Hydrolysis of hexose pentaacetate esters in rat pancreatic islets.

The pentaacetate esters of selected hexoses were recently found to stimulate insulin release. The kinetics of their hydrolysis was now investigated in both rat pancreatic islet homogenates and intact islets. In islet homogenates, the hydrolysis of alpha-d-glucose pentaacetate, as judged from the measurement of acetate production, displayed a pH optimum of 7.4 and a Km for the ester of 0.95 mM. At pH 7.4, the reaction velocity was about 5 times higher than the rate of alpha-d-glucose pentaacetate hydrolysis by intact islets, as judged from the ester-induced increase in the acetate content of both the islet and surrounding incubation medium. Comparable results were obtained in intact islets exposed to either beta-l-glucose pentaacetate or beta-d-galactose pentaacetate. The ester content of the islets after 120 min incubation was close to 0.1 nmol/islet, yielding an apparent intracellular concentration at least one order of magnitude higher than the extracellular concentration (1.7 mM). These findings indicate that hexose esters that either stimulate insulin release or fail to do so are equally well taken up and hydrolyzed by islet cells. They are compatible, therefore, with the view that the insulinotropic action of some of these esters may be favored by the catabolism of their hexose moiety, although some other mechanisms for stimulation of insulin release must be operative in the case of beta-l-glucose pentaacetate.

Interference of D-mannoheptulose with D-glucose phosphorylation, metabolism and functional effects: comparison between liver, parotid cells and pancreatic islets.

D-mannoheptulose is currently used as a tool to inhibit, in a competitive manner, D-glucose phosphorylation, metabolism and functional effects in the pancreatic islet B-cell. In order to better understand the mode of action of the heptose, we have explored its effect upon D-glucose phosphorylation in liver, parotid cells and islet homogenates, this allowing to characterize the interference of the heptose with glucokinase and/or hexokinase. The effect of D-mannoheptulose upon the metabolism of D-glucose was also examined in both intact parotid cells and pancreatic islets. Last, the effect of D-mannoheptulose upon glucose-stimulated insulin release was reinvestigated over large concentration ranges of both the heptose and hexose. The experimental data revealed a mixed type of D-mannoheptulose inhibitory action upon D-glucose phosphorylation, predominantly of the non-competitive and competitive type, in liver and parotid homogenates, respectively. Despite efficient inhibition of hexose phosphorylation in both parotid cell and islet homogenates, the heptose suppressed the metabolic and functional responses to D-glucose only in pancreatic islets, whilst failing to affect adversely D-glucose catabolism in parotid cells. These findings suggest that factors such as the intracellular transport and availability of the heptose may interfere with the expression of its antagonistic action upon D-glucose metabolism.

Energy-dependent intracellular translocation of glucokinase in rat pancreatic islets.

It was recently reported that hyperglycemia provokes a rapid and sustained translocation of glucokinase in rat pancreatic B-cells, and it was speculated that this may be associated with enhancement of its catalytic activity, as possibly attributable to the mitochondrial binding of the enzyme. In the present work, the activities of both hexokinase and glucokinase were measured in particulated and cytosolic subcellular fractions prepared from islets first incubated for 60 min either in the absence of exogenous nutrient or in the presence of D-glucose, tested at both low (2.8 mmol/L) and high (16.7 mmol/L) concentrations. The relative contribution of the cytosolic domain to the total activity of glucokinase recovered in the two subcellular fractions was higher in islets deprived of exogenous nutrient than in islets first exposed to 2.8 or 16.7 mmol/L D-glucose, the results obtained at each of the latter two hexose concentrations being comparable to one another. The subcellular distribution of hexokinase, however, was not significantly different in islets deprived of D-glucose or exposed to the hexose. These findings are interpreted as indicative of an energy-dependent translocation of glucokinase in the B-cell, distinct from the redistribution of the enzyme occurring in response to a rise in D-glucose concentration above its physiological value.

A mechanical device for studying mechanical properties of human muscles in vivo.

A mechanical device, primarily devoted to biomechanical studies of human calf muscles during microgravity experiments is presented. It allows investigation of both contractile and visco-elastic properties of musculo-articular systems using, respectively, isokinetic movements, quick-release tests and sinusoidal perturbations. This device is a specifically designed ergometer associated to an experimental protocol designed for pre- and post-flight tests. The protocol was evaluated on 22 healthy subjects and typical results are briefly presented. Preliminary results are discussed in terms of agreement with currently available data and a detailed evaluation of test-retest measurements is provided for quick-release experiments. Complementary investigations are suggested and potential fields of research are indicated.

Subcellular distribution of hexokinase isoenzymes in pancreatic islet cells exposed to digitonin after incubation at a low or high concentration of D-glucose.

It was recently proposed that stimulation of pancreatic islet by D-glucose results in the translocation of glucokinase from the perinuclear area to the cell periphery, where the enzyme might conceivably interact with either the glucose transporter GLUT-2 or some other proteins and, by doing so, become better able to express its full catalytic activity. To explore the possible interaction between glucokinase and the cell boundary, dispersed rat pancreatic islet cells were preincubated for 60 min at a low (2.8 mM) or high (16.7 mM) concentration of D-glucose, then exposed for 1 min to digitonin (0.5 mg/ml) and eventually centrifuged through a layer of oil for separation of the cell pellet from the supernatant fraction containing the material released by digitonin. Under these conditions, the bulk of lactate dehydrogenase and glutamate dehydrogenase activities were recovered in the supernatant fraction and cell pellet, respectively. The measurement of hexokinase isoenzyme activities in the two subcellular fractions, as conducted at low or high hexose concentrations and in either the absence or presence of exogenous hexose phosphates (3.0 mM glucose 6-phosphate and 1.0 mM fructose 1-phosphate) indicated a preferential location of the low-Km hexokinase in the cell pellet and of the high-Km glucokinase in the cytosolic fraction. Such a distribution pattern failed to be significantly affected by the concentration of D-glucose used during the initial incubation of the dispersed islet cells. These findings argue against the view that the glucose-induced translocation of glucokinase would result in any sizeable binding of the enzyme to a plasma membrane-associated protein.

Potentiation by its esterification of the inhibitory action of 2-deoxy-D-glucose on D-glucose metabolism and insulinotropic action.

It was recently reported that selected esters of D-glucose are more efficiently metabolized and display a greater insulinotropic action in pancreatic islets than the unesterified hexose. Likewise, in the present study, the inhibitory action of 2-deoxy-D-glucose tetraacetate upon D-glucose metabolism in rat erythrocytes and pancreatic islets was found to be more pronounced than that of unesterified 2-deoxy-D-glucose. At a concentration of 10 mM, the ester also inhibited more severely than unesterified 2-deoxy-D-glucose the release of insulin evoked by D-glucose in isolated islets. It is proposed that 2-deoxy-D-glucose tetraacetate represents a useful tool to facilitate the intracellular delivery of the glucose analog and to increase its efficiency as a potential therapeutic agent.

Anomalies of fetal development in GK rats.

Fetal development was investigated in Goto-Kakizaki (GK) rats. Between days 15 and 20 after identification of a positive sperm plug, the GK rats gained less weight than control animals. The number of conceptuses in each litter was not significantly different in control and GK rats. The incidence of abortive fetal development, however, averaged 39.7% +/- 9.1% in GK rats, compared with only 5.6% +/- 0.2% in control animals. The placental weight in living fetuses was slightly lower in GK rats than control rats. The crown-rump length was identical in the fetuses of control and diabetic mothers. The number of ossification points in the lumbosacral spine, pelvic girdle and anterior and posterior limbs was significantly lower in fetuses of GK rats than control animals. These anomalies could not be blamed on a lower plasma insulin concentration in GK than control animals, whether before or during (days 15-20) pregnancy. Moreover, in both control and GK rats, the insulinogenic index was raised during pregnancy. These findings indicate that GK rats represent a new model for the study of diabetes-related fetal anomalies, their pathogenesis and prevention.

D-glucose metabolism in dimethyl suberimidate-treated tumoral pancreatic islet cells.

After protein cross-linking by dimethyl suberimidate, tumoral insulin-producing cells of the RINm5F line were either exposed to digitonin for measurement of hexokinase activity in the resulting cell pellet and supernatant, or incubated in the presence of D-[5-3H]glucose, D-[U-14C]glucose or L-[U-14C]glutamine to assess the metabolism of these nutrients. After digitonin treatment, the activity of hexokinase recovered in the cell pellet was about 40% higher in cross-linked than control RINm5F cells. Although failing to affect the metabolism of L-[U-14C]glutamine, and severely decreasing the oxidation of D-[U-14C]glucose, the cross-linking of proteins accentuated the increase in D-[5-3H]glucose utilization and D-[U-14C]glucose conversion to acidic metabolites resulting from a rise in hexose concentration from 2.8 to 16.7 mM. The latter change represents a mirror image of that previously found in cross-linked pancreatic islets. Taking into account the vastly different participation of glucokinase to hexose phosphorylation in RINm5F cells and normal islet cells, the present findings further support, therefore, the regulatory role of protein-to-protein interaction in the control of glucokinase catalytic activity in these fuel-sensing cells.

Citrulline malate limits increase in muscle fatigue induced by bacterial endotoxins.

Citrulline malate is known to improve performance in weakened muscles. The present experiment was designed to test the hypothesis that citrulline malate can limit the effect of endotoxins on muscle fatigability. Endotoxemia was induced in rats by injection of lipopolysaccharides from Klebsiella pneumoniae. Resistance to fatigue was quantified by measuring tension production during repetitive electrical stimulation of the isolated epitrochlearis muscle. Oral treatment by citrulline malate was found to increase resistance to fatigue in infected rats, whereas twitch tension was not modified. This demonstrates the efficacy of citrulline malate for limiting an increase in muscle fatigue elicited with bacterial endotoxins.

Hexokinase and glucokinase activity in cross-linked and permeabilized pancreatic islets.

The activities of hexokinase and glucokinase were measured in cross-linked and permeabilized rat pancreatic islets. After exposure to dimethyl suberimidate (20 mM) and digitonin (0.4 mM), the activity of hexokinase represented about half of that found in homogenates of freshly isolated islets. The K(m) of hexokinase for D-glucose and the Ki for its inhibition by D-glucose-6-phosphate were similar, however, in the cross-linked and permeabilized islets and in homogenates of freshly isolated islets. Glucokinase activity also was documented in the cross-linked and permeabilized islets, it being less sensitive than hexokinase activity to inhibition by D-glucose-6-phosphate. At a high concentration of D-glucose (16.7 mM), the phosphorylation of the hexose failed to be increased by D-fructose-1-phosphate, whether in the absence or presence of D-glucose-6-phosphate. These findings indicate that the intrinsic properties of hexokinase isoenzymes are preserved in cross-linked islets, but suggest that the cross-linking of proteins prevents the activation of glucokinase by its regulatory protein.

Surface electromyogram power spectrum changes in human leg muscles following 4 weeks of simulated microgravity.

Surface electromyogram (EMG) spectrum changes in human tibialis anterior (TA) and gastrocnemius medialis (GM) muscles were studied to investigate the effect of 4-week bed rest (BR) on muscle fatigability. An exhausting isometric test at 50% of the maximal voluntary contraction was performed by 12 clinically healthy men before and after BR. During this test, mean power frequency (MPF) calculated from surface EMG decreased linearly for TA and GM. When changes in MPF were expressed in terms of rate of decrease a significant difference appeared between TA and GM. Furthermore, as a result of BR, the shift in MPF increased significantly for GM (6.1% vs 10.4%) whereas it was not significantly changed for TA (28.6% vs 20.95%). Alterations in maximal torque were also observed with a more pronounced decrease for plantar-flexor (20.5%) compared with dorsiflexor (15.1%) muscles. These results would seen: to indicate that simulated microgravity preferentially affects muscles having an antigravity function.

Relative contributions of the long and short heads of the biceps brachii during single or dual isometric tasks.

Electromyograms (EMG) of the long head (LH) and the short head (SH) of the biceps brachii were recorded during linearly increasing isometric elbow torques. These ramp contractions concerned flexion or supination of the elbow performed separately (flexion or supination single tasks) and simultaneously (flexion/supination dual tasks). EMG-torque relationships and EMG-EMG relationships were established. The EMG-torque relationships of the SH and LH show a more increased activity in supination than in flexion single task. The highest levels of LH and SH activations were found in the dual tasks. For the majority of subjects, the maximal flexion and supination torques reached in dual tasks were identical to those developed in single tasks. In the single tasks, the LH-SH relative contributions varied from one subject to another as attested by the EMG LH-EMG SH relationships, whereas in dual tasks, the EMG LH-EMG SH relationship was close to the identity line for all subjects, indicating a higher and homogeneous activation of both heads of the biceps. It is concluded that dual tasks are indicated in order to spread the motor unit (MU) recruitment or to activate fully a bifunctional muscle.

D-glucose metabolism in cross-linked pancreatic islets.

D-glucose metabolism was investigated in pancreatic islets that underwent cross-linking of intracellular proteins by dimethyl suberimidate. Both the utilization of D-[5-3H]glucose and oxidation of D-[U-14C]glucose were much more severely affected at high (16.7 mM) than at low (2.8 mM) concentration of the hexose, when comparing cross-linked to control islets. The preferential stimulation of D-[U-14C]glucose oxidation relative to D-[5-3H]glucose utilization, resulting from an increase in hexose concentration, was not abolished, however, in cross-linked islets. Likewise, the activation of phosphofructokinase in glucose-stimulated islets did not appear to be impaired in cross-linked islets, the islet content in tritiated acidic metabolites generated from D-[5-3H]glucose failing to be increased in cross-linked islets. It is proposed, therefore, that the impaired responsiveness of cross-linked islets to a rise in D-glucose concentration might be attributable to an altered regulation of glucokinase, as possibly resulting from a missing interaction of the enzyme with GLUT-2.

The ankle ergometer: a new tool for quantifying changes in mechanical properties of human muscle as a result of spaceflight.

A mechanical device for studying changes in mechanical properties of human muscle as a result of spaceflight is presented. Its main capacities are to allow during a given experiment investigation of both contractile and visco-elastic properties of a musculo-articular complex using respectively isometric contractions, isokinetic movements, quick-release tests and sinusoidal perturbations. This device is a motor driven ergometer associated to an experimental protocol designed for pre- and post-flight experiments. As microgravity preferentially affects postural muscles, the apparatus was designed to test muscle groups crossing the ankle joint. Three subjects were tested during the Euromir' 94 mission. Preliminary results obtained on the european astronaut are briefly reported. During the next two years the experiments will be performed during six missions.