Insulin secretion by anomers of d-glucose.

Isolated rat islets were incubated for 5 minutes in the media containing either the alpha or beta 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 alpha-D-glucose as compared with beta-D-glucose.

Glyceraldehyde is present in rat lens and its level is increased in diabetes mellitus.

PURPOSE: OP-lysine, a glycation product of lysine residues of proteins, has been reported to be formed with glyceraldehyde and glycolaldehyde as precursors in the lens, and has been suggested to play a role in senile cataracts. However, there has been no reliable information regarding the content of glyceraldehyde in tissues. This study determined the glyceraldehyde levels in the lenses of normal and diabetic rats. METHODS: Glyceraldehyde was derivatized to a fluorescent compound, and the compound was then quantified by high-performance liquid chromatography. RESULTS: The lens glyceraldehyde levels in normal and diabetic rats were 0.75 +/- 0.06 and 1.26 +/- 0.21 nmol/g wet weight (means +/- standard deviations of 6 animals, p < 0.01), respectively. Isolated rat lenses accumulated a higher level of glyceraldehyde when cultured for 6 days in 25.5 mM glucose than when cultured in 5.5 mM glucose. CONCLUSIONS: Glyceraldehyde was found to be present in the lens and was increased in diabetes mellitus. OP-lysine is thus likely to be a potential risk factor for senile and diabetic cataracts.

Different changes in resistance index between uterine artery and uterine radial artery during early pregnancy.

BACKGROUND: Changes in blood flow impedance of the uterine artery (UA) and uterine radial artery (RA) which is in the lower-extremity of the UA were examined during early pregnancy. METHODS: Blood flow impedance was assessed by transvaginal color-pulsed-Doppler-ultrasonography in 72 women from weeks 4-16 of pregnancy and expressed as a resistance index (RI). RESULTS: RA-RI remained at the late-luteal phase level until the 5th week of pregnancy, decreased until the 7th week, and remained low until the 10th week. UA-RI remained at the late-luteal phase level until the 10th week, and then gradually decreased until the 16th week. In nine women with spontaneous abortion, five out of six women with impaired growth of the gestational sac showed high RA-RI at the 6th week of pregnancy, whereas all three women with loss of fetal heart beat at the 8th week showed normal changes in RA-RI. CONCLUSIONS: Our results show different changes in blood flow impedance between the UA and RA during early pregnancy. A significant decrease of RA-RI after the 5th week may reflect vascular remodeling in the maternal-fetal interface at placentation, whereas a significant decrease of UA-RI after the 10th week may reflect changes of the whole uterine blood flow associated with uterine growth.

Kinetic difference between hydrolyses of gamma-cyclodextrin by human salivary and pancreatic alpha-amylases.

gamma-Cyclodextrin was found to be hydrolyzed by human salivary and pancreatic alpha-amylases (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1) at appreciable rates. The optimum pH for the enzyme reactions at 37 degrees C in the presence of 0.1 M NaCl was at around pH 5, which was remarkably different from the optimum pH (pH 6.9) of the enzymes for starch. The Km value (2.9 mg/ml) of pancreatic alpha-amylase for gamma-cyclodextrin was smaller than that (5.3 mg/ml) of salivary alpha-amylase at pH 5.3, while the V value of the former was 3.7-times larger than that of the latter. The hydrolyses of gamma-cyclodextrin by both enzymes took place via the multiple attack mechanism. The degrees of multiple attack by salivary and pancreatic alpha-amylases for gamma-cyclodextrin at pH 5.3 were 2.0 and 1.1, respectively. The distribution of maltodextrins produced by hydrolysis of gamma-cyclodextrin by salivary alpha-amylase was suggested to be independent of the substrate concentration, while that produced by pancreatic alpha-amylase was presumably dependent on the substrate concentration.

Glucose transport into human erythrocytes treated with phospholipase A2 or C.

Phospholipase A2 induced crenation of human erythrocytes and decreased glucose transport activity (influx rate) by 40% when 51% of phosphatidylcholine (PC) in the membrane was hydrolyzed. On the other hand, phospholipase C induced invagination of the cells and negligibly affected the glucose transport in the case of 21% hydrolysis of the PC. By altering the pH of the medium for suspending cells treated with phospholipase A2 from 7.4 to 6.0, cell shape was changed from clear crenation to slight invagination, but glucose transport activity was not affected. Cells that were treated with phospholipase A2 and then washed with albumin to remove free fatty acids produced in the cell membrane showed an almost normal cell shape and slightly higher glucose transport activity than did untreated cells. The ratios of beta-D-glucose transport rate to alpha-D-glucose transport rate in untreated cells, cells treated with phospholipase A2 and cells treated with phospholipase C were 1.13, 1.04, and 1.20, respectively. These results demonstrate that the drastic morphological change (invagination or crenation) induced by the treatment with phospholipases bears no clear relationship to the activity of glucose transport and suggest that the increase in the volume of the outer half of the lipid bilayer might reduce the rate of glucose transport across the human erythrocyte membrane and change the anomeric preference of glucose transport.

Stimulatory effect of fatty acid treatment on glucose utilization in human erythrocytes.

We previously reported that treatment of human erythrocytes with bee venom phospholipase A2 increased the rate of lactate production from glucose. This increase was suggested to be mediated through liberation of free fatty acids from membrane phospholipids. So, in the present study we examined the mechanism of stimulation of glycolysis by fatty acids. Treatment of intact erythrocytes with most of the 15 fatty acids tested resulted in stimulation of lactate production from glucose. Among the fatty acids tested, myristoleic acid showed the highest stimulatory activity. The ratio of moles of lactate produced to those of glucose utilized was about 1.9 in both myristoleic acid-treated and untreated cells. Treatment of erythrocytes with myristoleic acid did not affect the amount of 2,3-bisphosphoglycerate. Lactate production from D-glyceraldehyde, which is thought to be phosphorylated to D-glyceraldehyde 3-phosphate and then metabolized in the glycolytic pathway, was not at all affected by treatment of cells with myristoleic acid. The cross-over plot of glycolytic intermediates suggested that the enhancement of glycolysis was induced by activation of the 6-phosphofructokinase (PFK) step. Fatty acids incorporated into erythrocytes were found to be present predominantly in the cytoplasm rather than in the plasma membrane. The PFK activity, but not the hexokinase activity, in hemolysates was clearly increased by a set of fatty acids, and myristoleic acid was again the most potent. However, partially purified human erythrocyte PFK was not activated by the acid. We conclude that fatty acids stimulate glycolysis through activation of PFK in cooperation with some other component(s) in erythrocytes.

Stimulatory effect of phospholipase A2 treatment on glucose utilization in human erythrocytes.

We examined whether modification of membrane phospholipids of human erythrocytes by hydrolysis with phospholipase A2 (PLA2 from bee venom) would affect glucose utilization, chosen as a typical model of intracellular metabolism, and, if so, intended to clarify the mechanism of the alteration of glycolysis. Treatment of erythrocytes with PLA2 induced a marked shape change (i.e., crenation) and significantly increased the rate of lactate production from glucose. Available evidence indicated that there is no relevance of this cell-shape change to the alteration of glycolysis. The lack of a detectable effect of papain treatment on glycolysis in PLA2-treated cells suggested that the increase in glycolysis by PLA2 treatment might not be caused by the conformational change of band-3 protein through modulation of membrane phospholipids. The result of the measurement of lactate production in the presence and absence of ouabain did not support the idea that hydrolysis of phospholipids by PLA2 treatment makes plasma membranes leaky to Na+ and consequently enhances glycolysis through activation of Na+/K(+)-ATPase. The action of PLA2 on glycolysis was abolished by extraction of free fatty acids in the cell membrane with bovine serum albumin. Loading erythrocytes with free fatty acid (oleic acid, linoleic acid, or arachidonic acid) caused a significant increase in glycolysis. Analysis of glycolytic intermediates suggested that the enhancement of glycolysis was induced by activation of 6-phosphofructokinase. The data, thus, indicate that treatment of human erythrocytes with PLA2 significantly accelerates glucose utilization and suggest that the stimulation of glycolysis is caused by activation of 6-phosphofructokinase through liberation of free fatty acids of membrane phospholipids by PLA2.

Inhibition of glucose-induced insulin secretion through inactivation of glucokinase by glyceraldehyde.

D-Glyceraldehyde irreversibly inhibited rat liver glucokinase in a concentration-dependent manner. The inactivation of glucokinase by glyceraldehyde was blocked by the presence of its substrates such as glucose and mannose. Glucokinase was highly sensitive to glyceraldehyde compared with some other glycolytic enzymes (from animal tissues) including hexokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate kinase. The amino acid analysis of untreated and glyceraldehyde-treated glucokinase suggested that glyceraldehyde-induced inactivation of glucokinase is caused by glycation of Lys residues of the enzyme by the triose. Treatment of pancreatic islets with 6 mM glyceraldehyde for 1 h at 37 degrees C caused both inactivation of glucokinase and inhibition of glucose-induced insulin secretion. Another glucose-phosphorylating enzyme (hexokinase) in pancreatic islets, however, was little affected by glyceraldehyde. In addition, glyceraldehyde did not affect the insulin secretory responses of islets to nonglucose secretagogues such as glyceraldehyde and Leu. When pancreatic islets were cultured with a lower concentration (1 mM) of glyceraldehyde for a longer time (17 h) in the presence of 10 mM glucose to mimic the in vivo conditions, both glucokinase activity and glucose-induced insulin secretion were again decreased. This study demonstrates that glucose-induced insulin secretion is impaired by glyceraldehyde through the inactivation of glucokinase. The implication of this finding in the pathophysiology of type II diabetes is discussed.

Participation of glucokinase inactivation in inhibition of glucose-induced insulin secretion by 2-cyclohexen-1-one.

We assessed our speculation that 2-cyclohexen-1-one (CHX) impairs glucose-induced insulin secretion through inactivation of glucokinase. Treatment of pancreatic islets with CHX at concentrations (0-5 mM) that caused a dose-dependent inactivation of glucokinase activity similarly inhibited glucose-induced insulin secretion. Another glucose-phosphorylating enzyme (hexokinase) in pancreatic islets was little affected by CHX. CHX-induced inactivation of glucokinase was blocked by the presence of its substrates (glucose and mannose) and an inhibitor (N-acetylglucosamine), all of which also protected against the inhibitory effect of the drug on glucose-induced insulin secretion. CHX also impaired insulin secretion induced by D-glyceraldehyde and dimethyl succinate, which are believed to stimulate the release of the hormone by being directly oxidized by glyceraldehyde-3-phosphate dehydrogenase, by entering the midstream of the glycolytic pathway as glyceraldehyde 3-phosphate, or by entering the tricarboxylic acid cycle in mitochondria after intracellular hydrolysis. The inhibitory effect of CHX on glucose-induced insulin secretion, however, was far more marked than that on insulin secretion evoked by D-glyceraldehyde and dimethyl succinate at any CHX concentrations used. Our study revealed that the inhibitory action of CHX on glucose-induced insulin secretion is exerted mainly, but not solely, through inactivation of glucokinase. This conclusion supports the view that glucokinase is a key enzyme in the recognition of glucose as an insulin secretagogue in pancreatic islets.

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 alpha or beta 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 alpha anomer of D-glucose than the beta 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 alpha anomer of D-glucose, may exist for both insulin secretion and biosynthesis.

Circadian rhythmicity within single cells of Paramecium bursaria.

Cell populations of Paramecium bursaria show mating reactivity in the light period, but not in the dark period, when exposed to a light-dark cycle (LD 12:12). After they are transferred to constant-light (LL) conditions (1,000 lux), they continue to show a circadian rhythm of mating reactivity. The rhythm gradually dampens in LL so that mating reactivity in populations becomes arrhythmic in LL within 2 weeks. We wanted to know whether the arrhythmicity of this population was due to the absence of circadian rhythmicity within each individual cell, or merely due to asynchrony of a population of individually rhythmic cells. Therefore, single cells were isolated randomly from an arrhythmic population that had been in LL for a long time. Then the mating reactivity of these single cells was individually tested every 3 hr for 2 days. Each single cell showed a circadian mating rhythm in LL. This shows that the disappearance of the mating rhythm in cell populations under LL is not caused by disappearance of circadian rhythmicity within individual cells, but is due to desynchronization among cells in a population. When an arrhythmic population in LL is darkened for 9 hr, the mating reactivity rhythm of the cell population reappears. This occurs by resynchronization of the rhythms among individual cells, as can be shown by exposing single cells to pulses of 9 hr of darkness. This dark treatment causes phase shifts of single-cell rhythms, and a phase response curve is obtained for this stimulus. This phase-shifting behavior explains the efficacy of 9-hr dark pulses in restoring the population's rhythm.

Circadian rhythm of photoaccumulation in Paramecium bursaria.

Populations of Paramecium bursaria cells display a circadian rhythm of photoaccumulation. Both Chlorella-containing cells and Chlorella-free cells exhibit this rhythm. Several other species of Paramecium do not express rhythmic photoaccumulation when tested under the same conditions. In P. bursaria, photoaccumulation rhythms persist in continuous conditions (constant temperature and either continuous light or continuous darkness). The period of this rhythm is "temperature-compensated," with a Q10 of 1.10-1.12. The rhythm can be reset by pulses of light or darkness in a phase-dependent manner. Therefore, an endogenous circadian oscillator controls photoaccumulation behavior in P. bursaria.

Inhibition of glucose-induced insulin secretion by 4-hydroxy-2-nonenal and other lipid peroxidation products.

Lipid peroxidation due to oxidative stress is accelerated under hyperglycemic conditions such as diabetes mellitus. The effect of 4-hydroxy-2-nonenal (HNE) and other lipid peroxidation products on the ability of isolated rat pancreatic islets to secrete insulin was examined in this study. HNE concentration- and time-dependently deteriorated glucose-induced insulin secretion: insulin secretion was decreased by 50% when measured after incubation of islets with 100 microM HNE for 1 h. Other lipid peroxidation products, e.g. 2-hexenal and 2-butenal, also inhibited glucose-induced insulin secretion. HNE at 100 microM lowered alpha-ketoisocaproate-induced insulin secretion, whereas leucine-induced insulin secretion was stimulated. Insulin secretion induced by 10 mM glyceraldehyde was slightly decreased by HNE. On the other hand, HNE severely decreased insulin secretion induced by 10 mM glyceraldehyde and 2.8 mM glucose. Glucose utilization and glucose oxidation were significantly lowered in islets treated with HNE. The amounts of fructose 1,6-bisphosphate and dihydroxyacetone phosphate in islets were decreased by treatment with HNE, whereas the amount of fructose 6-phosphate was increased. Our study indicates that HNE and other lipid peroxidation products impair insulin secretion induced by glucose probably through affecting both the glycolytic pathway and the citric acid cycle.

Localization of glucokinase-like immunoreactivity in the rat lower brain stem: for possible location of brain glucose-sensing mechanisms.

Pancreatic glucokinase (GK) is considered an important element of the glucose-sensing unit in pancreatic beta-cells. It is possible that the brain uses similar glucose-sensing units, and we employed GK immunohistochemistry and confocal microscopy to examine the anatomical distribution of GK-like immunoreactivities in the rat brain. We found strong GK-like immunoreactivities in the ependymocytes, endothelial cells, and many serotonergic neurons. In the ependymocytes, the GK-like immunoreactivity was located in the cytoplasmic area, but not in the nucleus. The GK-positive ependymocytes were found to have glucose transporter-2 (GLUT2)-like immunoreactivities on the cilia. In addition, the ependymocytes had GLUT1-like immunoreactivity on the cilia and GLUT4-like immunoreactivity densely in the cytoplasmic area and slightly in the plasma membrane. In serotonergic neurons, GK-like immunoreactivity was found in the cytoplasm and their processes. The present results raise the possibility that these GK-like immunopositive cells comprise a part of a vast glucose-sensing mechanism in the brain.

4-Hydroxy-2-nonenal hardly affects glycolysis.

4-Hydroxy-2-nonenal (HNE), one of the major products of lipid peroxidation, inactivated the rate-limiting enzymes (from animal sources) of the glycolytic pathway and the pentose phosphate pathway when incubated at 37 degrees C for 1 h in the absence of glutathione (GSH). The HNE concentration for half-maximal inactivation of 6-phosphofructokinase (PFK) and glyceraldehyde-3-phosphate dehydrogenase was 3-10 microM; and that value for pyruvate kinase, glucose-6-phosphate dehydrogenase, and hexokinases I and II was 0.15-0.6 mM. In the presence of 5 mM GSH, however, only PFK, irrespective of the source (muscle, liver, or erythrocyte), was inactivated by 40-50% when incubated with 0.1 mM HNE for 1 h. Even PFK was not inactivated in the presence of both GSH and its substrate, ATP (2 mM). Glycolysis in human erythrocytes was not affected by treatment of cells with 0.1 mM HNE at 37 degrees C for 30 min. The results suggest that HNE, at concentrations observable under physiological and pathological conditions, hardly affects glycolysis in cells.

Changes in subcellular and zonal distribution of glucokinase in rat liver during postnatal development.

Subcellular and zonal distribution of glucokinase in rat liver during postnatal development was examined immunohistochemically. Before day 11 after birth, only some hepatocytes were immunostained, and a positive immunostaining was found in the cytoplasm but not in the nucleus. No zonal distribution of glucokinase was observed in livers of such pups. From day 15, at which time a dietary change from milk to laboratory chow begins to take place, glucokinase immunoreactivity increased; this increase was associated with increases in glucokinase activity and in glucokinase protein, and also the immunostaining was observed mainly in the nuclei. At day 21, the glucokinase immunoreactivity was found almost exclusively in the perivenous zone. At day 30, an intense immunostaining was seen both in the perivenous zone and in the periportal zone, being slightly predominant in the former. The present results indicate that dramatic changes in the distribution of glucokinase in developing rat liver may be related to dietary change.

Glucokinase is located in secretory granules of pancreatic D-cells.

We immunohistochemically examined the distribution of glucokinase in rat pancreatic islets. Glucokinase immunoreactivity under light microscopy was detected in the cytoplasm of somatostatin cells as well as in that of insulin cells. No specific immunoreactivity was detected in glucagon and pancreatic polypeptide cells. In somatostatin cells, glucokinase immunoreactivity was located by electron microscopy exclusively within secretory granules.

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 beta-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.

Non-enzymatic reduction of alloxan by reduced nicotinamide nucleotide.

Much evidence has been reported that the diabetogenic action of alloxan is caused by the formation of cytotoxic free radicals during the autoxidation of dialuric acid, a reduction product of alloan, to alloxan. The mechanism by which alloxan is reduced in vivo to dialuric acid, however, is unknown. The non-enzymatic reaction of alloxan with NAD(P)H was studied as a possible candidate for the reduction of alloxan. The reaction was carried out at 37 degrees in 50 mM phosphate buffer (mostly at pH 7.0) and was followed by measuring the decrease in absorbance at 340 nm. NADH and NADPH were found to be stoichiometrically oxidized by alloxan to NAD and NADP respectively. When the alloxan concentration (1.0 mM) was kept constant and the concentration of NAD(P)H (0.05 to 0.2 mM) was varied, the rate of decrease in the relative concentration of NAD(P)H was almost constant, suggesting that the autoxidation of dialuric acid by O2 was rapid enough to neglect its presence in the medium. The reaction between alloxan and NAD(P)H was accelerated by decreasing the pH. Both the rate of decrease in NAD(P)H concentration and the rate of O2 consumption resulting from autoxidation of the dialuric acid formed by reduction of alloxan were not affected by the presence of 20 mM D-glucose. Ethylene formation by the reaction of methional with . OH, one of the autoxidation products of dialuric acid, was clearly reduced by the presence of alpha- or beta-D-glucose (20 mM), but there was no significant difference between the effects of the two anomers. These results with D-glucose ruled out the possibility that the protection of beta-cells by D-glucose against the diabetogenicity of alloxan can be explained either by its inhibitory action on dialuric acid formation or by its scavenging effect on . OH.

In vivo formation of a Schiff base of aminoguanidine with pyridoxal phosphate.

Aminoguanidine (AG) is considered to be a promising compound for the treatment of diabetic complications. We examined the in vitro and in vivo formation of Schiff bases of AG with pyridoxal 5'-phosphate (PLP) and pyridoxal (PL). AG reacted in vitro far more rapidly with PLP to form a Schiff base (PLP-AG) than with PL to form another Schiff base (PL-AG). Administration of AG at 7 mM in drinking water for 18 weeks caused the formation of PLP-AG in the liver and kidney of mice (12.1 +/- 1.6 and 3.8 +/- 0.64 nmol/g of tissue, respectively, mean +/- SD, N = 6). The amount of PLP in the liver of mice AG administered was significantly lower than that of control mice (4.0 +/- 1.4 vs 17.4 +/- 1.3 nmol/g of wet tissue, mean +/- SD, N = 6). Simultaneous administration of pyridoxine (1 mM in drinking water) with AG (7 mM in drinking water) did not ameliorate the decrease in tissue PLP and caused the excess formation of PLP-AG. The results suggest that attention should be paid to the deficiency of tissue PLP in the clinical use of AG.