Microanalytical measurements of insulin-stimulated glucose transport in single cells

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CELL & DEVELOPMENTAL BIOLOGY, Vol 7, 1996: pp 279–285

Microanalytical measurements of insulin-stimulated glucose transport in single cells Jill Manchester and John C. Lawrence Jr.

tion of immunolabeled glucose transporters, either by fluorescence or electron microscopy, are useful for detecting heterogeneity in GLUT4 levels. However, such methods do not provide a measure of glucose transport activity. In this review, we summarize results of recent experiments in which ultrasensitive microanalytical methods developed by Lowry and coworkers4 have been used to measure directly insulin-stimulated glucose transport in single mammalian cells. An advantage of the methodology is that it is also possible to measure the activities of many enzymes and levels of numerous metabolites at the single cell level. Thus, microanalysis provides new avenues for investigating relationships between insulin-stimulated glucose transport and metabolism. An exciting development is the use of microanalysis in combination with microinjection to investigate transduction pathways involved in insulin action.

Ultrasensitive microanalytical methods provide a means to measure insulin-stimulated glucose transport in single cells, and have allowed investigation of the functional relationships between GLUT4 and hexokinase. Results obtained using microanalysis demonstrate that depending on the insulin concentration either glucose transport or glucose phosphorylation may limit glucose metabolism in cardiac myocytes. Evidence for coordinate regulation of GLUT4 and hexokinase expression in skeletal muscle fibers has also been obtained. In combination with microinjection, microanalysis is proving useful in investigating the signal transduction pathways involved in the stimulation of glucose transport and glycogen synthase by insulin. Key words: cardiac myocytes / GLUT4 / hexokinase / microinjection / Ras / MAP kinase ©1996 Academic Press Ltd

GLUCOSE TRANSPORT is most dramatically stimulated by insulin in cardiac myocytes, skeletal muscle fibers and adipocytes — cell types that express the highest levels of the glucose transporter isoform, GLUT4.1,2 In nonstimulated cells, most of the GLUT4 is found in intracellular vesicular compartments.1,2 Insulin increases the concentration of transporters found in the plasma membrane, accounting for much, if not all, of the increase in transport produced by the hormone.1-3 The signal transduction pathways involved in the stimulation of glucose transport by insulin have not been defined. Most investigations of the regulation of glucose transport have been conducted using tissues or whole populations of cells. Such studies have contributed greatly to our understanding of the regulation of glucose transport. Nevertheless, it is important to keep in mind that the responses observed represent population averages and that heterogeneity in glucose transport is likely to exist. Methods involving detec-

Microanalytical measurement of glucose transport Glucose transport is most often measured by monitoring the initial rate of uptake of radiolabeled 2-deoxyglucose (DG). With low extracellular concentrations of DG, essentially all of the sugar that enters cells initially accumulates as DG6P, because further metabolism is limited. Microanalytical methods to measure DG6P were originally developed for using nonradioactive DG to study the compartmentation of brain glucose metabolism and changes in neuronal activity.5,6 Measuring insulin-stimulated glucose transport in single cells is made possible by a relatively straightforward adaptation of these methods (Figure 1). Cells are first incubated with hormones and DG.7 Except for the use of unlabeled DG, the conditions for this incubation are the same as would be used for measuring transport with radiolabeled DG. However, subsequent steps differ from the isotopic method, as DG6P is measured enzymatically rather than by scintillation counting. After rinsing, the cells are snap

From the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St Louis, MO 63110, USA ©1996 Academic Press Ltd 1084-9521/96/020279 + 07 $18.00/0

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J. Manchester and J. C. Lawrence, Jr. frozen in liquid Freon chilled to –185° C, then freezedried at –30° C. Individual freeze dried cells, which are visible with a dissecting microscope, are picked up and weighed by using a quartz fiber ‘fish-pole’ balance.4 Placing a cell on the end of the fiber causes a downward deflection, the magnitude of which is related to the weight of the cell. With the most sensitive of these balances, it is possible to measure with a high degree of precision (–/ + 1%) weights as low as 100 pg, the approximate dry weight of a fibroblast.4 After weighing, the cells are transferred to a droplet of HCl to denature cellular enzymes and to extract DG6P. The construction of the ‘fish-pole’ balances and the other specialized equipment used in microanalysis have been described in detail by Lowry and Passoneau.4 DG6P is measured in four steps (Figure 2). In Step 1 the endogenous glucose-6-phosphate (G6P) is enzymatically removed.8 Step 2 is the specific reaction in which DG6P is oxidized by using G6P dehydrogenase and NADP + . In view of the metabolic stability of DG6P, it may seem surprising that G6P dehydrogenase can be used to measure the metabolite. However, DG6P is metabolized in cells, albeit very slowly; and G6P dehydrogenase from several sources will utilize DG6P as substrate, although at rates that are approximately 100 times slower than those with G6P.9 It is important to use very small volumes in Steps 1 & 2 to minimize the background caused by contaminants that are inevitably present in the reagents. For this reason, the reactions are conducted in 10–100 nl incubation mixtures under oil to prevent evaporation and dispersion of the droplets.4 The key to single cell analysis is amplification of the signal through the controlled cycling of the pyridine nucleotide product from Step 2.4 For DG6P this is accomplished by preparing a reaction mixture containing the opposing dehydrogenases, G6P dehydrogenase and glutamate dehydrogenase, and saturat-

ing concentrations of the respective substrates, G6P and α-ketoglutarate plus NH4 + .10 Until pyridine nucleotide is added, nothing happens. However, with the addition of NADPH, the nucleotide is cycled between the oxidized and reduced form. Each turn of the cycle generates 6-phosphogluconate and amplifies the signal. Thus, 10 cycles produce a 10-fold amplification, 1000-cycles produce a 1000-fold amplification, and so forth. Because either NADP + or NADPH will result in cycling, it is necessary to destroy the NADP + substrate that remains after Step 2 before proceeding to Step 3. NADP + is destroyed by heating in the presence of NaOH, a treatment to which NADPH is quite insensitive.4 After the cycling reactions are terminated, the amounts of 6-phosphogluconate are measured in Step 4. Amplification is such that the NADPH formed in Step 4 can be measured using a bench-top fluorometer. The DG6P content of single cells is determined from a curve generated with DG6P standards. These measurements require amplification of approximately 200,000-fold,7 which is by no means the maximum. Indeed, by having the potential for almost unlimited amplification, pyridine nucleotide cycling is comparable to thermal cycling (PCR) used to amplify segments of DNA. With both methods the actual amplification achieved is limited by contamination, depletion of substrates/primers, or inactivation of cycling enzymes.

Figure 1. Approach for measuring glucose transport activity in single cells.

Figure 2. Steps in the microanalytical measurements of DG6P.

Glucose transport in single cardiac myocytes The heart is one of the major insulin responsive tissues, and ventricular myocytes from adult rats retain insulin responsiveness in short-term primary cul-

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Glucose transport in single cells ture.11 The cells are relatively large (8 ng average dry weight) and are loosely attached to the culture surface.7 These two properties are helpful as single cells are picked up and weighed during the course of analyses. In the experiment presented in Figure 3, DG6P accumulation was measured in individual myocytes from cultures that had been incubated with increasing concentrations of insulin for 20 min, followed by a 10 min incubation with the same concentrations of insulin plus 2DG. Under these conditions 2DG6P accumulation has been shown to proceed in a linear manner for at least 20 min, in both the absence and presence of insulin.7 Moreover, accumulation was found to be blocked by cytochalasin B,7 an inhibitor of glucose transport.3 Thus, the changes in DG6P levels depicted in Figure 3 are indicative of changes in glucose transport activity. Even with a limited sampling of cells, the stimulation of DG6P accumulation by insulin is clear (Figure 3). The half maximum effect of the hormone occurred at approximately 70 µU/ml, which is well within the physiological range of insulin concentrations. Cardiac myocytes express relatively high levels of GLUT4, which was readily detected by immunoblotting of samples of individually dissected myocytes.12 Some GLUT1 was found in cultures,7 but not in

dissected myocytes.12 Thus, some of the GLUT1 found in cultures is probably found in fibroblasts and other contaminating cell types. Indeed one advantage of microanalysis is that non-muscle cells and obviously damaged (i.e. rounded-up) myocytes are excluded from the analyses, an operation that is not feasible when transport assays are performed using whole cultures. This may partly explain why the stimulatory effect of insulin in the single cell analyses (10- to 30-fold) is greater than observed previously with whole cultures of myocytes.11 The magnitude of the insulin-stimulated formation of DG6P approaches the increase in the myocyte plasma membrane content of GLUT4 observed by immunoelectron microscopy after injecting rats with insulin.13 DG6P accumulation provides an accurate index of glucose transport only if phosphorylation is not ratelimiting. With low extracellular concentrations of DG, direct measurements indicated that accumulation of intracellular DG in myocytes was negligible over a 20 min incubation period,7 although significant accumulation was noted after longer times ( > 1 h). Failure to detect intracellular glucose has led to the dogmatic view that glucose transport is rate-limiting for glucose metabolism in insulin-responsive cell types. A problem in measuring intracellular glucose in intact tissues

Figure 3. Effect of increasing concentrations of insulin on glucose transport in single cardiac myocytes. Cells were incubated at 37° C with increasing concentrations of insulin for 20 min. After an additional 10 min incubation with the same concentrations of insulin plus 100 µM DG, the cells were rinsed and snap-frozen at –150° C (see ref 7 for details) before DG6P accumulation was measured.

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J. Manchester and J. C. Lawrence, Jr. becomes limiting with insulin stimulation in skeletal muscle is still not clear, and there is evidence both pro17,18 and con.19,20 Skeletal muscle is a heterogeneous mixture of fibers that may have markedly different contractile and metabolic properties.21 Direct measurements of GLUT4 and hexokinase in single, manually dissected, fibers have revealed a very strong correlation between

is the necessity of making relatively large corrections for glucose present in the interstitial space. This is not a serious problem with cultured myocytes which can be rinsed to remove medium glucose; and, under appropriate conditions accumulation of intracellular glucose can be readily demonstrated.7 Values for intracellular glucose and G6P from individual myocytes that had been incubated in a medium containing 5 mM glucose and low, intermediate, and maximally effective concentrations of insulin are presented in Figure 4. Increasing insulin from 3 µU/ml to 100 µU/ml resulted in three-fold increases in both glucose and G6P. When insulin concentrations were raised from 100 µU/ml to the maximally effective level, G6P did not significantly change, but intracellular glucose increased dramatically, approaching 60% of the extracellular concentration. In a detailed study in which steady-state levels of glucose and glucose-6-P levels were varied by incubating cells in a medium containing insulin and different concentrations of glucose, G6P reached a plateau when the intracellular glucose reached 300 µM, or approximately 10-times the Km of hexokinase for glucose.7 Thus, at the normal blood glucose concentration and low insulin, little intracellular glucose accumulates as hexokinase is able to keep pace with the glucose entering via GLUT4 (Figure 5). With increasing insulin, more glucose enters the cells, and as concluded a number of years ago by Morgan and coworkers14, the rate limiting step shifts from transport to phosphorylation (Figure 5). In terms of maintaining blood glucose levels, skeletal muscle is the most important site for insulin-stimulated glucose uptake.15,16 Whether the hexokinase reaction

Figure 4. Insulin increases intracellular glucose in cardiac myocytes. Cells were incubated at 37° C in media containing 5 mM glucose plus 3 µU/ml, 100 µU/ml, or 10 mU/ml for 20 min. The cells were then rinsed and snap-frozen at –150° C. Glucose and G6P were measured in the same single cell extracts as described previously.7 To estimate the intracellular concentration, it was assumed that dry weight equals 20% of wet weight.

Figure 5. Insulin shifts the rate-limiting step from glucose transport to glucose phosphorylation in cardiac myocytes.

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Glucose transport in single cells the levels of GLUT4 and hexokinase among fibers,22 suggesting that their expression is coordinately controlled. Moreover, three weeks of electrical stimulation increased by 14-fold the levels of both GLUT4 and hexokinase.22 With acute electrical stimulation the intracellular glucose concentration may approach that of the blood,23 and thus appears to represent a situation in which glucose phosphorylation is limiting. Nevertheless, the tight correlation between GLUT4 and hexokinase indicates that the fibers place a premium on maintaining a balance in the capacities for glucose transport and phosphorylation.

insulin.12 In contrast, a Ras protein lacking the COOH-terminal site of fatty acylation required for Ras function was without effect. Introducing the neutralizing Ras Ab, Y13-259, into cells attenuated the effect of insulin.12 It seems clear from these findings, and results obtained from gene transfection studies,25,26 that activated Ras can increase glucose transport; however, it can not be concluded that Ras mediates the activation of transport by insulin. That Y13-259 decreased insulin-stimulated transport by only 60% is indicative of another pathway, as is the observation that microinjecting RasG12V potentiated the increase in transport produced by a maximally effective concentration of insulin.12 One model is that glucose transport in myocytes may be activated via two pathways, one of which is independent of Ras. Experiments have been performed to investigate the role of downstream elements in the Ras signaling pathway on glucose transport. Injecting Xenopus oocytes with MAP kinase activates glucose transport,27 although the relevance of this effect to the regulation of transport in mammalian muscle and fat cells is not clear. Injecting thiophosphorylated MAP kinase into cardiac myocytes was without effect on glucose transport, in either the absence or presence of insulin (Figure 6). This finding is consistent with results obtained in adipocytes where it was demonstrated that MAP kinase activity could be dramatically increased without activating glucose transport.28,29

Microinjection/microanalysis: a new strategy to investigate insulin-stimulated glucose transport The mechanism of insulin action is still not known, in part because of the difficulty in selectively changing the activities of signaling intermediates within the cell. An increasingly common approach involves overexpression of potential signaling enzymes in cells or animals. In interpreting the results from such studies it is important to keep in mind the tremendous discrepancy that exists between the time required to increase an enzyme by gene transfection and the time course of insulin action. Insulin stimulates glucose transport in sec to min; whereas, many hours to several weeks may be required for gene expression studies. Long-term changes in signaling molecules are likely to lead to adaptive changes that have little to do with the important rapid responses to insulin. Microinjection provides a means to rapidly change the intracellular levels of almost any substance, provided it is soluble. A major problem in applying microinjection to study the acute regulation of metabolism by insulin has been in analysing the effect of the injected substance. Microanalysis offers a solution to this problem, as glucose transport can be measured in the single cells that have been microinjected.12 The first combined use of microinjection and mircoanalysis was to investigate the role of the GTPbinding protein, Ras, in the regulation of glucose transport by insulin in cardiac myocytes.12 Insulin is known to increase the amount of Ras in the active, GTP-bound, state.24 Accumulation of DG6P was increased several-fold by microinjecting the nonhydrolyzable GTP analogue, GTP-γ-S,12 which activates members of the Ras superfamily of GTP-binding proteins. Injecting a persistently activated H-Ras protein (RasG12V) also increased transport severalfold, and potentiated the activation of transport by

Figure 6. Microinjecting MAP kinase does not activate glucose transport in cardiac myocytes. The recombinant ERK-2 (2 mg/ml) isoform of MAP kinase that had been activated by incubation with ATPγS and MAP kinase kinase was supplied by Dr Timothy Haystead, University of Virginia. Microinjection was performed under the conditions described previously.12 Accumulation of DG6P was measured after 10 min incubation with 100 µM DG. The results are mean values ± S.E. from three experiments.

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Regulation of glycogen synthase

injected cells are transferred to an extraction buffer containing EDTA and KF to inhibit protein kinases and phosphatases. Because of the metabolic stability of DG6P, it is possible to extract the metabolite with this buffer. Thus, a single cell extract can be prepared for measuring both DG6P and glycogen synthase. It is widely accepted that glycogen synthase involves Ras and MAP kinase in a pathway that leads to activation PP1G, the glycogen bound form of Type 1 protein phosphatase.33 PP1G is phosphorylated and activated by Rsk-2 in vitro.34,35 Ras activates the protein kinase Raf-1, triggering the sequential phosphorylation and activation of the downstream protein kinases, MEK, MAP kinase and Rsk-2.36 If it is assumed that this cascade is involved in synthase activation, then injecting activated forms of either Ras or MAP kinase should activate synthase. Injection of neither activated the enzyme in myocytes; however, interpretation of these results are complicated as insulin itself did not activate synthase in these cells. More definitive results have been obtained with 3T3-L1 adipocytes, where synthase activity ratios were increased approximately three-fold and DG6P levels were increased by approximately 10-fold in response to insulin (Figure 7).These results agree well with those obtained when synthase activity and transport were measured by conventional methods in whole culture of 3T3-L1 adipocytes.28 Injecting HRasG12V did not affect glycogen synthase in either the presence or absence of insulin (Figure 7); however, the activated Ras increased DG6P accumulation by approximately three-fold. The results in Figure 7 support the previous conclusion that the Ras/MAP kinase signaling pathway does not mediate the activation of glycogen synthase by insulin in adipocytes.28,29 Thus, the mechanisms by which insulin activates glucose transport and glycogen synthase are still not known. By providing a means to investigate the acute effects of almost any soluble substance, microinjection/microanalysis should prove useful in identifying the signal transduction pathways involved in these and other actions of insulin.

GLUT4 and glycogen synthase are partners in mediating the increase in glycogen synthesis, which accounts for the bulk of the glucose taken up in response to insulin in vivo.30 Insulin stimulates the dephosphorylation and activation of glycogen synthase.31 Microinjection can be used to investigate the regulation of glycogen synthase by using microanalytical methods to measure synthase activity.32 In this case, micro-

Figure 7. Effect of microinjecting HRasG12V on glucose transport and glycogen synthase in 3T3L1 adipocytes. Adipocytes were injected with buffer or with HRasG12V (0.8 mg/ml) and incubated for 3 h. The cells were then incubated with or without insulin (2.5 mU/ml) for 20 min. Glycogen synthase activity and DG6P accumulation were measured in groups of 20–30 cells after a final 20 min incubation in buffer containing 200 µM DG. To correct for unequal cell numbers, the DG6P level in each sample was divided by total glycogen synthase activity (measured in the presence of 10 mM G6P). The results are expressed as a % maximum for DG6P accumulation and as activity ratios for glycogen synthase activity. Mean values ± S.E. from three experiments are presented.

Acknowledgements We thank Dr Oliver H. Lowry for his enthusiastic support and for developing the elegant microanalytical methods that make measurements of insulin-stimulated glucose transport at the single cell level possible.

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Glucose transport in single cells 19. Ziel FH, Venkatesan N, Davidson MB (1988) Glucose utilization is rate-limiting for skeletal muscle glucose utilization in normal and STZ-induced diabetic rats. Diabetes 37:885-890 20. Berger M, Hagg S, Ruderman NB (1975) Glucose metabolism in perfused skeletal muscle. Interaction of insulin and exercise on glucose uptake. Biochem J 146:231-238 21. Peter JB, Barnard RJ, Edgerton VR, Gillespie CA, Stempel DE (1976) Metabolic profiles of three fiber types of skeletal muscle in guinea pigs and rabbits. Biochemistry 11:2627-2633 22. Kong X, Manchester J, Salmons S, Lawrence JC, Jr (1994) Glucose transporters in single skeletal muscle fibers. Relationship to hexokinase and regulation by contractile activity. J Biol Chem 269:12963-12967 23. Hintz CS, Chi MM-Y, Fell RD, Ivy JL, Kaiser KK, Lowry CV, Lowry OH (1982) Metabolite changes in individual rat muscle fibers during stimulation. Am J Physiol 242:C218-C228 24. Osterop AP, Medema RH, Bos JL, van de Zon GCM, Moller DE, Flier JS, Moller W, Massen JA (1992) Relation between the insulin receptor number in cells, autophosphorylation, and insulin-stimulated RasGTP formation. J Biol Chem 267:14647-14653 25. Quon MJ, Chen H, Ing BL, Liu M-L, Zarnowski MJ, Cushman SW, Taylor SI (1995) Constitutively active Ras recruits GLUT4 to the cell surface by an insulin-independent pathway in transfected rat adipose cells. Diabetes 44 (Suppl. 1):19A(Abstract) 26. Kozma L, Baltensperger K, Klarlund J, Porras A, Santos E, Czech MP (1993) The ras signaling pathway mimics insulin action on glucose transporter translocation. Proc Natl Acad Sci USA 90:4460-4464 27. Merrall NW, Plevin RJ, Stokoe D, Cohen P, Nebreda AR, Gould GW (1993) Mitogen-activated protein kinase (MAP kinase), MAP kinase kinase and c-Mos stimulate glucose transport in Xenopus oocytes. Biochem J 295:351-355 28. Robinson LJ, Razzack ZF, Lawrence JC, Jr, James DE (1993) Mitogen-activated protein kinase activation is not sufficient for stimulation of glucose transport or glycogen synthase in 3T3-L1 adipocytes. J Biol Chem 268:26422-26427 29. Lin T-A, Lawrence JC, Jr (1994) Activation of ribosomal protein S6 kinases does not increase glycogen synthesis or glucose transport in rat adipocytes. J Biol Chem 269:21255-21261 30. Jue T, Rothman DL, Shulman GI, Tavitian BA, DeFronzo RA, Shulman RG (1989) Natural-abundance 13C NMR study of glycogen repletion in human liver and muscle. Proc Natl Acad Sci 86:1439-1442 31. Lawrence JC, Jr (1992) Signal transduction and protein phosphorylation in the regulation of cellular metabolism by insulin. Annu Rev Physiol 54:177-193 32. Henry CG, Lowry OH (1985) Enzymes and metabolites of glycogen metabolism in canine cardiac Purkinje fibers. Am J Physiol 248:H599-H605 33. Cohen P (1989) The structure and regulation of protein phosphatases. Annu Rev Biochem 58:453-508 34. Sutherland C, Campbell DG, Cohen P (1993) Identification of insulin-stimulated protein kinase- 1 as the rabbit equivalent of rskmo-2. Identification of two threonines phosphorylated during activation by mitogen-activated protein kinase. Eur J Biochem 212:581-588 35. Dent P, Lavoinne A, Nakielny S, Caudwell FB, Watt P, Cohen P (1990) The molecular mechanism by which insulin stimulates glycogen synthesis in mammalian skeletal muscle. Nature 348:302-308 36. Davis RJ (1993) The mitogen-activated protein kinase signal transduction pathway. J Biol Chem 268:14553-14556

References 1. James DE, Piper RC (1994) Insulin resistance, diabetes, and the insulin-regulated trafficking of GLUT-4. J Cell Biol 126:1123-1126 2. Kasanicki MA, Pilch PF (1990) Regulation of glucose-transporter function. Diabetes Care 13:219-227 3. Simpson IA, Cushman SW (1986) Hormonal regulation of mammalian glucose transport. Annu Rev Biochem 55:1059-1089 4. Lowry OH, Passonneau JV (1992) Enzymatic Analysis: A Practical Guide. Human Press, Clifton, New Jersey 5. McDougal DB, Jr, Ferrendelli JA, Yip V, Pusateri ME, Carter JG, Chi M-Y, Norris B, Manchester J, Lowry OH (1990) Use of nonradioactive 2-deoxyglucose to study compartmentation of brain metabolism and rapid regional changes in rate. Proc Natl Acad Sci USA 87:1357-1361 6. McDougal DB, Jr, Carter JG, Pusateri ME, Manchester JK, Lowry OH (1992) Glucose metabolism assessed with 2-deoxyglucose and the effect of glutamate in subdivisions of rat hippocampal slices. J Neurochem 59:1915-1924 7. Manchester J, Kong X, Nerbonne J, Lowry OH, Lawrence JC, Jr (1994) Glucose transport and phosphorylation in single cardiac myocytes: rate limiting steps in glucose metabolism. Am J Physiol 266:E326-E333 8. Chi MM-Y, Manchester JK, Carter JG, Pusateri ME, McDougal DB, Lowry OH (1993) A refinement of the Akabayashi-SaitoKato modification of the enzymatic methods for 2-deoxyglucose and 2-deoxyglucose 6-phosphate. Analyt Biochem 209:335-338 9. Manchester JK, Chi MM-Y, Carter JG, Pusateri ME, McDougal DB, Lowry OH (1990) Measurement of 2-deoxyglucose and 2-deoxyglucose 6-phosphate in tissues. Analyt Biochem 185:118-124 10. Chi MM-Y, Lowry CV, Lowry OH (1978) An improved enzymatic cycle for nicotinamide-adenine dinucleotide phosphate. Analyt Biochem 89:119-129 11. Shanahan MF, Edwards BM, Ruoho AE (1986) Interactions of insulin, catecholamines, and adenosine in the regulation of glucose transport in isolated rat cardiac myocytes. Biochim Biophys Acta 887:121-129 12. Manchester J, Kong X, Lowry OH, Lawrence JC, Jr (1994) Ras signaling in the activation of glucose transport by insulin. Proc Natl Acad Sci USA 91:4644-4648 13. Slot JW, Gueze HJ, Gigengack S, James DE, Lienhard GE (1991) Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc Natl Acad Sci USA 88:7815-7819 14. Morgan HE, Henderson MJ, Park CR (1961) Regulation of glucose uptake in muscle. I. The effects of insulin and anoxia on glucose transport and phosphorylation in the isolated, perfused heart of normal rats. J Biol Chem 236:253-261 15. Baron AD, Brechtel G, Wallace P, Edelman SV (1988) Rates and tissue sites of non-insulin- and insulin-mediated glucose uptake in humans. Am J Physiol 255:E769-E774 16. Daniel PM, Love ER, Pratt OE (1975) Insulin-stimulated entry of glucose into muscle in vivo as a major factor in the regulation of blood glucose. J Physiol Lond 247:273-288 17. Kubo K, Foley JE (1986) Rate-limiting steps for insulinmediated glucose uptake into perfused rat hindlimb. Am J Physiol 250:E100-E102 18. Yki-Jarvinen H, Young AA, Lamkin C, Foley JE (1987) Kinetics of glucose disposal in whole body and across the forearm in man. J Clin Invest 79:1713-1719

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