

Isolated rat islets were incubated for 5 minutes in the media containing either the α or β anomer of D-glucose (2 milligrams per milliliter). The amounts of secreted insulin and changes of anomers ratio were concomitantly determined. In spite of rapid mutarotation, significantly greater stimulation of insulin secretion was observed by α-D-glucose as compared with β-D-glucose.

Mutarotase effect on micro determinations of d-glucose and its anomers with β-d-glucose oxidase

Abstract The polarographic micro determinations of d -glucose and its anomers with β- d -glucose oxidase were improved in rapidity and sensitivity by using mutarotase, an enzyme that catalyzes the interconversion of d -glucose anomers. The present method consists of measurements with a polarographic oxygen analyzer of oxygen consumption due to the oxidation of β- d -glucose in the presence of β- d -glucose oxidase and mutarotase.

Affinity purification and glucose specificity of aldose reductase from bovine lens

Abstract Aldose reductase, a possible key enzyme of sugar-cataract formation in diabetes, has been purified from bovine lens by a five-step procedure including affinity chromatography with Mātrex gel red A. The enzyme was purified 12,600-fold and was apparently homogeneous by polyacrylamide gel electrophoresis. The glucose specificity of the purified enzyme was studied with d -glucose anomers and d -glucitol as substrates. The ratios of the reduction rate of α- d -glucose to that of β- d -glucose at 10, 13, and 20 m m were 1.90, 1.76, and 1.72, respectively. These values were in good agreement with the ratios (1.92, 1.81, and 1.66) calculated on the basis of the rate constants reported for d -glucose mutarotation equilibrium ( J. M. Los, L. B. Simpson, and K. Wiesner, 1956, J. Amer. Chem. Soc.78, 1564–1568 ) and the assumption that aldose reductase acts on the aldehyde form of d -glucose. In addition, the composition of d -glucose produced from d -glucitol in the reverse reaction was 63% α anomer and 37% β anomer, which also agreed well with the values, 65 and 35%, respectively, calculated from the rate constants in reactions from the aldehyde form to both the α anomer and the β anomer. It was suggested from these kinetic analyses that aldose reductase acts on the aldehyde form of d -glucose (Km = 0.66 μm) but not on either the α or the β anomer of d -glucose.

Inhibition of advanced protein glycation by a Schiff base between aminoguanidine and pyridoxal.

Aminoguanidine is a well-known inhibitor of the formation of advanced glycation end products and is considered to be promising for the treatment of diabetic complications. We recently reported, however, that administration of aminoguanidine caused the formation of a Schiff base adduct between aminoguanidine and pyridoxal phosphate in the liver and kidney of mice and a concomitant decrease in the amount of liver pyridoxal phosphate. Our study led us to hypothesize that the Schiff base adduct and/or another Schiff base adduct formed from aminoguanidine and pyridoxal might be a better compound than aminoguanidine. In the present study, we examined the in vitro inhibitory potency of the latter adduct against advanced glycation end product formation and its effect on the tissue contents of pyridoxal and its phosphate. Aminoguanidine-pyridoxal phosphate adduct was not employed in this study because of its poor solubility in water. Aminoguanidine-pyridoxal adduct was hydrolyzed by only about 15% during 10 days at pH 7.4 and 37 degrees C. The adduct at 1 mM did not inhibit Amadori product formation induced by incubation of albumin with 100 mM mannose for 10 days. The adduct, when tested at 1 and 2 mM, dose-dependently inhibited advanced glycation end product formation induced by incubation of albumin with mannose; and the inhibitory potency of the adduct was similar to or higher than that of aminoguanidine. The presence of an appreciable amount of aminoguanidine-pyridoxal adduct in the kidney of mice given the adduct suggested that at least part of the adduct administered was absorbed from the gastrointestinal duct. The amounts of pyridoxal and its phosphate in tissues were not at all decreased by administration of the aminoguanidine-pyridoxal Schiff base. We conclude that the Schiff base may be a more promising inhibitor of advanced protein glycation than aminoguanidine.

Analysis of sorbitol, galactitol, and myo-inositol in lens and sciatic nerve by high-performance liquid chromatography

Accumulation of sorbitol or galactitol and depletion of myo-inositol in hyperglycemic conditions such as diabetes and galactosemia involve the activity of aldose reductase and are implicated in hyperglycemia-induced complications such as cataract and neuropathy. A high-performance liquid chromatographic method has been developed for the measurement of polyols in the lens and sciatic nerve of rats. This method comprises polyol extraction from tissues, lyophilization of extracts, derivatization of polyols by the reaction with phenylisocyanate, and HPLC of derivatives with detection at 240 nm. The time needed for each run is less than 25 min, which allows the testing of a large number of samples per day. Sensitivity is very high: as low as 0.5 nmol each of sorbitol, galactitol, and myo-inositol in lyophilized extracts of tissues can be determined. The present method offers a reliable tool to evaluate the in vivo activities of aldose reductase and its inhibitors.

Rapid and sensitive, colorimetric determination of the anomers of D-glucose with D-glucose oxidase, peroxidase, and mutarotase.

A modification, utilising mutarotase, of an enzymic, colorimetric system for determining D-glucose with D-glucose oxidase, peroxidase, and ABTS was satisfactory for the assay of the anomers of D-glucose in aqueous solution. The time required for a single assay is approximately 10 min, and the lower limit is 0.4 microgram of D-glucose. The method is applicable to the anomer analysis of D-glucose released by enzymic hydrolysis of D-glucosides.

Co-localization of glucokinase with actin filaments

A portion of glucokinase appeared to be co‐localized with actin filaments in the cytoplasm of cultured rat hepatocytes incubated with 25 mM glucose. When liver‐ or islet‐type glucokinase was transiently expressed in COS‐7 cells, the expressed glucokinase was also co‐localized with actin filaments in the cytoplasm of these transfected cells. Although co‐localization of glucokinase with actin filaments was not clearly demonstrated in the pancreatic β‐cell line MIN6, islet glucokinase was found to be present in both the nucleus and the cytoplasm, though predominantly in the nucleus. These findings suggest that subcellular localization of glucokinase, including co‐localization with actin filaments, may have an important physiological role in metabolic regulation.

Interaction of Alloxan and Anomers of D-Glucose on Glucose-induced Insulin Secretion and Biosynthesis in Vitro

The direct effects of alloxan on glucose-induced insulin secretion and biosynthesis and the interaction of alloxan and D-glucose anomers were studied in vitro by use of isolated islets from rat pancreas. Islets were pretreated by incubation for five minutes in media containing alloxan (0.2 mg./ml.) alone or alloxan with either the α or β anomer of D-glucose (3 mg./ml.). After washing, batches of five islets were incubated in the medium supplemented with glucose (1.8 mg./ml.) for 60 minutes to observe insulin secretion and for 90 minutes to observe insulin biosynthesis. Prior exposure to alloxan alone produced marked inhibition of subsequent glucose-induced insulin secretion and biosynthesis. A significantly greater protection against these inhibitory effects of alloxan was observed by using the α anomer of D-glucose than the β anomer. The anomeric preference of D-glucose for protecting islet cells from the inhibitory effect of alloxan on glucose-induced insulin secretion and biosynthesis was similar to that for triggering insulin secretion. Possible mechanisms of the inhibitory effect of alloxan and the protective effect of D-glucose anomers in connection with those of other sugars are discussed. It is suggested that a glucoreceptor, stereospecific to the α anomer of D-glucose, may exist for both insulin secretion and biosynthesis.