Richard R. Whitesell

Ascorbate recycling in human erythrocytes: Role of GSH in reducing dehydroascorbate

Human erythrocytes regenerate ascorbate from its oxidized product, dehydroascorbate. The extent to which such ascorbate recycling occurs by a GSH-dependent mechanism was investigated. In the presence of glucose, erythrocytes took up over 90% of extracellular [14C]dehydroascorbate and rapidly converted it to [14C]ascorbate, which was trapped within the cells. Dehydroascorbate uptake and reduction was not associated with generation of a monoascorbyl free radical intermediate. Uptake and reduction of dehydroascorbate by glucose-depleted erythrocytes coordinately decreased GSH and raised GSSG concentrations in erythrocytes. This effect was reversed by D-glucose, but not by L-lactate. Conversely, depletion of cellular GSH decreased the ability of cells to recycle dehydroascorbate to ascorbate, as reflected in the extent to which cells were able to reduce extracellular ferricyanide. Monoascorbyl free radical was formed during the reduction of extracellular ferricyanide, indicating that one electron transfer steps were involved in this process. In GSH-depleted cells, addition of L-lactate as an energy source for glycolysis-dependent NADH regeneration did cause a partial recovery of the ability of cells to reduce ferricyanide. However, in resealed erythrocyte ghosts containing either 4 mM GSH or 400 mu M NADH, only the GSH-containing ghosts supported regeneration of ascorbate from added dehydroascorbate. These results suggest that in human erythrocytes ascorbate regeneration from dehydroascorbate is largely GSH dependent, and that it occurs through either enzymatic or nonenzymatic reactions not involving the monoascorbyl free radical.

Uptake, recycling, and antioxidant actions of α-lipoic acid in endothelial cells

α-Lipoic acid, which becomes a powerful antioxidant in its reduced form, has been suggested as a dietary supplement to treat diseases associated with excessive oxidant stress. Because the vascular endothelium is dysfunctional in many of these conditions, we studied the uptake, reduction, and antioxidant effects of α-lipoic acid in cultured human endothelial cells (EA.hy926). Using a new assay for dihydrolipoic acid, we found that EA.hy926 cells rapidly take up and reduce α-lipoic acid to dihydrolipoic acid, most of which is released into the incubation medium. Nonetheless, the cells maintain dihydrolipoic acid following overnight culture, probably by recycling it from α-lipoic acid. Acute reduction of α-lipoic acid activates the pentose phosphate cycle and consumes nicotinamide adenine dinucleotide phosphate (NADPH). Lysates of EA.hy926 cells reduce α-lipoic acid using both NADPH and nicotinamide adenine dinucleotide (NADH) as electron donors, although NADPH-dependent reduction is about twice that due to NADH. NADPH-dependent α-lipoic acid reduction is mostly due to thioredoxin reductase. Pre-incubation of cells with α-lipoic acid increases their capacity to reduce extracellular ferricyanide, to recycle intracellular dehydroascorbic acid to ascorbate, to decrease reactive oxygen species generated by redox cycling of menadione, and to generate nitric oxide. These results show that α-lipoic acid enhances both the antioxidant defenses and the function of endothelial cells.

Ascorbate is the major electron donor for a transmembrane oxidoreductase of human erythrocytes

Ascorbic acid is an important antioxidant in human blood. Erythrocytes contribute to the antioxidant capacity of blood by regenerating ascorbate and possibly by exporting ascorbate-derived reducing equivalents through a transmembrane oxidoreductase. The role of ascorbate as an electron donor to the latter enzyme was tested in human erythrocytes and ghosts using nitroblue tetrazolium as an electron acceptor. Although nitroblue tetrazolium was not directly reduced by ascorbate, erythrocyte ghosts facilitated reduction of nitroblue tetrazolium in the presence of ascorbate and ascorbate derivatives containing a reducing double bond. The resulting blue monoformazan product was deposited directly in ghost membranes. Ascorbate-induced monoformazan deposition showed several features of an enzyme-mediated process, including hyperbolic dependence on substrate and acceptor concentrations, as well as sensitivity to enzyme proteolysis, detergent solubilization, and sulfhydryl reagents. Incubation of intact erythrocytes with nitroblue tetrazolium caused deposition of the monoformazan in ghost membranes prepared from the cells. This deposition reflected the intracellular ascorbate content and was inhibited by extracellular ferricyanide, a known electron acceptor for the transmembrane oxidoreductase. Although nitroblue tetrazolium did not cross the cell membrane, like the cell-impermeant ferricyanide, it oxidized intracellular [14C]ascorbate to [14C]dehydroascorbate, which then exited the cells. In resealed ghosts, both monoformazan deposition and ferricyanide reduction were proportional to the intravesicular ascorbate concentration. NADH was only about half as effective as a donor for the enzyme as ascorbate in both open and resealed ghosts. These results suggest that not only can ascorbate donate electrons to a transmembrane oxidoreductase, but that it may be the major donor in intact erythrocytes.

Coupled glucose transport and metabolism in cultured neuronal cells : determination of the rate-limiting step

In brain and nerves the phosphorylation of glucose, rather than its transport, is generally considered the major rate-limiting step in metabolism. Since little is known regarding the kinetic coupling between these processes in neuronal tissues, we investigated the transport and phosphorylation of [2-3H]glucose in two neuronal cell models: a stable neuroblastoma cell line (NCB20), and a primary culture of isolated rat dorsal root ganglia cells. When transport and phosphorylation were measured in series, phosphorylation was the limiting step, because intracellular glucose concentrations were the same as those outside of cells, and because the apparent Km for glucose utilization was lower than expected for the transport step. However, the apparent Km was still severalfold higher than the Km of hexokinase I. When [2-3H]glucose efflux and phosphorylation were measured from the same intracellular glucose pool in a parallel assay, rates of glucose efflux were three- to-fivefold greater than rates of phosphorylation. With the parallel assay, we observed that activation of glucose utilization by the sodium channel blocker veratridine caused a selective increase in glucose phosphorylation and was without effect on glucose transport. In contrast to results with glucose, both cell types accumulated 2-deoxy-d-[14C]glucose to concentrations severalfold greater than extracellular concentrations. We conclude from these studies that glucose utilization in neuronal cells is phosphorylation-limited, and that the coupling between transport and phosphorylation depends on the type of hexose used.

Generation of oxidant stress in cultured endothelial cells by methylene blue: protective effects of glucose and ascorbic acid.

The thiazine dye methylene blue has long been used to stimulate cellular redox metabolism. To determine the extent to which it also generates oxidant stress in cells, its effects in cultured human-derived endothelial cells were studied. As expected, low concentrations of the dye (2-20 microM) activated the pentose phosphate pathway and oxidized both NADPH and NADH. Methylene blue enhanced extracellular ferricyanide reduction, indicating that the reduced form of the dye was present outside the cells. This reduction was greater when ferricyanide was added just before rather than 15 min after methylene blue, confirming that the dye is at least initially reduced at the cell surface. In the absence of glucose, methylene blue at concentrations above 5 microM increased intracellular oxidant stress, as manifest by oxidation of dihydrofluorescein and cellular GSH. Inclusion of glucose protected against these effects. In cells that had been loaded with ascorbate, the dye caused progressive oxidation of ascorbate, even in the presence of D-glucose. Loading cells with ascorbate also partially prevented oxidation of dihydrofluorescein by methylene blue. These results suggest that concentrations of the dye above 5 microM generated intracellular reactive oxygen species that were scavenged by ascorbate and GSH. Further, although D-glucose enhanced reduction of methylene blue, it ameliorated the oxidant stress generated by the dye.

Functional Interaction between the N- and C-terminal Halves of Human Hexokinase II

Mammalian hexokinases (HKs) I–III are composed of two highly homologous ∼50-kDa halves. Studies of HKI indicate that the C-terminal half of the molecule is active and is sensitive to inhibition by glucose 6-phosphate (G6P), whereas the N-terminal half binds G6P but is devoid of catalytic activity. In contrast, both the N- and C-terminal halves of HKII (N-HKII and C-HKII, respectively) are catalytically active, and when expressed as discrete proteins both are inhibited by G6P. However, C-HKII has a significantly higherK i for G6P (K i G6P) than N-HKII. We here address the question of whether the highK i G6P of the C-terminal half (C-half) of HKII is decreased by interaction with the N-terminal half (N-half) in the context of the intact enzyme. A chimeric protein consisting of the N-half of HKI and the C-half of HKII was prepared. Because the N-half of HKI is unable to phosphorylate glucose, the catalytic activity of this chimeric enzyme depends entirely on the C-HKII component. The K i G6P of this chimeric enzyme is similar to that of HKI and is significantly lower than that of C-HKII. When a conserved amino acid (Asp209) required for glucose binding is mutated in the N-half of this chimeric protein, a significantly higher K i G6P (similar to that of C-HKII) is observed. However, mutation of a second conserved amino acid (Ser155), also involved in catalysis but not required for glucose binding, does not increase theK i G6P of the chimeric enzyme. This resembles the behavior of HKII, in which a D209A mutation results in an increase in the K i G6P of the enzyme, whereas a S155A mutation does not. These results suggest an interaction in which glucose binding by the N-half causes the activity of the C-half to be regulated by significantly lower concentrations of G6P.

Sugar-transport kinetics of the rat thymocyte.

Abstract Suspensions of 2 to 5% rat thymocytes were incubated at 35 °C in buffered balanced salt solution (pH 7.3) with lactate and β-hydroxybutyrate as fuels. The dependence of 3- O -[ Me - 3 H]methylglucose influx on external and internal 3- O -methylglucose concentrations was studied. Entry was almost rectilinear during the first minute. From the dependence of methylglucose entry (into sugar-free cells) on external methylglucose concentration, we judged the entry K m to be about 7.7 m m and the entry V to be about 0.64 μmol · min −1 · (ml of packed cell volume) −1 . Methylglucose inside the cell enhanced influx, hence equilibrium exchange was faster than entry. The dependence of equilibrium exchange on methylglucose concentration (inside and outside being equal) indicated a K m of about 25 m m and a V of about 2.1 μmol · (min) −1 · (ml of cell volume) −1 . This effect of internal sugar indicated that entry into sugar-free cells is limited mainly by the return of empty carrier to the outside surface and that loading the carrier on the inside enhances its outward mobility. The K m and V for influx into cells containing 21 m m methylglucose were 5.9 m m and 1.17 μmol · min −1 · (ml of packed cells) −1 . The effect of 21 m m internal sugar on lowering the influx K m from about 7.7 m m to about 6 m m was reproducible and contributed to the evaluation of the constants of the transport rate law. It indicated that loading of the carrier at the external surface reduces its mobility, in contrast to the effect of loading on the inside. Mechanical explanations for this behavior are discussed.

Control of glucose phosphorylation in L6 myotubes by compartmentalization, hexokinase, and glucose transport

In muscle, insulin enhances influx of glucose and its conversion to glucose 6-phosphate (G6P) by hexokinase (HK). While effects of insulin on glucose transport have been demonstrated, its effect on the activity of HK of cells has not. In L6 myotubes treated for 24 h with insulin there was increased expression of the HK isoform, HKII, and increased glucose phosphorylation without a concomitant increase in glucose transport, indirectly suggesting that phosphorylation of glucose was a target of insulin action [Osawa, Printz, Whitesell and Granner (1995) Diabetes 44, 1426-1432]. In the present work the same treatment led to a 2-fold rise in G6P, suggesting that transport and/or HK were important targets of insulin action. We used a method to identify the site of rate control involving the specificity of phosphorylation towards 2-deoxy-[1-14C]glucose and D-[2-3H]glucose. Glucose transport does not greatly discriminate between these two tracers while HK shows increased specificity for glucose. Specificity of the glucose phosphorylation of the cells increased with addition of insulin and when extracellular glucose was raised. Specificity was reduced at low glucose concentrations or when the inhibitor of transport, cytochalasin B, was added. We conclude that transport and HK share nearly equal control over glucose phosphorylation in these cells. A computer program was used to test models for compatibility with the different types of experiments. The predicted intracellular glucose and transport rates associated with phosphorylation activity were lower than their measured values for the whole cell. In the most likely model, 15+/-4% of the glucose transporters serve a proportionate volume of the cytoplasm. Insulin activation of glucose phosphorylation might then result from stimulation of these transporters together with HK recruitment or relief from inhibition by G6P.

Coexpression of glucose transporters and glucokinase in Xenopus oocytes indicates that both glucose transport and phosphorylation determine glucose utilization.

A Xenopus oocyte expression system was used to examine how glucose transporters (GLUT 2 and GLUT 3) and glucokinase (GK) activity affect glucose utilization. Uninjected oocytes and low rates of both glucose transport and phosphorylation; expression of GLUT 2 or GLUT 3 increased glucose phosphorylation approximately 20-fold by a low Km, endogenous hexokinase at glucose concentrations < or = 1 mM, but not at higher glucose concentrations. Coexpression of functional GK isoforms with GLUT 2 or 3 increased glucose utilization approximately an additional two- to threefold primarily at the physiologic glucose concentrations of 5-20 mM. The Km for glucose of both the hepatic and beta cell isoforms of GK, determined in situ, was approximately 5-10 mM when coexpressed with either GLUT 2 or GLUT 3. The increase in glucose utilization by coexpression of GLUT 3 and GK was dependent upon glucose phosphorylation since two missense GK mutations linked with maturity-onset diabetes, 182: Val-->Met and 228:Thr-->Met, did not increase glucose utilization despite accumulation of both a similar amount of immunoreactive GK protein and glucose inside the cell. Coexpression of a mutant GK and a normal GK isoform did not interfere with the function of the normal GK enzyme. Since the coexpression of GK and a glucose transporter in oocytes resembles conditions in the hepatocyte and pancreatic beta cell, these results indicate that increases in glucose utilization at glucose concentrations > 1 mM depend upon both a functional glucose transporter and GK.

Evidence that downregulation of hexose transport limits intracellular glucose in 3T3-L1 fibroblasts.

Measurements of initial glucose entry rate and intracellular glucose concentration in cultured cells are difficult because of rapid transport relative to intracellular volume and a substantial extracellular space from which glucose cannot be completely removed by quick exchanges of medium. In 3T3-L1 cells, we obtained good estimates of initial entry of [14C]methylglucose and D-[14C]glucose with 1) L-[3H]glucose as an extracellular marker together with the [14C]glucose or [14C]methylglucose in the substrate mixture, 2) sampling times as short as 2 s, 3) ice-cold phloretin-containing medium to stop uptake and rinse away the extracellular label, and 4) nonlinear regression of time courses. Methylglucose equilibrated in two phases--the first with a half-time of 1.7 s and the second with a half-time of 23 s; it eventually equilibrated in an intracellular space of 8 microliters/mg protein. Entry of glucose remained almost linear for 10 s, making its transport kinetics easier to study (Km = 5.7 mM, Vmax = 590 nmol.s-1.ml-1 cell water). Steady-state intracellular glucose concentration was 75-90% of extracellular glucose concentration. Cells grown in a high-glucose medium (24 mM) exhibited a 67% reduction of glucose-transport activity and a 50% reduction of steady-state ratio of intracellular glucose to extracellular glucose.