Thomas J. Wheeler

TRANSLOCATION OF TWO GLUCOSE TRANSPORTERS IN HEART : EFFECTS OF ROTENONE, UNCOUPLERS, WORKLOAD, PALMITATE, INSULIN AND ANOXIA

Our previous studies on the acute regulation of glucose transport in perfused rat hearts were extended to explore further the mechanism of regulation by anoxia; to test the effects of palmitate, a transport inhibitor; and to compare the translocation of two glucose transporter isoforms (GLUT1 and GLUT4). Following heart perfusions under various conditions, glucose transporters in intracellular membranes were quantitated by reconstitution of transport activity and by Western blotting. Rotenone stimulated glucose uptake and decreased the intracellular contents of glucose transporters. This indicates that it activates glucose transport via net outward translocation, similarly to anoxia. However, two uncouplers of oxidative phosphorylation produced little or no effect. Increased workload (which stimulates glucose transport) reduced the intracellular contents of transporters, while palmitate increased the contents, indicating that these factors cause net translocation from or to the intracellular pool, respectively. Relative changes in GLUT1 were similar to those in GLUT4 for most factors tested. A plot of changes in total intracellular transporter content vs. changes in glucose uptake was roughly linear, with a slope of -0.18. This indicates that translocation accounts for most of the changes in glucose transport, and the basal pool of intracellular transporters is five times as large as the plasma membrane pool.

Kinetics of glucose transport in human erythrocytes: zero-trans efflux and infinite-trans efflux at 0°C

Abstract The kinetic features of glucose transport in human erythrocytes have been the subject of many studies, but no model is consistent with both the kinetic observations and the characteristics of the purified transporter. In order to reevaluate some of the kinetic features, initial rate measurements were performed at 0°C. The following kinetic parameters were obtained for fresh blood: zero- trans efflux K m = 3.4 mM, V max = 5.5 mM/min; infinite- trans efflux K m = 8.7 mM, V max = 28 mM/min. For outdated blood, somewhat different parameters were obtained: zero- trans efflux K m = 2.7 mM, V max = 2.4 mM/min; infinite- trans efflux K m = 19 mM, V max = 23 mM/min. The K m values for fresh blood differ from the previously reported values of 16 mM and 3.4 mM for zero- trans and infinite- trans efflux, respectively (Baker, G.F. and Naftalin, R.J. (1979) Biochim. Biophys. Acta 550, 474–484). The use of 50 mM galactose rather than 100 mM glucose as the infinite- trans sugar produced no change in the infinite- trans efflux K m values but somewhat lower V max values. Simulations indicate that initial rates were closely approximated by the experimental conditions. The observed time courses of efflux are inconsistent with a model involving rate-limiting dissociation of glucose from hemoglobin (Naftalin, R.J., Smith, P.M. and Roselaar, S.E. (1985) Biochim. Biophys. Acta 820, 235–249). The results presented here support the adequacy of the carrier model to account for the kinetics.

Reconstitution of the glucose transporter from bovine heart

Reconstitution of the glucose transporter from heart should be useful as an assay in its purification and in the study of its regulation. We have prepared plasma membranes from bovine heart which display D-glucose reversible binding of cytochalasin B (33 pmol sites/mg protein; Kd = 0.2 muM). The membrane proteins were reconstituted into liposomes by the freeze-thaw procedure. Reconstituted liposomes showed D-glucose transport activity which was stereospecific, saturable and inhibited by cytochalasin B, phloretin, and mercuric chloride. Compared to membrane proteins reconstituted directly, proteins obtained by dispersal of the membranes with low concentrations of cholate or by cholate solubilization showed 1.2- or 2.3-fold higher specific activities for reconstituted transport, respectively. SDS-polyacrylamide gel electrophoresis followed by electrophoretic protein transfer and labeling with antisera prepared against the human erythrocyte transporter identified a single band of about 45 kDa in membranes from both dog and bovine hearts, a size similar to that reported for a number of other glucose transporters in various animals and tissues.

Stimulation of cardiac glucose transport by inhibitors of oxidative phosphorylation

Previously we showed that hypoxia in heart stimulates glucose transport via translocation of glucose transporters from intracellular membranes to the plasma membrane. We later showed that rotenone, an inhibitor of oxidative phosphorylation, also decreased intracellular transporters. Here, using another membrane fractionation technique, we show that rotenone increases plasma membrane transporters, and that another respiratory chain inhibitor, azide, acts similarly. Thus, they likely activate the same signaling pathway as hypoxia. Genistein, a tyrosine kinase inhibitor, inhibited insulin- and azide-stimulated 3-O-methylglucose transport similarly in cardiac myocytes. It also increased glucose transporters in the plasma membranes of perfused hearts even though it inhibited glucose uptake, suggesting effects on membrane trafficking. Another tyrosine kinase inhibitor, lavendustin A, and the cyclic nucleotide-dependent protein kinase inhibitors H-8 and H-7 had little effect on basal or azide-stimulated transport. Polymyxin B was a weak inhibitor of basal, insulin-stimulated, and azide-stimulated transport. A nitric oxide donor and a nitric oxide synthase inhibitor had no effect on basal and azide-stimulated transport. The results indicate that tyrosine kinases; protein kinases A, G, and C; and nitric oxide are not involved in the hypoxic activation of cardiac glucose transport.

Effects of three proposed inhibitors of adipocyte glucose transport on the reconstituted transporter

Three compounds which inhibit glucose transport in rat adipocytes have been proposed to act directly on the glucose transporter protein. We tested these proposals by examining the effects of the compounds on the stereospecific glucose uptake catalyzed by adipocyte membrane proteins after reconstitution into liposomes. Effects on the transport activity reconstituted from human erythrocyte membranes were also examined. Glucose 6-phosphate, which was suggested to inhibit the transporter noncompetitively (Foley, J.E. and Huecksteadt, T.P. (1984) Biochim. Biophys. Acta 805, 313-316), had no effect on either type of reconstituted transporter, even when present at 5 mM on both sides of the liposomal membranes. Thus, it is unlikely to act directly on the transporter. The metalloendoproteinase substrate dipeptide Cbz-Gly-Phe-NH2, which inhibited insulin-stimulated but not basal glucose uptake in adipocytes (Aiello, L.P., Wessling-Resnick, M. and Pilch, P.F. (1986) Biochemistry 25, 3944-3950), inhibited the reconstituted erythrocyte transporter noncompetitively with a Ki of 1.5-2 mM. The inhibition of the erythrocyte transporter was identical in liposomes of soybean and egg lipid. Transport reconstituted using adipocyte membrane fractions was also inhibited by the dipeptide, with the activity from basal microsomes more sensitive than that from insulin-stimulated plasma membranes. These results indicate that the dipeptide interacts directly with the transporter, and may be a potentially useful probe for changes in transporter structure accompanying insulin action. Phenylarsine oxide, which was suggested to act directly on the adipocyte transporter (Douen, A.G., and Jones, M.N. (1988) Biochim. Biophys. Acta 968, 109-118), produced only slight (about 10%) inhibition of the reconstituted adipocyte and erythrocyte transporters, even when present at 100-200 microM and after 30 min of pretreatment. These results suggest that the major actions of phenylarsine oxide observed in adipocytes are not direct effects on the transporter, but rather effects on the pathways by which insulin regulates glucose transport activity (Frost, S.C. and Lane, M.D. (1985) J. Biol. Chem. 260, 2646-2652).

CHARACTERIZATION OF GLUCOSE TRANSPORT ACTIVITY RECONSTITUTED FROM HEART AND OTHER TISSUES

We examined several aspects of glucose transport reconstituted in liposomes, with emphasis on transporters of rat heart (mostly GLUT4) compared to those of human erythrocytes (GLUT1), and on effects of agents that modulate transport in intact cells. Several types of samples gave higher reconstituted activity using liposomes of egg lipids rather than soybean lipids. Diacylglycerol, proposed to activate transporters directly as part of the mechanism of insulin action, increased the intrinsic activity of heart transporters by only 25%, but increased the size of the reconstituted liposomes by 90%. The dipeptide Cbz-Gly-Phe-NH2 inhibited GLUT4 with a Ki of 0.2 mM, compared to 2.5 mM for GLUT1, which explains its preferential inhibition of insulin-stimulated glucose transport in adipocytes. Verapamil, which inhibits insulin- and hypoxia-stimulated glucose transport in muscle, had no effect on reconstituted transporters. Heart transporters had a higher Km for glucose uptake (13.4) than did GLUT1 (1.6 mM), in agreement with a recent study of GLUT1 and GLUT4 expressed in yeast and reconstituted in liposomes. Transporters reconstituted from heart and adipocytes were 40-70% inactivated by external trypsin, suggesting the presence of trypsin-sensitive sites on the cytoplasmic domain of GLUT4. NaCl and KCl both reduced reconstituted transport activity, but KCl had a much smaller effect on the size of the liposomes.

Permeability of fructose-1,6-bisphosphate in liposomes and cardiac myocytes

Fructose-1,6-bisphosphate (FBP) helps preserve heart and other organs under ischemic conditions. Previous studies indicated that it can be taken up by various cell types. Here we extended observations from our group that FBP could penetrate artificial lipid bilayers and be taken up by cardiac myocytes [8, 10], comparing the uptake of FBP to that of L-glucose. Using liposomes prepared by the freeze-thaw method, FBP entered about 200-fold slower than L-glucose. For liposomes of either soybean or egg lipids, 50 mM FBP enhanced the permeability of FBP itself, with little effect on general permeability (measured by uptake of L-glucose). In experiments with isolated cardiac myocytes at 21°C, FBP uptake exceeded the uptake of L-glucose by several fold and appeared to equilibrate by 60 min. There was both a saturable component at micromolar levels and a nonsaturable component which dominated at millimolar levels. The saturable component was inhibited by Pi and by other phosphorylated sugars, though with lower affinity than FBP. Both saturable and nonsaturable uptakes were also observed at 3°C. The results indicate that FBP enters myocytes not by simple penetration through the lipid bilayer, but via at least two distinct protein-dependent processes. The uptake could lead to intracellular effects important in hypothermic heart preservation.

Reconstitution of glucose transport activity from erythrocyte membranes without detergent and its use in studying effects of ATP depletion

The direct reconstitution of unsolubilized membrane proteins by the freeze-thaw procedure avoids possible changes in properties produced by detergent solubilization and fractionation. Glucose transport activity was reconstituted using human erythrocyte membranes, with about 2/3 of the glucose uptake being stereo-specific. The highest specific activity occurred at low ratios of protein to lipid in the reconstitution, where most transport was due to liposomes containing single transporter molecules. Transporters were reconstituted with a scrambling of orientations, indicated by a 50% inactivation by added trypsin. Separation of unreconstituted protein doubled the specific activity. Similar results were obtained using the purified transporter (Wheeler, T.J. and Hinkle, P.C. (1981) J. Biol. Chem. 256, 8907-8914). The same ratio of net uptake to equilibrium exchange was observed for the two preparations. Their relative reconstituted transport activities and cytochalasin B binding activities were equal, indicating that the two were reconstituted with similar efficiencies. The decrease in glucose transport in erythrocytes produced by ATP depletion and the stimulation produced by resealing with ATP (Jacquez, J.A. (1983) Biochim. Biophys. Acta 727, 367-378) were confirmed. However, no difference was observed in reconstituted transport activity using ghosts resealed with or without ATP, indicating that ATP produces indirect effects rather than modifications of the transporter.

Protection of Rat Cardiac Myocytes by Fructose-1,6-Bisphosphate and 2,3-Butanedione

Earlier studies by our group showed that fructose-1,6-bisphosphate (FBP) enhances the hypothermic preservation of rat cardiac myocytes and the functional recovery of animal hearts after hypothermic storage. However, the mechanisms involved were not clear. We extended the cardiomyocyte studies by testing whether the FBP effects were due to chelation of extracellular calcium, leading to lower intracellular levels. We also tested effects of 2,3-butanedione monoxime (BDM), pyruvate, and adenine nucleotide precursors. Cardiomyocytes were incubated in ischemic suspension at 3°C, and aliquots examined over 48 to 72 hours for retention of rod-shaped morphology, a measure of viability. Cytosolic Ca2+ levels were measured in some experiments. FBP at 5 mM reduced the death rate even when added after one or two days of incubation. It caused cytosolic calcium levels that were 33% lower than controls in freshly-isolated cells and 70% lower after one day of incubation. EGTA protected against cell death similarly to FBP. These results indicated that one of the mechanisms by which FBP exerts protective effects is through chelation of extracellular calcium. BDM was strongly protective and reduced cytosolic calcium by 30% after one day of incubation. As with FBP, BDM was effective when added after one or two days of incubation. BDM may be useful in combination with FBP in preserving heart tissue. Pyruvate, adenine, and ribose provided little or no protection during hypothermia.

Accelerated net efflux of 3-O-methylglucose from rat adipocytes: a reevaluation

In a study of 3-O-methylglucose transport in insulin-stimulated rat adipocytes (catalyzed primarily by the GLUT4 isoform), it was reported that at 37 degrees C the Km and Vmax were 2.8-fold higher for net efflux than for equilibrium exchange (Vinten, J. (1984) Biochim. Biophys. Acta 772, 244-250). Because of its implications for the relative sizes of steps in the transport cycle, we reinvestigated this phenomenon. Accelerated net efflux was apparent when the extracellular methylglucose was diluted 26-fold but not when it was diluted 11-fold. When analyzed according to the one-site alternating conformation model, the data indicate about a 1.7-fold higher Vmax for efflux than for exchange, only about 40% of the difference reported previously. Together with other results in the literature, the accelerated net flux indicates that the conformational change of the loaded transporter from its outward-facing to its inward-facing form is likely the slowest step in the transport cycle, in contrast to the case for GLUT1. Experiments at 25 degrees C indicate a lower degree of accelerated net flux than at 37 degrees C. This is consistent with the above conformational change being the step with the lowest activation energy, as for GLUT1.