Point of View
July 7992: 214-215
The Viable Serotonin Hypothesis I read the review “Brain Neurochemistry and Macronutrient Selection: A Role for Serotonin Feedback?’” with interest. As was pointed out accurately, the theory that serotonin is part of a feedback loop in the control of diet selection has been the subject of a great controversy in the past decade (see reference 2 for review). I believe, however, that results from the investigation by Holder and Huether3 have not put an end to this controversy. Instead, their investigation serves as a typical example of how one can easily come up with negative findings and claim to “disprove” a hypothesis. The Holder and Huether3 investigation was divided into two components: biochemical and behavioral. It was the authors’ intention that results from these two series of experiments would complement one another. There were, however, numerous designs and methodological problems that did not allow them to test the hypothesis adequately. Thus, readers should take extreme caution in assessing the relevance of their interpretations and conclusion. With the exception of tryptophan (TRP) injection, results of the first experiment were basically negative. In contrast to many published reports (see reference 4 for review), glucose (carbohydrate) and insulin failed to alter the plasma ratio of TRP to other large neutral amino acids (LNAA), brain TRP, and 5-HT (5-hydroxytryptamine) levels. This could have been caused by any one or more of the following factors: 1) No attempt was made to control for food intake prior to or after treatments. It is important to realize that rats were fed only during the light phase of the lightldark cycle so that they were likely to engage in active feeding activity at the time of experimentation. Since food ingestion has a significant physiologic impact on central nervous system functions, this would undoubtedly increase variability within a group. 2) The design of the experiment clearly lacks statistical power. There was a maximum of four rats per time point and, more importantly, only two rats were allocated to each time point in the three treatment groups (glucose, insulin, and tryptophan). Thus, any “reasonable physiologic response” would be masked by a presumably big variance (note that error terms were not reported in the paper). 3) Although brain
214
5-hydroxyindole levels increased after TRP injection, there are reasons to suspect the validity of the brain 5-HT values. Peculiar fluctuations in forebrain 5-HT levels were observed that could only be explained by errors in procedure, storage, or analysis. The average 5-HT level (seven time points) in the forebrain of rats receiving no treatment was 222 ng/g (range: 106-384) with a rather large coefficient of variation (42%). The 5-HT levels of the glucoseinjected rats at 15, 30, 120, and 180 minutes were greater than 500 nglg, but were at 198 ng/g at 60 minutes. Furthermore, 5-HT levels in insulininjected rats dropped to 12 ng/g at 120 minutes, with levels at other time points ranging from 210 to 89 nglg. These phenomenal changes (compared to levels at the adjacent time points) are unlikely genuine biologic responses to treatments. Some of the observations the authors reported in their behavioral experiment can be rationalized on the basis of nutritional influences. Rats were trained to select from two types of food pellets: one contained only carbohydrate (no protein and no fat), whereas the other diet contained 55.5% carbohydrate, -28.1% protein, and 4.8% fat. The training session lasted for 2.5 hours, and after each session rats were given free access to standard laboratory rat food until 18:15. Food was then removed until the next session so that each rat was food-deprived overnight. It is possible that the high preference of the rats for the protein pellets was caused by the overnight food deprivation in combination with the low-protein content of the protein pellets. Within that 2.5 hours, rats basically assembled a diet that simulated the protein content of the standard laboratory food. Failure to alter food choice was not at all surprising if no significant neurochemical changes actually took place (experiment 1). Taking these data at face value, one should also be able to claim that the glucostatic theory’ and insulin theory6 of food intake are not supported, since these treatments did not affect food intake. Indeed, if the “significant” decrease in brain 5-HT after insulin injection were to have a behavioral consequence, one would expect an increase in food intake, since 5-HT is well recognized as an inhibitor of food intake. Increases in brain 5-HT after TRP injection were also not ac-
Nutrition Reviews, Vol. 50,No. 7
companied by decreases in food intake as observed by others (see ref. 7, 8 for review). Perhaps the most interesting point of the paper relates to the observation that after carbohydrate prefeeding, rats preferred the pellets containing protein to the protein-free pellets. Similarly, after protein prefeeding, rats preferred the pellets containing just carbohydrate to the protein-containing pellets. These findings are not and the possibility that such a phenomenon may have resulted from diet discrimination based on the sensory quality of the foods has been ruled out by studies showing nutrient-specific appetite adjustments after macronutrients were delivered by gavage, l 1 thus bypassing orosensory cues. The controversy on the hypothetical 5-HT feedback loop hinges on the notion that diet-induced changes in brain neurochemistry are meaningful to the animal and are involved in the process of food or macronutrient selection. Critics of this working hypothesis have provided data to suggest that neither the control of macronutrient intake nor dietinduced changes in neurochemistry are robust and readily demonstrable. '*,I3 While some of us are still involved in this argument, others have not disputed the presence of nutrient-specific appetite and have forged ahead to demonstrate that serotonin,' dopamine,14 and neuropeptide Y15 are likely to be involved in the control of carbohydrate intake and that abnormalities in brain ~ h e m i s t r y ' ~are " ~ reported in genetically obese Zucker rats that overeat carbohydrate. '',18 It is obvious that the debate in the 1980s has already carried over into the 1990s. Undoubtedly, testing the physiologic-feedback hypothesis in an unbiased and scientifically sound manner is a formidable task since mechanisms controlling ingestive behavior are so complex and redundant .19,20 Edmund T.S. Li, Ph.D. Department of Nutritional Sciences Faculty of Medicine University of Toronto Toronto, Ontario M5S 1A8 Canada 1. Anon. Brain neurochemistry and macronutrient selection: a role for serotonin feedback? Nutr Rev 1992;50:21-2 2. Appetite 1987;8:163-219 3. Holder MD, Huether G. Role of prefeedings, plasma amino acid ratios and brain serotonin levels in carbohydrate and protein selection. Physiol Behav 1990;47:113-9
Nutrition Reviews, Vol. 50, No. 7
4. Wurtman RJ, Hefti F, Melamed E. Precursor control of neurotransmitter synthesis. Pharmacol Rev 1981;32:315-35 5. Mayer J. Regulation of energy intake and body weight: the glucostatic theory and the lipostatic hypothesis. Ann NY Acad Sci 1955;63:1542 6. Woods SC, Porte D. The role of insulin as a satiety factor in the central nervous system. Adv Metab Dis 1983;10:457-68 7. Blundell J. Pharmacological approaches to appetite suppression. TiPS 1991;12:147-57 8. Leibowitz SF. The role of serotonin in eating disorders. Drugs 1990;39 (supp 3):33-48 9. Li ETS, Anderson GH. Meal composition influences subsequent food selection in the rat. Physiol Behav 1982;29:779-83 10. Wurtman JJ, Moses PL, Wurtman RJ. Prior carbohydrate consumption affects the amount of carbohydrate that rats choose to eat. J Nutr 1983;113: 70-8 11. van Zeggeren A, Li ETS. Food intake and choice in lean and obese Zucker rats after intragastric carbohydrate preloads. J Nutr 1990;120:309-16 12. Fernstrom JD. Food-induced changes in brain serotonin synthesis: is there a relationship to appetite for specific macronutrients? Appetite 1987;8: 163-82 13. Harper AE, Peters JC. Protein intake, brain amino acid and serotonin concentrations and protein self-selection. J Nutr 1989;119:677-89 14. Evans KR, Vaccarino FJ. Amphetamine- and morphine-induced feeding: evidence for involvement of reward mechanisms. Neurosci Biobehav Rev , 1990;14:9-22 15. Bray GA. Peptides affect the intake of specific nutrients and the sympathetic nervous system. Am J Clin Nutr 1992;55(supp):265S-71S 16. Routh VH, Murakami DM, Stern JS, Fuller CA, Horwitz BA. Neuronal activity in hypothalamic nuclei of obese and lean Zucker rats. Int J Obes 1990;14:879-92 17. McKibbin PE, Cotton SJ, McMillan S, et al. Altered neuropeptide Y concentrations in specific hypothalamic regions of obese (fdfa) Zucker rats. Diabetes 1991;40:1423-9 18. Enns MP, Grinker JA. Dietary self-selection and meal patterns of obese and lean Zucker rats. Appetite 1983;4:281-93 19. Anderson GH, Li ETS. Glanville NT. Brain mechanisms and the quantitative and qualitative aspects of food intake. Brain Res Bull 1984;12:167-73 20. Anderson GH, Black RM, Li ETS. Physiologic determinants of food selection: association with protein and carbohydrate. In: Anderson GH, Kennedy S , eds. The biology of feast and famine. New York: Academic Press, 1992:73-91
215
July 7992: 214-215
The Viable Serotonin Hypothesis I read the review “Brain Neurochemistry and Macronutrient Selection: A Role for Serotonin Feedback?’” with interest. As was pointed out accurately, the theory that serotonin is part of a feedback loop in the control of diet selection has been the subject of a great controversy in the past decade (see reference 2 for review). I believe, however, that results from the investigation by Holder and Huether3 have not put an end to this controversy. Instead, their investigation serves as a typical example of how one can easily come up with negative findings and claim to “disprove” a hypothesis. The Holder and Huether3 investigation was divided into two components: biochemical and behavioral. It was the authors’ intention that results from these two series of experiments would complement one another. There were, however, numerous designs and methodological problems that did not allow them to test the hypothesis adequately. Thus, readers should take extreme caution in assessing the relevance of their interpretations and conclusion. With the exception of tryptophan (TRP) injection, results of the first experiment were basically negative. In contrast to many published reports (see reference 4 for review), glucose (carbohydrate) and insulin failed to alter the plasma ratio of TRP to other large neutral amino acids (LNAA), brain TRP, and 5-HT (5-hydroxytryptamine) levels. This could have been caused by any one or more of the following factors: 1) No attempt was made to control for food intake prior to or after treatments. It is important to realize that rats were fed only during the light phase of the lightldark cycle so that they were likely to engage in active feeding activity at the time of experimentation. Since food ingestion has a significant physiologic impact on central nervous system functions, this would undoubtedly increase variability within a group. 2) The design of the experiment clearly lacks statistical power. There was a maximum of four rats per time point and, more importantly, only two rats were allocated to each time point in the three treatment groups (glucose, insulin, and tryptophan). Thus, any “reasonable physiologic response” would be masked by a presumably big variance (note that error terms were not reported in the paper). 3) Although brain
214
5-hydroxyindole levels increased after TRP injection, there are reasons to suspect the validity of the brain 5-HT values. Peculiar fluctuations in forebrain 5-HT levels were observed that could only be explained by errors in procedure, storage, or analysis. The average 5-HT level (seven time points) in the forebrain of rats receiving no treatment was 222 ng/g (range: 106-384) with a rather large coefficient of variation (42%). The 5-HT levels of the glucoseinjected rats at 15, 30, 120, and 180 minutes were greater than 500 nglg, but were at 198 ng/g at 60 minutes. Furthermore, 5-HT levels in insulininjected rats dropped to 12 ng/g at 120 minutes, with levels at other time points ranging from 210 to 89 nglg. These phenomenal changes (compared to levels at the adjacent time points) are unlikely genuine biologic responses to treatments. Some of the observations the authors reported in their behavioral experiment can be rationalized on the basis of nutritional influences. Rats were trained to select from two types of food pellets: one contained only carbohydrate (no protein and no fat), whereas the other diet contained 55.5% carbohydrate, -28.1% protein, and 4.8% fat. The training session lasted for 2.5 hours, and after each session rats were given free access to standard laboratory rat food until 18:15. Food was then removed until the next session so that each rat was food-deprived overnight. It is possible that the high preference of the rats for the protein pellets was caused by the overnight food deprivation in combination with the low-protein content of the protein pellets. Within that 2.5 hours, rats basically assembled a diet that simulated the protein content of the standard laboratory food. Failure to alter food choice was not at all surprising if no significant neurochemical changes actually took place (experiment 1). Taking these data at face value, one should also be able to claim that the glucostatic theory’ and insulin theory6 of food intake are not supported, since these treatments did not affect food intake. Indeed, if the “significant” decrease in brain 5-HT after insulin injection were to have a behavioral consequence, one would expect an increase in food intake, since 5-HT is well recognized as an inhibitor of food intake. Increases in brain 5-HT after TRP injection were also not ac-
Nutrition Reviews, Vol. 50,No. 7
companied by decreases in food intake as observed by others (see ref. 7, 8 for review). Perhaps the most interesting point of the paper relates to the observation that after carbohydrate prefeeding, rats preferred the pellets containing protein to the protein-free pellets. Similarly, after protein prefeeding, rats preferred the pellets containing just carbohydrate to the protein-containing pellets. These findings are not and the possibility that such a phenomenon may have resulted from diet discrimination based on the sensory quality of the foods has been ruled out by studies showing nutrient-specific appetite adjustments after macronutrients were delivered by gavage, l 1 thus bypassing orosensory cues. The controversy on the hypothetical 5-HT feedback loop hinges on the notion that diet-induced changes in brain neurochemistry are meaningful to the animal and are involved in the process of food or macronutrient selection. Critics of this working hypothesis have provided data to suggest that neither the control of macronutrient intake nor dietinduced changes in neurochemistry are robust and readily demonstrable. '*,I3 While some of us are still involved in this argument, others have not disputed the presence of nutrient-specific appetite and have forged ahead to demonstrate that serotonin,' dopamine,14 and neuropeptide Y15 are likely to be involved in the control of carbohydrate intake and that abnormalities in brain ~ h e m i s t r y ' ~are " ~ reported in genetically obese Zucker rats that overeat carbohydrate. '',18 It is obvious that the debate in the 1980s has already carried over into the 1990s. Undoubtedly, testing the physiologic-feedback hypothesis in an unbiased and scientifically sound manner is a formidable task since mechanisms controlling ingestive behavior are so complex and redundant .19,20 Edmund T.S. Li, Ph.D. Department of Nutritional Sciences Faculty of Medicine University of Toronto Toronto, Ontario M5S 1A8 Canada 1. Anon. Brain neurochemistry and macronutrient selection: a role for serotonin feedback? Nutr Rev 1992;50:21-2 2. Appetite 1987;8:163-219 3. Holder MD, Huether G. Role of prefeedings, plasma amino acid ratios and brain serotonin levels in carbohydrate and protein selection. Physiol Behav 1990;47:113-9
Nutrition Reviews, Vol. 50, No. 7
4. Wurtman RJ, Hefti F, Melamed E. Precursor control of neurotransmitter synthesis. Pharmacol Rev 1981;32:315-35 5. Mayer J. Regulation of energy intake and body weight: the glucostatic theory and the lipostatic hypothesis. Ann NY Acad Sci 1955;63:1542 6. Woods SC, Porte D. The role of insulin as a satiety factor in the central nervous system. Adv Metab Dis 1983;10:457-68 7. Blundell J. Pharmacological approaches to appetite suppression. TiPS 1991;12:147-57 8. Leibowitz SF. The role of serotonin in eating disorders. Drugs 1990;39 (supp 3):33-48 9. Li ETS, Anderson GH. Meal composition influences subsequent food selection in the rat. Physiol Behav 1982;29:779-83 10. Wurtman JJ, Moses PL, Wurtman RJ. Prior carbohydrate consumption affects the amount of carbohydrate that rats choose to eat. J Nutr 1983;113: 70-8 11. van Zeggeren A, Li ETS. Food intake and choice in lean and obese Zucker rats after intragastric carbohydrate preloads. J Nutr 1990;120:309-16 12. Fernstrom JD. Food-induced changes in brain serotonin synthesis: is there a relationship to appetite for specific macronutrients? Appetite 1987;8: 163-82 13. Harper AE, Peters JC. Protein intake, brain amino acid and serotonin concentrations and protein self-selection. J Nutr 1989;119:677-89 14. Evans KR, Vaccarino FJ. Amphetamine- and morphine-induced feeding: evidence for involvement of reward mechanisms. Neurosci Biobehav Rev , 1990;14:9-22 15. Bray GA. Peptides affect the intake of specific nutrients and the sympathetic nervous system. Am J Clin Nutr 1992;55(supp):265S-71S 16. Routh VH, Murakami DM, Stern JS, Fuller CA, Horwitz BA. Neuronal activity in hypothalamic nuclei of obese and lean Zucker rats. Int J Obes 1990;14:879-92 17. McKibbin PE, Cotton SJ, McMillan S, et al. Altered neuropeptide Y concentrations in specific hypothalamic regions of obese (fdfa) Zucker rats. Diabetes 1991;40:1423-9 18. Enns MP, Grinker JA. Dietary self-selection and meal patterns of obese and lean Zucker rats. Appetite 1983;4:281-93 19. Anderson GH, Li ETS. Glanville NT. Brain mechanisms and the quantitative and qualitative aspects of food intake. Brain Res Bull 1984;12:167-73 20. Anderson GH, Black RM, Li ETS. Physiologic determinants of food selection: association with protein and carbohydrate. In: Anderson GH, Kennedy S , eds. The biology of feast and famine. New York: Academic Press, 1992:73-91
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![[The serotonin hypothesis of depression].](/assets/img/hold.png)
