Preference of glucokinase for the alpha anomer of hexose: relation to alpha-anomer preference in hexose-induced insulin release by pancreatic islets

The alpha anomers of glucose and mannose were more rapidly phosphorylated than the beta anomers by rat liver glucokinase, whose chromatographic and kinetic properties are known to be quite similar to those of pancreatic islet glucokinase. This result may explain our previous findings of the alpha-anomeric preference in glucose- and mannose-stimulated insulin release, and therefore suggests to support the hypothesis that glucokinase in islets functions as a crucial hexose sensing enzyme for insulin release induced by glucose and mannose.     

Heat shock restores insulin secretion after injury by nitric oxide by maintaining glucokinase activity in rat islets

Heat shock protein (hsp), including hsp70, has been reported to restore the glucose-induced insulin release suppressed by nitric oxide (NO). However, the mechanism underlying this recovery remains unclear. In the present study, we examine the effects, in rat islets, of heat shock on insulin secretion inhibited by a small amount of NO and also on glucose metabolism, the crucial factor in insulin release. Exposure to a higher dose (15 U/ml) of interleukin-1beta (IL-1beta) abolished the insulin release by stimulation of glucose or KCl in both control and heat shocked islets. In rat islets exposed to a lower dose (1.5 U/ml) of IL-1beta, insulin secretion in response to glucose, but not to glyceraldehydes (GA), ketoisocaproate (KIC), or KCl, was selectively impaired, concomitantly with lower ATP concentrations in the presence of 16.7 mM glucose, while such suppression of insulin secretion and ATP content was not observed in heat shock-treated islets. NO production in islets exposed to 1.5 U/ml IL-1beta was significantly, but only partly, decreased by heat shock treatment. The glucose utilization rate measurement using [5-3H]-glucose and [2-3H]-glucose and the glucokinase activity in vitro were reduced in islets treated with 1.5 U/ml IL-1beta. In heat shock-treated islets, glucose utilization and glucokinase activity were not affected by 1.5 U/ml IL-1beta. These data suggest that heat shock restores glucose-induced insulin release inhibited by NO by maintaining glucokinase activity and the glucose utilization rate in islets in addition to reducing endogenous NO production.     

Precocious induction of hepatic glucokinase and malic enzyme in artificially reared rat pups fed a high-carbohydrate diet

Glucokinase and NADP:malate dehydrogenase (malic enzyme) first appear in liver when rat pups are weaned from milk which is high in fat to lab chow which is high in carbohydrate. To examine the influence of diet during the early neonatal period, before developmental changes in the circulating concentrations of thyroid and adrenocortical hormones occur, high-carbohydrate formula (56% of calories from carbohydrate), isocaloric and isonitrogenous with rat milk, was intermittently infused via gastrostomy starting on the second day of life. Pups had no further access to their dams. Body weights attained by these pups were at least 90% of those attained by mother-fed pups, which served as controls. In artificially reared rats fed the high-carbohydrate formula, on Day 4, glucokinase and malic enzyme were 30 and 18% of adult activity, respectively; on Day 10, glucokinase and malic enzyme were 71 and 96% of adult activity, respectively. On Days 4 and 10 glucose-6-phosphate dehydrogenase was elevated four- to fivefold in pups fed the high-carbohydrate formula compared to mother-fed pups. A second isocaloric formula, with 22% of calories from carbohydrate but low in protein, resulted in intermediate levels of all three enzymes on Day 10. Pups fed the high-carbohydrate formula has plasma insulin concentrations four- to fivefold greater than mother-fed pups on both Days 4 and 10. Triiodothyronine administration (1 microgram/g body wt) on Day 1 enhanced the induction of malic enzyme but not glucokinase on Day 4 in pups fed the high-carbohydrate formula. The results demonstrate that neonatal rat liver is competent to respond to high carbohydrate intake by induction of glucokinase and malic enzyme.     

Time-dependent Mechanisms in Beta-cell Glucose Sensing

The relation between plasma glucose and insulin release from pancreatic beta-cells is not stationary in the sense that a given glucose concentration leads to a specific rate of insulin secretion. A number of time-dependent mechanisms appear to exist that modify insulin release both on a short and a longer time scale. Typically, two phases are described. The first phase, lasting up to 10 min, is a pulse of insulin release in response to fast changes in glucose concentration. The second phase is a more steady increase of insulin release over minutes to hours, if the elevated glucose concentration is sustained. The paper describes the glucose sensing mechanism via the complex dynamics of the key enzyme glucokinase, which controls the first step in glucose metabolism: phosphorylation of glucose to glucose-6-phosphate. Three time-dependent phenomena (mechanisms) are described. The fastest, corresponding to the first phase, is a delayed negative feedback regulating the glucokinase activity. Due to the delay, a rapid glucose increase will cause a burst of activity in the glucose sensing system, before the glucokinase is down-regulated. The second mechanism corresponds to the translocation of glucokinase from an inactive to an active form. As the translocation is controlled by the product(s) of the glucokinase reaction rather than by the substrate glucose, this mechanism gives a positive, but saturable, feedback. Finally, the release of the insulin granules is assumed to be enhanced by previous glucose exposure, giving a so-called glucose memory to the beta-cells. The effect depends on the insulin release of the cells, and this mechanism constitutes a second positive, saturable feedback system. Taken together, the three phenomena describe most of the glucose sensing behaviour of the beta-cells. The results indicate that the insulin release is not a precise function of the plasma glucose concentration. It rather looks as if the beta-cells just increase the insulin production, until the plasma glucose has returned to normal. This type of integral control has the advantage that the precise glucose sensitivity of the beta-cells is not important for normal glucose homeostasis.     

Identification of two forms of glutamine synthetase in barley (Hordeum vulgare).

Hepatocytes of 14-day-old rats have no detectable glucokinase activity $\text{in}$$\text{vivo}$, but it was induced by insulin (10^?8M) in primary cultures of these hepatocytes. The glucokinase induced by insulin was separated by electrophoresis on a cellulose acetate membrane and identified by its low affinity for glucose. This precocious induction of glucokinase was completely prevented by the presence of either actinomycin D or cycloheximide. Glucagon also inhibited its induction by insulin. Dexamethasone and testosterone, which alone had no inductive effect, strongly enhanced the induction by insulin. When hepatocytes of 14-day-old rats were cultured with 10^?7M insulin, 10^?6M dexamethasone and 10^?7M testosterone for 48 hr, their glucokinase activity increased to the non-induced level in hepatocytes of adult rats. Estrogen, thyroxine or growth hormone did not induce glucokinase precociously. Testosterone did not enhance induction of glucokinase by insulin in cultured hepatocytes of adult rats.     

Adaptive character of liver glucokinase

1. Glucokinase is one of four glucose phosphorylating enzymes present in rat liver. Its distinctive features are a high K-m for glucose (high-K-m isozyme) and a rather narrow substrate specificity. In contrast, the other three enzymes, collectively called hexokinases or low-K-m isozymes, exhibit low K-m values for glucose and a wider substrate specificity. 2. Glucokinase is present in the liver os mammals (with some exceptions), amphibians and lower reptiles; It is absent from higher reptiles and birds. The presence or absence of glucokinase may represent an evolutionary adaptation to feeding habits and other physiological peculiarities. Differences in the immunological behavior and in the kinetic parameters of glucokinases from different taxa suggest the operation of divergent evolution. 3. The levels of glucokinase in rat liver depend strictly on the supply of carbohydrate in the diet. Glycogen phosphorylase and glycogen synthetase behave similarly, whereas other carbohydrate-metabolizing enzymes depend on the provision of either protein or protein plus carbohydrate. Glucokinase decays with a half-life of 33 hr when rats are starved or fed a carbohydrate-free diet, and is induced by the administration of glucose. The adaptive character is not exhibited by all mammals, indicating evolutionary discrimination within the same class and even within the same single order Rodentia. Enzyme adaptation in the liver may partially explain the condition known as 'hunger diabetes'. 4. The endocrine system plays a paramount role in glucokinase adaptation, since insulin is essential for glucose-dependent glucokinase induction and, on the other hand, glucagon, catecholamines and cyclic AMP prevent the induction. Glucocorticoids and some pituitary hormones modulate the rate of induction. The mechanisms underlying the hormonal regulation of glucokinase levels are not well known. 5. The variations in liver glucokinase correspond to changes in the amount of enzyme protein as assessed by immunochemical titration. This fact agrees with the effects of inhibitors of protein synthesis on glucokinase induction. 6. An antiserum against rat glucokinase reacts with the enzyme from mammals and turtles but not with the amphibian enzyme. It does not react with low-K-m hexokinases from different sources. 7. The saturation function for glucose is sigmoidal in mammalian and amphibian glucokinases but not in glucokinase from lower reptiles. The Hill's coefficient is very constant with values about 1.6. The K0.5 (concentration for half saturation) values in the different species studied vary between 1.5 and 8 mM. These kinetic parameters may be considered as another adaptive feature aimed to give maximal efficiency to the liver uptake of glucose at the changeable concentrations in the blood resulting from variations in the amount of dietary glucose.     

Hexose metabolism in pancreatic islets. — Galactose transport,phosphorylation and oxidation

Summary In rat pancreatic islets, the apparent space of distribution of galactose is not different from that of other hexoses. In homogenates of islets or tumoral insulin-producing cells, galactose is phosphorylated at a very low rate relative to either glucose phosphorylation in the same tissues or galactose phosphorylation by liver homogenates. In intact islets, galactose increases modestly the glucose 6-phosphate content and is oxidized at a much lower rate than glucose. Galactose slightly increases insulin output in the presence of a stimulatory concentration of glucose but fails to provoke insulin release in the absence of glucose, whether in islets removed from rats fed a normal or galactose-rich diet. The low rate of galactose oxidation and its poor insulinotropic capacity appear attributable to the weak activity of galactokinase in pancreatic islets.     

Regulation of the gene expression of glucokinase and L-type pyruvate kinase in primary cultures of rat hepatocytes by hormones and carbohydrates

The regulation of the gene expression of two important glycolytic enzymes, glucokinase and L-type pyruvate kinase, by hormones and carbohydrates was studied, in primary cultures of adult rat hepatocytes. Insulin caused time- and dose-dependent increases in the amounts of the mRNAs of the two enzymes in hepatocytes, although glucokinase responded to this hormone faster than L-type pyruvate kinase. The induction of glucokinase mRNA by insulin did not require the presence of glucose itself, but that of the L-type isozyme was dependent on the glucose concentration. For this effect, fructose and glycerol could partially substitute for glucose, but pyruvate and 2-deoxyglucose, a nonmetabolizable glucose analog, could not. The time course of insulin induction in the presence of fructose, but not of glycerol, was similar to that in the presence of glucose. In the presence of glycerol, the mRNA increased in a diphasic manner: the first increase, which probably reflected the effects of fructose and glycerol in normal liver, reached a maximum after 3 h, whereas the second increase corresponded to the increase in the presence of glucose. These results suggested that some metabolite of glucose was required for the insulin-induced increase in L-type pyruvate kinase mRNA. Cycloheximide inhibited the effects of insulin on the two mRNAs, suggesting that ongoing protein synthesis is required in both cases. The addition of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine, an inhibitor of protein kinase C, also inhibited the effects of insulin. However, phorbol 12-myristate 13-acetate alone did not induce the two mRNAs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of glucose-stimulated insulin release by pseudo-alpha-DL-glucose as a glucokinase inhibitor

Pseudo-alpha- and pseudo-beta-DL-glucose, the isomers of 5-hydroxymethyl-1,2,3,4-cyclohexanetetrol with alpha-gluco and beta-gluco configurations, were used as synthetic analogs of glucose anomers to study the mechanism of glucose-stimulated insulin release by pancreatic islets. Neither isomer was phosphorylated by liver glucokinase nor stimulated insulin release from islets. Incubation of islets with pseudo-alpha-DL-glucose resulted in a considerable accumulation of the glucose analog, probably the D form, in islets. The alpha-isomer, but not the beta-isomer, inhibited both glucose-stimulated insulin release (44% inhibition at 20 mM) and islet glucokinase activity (36% inhibition at 20 mM) in a concentration-dependent manner and to a comparable degree. These results strongly suggest that the inhibition of glucose-stimulated insulin release by pseudo-alpha-DL-glucose is due to the inhibition of islet glucokinase by the glucose analog, providing additional evidence for the essential role of islet glucokinase in glucose-stimulated insulin release.     

Glucose regulates glucokinase activity in cultured islets from rat pancreas

In this study, we have used isolated pancreatic islets cultured for 7 days in 3 or 30 mM glucose to explore whether glucokinase is induced or activated by high glucose concentrations and has related enzyme activity to glucose-stimulated insulin release. Islets cultured in low glucose medium or low glucose medium plus 350 ng/ml insulin did not respond to high glucose stimulation. Islets cultured in medium containing high glucose concentrations showed a high rate of basal insulin secretion when perifused with 5 mM glucose, and the insulin release was greatly augmented in a biphasic secretion profile when the glucose concentration was raised to 16 mM. Islet glucokinase and hexokinase activities were determined by a sensitive and specific fluorometric method. Glucokinase activity was reduced to approximately 50% in islets cultured in low glucose medium with or without insulin present compared to results with fresh islets. However, islets cultured in 30 mM glucose showed that glucokinase activity was elevated to 236% compared to results with fresh islets. It is concluded that (a) glucose is the physiological regulator of glucokinase in the islet of Langerhans and that (b) the activity of glucokinase plays a crucial role in glucose-induced insulin secretion.     

Insulin and tri-iodothyronine induce glucokinase mRNA in primary cultures of neonatal rat hepatocytes.

Glucokinase (EC 2.7.1.2) first appears in the liver of the rat 2 weeks after birth and increases after weaning on to a high-carbohydrate diet. We investigated the hormonal regulation of glucokinase (GK) mRNA in primary cultures of hepatocytes from 10-12-day-old suckling rats. GK mRNA was undetectable in such cells after 48 h of culture in serum-free medium devoid of hormones. Addition of insulin or tri-iodothyronine (T3) to the medium resulted in induction of GK mRNA. The effects of insulin and T3 were dose-dependent and additive. Dexamethasone alone did not induce GK mRNA, but enhanced the response to insulin and decreased the response to T3. Induction of GK mRNA by insulin was not affected when the medium glucose concentration was varied between 5 and 15 mM, nor when culture was conducted in glucose-free medium supplemented with lactate and pyruvate or galactose. The time course of initial accumulation of GK mRNA in response to insulin was characterized by a lag of 12 h and an induction plateau reached after 36 h. If hepatocytes were then withdrawn from insulin for 24 h and subsequently subjected to a secondary stimulation by insulin, GK mRNA re-accumulated with much faster kinetics and reached the fully induced level within 8 h. Both primary and secondary responses to insulin were abolished by actinomycin D. These results provide insight into the role of hormonal stimuli in the ontogenic development of hepatic glucokinase.     

Inhibition of glucose-induced insulin release by 3-O-methyl-D-glucose: Enzymatic,metabolic and cationic determinants

The analog of D-glucose, 3-O-methyl-D-glucose, is thought to delay the equilibration of D-glucose concentration across the plasma membrane of pancreatic islet B-cells, but not to exert any marked inhibitory action upon the late phase of glucose-stimulated insulin release. In this study, however, 3-O-methyl-D-glucose, when tested in high concentrations (30-80 mM) was found to cause a rapid, sustained and not rapidly reversible inhibition of glucose-induced insulin release in rat pancreatic islets. In relative terms, the inhibitory action of 3-O-methyl-D-glucose was more marked at low than high concentrations of D-glucose. It could not be attributed to hyperosmolarity and appeared specific for the insulinotropic action of D-glucose, as distinct from non-glucidic nutrient secretagogues. Although 3-O-methyl-D-glucose and D-glucose failed to exert any reciprocal effect upon the steady-state value for the net uptake of these monosaccharides by the islets, the glucose analog inhibited D-[5-3H]glucose utilization and D-[U-14C]glucose oxidation. This coincided with increased 86Rb outflow and decreased 45Ca outflow from prelabelled islets, as well as decreased 45Ca net uptake. A preferential effect of 3-O-methyl-D-glucose upon the first phase of glucose-stimulated insulin release was judged compatible with an altered initial rate of D-glucose entry into islet B-cells. The long-term inhibitory action of the glucose analog upon the metabolic and secretory response to D-glucose, however, may be due, in part at least, to an impaired rate of D-glucose phosphorylation. The phosphorylation of the hexose by beef heart hexokinase and human B-cell glucokinase, as well as by parotid and islet homogenates, was indeed inhibited by 3-O-methyl-D-glucose. The relationship between insulin release and D-glucose utilization or oxidation in the presence of 3-O-methyl-D-glucose was not different from that otherwise observed at increasing concentrations of either D-glucose or D-mannoheptulose. It is concluded, therefore, that 3-O-methyl-D-glucose adversely affects the metabolism and insulinotropic action of D-glucose by a mechanism largely unrelated to changes in the intracellular concentration of the latter hexose.     

A computer model of pancreatic islet glycolysis

We have modeled an experiment with perifused pancreatic islet cells using our BIOSSIM language. The experiment and the resulting model are concerned with glucose uptake and glycolysis by the beta-cells of pancreatic islets. Although glycolysis appears to be involved in insulin release, we do not have enough information to represent insulin release in detail. The rapid entry of glucose into the beta-cell is promoted by a carrier having a very high tissue capacity. Phosphorylation of glucose by the low affinity enzyme glucokinase appears to be limiting for glycolysis. The effects of several hexose diphosphate activators of phosphofructokinase are modeled. Model behavior is described. The kinetic parameters of the enzyme submodels are given. Because of the difficulties of preparing large amounts of experimental material, information on pancreatic islet metabolism is limited. This model is a plausible explanation of the experimental results. Recent work on the genetically engineered glucose transporter and glucokinase is discussed.     

Effect of dichlorvos on hepatic and pancreatic glucokinase activity and gene expression, and on insulin mRNA levels

Several studies have shown that organophosphate pesticides affect carbohydrate metabolism and produce hyperglycemia. It has been reported that exposure to the organophosphate pesticide dichlorvos affects glucose homeostasis and decreases liver glycogen content. Glucokinase (EC 2.7.1.1) is a tissue-specific enzyme expressed in liver and in pancreatic beta cells that plays a crucial role in glycogen synthesis and glucose homeostasis. In the present study we analyzed the effect of one or three days of dichlorvos administration [20 mg/kg body weight] on the activity and mRNA levels of hepatic and pancreatic glucokinase as well as on insulin mRNA abundance in the rat. We found that the pesticide affects pancreatic and hepatic glucokinase activity and expression differently. In the liver the pesticide decreased the enzyme activity; on the contrary glucokinase mRNA levels were increased. In contrast, pancreatic glucokinase activity as well as mRNA levels were not affected by the treatment. Insulin mRNA levels were not modified by dichlorvos administration. Our results suggest that the decreased activity of hepatic glucokinase may account for the adverse effects of dichlorvos on glucose metabolism.     

Induction of glucokinase by insulin under the permissive action of dexamethasone in primary rat hepatocyte cultures.

Glucokinase, the organ specific key enzyme of glucose metabolism in liver, was studied in primary cultures of adult rat hepatocytes during the first two days after cell preparation. In the presence of dexamethasone low concentrations of insulin (10^?9 mol/l) prevented the observed time dependent decrease of glucokinase activity while higher insulin concentrations (10^?8 and 10^?7 mol/l) led to a twofold increase of enzyme activity. The enhancement of glucokinase activity was completely blocked by either actinomycin D or cycloheximide. The degree of this insulin dependent induction was correlated with the concentration of added dexamethasone, which seemed to perform a permissive function. The induction of glucokinase activity could be prevented by addition of glucagon (2 × 10^?7 mol/l).     

Inhibitory effect of PGE1 on the liberation of insulin induced by glibenclamide

Glycemia and insulinemia in rat blood samples have been determined at different times before and after administration of glibenclamide, PGE1, glibenclamide and PGE1, glibenclamide and glucose, PGE1 and glucose, and glibenclamide, PGE1 and glucose. PGE1 led to a partial inhibition of glibenclamide induced insulin release, with and without glucose administration, but a total inhibition did not occur. The inhibitory action of PGE1 on insulin secretion was also reflected on the glycemia curves. Defects in insulin release in diabetes could be due in part to an excessive production of PGs, that involve a failure in the beta-cells to respond to glucose signals. The present paper shows that glibenclamide secretory action was not cancelled out by PGE1. These results could explain the availability of glibenclamide in the treatment of diabetes mellitus.     

The insulin-mimetic action of Mn2+: involvement of cyclic nucleotides and insulin in the regulation of hepatic hexokinase and glucokinase

Manganese causes a significant rise in hepatic glucokinase and hexokinase in 16-day-old suckling rats, and has an insulinomimetic effect in producing a precocious emergence of glucokinase (EC 2.7.1.2) and a rise in the low Km, hexokinases (EC 2.7.1.1) activities. These enzyme changes occur within 6 hr of manganese administration and there are accompanying increases in plasma insulin and hepatic cyclic GMP. That the effect of manganese is at a site other than, or in addition to, insulin secretion is suggested by the significant increases in glucokinase and hexokinase in 16-day-old streptozotocin-diabetic rats; in this group there is also an increase in hepatic cGMP similar in time scale to that of the normal-manganese-treated group. The effects of manganese and insulin were not additive. It is proposed that one site of action of manganese may be at the level of cyclic GMP systems. The results are also discussed in relation to the known action of manganese at the level of the protein phosphatases.