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Inhibition of fatty acid oxidation and glucose metabolism does not affect food intake or hunger motivation in syrian hamsters
Physiology & Behavior, Vol. 44, pp. 209-213. Copyright©Pergamon Press plc, 1988. Printed in the U.S.A.
0031-9384/88 $3.00 + .00
Inhibition of Fatty Acid Oxidation and Glucose Metabolism Does Not Affect Food Intake or Hunger Motivation in Syrian Hamsters S A N D R A J. L A Z Z A R I N I ,
J I L L E. S C H N E I D E R
AND GEORGE
N. WADE
N e u r o s c i e n c e and Behavior Program and D e p a r t m e n t o f Psychology University o f Massachusetts, Amherst, M A 01003 R e c e i v e d 4 M a r c h 1988 LAZZARINI, S. J., J. E. SCHNEIDER AND G. N. WADE. Inhibition of fatty acid oxidation and glucose metabolism does not affect food intake or hunger motivation in Syrian hamsters. PHYSIOL BEHAV 44(2) 209-213, 1988.--We examined the interaction of the metabolic fuels, glucose and free fatty acids (FFA), in the control of food intake in Syrian hamsters. Hamsters were treated with a 2-deoxy-D-glucose (2DG) which inhibits glucose utilization, and methyl palmoxirate (MP), which inhibits fatty acid oxidation. The 2DG and MP, alone or in combination did not enhance food intake in hamsters fed a standard rodent chow diet. Determination of the circulating glucose, FFAs, and ketones confirmed that the drugs were having the intended metabolic effects. The 2DG caused marked hyperglycemia and decreased ketones consistent with an inhibition of glycolysis, and the MP caused increased FFAs and decreased ketones indicating inhibition of fatty acid oxidation. A third experiment examined the hamsters' willingness to ingest a diet made highly unpalatable with NaCI, another measure of hunger motivation. Although food-deprived hamsters ingested more of a salt-adulterated diet than did control animals, hamsters treated with MP and 2DG did not. These experiments provide further evidence that the control of food intake in Syrian hamsters is appreciably different than that of laboratory rats. 2-Deoxy-D-glucose Methyl palmoxirate Metabolism Syrian hamster
Fatty acid oxidation
T H E availability of metabolic fuels appears to signal changes in food intake in many commonly studied animals (1, 12-14, 18, 33, 36). In rats, food intake is responsive to variations in utilization of glucose and free fatty acids (FFA). They become hyperglycemic and rapidly increase their food intake when injected either systemically or intracerebroventricularly with 2-deoxy-D-glucose (2DG, a glucose analogue that inhibits glycolysis) (1, 5, 15-17, 21, 36). Similar effects are produced by injections of 5-thio-glucose, another glucose antimetabolite (24). Inhibition of fatty acid oxidation by injections o f mercaptoacetate also increases food intake up to 120% in rats fed a high-fat diet (30). The interaction of glucose and fatty acids in the control of feeding behavior was demonstrated by Friedman, et al. (15,16) with the use of methyl palmoxirate [MP, an inhibitor of fatty acid oxidation at the mitochondrial transport level (19,37)]. When MP was given with 2DG, rats ate more than with either drug given alone. Syrian hamsters, Mesocricetus auratus, increase food intake during cold exposure (28), voluntary exercise (4,6), lactation (11). and dilution of a liquid or solid diet (35), which suggests they are capable o f controlling food intake in response to changes in the availability of metabolic fuels.
Food intake
Hunger motivation
However, unlike rats (10), they do not show a postfast hyperphagia even after long periods of deprivation (3, 4, 8, 9, 26, 29, 35). Injections of 2DG (23, 25, 27, 32, 34) or 5thio-glucose do not cause an increase in food intake (7), nor is food intake enhanced in MP-treated hamsters fed a high fat diet (31). Some enhancement of food intake is seen in hamsters treated with high doses of insulin which decrease both glucose and F F A availability (8, 23, 25). It is possible that 2DG and MP do not increase food intake when given alone because there may be compensatory utilization of the other substrate. The following experiments examined the possibility that blocking glucose metabolism and fatty acid oxidation simultaneously would lead to increased food intake. METHOD
Subjects Female L V G Syrian hamsters (90-100 g) were purchased from Charles River Breeding Laboratories, Wilmington, MA. They were housed in hanging wire cages in a room maintained on a 16:8 hr light:dark cycle at 23-+2°C. They were fed chow pellets (Purina Laboratory Rodent Chow No. 5001) unless otherwise noted. Prior to each experiment, the
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hamsters were adapted to procedures and handling required for that experiment. Water was available ad lib.
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General Procedures
Hours
The animals were divided into groups matched for baseline food intake and body weight. Hamsters were given either methyl palmoxirate (MP) suspended in 0.5% methyl cellulose or methyl cellulose vehicle alone by gavage. Injections 2DG were given at 750 mg/kg body weight, because this dose produces marked hyperglycemia in hamsters without neurological impairment (27). Either 2DG or 0.9% saline vehicle was injected intraperitoneally 2 hours after MP to allow time for the MP to take effect (15,16). Food intake, carefully corrected for spillage and pouching, was measured to the nearest 0.1 g at the time periods indicated. Data were analyzed using one-way analyses of variance (for each time point where necessary) followed by Tukey's HSD post hoc test if the main effects were significant at p<0.05.
After MP Treatment
FIG. 2. Time course of the plasma metabolic fuels after administration of metabolic inhibitors. MP was given at the 0 time point. Administration of 2DG is indicated by the arrows.
RESULTS Neither MP nor 2DG, given singly or in combination, stimulated food intake (Fig. 1). Although MP10/2DG and MP10 suppressed food intake 3 hours after MP administration, F(5,50)=2.62, p<0.05, and MPI0/2DG suppressed food intake 5 hours MP administration, F(5,50)=4.28, p<0.002, cumulative food intakes were not significantly different at any time point.
EXPERIMENT 1 The first experiment examined the effects of 2DG and MP, given alone or in combination, on ad lib food intake. Procedures
Fifty-six hamsters were divided into six groups and given either MP at 1 mg/kg (MP1), MP at 10 mg/kg (MP10), or the vehicle alone. Two hours later, they were given 2DG at 750 mg/kg or the saline vehicle. Food intake was measured 1 hour before MP injection, at the time of MP injection, and 2, 3, 5, and 7 hours after administration of MP (1, 3, and 5 hours after 2DG).
EXPERIMENT 2 The results of Experiment 1 indicated that hamsters, unlike rats, did not increase their food intake when treated with the metabolic inhibitors 2DG and MP, either alone or in combination. However, it was possible that the drugs did not produce the desired physiological effects in hamsters. Experiment 2 was designed to test this possibility. Procedures
Forty-eight hamsters were divided into five groups and received the following treatments: methyl cellulose plus
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FIG. 4. Cumulative food intake of a salt-adulterated diet of hamsters treated with MP and 2DG. Time points are hours after MP treatment. Administration of 2DG is indicated by the arrow. saline, methyl cellulose plus 2DG, MPI plus saline, MPI0 plus saline, and MP1 plus 2DG. The 2DG or saline was administered 2 hours after MP treatment. Animals were food deprived following administration of MP or methyl cellulose to control for the metabolic effects of food intake. Blood was drawn by cardiac puncture under light methoxyfluorane (Metofane) anesthesia at time of MP injection, 3, and 5 hours after MP treatment which corresponded to 1 and 3 hours after 2DG treatment. Plasma glucose was measured with a Yellow Springs Glucose Analyzer model 23A. Free fatty acids, ketone bodies, and glycerol were determined using an enzymatic method with fluorimetric detection (2, with modifications). RESULTS Treatment with 2DG caused marked hyperglycemia 1 and 3 hours after treatment when given alone or in combination with MP1, F(4,43)=27.9, p<0.001 (Fig. 2). The MPI did not affect the degree of hyperglycemia caused by 2DG. The MP did not alter glucose levels at either dose at any time period. The MP1, MP10, and MP1/2DG raised plasma F F A s significantly by 3 hours, F(4,43)=3.69, p<0.01, and there was no difference among the MP-treated groups. Plasma F F A s continued to rise in these groups 5 hours after administration, F(4,43)=4.73, p<0.003. Treatment with 2DG did not affect plasma levels of F F A s . Both doses of MP lowered plasma ketone bodies by 3 hours, and 2DG lowered ketones 1 hour after injection, F(4,38)=2.63, p<0.05. The 2DG group continued to show decreased ketone levels 3 hours after injection, F(4,38)= 3.19, p<0.05. All other groups returned to control levels. EXPERIMENT 3 It is possible that MP and 2DG affect hunger motivation, even though they do not increase food intake. DiBattista and Bedard (9) found that even though food-deprived hamsters do not compensate by increasing food intake when refed, they exhibit several signs of increased hunger, including ingestion of an unpalatable diet. Experiment 3 tested the effects of 2DG and MP in hamsters fed an unpalatable diet adulterated with salt in order to determine whether the drugs would increase the animals' motivation to eat.
Procedures
Forty-eight hamsters were allowed ad lib access to rodent chow ground to a fine powder. The animals were divided into five groups and received the following: methyl cellulose and saline vehicles, methyl cellulose plus 2DG, MP1 plus saline, MP10 plus saline, and MP1 plus 2DG. On the test day, the animals received a 2:1 mixture of powdered chow and NaCl. Food intake and spillage were measured to the nearest 0.1 g one hour prior to administration of MP, at the time of MP injection, and 2, 4, 6, and 8 hours after MP treatment. The 2DG or saline vehicle was given 2 hours after administration of MP. In a preliminary study, hamsters who were food deprived for 18 hours ate significantly more salt-adulterated diet during a 3 hour feeding test than nondeprived hamsters (p<0.001) (Fig. 3). RESULTS Neither MP or 2DG, alone or in combination, caused the hamsters to ingest more of an unpalatable diet than control animals at any time period (Fig. 4). GENERAL DISCUSSION The results of these experiments do not support the hypothesis that glucose metabolism and fatty acid oxidation are strong signals controlling food intake in Syrian hamsters. Previous work indicated that 2DG did not increase food intake in hamsters fed a high-carbohydrate chow diet (23, 25, 27, 32, 34), nor did MP stimulate consumption o f a high-fat diet (31). In those experiments, it was possible that hamsters responded to each drug by compensatory utilization of alternative substrates. This hypothesis was not supported by Experiment 1 or 3, because treatment with both metabolic inhibitors simultaneously did not enhance food intake or willingness to ingest an unpalatable diet (Figs. 1 and 4). These results cannot be explained by a general ineffectiveness of MP and 2DG in hamsters. The 2DG increased plasma glucose and decreased ketones which indicates inhibition of glycolysis and compensatory utilization of ketones. Circulating levels of free fatty acids were increased (15) and
212
LAZZARINI, SCHNEIDER AND WADE
ketones were decreased (15,31) in hamsters treated with MP indicating inhibition of fatty acid oxidation and ketogenesis (15, 31, 37). Although MP and 2DG produce similar physiological responses in rats and hamsters, the physiological changes do not give rise to similar behavioral responses. Other researchers have suggested that hamsters simply lack feeding mechanisms in the brain or periphery that are sensitive to food deprivation and glucoprivation [e.g., (23-25)]. Our data are consistent with this hypothesis and e x p a n d it to include inhibition of fatty acid utilization alone or its interaction with glucoprivation. Although pharmacological modification of metabolic fuel availability did not affect food intake or hunger motivation in our experiments, natural increases in metabolic d e m a n d do increase food intake (i.e., cold exposure, exercise, lactation), (4, 6, 11, 22, 35). It is possible that MP and 2DG do not produce metabolic changes comparable in quality or mag-
nitude to those p r o d u c e d by cold exposure, exercise, and lactation. A n o t h e r possibility is that postingestinal satiety signals in hamsters may differ from those o f m o s t o t h e r animals either in quality or degree (9). Although this has yet to be determined by research focusing on the nature of satiety signals in the hamster (20), it is possible that peripheral satiety signals override the effects of the signals for feeding that result from deprivation (26), glucoprivation, or the interaction of fatty acid oxidation and glucoprivation. ACKNOWLEDGEMENTS We thank Jay Alexander and Lynn Bengston for expert technical assistance, Charles Bowden and McNeil Pharmaceuticals for providing the methyl palmoxirate, and Israel Ramirez for advice on the assays. This research was supported by NIH Research Grants NS 10873, DK 32976, and NIMH Research Scientist Development Award MH 00321.
REFERENCES 1. Balagura, S.; Kanner, M. Hypothalamic sensitivity to 2-deoxyD-glucose and glucose: Effects on feeding behavior. Physiol. Behav. 7:251-255; 1971. 2. Bates, M.; Krebs, H.; Williamson, D. H. Turnover rates of ketone bodies in normal, starved and alloxan diabetic rats. Biochem. J. 110:655-661; 1968 3. Borer, K. T.; Rowland, N.; Mirow, A.; Borer, R.C., Jr.; Kelch, R. P. Physiological and behavioral responses to starvation in the golden hamsters. Am. J. Physiol. 236(2):E105-E112; 1979. 4. Borer, K. T. Regulation of energy balance in the golden hamster. In: Seigel, H. I., ed. The hamster: Reproduction and behavior. New York: Plenum Press; 1984. 5. Brown, J. Effects of 2-deoxyglucose on carbohydrate metabolism: Review of the literature and studies in rats. Physiol. Behay. 11:1098-1112; 1962. 6. Browne, S. A. H.; Borer, K. T. Basis for the exercise-induced hyperphagia in adult hamsters. Physiol. Behav. 20:553-557; 1978. 7. DiBattista, D. Effects of 5-thioglucose on feeding and glycemia in the hamster. Physiol. Behav. 29:803-806; 1982. 8. DiBattista, D. Food-deprivation and insulin-induced feeding in the hamster. Physiol. Behav. 30:683-687; 1983. 9. DiBattista, D.; Bedard, M. Effects of food deprivation on hunger motivation in golden hamsters (Mesocricetus auratus). J. Comp. Psychol. 101:183-189; 1987. 10. Fabry, P. Feeding patterns and nutritional adaptation. London: Butterworth; 1969. 11. Fleming, A. S.; Miceli, M. Effects of diet on feeding and body weight regulation during pregnancy and lactation in the golden hamster (Mesocricetus auratus). Behav. Neurosci. 97:246-254; 1983. 12. Friedman, M. I. Hyperphagia in rats with experimental diabetes mellitus; A response to a decreased supply of utilizable fuels. J. Comp. Physiol. Psychol. 92:109-117; 1978. 13. Friedman, M. I.; Ramirez, I. Relationship of fat metabolism to food intake. Am. J. Clin. Nutr. 42:1093-1098; 1985. 14. Friedman, M. I.; Ramirez, I.; Edens, N. K.; Granneman, J. Food intake in diabetic rats: isolation of primary metabolic effects of fat feeding. Am. J. Physiol. 249:R44-R51; 1985. 15. Friedman, M. I.; Tordoff, M. G. Fatty acid oxidation and glucose utilization interact to control food intake in rats. Am. J. Physiol. 251:R840-845; 1987. 16. Friedman, M. I.; Tordoff, M. G.; Ramirez, I. Integrated metabolic control of food intake. Brain Res. Bull. 17:855-859; 1986.
17. Gonzalez, M. F. ; Novin, D. Feeding induced by intracranial and intravenously administered 2-deoxy-D-glucose. Physiol. Psychol. 2:326-330; 1974. 18. Houpt, T. R.; Hance, H. E. Stimulation of food intake in the rabbit and rat by inhibition of glucose metabolism with 2-deoxy-d-glucose. J. Comp. Physiol. Psychol. 76(3):395--400; 1971. 19. Kiorpes, T.C.; Hoerr, D.; Ho, W.; Weaner, L. E.; Inman, M. G.; Tutwiler, G. F. Identification of 2-tetradecylglycidyl coenzyme A as the active form of methyl 2-tetradecylglycidte (methyl palmoxirate) and its charaterization as an irreversible, active site-directed inhibitor of carnitine palmitoyltransferase A in isolated rat liver mitochondria. J. Biol. Chem. 259:97509755; 1984. 20. Miceli, M. O.; Malsbury, C. W. Effects of putative satiety peptides on feeding and drinking behavior in golden hamsters. Behav. Neurosci. 99:1192-1207; 1985. 21. Miselis, R. R.; Epstein, A. N. Feeding induced by intracerebroventricular 2-deoxy-D-glucose in the rat. Am. J. Physiol. 229:1438-1447; 1975. 22. Murphy, M. R. Natural history of the Syrian golden hamster--A reconnaissance expedition Am. Zool. 11:632, 1971. 23. Ritter, R. C.; Balch, O. K. Feeding in response to insulin but not to 2-deoxy-D-glucose in the hamster. Am. J. Physiol. 234:E20-E24; 1978. 24. Ritter, R. C.; Slusser, P. 5-Thio-D-glucose causes increased feeding and hyperglycemia in the rat. Am. J. Physiol. 238:E141-E144; 1980. 25. Rowland, N. Effects of insulin and 2-deoxy-D-glucose on feeding in hamsters in gerbils. Physiol. Behav. 21:291-294; 1978. 26. Rowland, N. Failure by deprived hamsters to increase food intake: Some behavioral and physiological determinants. J. Comp. Physiol. Psychol. 96:591-603; 1982. 27. Rowland, N. Physiological and behavioral responses to glucoprivation in the golden hamster. Physiol. Behav. 30:743-747; 1983. 28. Rowland, N. Effects of chronic cold exposure on wheel running, food intake, and fatty acid synthesis in Syrian hamsters. Physiol. Behav. 33:254-256; 1984. 29. rowland, N. Ingestive behavior of Syrian hamsters: Advantages of the comparative approach. Brain Res. Bull. 15:417-423; 1985. 30. Scharrer, E.; Langhans, W. Control of food intake by fatty acid oxidation. Am. J. Physiol. 250:R1003-1006; 1986. 31. Schneider, J. E.; Lazzarini, S. J.; Friedman, M. I.; Wade, G. N. Effects of fatty acid oxidation in food intake and hunger motivation in Syrian hamsters. Physiol. Behav., in press; 1988.
METABOLIC INHIBITORS AND FOOD INTAKE 32. Sclafani, A.; Eisenstadt, D. 2-deoxy-D-glucose fails to induce feeding in hamsters fed a preferred diet. Physiol. Behav. 24:641-643; 1980. 33. Seoane, J. R.; Baile, C. A. Effects of intraventricular (III ventricle) injections of 2-deoxy-D-glueose, glucose and xylose on feeding behavior of sheep. Physiol. Behav. 9:423-428; 1972. 34. Silverman, J. H. Failure of 2-deoxy-D-glucose to increase feeding in the golden hamster. Physiol. Behav. 21:859-864; 1978.
213 35. Silverman, H. J.; Zucker, I. Absence of post-fast food compensation in the golden hamster (Mesocricetus auratus). Physiol. Behav. 17:271-285; 1976. 36. Smith, G. P. ; Epstein, A. N. Increased feeding in response to decreased glucose utilization in the rat and monkey. Am. J. Physiol. 217:1083--1087; 1969. 37. Tutwiler, G. F.; Brentzel, H. J.; Kiorpes, T. C. Inhibition of mitochondrial carnitine palmitoyl transferase A in vivo with methyl 2-tetradecylglycidate (methyl palmoxirate) and its relationship to ketonemia and glycemia. Proc. Soc. Exp. Biol. Med. 178:288-196; 1985.
0031-9384/88 $3.00 + .00
Inhibition of Fatty Acid Oxidation and Glucose Metabolism Does Not Affect Food Intake or Hunger Motivation in Syrian Hamsters S A N D R A J. L A Z Z A R I N I ,
J I L L E. S C H N E I D E R
AND GEORGE
N. WADE
N e u r o s c i e n c e and Behavior Program and D e p a r t m e n t o f Psychology University o f Massachusetts, Amherst, M A 01003 R e c e i v e d 4 M a r c h 1988 LAZZARINI, S. J., J. E. SCHNEIDER AND G. N. WADE. Inhibition of fatty acid oxidation and glucose metabolism does not affect food intake or hunger motivation in Syrian hamsters. PHYSIOL BEHAV 44(2) 209-213, 1988.--We examined the interaction of the metabolic fuels, glucose and free fatty acids (FFA), in the control of food intake in Syrian hamsters. Hamsters were treated with a 2-deoxy-D-glucose (2DG) which inhibits glucose utilization, and methyl palmoxirate (MP), which inhibits fatty acid oxidation. The 2DG and MP, alone or in combination did not enhance food intake in hamsters fed a standard rodent chow diet. Determination of the circulating glucose, FFAs, and ketones confirmed that the drugs were having the intended metabolic effects. The 2DG caused marked hyperglycemia and decreased ketones consistent with an inhibition of glycolysis, and the MP caused increased FFAs and decreased ketones indicating inhibition of fatty acid oxidation. A third experiment examined the hamsters' willingness to ingest a diet made highly unpalatable with NaCI, another measure of hunger motivation. Although food-deprived hamsters ingested more of a salt-adulterated diet than did control animals, hamsters treated with MP and 2DG did not. These experiments provide further evidence that the control of food intake in Syrian hamsters is appreciably different than that of laboratory rats. 2-Deoxy-D-glucose Methyl palmoxirate Metabolism Syrian hamster
Fatty acid oxidation
T H E availability of metabolic fuels appears to signal changes in food intake in many commonly studied animals (1, 12-14, 18, 33, 36). In rats, food intake is responsive to variations in utilization of glucose and free fatty acids (FFA). They become hyperglycemic and rapidly increase their food intake when injected either systemically or intracerebroventricularly with 2-deoxy-D-glucose (2DG, a glucose analogue that inhibits glycolysis) (1, 5, 15-17, 21, 36). Similar effects are produced by injections of 5-thio-glucose, another glucose antimetabolite (24). Inhibition of fatty acid oxidation by injections o f mercaptoacetate also increases food intake up to 120% in rats fed a high-fat diet (30). The interaction of glucose and fatty acids in the control of feeding behavior was demonstrated by Friedman, et al. (15,16) with the use of methyl palmoxirate [MP, an inhibitor of fatty acid oxidation at the mitochondrial transport level (19,37)]. When MP was given with 2DG, rats ate more than with either drug given alone. Syrian hamsters, Mesocricetus auratus, increase food intake during cold exposure (28), voluntary exercise (4,6), lactation (11). and dilution of a liquid or solid diet (35), which suggests they are capable o f controlling food intake in response to changes in the availability of metabolic fuels.
Food intake
Hunger motivation
However, unlike rats (10), they do not show a postfast hyperphagia even after long periods of deprivation (3, 4, 8, 9, 26, 29, 35). Injections of 2DG (23, 25, 27, 32, 34) or 5thio-glucose do not cause an increase in food intake (7), nor is food intake enhanced in MP-treated hamsters fed a high fat diet (31). Some enhancement of food intake is seen in hamsters treated with high doses of insulin which decrease both glucose and F F A availability (8, 23, 25). It is possible that 2DG and MP do not increase food intake when given alone because there may be compensatory utilization of the other substrate. The following experiments examined the possibility that blocking glucose metabolism and fatty acid oxidation simultaneously would lead to increased food intake. METHOD
Subjects Female L V G Syrian hamsters (90-100 g) were purchased from Charles River Breeding Laboratories, Wilmington, MA. They were housed in hanging wire cages in a room maintained on a 16:8 hr light:dark cycle at 23-+2°C. They were fed chow pellets (Purina Laboratory Rodent Chow No. 5001) unless otherwise noted. Prior to each experiment, the
209
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LAZZARINI, SCHNEIDER AND WADE
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hamsters were adapted to procedures and handling required for that experiment. Water was available ad lib.
0
General Procedures
Hours
The animals were divided into groups matched for baseline food intake and body weight. Hamsters were given either methyl palmoxirate (MP) suspended in 0.5% methyl cellulose or methyl cellulose vehicle alone by gavage. Injections 2DG were given at 750 mg/kg body weight, because this dose produces marked hyperglycemia in hamsters without neurological impairment (27). Either 2DG or 0.9% saline vehicle was injected intraperitoneally 2 hours after MP to allow time for the MP to take effect (15,16). Food intake, carefully corrected for spillage and pouching, was measured to the nearest 0.1 g at the time periods indicated. Data were analyzed using one-way analyses of variance (for each time point where necessary) followed by Tukey's HSD post hoc test if the main effects were significant at p<0.05.
After MP Treatment
FIG. 2. Time course of the plasma metabolic fuels after administration of metabolic inhibitors. MP was given at the 0 time point. Administration of 2DG is indicated by the arrows.
RESULTS Neither MP nor 2DG, given singly or in combination, stimulated food intake (Fig. 1). Although MP10/2DG and MP10 suppressed food intake 3 hours after MP administration, F(5,50)=2.62, p<0.05, and MPI0/2DG suppressed food intake 5 hours MP administration, F(5,50)=4.28, p<0.002, cumulative food intakes were not significantly different at any time point.
EXPERIMENT 1 The first experiment examined the effects of 2DG and MP, given alone or in combination, on ad lib food intake. Procedures
Fifty-six hamsters were divided into six groups and given either MP at 1 mg/kg (MP1), MP at 10 mg/kg (MP10), or the vehicle alone. Two hours later, they were given 2DG at 750 mg/kg or the saline vehicle. Food intake was measured 1 hour before MP injection, at the time of MP injection, and 2, 3, 5, and 7 hours after administration of MP (1, 3, and 5 hours after 2DG).
EXPERIMENT 2 The results of Experiment 1 indicated that hamsters, unlike rats, did not increase their food intake when treated with the metabolic inhibitors 2DG and MP, either alone or in combination. However, it was possible that the drugs did not produce the desired physiological effects in hamsters. Experiment 2 was designed to test this possibility. Procedures
Forty-eight hamsters were divided into five groups and received the following treatments: methyl cellulose plus
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FIG. 3. Effects of food deprivation for 18 hr on intake of a saltadulterated diet (2 parts chow: 1 part NaC1) in hamsters.
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i 8
Hours After MP Treatment
FIG. 4. Cumulative food intake of a salt-adulterated diet of hamsters treated with MP and 2DG. Time points are hours after MP treatment. Administration of 2DG is indicated by the arrow. saline, methyl cellulose plus 2DG, MPI plus saline, MPI0 plus saline, and MP1 plus 2DG. The 2DG or saline was administered 2 hours after MP treatment. Animals were food deprived following administration of MP or methyl cellulose to control for the metabolic effects of food intake. Blood was drawn by cardiac puncture under light methoxyfluorane (Metofane) anesthesia at time of MP injection, 3, and 5 hours after MP treatment which corresponded to 1 and 3 hours after 2DG treatment. Plasma glucose was measured with a Yellow Springs Glucose Analyzer model 23A. Free fatty acids, ketone bodies, and glycerol were determined using an enzymatic method with fluorimetric detection (2, with modifications). RESULTS Treatment with 2DG caused marked hyperglycemia 1 and 3 hours after treatment when given alone or in combination with MP1, F(4,43)=27.9, p<0.001 (Fig. 2). The MPI did not affect the degree of hyperglycemia caused by 2DG. The MP did not alter glucose levels at either dose at any time period. The MP1, MP10, and MP1/2DG raised plasma F F A s significantly by 3 hours, F(4,43)=3.69, p<0.01, and there was no difference among the MP-treated groups. Plasma F F A s continued to rise in these groups 5 hours after administration, F(4,43)=4.73, p<0.003. Treatment with 2DG did not affect plasma levels of F F A s . Both doses of MP lowered plasma ketone bodies by 3 hours, and 2DG lowered ketones 1 hour after injection, F(4,38)=2.63, p<0.05. The 2DG group continued to show decreased ketone levels 3 hours after injection, F(4,38)= 3.19, p<0.05. All other groups returned to control levels. EXPERIMENT 3 It is possible that MP and 2DG affect hunger motivation, even though they do not increase food intake. DiBattista and Bedard (9) found that even though food-deprived hamsters do not compensate by increasing food intake when refed, they exhibit several signs of increased hunger, including ingestion of an unpalatable diet. Experiment 3 tested the effects of 2DG and MP in hamsters fed an unpalatable diet adulterated with salt in order to determine whether the drugs would increase the animals' motivation to eat.
Procedures
Forty-eight hamsters were allowed ad lib access to rodent chow ground to a fine powder. The animals were divided into five groups and received the following: methyl cellulose and saline vehicles, methyl cellulose plus 2DG, MP1 plus saline, MP10 plus saline, and MP1 plus 2DG. On the test day, the animals received a 2:1 mixture of powdered chow and NaCl. Food intake and spillage were measured to the nearest 0.1 g one hour prior to administration of MP, at the time of MP injection, and 2, 4, 6, and 8 hours after MP treatment. The 2DG or saline vehicle was given 2 hours after administration of MP. In a preliminary study, hamsters who were food deprived for 18 hours ate significantly more salt-adulterated diet during a 3 hour feeding test than nondeprived hamsters (p<0.001) (Fig. 3). RESULTS Neither MP or 2DG, alone or in combination, caused the hamsters to ingest more of an unpalatable diet than control animals at any time period (Fig. 4). GENERAL DISCUSSION The results of these experiments do not support the hypothesis that glucose metabolism and fatty acid oxidation are strong signals controlling food intake in Syrian hamsters. Previous work indicated that 2DG did not increase food intake in hamsters fed a high-carbohydrate chow diet (23, 25, 27, 32, 34), nor did MP stimulate consumption o f a high-fat diet (31). In those experiments, it was possible that hamsters responded to each drug by compensatory utilization of alternative substrates. This hypothesis was not supported by Experiment 1 or 3, because treatment with both metabolic inhibitors simultaneously did not enhance food intake or willingness to ingest an unpalatable diet (Figs. 1 and 4). These results cannot be explained by a general ineffectiveness of MP and 2DG in hamsters. The 2DG increased plasma glucose and decreased ketones which indicates inhibition of glycolysis and compensatory utilization of ketones. Circulating levels of free fatty acids were increased (15) and
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ketones were decreased (15,31) in hamsters treated with MP indicating inhibition of fatty acid oxidation and ketogenesis (15, 31, 37). Although MP and 2DG produce similar physiological responses in rats and hamsters, the physiological changes do not give rise to similar behavioral responses. Other researchers have suggested that hamsters simply lack feeding mechanisms in the brain or periphery that are sensitive to food deprivation and glucoprivation [e.g., (23-25)]. Our data are consistent with this hypothesis and e x p a n d it to include inhibition of fatty acid utilization alone or its interaction with glucoprivation. Although pharmacological modification of metabolic fuel availability did not affect food intake or hunger motivation in our experiments, natural increases in metabolic d e m a n d do increase food intake (i.e., cold exposure, exercise, lactation), (4, 6, 11, 22, 35). It is possible that MP and 2DG do not produce metabolic changes comparable in quality or mag-
nitude to those p r o d u c e d by cold exposure, exercise, and lactation. A n o t h e r possibility is that postingestinal satiety signals in hamsters may differ from those o f m o s t o t h e r animals either in quality or degree (9). Although this has yet to be determined by research focusing on the nature of satiety signals in the hamster (20), it is possible that peripheral satiety signals override the effects of the signals for feeding that result from deprivation (26), glucoprivation, or the interaction of fatty acid oxidation and glucoprivation. ACKNOWLEDGEMENTS We thank Jay Alexander and Lynn Bengston for expert technical assistance, Charles Bowden and McNeil Pharmaceuticals for providing the methyl palmoxirate, and Israel Ramirez for advice on the assays. This research was supported by NIH Research Grants NS 10873, DK 32976, and NIMH Research Scientist Development Award MH 00321.
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