Glucose uptake and metabolism by cultured human skeletal muscle cells: rate-limiting steps.

To use primary cultures of human skeletal muscle cells to establish defects in glucose metabolism that underlie clinical insulin resistance, it is necessary to define the rate-determining steps in glucose metabolism and to improve the insulin response attained in previous studies. We modified experimental conditions to achieve an insulin effect on 3-O-methylglucose transport that was more than twofold over basal. Glucose phosphorylation by hexokinase limits glucose metabolism in these cells, because the apparent Michaelis-Menten constant of coupled glucose transport and phosphorylation is intermediate between that of transport and that of the hexokinase and because rates of 2-deoxyglucose uptake and phosphorylation are less than those of glucose. The latter reflects a preference of hexokinase for glucose over 2-deoxyglucose. Cellular NAD(P)H autofluorescence, measured using two-photon excitation microscopy, is both sensitive to insulin and indicative of additional distal control steps in glucose metabolism. Whereas the predominant effect of insulin in human skeletal muscle cells is to enhance glucose transport, phosphorylation, and steps beyond, it also determines the overall rate of glucose metabolism.

Functional interaction between the N- and C-terminal halves of human hexokinase II.

Mammalian hexokinases (HKs) I-III are composed of two highly homologous approximately 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 higher Ki for G6P (KiG6P) than N-HKII. We here address the question of whether the high KiG6P 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 KiG6P 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 KiG6P (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 the KiG6P of the chimeric enzyme. This resembles the behavior of HKII, in which a D209A mutation results in an increase in the KiG6P 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.

GLUT1 is adequate for glucose uptake in GLUT2-deficient insulin-releasing beta-cells.

GLUT2 may play an important role in pancreatic beta-cell glucose metabolism. A decrease in glucose uptake due to underexpression of GLUT2 has been considered as the cause of beta-cell dysfunction in diabetes with different pathogenesis. However, this view has been challenged by recent studies, in which the underexpression of GLUT2 was not accompanied by a decrease in glucose uptake. Our present aim is to evaluate the presumed importance of GLUT2 in maintaining the efficiency of beta-cell glucose uptake. We studied the kinetic characteristics of 3-O-methylglucose uptake in two beta-cell lines. One of these is the beta TC3 cell line which expresses GLUT1 and the other is the beta HC9 cell line which expresses both GLUT1 and GLUT2. Under equilibrium exchange conditions, 3-O-methylglucose transport in these two cell lines showed similar values of K(m) and V(max). The apparent IC50 of cytochalasin B for inhibiting 3-O-methylglucose transport in beta HC9 cells was nine times as high as in beta TC3 cells, indicating that GLUT1 is the critically important glucose transporter in the beta TC3 cell line and GLUT2 in the beta HC9 cell line. In both cell lines, the rates of glucose uptake were at least three times as fast as that of glucose phosphorylation. Our results suggest that GLUT1 is able to compensate for GLUT2 loss as it occurs in beta TC3 and maintains a commensurately high capacity of glucose uptake to sustain glucose metabolism in pancreatic beta-cells.

Functional organization of mammalian hexokinase II. Retention of catalytic and regulatory functions in both the NH2- and COOH-terminal halves.

The mammalian hexokinase (HK) family includes three closely related 100-kDa isoforms (HKI-III) that are thought to have arisen from a common 50-kDa precursor by gene duplication and tandem ligation. Previous studies of HKI indicated that a glucose 6-phosphate (Glu-6-P)-regulated catalytic site resides in the COOH-terminal half of the molecule and that the NH2-terminal half contains only a Glu-6-P binding site. In contrast, we now show that proteins representing both halves of human and rat HKII have catalytic activity and that each is inhibited by Glu-6-P. The intact enzyme and the NH2- and COOH-terminal halves of the enzyme each increase glucose utilization when expressed in Xenopus oocytes. Mutations corresponding to either Asp-209 or Asp-657 in the intact enzyme completely inactivate the NH2- and COOH-terminal half enzymes, respectively. Mutation of either of these sites results in a 50% reduction of activity in the 100-kDa enzyme. Mutation of both sites results in a complete loss of activity. This suggests that each half of the HKII molecule retains catalytic activity within the 100-kDa protein. These observations indicate that HKI and HKII are functionally distinct and have evolved differently.

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.

Regulation of hexokinase II gene transcription and glucose phosphorylation by catecholamines, cyclic AMP, and insulin.

The hexokinases, by converting glucose to glucose-6-phosphate, help maintain the downhill gradient that results in movement of glucose into cells through the facilitative glucose transporters. GLUT4 and hexokinase (HK) II are the major transporter and hexokinase isoforms in skeletal muscle, heart, and adipose tissue, wherein insulin promotes glucose utilization. To understand whether hormones influence the contribution of phosphorylation to cellular glucose utilization, we investigated the effects that catecholamines, cyclic AMP (cAMP), and insulin have on HKII gene expression in cells representative of muscle (L6 cells) and brown (BFC-1B cells) and white (3T3-F442A cells) adipose tissues. Isoproterenol or the cAMP analog 8-chlorophenylthio-cAMP selectively increase HKII gene transcription in L6 cells, as does insulin (Printz RL, Koch S, Potter LP, O'Doherty RM, Tiesinga JJ, Moritz S, Granner DK: Hexokinase II mRNA and gene structure, regulation by insulin, and evolution. J Biol Chem 268:5209-5219, 1993), and cause a concentration- and time-dependent increase of HKII mRNA in both muscle and fat cell lines without changing HKI mRNA. Isoproterenol and insulin also increase the rate of synthesis of HKII protein and increase glucose phosphorylation and glucose utilization in L6 cells.

Ascorbic acid recycling enhances the antioxidant reserve of human erythrocytes.

The role of ascorbate transport and metabolism in the response of human erythrocytes to an extracellular oxidant stress was investigated. Rates of entry and exit of [14C]dehydroascorbate from erythrocytes were more than 10-fold greater than those of [14C]ascorbate. Both the reduced and oxidized forms of the vitamin were transported largely by the glucose transporter. Inside erythrocytes, dehydroascorbate was converted to ascorbate, increasing intracellular ascorbate concentrations 2-3-fold over those in the medium. In such ascorbate-loaded cells, the membrane-impermeant oxidant ferricyanide induced a transmembrane oxidation of intracellular ascorbate to dehydroascorbate. The latter escaped the cells on the glucose transporter, which resulted in a halving of the net entry of [14C]dehydroascorbate in the presence of ferricyanide. Treatment of ascorbate-loaded cells with H2O2 and Cu2+ also oxidized ascorbate and induced efflux of [14C]dehydroascorbate. Ferricyanide-dependent intracellular oxidation of ascorbate resulted in a corresponding reduction of extracellular ferricyanide, which served as an integrated measure of ascorbate recycling. Ferricyanide reduction was proportional to the loading concentration of dehydroascorbate and was enhanced when loss of dehydroascorbate from cells was decreased, either by blockade of the glucose transporter or by concentrating the cells. Selective depletion of cellular ascorbate lowered rates of ferricyanide reduction by two-thirds, suggesting that ascorbate rather than NADH is the major donor for the transmembrane ferricyanide oxidoreductase activity. On the basis of the ascorbate-dependent rate of ferricyanide reduction, erythrocytes at a 45% hematocrit can regenerate the ascorbic acid present in whole blood every 3 min. Erythrocyte ascorbate recycling may thus contribute more to the antioxidant reserve of blood than is evident from plasma ascorbate concentrations alone.

Two regions of GLUT 2 glucose transporter protein are responsible for its distinctive affinity for glucose.

The glucose transporter in the hepatocyte and pancreatic beta-cell (GLUT 2) has a lower affinity for glucose than other members of the glucose transporter family. To investigate the molecular mechanism for the distinctive affinity of GLUT 2 for glucose, we expressed chimeric GLUT 2 and GLUT 4 proteins in Xenopus oocytes and measured 3-O-methyl-D-glucose transport. In the oocyte system, GLUT 2 had a Km of 31.8 +/- 2.8 mM for 3-O-methyl-D-glucose, whereas GLUT 4 had a Km of 7.2 +/- 2.4 mM under equilibrium exchange conditions. GLUT 4/GLUT 2 chimera that contained the intracellular loop and transmembrane domains 7-12 of GLUT 2 (amino acids 239-497) had a Km similar to that of wild-type GLUT 2. A GLUT 4/GLUT 2 chimera in which the COOH-terminal 30 amino acids of GLUT 4 were replaced with the corresponding region of GLUT 2 had a 2-fold higher Km than GLUT 4, but still had a much lower Km than GLUT 2. These results indicate that both transmembrane domains 7-12 and the COOH-terminus of the protein are responsible for the distinctive glucose affinity of GLUT 2.

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.

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.

Coupling of glucose transport and phosphorylation in Xenopus oocytes and cultured cells: determination of the rate-limiting step.

The initial events in glucose metabolism by all cells are the transport and phosphorylation of glucose. To quantify the relative contributions of these two processes to overall glucose utilization, we have developed an experimental approach for their in situ measurement as parallel processes. The method is based on the use of intracellular [2-3H]glucose as a substrate for both the transporter and hexokinase, and involves simultaneous measurement of [2-3H]glucose efflux and of 3H2O released by phosphorylation. The Xenopus oocyte expression system was used to test the method, since in these cells transport and phosphorylation activities can be regulated by expression of mRNA or injection of foreign protein. Oocytes microinjected with [2-3H]glucose showed no release of injected glucose, but did have saturable phosphorylation kinetics, with a Km of 40 microM and a Vmax of 0.1 nmol/min/oocyte. Co-injection of yeast hexokinase increased glucose phosphorylation by five-fold. Expression of human glucose transporter (GLUT1) mRNA resulted in a 25-30-fold increase in the rate of saturable efflux of microinjected glucose compared to control oocytes. The kinetics of transport and phosphorylation of [2-3H]glucose were analyzed by a multiple curve-fitting program that provided estimates of kinetic coefficients for both processes from a single time course. The analysis showed that expression of GLUT1 shifted the rate-limiting step in glucose utilization from transport to phosphorylation. A similar shift occurred at a three-fold lower extracellular concentration of 2-deoxyglucose. In a pancreatic beta cell line both transport and phosphorylation showed high Km values, with phosphorylation as the limiting step. The in situ measurement of glucose transport and phosphorylation as parallel processes should be useful in defining the relative contributions of each step to overall glucose metabolism in other cell and tissue models.

Induction of aP2 gene expression by nonmetabolized long-chain fatty acids.

Long-chain fatty acids (FA) have been shown to regulate expression of the gene for the adipocyte FA-binding protein aP2. We examined whether this effect was exerted by FA themselves or by a FA metabolite. The alpha-bromo derivative of palmitate, an inhibitor of FA oxidation, was synthesized in the radioactive form, and its metabolism was investigated and correlated with its ability to induce aP2 in Ob1771 preadipocytes. alpha-Bromopalmitate was not utilized by preadipocytes. It was not cleared from the medium over a 24-hr period and was not incorporated into cellular lipids. Short incubations indicated that alpha-bromopalmitate exchanged across the preadipocyte membrane but remained in the free form inside the cell. In line with this, preadipocyte homogenates did not activate alpha-bromopalmitate to the acyl form. However, although it was not metabolized, bromopalmitate was much more potent than native FA in inducing aP2 gene expression. Induction exhibited the characteristics previously described for native FA, indicating that a similar if not identical mechanism was involved. The data indicated that induction of aP2 was exerted by unprocessed FA. Finally, in contrast to preadipocytes, adipocytes metabolized bromopalmitate. This reflected increased activity with cell differentiation of a palmitoyl-CoA synthase that could activate palmitate and bromopalmitate at about one-fifth the rate for palmitate. In preadipocytes, the predominant fatty-acyl-CoA synthase, arachidonyl-CoA synthase, had very low affinity for both FA. Increased activity of the palmitoyl-CoA synthase, which has a wider substrate range, is likely to be important for initiation of lipid deposition.

Transport and metabolism of glucose in an insulin-secreting cell line, beta TC-1.

Kinetic characteristics of glucose transport and glucose phosphorylation were studied in the islet cell line beta TC-1 to explore the roles of these processes in determining the dependence of glucose metabolism and insulin secretion on external glucose. The predominant glucose transporter present was the rat brain/erythrocyte type (Glut1), as determined by RNA and immunoblot analysis. The liver/islet glucose transporter (Glut2) RNA was not detected. The functional parameters of zero-trans glucose entry were Km = 9.5 +/- 2 mM and Vmax = 15.2 +/- 2 nmol min-1 (microL of cell water)-1. Phosphorylation kinetics of two hexokinase activities were characterized in situ. A low-Km (0.036 mM) hexokinase with a Vmax of 0.40 nmol min-1 (microL of cell water)-1 was present along with a high-Km (10 mM) hexokinase, which appeared to conform to a cooperative model with a Hill coefficient of about 1.4 and a Vmax of 0.3 nmol min-1 (microL of cell water)-1. Intracellular glucose at steady state was about 80% of the extracellular glucose from 3 to 15 mM, and transport did not limit metabolism in this range. In this static (nonperifusion) system, 2-3 times more immunoreactive insulin was secreted into the medium at 15 mM glucose than at 3 mM. The dependence of insulin secretion on external glucose roughly paralleled the dependence of glucose metabolism on external glucose. Simulations with a model demonstrated the degree to which changes in transport activity would affect intracellular glucose levels and the rate of the high-Km hexokinase (with the potential to affect insulin release).

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.

Evidence for functionally distinct glucose transporters in basal and insulin-stimulated adipocytes.

The activity and Km of glucose transport of rat adipocytes are quite variable in the basal state. This could be due to differing levels of highly saturable transport against a background of less saturable transport. Such heterogeneity could lead to differing conclusions as to the Km of basal cells compared to insulin-stimulated cells depending on the choice of substrate, the range of concentrations tested, and the rigor of data analysis. In the present work, we used a cell preparation which was stable and partially activated by constant agitation. We used a two-component model to fit the concentration dependence of D-glucose uptake. We defined two parallel pathways of glucose entry, a high-affinity/low-capacity pathway and a low-affinity/high-capacity pathway. Both pathways were stereospecific and were inhibited by cytochalasin B. The low-affinity pathway in basal cells had 97% of the total capacity (Vmax) with a high Km (greater than 50 mM). A second pathway had a very low Km (less than 1 mM) and only 3% of the total capacity, but contributed to 30-60% of glucose uptake at 8 mM glucose. In insulin-stimulated cells, a pathway with a Km of 4-5 mM dominated and contributed 85% of glucose transport. The low-affinity but not the very high affinity pathway persisted in stimulated cells, but its contribution was only 10-15% of transport at 8 mM glucose. These results suggest the presence of at least two functionally distinct transporters whose respective contributions can be characterized by nonlinear regression of data over a wide range of glucose concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation energy of the slowest step in the glucose carrier cycle: break at 23 degrees C and correlation with membrane lipid fluidity.

Glucose transport in the rat erythrocyte is subject to feedback regulation by sugar metabolism at high but not at low temperatures [Abumrad et al. (1988) Biochim. Biophys. Acta 938, 222-230]. This indicates that temperature, which is known to alter membrane fluidity, also alters sensitivity of transport to regulation. In the present work, we have investigated a possible correlation between the effects of temperature on rate-limiting steps of glucose transport and on membrane fluidity. The dependences of methylglucose efflux and influx on cis and trans methylglucose concentrations were studied at temperatures between 17 and 37 degrees C. Membrane fluidity was monitored over the same temperature range by using electron paramagnetic resonance spectroscopy. External sugar did not affect efflux, and the Km and Vmax of sugar exit were respectively the same as the Km and Vmax of equilibrium exchange. These Km's were relatively temperature independent, but the Vmax's increased sharply with temperature. The Km and Vmax of methylglucose entry were respectively much lower than the Km and Vmax of exit and exchange. Consistent with the above, intracellular sugar greatly enhanced sugar influx, and did so by increasing the influx Vmax without affecting the influx Km. Both lines of evidence indicated that the conformational change of the empty sugar-binding site from in-facing to out-facing orientation is the rate-limiting step of sugar entry into the rat erythrocyte. This was the case at all temperatures; however, the discrepancies of coefficients declined significantly with increasing temperature.2+ The temperature dependence of the slowest step (change from in- to out-facing empty carrier) was evaluated.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin antagonism of catecholamine stimulation of fatty acid transport in the adipocyte. Studies on its mechanism of action.

Insulin at physiological concentrations can suppress catecholamine activation of the membrane transport of long chain fatty acids in the adipocyte. We have previously shown that the stimulatory effect of catecholamines was mediated by a beta-receptor interaction and cAMP (Abumrad, N.A., Park, C.R., and Whitesell, R. R. (1986) J. Biol. Chem. 261, 13082-13086). In this study we have investigated the mechanism of insulin action to antagonize transport activation. Fatty acid transport was stimulated using different cAMP derivatives with varying susceptibilities to hydrolysis by the cAMP-degrading enzyme phosphodiesterase. Insulin was effective in antagonizing the effect of cAMP analogs which were good substrates for the phosphodiesterase and failed to suppress the effect of those which were poorly hydrolyzed by the enzyme. Addition of increasing concentrations (1-100 microM) of the phosphodiesterase inhibitor methylisobutylxanthine (MIX) to norepinephrine (0.1 microgram/ml) gradually abolished insulin's antagonism. Insulin was completely ineffective in inhibiting stimulation by norepinephrine and 20 microM methylisobutylxanthine. Also consistent with involvement of cAMP lowering in insulin action was the finding that adenosine removal greatly diminished insulin's responsiveness. Treatment of cells with adenosine deaminase (1 unit/ml) enhanced the effect of norepinephrine by about 30%. A 10-fold higher range of insulin concentrations was then required to produce inhibition of fatty acid transport. The effect of adenosine removal was reversed by addition of phenylisopropyladenosine (500 nM), which is resistant to hydrolysis by the deaminase. Finally, exposure of insulin-treated cells (1 nM for 5 min) to dinitrophenol (1 mM for 5 min) reversed insulin action, consistent with reports of reversal of insulin's activation of the phosphodiesterase. In conclusion, our studies support the involvement of cAMP lowering in insulin's antagonism of fatty acid transport stimulation in the adipocyte.

Temperature dependence of glucose transport in erythrocytes from normal and alloxan-diabetic rats.

Alloxan diabetes increased 3-O-methylglucose transport rates in rat red blood cells (RBC) at temperatures below 30 degrees C and decreased them above 30 degrees C. Preincubation of RBC from control rats with 20 mM glucose, 3-O-methylglucose, 2-deoxyglucose or xylose greatly elevated transport at 14 degrees C by increasing Vmax. The effect was slight at 40 degrees C. Preincubation with glucose or deoxyglucose alone caused a 50% depression of transport rates at 40 degrees C as a result of a rise in the Km, which is similar to findings in cells from alloxan-diabetic rats. Measurement of intracellular glucose metabolites suggested inhibition of glycolysis in cells from diabetic rats and a positive correlation between the level of intracellular hexose monophosphates and transport inhibition. Membrane fatty-acid and cholesterol composition and membrane lipid-ordering as monitored by electron paramagnetic resonance were not altered by alloxan diabetes. It is concluded that intracellular sugar and sugar metabolism alter the temperature dependence of glucose transport kinetics. Glucose metabolism can feed back to inhibit transport by increasing the transport Km at physiological temperatures only.

Modulation of basal glucose transporter Km in the adipocyte by insulin and other factors.

We have previously described experimental conditions where basal methylglucose transport in adipocytes exhibited an apparent Km of approximately 35 mM. Under those conditions insulin stimulated transport predominantly by decreasing the transport Km (Whitesell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, 2894-2899). Our findings were in contrast with earlier reports that the Km of basal glucose transport was low (3-5 mM) and similar to that of transport in insulin-treated cells. In this study we have investigated the effect of different experimental conditions on the kinetics of basal glucose transport in adipocytes. When transport was assayed at 37 degrees C, cell agitation for 10 min prior to the transport assay decreased the basal Km from 35 to 12 mM. Deprivation of metabolic substrate produced a further reduction down to 2 mM. Refeeding starved cells with 1 mM glucose returned the Km back up to 12 mM in agitated cells and to 40 mM in stabilized cells. The effects of agitation to lower and of glucose to raise the basal Km were prevented by preincubating cells with dinitrophenol. Cell agitation or substrate lack did not alter the Vmax of basal transport and were without effect on both Km and Vmax in insulin-treated cells. The temperature dependencies of the kinetics of basal and stimulated transport were studied. A decrease in the assay temperature from 37 to 23 degrees C caused both basal Km and Vmax to drop proportionately from 25 to 5 mM, and 13 to 3.6 nmol/(microliter X min), respectively. In insulin-stimulated cells, only the Vmax was decreased (Km went from 3.5 to 3 mM, Vmax from 45 to 17 nmol/(microliter X min]. The results support the concept that experimental conditions can produce large changes in the Km of basal glucose transporters. Furthermore they explain why, under certain assay conditions (with temperatures around 23 degrees C or with deprivation of metabolic substrate), the effect of insulin on transport Km is not observed. Our data also suggest that basal transport characteristics do not persist in insulin-treated cells. We would propose that one of the actions of insulin (in addition to raising Vmax) is to change the characteristics of basal transporters by overriding metabolic factors which keep the Km high. Alternatively, insulin could cause the disappearance of basal transporters as new and different ones are recruited from intracellular stores.