Accelerative exchange diffusion kinetics of glucose between blood and brain and its relation to transport during anoxia

An earlier study showed that unidirectional glucose transport from blood to brain decreases during perfusion with anoxic blood (Betz, A.L., Gilboe, D.D. and Drewes, L.R. (1974) Brain Res. 67, 307–316). Brain glucose levels also decrease during anoxia. Therefore, the present study was designed to investigate whether the decreased transport might be the result of decreased accelerative exchange diffusion when brain glucose levels are low.The rate of unidirectional transport into brain (v) of d-[6-³H]glucose was studied in 22 isolated, perfused dog brains by means of an indicator dilution technique using ²²Na as the intravascular reference. The kinetics of transport were determined over a range of blood glucose concentrations (S₁) at each of live different brain glucose levels (S₂). The existence of accelerative exchange diffusion for glucose was indicated by a decrease in the intercept (increase of apparent V) of a double reciprocal plot ($\text{1}\text{v}$ versus $\text{1}\text{S}{}_1$) as S₂ increased. This phenomenon is consistent with a model for facilitated diffusion in which the mobility of the loaded carrier is greater than that of the unloaded carrier. Although the data predict a decrease in glucose transport during anoxia, the predicted decrease (5%) is less than the observed decrease (35%). It is concluded that the simple mobile-carrier model for facilitated diffusion cannot, by itself, describe all properties of blood-brain glucose transport.     

The relationship between active transport and the exchange diffusion effect

Dependences of unidirectional ionic fluxes across biological membranes on the trans concentrations of the same ion, commonly described as exchange diffusion, and the association of this phenomenon with active transport, are noted. It is suggested that this effect could arise as a result of energetic coupling between the movement of ions conveyed in each direction by the pump if the latter operates near thermodynamic equilibrium and if the rate of the energizing reactions are restricted. This hypothesis is supported by an analysis in which the transport step and the energizing reactions are separated and described according to the laws of chemical kinetics. A likely cause for such restriction of the maximum rate of energy supply is shown to lie in evolutionary optimization of the efficiency of active transport if the energizing reaction is not perfectly coupled. Similar optimization will produce gross ionic fluxes large compared with the net flux, especially if the transport step approaches perfect coupling, when restriction of the rate of energy supply will cause a large exchange diffusion effect. The range of validity of the analysis is examined with particular reference to the ionic exchanges between osmoregulating animals and their surroundings.     

The kinetics of glucose transport in human red blood cells

A quenched-flow apparatus and a newly developed automated syringe system have been used to measure initial rates of D-[14C]glucose transport into human red blood cells at temperatures ranging from 0 degrees to 53 degrees C. The Haldane relationship is found to be obeyed satisfactorily at both 0 and 20 degrees C, but Arrhenius plots of maximum D-[14C]glucose transport rates are non-linear under conditions of both equilibrium exchange and zero trans influx. Fitting of the data by non-linear regression to the conventional model for glucose transport gives values at 0 degrees C of 0.726 +/- 0.0498 s-1 and 12.1 +/- 0.98 s-1 for the rate constants governing outward and inward movements of the unloaded carrier molecule and 90.3 +/- 3.47 s-1 and 1113 +/- 494 s-1 for outward and inward movements of the carrier-glucose complex. Activation energies for these four rate constants are respectively 173 +/- 3.10, 127 +/- 4.78, 88.0 +/- 6.17 and 31.7 +/- 5.11 kJ X mol-1. These parameters indicate that at low temperatures the marked asymmetry of the transport mechanism arises mainly from the high proportion of inward-facing carriers and carrier-glucose complexes, and that there is a relatively small difference between the affinities of the carrier for glucose in the inward and outward-facing conformations. At high (physiological) temperatures the carrier is fairly evenly distributed between outward- and inward-facing conformations and the affinity for glucose is about 2.5-times greater outside than inside.     

The relation between osmotic flow and tracer solvent diffusion for single-file transport.

It is demonstrated that a reasonably general model for single-file passage of solvent through an ultra-narrow pore implies the equality of tracer diffusion and osmotic flow. This result is not trival, but follows from the exactly compensating effects of solvent-solvent interaction on the paritioning of bulk solvent into the pore and on the diffusion rate within the pore. A previous calculation of Longuet-Higgins and Austin is seen to be valid only in the absence of interactions among solvent molecules in the pore.     

Glucose monitoring at the thenar: evaluation of upper dermal blood glucose kinetics during rapid systemic blood glucose changes.

OBJECTIVE: We compared blood glucose measurements at the thenar with those at the fingertip during glucose increase and decrease that was rapid enough to induce glucose differences between the forearm and the fingertip. METHODS: A rapid glucose increase was induced by oral glucose; subsequently, a rapid glucose decrease was induced by intravenous insulin in 16 insulin-treated patients with diabetes. Capillary samples were taken in parallel from the thenar and fingertip. Different glucose monitors (FreeStyle, OneTouch Ultra, Soft-Sense) were used. Additional samples were taken from the forearm (n = 10 patients) in order to demonstrate that the blood glucose change achieved was rapid enough to principally induce glucose differences at alternative sites. RESULTS: Neither blood glucose at baseline (135 +/- 34 vs. 136 +/- 41 mg/dl, p = 0.86) nor glucose amplitude during increase (190 +/- 35 vs. 188 +/- 41 mg/dl, p = 0.65) or decrease (255 +/- 32 vs. 257 +/- 45 mg/dl, p = 0.83) differed significantly between the fingertip and the thenar. Intra-individual average thenar-fingertip glucose difference was - 2 +/- 12 (p = 1.00) and + 5 +/- 9 mg/dl (p = 0.11). In the subgroup, intra-individual average forearm-finger difference was - 50 +/- 19 (p < 0.01) and + 45 +/- 11 mg/dl (p < 0.01) during glucose-increase and decrease, respectively. There were no obvious device-specific differences. CONCLUSIONS: Blood glucose measurements at the thenar are a safe alternative to measurements at the fingertip at steady state as well as during blood glucose change that is sufficiently rapid to induce clinically relevant differences between forearm and fingertip.     

Muscle blood flow during isometric activity and its relation to muscle fatigue

The effect of isometric exercise on blood flow, blood pressure, intramuscular pressure as well as lactate and potassium efflux from exercising muscle was examined. The contractions performed were continuous or intermittent (5 s on, 5 s off) and varied between 5% and 50% maximal voluntary contraction (MVC). A knee-extensor and a hand-grip protocol were used. Evidence is presented that blood flow through the muscle is sufficient during low-level sustained contractions (less than 10% MVC). Despite this muscle fatigue occurs during prolonged contractions. One mechanism for this fatigue may be the disturbance of the potassium homeostasis. Such changes may also play a role in the development of fatigue during intermittent isometric contractions and even more so in the recovery from such exercise. In addition the role of impaired transport of substances within the muscle, due to long-lasting daily oedema formation, is discussed in relation to fatigue in highly repetitive, monotonous jobs.     

Inhibition of glucose transport into brain by phlorizin, phloretin and glucose analogues.

An indicator dilution technique with 22Na+ as the intravascular marker was used to measure unidirectional transport of D-[6-3H]glucose from blood into the isolated, perfused dog brain. 18 compounds which are structurally related to glucose were tested for their ability to inhibit glucose transport. The data suggest that no single hydroxyl group is absolutely required for glucose transport, but rather that glucose binding to the carrier probably occurs through hydrogen bonding at several sites (hydroxyls on carbons 1, 3, 4 and 6). In addition, alpha-D-glucose has higher affinity for the carrier than does beta-D-glucose. A separate series of experiments demonstrated that phlorizin and phloretin are competitive inhibitors of glucose transport into brain; however, phloretin is partially competitive and inhibits at lower concentrations than does phlorizin. Inhibition by phlorizin and phloretin is mutually competitive, indicating that these compounds compete for binding to the glucose carrier. Comparison with the results reported in the literature for similar studies using the human erythrocyte demonstrates a fundamental similarity between glucose transport systems in the blood-brain barrier and erythrocyte.     

Cytoplasmic Ca2+ during differentiation of 3T3-L1 adipocytes. Effect of insulin and relation to glucose transport

The cytoplasmic concentration of ionized Ca2+ [( Ca2+]i) was determined in 3T3-L1 cells during their differentiation from fibroblasts to adipocytes, suspended and loaded with the fluorescent Ca2+ indicators quin2 or indo-1. In undifferentiated fibroblasts, as well as in differentiated adipocytes up to day 9, [Ca2+]i was steady around 170 nM, and it increased significantly only in old adipocytes (day 12). During differentiation, stimulation of glucose uptake by insulin increased from a few percent to severalfold. Stimulation of uptake was already apparent after 10 min of addition of the hormone, and 10 nM insulin produced maximal stimulation in 30 min. Insulin (10(-6) M) added to quin2- or indo-1-loaded, suspended adipocytes had no detectable effect on [Ca2+]i for at least 10 min. In contrast, addition of the general anesthetic halothane increased [Ca2+]i from 172 to 251 nM in 3 min. In EGTA solution, the Ca2+ ionophore ionomycin elicited release of Ca2+ from intracellular stores that resulted in a transient increase in [Ca2+]i. A smaller but measurable Ca2+ release from intracellular stores (increasing [Ca2+]i by 20 nM) resulted upon addition of 20 micrograms/ml phosphatidic acid. In contrast, insulin did not produce any detectable release of Ca2+ from intracellular stores. Incubation of 3T3-L1 adipocytes with insulin in the presence of EGTA (the latter in excess over the Ca2+ concentration of the medium) did not prevent the stimulation of hexose uptake by the hormone, indicating that extracellular Ca2+ does not play a role in the insulin response. Furthermore, incubation of cells with quin2/AM in EGTA medium during exposure to insulin did not prevent stimulation of hexose uptake. Under these conditions it is demonstrated that intracellular quin2 suffices to chelate cytoplasmic Ca2+ even if releasable Ca2+ from intracellular stores were to pour into the cytoplasm. Thus, quin2 effectively lowers [Ca2+]i without impairing insulin action. It is concluded that insulin does not produce changes in [Ca2+]i and that chelating intracellular Ca2+ does not prevent stimulation of hexose uptake by insulin. These results suggest that it is unlikely that changes in [Ca2+]i may play a role in the transduction of information in insulin stimulation of glucose uptake in 3T3-L1 adipocytes.     

The relationship between transport kinetics and glucose uptake by Saccharomyces cerevisiae in aerobic chemostat cultures

The steady-state residual glucose concentrations in aerobic chemostat cultures of Saccharomyces cerevisiae ATCC 4126, grown in a complex medium, increased sharply in the respiro-fermentative region, suggesting a large increase in the apparent k_s value. By contrast, strain CBS 8066 exhibited much lower steady-state residual glucose concentrations in this region. Glucose transport assays were conducted with these strains to determine the relationship between transport kinetics and sugar assimilation. With strain CBS 8066, a high-affinity glucose uptake system was evident up to a dilution rate of 0.41 h^–1, with a low-affinity uptake system and high residual glucose levels only evident at the higher dilution rates. With strain ATCC 4126, the high-affinity uptake system was present up to a dilution rate of about 0.38 h^–1, but a low-affinity uptake system was discerned already from a dilution rate of 0.27 h^–1, which coincided with the sharp increase in the residual glucose concentration. Neither of the above yeast strains had an absolute vitamin requirement for aerobic growth. Nevertheless, in the same medium supplemented with vitamins, no low-affinity uptake system was evident in cells of strain ATCC 4126 even at high dilution rates and the steady-state residual glucose concentration was much lower. The shift in the relative proportions of the high and low-affinity uptake systems of strain ATCC 4126, which might have been mediated by an inositol deficiency through its effect on the cell membrane, may offer an explanation for the unusually high steady-state residual glucose concentrations observed at dilution rates above 52% of the wash-out dilution rate.     

A relation between (NAD+)/(NADH) potentials and glucose utilization in rat brain slices.

Changes in several parameters involved in the control of metabolism were correlated with changes in glucose utilization in rat brain slices incubated under conditions which reduced glucose oxidation by 40 to 70%. The parameters included: the concentrations of ATP, ADP, AMP, and the adenylate energy charge; the cytoplasmic oxidation-reduction state ([NAD+]/[NADH]), determined from the [pyruvate]/[lactate] equilibrium; the mitochondrial oxidation-reduction state, determined from the [NH4+] ]2-oxoglutarate]/[glutamate] Equilibrium; the cytoplasmic and mitochondrial oxidation-reduction potentials (in volts), calculated from the respective [NAD+]/ [NADH] ratios using the Nernst equation; and the difference between the cytoplasmic and mitochondrial [NAD+]/[NADH] potentials. The conversion of [3, 4-14C] glucose to 14CO2 and of [U-14C] glucose to acetylcholine and to lipids, proteins, and nucleic acids by the brain slices were also determined. The values obtained by subtracting the mitochondrial from the cytoplasmic [NAD+1/[NADH] potentials correlated more closely with glucose utilization than did other parameters, under the conditions studied. For the synthesis of acetylcholine, the correlation coefficient was 0.96, and for the production of 14CO2 from [3, 4-14C] glucose it was 0.82.     

A novel method to differentiate between ping-pong and simultaneous exchange kinetics and its application to the anion exchanger of the HL60 cell.

We have developed a new test to differentiate between ping-pong and simultaneous mechanisms for tightly coupled anion exchange. This test requires the use of a dead-end reversible noncompetitive inhibitor. As an example, we have applied the test to the anion exchanger of the HL60 cell using the salicylic acid derivative 3,5-diiodosalicylic acid (DIS), which reversibly inhibits HL60 cell Cl/Cl exchange. The concentration of DIS that causes 50% inhibition (ID50) increased only slightly as either intra- or extracellular chloride was increased, indicating that DIS inhibits HL60 anion exchange in a noncompetitive manner. In agreement with this observation, plots of the slope of the Dixon plot as a function of 1/[Clo] or 1/[Cli] were fit with straight lines with nonzero intercepts, indicating that DIS does not compete with either of the substrates ([Clo] and [Cli]). The secondary Dixon slope test is based on the fact that, for a dead-end inhibitor such as DIS, the slope of the Dixon plot slope vs. 1/[Cli] (secondary Dixon slope or SDS) is independent of extracellular Cl when the exchange mechanism follows ping-pong kinetics. Similarly, the SDS calculated from a plot as a function of 1/[Clo] is also independent of intracellular Cl for a ping-pong exchanger. In contrast to this prediction, we found that for DIS inhibition of Cl/Cl exchange in HL60 cells the slope of the Dixon plot slope vs. 1/[Cli] decreased by a factor of 2.5-fold when [Clo] was increased from 1 to 11 mM (P < 0.0001). This change in the SDS rules out ping-pong kinetics, but is consistent with a simultaneous model of Cl/Cl exchange in which there are extra- and intracellular anion binding sites, both of which must be occupied by suitable anions in order to allow simultaneous exchange of the ions.     

Equilibrium and kinetics of the thermal unfolding of alpha-lactalbumin. The relation to its folding mechanism.

The thermal unfolding of alpha-lactalbumin has been studied by equilibrium measurements of aromatic difference spectra, and by kinetic measurements of the Joule heating temperature-jump. The unfolding at neutral pH is a reversible two-state transition. The equilibrium transition curves are analyzed by the nonlinear squares method, which gives correct values of thermodynamic parameters based on the data in a wide range of temperature. The results are discussed in relation to the previous studies on the unfolding by guanidine hydrochloride or by acid. The thermally unfolded state, a partially unfolded species, is shown to be thermodynamically similar to but not identical with the acid state. The folding pathway deduced from the kinetic results is essentially consistent with the folding model of alpha-lactalbumin proposed previously. Large decreases in entropy and in heat capacity during the reversed activation suggest the packing of the folded segments by hydrophobic interactions, while the forward activation shows a marked temperature dependence, probably caused by the disruption of specific long-range interactions.     

Active transport and mediated diffusion of glucose and other monosaccharides inEndomyces magnusii

After growth on sucrose or glucose,Endomyces magnusii possesses a monosaccliaride uptake which resembles that ofSaccharomyces cerevisiae (a high K_Tof uptake, preference for α-anomers of D-xylose and D-glucose, enhanced uptake during anaerobiosis, attainment of a diffusion equilibrium). The uptake is inhibited by other monosaccharides and especially strongly by D-galactose. In the absence of high concentrations of metabolizable sugars,E. magnusii develops a capacity to accumulate 3-O-methyl-D-glucose and D-xylose against a concentration gradient the new system displaying a high affinity for glucose (K_T < 0.1 mM), repression by glucose, mannose or galactose. Cycloheximide (0.2 %) blocks the formation of the active system.