Simultaneous measurement of glucose transport and utilization in the human brain

Glucose is the primary fuel for brain function, and determining the kinetics of cerebral glucose transport and utilization is critical for quantifying cerebral energy metabolism. The kinetic parameters of cerebral glucose transport, K(M)(t) and V(max)(t), in humans have so far been obtained by measuring steady-state brain glucose levels by proton ((1)H) NMR as a function of plasma glucose levels and fitting steady-state models to these data. Extraction of the kinetic parameters for cerebral glucose transport necessitated assuming a constant cerebral metabolic rate of glucose (CMR(glc)) obtained from other tracer studies, such as (13)C NMR. Here we present new methodology to simultaneously obtain kinetic parameters for glucose transport and utilization in the human brain by fitting both dynamic and steady-state (1)H NMR data with a reversible, non-steady-state Michaelis-Menten model. Dynamic data were obtained by measuring brain and plasma glucose time courses during glucose infusions to raise and maintain plasma concentration at ～17 mmol/l for ～2 h in five healthy volunteers. Steady-state brain vs. plasma glucose concentrations were taken from literature and the steady-state portions of data from the five volunteers. In addition to providing simultaneous measurements of glucose transport and utilization and obviating assumptions for constant CMR(glc), this methodology does not necessitate infusions of expensive or radioactive tracers. Using this new methodology, we found that the maximum transport capacity for glucose through the blood-brain barrier was nearly twofold higher than maximum cerebral glucose utilization. The glucose transport and utilization parameters were consistent with previously published values for human brain.     

Rhodopseudomonas cryptolactis, sp. nov., a new thermotolerant species of budding phototrophic purple bacteria

Abstract A proton motive force (Δp) generated by oxidation of CO in membrane vesicles of Clostridium thermoautotrophicum drove active transport of l -alanine, glycine and l -serine. The maximum rate ( V _max) for l -alanine transport was 12 × higher at 50°C than at 25°C. The apparent transport constant ( K _t) for l -alanine uptake was 30–40 μM and independent of the temperature. Glycine was a substrate for the l -alanine transport system as demonstrated by the competitive inhibition of l -alanine uptake by glycine ( K _i= 6 μ M), by the kinetics of glycine uptake ( K _t= 7 μ M) and by the inhibiton of glycine uptake by l -alanine. The uptake kinetics of glycine was biphasic. l -Serine inhibited competitively also l -alanine and glycine transport but it was taken up by a separate transport system. The rate of amino acid transport, but not the K _t, was dependent on the value of the proton motive force.     

Mechanisms of Sodium Transport at the Blood-Brain Barrier Studied with In Situ Perfusion of Rat Brain

Abstract: The mechanism of unidirectional transport of sodium from blood to brain in pentobarbital-anesthetized rats was examined using in situ perfusion. Sodium transport followed Michaelis-Menten saturation kinetics with a V _max of 50.1 nmol/g/min and a K _m of 17.7 m M in the left frontal cortex. The kinetic analysis indicated that, at a physiologic sodium concentration, ∼26% of sodium transport at the blood-brain barrier (BBB) was carrier mediated. Dimethylamiloride (25 µ M ), an inhibitor of Na⁺/H⁺ exchange, reduced sodium transport by 28%, whereas phenamil (25 µ M ), a sodium channel inhibitor, reduced the transfer constant for sodium by 22%. Bumetanide (250 µ M ) and hydrochlorothiazide (1.5 m M ), inhibitors of Na⁺-K⁺-2Cl⁻/NaCl symport, were ineffective in reducing blood to brain sodium transport. Acetazolamide (0.25 m M ), an inhibitor of carbonic anhydrase, did not change sodium transport at the BBB. Finally, a perfusate pH of 7.0 or 7.8 or a perfusate P co ₂ of 86 mm Hg failed to change sodium transport. These results indicate that 50% of transcellular transport of sodium from blood to brain occurs through Na⁺/H⁺ exchange and a sodium channel in the luminal membrane of the BBB. We propose that the sodium transport systems at the luminal membrane of the BBB, in conjunction with Cl⁻/HCO₃⁻ exchange, lead to net NaCl secretion and obligate water transport into the brain.     

Kinetics of Regional Blood–Brain Barrier Transport and Brain Phosphorylation of Glucose and 2-Deoxyglucose in the Barbiturate–Anesthetized Rat

Abstract: Recent studies indicate the lumped constant (LC), which defines the relative rates of brain utilization of glucose and 2-deoxyglucose (2-DG), doubles to values > 1.0 under conditions of hypoglycemia. Since changes in the LC should be predictable given the kinetic parameters of blood-brain barrier (BBB) transport and brain phosphorylation of glucose and 2-DG, the present studies were designed to measure the necessary kinetic parameters. The carotid injection technique was used to determine cerebral blood flow and the K_m , V_max, and K _D of glucose and 2-DG transport through the BBB in seven brain regions in rats anesthetized with 50 mg/kg i.p. pentobarbital. Regional glucose transport through the BBB was characterized by an average K_m = 6.3 m m , average V_max = 0.53 μmol min⁻¹g⁻¹, and average K _D= 0.022 ml min⁻¹g⁻¹. The nonsaturable route of transport of glucose represented on the average 40% of the total glucose influx into brain regions at an arterial glucose concentration of 10 m m . In addition, the rate constants of phosphorylation of glucose and 2-DG were measured for each region. Substitutions of the measured kinetic parameters for sugar transport and phosphorylation into equations defining the LC confirm the observation that the LC would be expected to vary under extreme conditions such as hypoglycemia and to exceed values of 1.0 under these conditions.     

A MODEL OF HIGH AFFINITY GLUTAMIC ACID TRANSPORT BY CORTICAL SYNAPTOSOMES FROM THE LONG-EVANS RAT

Abstract— The initial velocity of uptake of L-glutamic acid by cortical synaptosomes from the Long-Evans rat has been measured as a function of sodium and glutamic acid concentration. These data were then fitted to the rate equation from each of several possible models, and the model giving minimum error identified. The major predictions from the best fit model are as follows: (1) The order of combination with the carrier should be sodium, sodium, glutamic acid. (2) Uptake should be 100% dependent on the presence of sodium in the incubation medium. (3) At a finite concentration of sodium, V_mx should be independent of the sodium concentration, which implies that translocation of the carrier across the membrane is independent of the sodium concentration. (4) Lineweaver-Burk plots should be linear with slopes depending on the sodium Concentration. (5) There should be co-transport of two sodium ions with each glutamic acid molecule. (6) The dependence of K _t, on the sodium concentration should have the following form: K _t= A/[Na]²+ 8/[Na] + C , where A, B , and C are constants. The results differ substantially from those previously reported for Sprague-Dawley rats. K _t, is 2–5 times less than that for Sprfague-Dawley rats, and the relation of sodium to K _t, is basically different. We also find a coupling ratio of two, whereas previous studies found a coupling ratio of one. Thus the results raise the possibility that there are fundamental differences between Sprague-Dawley and Long-Evans rats with regard to the mechanism by which sodium participates in amino acid transport.     

Glutathione Efflux from Cultured Astrocytes

Abstract: The characteristics and kinetics of GSH efflux from the monolayer culture of rat astrocytes were investigated. GSH efflux was dependent on temperature, with a Q ₁₀ value of 2.0 between 37 and 25°C. The GSH efflux rate showed a hyperbolic dependency on the intracellular GSH concentration. The data were fitted well to the Michaelis-Menten model, giving the following kinetic parameter values: K _m = 127 nmol/mg of protein; V _max = 0.39 nmol/min/mg of protein. p -Chloromercuribenzenesulfonic acid, a thiol-reactive agent impermeable to the cell membrane, lowered the GSH efflux rate by 25% without affecting the intracellular GSH content. These results suggest that a carrier is involved in the efflux of GSH. The GSH content of cultured astrocytes showed a marked increase when the cells were exposed to insults, such as sodium arsenite, cadmium chloride, and glucose/glucose oxidase that lead to the generation of hydrogen peroxide. The increase in GSH content was attributed to the induction of the cystine transport activity by the agents. Although the intracellular GSH concentration and GSH efflux were increased, the kinetics of GSH efflux were not affected by those agents that imposed the oxidative stress. Because the K _m value is very large, it is suggested that astrocytes release GSH depending on their GSH concentration in a wide range.     

KINETICS OF ADENOSINE UPTAKE INTO ASTROCYTES

Abstract— Kinetics for uptake of adenosine, a putative inhibitory transmitter, were measured in normal, i.e. non-transformed, astrocytes in cultures obtained from the dissociated, cortex-enriched superficial parts of the brain hemispheres of newborn DBA mice. The uptake kinetics indicated a minor, unsaturable component together with a rather intense (V_max 0.36nmol/min per mg protein) high affinity ( K _m 3.4 μ m ) uptake following Michaelis-Menten kinetics and inhibited by 100 μ m -papaverine. The V_max was about two times higher than that reported in the literature for brain slices suggesting that a considerable part of the adenosine uptake in brain slices occurs into glial cells. Such an accumulation of adenosine into normal astrocytes may play a major role in nucleoside and nucleotide metabolism in the brain and help in regulating the extracellular adenosine concentration.     

Characterization of cerebral glucose dynamics in vivo with a four-state conformational model of transport at the blood-brain barrier

Determination of brain glucose transport kinetics in vivo at steady-state typically does not allow distinguishing apparent maximum transport rate (T(max)) from cerebral consumption rate. Using a four-state conformational model of glucose transport, we show that simultaneous dynamic measurement of brain and plasma glucose concentrations provide enough information for independent and reliable determination of the two rates. In addition, although dynamic glucose homeostasis can be described with a reversible Michaelis-Menten model, which is implicit to the large iso-inhibition constant (K(ii)) relative to physiological brain glucose content, we found that the apparent affinity constant (K(t)) was better determined with the four-state conformational model of glucose transport than with any of the other models tested. Furthermore, we confirmed the utility of the present method to determine glucose transport and consumption by analysing the modulation of both glucose transport and consumption by anaesthesia conditions that modify cerebral activity. In particular, deep thiopental anaesthesia caused a significant reduction of both T(max) and cerebral metabolic rate for glucose consumption. In conclusion, dynamic measurement of brain glucose in vivo in function of plasma glucose allows robust determination of both glucose uptake and consumption kinetics.     

KINETICS OF BLOOD-BRAIN BARRIER TRANSPORT OF PYRUVATE, LACTATE AND GLUCOSE IN SUCKLING, WEANLING AND ADULT RATS

Abstract— The kinetics of the uptake from blood to brain of pyruvate, lactate and glucose have been determined in rats of different ages. The carotid artery single injection technique was used in animals anaesthetized with pentobarbital. The rates of influx for each substrate were determined over a range of concentrations for the different age-groups. Data were analysed in terms of the Michaelis-Menten equation with a component to allow for non-saturable diffusion. Values are given for K _m, V _max and K _d. In suckling rats (15-21 days) the V _max values for both pyruvate and lactate were 2.0 μmol g⁻¹ min⁻¹. In 28-day-old rats the V _max values had fallen to one-half and in adults they were less than one-tenth. K _m, values were higher in the younger animals. The rate of glucose transport in suckling rats was half that of 28-day-old and adults although there was no difference with age in the K _m values.
The results are discussed in relation to the net flux of these substrates in and out of brain during different stages of post-natal development.     

ESTIMATION OF THE KINETIC PARAMETERS OF UNIDIRECTIONAL TRANSPORT ACROSS THE BLOOD-BRAIN BARRIER

Abstract— The unidirectional transport of metabolic substrates from blood to brain may be defined in terms of Michaelis-Menten saturable ( K m, V _max) and non-saturable ( K _d) components of influx. Various computation procedures have been previously reported to estimate the kinetic parameters when an intracarotid injection technique is used. Transformations of the influx data which allow linear plots to obtain estimates were compared with estimates obtained directly from a best fit on a least means squares criterion for both experimental and simulated data. Large discrepancies were apparent between the various estimates of the kinetic parameters when an equal weight was given to transformed data. For pyruvate (21-day-old rats), K _m, values varied between 1.02 and 6.25 mM and V _max varied between 0.68 and 2.30 μmol g⁻¹ min⁻¹. The estimates were almost equivalent when pyruvate data was re-analysed using a weighting scheme based on the finding that the absolute value of the S.D. of influx increased in proportion to influx. It is recommended that estimates of kinetic parameters be obtained by an iterative, non-linear least squares method to fit appropriately weighted data directly.     

Transport of Zinc-65 at the Blood-Brain Barrier During Short Cerebrovascular Perfusion in the Rat: Its Enhancement by Histidine

Abstract: Zinc-65 transport into different regions of rat brain has been measured during short vascular perfusion of one cerebral hemisphere with an oxygenated HEPES-containing physiological saline at pH 7.40. The [Zn²⁺] was buffered with either bovine serum albumin or histidine. In each case uptake was linear with time up to 90 s. ⁶⁵Zn flux into brain in the presence of albumin followed Michaelis-Menten kinetics and for parietal cortex had a K _m of 16 n M and a V _max of 44 nmol/kg/min. Increasing concentrations of l -histidine enhanced ⁶⁵Zn flux into brain at [Zn²⁺] values between 1 and 1,000 n M . The combined effect of [histidine] and [Zn²⁺] was best accounted for by a function of [ZnHis⁺], i.e., flux = 64.4 · [ZnHis⁺]/(390 + [ZnHis⁺]) + 0.00378 · [ZnHis⁺], with concentrations being nanomolar. d -Histidine had an influence similar to that of l -histidine. ⁶⁵Zn flux in the presence of 100 µ M l -histidine was not affected by either 500 µ M l -arginine or 500 µ M l -phenylalanine. The results indicate specific transport of Zn²⁺ across the plasma membranes of brain endothelium. The enhancement due to histidine has been attributed to diffusion of ZnHis⁺ across unstirred layers "ferrying" zinc to and from transport sites.     

Induction of macrophage apoptosis by Bordetella pertussis adenylate cyclase-hemolysin

Abstract The addition of 1 mM glycine betaine to the growth medium of Chromatium sp. NCIMB 8379 relieved growth inhibition caused by exposure to supra-optimal Nad concentrations. Intracellular glycine betaine concentrations were dependent upon the NaCl concentration of the growth medium up to 3 M exogenous Nad. Kinetic data for the accumulation of [methyl-¹⁴C]-glycine betaine demonstrated that Chromatium sp. NCIMB 8379 possesses a constitutively expressed active transport system for glycine betaine. The transport system was saturable with respect to glycine betaine concentration and exhibited typical Michaelis-Menten type kinetics: K _m= 24 μ M, V _max= 306 nmol min⁻¹ mg protein⁻¹ at an external NaCl concentration of 1 M. The rate of glycine betaine transport decreased progressively with increasing growth medium NaCl concentration. This transport system may represent an adaptive response to growth in high osmolarity environments in this halotolerant isolate, allowing accumulation of glycine betaine from the external cell environment or recycling synthesised glycine betaine which has passively diffused from the cell.     

NADP+-dependent glutamate dehydrogenase from the facultative methylotroph Hyphomicrobium X

Abstract NADP⁺-dependent glutamate dehydrogenase (GDH; EC 1.4.1.4) was purified using acetone precipitation, heat, DEAE-cellulose and dye-ligand Ramazol Red column chromatography. The M _r of the native enzyme was estimated to be 380 000 (± 10 000) by polyacrylamide gel electrophoresis. The same technique in the presence of sodium dodecyl sulphate (SDS) gave one subunit band with an M _r of 63 400 (±4000). Thus the enzyme has a hexameric structure. The enzyme has a pH optimum of 8.5 and has K _m apparent values of 1.6 mM, 0.015 mM and 10.2 mM for α-ketoglutarate, N NADPH and L -glutamate, respectively. Michaelis-Menten kinetics were not observed when the ammonium concentration was increased. A progressive increase in the ammonium concentration resulted in a progressively increasing K _m value. The enzyme was highly specific for all substrates and markedly insensitive to inhibitors.     

Phosphate Ion Transport in Rabbit Brain Synaptosomes

Abstract: Synaptosomes (vesicles of nerve endings) isolated from rabbit brain were studied as a model system for the uptake of inorganic phosphate. The phosphate uptake showed a sodium-dependent, saturable component with a K _t of 0.29 m m , The sodium-dependent component was larger at pH 6 than at pH 7.4 or 8. Application of potassium salts, ouabain, monensin, nigericin or FCCP decreased the uptake. The results indicate that the sodium-sensitive phosphate influx is dependent on the Na⁺ gradient and on the membrane potential, which might act, preferentially, on the transport of the monovalent phosphate ion.     

Glucose transport in a methylotrophic yeast Hansenula polymorpha

Glucose transport was studied in a methylotrophic yeast Hansenula polymorpha . Two kinetically different glucose transport systems were revealed in cells grown under different growth conditions. Glucose-repressed cells exhibited a low-affinity transport system ( K _m for glucose 1.75 mM) while glucose-derepressed and ethanol-grown cells had a high-affinity transport system ( K _m for glucose 0.05–0.06 mM). The high- and low-affinity transport systems differed in substrate specificity, sensitivity to pH, dinitrophenol and protonophore carbonyl cyanide- m -chlorophenyl-hydrazone. The kinetic rearrangement of the glucose transport system in response to altered growth conditions was dependent on de novo protein synthesis.     

The Interaction of Transport and Metabolism on Brain Glucose Utilization: A Reevaluation of the Lumped Constant

Abstract: The relative cerebral cortical metabolism of glucose (GLU) and 2-deoxy-D-glucose (DG) was measured in vivo in control and insulin-treated hypoglycemic rats. The ratio of the utilization rate constants for the two hexoses, i.e., K _DG/ K _CLU is defined as the Hexose Utilization Index (HUI). The HUI was found to be invariant in rats whose cerebral glucose content exceeded 1 μmo1.g⁻¹ wet weight (HUI = 0.48 ± 0.07). Severe hypoglycemia (plasma glucose <2 mM) effected a shift in the HUI to 1.04 ± 0.21. The results are consistent with a model in which the interpretation of the HUI is determined by the rate of transport into brain, or subsequent phosphorylation, as the rate-limiting step for hexose utilization.     

DEMONSTRATION OF HIGH AFFINITY HEXOSE UPTAKE IN CEREBRAL CORTEX SLICES

Abstract— The high-affinity glucose transport system ( K _m 0.2–0.4 m m ), previously detected in synaptosome preparations, has now been demonstrated to be present in slices of the cerebral cortex incubated in vitro. The kinetic properties of this undirectional uptake process in slices were similar to those exhibited by synaptosomes. The results are discussed with respect to the possible sites of the high affinity and low affinity glucose transport processes in the brain.     

Competitive Inhibition of Rat Brain Hexokinase by 2-Deoxyglucose, Glucosamine, and Metrizamide

Abstract: The effects of metrizamide on the kinetics of rat brain hexokinase were compared in vitro with those of 2-deoxyglucose and glucosamine. Although metrizamide, 2-deoxyglucose, and glucosamine are known to be competitive inhibitors of approximately equal potency for glucose of yeast hexokinase ( K ₁ approximately 0.7 m m for all three), metrizamide is a much weaker competitive inhibitor ( K _i about 20 m m ) of rat brain hexokinase than either 2-deoxyglucose or glucosamine ( K _i about 0.3 m m for both). This indicates a greater active site specificity of rat brain hexokinase than of yeast hexokinase. Rat brain hexokinase activity is enhanced approximately threefold in the presence of 0.05, 0.2, and 0.8 mg/ml bovine serum albumin, while yeast hexokinase is only enhanced by 50% under these conditions. Despite the high K _i value for metrizamide, interference with glucose metabolism may occur whenever metrizamide is present in much higher concentrations than glucose. Myelography in humans is one such situation.     

Transport of G AB A, β-Alanine and Glutamate into Perikarya of Postnatal Rat Cerebellum

Abstract: Cells dissociated from the postnatally developing rat cerebellum retain their high-affinity carrier-mediated transport systems for [³H]GABA ( K _t=1.9 μM, V = 1.8 pmol/10⁶ cells/min) and [³H]glutamate ( K _t= 10 μM, V = 7.9 pmol/10⁶ cells/min). Using a unit gravity sedimentation technique it was demonstrated that [3H]GABA was taken principally into fractions that were enriched in inhibitory neurons (Purkinje, stellate and basket cells). [³H]β-alanine (which is taken up specifically by the glial GABA transport system) and [³H]glutamate were concentrated by glial-enriched fractions. However [³H]glutamate uptake was minimal in fractions enriched in precursors of granule cells, which may utilise this amino acid as their neurotransmitter. These results are discussed in relation to reports of high-affinity [³H]glutamate uptake by glia. The role of glutamate transport in glutamatergic cells is also considered. The data suggest that high-affinity glutamate transport is a property of glial cells but not granule neurons.