Differential regulation of glucokinase activity in pancreatic islets and liver of the rat

The differential tissue-specific regulation of glucokinase activity in liver and pancreatic islet cells was investigated in the insulinoma-bearing rat. A transplantable insulinoma caused hyperinsulinemia and hypoglycemia in the host by 2-3 months after implantation. Suppression of the pancreatic B-cells by the high insulin and/or low glucose manifested itself by a decrease of insulin in islet tissue. Removal of the tumor initiated transient insulin deficiency and hyperglycemia with extremes of these changes at 24 h after tumor resection. These conditions markedly affected glucose phosphorylation in the islet cells: glucokinase activity was reduced 71% in islet samples from insulinoma-bearing rats, and the enzyme fully recovered within 24 h after tumor resection. Hexokinase activity, by contrast, was not affected by these manipulations. To evaluate the relative contributions of hypoglycemia and hyperinsulinemia in islet glucokinase adaptation, glucose was intravenously infused to insulinoma-bearing rats; glycemia in excess of 150 mg/100 ml combined with excessive hyperinsulinemia resulted in a partial recovery of islet glucokinase activity, first apparent after 9 h of glucose infusion and with doubling of the activity after 24 h after glucose loading. In contrast, liver glucokinase was increased nearly 4-fold at the time of extreme hypoglycemia and hyperinsulinemia and rapidly fell to control rates following tumor removal. Intravenous infusion of glucose for 24 h into the tumor-bearing rat (i.e. hyperglycemia combined with excessive plasma insulin) had no influence on liver glucokinase activity. Liver hexokinase was not influenced by any of these experimental manipulations. The data indicate that the activities of pancreatic islet and liver glucokinase are regulated in a differential manner. Insulin is apparently the primary determinant of liver glucokinase and glucose seems to control islet glucokinase. Biochemical mechanisms for differential organ-specific regulation of glucokinase activity seem to have evolved such that this enzyme may play a dual role in glucose homeostasis, namely to serve as insulin-dependent glucose sensor in the B-cells and as insulin-sensitive determinant of hepatic glucose use.     

Tacrolimus suppresses glucose-induced insulin release from pancreatic islets by reducing glucokinase activity

Tacrolimus is widely used for immunosuppressant therapy, including various organ transplantations. One of its main side effects is hyperglycemia due to reduced insulin secretion, but the mechanism remains unknown. We have investigated the metabolic effects of tacrolimus on insulin secretion at a concentration that does not influence insulin content. Twenty-four-hour exposure to 3 nM tacrolimus reduced high glucose (16.7 mM)-induced insulin secretion (control 2.14 +/- 0.08 vs. tacrolimus 1.75 +/- 0.02 ng.islet(-1).30 min(-1), P < 0.01) without affecting insulin content. In dynamic experiments, insulin secretion and NAD(P)H fluorescence during a 20-min period after 10 min of high-glucose exposure were reduced in tacrolimus-treated islets. ATP content and glucose utilization of tacrolimus-treated islets in the presence of 16.7 mM glucose were less than in control (ATP content: control 9.69 +/- 0.99 vs. tacrolimus 6.52 +/- 0.40 pmol/islet, P < 0.01; glucose utilization: control 103.8 +/- 6.9 vs. tacrolimus 74.4 +/- 5.1 pmol.islet(-1).90 min(-1), P < 0.01). However, insulin release from tacrolimus-treated islets was similar to that from control islets in the presence of 16.7 mM alpha-ketoisocaproate, a mitochondrial fuel. Glucokinase activity, which determines glycolytic velocity, was reduced by tacrolimus treatment (control 65.3 +/- 3.4 vs. tacrolimus 49.9 +/- 2.8 pmol.islet(-1).60 min(-1), P < 0.01), whereas hexokinase activity was not affected. These results indicate that glucose-stimulated insulin release is decreased by chronic exposure to tacrolimus due to reduced ATP production and glycolysis derived from reduced glucokinase activity.     

Calcium and glycolysis mediate multiple bursting modes in pancreatic islets

Pancreatic islets of Langerhans produce bursts of electrical activity when exposed to stimulatory glucose levels. These bursts often have a regular repeating pattern, with a period of 10-60 s. In some cases, however, the bursts are episodic, clustered into bursts of bursts, which we call compound bursting. Consistent with this are recordings of free Ca2+ concentration, oxygen consumption, mitochondrial membrane potential, and intraislet glucose levels that exhibit very slow oscillations, with faster oscillations superimposed. We describe a new mathematical model of the pancreatic beta-cell that can account for these multimodal patterns. The model includes the feedback of cytosolic Ca2+ onto ion channels that can account for bursting, and a metabolic subsystem that is capable of producing slow oscillations driven by oscillations in glycolysis. This slow rhythm is responsible for the slow mode of compound bursting in the model. We also show that it is possible for glycolytic oscillations alone to drive a very slow form of bursting, which we call "glycolytic bursting." Finally, the model predicts that there is bistability between stationary and oscillatory glycolysis for a range of parameter values. We provide experimental support for this model prediction. Overall, the model can account for a diversity of islet behaviors described in the literature over the past 20 years.     

Transglutaminase activity in pancreatic islets

1. Pancreatic islet homogenates catalyze, in a Ca²⁺-dependent fashion, the incorporation of [2,5-³H]histamine, [1,4-¹⁴C]putrescine, [1,2-³H]agmatine, [¹⁴C]methylamine, L-[U-¹⁴C]lysine in N,N-dimethylcasein. 2. Using [2,5-³H]histamine as the amine donor, the K_m for Ca²⁺ and histamine amounts to 90μM and 0.7 mM, respectively. 3. The incorporation of [2,5-³H]histamine into N,N-dimethylcasein is inhibited by monodansylcadaverine, N-p-tosyl glycine, bacitracin and methylamine, the relative extent of inhibition depending on the respective concentrations of Ca²⁺, inhibitor and amine donor. 4. Bacitracin and methylamine, but not N-p-tosyl glycine, cause a dose-related inhibition of glucose-stimulated insulin release. 5. It is concluded that, in pancreatic islets, the Ca²⁺-responsive transglutaminase activity plays a critical role in the process of glucose-induced insulin release.     

Fasting-induced dissociation of cationic and secretory events in pancreatic islets

In pancreatic islets removed from 48 h-fasted rats, as distinct from fed animals, the release of insulin evoked by D-glucose is more severely impaired than that evoked by 2-ketoisocaproate. This decreased secretory response to D-glucose contrasts with an unimpaired cationic response to the sugar in terms of the glucose-induced decrease in both 86Rb and 45Ca outflow from pre-labelled islets. Likewise, fasting only causes a modest decrease of the secondary rise in 45Ca outflow evoked by D-glucose in islets perifused at normal Ca2+ concentration. The latter decrease appears more marked, however, if the cationic response to glucose is expressed relative to that evoked by 2-ketoisocaproate in islets removed from rats in the same nutritional state. It is concluded that, in the process of nutrient-stimulated insulin release, neither the decrease in K+ conductance (inhibition of 86Rb outflow) nor the sequestration of Ca2+ by intracellular organelles and/or direct inhibition of Ca2+ outward transport (decrease in 45Ca outflow) represent the sole determinant(s) of the subsequent gating of Ca2+ channels (secondary rise in 45Ca efflux).     

Regulation of glucokinase by a fructose-1-phosphate-sensitive protein in pancreatic islets

In the post-microsomal supernatant of pancreatic islets, prepared from fasted or fed rats, D-fructose 1-phosphate increased the activity of glucokinase by 20-30% as measured in the presence of D-glucose 6-phosphate and D-fructose 6-phosphate. Such an activation was less marked than that found in liver extracts. The islet cytosol was also found to inhibit purified liver glucokinase, and this effect was antagonized by D-fructose 1-phosphate. In the presence of hexose 6-phosphates, partially purified islet glucokinase was inhibited by the hepatic glucokinase regulatory protein in a D-fructose-1-phosphate-sensitive manner. In intact islets, D-glyceraldehyde stimulated the generation of 14C-labelled D-fructose 1-phosphate from D-[U-14C]glucose and increased the production of 3H2O from D-[5-3H]glucose. These findings suggest that the activity of glucokinase in islet cells may be regulated by a protein mediating the antagonistic effects of D-fructose 6-phosphate and D-fructose 1-phosphate in a manner qualitatively similar to that operating in hepatocytes, but with lower efficiency.     

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.     

Ionophoretic activity in pancreatic islets.

Extracts of pancreatic islets stimulate the translocation of calcium from an aqueous into an organic immiscible phase. This ionophoretic activity, which is derived mainly from membrane-rich subcellular fractions, displays several features in common with that of A23187 in the same model. The phenomenon of calcium translocation caused by either the islet extract or the antibiotic ionophore represents a power function of the concentration of ionophoretic material; it is saturable at high calcium concentrations, affected by the concentration of Na⁺ and pH of the aqueous phase, increased at low temperature, and inhibited by suloctidil, the latter inhibitory effect being antagonized by calcium itself. These findings underline the potential significance of native ionophores in the regulation of calcium movements across membrane systems in the islet cells.     

Enzyme-to-enzyme channelling in the early steps of glycolysis in rat pancreatic islets

The metabolism of D-glucose displays anomeric specificity in rat pancreatic islets. The aim of the present report is to investigate whether such a situation implies enzyme-to-enzyme tunnelling of metabolites in the early steps of glycolysis. For such a purpose, the modelling of alpha- and beta-D-glucose catabolism, itself based on available information concerning both the utilisation of these two anomers and the intrinsic properties of phosphoglucoisomerase, was first examined. According to a theoretical model with enzyme-to-enzyme channelling, the generation of 3HOH from D-[2-3H]glucose should be higher in islets exposed to beta-D-glucose rather than alpha-D-glucose, whilst the opposite situation should prevail in the case of D-[5-3H]glucose conversion to 3HOH. Experimental data collected in rat islets incubated for 60 min at 4 degrees C in the presence of either alpha- or beta-D-glucose mixed with tracer amounts of either alpha- or beta-D-[2- 3H]glucose and alpha- or beta-D-[5-3H]glucose indicate that the beta/alpha ratio for D-[2-3H]glucose conversion to 3HOH is indeed higher than the beta/alpha ratio for D-[5-3H]glucose conversion to 3HOH. These findings are consistent with the postulated enzyme-to-enzyme tunnelling of glycolytic intermediates between hexokinase isoenzyme(s), phosphoglucoisomerase and, possibly, phosphofructokinase.     

Anomeric specificity of human liver and B-cell glucokinase: modulation by the glucokinase regulatory protein

The anomeric specificity of the wild-type recombinant forms of human liver and B-cell glucokinase was investigated using radioactive anomers of d-glucose as tracers. With d-glucose at anomeric equilibrium and at 30 degrees C, the maximal velocity, Hill number, and K(s) amounted, respectively, to 16 micromol min(-1) mg(-1), 1.8 and 6.9 mM in the case of liver glucokinase, and 7.3 micromol min(-1) mg(-1), 2.0 and 7.1 mM in the case of B-cell glucokinase. Whether at 20-22 or 30 degrees C, the maximal velocity, Hill number, and K(m) were significantly lower with alpha-d-glucose than with beta-d-glucose in both liver and B-cell glucokinase. As a result of these differences, the reaction velocity was higher with alpha-d-glucose at low hexose concentrations, while the opposite situation prevailed at high hexose concentrations. In the presence of 0.2 mM d-fructose 6-phosphate, the glucokinase regulatory protein caused a concentration-related inhibition of d-glucose phosphorylation, such an effect fading out at high concentrations of either d-glucose or glucokinase relative to that of its regulatory protein. The phosphorylation of alpha-d-glucose by liver glucokinase appeared more resistant than that of beta-d-glucose to the inhibitory action of d-fructose 6-phosphate, as mediated by the glucokinase regulatory protein. Such a phenomenon failed to achieve statistical significance in the case of the B-cell glucokinase. It is proposed that this information, especially the novel findings concerning the anomeric difference in both Hill number and sensitivity to the glucokinase regulatory protein, should be taken into account when considering the respective contributions of alpha- and beta-d-glucose to the overall phosphorylation of equilibrated d-glucose by glucokinase.     

Anomeric preference of glucose phosphorylation and glycolysis in human erythrocytes

Lactate output from the alpha and beta anomers of glucose was measured in intact human erythrocytes at 37 degrees C; and glucose anomer phosphorylation, in human erythrocyte homogenates. The rates of both glucose metabolism (lactate output) and phosphorylation were higher in the presence of beta-D-glucose as distinct from alpha-D-glucose at three glucose concentrations used (2, 5, and 10 mM). Thus, the v beta/v alpha ratios of metabolism and phosphorylation of glucose at 2 mM were 1.24 and 1.22, respectively. The results indicate that the beta preference of hexokinase, a rate-limiting enzyme in glycolysis, is reflected in beta-preferential glycolysis.     

Aromatic-L-amino-acid decarboxylase activity in mouse pancreatic islets

Aromatic-L-amino-acid decarboxylase activity has been measured in intact or homogenised pancreatic islets of ob/ob mice (Ume? ob/ob). The method used involves the trapping and measuring of the 14CO2 released from L-[1-14C]dihydroxyphenylalanine (L-dopa). Islets showed a decarboxylase activity which was dependent on pyridoxal phosphate and inhibitable by 0.1 mM benserazide or 0.1 mM alpha-monofluoromethyldopa. Maximum activity in intact islets was about 330 mmol/kg dry islet per h with an apparent Km of 3.3 mM. Islet homogenates had a Vmax of about 120 mmol/kg per h with a Km of 0.3 mM. L-5-Hydroxytryptophan, m-tyrosine and o-tyrosine interfered with the decarboxylation of L-dopa in a way that suggested a high activity also towards those substrates. L-Phenylalanine, L-tyrosine and D-glucose had no effect. At 0.05 mM L-dopa islet homogenates showed a much higher activity than homogenates of liver, kidney, or spleen. Islet uptake of L-[3H]dopa was well in excess of the decarboxylation rate and thus probably not rate-limiting. It is concluded that mouse pancreatic islets have a high activity of aromatic-L-amino-acid decarboxylase. This is in accordance with previous suggestions of a stimulatory effect of this enzyme on insulin secretion.     

Hexose metabolism in pancreatic islets. Regulation of aerobic glycolysis and pyruvate decarboxylation.

1. D-Glucose (0.5-16.7 mM) preferentially stimulates aerobic glycolysis and D-[3,4-14C]glucose oxidation, relative to D-[5-3H]glucose utilization in rat pancreatic islets, the concentration dependency of such a preferential effect displaying a sigmoidal pattern. 2. Inorganic and organic calcium antagonists, as well as Ca2+ deprivation, only cause a minor decrease in the ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization in islets exposed to a high concentration of the hexose (16.7 mM). 3. Non-glucidic nutrient secretagogues such as 2-aminobicyclo[2,2,1]heptane-2-carboxylate (BCH), 2-ketoisocaproate and 3-phenylpyruvate fail to stimulate aerobic glycolysis and D-[3,4-14C]glucose oxidation in islets exposed to 6.0 mM D-glucose. Nevertheless, BCH augments [1-14C]pyruvate and [2-14C]pyruvate oxidation. 4. The glucose-induced increment in the paired ratio between D-[3,4-14C]glucose oxidation and D-[5-3H]glucose utilization is impaired in the presence of either cycloheximide or ouabain. 5. These findings suggest that the preferential effect of D-glucose upon aerobic glycolysis and pyruvate decarboxylation is not attributable solely to a Ca(2+)-induced activation of FAD-linked glycerophosphate dehydrogenase and/or pyruvate dehydrogenase, but may also involve an ATP-modulated regulatory process.     

Anomeric specificity of glycolysis in a non glucokinase-containing cell

In rat erythrocyte homogenates, the phosphorylation of D-glucose measured at 30 degrees C over a wide range of glucose concentrations (50 microM to 20 mM) yielded in a double reciprocal plot a single straight line with a Km close to 0.06 mM and a maximal velocity close to 47 nmol/60 min per mg hemoglobin. At 8 degrees C, the rate of glucose phosphorylation was 60% higher in the presence of beta-D-glucose than alpha-D-glucose. Yet, in intact erythrocytes incubated at 8 degrees C in the presence of beta-D-glucose (4 or 7 mM), the glucose-induced increment in lactic acid output represented no more than 39 to 74% of that found in the presence of alpha-D-glucose. Thus, a greater rate of glycolysis in the presence of alpha-D-glucose was observed in a cell devoid of glucokinase and containing a hexokinase with preference for beta-D-glucose. These findings indicate that the anomeric specificity of glycolysis in intact cells cannot be predicted and does not necessarily depend on the anomeric preference of glucose-phosphorylating enzyme(s).     

Anomeric preference of glucose utilization in human erythrocytes loaded with glucokinase

Human erythrocytes were loaded with homogeneous rat liver glucokinase by an encapsulation method based on hypotonic hemolysis and isotonic resealing. As assayed at 10 mM glucose, glucokinase and hexokinase activities in glucokinase-loaded erythrocytes were 218 and 384 nmol/min/gHb, respectively; whereas hexokinase activity in both intact and unloaded red cells, which contain no glucokinase activity, was about 400 nmol/min/gHb. No difference in the rate of lactate production from glucose anomers between intact and unloaded erythrocytes suggested that the encapsulation procedure itself did not affect glucose utilization in red cells. Alpha-anomeric preference in lactate production from glucose was observed in glucokinase-loaded erythrocytes, whereas the beta anomer of glucose was more rapidly utilized than the alpha anomer in intact and unloaded erythrocytes. The results indicate that the step of glucose phosphorylation determines the anomeric preference in glucose utilization by human erythrocytes, since glucokinase and hexokinase are alpha- and beta-preferential, respectively, in glucose phosphorylation.     

Anomeric specificity of hexokinase and glucokinase activities in liver and insulin-producing cells.

Conflicting data have been reported concerning the anomeric specificity of glucokinase. In the present study, liver hexokinase (Km for D-glucose 0.4 mM) displayed a higher affinity for but lower Vmax. with alpha- than with beta-D-glucose. The velocity of the reaction catalysed by liver glucokinase was higher with with beta- than with alpha-D-glucose, whatever the glucose concentration. The apparent Km of glucokinase was somewhat lower, however, with alpha- than with beta-D-glucose. Comparable results were obtained for the high-Km glucokinase-like enzymic activity present in normal pancreatic islets or insulin-producing tumoral cells. These results suggest that the anomeric specificity of glucokinase cannot account for the higher rate of glycolysis found in islets exposed to alpha- as distinct from beta-D-glucose.