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The inhibition of GLUT1 glucose transport and cytochalasin B binding activity by tricyclic antidepressants
PII SOO24-3205(9!9)0090-1
Life Sciaxas, Vol. 66, No. 3, pp. 271-278.2000 Cbpyi&tC’ 1999 Elwvk Scii Inc. RinkdintbUSA. Allrighbrosaved 0024-3205&O/S-a liunt mat&z
THE INHIBITION OF GLUT1 GLUCOSE TRANSPORT AND CYTOCHALASIN B BINDING ACTIVITY BY TRICYCLIC ANTIDEPRESSANTS Harold B. Pinkofsky, Donard S. Dwyer, Ronald J. Bradley Department of Psychiatry, Louisiana State University Health Sciences Center, P.O. Box 33932, Shreveport, LA 71130-3932 (Receivedin finalform August31, 1999) Summary
Under normal metabolic conditions glucose is an important energy source for the mammalian brain. Positron Emission Tomography studies of the central nervous system have demonstrated that tricyclic antidepressant medications alter cerebral metabolic tknction. The mode by which these drugs perturb metabolism is unknown. In the present study the interactions of tricyclic antidepressants with the GLUT1 glucose transport protein is examined. Amitriptyline, nortriptyline, desipramine, and imipramine all inhibit the intlux of 3-O-methyl glucose into resealed erythrocytes. This inhibition is observed with drug concentrations in the millimolar range. All four antidepressants also noncompetitively displace cytochalasin B binding to GLUTl. The KI for this displacement ranges from 0.56 to 1.43 millimolar. This value is in a range greater than that associated with clinical doses and this effect may not be directly applicable to side effects observed with normal use. The observed interaction of these drugs with GLUT1 may reflect an afhnity for other glucosetransport or glucose-binding proteins, and may possibly contribute to tricyclic antidepressant toxicity. Key F+‘o&: glucose,met&&m, tricyclicantidePressants, GLUT1 , erythrocytes, cytochalasinB, glucose u=Wn The mammalian brain, under normal conditions, relies on glucose as its primary source of energy. Glucose transport into cerebral tissue occurs via facilitative transporters. The family of facilitative glucose transporters, termed GLUT1 through GLUT7, displays a high degree of homology (1). In neurons transmembrane glucose transport is mediated predominantly by GLUT3, and to a lesser extent by GLUT1 (2,3,4). GLUT1 appears to be the primary glucose transporter protein in cerebral microvessels as well as astrocytes (2). GLUT5 has been associated with microglia (2). In-vivo cerebral glucose utilization has been studied using positron emission tomography (PET) and single photon emission computerized tomography (SPECT) technology. Mood disorders have been associated with regional reductions in cerebral glucose metabolism as measured by these techniques. Antidepressant treatment has been observed to normalize glucose metabolism (5). This e&ct is believed to be secondary to the action of antidepressants on neurotransmission. corresponding author: Harold Pit&of&y, MD, PhD., Department of Psychiatry, PO Box 33932, Shreveport, LA 71130-3932. Telephone: (318) 675-6042, FAX: (318) 675-6148
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Other studies have supported these initial PET and SPECT findings. In studies utilizing rats, acute administration of desmethylimipramine, a tricyclic antidepressant, was found to differentially increase cerebral glucose utilization, whereas chronic administration decreased glucose utilization (6). In-vitro experiments demonstrated that imipramine, another tricyclic antidepressant, can inhibit brain tissue respiration (7). Tricyclic antidepressants have also been found to modulate other modalities of glucose regulation. For instance, high concentrations of imipramine (8) and amitriptyline (9) inhibit insulin release from pancreas cells. Paradoxically amitriptyline can produce hyperinsulinemia, with normoglycemia, in rats (10). Imipramine treatment has been demonstrated to produce a reduction in glucose serum levels (11) and there have been case-reports of imipramine producing a hypoglycemic effect in diabetic patients (12). Tricyclic antidepressants have been reported to influence glucose transport into platelets (13). In the present work the influence of tricyclic antidepressants on metabolism was tm-ther investigated using purified erythrocyte ghosts. Human erythrocytes are easily obtainable, lack organelles, and possess only the GLUT1 isoform, which accounts for approximately 6% of the total erythrocyte membrane protein (14). The transport of glucose across the cell membrane may be considered to be one of the early steps in the energy metabolism cascade.
Methods Fresh human blood was collected from healthy donors into KzEDTA containing vacutainers. Ghosts were prepared from erythrocytes by the method of Dodge et al (15). When used for transport studies ghosts were resealed in a 1: 10 balanced salt solution (containing 12.5 mM NaCl, 0.5 mM KCL, 0.38 mM CaC12, and 0.25 mM MgC12 but&red at pH 7.4 with Tris) as previously outlined (16). Transport assays and cytochalasin B binding studies were conducted as previously described (17, 18) and membrane protein was determined by the method of Lowry et al (19). Prior to transport assays, 2 ml of packed, resealed ghosts pre-equilibrated with 0.5 mM 3-O-methyl glucose (3-OMG) were added to 10 ml of a 1:lO balanced salt solution containing 0.5 mM 3-OMG. This suspension was then incubated for 30 minutes at room temperature (25” C). Transport measurements were initiated by adding tracer concentrations of tritiated 3-OMG. At fixed time intervals a 2 ml aliquot was removed from this suspension and added to 10 ml of chilled 1: 10 basic salt solution (0’ C) containing 2 mM HgC&. The samples were then centrifuged at 3 1,000 x g for 15 minutes. The pellets and supematants were separated and assayed for radioactivity. Transport rates were extrapolated from the linear component of uptake at the early transport times by fitting the results to a straight line using a least-means square regression analysis. Transport assays were calculated using measurements obtained from a total transport time course lasting % to 1 minute (Figures la and lb).
Materials Cytochalasin B, mercuric chloride, nortriptyline, desipramine, imipramine and amitriptyline were obtained from Sigma Chemical Company (St. Louis, MO). 3H-Cytochalasin B and “H-3OMG were obtained from Aldrich Chemical Company (Milwaukee, WI).
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Fig. la and lb Inhibition of 3-OMG transport by tricyclic antidepressants. In Figure la resealed erythrocyte ghosts were preincubated in the presence of 0.5 mM 3-OMG and varying concentrations (0.2 mM, 1 mM, and 3 mM) of tricyclic antidepressants (see Methods). The transport rates were normal&d to those observed for control erythrocytes not exposed to tricyclic antidepressants. Error bars display the observed standard error (s3). Transport rates in the presence of antidepressants were compared to rates observed for a control in which transport was assayed in the absence of any antidepressant using the identical preparation of erythrocyte ghosts. A paired Student-t test demonstrated that the transport rate observed in the presence of 1 mM or 3 mM antidepressant concentrations was significantly difkent (pc.05) Corn that observed in control preparations. No significant diflkrence was found for transport in the presence of 0.2 mM antidepressant concentrations (p>.OS). In Figure la, in order to avoid the results being superimposed, the measurements are shown clwtered adjacent to each other for the purpose of clarity. Figure lb shows a typical 3-OMG uptake time course for resealed ghosts in the presence of 0.5 mM 3-O-methyl glucose. 3-OMG uptake is normalized to mg of membrane protein and displayed as a tbnction of time.
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Results Resealed erythrocyte ghost membranes were pre-equilibrated with 0.5 mM 3-OMG as outlined above. Resealed erythrocyte ghosts, in the absence of antidepressants, exhibited a rate of 3OMG transport of 0.294 _t (0.096) nm 3-OMG/mg membrane protein x second (mean + SE, n=3). The observed variability resulted from the different uptake rates displayed by different batches of resealed ghosts. Tricyclic antidepressants were found to inhibit the uptake of tritiated 3-OMG by resealed erythrocyte ghosts as shown in Figure 1. All four tricyclic antidepressants studied (imipramine, desipramine, amitriptyhne, and nortriptyline) exhibited a 30% to 60% inhibition of influx at concentrations of approximately 1 n&I. A similar inhibition pattern was also seen with resealed erythrocyte ghosts using higher concentrations of 3-OMG (not shown). The interaction of tricyclic antidepressants with the erythrocyte glucose transporter was huther studied using cytochalasin B binding assays. Cytochalasin B has been reported to bind the human erythrocyte glucose transporter in erythrocyte ghosts with a dissociation constant of approximately 3-5 x lo-’ M (17, 18). Cytochalasin B binding at this site is displaced by Dglucose. There also exist two other sites in erythrocyte membranes which are not related to the glucose transporter but bind cytochalasin B. The non-transporter associated cytochalasin B binding is normally not altered by D-glucose (17, 18). The cytochalasin B binding at one of the non-transporter sites is inhibited by cytochalasin E. All four antidepressants were found to displace cytochalasin B binding to erythrocyte ghosts (Figure 2). The extent of the tricyclic antidepressant displacement on this binding was greatly diminished when 0.5 M D-glucose was present. The magnitude of tricyclic drug displacement of cytochalasin B binding did not appear altered by the presence of 10-r M cytochalasin E. Furthermore, the inhibition of cytochalasin B binding by tricyclic antidepressants has been observed to be reversible (not shown). The cytochalasin B bmding component sensitive to the presence of tricyclic-antidepressants was analyzed using a Lineweaver-Burk plot (Figures 3a and 3b). Noncompetitive inhibition kinetics were observed for ail four tricyclic antidepressants studied. The averaged inhibition constant (Kr) observed using this analysis ranged from 0.56 to 1.4 mM (see Table I). The averaged cytochalaain B dissociation constant derived from this bmding component was 4.4 (k1.5) x lo-’ M, similar to the range of the dissociation constant for cytochalasin B with the glucose transporter in erythrocyte ghosts (17).
Discussion Prior research has shown that some psychotropic medications exhibit an effect on energy metabolism. Barbiturates have been found to reduce glucose metabolism in the rat brain (21) and hexose transport rates in human erythrocytes (22). Chlorpromazine, an antipsychotic medication, has been found to accelerate glucose ef%lux from erythrocytes at low drug concemrations but inhibit eBIux at higher drug concemrations (23). Recently, we have shown that chlorpromazine and tiphenazine inhiiit glucose uptake in the PC12 neuronal cell line (24). The antidepressant trazodone was found to reduce oxygen consumption in rat brain homogenates (25). Tianeptine, a tricyclic antidepressant, has been reported to inhibit mitochondrial oxidation (26). The role played by t&y&c antidepressants in promoting weight gain and enhan&g fbod consumption, in both human and animal models may be a side effect of the infhrence that these drugs exert on energy metabolism (10,27).
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1T L
1.5 t!b u" 5% oh a
1 0.5 0
L
ii
IIf IDA
OIDAN.
OIDAN
Antidepressant Fig. 2 Cyto~halasin B binding in the presence or absence of tricyclic antidepressants. Erythrocyte ghosts were preincubated for 30 minutes in the presence of 2x10“ M cytochalasin B and the presence or absence of different tricyclic antidepressants at 3 mM concentration (labeled as I, D, A, or N). 0 represents bmding in the absence of antidepressants. I, D, A, and N represent binding in the presence of imipramine, despramine, amitriptyline, and nortiptyline respectively. When present the ticyclic antidepressants are at concentrations of 0.1 n&I, 1 mM, and 3 mM. ‘Bound CB/Free CB” is expressed in units of lo3 liters/mg protein. This binding was studied in the absence of any additional additives (open bars) the presence of 0.5 M D-glucose [dark bars] or the presence of 1OS5 M cytochahtsin E [striped bars]. The binding shown represents the total cytochalasin B binding observed. The nonspecific unsaturated cytochalasin B binding component has not been subtracted. The inhiiition of cytochalasin B bmding in the presence of each antidepressant is statistically sign&ant when compared to parallel controls in the absence of additional additives or the presence of 10% cytochalasin E (p<.OS). In the presence of OSm D-glucose, the binding observed in the presence or absence of antidepressants was not significantly different w.05). Tricyclic antidepressams were not found to have any etTixt on the nonspecific binding component. Error bars display the observed standard error (n=3).
In the present work ticyclic antidepressant medications were observed to inhibit ghmose tranpwrt t%nction and noncompetitively displace cytochalasin B from its glucose-displaceable binding ate. Alterations in membrane fluidity do not appear responsible for the observed changes. Prior studies have found that pertubations in erythrocyte membrane tluidity have little etfect on the monosaccharide transport system (28, 29). The observed noncompetitive kinetics of cytochalasin B displacement suggest that tricyclic antidepressants are. interact& directly with the transport protein. Although tricyclic antidepressants have been previously reported to exhibit metabolic et&&s, this is the tlrst report of an interaction with the glucose transport protein in human erythrocytes. The inhiion constants observed for the trlcyclic
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I= 3mM
o=o 0 = 1 mM A =2mM o=3mM
a
I
2mM
/
/
1 Bound
I
=I =o
r
-1
1
0
2
3
6
5
4
1 Free 20 -
I -1
0
I 1
I
I
2
3
lmipramine concentration Fig. 3a and 3b Inhibition of cytochalasin B binding by imipramine. In Figure 3a the component of cytochalasin B binding which was displaceable by the tricyclic antidepressant was analyzed using a Lineweaver-Burk plot at varying imipramine concentrations (I). Fitted lines were obtained using a least squares analysis (?=.90). The apparently common intercepts on the “l/Free CB” axis suggests The Kr was that imipramine displaces cytochalasin B binding noncompetitively. analyzed for noncompetitive inhibition using the equation “Slope= [K&-l [l + ([I]/&)” (26). Figure 3B shows a graph of the slope versus imipramine concentration with a least squares fit to the data. “l/Bound CB” is expressed in units of 10” mg protein/mole CB. “l/Free CB” is expressed in units of IO’ liter/mole CB. The concentration of imipramine present is displayed to the right of each fitted line.
mM
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Table I The dissociation constants for the tricyclic antidepressant inhibition of cytochalasin B biding. Tricyclic Antidepressant
Kr(mM) (Mea&SE)
Imipramine (n=3) Amitriptyline (n=3) Desipramine (n=3) Nortrintvline (n=3)
0.56*0.12 1.43 f 0.63 0.72 f 0.02 0.98 f 0.10
Inhibition of cytochalasin B binding by tricyclic antidepressants was analyzed as in Figure 3. The dissociation constants shown for the various antidepressants are the average of three separate All of the tricyclic binding assays. antidepressants assayed displayed noncompetitive displacement of cytochalasin B binding.
effect on the glucose transporter are however, within a concentration range well above normal serum levels (30). Therapeutic serum concentrations for most tricyclic antidepressants is between 100 to 260 nanograms/milliliter; lethal tricyclic antidepressant levels have been reported to range from 1,100 to 21,800 nanograms/milliliter (4,000 to 80,000 nanomolefliter) (3 1). The present results were obtained using the GLUT1 isoform and it is possible that other GLUT proteins may have a different sensitivity to the tricyclic antidepressants assayed. Moreover, alterations in the glucose transporter, such as those that occur during development (32), or due to phosphorylation (33) may change the sensitivity to these drugs. It is also possible that the observed effect on glucose transport may reflect binding of the tricyclic Some of the reported antidepressant medications to associated regulatory proteins. physiological changes observed with tricyclic medications may be the result of such an interaction. Conceivably tricyclic antidepressants may act on neuronal cells or at sites in the blood brain barrier. Our findings suggest that alterations in glucose transport may contribute to the toxic properties of tricyclic antidepressants, including those present in clinical overdoses. References
1. 2. 3. 4.
G. W. GOULD and G.D. HOLMAN, Biochem. J. 295 329-341 (1993). F. MAHER, S.J. VANNUCCI, and I.A. SIMPSON, FASEB 8 1003-lOll(1994). F. MAHER and I.A. SIMPSON, Biochem. J. 301 379-384 (1994). A.L. MCCALL, A.M. VANBUEREN, M. MOHOLT-SIEBERT, N.J. CHERRY and W.R. WOODWARD, Brain Res. 659 292-297 (1994). 5. H.S. MAYBERRY, R.K. MAHURIN , and S.K. BRANNAN, Textbook of Neurology. Yudofsky S.L. and Hales R.E. eds. 885-890, American Psychiatric Press Inc, Washington DC (1997). 6. J.C. GERBER, J. CHOKI, D.F. BRUNSWICK, M. REIVICH, and A. FRAZIER Brain Research 269 319-325 (1983) 7. M. NAG andN. NANDI, Biosci. Rep. 11 11-14 (1991).
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8. M. EL DAKHAKHRY, EL ABDEL, H.A. LATIF , and A. HERMANN, Arzneim. Fort/Drug Res. 46(B) 667-669 (1996). 9. H.G. JOOST, W. POSER and U. PANTEN, Naunym-Schmiedeberg’s Arch. Pharmakol. 285 99-102 (1974). 10. L.H. STORLIEN, F.M. HIGSON, R.M. GLEESON, G.A. SMYTHE, and D.M. ATRENS, Biochem. Behav. 22 119-125 (1985). Il. R.G. KATHOL, R. JOECKE, C. WYSMAN, and B.M. SHERM, Psychiatry Res. 41 4552 (1992). 12. SM. KAPLAN, J.W. MASS, J.M. PIXLEY, and W.D. ROSS, JAMA 174 511-517 (1960). 13. A.N. GAUT, J. Pharmacol. Exp. Ther. 190 180-186 (1974). 14. S.A. BALDWIN, Biochem. Biophys. Acta 1154 17-49 (1993). Arch. Biochem. Biophys. 100 11915. J.T. DODGE, C. MITCHELL, and D. J. HANAHAN, 130 (1963). 16. C.Y. JUNG, L.M. CARLSON, and D.A. WHALEY, Biochem. Biophys. Acta 241 613627 (1971). 17. C.Y. JUNG and A.L. RAMPAL, J. Biol. Chem. 252 5456-5463 (1977). 18. H.B. PINKOFSKY, A.L. RAMPAL, M.A. COWDEN, and C.Y. JUNG, J. Biol. Chem. 253 4930-4937 (1978). 19. O.H. LOWRY, N.J. ROSENBROUGH, A.L. FARR, and R.J. RANDALL, J. Biol. Chem. 193 265-275 (1951). 20. I. SEGAL Enzyme Kinetics, John Wiley and Sons, NY 132-136 (1975). 21. T. OTSUKA, L. WEI, D. BERCEZK, V. ACUFF, C. PATLAH, and J. FENSTERMACHER, Am. J. Physiol261 R265-275 (1991). 22. A. EL-BARBARY, J.D. FESTERMACHER, and H.C. HESPEL, Biochemistry 35 15222-I 5227 (1996). 23. G.F. BAKER and H.J. ROGERS, Biochem. Pharmacol. 21 1871-1878 (1972) 24. D.S. DWYER, H.B. PINKOFSKY, YE LIU, R. J. BRADLEY, Prog. NeuroPsychopharmacol. Biol. Psychiatry 23 69-80 (1999). 25. A. VELASCO, V. GONZALEZ-CALVO, F.J. ALVEREZ , A. DUENAS, and J.L. GARCIA-ROLDAN, Rev. Sp. Fisiol. 41 201-206 (1985). 26. B. FROMENTY, E. FRENEAUX, G. LABBE, D. DESCHAMPS, D. LARREY, P. LETTERON, and D. PESSAYRE, Biochem. Ph armacol. 38 3743-3751 (1989). 27. F. WINSTON and M.L. MCCANN, Br. J. Psychiatry 120 693-697 (1972). 28. H. KUTCHAI, L.H. CHANDLER, and L.M.GEDDO, Biochem. Biophys. Acta 600 870881 (1980). 29. B.D. READ and R.N. MCELHANEY, Biochem. Biophys. Acta 419 33 1-341 (1976). 30. B.M. POWER, L.P. HACKETT, L.F. DUSCI, and K.F. ILETT, Clin. Pharmacokinet. 29 154-171 (1995). 3 1. Poisindex @ Editorial Stat? Antidepressants, Tricyclic (Toxicologic Management). In: Rumack BH, Toll LL and Gelman CR (Eds): Poisindex @ System. Micromedex, Inc., Englewood, Colorado V.98 (Edition Expires [December 19981). 32. S.J. VANNUCCI, J. Neurochem. 62 240-246 (1994). 33. J.E. REUSCH, K.E. SUSSMAN and B. DRAZNIN, J. Biol Chem 268 3348-3351 (1993).
Life Sciaxas, Vol. 66, No. 3, pp. 271-278.2000 Cbpyi&tC’ 1999 Elwvk Scii Inc. RinkdintbUSA. Allrighbrosaved 0024-3205&O/S-a liunt mat&z
THE INHIBITION OF GLUT1 GLUCOSE TRANSPORT AND CYTOCHALASIN B BINDING ACTIVITY BY TRICYCLIC ANTIDEPRESSANTS Harold B. Pinkofsky, Donard S. Dwyer, Ronald J. Bradley Department of Psychiatry, Louisiana State University Health Sciences Center, P.O. Box 33932, Shreveport, LA 71130-3932 (Receivedin finalform August31, 1999) Summary
Under normal metabolic conditions glucose is an important energy source for the mammalian brain. Positron Emission Tomography studies of the central nervous system have demonstrated that tricyclic antidepressant medications alter cerebral metabolic tknction. The mode by which these drugs perturb metabolism is unknown. In the present study the interactions of tricyclic antidepressants with the GLUT1 glucose transport protein is examined. Amitriptyline, nortriptyline, desipramine, and imipramine all inhibit the intlux of 3-O-methyl glucose into resealed erythrocytes. This inhibition is observed with drug concentrations in the millimolar range. All four antidepressants also noncompetitively displace cytochalasin B binding to GLUTl. The KI for this displacement ranges from 0.56 to 1.43 millimolar. This value is in a range greater than that associated with clinical doses and this effect may not be directly applicable to side effects observed with normal use. The observed interaction of these drugs with GLUT1 may reflect an afhnity for other glucosetransport or glucose-binding proteins, and may possibly contribute to tricyclic antidepressant toxicity. Key F+‘o&: glucose,met&&m, tricyclicantidePressants, GLUT1 , erythrocytes, cytochalasinB, glucose u=Wn The mammalian brain, under normal conditions, relies on glucose as its primary source of energy. Glucose transport into cerebral tissue occurs via facilitative transporters. The family of facilitative glucose transporters, termed GLUT1 through GLUT7, displays a high degree of homology (1). In neurons transmembrane glucose transport is mediated predominantly by GLUT3, and to a lesser extent by GLUT1 (2,3,4). GLUT1 appears to be the primary glucose transporter protein in cerebral microvessels as well as astrocytes (2). GLUT5 has been associated with microglia (2). In-vivo cerebral glucose utilization has been studied using positron emission tomography (PET) and single photon emission computerized tomography (SPECT) technology. Mood disorders have been associated with regional reductions in cerebral glucose metabolism as measured by these techniques. Antidepressant treatment has been observed to normalize glucose metabolism (5). This e&ct is believed to be secondary to the action of antidepressants on neurotransmission. corresponding author: Harold Pit&of&y, MD, PhD., Department of Psychiatry, PO Box 33932, Shreveport, LA 71130-3932. Telephone: (318) 675-6042, FAX: (318) 675-6148
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Other studies have supported these initial PET and SPECT findings. In studies utilizing rats, acute administration of desmethylimipramine, a tricyclic antidepressant, was found to differentially increase cerebral glucose utilization, whereas chronic administration decreased glucose utilization (6). In-vitro experiments demonstrated that imipramine, another tricyclic antidepressant, can inhibit brain tissue respiration (7). Tricyclic antidepressants have also been found to modulate other modalities of glucose regulation. For instance, high concentrations of imipramine (8) and amitriptyline (9) inhibit insulin release from pancreas cells. Paradoxically amitriptyline can produce hyperinsulinemia, with normoglycemia, in rats (10). Imipramine treatment has been demonstrated to produce a reduction in glucose serum levels (11) and there have been case-reports of imipramine producing a hypoglycemic effect in diabetic patients (12). Tricyclic antidepressants have been reported to influence glucose transport into platelets (13). In the present work the influence of tricyclic antidepressants on metabolism was tm-ther investigated using purified erythrocyte ghosts. Human erythrocytes are easily obtainable, lack organelles, and possess only the GLUT1 isoform, which accounts for approximately 6% of the total erythrocyte membrane protein (14). The transport of glucose across the cell membrane may be considered to be one of the early steps in the energy metabolism cascade.
Methods Fresh human blood was collected from healthy donors into KzEDTA containing vacutainers. Ghosts were prepared from erythrocytes by the method of Dodge et al (15). When used for transport studies ghosts were resealed in a 1: 10 balanced salt solution (containing 12.5 mM NaCl, 0.5 mM KCL, 0.38 mM CaC12, and 0.25 mM MgC12 but&red at pH 7.4 with Tris) as previously outlined (16). Transport assays and cytochalasin B binding studies were conducted as previously described (17, 18) and membrane protein was determined by the method of Lowry et al (19). Prior to transport assays, 2 ml of packed, resealed ghosts pre-equilibrated with 0.5 mM 3-O-methyl glucose (3-OMG) were added to 10 ml of a 1:lO balanced salt solution containing 0.5 mM 3-OMG. This suspension was then incubated for 30 minutes at room temperature (25” C). Transport measurements were initiated by adding tracer concentrations of tritiated 3-OMG. At fixed time intervals a 2 ml aliquot was removed from this suspension and added to 10 ml of chilled 1: 10 basic salt solution (0’ C) containing 2 mM HgC&. The samples were then centrifuged at 3 1,000 x g for 15 minutes. The pellets and supematants were separated and assayed for radioactivity. Transport rates were extrapolated from the linear component of uptake at the early transport times by fitting the results to a straight line using a least-means square regression analysis. Transport assays were calculated using measurements obtained from a total transport time course lasting % to 1 minute (Figures la and lb).
Materials Cytochalasin B, mercuric chloride, nortriptyline, desipramine, imipramine and amitriptyline were obtained from Sigma Chemical Company (St. Louis, MO). 3H-Cytochalasin B and “H-3OMG were obtained from Aldrich Chemical Company (Milwaukee, WI).
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0
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5
10
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Time (seconds)
Fig. la and lb Inhibition of 3-OMG transport by tricyclic antidepressants. In Figure la resealed erythrocyte ghosts were preincubated in the presence of 0.5 mM 3-OMG and varying concentrations (0.2 mM, 1 mM, and 3 mM) of tricyclic antidepressants (see Methods). The transport rates were normal&d to those observed for control erythrocytes not exposed to tricyclic antidepressants. Error bars display the observed standard error (s3). Transport rates in the presence of antidepressants were compared to rates observed for a control in which transport was assayed in the absence of any antidepressant using the identical preparation of erythrocyte ghosts. A paired Student-t test demonstrated that the transport rate observed in the presence of 1 mM or 3 mM antidepressant concentrations was significantly difkent (pc.05) Corn that observed in control preparations. No significant diflkrence was found for transport in the presence of 0.2 mM antidepressant concentrations (p>.OS). In Figure la, in order to avoid the results being superimposed, the measurements are shown clwtered adjacent to each other for the purpose of clarity. Figure lb shows a typical 3-OMG uptake time course for resealed ghosts in the presence of 0.5 mM 3-O-methyl glucose. 3-OMG uptake is normalized to mg of membrane protein and displayed as a tbnction of time.
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Results Resealed erythrocyte ghost membranes were pre-equilibrated with 0.5 mM 3-OMG as outlined above. Resealed erythrocyte ghosts, in the absence of antidepressants, exhibited a rate of 3OMG transport of 0.294 _t (0.096) nm 3-OMG/mg membrane protein x second (mean + SE, n=3). The observed variability resulted from the different uptake rates displayed by different batches of resealed ghosts. Tricyclic antidepressants were found to inhibit the uptake of tritiated 3-OMG by resealed erythrocyte ghosts as shown in Figure 1. All four tricyclic antidepressants studied (imipramine, desipramine, amitriptyhne, and nortriptyline) exhibited a 30% to 60% inhibition of influx at concentrations of approximately 1 n&I. A similar inhibition pattern was also seen with resealed erythrocyte ghosts using higher concentrations of 3-OMG (not shown). The interaction of tricyclic antidepressants with the erythrocyte glucose transporter was huther studied using cytochalasin B binding assays. Cytochalasin B has been reported to bind the human erythrocyte glucose transporter in erythrocyte ghosts with a dissociation constant of approximately 3-5 x lo-’ M (17, 18). Cytochalasin B binding at this site is displaced by Dglucose. There also exist two other sites in erythrocyte membranes which are not related to the glucose transporter but bind cytochalasin B. The non-transporter associated cytochalasin B binding is normally not altered by D-glucose (17, 18). The cytochalasin B binding at one of the non-transporter sites is inhibited by cytochalasin E. All four antidepressants were found to displace cytochalasin B binding to erythrocyte ghosts (Figure 2). The extent of the tricyclic antidepressant displacement on this binding was greatly diminished when 0.5 M D-glucose was present. The magnitude of tricyclic drug displacement of cytochalasin B binding did not appear altered by the presence of 10-r M cytochalasin E. Furthermore, the inhibition of cytochalasin B binding by tricyclic antidepressants has been observed to be reversible (not shown). The cytochalasin B bmding component sensitive to the presence of tricyclic-antidepressants was analyzed using a Lineweaver-Burk plot (Figures 3a and 3b). Noncompetitive inhibition kinetics were observed for ail four tricyclic antidepressants studied. The averaged inhibition constant (Kr) observed using this analysis ranged from 0.56 to 1.4 mM (see Table I). The averaged cytochalaain B dissociation constant derived from this bmding component was 4.4 (k1.5) x lo-’ M, similar to the range of the dissociation constant for cytochalasin B with the glucose transporter in erythrocyte ghosts (17).
Discussion Prior research has shown that some psychotropic medications exhibit an effect on energy metabolism. Barbiturates have been found to reduce glucose metabolism in the rat brain (21) and hexose transport rates in human erythrocytes (22). Chlorpromazine, an antipsychotic medication, has been found to accelerate glucose ef%lux from erythrocytes at low drug concemrations but inhibit eBIux at higher drug concemrations (23). Recently, we have shown that chlorpromazine and tiphenazine inhiiit glucose uptake in the PC12 neuronal cell line (24). The antidepressant trazodone was found to reduce oxygen consumption in rat brain homogenates (25). Tianeptine, a tricyclic antidepressant, has been reported to inhibit mitochondrial oxidation (26). The role played by t&y&c antidepressants in promoting weight gain and enhan&g fbod consumption, in both human and animal models may be a side effect of the infhrence that these drugs exert on energy metabolism (10,27).
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1T L
1.5 t!b u" 5% oh a
1 0.5 0
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ii
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Antidepressant Fig. 2 Cyto~halasin B binding in the presence or absence of tricyclic antidepressants. Erythrocyte ghosts were preincubated for 30 minutes in the presence of 2x10“ M cytochalasin B and the presence or absence of different tricyclic antidepressants at 3 mM concentration (labeled as I, D, A, or N). 0 represents bmding in the absence of antidepressants. I, D, A, and N represent binding in the presence of imipramine, despramine, amitriptyline, and nortiptyline respectively. When present the ticyclic antidepressants are at concentrations of 0.1 n&I, 1 mM, and 3 mM. ‘Bound CB/Free CB” is expressed in units of lo3 liters/mg protein. This binding was studied in the absence of any additional additives (open bars) the presence of 0.5 M D-glucose [dark bars] or the presence of 1OS5 M cytochahtsin E [striped bars]. The binding shown represents the total cytochalasin B binding observed. The nonspecific unsaturated cytochalasin B binding component has not been subtracted. The inhiiition of cytochalasin B bmding in the presence of each antidepressant is statistically sign&ant when compared to parallel controls in the absence of additional additives or the presence of 10% cytochalasin E (p<.OS). In the presence of OSm D-glucose, the binding observed in the presence or absence of antidepressants was not significantly different w.05). Tricyclic antidepressams were not found to have any etTixt on the nonspecific binding component. Error bars display the observed standard error (n=3).
In the present work ticyclic antidepressant medications were observed to inhibit ghmose tranpwrt t%nction and noncompetitively displace cytochalasin B from its glucose-displaceable binding ate. Alterations in membrane fluidity do not appear responsible for the observed changes. Prior studies have found that pertubations in erythrocyte membrane tluidity have little etfect on the monosaccharide transport system (28, 29). The observed noncompetitive kinetics of cytochalasin B displacement suggest that tricyclic antidepressants are. interact& directly with the transport protein. Although tricyclic antidepressants have been previously reported to exhibit metabolic et&&s, this is the tlrst report of an interaction with the glucose transport protein in human erythrocytes. The inhiion constants observed for the trlcyclic
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I= 3mM
o=o 0 = 1 mM A =2mM o=3mM
a
I
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/
/
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lmipramine concentration Fig. 3a and 3b Inhibition of cytochalasin B binding by imipramine. In Figure 3a the component of cytochalasin B binding which was displaceable by the tricyclic antidepressant was analyzed using a Lineweaver-Burk plot at varying imipramine concentrations (I). Fitted lines were obtained using a least squares analysis (?=.90). The apparently common intercepts on the “l/Free CB” axis suggests The Kr was that imipramine displaces cytochalasin B binding noncompetitively. analyzed for noncompetitive inhibition using the equation “Slope= [K&-l [l + ([I]/&)” (26). Figure 3B shows a graph of the slope versus imipramine concentration with a least squares fit to the data. “l/Bound CB” is expressed in units of 10” mg protein/mole CB. “l/Free CB” is expressed in units of IO’ liter/mole CB. The concentration of imipramine present is displayed to the right of each fitted line.
mM
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Table I The dissociation constants for the tricyclic antidepressant inhibition of cytochalasin B biding. Tricyclic Antidepressant
Kr(mM) (Mea&SE)
Imipramine (n=3) Amitriptyline (n=3) Desipramine (n=3) Nortrintvline (n=3)
0.56*0.12 1.43 f 0.63 0.72 f 0.02 0.98 f 0.10
Inhibition of cytochalasin B binding by tricyclic antidepressants was analyzed as in Figure 3. The dissociation constants shown for the various antidepressants are the average of three separate All of the tricyclic binding assays. antidepressants assayed displayed noncompetitive displacement of cytochalasin B binding.
effect on the glucose transporter are however, within a concentration range well above normal serum levels (30). Therapeutic serum concentrations for most tricyclic antidepressants is between 100 to 260 nanograms/milliliter; lethal tricyclic antidepressant levels have been reported to range from 1,100 to 21,800 nanograms/milliliter (4,000 to 80,000 nanomolefliter) (3 1). The present results were obtained using the GLUT1 isoform and it is possible that other GLUT proteins may have a different sensitivity to the tricyclic antidepressants assayed. Moreover, alterations in the glucose transporter, such as those that occur during development (32), or due to phosphorylation (33) may change the sensitivity to these drugs. It is also possible that the observed effect on glucose transport may reflect binding of the tricyclic Some of the reported antidepressant medications to associated regulatory proteins. physiological changes observed with tricyclic medications may be the result of such an interaction. Conceivably tricyclic antidepressants may act on neuronal cells or at sites in the blood brain barrier. Our findings suggest that alterations in glucose transport may contribute to the toxic properties of tricyclic antidepressants, including those present in clinical overdoses. References
1. 2. 3. 4.
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