Brain tryptophan concentrations and serotonin synthesis ... · raise brain tryptophan and serotonin...
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312 Am J Clin Nutr 1995;61:312-9. Printed in USA. © 1995 American Society for Clinical Nutrition
Brain tryptophan concentrations and serotonin synthesisremain responsive to food consumption after the ingestionof sequential meals13
Madelyn H Fernstrom and John D Fernstrom
ABSTRACT The response of brain tryptophan concentra-tion and serotonin synthesis to the ingestion of two sequentialmeals was examined in rats. Fasted rats ingested a carbohy-
drate meal followed 2 h later by a protein-containing meal andwere examined 2 or 4 h after the first meal. Other rats ingesteda protein meal first, followed by a carbohydrate meal. Whenthe carbohydrate meal was fed first, brain tryptophan concen-
trations and serotonin synthesis increased at 2 h; these changeswere reversed at 4 h if the second meal contained protein.When the protein meal was fed first, there were no changes inbrain tryptophan or serotonin at 2 h, and a second carbohydratemeal at 2 h did not raise brain tryptophan or serotonin 2 h later.Carbohydrate ingestion 3 h after a protein meal, however, didraise brain tryptophan and serotonin 2 h later. Brain tryptophan
concentrations and serotonin synthesis are thus responsive tothe sequential ingestion of protein and carbohydrate meals ifthere is a sufficient interval between meals. Am J Clin Nutr
1995;61 :312-9
KEY WORDS Tryptophan, serotonin, brain, rat, diet, di-
etary protein, plasma amino acids, large neutral amino acids
Introduction
The synthesis and release of serotonin by brain neurons is
rapidly influenced by the local tryptophan (Trp) concentration
( 1 , 2). Brain Trp concentrations, in turn, appear to reflect Trpuptake from the circulation; uptake occurs via a transportcarrier, located at the blood-brain barrier (the brain capillaryendothelial cells). The carrier is shared between Trp and sev-
eral other large neutral amino acids (LNAAs) and is competi-tive (3). Changes in the blood concentration of Trp or any of
the other LNAAs can thus alter competition at the transport
sites and thereby influence Trp uptake into brain and brain Trp
concentrations (1).
The ingestion of food is one of the most potent physiological
processes to alter the blood concentrations of Trp and its
LNAA competitors (principally leucine, isoleucine, valine, ty-
rosine, and phenylalanine). By this mechanism, food ingestion
influences serotonin synthesis in brain. It has been observedthat the ingestion of a largely carbohydrate, protein-free meal[the fat content is not important (4)] by fasting rats rapidlyraises brain Trp concentrations and stimulates serotonin syn-
thesis, whereas the consumption of a protein-containing meal
(typically 18-40% protein by wt) fails to raise brain Trp
concentrations or to stimulate serotonin synthesis, despite very
large increments in serum Trp concentrations (1). The carbo-hydrate meal is known to produce its effects on brain Trp and
serotonin via insulin secretion (5), which raises serum Trp
concentrations and lowers the serum concentrations of the
other LNAAs, thereby giving Trp a competitive advantage forbrain transport (1). The protein-containing meal fails to raisebrain Trp concentrations because its ingestion causes serum
Trp concentrations and the concentrations of its transport com-petitors to rise by proportionally similar amounts, resulting in
no net change in competition for uptake. Meal-induced changesin brain Trp concentrations thus appear to be predicted by thealterations produced by the meal in the serum concentration ofTrp relative to that of the other LNAAs, a relationship thatcan be simply expressed as a serum ratio of the Trp con-centration to the sum of the concentrations of the other
LNAAs (Trp/ILNAA); this ratio rises when carbohydrates
are ingested and fails to change after the consumption of
protein-containing meals (1).Over the past decade, the recognition that single meals can
influence serum LNAA concentrations, and thus ultimatelybrain serotonin synthesis, has led to the suggestion that thebrain may use this metabolic and neurochemical cascade tomonitor the recent history of carbohydrate and/or protein
consumption. Indeed, in some hypotheses of macronutrientappetite regulation, brain Trp concentrations and serotonin
synthesis are viewed as fluctuating from meal to meal as a
function of the protein and carbohydrate contents of themeal (6, 7). These food-induced neurochemical changes are
then said to be used by the brain to decide the macronutrient
selection at the next meal.
Appealing though such hypotheses are, they involve a majorassumption regarding food intake and serotonin synthesis,namely that brain Trp uptake and serotonin synthesis can vary
1 From the Departments of Psychiatry, Pharmacology, and Behavioral
Neuroscience, University of Pittsburgh School of Medicine.2 Supported by a grant from the National Institutes of Health (HD24730)
and an NIMH Research Scientist Award (MH00254) to JDF.
3 Address reprint requests to MH Fernstrom, Department of Psychiatry,
University of Pittsburgh School of Medicine, Western Psychiatric Institute
and Clinic, 3811 O’Hara Street, Pittsburgh, PA 15213.
Received November 29, 1993.
Accepted for publication September 8, 1994.
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FOOD INGESTION AND BRAIN SEROTONIN 313
on a meal-to-meal basis. However, the only studies that have
examined the relationship of food ingestion to brain serotonin
synthesis have involved feeding large, single meals to rats after
an overnight fast. No attempts have been made to determine
whether the ingestion of a carbohydrate- or protein-containing
meal by a nonfasting rat would produce the same effects on
brain Trp concentrations and serotonin synthesis as occur after
their ingestion by fasting animals.
The present studies therefore examine the issue of whether
nonfasting rats that ingest a carbohydrate- or protein-contain-
ing meal show changes in the serum LNAA pattern, brain Trp
concentration, and serotonin synthesis similar to those ob-
served in fasting rats. In particular, we conducted studies in rats
fed two sequential meals after an overnight fast. The meal size
(16 kcal, 67 kJ) and intermeal interval (2 h) were high, but in
the physiological range for rats (8, 9), and were chosen to
ensure that biochemical changes would be detected if they
occurred.
Materials and methods
All experimental procedures were approved by the
University of Pittsburgh Animal Care and Use Committee.
Male Sprague-Dawley rats weighing 175-200 g (Hilltop
Laboratories, Scottdale, PA) were acclimated to our animal
quarters for 7 d before experimentation. During this time, water
and food (Purina Rodent Laboratory Chow 5001; Purina Mills,
St Louis) were provided ad libitum. The animals were exposed
to 12 h of light daily (0700-1900) and an ambient temperature
of 22 #{176}C.At 1700 on the day before each experiment, rats were
deprived of food but not water. At 0900 the next morning,
groups of seven rats were given free access to a 4-g meal (dry
wt), or remained fasting. All food was consumed within 15
mm. In some experiments, rats received only one meal while in
others they received a second 4-g meal, presented 2 or 3 h after
the first meal. All rats were decapitated 2 h after their last meal.
Thirty minutes beforehand, they received an injection of m-
hydroxybenzylhydrazine (NSD-1015), an inhibitor of aromatic
L-amino acid decarboxylase, to allow estimation of serotonin
synthesis rate (10). The brains were rapidly removed and
placed on an ice-cold glass plate. The hypothalamus and pieces
of cerebral cortex were removed from each brain (1 1) and
rapidly frozen on dry ice. Blood was collected from the cervi-
cal wound into glass tubes and allowed to clot in an ice bath.
The sera were then harvested after low-speed centrifugation at
1200 x g for 20 mm at 4 #{176}Cand aliquoted into test tubes. All
samples were stored at -70 #{176}Cuntil assayed.
The meals were prepared by using ingredients obtained from
Teklad (Madison, WI). The 0% protein (carbohydrate) diet
contained (in g/kg dry wt) 250 g sucrose, 200 g dextrose, and
460 g dextrin. The 6% protein diet (6% protein by wt, 5.9% of
total energy) contained 60 g casein, 250 g sucrose, 200 gdextrose, and 400 g dextrin. The 12% protein diet (12% by wt,
I 1.7% of total energy) contained 120 g casein, 250 g sucrose,
200 g dextrose, and 340 g dextrin. The 24% protein diet (24%
by wt, 23.5% of total energy) contained 240 g casein, 250 g
sucrose, 200 g dextrose, and 220 g dextrin. The 40% protein
diet (40% by wt, 39. 1% oftotal energy) contained 400 g casein,
250 g sucrose, 200 g dextrose, and 60 g dextrin. In addition to
these ingredients, each diet contained 50 g Mazola Oil (CPC
International, Englewood Cliffs, NJ) and 40 g agar. The agar
was mixed with 1 L water, heated, and then homogenized with
the other ingredients. After cooling, the final food had a firm
consistency. The energy density of the diets was ‘�17.2 kJ/g
dry wt (4. 1 kcal/g dry wt).
Trp concentrations in serum and brain were quantitated
fluorometrically (12-14) with a Hitachi F-2000 (HitachiInstruments, Danbury, CT) spectrophotofluorometer. The
concentrations of other LNAAs in serum (tyrosine, phenyl-
alanine, leucine, isoleucine, and valine) were quantitated by
using a Beckman 6300 amino acid analyzer, equipped for
fluorometric detection of o-phthalaldehyde derivatives of
the amino acids (15). 5-Hydroxytryptophan (5-HTP) con-
centrations in brain were measured by using HPLC and
electrochemical detection (16).
The data were analyzed statistically by using one-way analysis
of variance and the Newman-Keuls test, or Student’s t test (17).
Results
In initial studies, rats were tested by using two diets: 0% and
40% protein. Animals were fasted overnight; the next morning
at 0900 they either remained without food or received a car-
bohydrate (0% protein) meal (4 g dry wt). Ninety minutes later,
one group of fasted rats and one group of rats consuming
carbohydrates received an injection of NSD-1015 and were
killed 30 mm later (2 h after the meal was presented). Other
groups of rats fed the carbohydrate meal at 0 h received a
second meal at 2 h, consisting of either 0% (carbohydrate) or
40% protein. Another group of fasted rats remained without
food. Ninety minutes later, all rats received NSD-l0l5 and
were killed 30 mm later (4 h after the first meal was presented).
The results are presented in Figure 1 . The rats consumed (dry
wt/rat) 3.55 ± 0.1 g (61.1 ± 1.7 kJ, 14.6 ± 0.4 kcal) ofthe first
carbohydrate meal, 2.5 ± 0.45 g (42.7 ± 7.5 kJ, 10.2 ± 1.8
kcal) of the second carbohydrate meal, and 3.45 ± 0.3 g (59 ±
5 Id, 14.1 ± 1.2 kcal) ofthe 40% protein meal. Ingestion of thefirst carbohydrate meal caused the serum Trp/�LNAA to rise
�=60% over fasting values at 2 h. This increase was accompa-
nied by significant increases over fasting values in Trp con-
centrations and in 5-HTP accumulation rates in both cortex and
hypothalamus (Fig 1). After ingestion of the second meal,
presented 2 h after the first meal, the serum Trp/INAA re-
mained high at 4 h if the second meal was carbohydrates, as did
Trp concentrations and 5-HiT synthesis rates in both cortex
and hypothalamus. However, if the second meal contained 40%
protein, the serum Trp/ILNAA, as well as Trp concentrations
and 5-HTP accumulation rates in cortex and hypothalamus, all
declined to or below fasting control values (Figure 1).
The next studies were designed to determine the dietary
protein concentration necessary in the second meal to reverse
the increments in brain Trp concentrations and 5-HTP synthe-
sis produced by an initial meal of carbohydrates. In these
experiments, rats were fasted overnight and presented at 0900
the next morning with either nothing or with a portion of the
carbohydrate diet (4 g dry wt). Two hours later, groups of rats
either remained without food or received a second meal
(4 g dry wt) of either carbohydrate or 6%, 12%, 24%, or 40%
protein. As before, they received NSD-1015 90 mm after the
second meal was presented and were killed 30 mm thereafter.
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A. . . .B
25
V0� 15
10
200
� 150
serun�TRPn;LNA:a�
In
Coilical TRP �.
CortIcal 5HTP
l� ‘!�
314 FERNSTROM AND FERNSTROM
0.40
______ ‘iid il� I�
10 � 5H1P
9 ** **
�: *
_____ � I�I!TTh�)
FIGURE 1. Effect of ingesting sequential meals of carbohydrates or
carbohydrates and 40% protein on brain tryptophan (Trp) concentrations
and 5-hydroxytryptophan (SHiP) synthesis. A, cerebral cortex; B, hypo-
thalamus. At 2 h: open bar = fasted; shaded bar carbohydrate meal. At
4 h: open bar = fasted; shaded bar two sequential carbohydrate meals;
black bar = carbohydrate meal followed by protein meal. * ,D < 0.05, ** P< 0.01 vs fasted groups at 2 h (t test). * �P < 0.05, ** P < 0.01 vs fastedvalues at 4 h (Newman-Keuls test). .1 ± SEM; n = 7/group.
In this study all animals rapidly consumed all of the food at
both meals. The ingestion of any of the meal pairs raised serum
Trp concentrations significantly over fasting values (Table 1).As in the initial experiments, the ingestion of two sequentialcarbohydrate meals caused the serum Trp/ILNAA to rise
(Table 1). Cortical and hypothalamic Trp concentrations and
TABLE 1
5-Hi? synthesis rates were also significantly elevated above
fasting control values. Also analogous to the first study, inges-tion of an initial carbohydrate meal followed by a 40% protein
meal caused the serum Trp/ILNAA, brain Trp concentrations,
and 5-Hi? synthesis to be at or below fasting control values
(Table 1). The failure of the ratio to exceed fasting values,despite the rise in serum Trp concentrations, was attributable to
the large increase in the serum concentrations of the other
LNAAS. Between these two endpoints, the serum Trp/ILNAAand each of the brain variables could be seen to fall gradually
from the highest values, obtained after two meals of carbohy-
drates, to the lowest values, obtained after ingestion of carbo-
hydrates followed by 40% protein. Of particular note, the
serum Trp/ILNAA and cortical and hypothalamic Trp and5-HTP concentrations were as high when carbohydrates were
followed by a 6% protein meal as the values obtained when two
meals of carbohydrates were ingested. At 12% protein, the
serum Trp/ILNAA and cortical Trp concentrations were sig-
nificantly elevated above fasting concentrations, but hypotha-
lamic Trp and cortical and hypothalamic 5-HTP accumulation
rates were not. At 24% protein, the serum Trp/ILNAA was not
significantly elevated over control values, because the second
meal elevated the serum concentrations of the other LNAAs by
almost as much, proportionally, as the serum Trp concentration
(Table 1). Brain Trp concentrations and 5-HTP synthesis were
also at fasting values. From these studies it is apparent that
when a protein-containing meal is consumed 2 h after a car-
bohydrate meal, the protein content of the second meal must
contain >6% protein to lower Trp concentrations and 5-HTP
synthesis rates in the brain.
Another series of studies examined the ability of a second
meal of carbohydrates to increase the serum Trp/ILNAA and
brain Trp and 5-Hi? concentrations after an initial meal con-
taming protein. As a preliminary step in this study, we evalu-ated the effects of single meals containing different amounts of
protein on the serum Trp/ILNAA, and on cortical and hypo-
thalamic concentrations of Trp and 5-I-ITP synthesis. As antic-
ipated, a single meal of carbohydrates caused all of thesevariables to rise significantly 2 h later (Table 2). Smaller but
nonetheless significant increases were also evident after inges-
Changes in tryptophan (Trp) concentrations and 5-hydroxytryptophan (5-HTP) synthesis rate in cerebral cortex and hypothalamus in rats ingesting a
carbohydrate (CHO) meal followed by a protein-containing meal’
Group Serum lipSerumLNAA
Serum Trp/�LNAA
Trp 5-HTP
Cortex Hypothalamus Cortex Hypothalamus
pjnollL p.snolIL nmol/g �i.molJg protein ng/g p.g/g protein
No food 96 ± 6 482 ± 14 0.19 ± 0.01 28 ± 2 0.34 ± 0.01 113 ± 7 4.1 ± 0.1
CHO-CHO 140 ± 62 376 ± 21 0.38 ± 0.022 36 ± 22 0.41 ± 0.032 159 ± 112 5.5 ± 0.32
CHO-�’6% protein 150 ± 52 437 ± 28 0.35 ± 0.022 36 ± 22 0.38 ± 0.01� 171 ± 112 5.8 ± 0.42
CHO-�12% protein 129 ± 62 385 ± 27 0.33 ± 0.032 35 ± 12 0.35 ± 0.01 136 ± 8 4.4 ± 0.3
CHO-�24% protein 158 ± 10� 675 ± 432 0.24 ± 0.01 28 ± 1 0.30 ± 0.0i3 110 ± 5 4.1 ± 0.2
CHO-+40% protein 159 ± 9� 976 ± 472 0.16 ± 0.01 23 ± 12 0.28 ± 0.012 95 ± 7 3.4 ± 0.2
F 10.74� 45�474 26.67� 17.90� 22.35� 13.22� 13.36�
‘ .1 ± SE. Groups of seven rats, fasted overnight, ingested at 0 h either no food or CHO (4 g dry wt). At 2 h the animals received a second meal: 4 g
dry wt of 0%, 6%, 12%, 24%, or 40% protein (except for the fasted group); 90 mm thereafter, all rats received NSD-1015 and were killed 30 mm later.
LNAA, large neutral amino acids.
2.3 Statistically significant vs no food values (Newman-Keuls test): 2 p < 0.01, -� p < 0.05.
4 Statistically significant vs no food values, P < 0.01 (ANOVA).
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FOOD INGESTION AND BRAIN SEROTONIN 315
TABLE 2
Changes in tryptophan (Trp) concentrations and 5-hydroxytryptophan (5-HTP) synthesis rate in cerebral cortex and hypothalamus in rats ingesting
single meals containing different amounts of protein’
Group Serum TrpSerumLNAA
Serum Trp/�LNAA
Trp 5-HTP
Cortex Hypothalamus Cortex Hypothalamus
pinoliL pinoliL nmol/g junol/g protein ng/g p.g/g protein
No food 85 ± 8 603 ± 16 0.15 ± 0.01 25 ± 1 0.39 ± 0.01 124 ± 5 4.3 ± 0.2
CHO 128 ± 72 454 ± 28 0.30 ± 0.042 34 ± 22 0.54 ± 0.012 184 ± 82 6.3 ± 0.22
6% Protein 130 ± 42 462 ± 14 0.29 ± 0.012 29 ± 2 0.48 ± 0.012 149 ± 6� 5.2 ± 0.22
12% Protein 123 ± 92 578 ± 48 0.21 ± 0.02� 23 ± 2 0.42 ± 0.01 136 ± 10 4.5 ± 0.2
24% Protein 137 ± 122 728 ± 392 0.17 ± 0.01 27 ± 1 0.38 ± 0.01 133 ± 5 4.1 ± 0.1
40% Protein 170 ± 112 1223 ± 59� 0.14 ± 0.01 20 ± 12 0.35 ± O.Oi� 111 ± 5 3.9 ± 0.1
F 9.21� 49.58� 14.23� 12.37� 35.85� 14.21� 20.82�
‘ .1 ± SE. Groups of seven male rats, fasted overnight, ingested at 0 h either no food or 4 g dry wt of one of the diets indicated in the table (all 4 g was
consumed); 90 mm thereafter, all rats received NSD-1015 and were killed 30 mm later. LNAA, large neutral amino acids; CHO, carbohydrate.
2.3 Statistically significant vs no food values (Newman-Keuls test): 2 p < 0.01, �? p < 0.05.
4 Statistically significant vs no food values, P < 0.01 (ANOVA).
tion of the 6% protein meal (except for cortical Trp, which
increased but not significantly so in this experiment). Con-
sumption of the 12%, 24%, or 40% protein meals did not
elevate any of the brain variables above fasting control values,though ingestion of 12% protein did cause a small rise in theserum Trp/ILNAA. Also, the 40% protein meal caused noincrease over fasting values in the serum Trp/�LNAA despitethe large rise in serum Trp concentrations, because of thesubstantial increments in the serum concentrations of the otherLNAAs. Significant reductions in cortical and hypothalamic
concentrations of Trp also occurred (5-Hi? synthesis also
declined, but not significantly so).Other groups of fasted rats were then given an initial meal of
either carbohydrates or 6%, 12%, 24%, or 40% protein. Two
hours later a second meal was offered that consisted of carbo-hydrates only. The rats were killed 2 h after the second meal,
and had received NSD-1015 30 mm beforehand. As in theother studies, serum Trp concentrations rose after the ingestionof each of the meals (Table 3). The serum Trp/ILNAA and
cortical and hypothalamic concentrations of Tip and 5-HTP
were increased at the 4-h time point in animals consuming two
consecutive carbohydrate meals (Table 3). If the rats consumed
TABLE 3
6% protein as their first meal, the serum Trp/ILNAA and all of
the brain variables measured were almost as high after the
ingestion of the second carbohydrate meal as they were when
both meals had been carbohydrate. When the initial meal
contained 12% or 24% protein, the second (carbohydrate) meal
raised the serum Trp/ILNAA above fasting values, but cortical
and hypothalamic Trp concentrations were not significantly
increased, and 5-Hi? accumulation did not rise significantly
over fasting values (for cortex or hypothalamus). At 40%
protein, neither the serum Trp/ILNAA nor any of the brainvariables was significantly increased over fasting values (Table
3). The principal conclusion from these results is that a second
meal of carbohydrates, 2 h after an initial meal of 12-40%
protein, does not significantly increase brain Trp concentra-tions or serotonin synthesis.
A final series of studies was conducted to determine whether
a longer interval between the first and second meals would
allow a second carbohydrate meal to elevate brain Trp and
5-HTP after an initial meal of high protein content (24%
protein). An intermeal period of 3 h was selected. As a pre-
liminary step, we measured cortical and hypothalamic Trp
concentrations and 5-HTP synthesis 3 h after fasting rats re-
Changes in tryptophan (Tm) concentrations and 5-hydroxytryptophan (5-HiP) synthesis rate in cerebral cortex and hypothalamus in rats ingesting a
protein-containing meal followed by a carbohydrate (CHO) meal’
Group
Serum
Trp
Serum
LNAASerum Trp:
�LNAA
Trp 5-HTP
Cortex Hypothalamus Cortex Hypothalamus
pinoliL p.mol/L nmol/g pinolig protein ng/g pg/g protein
No food 142 ± 3 731 ± 36 0.20 ± 0.01 26 ± 1 0.35 ± 0.01 125 ± 1 4.3 ± 0.1
CHO-CHO 162 ± 82 497 ± 442 0.33 ± 0.012 33 ± 12 0.41 ± 0.0i3 181 ± 102 5.4 ± 0.22
6%-’CHO 165 ± 72 533 ± 132 0.31 ± 0.012 31 ± i� 0.40 ± 0.02� 173 ± 10�� 5.2 ± 0.12
12%-*CHO 190 ± 92 521 ± 272 0.31 ± 0.022 27 ± 1 0.34 ± 0.02 152 ± 3 5.0 ± 0.2
24%-*CHO 184 ± 72 643 ± 122 0.28 ± 0.012 28 ± 1 0.33 ± 0.01 141 ± 6 4.6 ± 0.2
40%-CHO 194 ± 62 814 ± 362 0.24 ± 0.01 25 ± 1 0.35 ± 0.01 141 ± 12 4.6 ± 0.5
F 8.86� 14.96� 11.45� 7.01� 4.00� 8.79� 4.90�
‘ .1 ± SE. Groups of seven male rats, fasted overnight, ingested at 0 h either no food or 4 g dry wt of 0%, 6%, 12%, 24%, or 40% protein. At 2 h the
animals received a second meal of CHO (4 g dry wt, except for the fasted group); 90 mm thereafter all rats received NSD-1015 and were killed 30 mm
later. LNAA, large neutral amino acids.
2.3 Statistically significant vs no food values (Newman-Keuls test): 2 p < 0.01, -� P < 0.05.
4 Statistically significant vs no food values, P < 0.01 (ANOVA).
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316 FERNSTROM AND FERNSTROM
ceived a 4-g meal (dry wt) of carbohydrates or 24% protein
(Table 4). The results were very similar to those obtained 2 h
after the ingestion of either meal (Table 2). That is, ingestion of
the carbohydrate meal raised Trp concentrations and 5-HTP
synthesis rates in brain, whereas the consumption of the 24%
protein meal did not (Table 4). (Both meals raised serum Trp
concentrations, with the protein-containing meal producing agreater effect, as in the 2-h studies; the serum concentrations of
the other LNAAs were not measured in this experiment.) Other
groups of fasted rats were then studied by using the two-meal
paradigm: one group ingested two meals of carbohydrates (0%
protein), a second group consumed two meals of 24% protein,and a third group consumed an initial meal of 24% protein
followed by a second meal of carbohydrates (Table 5). The
second meal was offered 3 h after the initial meal. As before,
all rats received NSD-1015 30 mm before being killed (at 5 h).
The serum Trp/ILNAA, cortical and hypothalamic Trp con-
centrations, and 5-HTP synthesis rates were all significantly
higher in rats ingesting two sequential carbohydrate meals than
in animals consuming two 24% protein meals. These variables
were all significantly elevated as well in rats ingesting the
protein meal followed by the carbohydrate meal, when com-
pared with values in animals consuming two 24% protein
meals. Thus, an intermeal interval of 3 h appears to be suffi-
cient to allow a meal of carbohydrates subsequent to a meal of
24% protein to raise brain Trp concentrations and 5-HTP
synthesis to values comparable with those obtained when two
meals of carbohydrates are consumed.
Discussion
These results demonstrate that brain Trp concentrations and
the rate of serotonin synthesis [as estimated via 5-HiP accu-mulation rate (10)] respond predictably to the sequential inges-
tion of two meals differing in protein content, though with
some qualifications. An initial meal of carbohydrates (0%
protein) raised brain Trp concentrations and serotonin synthesis
2 h after its ingestion. A second meal containing moderate to
large amounts of protein, ingested 2 h after the first, generally
reversed these effects 2 h later. If the initial meal contained
protein (� 12%), brain Trp concentrations and serotonin syn-
thesis were usually unchanged at 2 h (an anticipated result).
However, a second meal containing carbohydrates (0%
protein) offered at 2 h failed to raise brain Trp concentrations
TABLE 4
or to stimulate serotonin synthesis 2 h thereafter. However, if
the second meal of carbohydrates was offered 3 h after the
protein-containing meal, Trp concentrations and serotonin syn-
thesis rates 2 h later were as high as those seen in animals
consuming two meals of carbohydrates. We conclude that brain
Trp concentrations and the serotonin synthesis rate, at least in
the present context, can respond to the presence or absence of
protein in a meal, even in nonfasted animals, but that this
continued responsivity requires a significant intermeal interval.
Some of the present findings were not unexpected; namely,
that when a protein meal follows a carbohydrate meal, the
serum Trp/ILNAA would fall and brain Trp concentrations
and 5-HTP synthesis would decline. This expectation derived
from some [though not all (18)] earlier reports showing that
protein ingestion can lower the serum Trp/ILNAA and brain
Trp (19, 20). The same effect could be predicted in rats ingest-
ing protein after a carbohydrate meal: the influx of competitors
to Trp into the blood after the protein meal would lower the
already elevated serum Trp/�LNAA (Table 1). The unantici-
pated result was that a carbohydrate meal failed to raise brain
Trp concentrations and 5-HTP synthesis when consumed 2 h
after a protein meal (12-40% by wt; Table 3). This nonrespon-
siveness to carbohydrate might have been due to the lingeringof elevated serum concentrations of Trp’s LNAA competitors
after the first protein meal (Table 2). This would make it
difficult for the second carbohydrate meal (and insulin secre-
tion) to reduce LNAA concentrations sufficiently to raise the
serum Trp/ILNAA (and thus brain Trp concentrations and
serotonin synthesis). This possibility is consistent with the
results of an experiment in which the intermeal interval was
lengthened to 3 h. In this case, the serum Trp/ILNAA, brain
Trp concentrations, and Trp hydroxylation rate were all in-
creased by a carbohydrate meal that followed a 24% protein
meal (Table 5). Perhaps the longer intermeal interval allowed
time for serum LNAA concentrations and insulin concentra-
tions to fall sufficiently toward baseline to permit a subsequent
carbohydrate meal (and insulin secretion) to produce a marked
lowering of serum LNAA concentrations, and thus an increase
in the serum Trp/�LNAA (Table 5).
Is this temporal difference in the ability of a carbohydrate
meal to raise brain Trp concentrations and serotonin synthesis
compatible with current hypotheses regarding the role of meal-induced changes in brain serotonin synthesis in carbohydrate
intake regulation? The notion that carbohydrate intake is reg-
Changes in tryptophan (Tip) concentrations and 5-hydroxytryptophan (5-HTP) synthesis rate in cerebral cortex and hypothalamus in rats 3 h after
ingesting single meals containing different amounts of protein’
GroupSerum
Trp
Trp 5-HTP
Cortex Hypothalamus Cortex Hypothalamus
pnzollL nmol/g p.znol/g protein ng/g p.g/g protein
No food 88 ± 6 20 ± 1 0.45 ± 0.02 113 ± 5 4.0 ± 0.2
CHO 116 ± 62 26 ± 12 0.57 ± 0.022 156 ± 42 5.3 ± 0.22
24% Protein 140 ± 72 19 ± 1 0.47 ± 0.02 115 ± 5 4.2 ± 0.1
F 16.14� 17.50�’ 12.27� 23.59� 20.57�
‘ .t ± SE. Groups of seven male rats, fasted overnight, ingested at 0 h either no food or 4 g dry wt of one of the diets indicated in the table (all 4 g was
consumed); 150 mm thereafter all rats received NSD-1015 and were killed 30 mm later. CHO, carbohydrate.
2 Statistically significant vs no food values, P < 0.01 (Newman-Keuls test).
3 Statistically significant vs no food values, P < 0.01 (ANOVA).
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FOOD INGESTION AND BRAIN SEROTONIN 317
TABLES
Changes in tryptophan (Tm) concentrations and 5-hydroxytryptophan (5-HTP) synthesis rate in cerebral cortex and hypothalamus in rats ingesting a
24% protein meal or a carbohydrate (CHO) meal followed 3 h later by a second CHO or protein meal’
Group
Serum
Tip
Serum
LNAA
Serum Trp/
�LNAA
Trp 5-HTP
Cortex HypothalamusCortex Hypothalamus
j.unol/L j.unol/L nmol/g pinolig protein ng/g �g/g protein
Protein-+protein 155 ± 7 680 ± 18 0.24 ± 0.01 21 ± 2 0.39 ± 0.02 123 ± 5 3.7 ± 0.2
Protein-*CHO 157 ± 7 475 ± 252 0.34 ± 0.022 27 ± 12 0.49 ± 0.03� 169 ± 72 4�7 ± 0.22
CHO-*CHO 132 ± 33 408 ± 282 0.34 ± 0.03� 29 ± 12 0.52 ± 0.04� 186 ± 72 5.0 ± 0.12
F 5#{149}474 26.16� 5.88� 8.89� 6.10� 25.79� 22.08�
‘ I ± SE. Groups of seven male rats, fasted overnight, ingested at 0 h 4 g dry wt of either 0% (CHO) or 24% protein. At 3 h the animals received a
second 4-g meal of protein or CHO; 90 mm thereafter all rats received NSD-1015 and were killed 30 mm later. Each rat consumed all of the first and second
meals (4 g; 68.6 kJ, 16.4 kcal). LNAA, large neutral amino acids.
2.3 Statistically significant vs protein -� protein values (Newman-Keuls test): 2 p < 0.01, � p < 0.05.
4 Statistically significant vs protein -� protein values, P < 0.01 (ANOVA).
ulated, and that serotonin neurons participate in this regulation,
was assembled primarily from three sets of data: 1) that single
carbohydrate meals consumed by fasted rats would rapidly
elevate the serum Trp/ILNAA, brain Trp concentrations, and
serotonin synthesis, whereas ingestion of a protein-containing
meal would not (21); 2) that the injection of drugs that selec-
tively enhance transmission across serotonin synapses (ie, se-
rotonin agonists and re-uptake blockers) selectively reduces the
ingestion of carbohydrates by rats and humans (22-29); and 3)that rats can change their selection of carbohydrates from meal
to meal in both experimental (29, 30) and free-feeding (31, 32)
contexts. Together, these data have been synthesized into a
negative feedback model for regulating meal-to-meal appetite
for carbohydrates (30, 33). When a rat consumes carbohy-
drates, the serum Trp/ILNAA rises, causing brain Trp uptake
and serotonin synthesis to increase (21), which presumably
leads to an enhancement of neuronal serotonin release (2). The
result is a reduction in carbohydrate (and an increase in protein)
intake at the next meal. At the next meal, more protein (and less
carbohydrate) is ingested, leading to a reduction in the serum
Trp/�LNAA and a decline in brain Trp concentrations and
serotonin synthesis and release. The inhibition of carbohydrate
intake is thus relieved, and carbohydrate ingestion increases at
the succeeding meal. Thus, this feedback mechanism, as envi-
sioned, has both rats and humans proceeding to each meal,
choosing more carbohydrate or protein on the basis of whether
brain Trp concentrations and serotonin synthesis have been
raised or lowered at the preceding meal. Indeed, certain forms
of obesity in humans have been attributed to a breakdown in
this feedback loop, with obesity resulting from a deficiency of
serotonin in the brain and thus an excessive craving for
carbohydrates (23).
The present results probably detract from this hypothesis
because of the findings associated with feeding carbohydrate
meals after protein-containing meals. For the feedback loop to
work, it must be responsive to each meal. However, our data
show that a rat needs 3 h after a protein-containing meal to
exhibit an increase in brain serotonin synthesis when a subse-
quent carbohydrate meal is consumed. When the interval is 2 h,
carbohydrate ingestion does not stimulate serotonin synthesis
(Table 3). In a free-feeding context, rats consume many meals
during the night (the normal feeding period), sometimes sepa-
rated by 2 h but more often by less (8, 9, 32). Given such short
time intervals, it is difficult to envision in most cases that a
carbohydrate meal would be able to raise the serum Trp/
ILNAA when consumed after a protein meal. Such nonrespon-
siveness, if it exists, makes the model untenable. However, this
conclusion cannot be drawn definitively until macronutrient
intake patterns are followed temporally in a free-feeding con-
text, and accompanied by on-line measurements of serotonin
synthesis or release [such as might be possible using in vivo
microdialysis (34)].
The issue of the intermeal interval between protein and
carbohydrate meals is the same for humans. However, because
the rate of metabolism in rats is much greater than that in
humans (35), a minimum intermeal interval of 3 h in rats might
be substantially longer in humans. Normal-weight humans eat
on average 5-7 times/d (though some eat 10 times/d) (36, 37).
Hence, the intermeal-intersnack intervals in humans may be
too short at some times of day to allow a carbohydrate meal or
snack to raise the serum Trp/ILNAA if it follows a protein-
containing meal. In this case, the meal-to-meal feedback hy-
pothesis, as described above, could not reliably work in hu-
mans. A similar argument would apply to individuals (eg,
so-called “carbohydrate cravers”) who consume above normal
numbers of snacks each day (22). To clarify this issue further,
it would be of interest to conduct a study in which the normal
food-intake pattern of human subjects was recorded throughout
the day, in association with frequent measurements of the
serum Trp/ILNAA.
A broader question is whether the serum Trp/ILNAA is
sufficiently responsive to meals and snacks in general to pro-
duce notable changes in serotonin synthesis and attendant brain
functions. If not, then it is of little interest whether this ratio
rises after one carbohydrate meal but not after another. In both
cases there would be no effect on the brain (at least in relation
to serotonin synthesis and function). Indeed, some investigators
suggest as much. Teff et al (38) report that the modest changes
in the serum Trp/ILNAA that follow the ingestion of a car-
bohydrate or protein-containing breakfast are not sufficient to
influence cerebrospinal fluid concentrations of Trp (an index ofbrain Trp concentrations in humans). The ratio changes they
obtained are of similar magnitude to those observed by others
using similar dietary treatments (39, 40). Teff et al (38) con-elude from their studies that protein and carbohydrate meals
fail to alter human brain Trp concentrations sufficiently to
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318 FERNSTROM AND FERNSTROM
influence serotonin synthesis. Delgado et al (41), however,observed a short-term, substantial lowering of mood in remit-
ted depressed patients after the oral administration of an amino
acid mixture that reduces brain Trp concentrations in rats (42).
Such an effect is consistent with the well-established relation-
ship between affective state and brain serotonin (ie, that en-
hancing serotonin release elevates mood, whereas reducing it
depresses mood) (43). Their amino acid treatment causes a
short-term reduction in the serum Trp/ILNAA (44), the mag-
nitude of which is similar to that observed after the ingestion of
carbohydrate or protein meals by humans (39, 40, 45). This
recognition suggests that changes in the serum Trp/ILNAA of
this size might well produce changes in brain function that
signal significant alterations in brain Trp uptake and serotonin
synthesis. Regardless of the ultimate resolution of this issue,
note that this issue remains unresolved.
Three technical points deserve comment. First, we have
focused on Trp concentrations and serotonin synthesis in the
cerebral cortex and the hypothalamus. The hypothalamus was
selected for study because of its role in appetite regulation (46).
An examination of the ability of meals to influence biochem-
ically serotonin neurons projecting into this region was thus of
great interest, particularly because these serotonin projections
are likely to be involved in food-intake regulation (47). Cere-
bral cortex was examined because it is a major recipient of
serotonin axons and nerve terminals (48), but is not known tohave a key role in food-intake regulation. Using cortex, we
could thus explore whether serotonin synthesis in nerve termi-
nals not linked to appetite control is likely to be influenced by
meal-induced changes in Trp concentrations. Clearly, the re-
sults support the view that all serotonin projections are likely to
be influenced by meals, a finding consistent with an earlier
report that carbohydrate ingestion increases Trp concentrations
and serotonin synthesis in several brain regions (49). Though it
is not presently clear why all serotonin neurons should be
susceptible to food-induced changes in transmitter synthesis,
the effects on those projecting into the hypothalamus might be
to influence local appetite circuits.
Second, the present studies have used meals of large, but
normal size (8, 9). The issue of meal size is important because,
in previous single meal studies using fasting rats, animals
consumed a high proportion of their daily energy intake as asingle, large meal. The neurochemical responses, it could thus
be argued, might not be predictive of effects obtained after a
meal of normal size. The present results suggest that the
responses may be the same in the two dietary contexts. And
third, the present results indicate that a meal containing �6%
but < 12% protein raises the serum Trp/ILNAA, brain Trp
concentrations, and serotonin synthesis (Table 2). This findingdiffers from that of Yokogoshi and Wurtman (50), who re-
ported that a meal containing 5% casein was associated with nopostingestion increase in the serum Trp/ILNAA (neither brain
Trp concentrations nor serotonin synthesis were measured in
their study). We have no simple explanation for this difference,
except to note that several days before experimentation, they
entrained their animals to a feeding schedule that included an
extended period of fasting each day (20 h). Metabolic effects
may therefore have been present during their test meal that do
not occur in animals that are fasted only once, the night before
the test meal is presented.
Finally, although the present results focus on serotonin’s role
in food-intake sensing and regulation by the brain, serotonin
neurons are but one of many neurochemically specific cells in
the brain that participate in appetite regulation. Ultimately,
information regarding the participation of serotonin neurons in
brain circuits that monitor nutrient intake and influence appe-
tite must be integrated with knowledge regarding other neuro-
chemically specific neurons that function in the same brain
circuitry before a full understanding of the brain’s role infood-intake regulation can be achieved. El
We gratefully acknowledge the expert technical assistance of Alissa
Ebaugh, Karl Kovalkovich, and Terre Constantine.
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